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Investigations on the muscular dystrophy protein emerin: from nuclear mechanotransduction to molecular interactions

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Investigations on the muscular dystrophy protein emerin: from nuclear mechanotransduction to molecular interactions

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Content INVESTIGATIONS ON THE MUSCULAR DYSTORPHY PROTEIN EMERIN:
FROM NUCLEAR MECHANOTRANSDUCTION TO
MOLECULAR INTERACTIONS
by
Markville Bulosan Bautista
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
December 2024
Copyright 2024 Markville Bulosan Bautista

ii
DEDICATION
For Rodrigo and Patricia

iv
TABLE OF CONTENTS
Dedication ........................................................................................................................... ii
List of Tables ....................................................................................................................... vii
List of Figures...................................................................................................................... viii
Abstract ............................................................................................................................... xv
CHAPTER 1: INTRODUCTION .......................................................................................
1.1. THE NUCLEAR ENVELOPE ................................................................................
1.2. NUCLEAR ENVELOPE PROTEINS .....................................................................
1.2.1. Lamin B receptor ......................................................................................
1.2.2. LAP Proteins .............................................................................................
1.2.3. Nesprins ....................................................................................................
1.2.4. SUN domain proteins ................................................................................
1.2.5. Nuclear Pore Complexes ..........................................................................
1.2.6. Nuclear Lamina ........................................................................................
1.3. LEM DOMAIN PROTEINS ...................................................................................
1.4. EMERIN ..................................................................................................................
1.4.1. Emerin Synthesis and Localization ...........................................................
1.4.2. Interacting Partners ...................................................................................
1.4.2.1. Lamins ..............................................................................................
1.4.2.2. BAF ...................................................................................................
1.4.2.3. LINC Complex ..................................................................................
1.4.2.4. Actins and myosins ...........................................................................
1.4.2.5. HDAC3 .............................................................................................
1.4.2.6. Other Transcription Factors ...............................................................
1.4.2.7. Implication of emerin binding partners for its functions ....................
1.5. EMERY-DREIFUSS MUSCULAR DYSTROPHY (EDMD) .................................
1.6. AIMS OF THIS WORK ...........................................................................................
1.7. REFERENCES ........................................................................................................
1
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9
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CHAPTER 2: DEVELOPMENT OF A MICROPATTERNING STRATEGY TO STUDY
NUCLEAR MECHANOTRANSDUCTION ......................................................................
2.1. INTRODUCTION ..................................................................................................
2.2. RESULTS AND DISCUSSION ..............................................................................
2.2.1. Vapor-Phase Surface Silanization of Glass Coverslips ..........................
2.2.2. Optimization of Cell Micropatterning on Silane-Coated Coverslips ......
2.2.3. Micropatterning for the Study of Mechanotransduction ........................
2.3. CONCLUSIONS .....................................................................................................
2.4. MATERIALS AND METHODS .............................................................................
2.5. REFERENCES .......................................................................................................
32
32
34
34
37
44
53
54
57
CHAPTER 3: PROMITY LABELING METHODS FOR INVESTIGATING
BIOMOLECULAR INTERACTIONS ...............................................................................
3.1. INTRODUCTION ...................................................................................................
69
69

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3.2. CLASSICAL METHODS FOR STUDYING PROTEIN-PROTEIN
INTERACTIONS ....................................................................................................
3.2.1. Yeast Two-Hybrid System .....................................................................
3.2.2. Affinity Purification-Mass Spectrometry (AP-MS)/ CoImmunoprecipitation-Mass Spectrometry (Co-IP/MS) .........................
3.2.3. Chemical Crosslinking-Mass Spectrometry ..........................................
3.3. PROXIMITY LABELING METHODS FOR PROTEIN-PROTEIN
INTERACTIONS ....................................................................................................
3.3.1. Biotin Ligase-Based Methods ................................................................
3.3.2. Peroxidase-Based Methods ...................................................................
3.4. PROXIMITY LABELING METHODS FOR PROTEIN-NUCLEIC ACID
INTERACTIONS ....................................................................................................
3.4.1. DamID and Related Methods .................................................................
3.4.2. APEX2- and BioID-based Methods .......................................................
3.4.3. Photoactivated PL methods ....................................................................
3.5. SUMMARY .............................................................................................................
3.6. REFERENCES ........................................................................................................
69
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CHAPTER 4: EXPLORING THE EMERIN INTERACTOME BY
APEX2-PROXIMITY PROTEOMICS ...............................................................................
4.1. INTRODUCTION ...................................................................................................
4.2. RESULTS AND DISCUSSION ...............................................................................
4.2.1. Generating Stable V5-tagged APEX2-expressing Dermal Fibroblasts...
4.2.2. Rational design of APEX2 fusions to emerin ........................................
4.2.3. APEX2 fused to emerin localizes predominantly to the INM and
biotinylates endogenous proteins ...........................................................
4.2.4. Quantitative Proteomic Profiling of Emerin Interacting Partners by
APEX2-mediated Biotinylation .............................................................
4.2.4.1. Proteome From APEX2-EMD .......................................................
4.2.4.2. EMD-APEX2 .................................................................................
4.2.5. Potential of splitAPEX2 for Profiling the Interactors of Emerin
Oligomers ..............................................................................................
4.2.6. Comparison with our work with previous emerin proximity labeling ....
4.2.7. Search for the Missing BAF ...................................................................
4.3. CONCLUSIONS .....................................................................................................
4.4. MATERIALS AND METHODS .............................................................................
4.5. REFERENCES ........................................................................................................
94
94
95
95
96
100
104
106
113
118
122
124
126
126
134
CHAPTER 5: BENCHMARKING APEX2 FOR MAPPING EMERINASSOCIATED DOMAINS .................................................................................................
5.1 INTRODUCTION ...................................................................................................
5.2 RESULTS AND DISCUSSION ..............................................................................
5.2.1 Live cell biotinylation using BtAn .........................................................
5.2.2 HRP-Mediated Biotinylation of Plasmid DNA ......................................
5.2.3 Optimization of Biotinylation on Fixed Cells ........................................
139
139
142
142
144
145

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5.2.4 Quantitation of Biotinylation Levels in the Genomic DNA of Labeled
Cells .......................................................................................................
5.3 CONCLUSIONS .....................................................................................................
5.4 MATERIALS AND METHODS .............................................................................
5.5 REFERENCES .......................................................................................................
149
152
153
159
BIBLIOGRAPHY ............................................................................................................... 163
APPENDICES ....................................................................................................................
APPENDIX 1: Summary of Enriched Proteins from Proteomic Experiments ..................
APPENDIX 2: List of Enriched Proteins from Proteomic Experiments ...........................
191
191
197

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LIST OF TABLES
Table 1.1. Current classification of EDMD subtypes.126
..................................................... 17
Table 2.1. Quality assessment of fibronectin micropatterns. ............................................... 39

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LIST OF FIGURES
Figure 1.1. The nuclear envelope ..................................................................................... 2
Figure 1.2. The human LEM domain protein family ........................................................ 7
Figure 1.3. Binding domains of emerin ............................................................................ 11
Figure 2.1. Glass coverslip silanization by vapor coating. (A) schematic representation
of the vapor-phase silanization of glass coverslips with hexamethyldisilazane
(HMDS); (B) structures of (3-Aminopropyl)triethoxysilane (APTES) and (3-
Glycidyloxypropyl)trimethoxysilane (GPTMS); (C) optical image of water droplets
taken by contact angle goniometry and showing significant difference in contact angle
between bare glass coverslip and HMDS-coated coverslip; (D) kinetics of water
contact angles for glass coverslips coated with different silane reagents ........................ 35
Figure 2.2. Fibronectin stamping and cell micropatterning on HMDS-coated coverslips.
(A) schematic of microcontact printing of fibronectin on silane-modified glass
substrates; (B) microscopy images of Cy3B-stained fibronectin strips on HMDScoated coverslips before and after PF-127 treatment. Scale bar: 50 µm; (C) brightfield and wide-field fluorescence microscopy images of the nucleus (DAPI) and
SNAP-emerin fusion for U2OS cells grown on 210 × 15 µm rectangular
micropatterns. Scale bars: 50 µm .................................................................................. 38
Figure 2.3. Antifouling efficacy of PF-127 treatment on HMDS-, APTES- and
GPTMS-coated coverslips after fibronectin attachment. Top row: Dual-color
fluorescence confocal images of fibronectin microstamped from 210 × 10 μm
rectangular PDMS stamps (green) and of A647-BSA non-specifically binding outside
micropatterns (red) on HMDS-, APTES- and GPTMS-coated coverslips after
treatment with PF-127. All scale bars: 50 µm. Bottom row: Fluorescence intensity
profiles for fluorescently labeled fibronectin (green) and A647-BSA (red) along the
blue line in each of the corresponding confocal images from the top row ...................... 40
Figure 2.4. Effect of PF-127 treatment on fibronectin attachment to APTES- and
GPTMS-coated coverslips. (A) fluorescence confocal images of fibronectin
microstamped from 210 × 10 μm rectangular PDMS stamps on APTES-coated
coverslips before and after treatment with PF-127; (B) fluorescence confocal images
of fibronectin microstamped from 210 × 10 μm rectangular PDMS stamps on
GPTMS-coated coverslips before and after treatment with PF-127. Note that the
fluorescence intensity in the microscopy image after pluronic treatment for GPTMS
was multiplied by two to facilitate a visualization of the micropatterns. Scale bar for
all images: 50 μm ........................................................................................................... 41
Figure 2.5. Fibronectin stamping and cell micropatterning on APTES- and GPTMScoated coverslips. (A) microscopy images of Cy3B-stained fibronectin strips on
APTES-coated coverslips after PF-127 treatment and of cell distribution in

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micropatterned and non-micropatterned areas; (B) microscopy images of Cy3Bstained fibronectin strips on GPTMS-coated coverslips after PF-127 treatment and of
cell distribution in micropatterned and non-micropatterned areas. For both A and B,
scale bars: 50 µm (fibronectin patterns), 100 µm (patterned and non-patterned areas),
20 µm (zoomed areas) .................................................................................................... 42
Figure 2.6. Applying mechanical strains to the nucleus by micropatterning cells on
increasingly narrow rectangular fibronectin strips. (A) confocal images of wild type
fibroblasts immunostained for F-actin (red) and the nucleus (blue) after plating on
non-patterned or patterned rectangular fibronectin substrates having 15 µm, 10 µm
and 5 µm widths. Scale bar: 20 µm; (B) wide-field images of emerin-null fibroblasts
immunostained for F-actin (red) and the nucleus (blue) after plating on non-patterned
or patterned rectangular fibronectin substrates having 15 µm, 10 µm and 5 µm widths.
Scale bar: 20 µm, (C) changes in NSI as a function of micropattern width for wild
type human skin fibroblasts (Emd+/y), emerin-null skin fibroblasts (Emd−/y
) and
emerin-null skin fibroblasts rescued with wild type (WT) SNAP-emerin fusion
(Emd−/y + WT emerin-fusion). The box length represents the NSI interquartile range,
the central square represents the NSI mean, the central bar represents the NSI median,
and the error bars represent the standard deviation of the mean. N = 50–60 cell nuclei
per condition. T-test comparison to Emd+/y cells: ** p < 0.01, ns: non-significant ......... 44
Figure 2.7. Fluorescence confocal imaging of emerin distribution in wild type Emd+/y
human skin fibroblasts as a function of nuclear strains. The distribution of the entire
cellular pool of emerin (ER, ONE, and INE) is imaged in cells treated with Triton X100 as a detergent to permeabilize both the plasma and the nuclear membrane (left
panels). The pool of emerin only associated with the ER and the ONE is imaged in
cells treated with saponin to permeabilize only the plasma membrane (right panels).
Cells grown randomly on fibronectin (non-patterned) or on increasingly narrow
rectangular micropatterns (15–5 µm) are immunostained for emerin (green), lamin
A/C (red) and the nucleus (blue). Scale bar for all images: 20 µm ................................. 47
Figure 2.8. Quantification of emerin redistribution in response to nuclear mechanical
strains. (A) ratio of ER emerin to nuclear envelope (ONE and INE) emerin for nonpatterned Emd+/y skin fibroblasts (Triton X-100, n = 16 cells) or fibroblasts grown on
15 µm-wide (Triton X-100, n = 15 cells), 10 µm-wide (Triton X-100, n = 15 cells),
and 5 µm-wide (Triton X-100, n = 20 cells) fibronectin rectangular micropatterns. The
fluorescence intensity quantification is done on sum slices images of full cell confocal
z-scans obtained after Triton X-100 permeabilization and emerin immunostaining. For
each cell, the intensity ratio is normalized to the mean ER/nuclear envelope ratio of
non-patterned cells; (B) relative amount of ER and outer nuclear envelope emerin for
non-patterned Emd+/y skin fibroblasts (saponin, n = 17 cells) or fibroblasts grown on
15 µm-wide (saponin, n = 14 cells), 10 µm-wide (saponin, n = 18 cells), and 5 µmwide (saponin, n = 24 cells) fibronectin rectangular micropatterns. The fluorescence
intensity quantification is done on sum slices images of full cell confocal z-scans
obtained after saponin permeabilization and emerin immunostaining. For each cell,
the quantified emerin intensity is normalized to the mean emerin intensity for nonpatterned cells. For both (A) and (B), the thick bars and the error bars represent the

x
mean and the standard deviation of each distribution, respectively. T-tests: ** p < 0.01,
ns: non-significant ......................................................................................................... 48
Figure 2.9. Emerin mutations induce defective nuclear shape adaptation against
mechanical stress. (A) Diagram of emerin with binding and self-association domains
(self-ass.) and position of Δ95-99, Q133H and P183H mutations. (B) Nuclear shape
index as a function of micropattern width for EMD−/y HDFs expressing wild-type,
Q133H, Δ95-99 or P183H emerin. The box represents the 25–75th percentiles, and
the median is indicated. The whiskers mark the s.d. of the mean, and the squares
indicate the mean. *P<0.05; **P<0.01; ns, not significant (Wilcoxon test). (C)
Fluorescence imaging of actin (green) and the nucleus (blue) in micropatterned
EMD−/y HDFs expressing Q133H, Δ95-99 or P183H emerin. Images representative of
three experiments for each micropattern width. Scale bars: 50 µm ................................ 50
Figure 2.10. (A) Nuclear shape index as a function of micropattern width in EMD+/y
HDFs depleted for lamin A/C or nuclear actin. The box represents the 25–75th
percentiles, and the median is indicated. The whiskers mark the s.d. of the mean, and
the squares indicate the mean. Wilcoxon test, **P<0.01. (B) Confocal fluorescence
imaging of lamin A/C and the nucleus (DAPI) after siRNA-induced depletion of lamin
A/C and re-expression of exogenous and siRNA-resistant lamin A/C. Scale: 20 µm.
(C-D) Effects of IPO9 siRNA and XPO6 siRNA on nuclear actin organization. (C)
Examples of nuclear localization patterns for the short nuclear actin filament probe
Utr230-EN in HDF cells. Patterns are classified as: (i) small puncta and diffuse, (ii)
diffuse or (iii) large foci, reflecting variations in nuclear actin filament content across
cells. (D) Distribution of nuclear actin filament classes after control siRNA treatment
(n = 635 nuclei), IPO9 siRNA (n = 663 nuclei) or XPO6 siRNA (n = 625 nuclei).
Knockdown of IPO9 results in the majority of cells displaying a diffused Utr230-EN
pattern, indicative of lower nuclear actin filament contents. Inversely, knockdown of
XPO6 results in the majority of cells displaying larger and brighter foci compared to
control siRNA, indicative of increased nuclear actin filament contents ......................... 51
Figure 3.1. Traditional methods for studying protein-protein interactions. (A) Yeast
two-hybrid method: a protein of interest (POI) is fused to the DNA binding domain
while its potential interactor is fused to the activation domain of a functional
transcription factor. If the proteins interact, the transcription factor is activated
producing a color or allowing growth in selective media. (B) Co-immunoprecipitation
of protein interactors: The POI is purified by affinity chromatography with an
antibody or via an affinity tag, bringing along interactors. (C) Chemical crosslinking
of protein interactors: Bifunctional linkers are used to covalently crosslink protein
interactors that are then purified by affinity chromatography ........................................ 70
Figure 3.2. Mechanism and evolution of the most common methods for enzymemediated proximity labeling. (A) Schematic of a typical proximity labeling (PL)
workflow. An enzyme (a biotin ligase or a peroxidase) is fused to the protein of
interest (bait) and targeted to a specific subcellular location. Proximity labeling is
achieved through the in situ enzymatic synthesis of biotin-conjugated reactive
intermediates, which subsequently diffuse away and react with nearby

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proteins/nucleic acids (NAs). The nanometer-scale action radius of the intermediates
(shown as a red contour map) covers both proteins/NAs that tightly associate with the
bait and those that loosely interact in the same compartment, enabling PL to reach
over multiple layers of protein–protein/NA interactions. After cell lysis, biotinylated
proteins are collected by affinity purification and characterized by mass spectrometry.
Biotinylated RNAs are analyzed by high-throughput sequencing. (B) The mechanism
and technology development timeline of PL. In the presence of H2O2, APEX (green)
converts biotin phenol to phenoxyl free radical, which reacts with the
adjacent tyrosine residues. In the presence of ATP, BioID (cyan) activates biotin into
bio-AMP, which reacts with lysine residues of neighboring proteins. The timeline
describes a brief history of major APEX- and BirA-mediated PL techniques. Methods
highlighted in green, blue, and pink refer to protein-centered, RNA-centered, and
DNA-centered profiling, respectively ........................................................................... 73
Figure 4.1. Generation of emerin-knockdown fibroblasts by lentiviral transduction with
shRNA targeting the 3’-UTR of the EMD gene. (A) Confocal florescnce images of
HDF+/y transduced with scramble shRNA and EMD shRNA clearly showed a marked
decrease in endogenous emerin. Scale bars: 50 µm. (B) Confirmation of the emerin
knockdown efficiency by western blot. Data were normalized to β-actin expression.
The results presented are the mean ± SD of two biological replicates (each with three
technical replicates). Student’s t-test for statistical significance. **** p < 0.0001 ......... 96
Figure 4.2. Schematic of V5-tagged APEX2 fusion constructs and principles of
APEX2-mediated proximity labeling. The APEX2 biotinylation zone is expected to
be <20 nm ...................................................................................................................... 97
Figure 4.3. The V5-tagged APEX2 fusion constructs used in this work ........................... 98
Figure 4.4. Characterization of biotinylation by confocal fluorescence imaging of
human dermal fibroblasts stably expressing APEX2. Cells were treated with 0.5 mM
biotin-phenol (BP) for 30 min and the biotinylation reaction was triggered by the
addition of 1mM H2O2. Cells were fixed and stained with an anti-V5 antibody (green)
to visualize APEX2 localization and Streptavidin-Alexa Fluor 594 (red) to visualize
biotinylated proteins. All cell lines show correct localization of each fusion and
effective biotinylation Scale bars, 50 μm ....................................................................... 102
Figure 4.5. Characterization of biotinylation activity by western blotting for wild-type
and mutated N-terminal APEX2-emerin fusions and controls. (A) HDFs stably
expressing APEX2-EMD constructs, APEX2-LBR1TM, APEX2-NLS and APEX2-
NES were treated with 0.5 mM biotin-phenol (BP) for 30 min and 1mM H2O2 for 1
minute. This was followed by cell lysis, and clarified whole-cell lysates were
analyzed by streptavidin-HRP blotting. (B) Quantitation of emerin levels on Nterminus constructs by western blot probed by anti-emerin antibody. Normalization is
done with respect to β-actin expression levels. All statistical comparisons to HDF
EMD+/y with a student t-test: **p<0.01, ns: non-significant .......................................... 103

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Figure 4.6. Characterization of biotinylation activity by western blotting for wild-type
and mutated C-terminal emerin-APEX2 fusions and controls. (A) HDFs stably
expressing EMD-APEX2 constructs, LBR1TM-APEX2, APEX2-KDEL and APEX2-
NES were treated with 0.5 mM biotin-phenol (BP) for 30 min and 1mM H2O2 for 1
minute. This was followed by cell lysis, and clarified whole-cell lysates were
analyzed by streptavidin-HRP blotting. (B) Quantitation of emerin levels on Cterminus constructs by western blot probed by anti-emerin antibody. Normalization is
done with respect to β-actin expression levels. All statistical comparisons to HDF
EMD+/y with a student t-test: *** P<0.001, ns: non-significant ..................................... 104
Figure 4.7. Principle and experimental workflow of TMT-based MS proteomics. After
on-bead digestion, the tryptic peptides are labeled by a different Tandem Mass Tag
(TMT). The samples are pooled together and injected into the mass spectrometer for
identification and quantitation ....................................................................................... 106
Figure 4.8. (A) PCA plot and (B) correlation matrix plot to assess the quality of APEX2-
EMD samples sent for MS-based proteomics ................................................................ 107
Figure 4.9. Schematic showing the filtering criteria for selection of proteins enriched
in APEX2-EMDWT samples relative to APEX2-LBR1TM ............................................ 108
Figure 4.10. Proximity labeling proteomics results of APEX2-EMDWT. (A) Volcano
plot and list of all high confidence proteins identified where the APEX2-
EMDWT/APEX2-LBR1TM ratio was greater than 0.322. Highlighted in maroon/red
are the previously known emerin interacting partners. (B-C) Gene ontology analyses
for cellular component (B) and biological processes (C) of proteins from (A) that met
our selection criteria ...................................................................................................... 110
Figure 4.11. Venn diagrams showing the similarity of enriched proteins for the APEX2-
EMD compared to the emerin mutants ........................................................................... 111
Figure 4.12. Gene ontology analysis for the cellular components (B) and biological
processes (C) of the enriched proteins for the APEX2-EMD P183H, Q133H and Δ95-
99 mutants ..................................................................................................................... 113
Figure 4.13. (A) PCA plot and (B) correlation matrix plot to assess the quality of EMDAPEX2 samples sent for MS-based proteomics ............................................................. 114
Figure 4.14. Schematic showing the filtering criteria for selection of proteins enriched
in EMDWT
-APEX2 samples relative to LBR1TM-APEX2 ............................................ 115
Figure 4.15. Proximity labeling proteomics results of EMDWT
-APEX2. (A) Volcano
plot and list of all high confidence proteins identified where the APEX2-
EMDWT/APEX2-LBR1TM ratio was greater than 0.322. (B-C) Gene ontology
analyses for cellular component (B) and biological processes (C) of proteins from (A)
that met our selection criteria ......................................................................................... 116

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Figure 4.16. Venn diagrams showing the similarity of enriched proteins for the EMDAPEX2 compared to the emerin mutants ....................................................................... 117
Figure 4.17. Gene ontology analysis for the cellular components (B) and biological
processes (C) of the enriched proteins for the P183H, Q133H and Δ95-99 mutants of
EMD-APEX2 ................................................................................................................ 118
Figure 4.18. Confocal microscopy imaging on HDFs expressing splitAPEX2-EMD and
EMD-splitAPEX2. Proper localization was observed from all emerin fusions and
biotinylation activity was restored upon contact of the AP and EX fragments due to
emerin self-association .................................................................................................. 119
Figure 4.19. Characterization of biotinylation activity by western blotting for the
splitAPEX2-emerin fusion constructs. HDFs stably expressing (A) splitAPEX2-EMD
or (2) EMD-splitAPEX2 along with the other APEX2 constructs were treated with 0.5
mM biotin-phenol (BP) for 30 min and 1mM H2O2 for 1 minute. This was followed
by cell lysis, and clarified whole-cell lysates were analyzed by streptavidin-HRP
blotting .......................................................................................................................... 120
Figure 4.20. Proximity labeling proteomics results of splitAPEX2-EMD. (A) Volcano
plot and list of all high confidence proteins identified where the splitAPEX2-
EMD/APEX2-LBR1TM ratio was greater than 0.322. Highlighted in maroon/red are
the previously known emerin interacting partners. (B-C) Gene ontology analyses for
cellular component (B) and biological processes (C) of proteins from (A) that met our
selection criteria ............................................................................................................. 121
Figure 4.21. Venn diagram comparing the list of enriched proteins for emerin proximity
mapping in our work and previously published studies .................................................. 123
Figure 4.22. Utilizing splitAPEX2 to detect the interaction of emerin with barrier-toautointegration factor in emerin-null human skeletal myoblasts rescued with EX-FlagEMD, then treated with BAF shRNA following by transduction with BAF-V5-AP ...... 125
Figure 5.1. Screening of biotinylated arylamines for protein, DNA and RNA labeling .. 141
Figure 5.2. Labeling on live APEX2-expressing human skeletal myoblasts (Protocol 1). 143
Figure 5.3. Comparison of HRP-mediated labeling efficiency toward plasmid DNA.
(A) Representative dot blot image. 20 μg plasmid DNA were labeled with 0.5 mM BP
or Btn-An, HRP, and 1 mM H2O2 for different time points at RT. Following plasmid
DNA recovery, 1 μg plasmid DNA was dot blotted onto the membrane; (B)
Quantitative analysis of biotinylation intensity. Student’t t-test was used to test for
significance, ****p < 0.0001 ......................................................................................... 145
Figure 5.4. Labeling on fixed APEX2-expressing human skeletal myoblasts following
Protocol 2. Labeling time is extended to 5 minutes. Background streptavidin staining
on control myoblasts are reduced but still significant compared to live cell labeling .... 146

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Figure 5.5. Labeling on fixed APEX2-expressing human skin fibroblasts following
Protocol 2. Labeling time is 1 minute. Similar background streptavidin staining is
observed in non-APEX2 expressing fibroblasts, similar to control myoblasts ............... 146
Figure 5.6. Labeling on fixed APEX2-expressing human skeletal myoblasts following
Protocol 3. Perfoming the Triton-X100 permeabilization after BtAn labeling
significantly reduced background staining on control myoblasts even for extended
periods of biotinylation reaction .................................................................................... 148
Figure 5.7. Assay to determine the biotin levels in the gDNA sample. (A) Chemical
basis of the fluorescent biotin assay; (B) biocytin standard curve .................................. 149
Figure 5.8. Biotin levels of the labeled gDNA samples. (A) calculated and (B)
normalized biotin levels. Student’t t-test was used to test for significance, *p < 0.05 .. 151

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ABSTRACT
Emerin is an integral membrane protein of the inner nuclear membrane. Mutations in the EMD
gene can cause Emery-Dreifuss Muscular Dystrophy (EDMD), a disorder characterized by
progressive skeletal muscle wasting, irregular heart rhythms and contractures of major tendons.
Despite extensive research aimed at identifying the binding partners of emerin and understanding
its structural organization at the nuclear envelope, how the interacting partners of emerin influence
the nanoscale organization of emerin remains poorly defined.
First, we aimed to develop a simple platform based on cell micropatterning to study the role of
emerin during nuclear mechanotransduction By optimizing the microcontact printing of the
extracellular matrix protein fibronectin on HMDS-modifed glass coverslips and micropatterning
cells, we were able to imposing specific physical strains at the nuclear envelope to reveal some of
the mechanotransducing functions of emerin in response to mechanical cues. By utilizing twodimensional cell micropatterning on fibronectin substrates of varying widths, it is possible to
efficiently impose incremental steady-state mechanical strains at the cell nuclear envelope and to
reveal some of the mechanotransducing responses of emerin, as well as the effect of the
nucleoskeletal proteins lamin A/C and actin.
Next, we aimed to define the nanoscale proteomic landscape of wild-type as well as EDMDrelevant mutants by proximity-dependent biotinylation using the engineered ascorbate peroxidase
APEX2 combined with mass spectrometry-based proteomics. When activated by H2O2, APEX2
catalyzes the conversion of its substrate, biotin-phenol, into short-lived and highly reactive radicals
that can form covalent bonds with electron-rich amino acids, including tyrosine, present in nearby
endogenous proteins. By using APEX2 fusion to wild-type emerin and EDMD-causing mutants,

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we hypothesized that the emerin mutations result in altered interactions that could provide insight
into the mechanism of EDMD. Moreover, inspired by prior research employing splitGFP to
illustrate the self-association of emerin, we have adapted the splitAPEX2 platform to identify
proteins that interact specifically with emerin oligomers.
Finally, recent reports of the capability of APEX2 to biotinylate nucleic acids has led us to
explore its potential to map the genomic regions closely associating with emerin. By simply
switching to another substrate, biotin-aniline, that has a higher biotinylation specifity towards
nucleic acid, we have optimized protocols to confirm APEX2 biotinylation of the genomic DNA
by fluorescence imaging and quantitation of biotin levels of extracted genomic DNA.

1
CHAPTER 1: INTRODUCTION
The nucleus is often considered the mastermind of human cells as it houses the genetic
information. In our body, cells deform their nucleus in response to forces during critical functions
such as cell movements or muscle contraction. Failure of the cell nucleus to adapt to mechanical
constraints contributes to many human diseases including muscular dystrophies, cancers and
premature aging.
The structure and the organization of the nuclear envelope plays a key role in the cell nucleus's
capacity to respond to mechanical signals. Indeed, at the nuclear envelope, many proteins function
together to form molecular scaffolds that regulate nuclear shape remodeling and maintain the
structural integrity of this double membrane in response to forces. In this introduction, I describe
the nuclear envelope and the components that participate in its scaffolding, including emerin,
which mutation leads to EDMD.
1
1.1. THE NUCLEAR ENVELOPE
All living organisms are composed of cells, which are divided into two principal categories:
prokaryotic and eukaryotic. The defining feature that differentiates eukaryotic cells from
prokaryotic cells is the existence of a nucleus, the organelle that encases nearly all of the cell’s
genetic material.2
The nucleus is surrounded by a structure referred to as the nuclear envelope,
which consists of two phospholipid bilayers that functionally separate the nucleoplasm from the
cytoplasm (Figure 1.1).
2 The INM and ONM are separated by the perinuclear space (PNS), and
merge at numerous sites, leading to the formation of membrane pores that are filled by nuclear
pore complexes (NPCs), facilitating the exchange of materials between the nucleus and
cytoplasm.2, 3 The ONM is the exposed to the cytoplasm and is contiguous with the endoplasmic

2
Figure 1.1. The nuclear envelope. (Adapted from Ref. 3
).
reticulum (ER). Underlying the INM is a proteinaceous meshwork called the nuclear lamina,
which provides structural support to the nuclear envelope.
4
1.2. NUCLEAR ENVELOPE PROTEINS
Currently, a vast number of proteins have been identified to be associated to the nuclear
envelope, encompassing transmembrane (TM) proteins that are situated at the INM and ONM,
proteins forming the NPCs, nuclear lamins and lamin-associated proteins (Figure 1.1).2 To be
concise, the discussion will be restricted to nuclear envelope proteins relevant to this thesis work.
1.2.1. Lamin B receptor
Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane containing a
hydrophilic N-terminal end that extends into the nucleoplasm, eight hydrophobic transmembrane

3
(TM) domains and a short, nucleoplasmic C-terminal tail.5 LBR was first identified as a nuclear
envelope protein in avian cells, and was later shown to interact directly with lamin B.6, 7 Its Nterminal domain tethers heterochromatin to the nuclear periphery, while its transmembrane
domains exhibit sterol reductase activity.8, 9 Mutations within the TM segments result in defects in
cholesterol synthesis and are associated with diseases such as the Pelger–Huët anomaly and
Greenberg skeletal dysplasia, whereas no such harmful mutations related to the anchoring
properties of LBR have been reported so far.10, 11
1.2.2. LAP Proteins
The lamin associated polypeptide (LAP) family are type II integral membrane proteins divided
into LAP1 and LAP2 proteins.12, 13 As their name implies LAPs can bind lamins. The LAP1 family
comprises three isoforms: LAP1A, LAP1B, and LAP1C, and are expressed in most cells and
tissues.12 On the other hand, the LAP2 family consists of six alternatively spliced proteins in
mammalian cells and three isoforms in Xenopus.
13 LAP1 proteins (LAP1A, LAP1B and LAP1C)
as well as two out of six LAP2 proteins (LAP2β-ε) contain a C-terminal TM domain and are
localized to the INM. LAP2α and LAP2ζ lack the TM domain and are found in the nucleoplasm.14
Only LAP2 isoforms have a LEM domain which binds BAF.
15-17 LAP1A and LAP1B have
been shown to bind to A and B-type lamins, while LAP2C only inds lamin B.18 Lap2α directly
interacts with nucleoplasmic lamin A.13 In human fibroblasts, loss of Lap2a induces cell cycle
arrest, while in mice, the loss of Lap2α is associated with cell cycle progression.19, 20
1.2.3. Nesprins
Nesprins (Nuclear envelope spectrin repeat proteins) is a large protein family of spectrin repeat
proteins primarily embedded in the ONM and encoded by four genes (SYNE1-4).21 They are
characterized by a variable length central rod region, and an unique evolutionary conserved C-

4
terminal Klarsicht, ANC-1 and Syne Homology (KASH) transmembrane domain that specifically
recruits nesprins to the nuclear envelope and interacts with the SUN proteins (SUN1 and SUN2)
to form forming the LINC (linker of nucleoskeleton and cytoskeleton) complex.22-24 Nesprin-1 and
-2 are two giant isoforms with an N-terminal calponin homology (CH) domains that binds to Factin.22 Nesprin-3 has an N-terminal plectin-binding motif which interacts with cytoplasmic
intermediate filament proteins and nesprin-4 directly interacts with microtubules.25, 26 Due to
alternative splicing, shorter nesprin isoforms that lack the KASH domain, including some nesprin2 isoforms, are located in the nucleoplasm.27 Missense mutations in nesprin-1 and nesprin-2 have
been identified in patients, leading to skeletal muscle phenotype that are similar to Emery-Dreifuss
Muscular Dystrophy. (EDMD).1
1.2.4. SUN domain proteins
The SUN (Sad1, UNC-84)-domain proteins SUN1 and SUN2 represent a conserved family of
proteins located in the inner nuclear membrane (INM) that interacts with various KASH-domain
partners (e.g. nesprins) to facilitate physical interactions between the nucleoplasm and the
cytoskeletal framework across the nuclear envelope.28 The N-terminal domain of both SUN1 and
SUN2 is exposed to the nucleoplasm and was shown to interact directly with A-type lamins,
emerin and short nesprin-2 isoforms.29, 30 The C terminal SUN-domain extends into the perinuclear
space and interacts with the KASH domain of nesprins to form the LINC complex (Crisp et al.,
2006; Padmakumar et al., 2005) responsible for transmitting forces between the cytoskeleton and
the nucleoskeleton.23, 24
1.2.5. Nuclear Pore Complexes
NPCs are large macromolecular assemblies embedded into the nuclear envelope to form a
channel, allowing transport of molecules across the membrane. Consisting of multiple copies of

5
~30 distinct protein subunits called nucleoporins (Nups), NPCs are aqueous channels that show
eight-fold rotational symmetry with an outer diameter of about 100 nm and a central transport
channel diameter of about 40 nm, through which bidirectional exchange of proteins, RNA, and
ribonucleoprotein complexes between the nucleoplasm and cytoplasm occurs.31, 32 Proteins
embedded in the nuclear membrane and bearing nuclear localization sequences (NLS) or INM
sorting motifs can access or leave the INM via central or lateral channels of the NPC.33
1.2.6. Nuclear Lamina
The nuclear lamina is a proteinaceous meshwork underlying the INM and mainly composed
of nuclear lamins and associated proteins. Lamins are type V intermediate filament proteins that
play a crucial role in preserving the structural integrity and mechanical characteristics of the
nucleus.34 The A-type lamins (lamin A/C, and minor isoforms), encoded by the gene LMNA, are
expressed in a tissue- and development-specific manner, while the ubiquitously expressed B-type
lamins, lamin B1 and lamin B2 are encoded by LMNB1 and LMNB2, respectively.35 Lamins have
three structural regions: a globular amino-terminal head, a central α-helical ‘rod’ domain, and a
carboxy-terminal tail. The α-helical domain has four coiled coils that mediate dimer formation.
The carboxy-terminal tail includes a flexible tether with a nuclear localization signal (NLS), an Igfold domain, and a ‘CaaX box’ (except lamin C) that is modified by carboxylation and
farnesylation.35, 36 Through their interactions with a wide range of nuclear membrane proteins,
chromatin regulators, transcription factors, splicing factors, signaling molecules, and chromatin,
lamins play a crucial role in a wide range of cellular functions, including the maintenance of
nuclear integrity, the positioning of the nucleus, transcriptional regulation, DNA repair and
replication, splicing, signaling pathways, mechanotransduction, and the organization of the
genome.35, 36

6
1.3. LEM DOMAIN PROTEINS
The LEM domain proteins, named after its original members LAP2, emerin and MAN1, are
components of the nuclear lamina and mainly identified by the ~40 amino acid domain (the LEM
domain) composed of a three-residue turn at its N-terminus, and two parallel alpha helices
interacting through a conserved set of hydrophobic residues and connected by a long loop, forming
a helix-loop-helix fold (Figure 1.2).37, 38 The defining characteristic of the LEM domain is its
ability to bind barrier-to-autointegration factor (BAF), a DNA-bridging protein, highly conserved
in metazoans.39
LAP2, encoded by TMPO, carries both a LEM domain and a LEM-like domain allowing it to
bind DNA.40 Most LAP2 isoforms carry a carboxyl-terminal transmembrane domain and localize
to the inner nuclear membrane, such as LAP2β, whereas others such as LAP2α lack the TM
domain, localize to the nucleoplasm and associate with distinct partners.17
MAN1 (also known as LEMD3) is an interal membrane protein of the INM that is encoded by the
gene LEMD3. It has a LEM domain at the N-terminus, a winged helix domain at the C-terminus
that can bind DNA, and an RNA recognition motif which is the binding site for regulatory Smads
proteins, but not the inhibitory Smads.41 MAN1 is anchored to the INM via two transmembrane
domains meaning that both the N-and C-termini reside in the nucleoplasm. Mutations in MAN1
result in osteopoikilosis, Buschke-Ollendorff syndrome and is also associated with
laminopathies.42, 43
LEM domain-containing protein 2 (LEMD2) is a protein that is widely expressed and is notable
for containing a LEM domain and two transmembrane domains.44 Various in vitro studies revealed
its ability to interact with DNA-binding proteins such as lamins and barrier-to-autointegration
factor (BAF), thereby implicating LEMD2 as a key mediator between chromatin and the nuclear

7
Figure 1.2. The human LEM domain protein family. (Adapted from Ref. 37).
envelope.45, 46
LEM domain-containing protein 1 (LEMD1) is identified as a cancer testicular antigen (CTA)
that is present only in normal testicular cells and is linked to oncogenic characteristics.47 Previous
investigations have revealed that LEMD1 is abnormally expressed in a range of cancers, including
oral squamous cell carcinoma, colorectal cancer, prostate cancer, and anaplastic large cell
lymphoma.48-50
Ankyrin repeats and LEM domain containing protein 1 (Ankle1) lacks a transmembrane
domain and shuttles between the cytoplasm and the nucleoplasm, and it contains a C-terminal GIYYIG-type endonuclease domain.
51 The GIY-YIG domain of Ankle1 exhibits nuclease catalytic
activity, which enables it to cleave plasmid DNA in vitro and cause DNA damage in vivo.51 Studies
on C. elegans reveal that a mutation that inactivates the Ankle1 ortholog LEM3 causes the worm
mutants to exhibit increased sensitivity to various types of DNA damage, including ionizing

8
radiation, UV-C light, and agents that cause DNA crosslinking. As a result, Ankle1/LEM3 has been
proposed to be involved in the mechanisms of DNA damage repair.52 In mammals, it appears that
the functions of Ankle1 are largely redundant, given that Ankle1 knockout mice and cells maintain
normal characteristics and do not exhibit any impairment in their DNA damage response.
53
Conversely, Ankle2 is devoid of any identified enzymatic domains or activities, and instead
acts as a scaffold to regulate BAF phosphorylation and control nuclear envelope dynamics.
54, 55
Ankle2 has be shown to be essential for the disassembly of the nuclear envelope at the onset of
mitosis and its reassembly after the segregation of chromosomes, and Ankle2 dysfunctions are
correlated with abnormal nuclear morphology and disruptions in cell division.56
Emerin is encoded by the EMD gene (formerly STA gene) that is located on long arm of the X
chromosome at position 29 (Xq28).57 Sequencing of the EMD gene revealed that it is 2.1 kb long,
has six exons and has an open reading frame of 762 nucleotides encoding emerin.58 Emerin is
widely expressed across various tissues, with particularly elevated levels found in skeletal muscle
and cardiac tissue.59
Emerin being the principal protein studied in this work, it is being discussed in more details in
the section below. These LEM domain proteins interact with a numerous other proteins involved
in mechanotransduction, cellular architecture, transcriptional regulation, and chromatin
tethering.59 Interestingly, they also exhibit overlapping functions, as evidenced by several studies
showing that the loss of two LEM proteins causes phenotypes significantly more severe than loss
of a single LEM protein.60-63 Indeed, MAN1, LAP2α/β and LEMD2and emerin, are linked to
muscle contractures and myopathies
44, 64, 65 and they have overlapping functions for nuclear
morphology and nuclear envelope mechanics.
37 For instance, depletion of MAN1 exacerbates
nuclear envelope defects when combined with emerin depletion. LAP2β overexpression prevents

9
nuclear envelope rupture in mechanically challenged cells,66 while LAP2α regulates the formation
of dense lamin A/C structures.67 Both LAP2 isoforms are upregulated in EDMD patients.68 Loss
of LEMD2 also results in abnormal nuclear envelope shapes
69 and its over-expression induces
nuclear envelope folding by recruitment of emerin.45 Emerin, MAN1, LEM2, and more indirectly
LAP2 isoforms are required for efficient myogenic differentiation,46, 70, 71 with LEM2 and emerin
having overlapping roles in the maintenance of nuclear shape.72 Myogenesis also triggers
significant changes in expression levels of these LEM proteins
68, 73, 74 Considering the apparent
overlapping function of many of these LEM proteins, it was suggested that they could partially
compensate for mutations in other LEM proteins, including mutations in emerin.59
1.4. EMERIN
Emerin is a 34 kDa serine-rich type II integral membrane protein of nuclear envelope that is
made of 254 amino acids.57, 59 It is characterized by an N-terminal LEM domain, a large
intrinsically disordered domain, a transmembrane domain, and a small C-terminal domain75, 76
(Fig. 1.3.). The only structured region in emerin, beside the TM domain, is the LEM domain, as
revealed by solution state nuclear magnetic resonance (NMR) analysis of several emerin
fragments.77 The large intrinsically disordered domain of emerin provides it the flexibility to adopt
various conformations. In addition, emerin has been shown to self-associate and form oligomers
in vitro75, 78, 79 and at the INM. This self-assembly process appears to involve the partial unfolding
of the LEM domain and its binding to potentially two segments along the disordered region of
other emerins75, 79 in order to establish emerin/emerin contacts.75
The self-association of emerin
into nanodomains at the INM is a central determinant of nuclear shape adaptation against
mechanical challenges.75, 79, 80

10
1.4.1. Emerin Synthesis and Localization
Emerin is an integral membrane protein of the nuclear envelope. Despite the hypothesized
presence of a bipartite nuclear localization signal (NLS) in its N-terminal domain, the correct
localization of emerin to the INM is not facilitated by this signal.33 While soluble proteins are
targeted to the nucleus by an NLS through NPCs, the mechanism for nuclear targeting of integral
membrane proteins differs because they do not necessarily involve NLSs.81 Instead many INM
proteins reach the nuclear envelope via NPC side channels and accumulate at this membrane
following an ER diffusion/retention model. Through its TM domain, newly synthesized emerin in
the cytoplasm are inserted post-translationally into the ER membrane in a process mediated by the
ATP-dependent TRC40/Asna-1 complex.82 Emerin then diffuses laterally from the ER until it
reaches the INM as shown by fluorescence loss in photobleaching (FLIP) and fluorescence
recovery after photobleaching (FRAP) studies.83 At the INM, emerin gets retained by interacting
with other nuclear envelope components, like lamins or chromatin, through its nucleoplasmic
domain.81, 84
While it localizes predominantly at the INM in skin cells and in skeletal and cardiac muscles,
emerin has also been shown to sometime localize in other cellular regions in various tissues and
cell types. Emerin has been detected at the ONM and ER, consistent with its biogenesis.85, 86 It was
also detected at the on ER-Golgi intermediate compartment (ERGIC) vesicles in human dermal
fibroblasts85
1.4.2. Interacting Partners
In addition to binding itself and forming self-assembly nanodomain at the INM, emerin has
been reported to directly bind to at least 16 proteins, an indication that it serves a multitude of

11
Figure 1.3. Binding domains of emerin. (Adapted from 40 and 80).
functions within cellular environments, including maintenance of the nuclear lamina structure,
chromatin tethering, mechanotransduction, gene regulation, and signaling.59 Below is description
of some of emerin primary binders.
1.4.2.1. Lamins
Mutations in lamins cause a variety of human diseases called laminopathies, and hundreds of
missense mutations have been documented causing EDMD, Atypical Werner syndrome,
Hutchinson-Gilford progeria syndrome and lipodystrophy.87, 88 So far, several binding partners
have already been identified for Lamin A which include, emerin, MAN1 and BAF.89 Competition
studies by co-immunoprecipitation of lamins A, C and Bl with emerin in rabbit reticulocyte lysates
showed that emerin preferred interaction with lamin C, although emerin can interact in vitro all
lamins.90 In the same study, it was shown that in cell lines where lamin A is absent, lamin C is
mislocalized in the nucleolus while lamins Bl and B2 are normal, and that emerin is mislocalized
in the ER forming aggregates.90 Upon lamin A rescue, emerin relocalizes correctly to the nuclear
envelope while transfection of lamin Bl is not able to rescue emerin localisation.90. Lamin A/C
knock down by siRNA in cells was also shown to prevent the self-assembly of emerin at the INM.80

12
1.4.2.2. BAF
BAF is a highly conserved metazoan protein. It is a small (10 kDa; 89 residues) DNA-bridging
protein that forms an obligate dimer, which binds two dsDNA molecules.91 The interaction
between BAF and the LEM domain of emerin was examined through blot overlay experiments,
where emerin mutants were fixed onto blots and subsequently exposed to 35S-BAF.92 Evidence for
the direct interaction of BAF with emerin in living cells has been provided by Fluorescence
Resonance Energy Transfer (FRET) analysis. In this study, the repeated photobleaching of YFPemerin resulted in an increase in the fluorescence of CFP-BAF, which supports the notion of their
direct association.93 FRAP and FLIP analysis of BAF and its binding partners showed that BAF
exists in two separate pools in the cell – one nucleoplasmic and one cytoplasmic – that do not mix
with each other, and that BAF diffuses rapidly as opposed to emerin and LAP2, which appear
primarily immobile at the nuclear envelope.93 In addition a BAFL58R mutant that does not bind
emerin nor other LEM domain proteins94, 95 was shown to induce a significantly faster mobility of
emerin at both ONM and the INM,95 consistent with a direct interaction between cytosolic and
nucleoplasmic BAF with the emerin LEM domain. Thus, a “touch and go” model was proposed
according to which BAF interacts frequently but transiently with emerin in interphase.93
1.4.2.3. LINC Complex
The LINC complex (nesprins and SUN1 and SUN2 proteins) is considered the bridge that
connects the cytoskeleton to the nucleus and that relays the mechanical forces from the outside of
the nucleus, across the nuclear envelope to the nucleoskeleton.23, 24 Emerin interacts directly with
both SUN-domain proteins and with nesprins, cementing its role in mechanotransduction. Indeed,
isolated nuclei from emerin-null cells have a defective response in nuclear adaption to stress.
96
Emerin binding to nesprins was studied using relatively short isoforms, nesprin-1α and nesprin-

13
2β.
97, 98 Meanwhile, emerin was shown to bind to the nucleoplasmic domains of SUN1 (SUN1
residues 223–302) and SUN2.
30 Additional work showing that siRNA depletion of SUN1 or
disruption of SUN domain/KASH domain interactions between SUN proteins and nesprins result
in the absence of emerin self-assembly at the INM,95 further highlighted the connection between
the LINC complex and emerin organization, consistent with the direct interaction of SUN proteins
and emerin at the nuclear envelope.
1.4.2.4. Actins and myosins
Actin was first shown to co-immunoprecipitate with emerin in C2C12 myoblast extracts.99 In
another study, it also co-immunoprecipitated with emerin and lamin A/C in the late differentiation
stages of C2C12 myotubes and in mature muscle fibers, with those interactions seemingly
regulated by protein phosphorylation.100 Holaska et al. further showed that emerin binds and caps
the pointed end of growing actin filaments, promoting its polymerization in vitro, via an interaction
that requires the entire disordered domain of emerin.101 While those studies demonstrated that
emerin interacts with cytoplasmic actin, additional studies also showed that emerin likely binds to
nuclear actin. Indeed, in dermal fibroblasts, depletion or increase in nuclear actin content by knock
down of the actin nuclear import factor importin-9 or the actin nuclear export factor exportin 6,
respectively, directly impact the mobility and the nanoscale organization of emerin at the INM.95

Proteomic studies performed on HeLa cells nuclear lysates have also shown that, in vitro,
emerin binds directly to nuclear myosin I (NMI) and nuclear specific spectrin isoform αII (αII
spectrin).102 Nuclear myosin I, an isoform of myosin I that is specifically localized to the nucleus,
is involved in rRNA gene transcription and interacts with the nuclear pore basket, while spectrin
is a cytoskeleton protein present in eukaryotic cells at the intracellular side of the plasma

14
membrane. It maintains the cytoskeleton structure, plasma membrane integrity, and genomic
stability.103, 104
These interactions highlight emerin's contribution to cell architecture and to anchoring the
cortical and nuclear actin-myosin network to the nuclear envelope through its binding to
cytoskeletal actin, nucleoskeletal actin, and to actin-binding proteins.
1.4.2.5. HDAC3
Emerin interacts directly with the core components of the nuclear co-repressor (NCoR)
complex, which represses genes by stably binding chromatin.102 Histone deacetylase 3 (HDAC3)
is a key component of the NCoR complex, facilitating the deacetylation of specific lysine residues
on histone H4 tails. Emerin binds directly to HDAC3 and stimulates its activity.105 The affinity for
binding of emerin with HDAC3 was measured in-vitro (7.2 µM) and there was a 2.5 fold increase
in enzyme activity upon emerin binding.
105 LAP2β also interacts with HDAC3, an interaction that
result in reduced H4 acetylation and chromatin tethering to the nuclear envelope.106 This suggests
there may be overlapping roles of the two LEM domain proteins emerin and Lap2β with respect
to regulation of genome organization and tissue-specific gene expression.106
1.4.2.6. Other Transcription Factors
Germ cell-less (GCL) GCL is a transcription repressor that is conserved from Caenorhabditis
elegans to humans. It is not a nuclear envelope integral protein, but rather a nuclear matrix protein,
which binds several nuclear structures, such as: lamins, chromatin, and E2F-DP transcription
factor.107 GCL was shown to co-immunoprecipitate with emerin in nuclear extracts prepared from
HeLa cells through a microtiter well binding assay with full-length and mutant emerin fragments
immobilised on wells and incubated with 35S-GCL.
108 Results showed that GCL binds to emerin
residues 34-83, 175-196 and 207-217.108

15
Bcl-2 associated transcription factor (Btf) is a transcription repressor, which is expressed in
various tissues and markedly expressed in skeletal muscles.109 Biochemical studies have revealed
that emerin directly binds Btf with 100 µM affinity, and the Btf binding domains were mapped to
residues 45-83 and 175-217 on emerin.109
Lim-domain-only 7 (Lmo7) functions as a transcriptional activator that translocates between
the nucleus and the cell membrane.
110 The interaction between Emerin and Lmo7 revealed that
they serve as mutual regulators, with emerin surprisingly influencing Lmo7 both positively and
negatively.110
β-catenin is a transcriptional co-activator of the canonical Wnt signaling pathway and is
involved in several developmental processes.111 Markiewicz and coworkers have identified the
interaction between β-catenin and emerin through its conserved adenomatous polyposis coli
(APC)-like domain. Their results indicate that emerin plays a role in preventing the nuclear
accumulation of β-catenin.
111
1.4.2.7. Implication of emerin binding partners for its functions
Given this broad and probably incomplete list of emerin-binding proteins identified since its
discovery, it has been difficult to clearly delineate the many possible functions of emerin at the
nuclear envelope. Yet, based on the properties of those binding partners, it is clear that emerin is a
key component of the mechanotransduction machinery and gene regulation at the nuclear
envelope95, 96, 112 While emerin is expressed in all somatic cells, nonsense mutations in the EMD
gene and absence of emerin expression primarily affect cells exposed to extensive mechanical
stress, such as skeletal and cardiac muscle cells. Emerin-null muscle tissues display deformed and
disorganized nuclei, impaired myogenesis and improper muscle fiber formation, which result in
the muscle wasting and cardiac disease phenotypes of EDMD59, 113-115, as discussed below. In non-

16
muscle cultured cells, loss of emerin also leads to altered nuclear envelope elasticity, increased
nuclear fragility116, impaired expression of mechanosensitive genes and enhanced apoptosis after
continuous mechanical strain/
117. Two hypotheses have been advanced to explain the muscle
specific phenotypes of EDMD:118 (1) a mechanical hypothesis, where EDMD originates from the
altered structural integrity of nuclei and a loss in nuclear envelope adaptation to forces in muscle
cells and (2) a genomic architecture hypothesis, where EDMD is thought to stem from modified
gene expression profiles
119, 120 and abnormal cell differentiation in muscle tissues lacking emerin
or expressing mutated emerin. Those two hypotheses are not necessarily mutually exclusive and
altered genomic regulations may be a consequence of the impaired nuclear adaptability to
mechanical stresses.
1.5. EMERY-DREIFUSS MUSCULAR DYSTROPHY (EDMD)
EDMD is one of the nine main forms of muscular dystrophy, and was first described by Alan
Eglin H. Emery and Fritz E. Dreifuss in 1966.121 There are three main forms of EDMD depending
on the pattern of inheritance: X-linked, autosomal dominant and the rare autosomal recessive.122
A systematic meta-analysis determined that the overall prevalence of EDMD across all age
demographics was 0.39 cases per 100,000 individuals.123 As expected, the X‐linked form primarily
affects males, with some disease manifestations among female carriers, while in the autosomal
dominant and recessive forms, males and females are equally affected.
124 Classical clinical features
of EDMD include early contractures, progressive muscle weakness and atrophy, and cardiac
conduction defects. The pattern of contractures is most prominently observed during neck
extension, elbow flexion, and heel cord tightening.125, 126

17
Table 1.1. Current classification of EDMD subtypes.126
Type Gene Protein Inheritance
EDMD1 EMD emerin XLR
EDMD2
LMNA lamin A and lamin C
AD
EDMD3 AR
EDMD4 SYNE1 nesprin-1 AD
EDMD5 SYNE2 nesprin-2 AD
EDMD6 FHL1 four and a half LIM
domains 1 (FHL1)
XLR
EDMD7 TMEM43 transmembrane protein
43 (LUMA)
AD
XLR = X-linked recessive; AD = autosomal dominant; AR = autosomal recessive
The two most common subtypes of EDMD (EDMD1 and EDMD2) are caused by mutations
in the EMD gene that encodes emerin and the LMNA gene that encodes lamin A/C, respectively.
126
In addition to EMD and LMNA, several other genes have been implicated in the pathogenesis of
EDMD including SYNE1, SYNE2, FHL1, and TMEM43 (Table 1.1).126, 127 Because the genes
implicated in EDMD mostly code for proteins of the NE, EDMD is also considered a form of
nuclear envelopathy.
128
The X-linked EDMD (EDMD1, emerin mutation) affects about 1:100,000 males. Female
carriers of EDMD1 are generally asymptomatic; however, they may occasionally display clinical
symptoms, which can include cardiac arrhythmias like atrial fibrillation or atrioventricular (AV)
block, leading to sudden cardiac death.129-133 Symptoms in X-linked EDMD typically manifests in
early childhood, usually between the ages of 3 and 5 years, including an unstable gait, frequent
falls, and toe walking because of the contractures in the Achilles tendons. Additionally, there is
often a restriction in the flexion of the elbows and neck, which subsequently advances to involve
the entire spine, presenting clinical similarities to rigid spine syndrome.134 The onset of cardiac

18
defects generally occurs in the second decade of life, with a progressive worsening observed.
Cardiac conduction defects are the primary cause of death in patients, with sudden death being the
most common outcome. emphasizes their need for pacemaker later in life.
125
The large majority of EDMD-causing mutations in the EMD gene are null and result in a
complete loss of emerin.135 However, few sets of missense mutations and small deletions have
been identified and also cause EDMD in patients despite proper localization of these mutated
emerins at the NE82, 92, 99, 136, 137
. These include four missense mutations, which are single
substitutions (S54F, Q133H, P183H and P183T) and two in-frame deletions that resulted to
deletion of amino acids 95-99 (Δ95-99) and 236-241 (Δ236-241).59, 135 The mutations that produce
emerin variants (S54F, Q133H, P183H and ∆95-99) are particularly interesting because the
resulting mutant emerins were shown to be expressed at levels comparable to wildtype and they
all properly localize to the nuclear envelope.
138, 139 Another emerin mutation, P183T, has been
considered to result in a less severe phenotype in affected individuals, due to the delayed onset of
initial symptoms. Like P183H, P183T is also expressed in normal levels but with abnormal
subcellular distribution.135 Moreover, the dynamics of emerin mutants ∆95-99, ∆236-241, S54F,
P183H, and P183T was analyzed by transfecting C2C12 myoblasts with of GFP-tagged emerin
mutant variants.92 The study found that all mutants showed a decrease in both targeting and
retention at the nuclear envelope compared to the wild-type emerin, with the deletion mutations
being more adversely affected than the two missense mutations.92 In a more recent study, emerin
mutants ∆95-99, Q133H and P183H were also shown to display either insufficient or excessive
self-assemblies at the nuclear envelope, both of which resulted in defective nuclear shape
adaptation against mechanical challenges.
95 This study demonstrated that controlling the selfassembly of emerin is a central determinant of the nuclear envelope response to forces, since the

19
proper self-assembly of emerin prevents deleterious nucleus deformations typically observed in
EDMD.
95
1.6. AIMS OF THIS WORK
Although considerable research has been conducted to identify emerin binding partners and
understand the structural organization of emerin at the nuclear envelope, how most of emerin
binders influence the nanoscale organization of emerin remain poorly defined. In particular, the
specific proteomic environment at the nuclear envelope that allows for a regulation of emerin selfassemblies and their protective functions during nuclear mechanics is not clearly delineated.
Whether the local proteomic neighborhoods of emerin change as a function of EDMD-inducing
emerin mutations or as a function of cell myogenesis is also unknown. As such, identifying new
binders of emerin and defining the role of currently known partners during those processes could
significantly enhance our understanding of emerin's molecular functions and contribute towards
elucidating the pathogenesis of EDMD.
Hence, the objectives of this work are: (1) to develop a simple platform based on cell
micropatterning to study the role of emerin during nuclear mechanotransduction, (2) to
quantitatively define changes in the nanoscale proteomic neighborhoods of wild-type emerin and
EDMD-relevant emerin mutants by proximity-dependent biotinylation with the engineered
ascorbate peroxidase APEX2, and (3) to explore the potential of APEX2 as an alternative method
to also map genomic regions closely associating with emerin.
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10.1016/j.ceb.2003.11.012.

32
CHAPTER 2: DEVELOPMENT OF A MICROPATTERNING STRATEGY TO STUDY
NUCLEAR MECHANOTRANSDUCTION
2.1. INTRODUCTION
Over the past two decades, micropatterning methods have become popular in biological
studies. Initially used for the electronics industry, micropatterning now provides new tools for cell
biology, and, combined with surface chemistry, it is particularly useful to study the interactions of
cells with their microenvironment on different engineered surfaces.1,2 Several micropatterning
methods of varying complexity have been developed, such as microcontact printing, microfluidic
patterning, UV-based deep etching, and micro-stencils.
3-7 While each technique has its own
advantages and drawbacks, they all provide means to study crucial cellular processes including
cell adhesion and migration, cell polarity, cell shape dynamics, cytoskeletal rearrangement, and
spatial coordination between cell and nuclear shape.1,8-12
Moreover, different materials have been used to manipulate cell adhesion on several substrates.
For instance, polyethylene glycol (PEG) can be used to functionalize surfaces for cell attachment
on defined patterns, while other hydrogels are a common substrate for three-dimensional (3D) cell
growth and adhesion.13-18 Meanwhile, spin-coating a thin layer of polydimethylsiloxane (PDMS)
on surfaces have been extensively used in cell studies due to its ease of fabrication and low cost.19,20
PDMS is also highly hydrophobic which makes it unsuitable for prolonged cell adhesion and
proliferation.21-23 Engineering the PDMS surface can optimize its compatibility for cell growth;
however, due to its limitations in forming a very thin layer on a surface by spin coating and its
relatively low optical transparency, PDMS is not ideal for imaging purposes especially with
nanometer precision imaging by single molecule super-resolution microscopy.24

33
Over the last few years, microcontact printing has become a popular micropatterning technique
because of its simplicity and robustness in generating micropatterns that guide cell adhesion and
spreading. This approach offers the advantage of requiring only mild reagents and simple
equipment, allowing medical and biological laboratories to easily participate in micropatterning.
In general, microcontact printing is a soft lithographic technique that requires a transfer of an "ink"
from an elastomeric stamp onto a surface.25 Within a short contact time between the stamp and the
surface, the “ink” is transferred with high precision and control. Microcontact printing has been
utilized to transfer self-assembled monolayers of compounds such as alkylthiols on gold surfaces,26
while extra-cellular matrix proteins such as fibronectin or laminin have been used as “ink” to
generate cell and tissue-compatible substrates.27-29
Here, we tested different silanization strategies to modify glass surface and preserve its optical
transparency making it suitable for cell adhesion and imaging. By optimizing both the
microcontact printing of the extacellular matrix protein fibronectin on modified glass coverslips
and micropatterning cells, we aimed at imposing specific physical strains at the nuclear envelope
to reveal some of the mechanotransducing functions of emerin in response to mechanical cues.
Emerin is a ubiquitous integral protein that is primarily located at the inner nuclear envelope
(INE) and belongs to the LEM (LAP2, emerin, MAN1)-domain family of proteins.30,31 It is
encoded by the EMD gene and is composed of 254 amino acids with the LEM domain at the Nterminus, a large intrinsically disordered region, and a short C-terminal transmembrane domain.31
Emerin participates in nuclear mechanotransduction and maintenance of the nuclear architecture
by interacting with a variety of nucleoskeletal proteins such as lamins and nuclear actin, and with
structural elements of the NE.31-34 Nonsense mutations in the EMD gene and lack of emerin
expression lead to Emery–Dreifuss muscular dystrophy (EDMD), an X-linked recessive disease35

34
that causes degenerative skeletal muscle wasting, heart failure, and early death.36-38 While the exact
molecular mechanisms underlying the disease are still not fully established, one hypothesis to
explain the muscle-specific phenotypes of EDMD39 is the lost ability of the cell nucleus to adapt
to mechanical cues due to the loss of emerin. For instance, isolated nuclei from emerin-null cells
show defective nuclear adaptation to mechanical strains,40 whereas emerin-deficient mouse
embryo fibroblasts display abnormal nuclear shape and impaired expression of mechanosensitive
genes after mechanical challenges.33
Our study illustrates that by utilizing two-dimensional cell micropatterning on fibronectin
substrates with varying widths, it is possible to efficiently impose incremental steady-state
mechanical strains at the cell nuclear envelope and to reveal some of the mechanotransducing
responses of emerin, as well as the effect of the nucleoskeletal proteins lamin A/C and actin.
2.2. RESULTS AND DISCUSSION
2.2.1. Vapor-Phase Surface Silanization of Glass Coverslips
In order to optimize cell micropatterning, we first developed a simple approach for surface
functionalization of glass substrates via vapor deposition of silanes. It is crucial to ensure that the
silanization of the glass substrates preserve good optical coupling and minimal background
interference, in order for the silanized glass to remain suitable for optical microscopy. We thus
tested the vapor deposition of three simple organosilanes: hexamethyldisilazane (HMDS), 3-
aminopropylethoxysilane (APTES) and (3-glycidyloxypropyl)trimethoxysilane (GPTMS) (Figure
2.1A-B). To evaluate the efficacy of the silanization procedure, we used water contact angle
measurements. Bare glass coverslips that were cleaned with Milli-Q water in an ultrasonic bath

35
Figure 2.1. Glass coverslip silanization by vapor coating. (A) schematic representation of the
vapor-phase silanization of glass coverslips with hexamethyldisilazane (HMDS); (B) structures of
(3-Aminopropyl)triethoxysilane (APTES) and (3-Glycidyloxypropyl)trimethoxysilane (GPTMS);
(C) optical image of water droplets taken by contact angle goniometry and showing significant
difference in contact angle between bare glass coverslip and HMDS-coated coverslip; (D) kinetics
of water contact angles for glass coverslips coated with different silane reagents.
and dried at 100 °C displayed contact angles of about 66° (Figure 2.1C), indicative of a relatively
good wettability. The glass surface was then etched and activated with silanol groups using Piranha
solution. This resulted in a decrease in contact angles dropping to <10°, which can be attributed to
the introduction of extra hydrophilic –OH groups on the surface of the glass.24
Reaction of the silanol groups with HMDS vapor at 95 °C (Figure 2.1A) resulted in a due to
the formation of single siloxane bonds between the trimethysilyl groups of HMDS and the silanol
groups present on the glass surface and subsequent release of NH3 gas. Upon conducting a kinetic
study of HMDS deposition, we observed that a complete coating of the glass surface is rapidly
achieved within a reaction period of 45 to 90 minutes, as indicated by the saturation of the contact

36
indicated by the saturation of the contact angles at 87° ± 1° (Figure 2.1D). These contact angle
values are similar to those previously observed for monolayer depositions of HMDS on
substrates,41 and are also in very good agreement with the theoretical contact angle expected for a
homogeneous monolayer of HMDS on glass and calculated based on the molecular cross-section
of the trimethylsilyl group (Ф = 87°, see methods).42,43 Therefore, in the succeeding experiments,
we opted for a 60-minute incubation period to ensure a consistent and complete HMDS monolayer
deposition. The HMDS-functionalized coverslips also remained hydrophobic, without showing
any change in contact angle, for more than two weeks when stored under a dry N2 atmosphere.
For the vapor-phase deposition of GPTMS, the water contact angles rapidly increased from
24° ± 6° after 3 minutes of reaction to 40° ± 10° after 10 minutes (Figure 2.1D), suggesting a
successful reaction of GPTMS to the silanol groups present on the glass surface, as previously
observed by Tsukruk et al.
44 The contact angles exhibited a gradual increase as the reaction
progressed, eventually reaching a plateau at 50° ± 4° after 90 minutes (Figure 2.1D). This result is
comparable to the previous studies on the deposition of GPTMS self-assembled monolayers on
silicon wafers by vapor and solution-phase methods, where the water contact angles saturate at
around 52° upon full monolayer coverage.44,45 The reduction in the standard deviation of mean
contact angles from 25% error at 10 min to 8% error at 90 min indicates that monolayer coverage
becomes more homogeneous across the coverslip surface as the vapor-coating reaction proceeds.
Lastly, while the deposition kinetics of APTES closely resemble those of HMDS and GPTMS,
APTES has notably higher standard deviations in mean contact angle values (Figure 2.1D). This
can be attributed to the ability of the terminal amino group of APTES to form hydrogen bonds with
silanol groups, resulting in more ways to interact with the glass surface and potentially causing
differences in the surface orientation of APTES compared to the other two organosilanes.46,47

37
During APTES vapor-coating, contact angles rise rapidly to 45° ± 8° in the initial 20 min of
reaction, after which the increase is significantly slower (Figure 2.1D). The contact angles saturate
around 50° ± 9° after 90 minutes, which is higher than the expected 41° contact angle for an APTES
monolayer with the terminal amino groups uniformly oriented away from the glass surface.46,48
The measured angles of 50° ± 9° align with an APTES coating where both hydrophilic amines and
the relatively hydrophobic ethoxy groups are exposed, indicating an uneven orientation of APTES
molecules on the glass surface.
Contrary to the relatively homogeneous monolayer achieved with HMDS and GPTMS
coatings, APTES deposition resulted in glass surfaces that are more heterogeneous and
hydrophobic than expected for an optimal monolayer coverage. Overall, HMDS monolayer
deposition by vapor-coating makes for a significantly more hydrophobic and more homogeneous
layer on glass coverslips than similar deposition of GPTMS and APTES.
2.2.2. Optimization of Cell Micropatterning on Silane-Coated Coverslips
Figure 2.2A outlines the steps employed when preparing coverslips for cell micropatterning. A
polydimethylsiloxane (PDMS) stamp containing rectangular micropatterns is incubated with
fibronectin for 30 minutes. Upon drying and stamping onto the silane-functionalized glass
coverslip, rectangular fibronectin “islands” are printed. The non-patterned areas are then blocked
by physisorption of Pluronic F-127 (PF-127), a non-ionic triblock copolymer composed of a
hydrophobic poly(propylene glycol) chain flanked by two poly(ethylene glycol) domains24,49 that
acts as an antifouling agent to prevent cell adhesion (Figure 2.2A). To determine the effectiveness
and robustness of this stamping process, we took confocal images of Cy3B-stained fibronectin
patterns on silane-coated coverslips before and after PF-127 treatment, and analyzed the contrast

38
value between “ON” stamped areas and “OFF” non-stamped areas.42,50 Fibronectin was efficiently
transferred onto HMDS-coated coverslips, resulting in the micropatterns with dimensions
matching those of the PDMS stamps (Figure 2.2B). Fibronectin remained firmly and specifically
attached on the coverslips even after incubation with PF-127 and following several washes in PBS
to remove excess PF-127 (Figure 2.2B).
Figure 2.2. Fibronectin stamping and cell micropatterning on HMDS-coated coverslips. (A)
schematic of microcontact printing of fibronectin on silane-modified glass substrates; (B)
microscopy images of Cy3B-stained fibronectin strips on HMDS-coated coverslips before and
after PF-127 treatment. Scale bar: 50 µm; (C) bright-field and wide-field fluorescence microscopy
images of the nucleus (DAPI) and SNAP-emerin fusion for U2OS cells grown on 210 × 15 µm
rectangular micropatterns. Scale bars: 50 µm.

39
Table 2.1. Quality assessment of fibronectin micropatterns.
Coating Contrast Value ± Standard Deviation
Before PF-127 After PF-127
HMDS 0.71 ± 0.04 0.67 ± 0.07
APTES 0.57 ± 0.10 0.28 ± 0.11
GPTMS 0.79 ± 0.11 0.72 ± 0.06
The contrast values for HMDS microstamping were 0.71 ± 0.04 before and 0.67 ± 0.07 after PF127 treatment, indicative of a precise and reproducible fibronectin transfer42, and of the innocuity
of PF-127 on fibronectin attachment to HMDS-treated glass (Table 2.1). As discussed later, similar
contrast values were observed for GPTMS, but for APTES coatings, the binding of fibronectin
was reduced and fibronectin detachment was significant after incubation PF-127 which induced a
drop in contrast value to 0.28 ± 0.11 (Table 2.1). This suggests that GPTMS performs as well as
HDMS for fibronectin binding and resistance to detachment upon PF-127 treatment, while APTES
does not allow for a strong attachment of fibronectin to treated glass, as evidenced by its significant
detachment in the presence of PF-127 antifouling agent.
When seeded on the HMDS-treated and fibronectin-micropatterned coverslips further
blaocked by PF-127, U2OS cells adhered specifically on the rectangular fibronectin islands within
30 min of incubation (Figure 2.2C), and remained fully spread out on the dimensions of the patterns
after 6 h of incubation. No cell adhesion was observed in non-patterned and PF-127-blocked
regions of the coverslips. For long-term culture, cells remained confined on the micropatterns for
up to 72 h by alternating the use of serum-free cell media and daily 1–2 h feeding with 10%
serum.24
This effective transfer of fibronectin and the efficient physisorption of PF-127, which prevents
cell adhesion and cell spreading outside the micropatterns can be attributed to the balanced surface

40
Figure 2.3. Antifouling efficacy of PF-127 treatment on HMDS-, APTES- and GPTMS-coated
coverslips after fibronectin attachment. Top row: Dual-color fluorescence confocal images of
fibronectin microstamped from 210 × 10 μm rectangular PDMS stamps (green) and of A647-BSA
non-specifically binding outside micropatterns (red) on HMDS-, APTES- and GPTMS-coated
coverslips after treatment with PF-127. All scale bars: 50 µm. Bottom row: Fluorescence intensity
profiles for fluorescently labeled fibronectin (green) and A647-BSA (red) along the blue line in
each of the corresponding confocal images from the top row.
wettability provided by the HMDS monolayer. Materials exhibiting water contact angles between
80° and 100° possess adequate wettability49 for facilitating both the microcontact printing of the
hydrophilic fibronectin and the adsorption of the hydrophobic polypropylene glycol domain of PF127 on surfaces. The homogeneous monolayer of the hydrophobic trimethylsilyl groups in HMDS
allow stable interactions with the polypropylene glycol of PF-127, favoring an outward orientation
of its hydrophilic polyethylene glycol “arms” away from the glass surface to provide an efficient
antifouling coating and prevent cell adhesion.
We also verified the antifouling efficacy of PF-127 on HMDS-coated coverslips by incubating
the PF-127-blocked coverslips with Alexa Fluor 647-labeled bovine serum albumin (A647-BSA).

41
Figure 2.4. Effect of PF-127 treatment on fibronectin attachment to APTES- and GPTMS-coated
coverslips. (A) fluorescence confocal images of fibronectin microstamped from 210 × 10 μm
rectangular PDMS stamps on APTES-coated coverslips before and after treatment with PF-127;
(B) fluorescence confocal images of fibronectin microstamped from 210 × 10 μm rectangular
PDMS stamps on GPTMS-coated coverslips before and after treatment with PF-127. Note that the
fluorescence intensity in the microscopy image after pluronic treatment for GPTMS was multiplied
by two to facilitate a visualization of the micropatterns. Scale bar for all images: 50 μm.
We observed little to none non-specific binding of A647-BSA outside fibronectin micropatterns
(Figure 2.3.), confirming the strong anchoring of PF-127 on HMDS. While HMDS makes the
surface hydrophobic, it does not prevent cell adhesion unless combined with PF-127.
For the APTES- and GPTMS-coated coverslips, fibronectin can also be stamped onto the glass
surfaces, where it formed the expected 210 × 10 µm rectangular micropatterns (Figure 2.4).
However, the fibronectin transfer was not as homogeneous as for the HMDS-coated surfaces, with
contrast values of 0.57 ± 0.10 for APTES and 0.79 ± 0.11 for GPTMS, both of which have
significantly larger standard deviations than for HMDS (Figure 2.4A and Table 2.1).

42
Figure 2.5. Fibronectin stamping and cell micropatterning on APTES- and GPTMS-coated
coverslips. (A) microscopy images of Cy3B-stained fibronectin strips on APTES-coated coverslips
after PF-127 treatment and of cell distribution in micropatterned and non-micropatterned areas;
(B) microscopy images of Cy3B-stained fibronectin strips on GPTMS-coated coverslips after PF127 treatment and of cell distribution in micropatterned and non-micropatterned areas. For both A
and B, scale bars: 50 µm (fibronectin patterns), 100 µm (patterned and non-patterned areas), 20
µm (zoomed areas).
Significant desorption of the stamped fibronectin was observed for the APTES-coated
coverslips after PF-127 treatment and the rectangular micropatterns become very imprecise, with
a contrast value dropping to 0.28 ± 0.11 (Figure 2.5A and Table 2.1). Because of this loss in surface
fibronectin, cells seeded on APTES-coated coverslips are not reliably confined to the expected
rectangular patterns (Figure 2.5A). In addition, many cells attached to non-stamped areas of the
coverslips, indicative of an inefficient surface antifouling by PF-127 (Figure 2.5A). Consistent
with the inability of PF-127 to act as an efficient antifouling agent, A647-BSA displayed
significantly more non-specific binding outside micropatterned areas on APTES-coated coverslips
than on HMDS-coated coverslips (Figure 2.3).

43
For GPTMS-coated coverslips, we observed a partial loss of fibronectin upon incubation with
PF-127, but the rectangular micropatterns remained well defined, with a contrast value of 0.72 ±
0.06 (Figure 2.5B and Table 2.1). While cells seeded on GPTMS-coated coverslips adhere to the
rectangular fibronectin islands and adapt their overall shape to the dimensions of the patterns,
many cells also bind to non-stamped area (Figure 2.5B). Despite incubation with PF-127,
micropatterned cells rapidly escaped the confinement of the micropatterns and started growing
outside the fibronectin 2 hours after seeding (Figure 2.5B). Indeed, as for APTES-coated
coverslips, A647-BSA exhibited relatively high non-specific binding outside the fibronectin
micropatterns, indicating that PF-127 was not well attached to the GPTMS surface or insufficiently
well oriented to exhibit efficient antifouling properties (Figure 2.3). In contrast, we see no
attachment of cells outside micropatterns and no escape from fibronectin islands even 72 h after
cell seeding on HMDS-treated coverslips. Compared to HMDS-coated glass, the higher surface
wettability of GPTMS- and APTES-coated coverslips, outside the optimal 80°–100° contact angle
range,49 likely explains the inefficient physisorption of PF-127 and the issues in controlling longterm confinement of cells within the fibronectin micropatterns.
These data indicate that HMDS-treated coverslips provide a much more robust and
reproducible surface for cell patterning compared to GPTMS or APTES coatings. Indeed, the
balanced wettability of the HMDS monolayer not only allows homogeneous microcontact printing
of extra-cellular matrix proteins such as fibronectin, but also provides a good physisorption of the
PF-127 antifouling agent that prevents cell adhesion outside patterns and limits cell escape from
microstamped areas over long periods of time.

44
2.2.3. Micropatterning for the Study of Mechanotransduction
We then used cell micropatterning to evaluate some of the mechanotransducing functions of
emerin, an INE protein linked to EDMD. While emerin is expressed in all somatic cells, nonsense
mutations in the Emd gene and absence of emerin expression primarily affect cells exposed to
extensive mechanical stress, such as skeletal and cardiac muscle cells.51 We therefore studied the
influence of increasing mechanical strains on the nuclear shape of wild type (Emd+/y) and emerin
Figure 2.6. Applying mechanical strains to the nucleus by micropatterning cells on increasingly
narrow rectangular fibronectin strips. (A) confocal images of wild type fibroblasts immunostained
for F-actin (red) and the nucleus (blue) after plating on non-patterned or patterned rectangular
fibronectin substrates having 15 µm, 10 µm and 5 µm widths. Scale bar: 20 µm; (B) wide-field
images of emerin-null fibroblasts immunostained for F-actin (red) and the nucleus (blue) after

45
plating on non-patterned or patterned rectangular fibronectin substrates having 15 µm, 10 µm and
5 µm widths. Scale bar: 20 µm, (C) changes in NSI as a function of micropattern width for wild
type human skin fibroblasts (Emd+/y), emerin-null skin fibroblasts (Emd−/y
) and emerin-null skin
fibroblasts rescued with wild type (WT) SNAP-emerin fusion (Emd−/y + WT emerin-fusion). The
box length represents the NSI interquartile range, the central square represents the NSI mean, the
central bar represents the NSI median, and the error bars represent the standard deviation of the
mean. N = 50–60 cell nuclei per condition. T-test comparison to Emd+/y cells: ** p < 0.01, ns: nonsignificant.
null (Emd−/y
) human dermal fibroblasts by patterning them on increasingly narrow rectangular
fibronectin islands on HMDS-coated coverslips (Figure 2.6A,B). The fibronectin micropatterns
have widths of 15, 10, or 5 µm and constrain adhesion to regions smaller than the size of the
fibroblasts, effectively resulting in cell-shape modification and actin-driven modulation of the
nuclear shape index (NSI).11,24 The NSI is a measure of the roundness of the nucleus such that an
NSI of 1 corresponds to a circular nuclear shape.11 As shown in Figure 2.6C, Emd+/y fibroblasts
have a nearly circular nucleus with NSI of 0.96 ± 0.01 when grown randomly on fibronectin-coated
coverslips. As nuclear mechanical strains increase, the nucleus adopts an oval shape and the NSI
of Emd+/y fibroblasts gradually decreased to 0.88 ± 0.03, 0.81 ± 0.02 and 0.76 ± 0.03 for 15, 10,
and 5 µm-wide patterns, respectively (Figure 2.6A,C). On the other hand, Emd−/y
fibroblasts from
EDMD patients grown randomly on fibronectin display a slightly more deformed nucleus with an
NSI at 0.94 ± 0.01 (p < 0.01, Figure 2.6B,C), consistent with previous results in emerin-deficient
mouse fibroblasts33 and human muscle cells.52 The increased nuclear deformation and modified
nuclear envelope stiffness of Emd−/y
fibroblasts are more evident from the NSI values obtained in
micropatterns, all of which are significantly lower than those of normal Emd+/y fibroblasts (p <
0.01, Figure 2.6C).
These aberrant nuclear mechanoresponses of emerin-null fibroblasts are directly linked to the
absence of emerin. Interestingly, rescuing emerin expression in Emd−/y cells by lentiviral

46
transduction of a SNAP-tag N-terminal fusion to wild type emerin (SNAP-emerin) led to a
complete recovery of normal nuclear responses to forces, showing no significant difference in NSI
compared to wild type Emd+/y fibroblasts (ns, Figure 2.6C). These results confirm that the absence
of emerin led to abnormal nuclear envelope mechanics, emphasizing the crucial role of emerin in
maintaining the structural integrity of the nucleus.
While emerin primarily localizes at the INE, it was recently shown that external forces
cyclically applied to the nucleus of epidermal keratinocytes trigger a partial redistribution of
emerin to the ONE.53 When cells are patterned on narrow fibronectin strips, we also previously
observed that transiently expressed emerin fusions have increased association with the perinuclear
ER membrane, which is continuous with the ONE.24 Consequently, we investigated whether the
nuclear envelope deformation and maintenance of nuclear shape against steady-state mechanical
strains imposed by micropatterns would result in the relocation of endogenous emerin from the
INE toward the ONE and the ER membrane. We employed two different membrane
permeabilization methods with (1) Triton X-100 to selectively label the entire cellular pool of
emerin (ER, ONE, and INE), and with (2) saponin to specifically look at the pool of emerin
associated with the ER and ONE since saponin only permeabilizes the plasma membrane and not
the nuclear membrane.
As shown in Figure 2.7, fibroblasts grown randomly on fibronectin-coated coverslips showed
a clear enrichment of emerin at the INE, while the application of mechanical strains and ensuing
nuclear deformations led to a non-negligible relocation of emerin to the ONE and ER. In particular,
the cells permeabilized with Triton X-100 exhibited a 25% increase in the ratio of ER to total
nuclear envelope emerin levels as the nucleus adapts to strains, and this increase remained constant
across different nuclear deformations with no significant changes between 15, 10, and 5 µm

47
Figure 2.7. Fluorescence confocal imaging of emerin distribution in wild type Emd+/y human skin
fibroblasts as a function of nuclear strains. The distribution of the entire cellular pool of emerin
(ER, ONE, and INE) is imaged in cells treated with Triton X-100 as a detergent to permeabilize
both the plasma and the nuclear membrane (left panels). The pool of emerin only associated with

48
the ER and the ONE is imaged in cells treated with saponin to permeabilize only the plasma
membrane (right panels). Cells grown randomly on fibronectin (non-patterned) or on increasingly
narrow rectangular micropatterns (15–5 µm) are immunostained for emerin (green), lamin A/C
(red) and the nucleus (blue). Scale bar for all images: 20 µm.
Figure 2.8. Quantification of emerin redistribution in response to nuclear mechanical strains. (A)
ratio of ER emerin to nuclear envelope (ONE and INE) emerin for non-patterned Emd+/y skin
fibroblasts (Triton X-100, n = 16 cells) or fibroblasts grown on 15 µm-wide (Triton X-100, n = 15
cells), 10 µm-wide (Triton X-100, n = 15 cells), and 5 µm-wide (Triton X-100, n = 20 cells)
fibronectin rectangular micropatterns. The fluorescence intensity quantification is done on sum
slices images of full cell confocal z-scans obtained after Triton X-100 permeabilization and emerin
immunostaining. For each cell, the intensity ratio is normalized to the mean ER/nuclear envelope
ratio of non-patterned cells; (B) relative amount of ER and outer nuclear envelope emerin for nonpatterned Emd+/y skin fibroblasts (saponin, n = 17 cells) or fibroblasts grown on 15 µm-wide
(saponin, n = 14 cells), 10 µm-wide (saponin, n = 18 cells), and 5 µm-wide (saponin, n = 24 cells)
fibronectin rectangular micropatterns. The fluorescence intensity quantification is done on sum
slices images of full cell confocal z-scans obtained after saponin permeabilization and emerin
immunostaining. For each cell, the quantified emerin intensity is normalized to the mean emerin
intensity for non-patterned cells. For both (A) and (B), the thick bars and the error bars represent
the mean and the standard deviation of each distribution, respectively. T-tests: ** p < 0.01, ns: nonsignificant.
A B

49
patterns (Figure 2.8A). These results indicate that adjusting nuclear shapes against mechanical
strains involves an initial relocation of emerin to the ER membrane independent of the overall
emerin expression level.
Meanwhile, a similar analysis of saponin-permeabilized cells verified that when the nucleus is
subjected to mechanical strains in micropatterns, the amount of endogenous ER and ONE emerin
increases significantly (Figure 2.8B). Specifically, the amount of emerin associated with the ER
and the ONE grew by 50% in micropatterned fibroblasts, with normalized emerin quantities
shifting from 1.00 ± 0.29 (n = 17 cells) for non-patterned fibroblasts to 1.46 ± 0.42 (n = 13 cells,
p < 0.01), 1.52 ± 0.38 (n = 18 cells, p < 0.01) and 1.56 ± 0.44 (n = 24 cells, p < 0.01) for fibroblasts
on 15, 10, and 5 µm-wide patterns, respectively (Figure 2.8B). Together, these data demonstrate
that a key mechanotransducing function of emerin is to partially relocate from the INE to the ONE
and the ER membrane in order to guarantee adequate nuclear deformation when a cell nucleus is
exposed to mechanical challenges.
While a majority of EDMD patients carry nonsense mutations resulting in undetectable emerin
protein, four mutations (S54F, Q133H, Δ95–99, P183H) were found to cause EDMD despite the
mutant emerin protein localizing normally and being expressed at normal or near-normal
levels.54,55 We then studied how cells expressing three of these mutants (Q133H, Δ95–99, P183H)
respond to mechanical stress introduced by the micropatterns. Emd−/y
fibroblasts were transduced
with lentivirus for the expression of SNAP-emerin mutants for 48 hours, and the cells were then
plated on fibronectin-stamped HMDS coverslips. As shown in the changes in NSI in Figure 2.9B,
the three emerin mutants caused defective mechanical responses of the nuclei of Emd−/y
fibroblasts
as the micropattern width decreased. In randomly plated cells, the expression of mutant emerin
resulted in slightly less circular nuclei (lower NSI) compared to cells expressing wild-type emerin

50
(Figure 2.9B). Furthermore, cells expressing emerin mutants exhibited a notably higher NSI
compared to cells expressing wild-type emerin as the micropattern width decreased, suggesting an
inability to properly adapt the nuclear shape in response to increasing mechanical forces. These
deficient changes in nuclear shape were also accompanied by a mispositioning of the nucleus
relative to the cell major axis, nuclear crumpling, abnormal organization of the actin cytoskeleton
Figure 2.9. Emerin mutations induce defective nuclear shape adaptation against mechanical stress.
(A) Diagram of emerin with binding and self-association domains (self-ass.) and position of Δ95-
99, Q133H and P183H mutations. (B) Nuclear shape index as a function of micropattern width for
EMD−/y HDFs expressing wild-type, Q133H, Δ95-99 or P183H emerin. The box represents the
25–75th percentiles, and the median is indicated. The whiskers mark the s.d. of the mean, and the
squares indicate the mean. *P<0.05; **P<0.01; ns, not significant (Wilcoxon test). (C)
Fluorescence imaging of actin (green) and the nucleus (blue) in micropatterned EMD−/y HDFs
expressing Q133H, Δ95-99 or P183H emerin. Images representative of three experiments for each
micropattern width. Scale bars: 50 µm.
A B
C

51
Figure 2.10. (A) Nuclear shape index as a function of micropattern width in EMD+/y HDFs
depleted for lamin A/C or nuclear actin. The box represents the 25–75th percentiles, and the
median is indicated. The whiskers mark the s.d. of the mean, and the squares indicate the mean.
Wilcoxon test, **P<0.01. (B) Confocal fluorescence imaging of lamin A/C and the nucleus (DAPI)
after siRNA-induced depletion of lamin A/C and re-expression of exogenous and siRNA-resistant
lamin A/C. Scale: 20 µm. (C-D) Effects of IPO9 siRNA and XPO6 siRNA on nuclear actin
organization. (C) Examples of nuclear localization patterns for the short nuclear actin filament
probe Utr230-EN in HDF cells. Patterns are classified as: (i) small puncta and diffuse, (ii) diffuse
or (iii) large foci, reflecting variations in nuclear actin filament content across cells. (D)
Distribution of nuclear actin filament classes after control siRNA treatment (n = 635 nuclei), IPO9
siRNA (n = 663 nuclei) or XPO6 siRNA (n = 625 nuclei). Knockdown of IPO9 results in the
majority of cells displaying a diffused Utr230-EN pattern, indicative of lower nuclear actin
filament contents. Inversely, knockdown of XPO6 results in the majority of cells displaying larger
and brighter foci compared to control siRNA, indicative of increased nuclear actin filament
contents.

52
and failure of cells to properly fit within micropatterns, specifically in cell areas adjacent to the
misshaped nucleus (Figure 2.9C). This indicates that the expression of mutated emerin in Emd−/y
fibroblasts deformed as the patterns got narrower (p<0.01, Figure 2.9B).
To observe the effect of nuclear actin depletion, we first generated cells that are stably
expressing Utr230-EN, a probe based on a truncated mutant of utrophin that was shown to
selectively bind actin filaments and fused to enhanced green fluorescent protein (eGFP) and to a
nuclear localization sequence (NLS).56,57 Utr230-EN bind nuclear F-actin and show a distinct
pattern characterized by small, distributed puncta, and these localization patterns were found to be
similar in the nuclei of various mammalian cell types. 56,57 We observed the same puncta on our
EMD+/y HDFs expressing Utr230-EN. Furthermore, we characterized the distribution of nuclear
actin into three categories: (1) small, diffuse puncta, (2) diffuse, and (3) large and bright foci.56
The levels of nuclear actin filaments can then be controlled by knocking down importin 9 (IPO9)
and exportin 6 (XPO6), which shuttle actin into and out of the nucleus, respectively.56,58,59
Reduced expression levels of IPO9 caused a shift in the distribution of Utr230-EN to more
diffuse structures, indicating a lower concentration of nuclear actin filaments. Conversely, the
knocking down of XPO6 resulted in the opposite effect, that is: larger and brighter foci are present
in XPO6 knockdown cells. The results align closely with prior results obtained in U2OS cells.56
Once we established a method to control the nuclear actin levels, we then looked at how the nuclear
actin-depleted cells respond to mechanical stress. Interestingly, similar results are observed with
LMNA knockdown cells, where in non-patterned cells, the nuclei were less circular than control
cells, and significantly more deformed as the cell growth width decreased. Thus, both results from
LMNA and IPO9 depletion indicate that the emerin binding partners lamin A/C and nuclear actin

53
indicate the significant impact of the nucleoskeleton in influencing the response of the nucleus to
mechanical stress.
2.3. CONCLUSIONS
Cell patterning is an effective way to control cell shape and study cell and nuclear
mechanotransduction. We developed a technique that demonstrated that a monolayer deposition
of HMDS is possible by vapor coating on activated glass coverslips. This HMDS vapor deposition
method provides a surface that has balanced wettability, sufficient hydrophilicity for the
microstamping of fibronectin, and adequate hydrophobicity to maintain PF-127 on non-stamped
areas and provide good anti-fouling efficiency. Through a simple micropatterning strategy, we
investigated the function of emerin during nuclear mechanoresponses in human skin fibroblasts.
While cells might be shaped in many different geometries on two-dimensional micropatterns,
robust and stable cell confinement in rectangular fibronectin islands having widths smaller than
the dimension of a typical cell provides a simple means to impose and modulate steady-state
mechanical strains on the nucleus, as demonstrated by gradual changes in NSI. With different size
of the micropatterns, we showed that cellular expression of emerin is critical for the maintenance
of nuclear shape against increasing mechanical strains. We also demonstrated that nuclear
deformation against forces is associated with an initial redistribution of emerin from the INE
towards the ONE and the ER membrane. Furthermore, micropatterning studies involving cells
expressing the EDMD-causing emerin mutants showed aberrant nuclear response with mechanical
stress. These findings confirm that emerin plays a pivotal role in nuclear mechanotransduction
processes at the nuclear envelope. Finally, we also applied our micropatterning strategy to study
the impact of nucleoskeleton in the nuclear response to mechanical stress. Overall, cell

54
micropatterning by direct fibronectin microcontact printing on vapor-coated HMDS coverslips
provide a straightforward approach to impose steady-state forces to cells and study their molecular
mechanobiology in a quantitative manner in vitro.
2.4. MATERIALS AND METHODS
2.4.1. Surface Modification of Glass Coverslips
High precision microscope glass coverslips (Marienfeld, #1.5, Ø25 mm) were cleaned using a
Piranha solution made of a 3:1 (v/v) mixture of 18 M sulfuric acid and 30% hydrogen peroxide for
15 min and rinsed thoroughly with deionized (DI) water. Following drying with nitrogen gas, the
coverslips were heated to 95 °C in a sealed glass jar containing 100 μL of silane reagents for
incremental reaction times of 3 to 90 min (silanization kinetics) or for a fixed reaction time of 90
min (cell micropatterning). The silane reagents used were: hexamethyldisilazane (HMDS, SigmaAldrich, St. Louis, MO, USA), (3-Aminopropyl)triethoxysilane (APTES, Sigma-Aldrich,) and (3-
Glycidyloxypropyl)trimethoxysilane (GPTMS, Sigma-Aldrich). After vapor coating, the silanecoated coverslips were cleaned by sonication in water for 5 min and dried with N2 gas. The silanetreated coverslips were then stored in a separate and sealed glass container flushed with nitrogen.
Static contact angle measurements were done on a ramé-hart 290-F1 Contact Angle Goniometer
(ramé-hart instrument co., Succasunna, NJ, USA) with 5 μL water droplet volumes tested over 5–
8 different positions per coverslip on at least 6 coverslips per reaction points.
2.4.2. Fabrication of PDMS stamps
Polydimethylsiloxane (PDMS) stamps with rectangular micropatterns were produced from
silicon masters fabricated using a chrome photomask (Minnesota Nano Center, Minneapolis, MN,
USA). The micropatterns are 210 μm in length and have widths of 5 μm, 10 μm or 15 μm,

55
respectively, with a constant periodic intervals of 30 μm. The etching depth on these silicon
masters is 10 μm.
The silicon masters were treated with tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane
(Gelest Inc., Morrisville, PA, USA) for 90 min under vacuum to induce the formation of
fluorosilane vapors and surface fluorosilanization of the masters. A 10:1 mixture of PDMS and
curing agent (Sylgard 184 elastomer kit, Dow Corning, Midland, MI, USA) was combined in a
plastic beaker and thoroughly mixed with a glass stirring rod for 10 min. As this step generates
bubbles, the PDMS mixture is degassed under vacuum for 30 min (or by centrifugation at 3000
rpm for 10 min). The degassed PDMS mixture was then slowly poured onto the silicon masters,
and another degassing step was done to avoid unwanted bubbles. After curing for 3 h at 65 °C and
overnight at room temperature (RT), the PDMS stamps were removed slowly from the silicon
master using a razor blade. Before being used in microcontact printing, the PDMS stamps were
cleaned by 5 min sonication in water followed by 5 min sonication in ethanol. We routinely use
the PDMS stamps for a period of 3–4 months without loss of stamping efficiency.
2.4.3. Calculation of Theoretical Contact Angle for a Monolayer Formation with HMDS
The Cassie equation43 [1] describes the expected contact angle of a solute on a bifunctional
surface(in our case the piranha-treated coverslip glass surface and its HMDS layer) as follows:
cos Ф = f1 cos θ1 + f2 cos θ2, [1]
where Ф is the equilibrium contact angle of water on the bifunctional surface (glass coverslip +
HMDS), f1 is the molecular fraction coverage of HMDS on the bifunctional surface, θ1 is the
expected contact angle of water on a pure HMDS surface (without coverslip support), f2 is the
molecular fraction of the bifunctional surface not covered by HMDS, and θ2 is the expected
contact angle of water on the pure glass coverslip surface (without HMDS).

56
f1 and f2 depend:
(i) On the circular cross-sectional area taken up by the trimethylsilyl group n the
circular cross-sectional area taken up by the trimethylsilyl group (Si-(CH3)3) of
HMDS, which has been estimated by Herzberg et al. (2) at 27.7 Å2
, and
(ii) On the number per surface area of hydroxyl silanol groups (Si-OH) that are
available to react with HMDS on the coverslips. For a fully hydroxylated silica
surface, this number has been estimated at 5 per 100 Å2 by different groups,62-64
and it is assumed to be unchanged for glass.
Thus, for a perfectly modified glass coverslip substrate where coverage and reaction of HMDS
with silanol groups is optimal (monolayer), the surface molecular fraction coverage of
trimethylsilylshould be f1 = (100/27.7)/5 = 0.722. Consequently, we have a glass surface molecular
fraction non-covered by HMDS of f2 = 1 − 0.722 = 0.278.
The contact angle value of water on the piranha-treated glass coverslip surface (without
HMDS) was measured and reported in Fernandez et al. as θ2 = 3.5°.24 There is, however, no known
θ1 valuefor a pure HMDS surface (without substrate support) because HMDS cannot polymerize
on its own into a solid surface. However, the contact angle value of water on PDMS can be used
as an appropriate estimate of θ1 for HMDS because this polymer is extremely rich in methyl groups
similar to those found in HMDS. Here, we used a contact angle of water on PDMS of 108° as
determined by molecular simulation.66 This value is in good agreement with reported experimental
estimates between 98° and 112°. Thus, with values: f1 = 0.72, θ1 = 108°, f2 = 0.28 and θ2 = 3.5°
we can evaluate the expected equilibrium contact angle of water (Ф) for a perfect and monolayer
coverage of HMDS on a glass coverslip, from Cassie's equation. We obtain cos Ф = 0.054 and Ф
= 87°. This value is in excellent agreement with our measured value of 87 ± 1° after 90 min of

57
reaction, thus indicating that vapor coating provide a homogenous monolayer deposition of HMDS
on glass coverslips.
We note that multilayer polymerization of HMDS at the coverslip surface (although it is
improbable considering the nature of the chemical reaction) would result in water contact angles
that would increase toward 108° overtime. Similarly, sub-monolayer coverage would result in
water contact angles that would decrease from the theoretical 87° value towards the 3.5° angle
value measured for piranha-treated glass.
2.4.4. Microcontact Printing
For microcontact printing, 100 μL of 100 mg/mL fibronectin in phosphate-buffered saline
(PBS, 154 mM NaCl, 5.6 mM Na2HPO4, 1.1 mM KH2PO4, pH 7.5) were placed on the PDMS
stamps and incubated at 25 °C for 30 min. The fibronectin solution was then aspirated off and the
PDMS surface was rinsed twice with PBS. Upon drying, the inked PDMS surface was brought
into contact with the silane-functionalized glass coverslips for 1–2 min, applying light pressure to
guarantee good contact between the stamp and the glass. To block non-patterned areas, the
coverslips were immersed in 1% (m/v) Pluronic F-127 (PF-127, Sigma-Aldrich) in Milli-Q water
(EMD Millipore, Billerica, MA, USA) for 20 min, then rinsed three times with PBS prior to cell
seeding.
To evaluate the quality of the stamping before and after PF-127 treatment, the fibronectinstamped HMDS-, GPTMS- and APTES-coated coverslips were treated with 31 nM Cy3Bmaleimide (GE Healthcare Life Sciences, Marlborough, MA, USA) in PBS for 1 h and rinsed three
times with PBS, before imaging on a LSM 700 Confocal Laser Scanning Microscope (Zeiss, White
Plains, NY, USA). To assess the antifouling efficacy of PF-127, 100 µL of 10 mg/mL (150 µM)
of bovine serum albumin (BSA, Alfa Aesar-Thermo Fisher Scientific Chemicals, Ward Hill, MA,

58
USA) was fluorescently labeled with 10 µL of 10 mg/mL (8 mM) of Alexa-Fluor 647 Succinimidyl
Ester (Life Technologies, Carlsbad, CA, USA) for 1 h in PBS. The reaction was quenched with
150 µL of 20 mM Tris buffer (pH 7.8) for 1 h and the mixture was diluted to a final concentration
of 2.5 mg/mL (37 µM) of A647-BSA with PBS. A647-BSA at 2.5 mg/mL was applied for 1 h on
fibronectin-stamped coverslip previously labeled with Cy3B-maleimide and treated with PF-127.
After three rinses with PBS, the coverslips were imaged by fluorescence confocal microscopy.
After stamping, the PDMS stamps were immersed in DI water and cleaned in an ultrasonic
bath at 60 °C for 10 min, then immersed in 100% ethanol and sonicated for another 10 min at 60
°C before drying with nitrogen gas and storage.
2.4.5. Measurements of Contrast Values to Compare the Quality of Fibronectin Microcontact
Printing across Coverslips Coated with Different Organosilanes
To quantify the quality of the fibronectin micropatterns, fluorescence intensities of Cy3B
fibronectin along microcontact printed strips (n = 15–25 rectangular 10 × 210 µm strips) on
multiple coverslips were measured for the three organosilane coatings, before and after PF-127
treatments. Specifically, the mean contrast fluorescence values ± standard deviation were
calculated as previously described67 as:
𝐶 =
𝐼𝑂𝑁 − 𝐼𝑂𝐹𝐹
𝐼𝑂𝑁
where C is the mean contrast value, Ion is the mean fluorescence intensity within the rectangular
fibronectin strips where fibronectin transfer occurs, and Ioff is the mean fluorescence intensity in
between strips, where no transfer should occur. These results are reported in Table 2.1.
2.4.6. Cell Culture and Expression of Emerin Fusion
The cells used in this study are: human osteosarcoma U2OS cells, wild type human dermal
fibroblasts (Emd+/y) and emerin-null human dermal fibroblasts (Emd−/y). All cells were maintained

59
in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Walkersville, MD, USA) supplemented
with 10% fetal bovine serum (FBS; Gibco-Life Technologies, Gaithersburg, MD, USA) and 1%
penicillin/streptomycin (Lonza, Walkersville, MD, USA) in a humidified incubator at 37 °C,
supplied with 5% CO2. For micropatterning, trypsinized cells resuspended in DMEM + 10% FBS
were plated on fibronectin-stamped and PF-127-blocked coverslips, and were allowed to attach
onto the micropatterns for 1 h. The cell culture media was then replaced to remove excess
unattached cells. The cells were allowed to fully spread out for about 6 h at 37 °C before live
imaging or chemical fixation.
The lentiviral plasmid containing containing the genes encoding a SNAP-tag N-terminal fusion
to the wild type (SNAP-emerin) were produced by Fernandez et al. in Ref. 60. Similarly, sitedirected mutagenesis to produce the SNAP-emerin mutants are reported in Ref. 60. The lentiviral
particles for the expression SNAP–emerin were produced by the UCLA Vector Core. Cells grown
at 70% confluence on 6 well-plates were infected with 150 µL of a viral titer at 0.09 µg/mL in
complete medium containing 8 μg/mL polybrene for 48 h, after which the lentivirus was removed
and replaced with fresh complete medium. After another 24-h incubation, cells were trypsinized
and plated on micropatterns, as described above.
2.4.7. RNA interference
The Dicer siRNA for lamin A/C (labeled Lamin A/C DsiRNA #2, 5′-
GGAACUGGACUUCCAGAAGAACAtc-3′) was obtained from Integrated DNA Technologies
(IDT). Lamin A/C DsiRNA #2 was designed using IDT’s Custom Dicer-Substrate siRNA
(DsiRNA) Tool, and would work well with eGFP-lamin A/C expression, with the cDNA encoding
for an exogenous siRNA-resistant human lamin A/C was kindly provided by Dr Richard Frock,
Stanford University, USA.61 The control DsiRNA, 5′-CGUUAAUCGCGUAUAAUACGCGUat-

60
3′ (IDT#51-01-14-04) was also from IDT. Lamin A/C DsiRNA #2 and control DsiRNA were
transfected at 25 nM using X-tremeGENE HP (Roche). The cells are then fixed after 2 days.
The siRNA duplex for IPO9 was obtained from Ambion (ID: S31299) and siRNA for the
nuclear actin exporter XPO6 was obtained from Qiagen (ID: SI00764099). IPO9 and XPO6 siRNA
were transfected at 25 nM using X-tremeGENE HP (Roche). Every 2 days, the cells are retransfected with the IPO9 and XPO6 siRNA before fixation after 5 days.
2.4.8. Cloning and Lentiviral Production for Utr-230-EN
pEGFP-C1 Utr230-EGFP-3XNLS was a gift from Dyche Mullins (Addgene plasmid # 58466).
Utr-230-EGFP-3xNLS was subcloned into into pHR-SFFV-V5-APEX-NLS lentiviral plasmid
with BamHI and NdeI restriction sites. For PCR amplification, the following primers were used:
BamHI-Utr230_F: 5’-atagGATCCgccaccatggccaagtatggagaaca-3’
EGFP-NdeI_R: 5’-atatCATATGgattgagctcgagatctgag-3’
Correct insertion of Utr230-EGFP-3XNLS into pHR-SFFV lentiviral backbone was verified
by Sanger sequencing (Genewiz).
To generate lentiviruses, HEK 293T cells plated in a T25 flask (Falcon) were transfected at
∼80% confluency with 0.5 mL of serum-free DMEM containing the lentiviral plasmid encoding
the Utr230-EN, the lentiviral packaging plasmids psPAX2 (Addgene #12260) and pCMV-VSV-G
(Addgene #8454) at 2:1.5:1 mass ratio, respectively, and 12 µL X-tremeGENE HP (transfection
protocol was followed according to manufacturer’s instructions). The next day, the media was
changed to 5 mL DMEM containing 10% FBS and 1% bovine serum albumin (Gold
Biotechnology). After 24 hours, the medium containing lentivirus particles was harvested and
stored at 4 °C, and replaced with another 5 mL DMEM containing 10% FBS and 1% bovine serum

61
albumin. A second harvest was performed after another 24 hours and combined with the previous
harvest, filtered through a 0.45 μm filter, aliquoted and stored at -80 °C until further use.
2.4.9. Immunofluorescence
For nuclear shape index (NSI) measurements, cells were fixed with 4% paraformaldehyde in
PBS for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 4% bovine
serum albumin (BSA) + 0.1% Tween-20 for 1 h, all at RT. Wild type and emerin-null fibroblasts
were then stained with Rhodamine Phalloidin (1:1000, Abcam Inc., Cambridge, MA, USA) for 1
h, and washed three times with PBS for 5 min each. Coverslips were mounted on a glass slide
using DAPI-Fluoromount G (Electron Microscopy Sciences, Hatfield, PA, USA) and sealed with
clear nailpolish. U2OS cells and emerin-null human dermal fibroblasts expressing SNAP-emerin
were fixed, permeabilized, and blocked as mentioned. Before mounting in DAPI-Fluoromount G,
cells were then additionally stained with 1:1000 of SNAP-Surface® Alexa Fluor 647
benzylguanine substrate (BG-A647; New England Biolabs, Ipswich, MA, USA) in 4% BSA +
0.1% Tween-20 for 1 h at 37 °C and washed three times with PBS for 5 min each. Microscopy
images were acquired by wide-field on an inverted Eclipse Ti-E microscope (Nikon Instruments
Inc., Melville, NY, USA) or by confocal imaging on a Zeiss LSM 700 Confocal Laser Scanning
Microscope.
For experiments involving emerin redistribution as a function of nuclear mechanical strains on
micropatterns, wild type human dermal fibroblasts were fixed with 4% paraformaldehyde in PBS
for 15 min, permeabilized with either 0.1% Triton X-100 (cell membrane and nuclear
permeabilization, Sigma-Aldrich) or 0.1 % saponin (cell membrane permeabilization only, EMD
Millipore) for 10 min, and blocked with 2% BSA + 1% normal goat serum (NGS) for 1 h, all at
RT. Cells were stained with rabbit anti-emerin (1 μg/mL, Abcam Inc., Cambridge, MA, USA) and

62
mouse anti-Lamin A/C (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) primary antibodies
for 1 h at RT, then rinsed 3x with blocking buffer for 5 min. Staining with a goat anti rabbit-Alexa
Fluor 488 (1:500, Life Technologies) and a goat anti mouse-Alexa Fluor 647 (1:1000,
ThermoFisher, Life Technologies) secondary antibodies was then done for 1 h. Following three
washing steps of 5 min in blocking buffer and three additional washing steps of 5 min in PBS, the
coverslips were mounted and sealed as stated before. Images and z-scans through each cell were
acquired on a Zeiss LSM 700 Confocal Laser Scanning Microscope.
2.4.10. Microscopy Imaging
Confocal microscopy images were acquired on a Zeiss LSM 700 confocal laser scanning
microscope equipped with a C-Apochromat 40×/1.2 W Korr objective, excitation lasers at 405 nm,
488 nm, 555 nm, and 639 nm, a multiband 405/488/555/639 beam splitter and appropriate emission
filters for the detection of DAPI, Alexa Fluor 488, Cy3B, Rhodamine and Alexa Fluor 647. Images
were acquired in 12-bit mode and the same settings were used across all samples. Confocal zstacks were collected over the entire thickness of each cell in 0.34-μm slice intervals.
Wide field microscopy images were acquired on a Nikon Eclipse Ti-E microscope equipped
with a 40× objective (Nikon), an X-Cite 120XL fluorescence illumination system, an iXon Ultra
EMCCD camera (Andor) and appropriate filter sets for DAPI (Ex: 430DF24, Dich.:458DiO2,Em:
483DF32, Semrock) and A647 (Ex: 628DF40, Dich.:660DiO2,Em: 692DF40, Semrock)
detections.
2.4.11. Image Analysis and Statistics
All image analyses were performed using FIJI software (version 1.52, National Institutes of
Health, Bethesda, MD, USA). The nuclear shape index (NSI) for each cell was determined by

63
measuring the nuclear cross-sectional area and the nuclear perimeter of DAPI-stained nuclei, and
by calculating the ratio:
𝑁𝑆𝐼 =
4𝜋 × 𝑎𝑟𝑒𝑎
𝑝𝑒𝑟𝑖𝑚𝑒𝑡𝑒𝑟2
(1)
The NSI is a measure of the roundness of the nucleus such that an NSI of 1 corresponds to a
circular nuclear shape. Mean NSI values ± standard deviation of the mean are reported for n = 50–
60 cell nuclei per condition.
Total ER and ONE emerin intensities were obtained from sum slices Z projection of each cell
treated with saponin. After correction for background signal, the emerin intensity for each cell was
normalized to the mean emerin intensity for non-patterned cells. ER to nuclear envelope emerin
ratios were also measured from sum slices Z projection of cells treated with Triton X-100. For
each cell, emerin intensities in the ER and the nuclear envelope were separately measured using
region-of-interests drawn over the entire cell or over the cell nucleus only, using DAPI signal as a
template. After correction for background signal, the ER to nuclear envelope intensity ratio for
each cell was normalized to the mean ER to nuclear envelope intensity ratio for non-patterned
cells. OriginPro (version 2019b, OriginLab, Northampton, MA, USA) was used to plot
distribution, means and standard deviations of measured quantities. Statistical significances were
assessed by two-tailed student’s t-tests. p-values < 0.05 were considered significant.
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CHAPTER 3: PROMITY LABELING METHODS FOR INVESTIGATING
BIOMOLECULAR INTERACTIONS
3.1. INTRODUCTION
Cellular activities are tightly regulated by molecular interactions among biomolecules
including proteins, nucleic acids and small molecules.1-3 These intricate molecular interaction
networks vary from stable to transient interactions, and while they play a crucial role in a wide
array of biological processes, their perturbation or disruption is associated with numerous human
diseases. Because of the complex nature of these molecular interactions, numerous techniques that
facilitate their identification within living cells have significantly advanced our understanding of
the biological mechanisms, signaling pathways and subcellular spatial localization of
biomolecules.4-6
3.2. CLASSICAL METHODS FOR STUDYING PROTEIN-PROTEIN INTERACTIONS
To elucidate protein-protein interactions (PPIs), conventional approaches such as yeast twohybrid (Y2H), affinity purification (AP), co-immunoprecipitation (Co-IP) and protein crosslinking
are often coupled to mass spectrometry (MS)-based proteomics (Figure 3.1).7
3.2.1. Yeast Two-Hybrid System
The yeast two-hybrid system (Y2H) is a protein fragment complementation assay used for
mapping protein-protein and protein-nucleic acid interactions.8
In general, a transcription factor is
split into two separate fragments, called the DNA-binding domain (BD) and the activating domain
(AD). The protein of interest (POI) and its potential binding partners are then expressed as fusions
to the BD and AD of the transcription factor, respectively, in genetically modified yeast (Figure

70
3.1A). The interaction between the two proteins leads to the reconstitution of the activation and
DNA binding domains of the transcription factor, which are positioned upstream of a reporter gene.
This then may lead to a color change, or it may allow the yeast to thrive in selective media.7, 8
While this method is often cost-effective and high throughput, several limitations include issues
in expression levels of the POI, possible protein misfolding in yeast, cell type and organelle type
restrictions in yeast, lack of or abnormal posttranslational modifications, and the inability to
determine multiprotein complexes.7, 8
Figure 3.1. Traditional methods for studying protein-protein interactions. (A) Yeast two-hybrid
method: a protein of interest (POI) is fused to the DNA binding domain while its potential
interactor is fused to the activation domain of a functional transcription factor. If the proteins
interact, the transcription factor is activated producing a color or allowing growth in selective
media. (B) Co-immunoprecipitation of protein interactors: The POI is purified by affinity
chromatography with an antibody or via an affinity tag, bringing along interactors. (C) Chemical
crosslinking of protein interactors: Bifunctional linkers are used to covalently crosslink protein
interactors that are then purified by affinity chromatography. (Adapted from Ref. 7
).

71
3.2.2. Affinity Purification-Mass Spectrometry (AP-MS)/ Co-Immunoprecipitation-Mass
Spectrometry (Co-IP/MS)
AP-MS and Co-IP/MS are methods based on the principle that interacting proteins can be
effectively pulled-down with the protein of interest.9
In AP-MS, also referred to as
immunoprecipitation (IP)-MS, a specific protein or molecule of interest bound to a solid support
or a matrix serves as a bait. A mixture of proteins is introduced to the matrix, which allows the bait
protein to capture its interacting partners (preys). The identity of the interacting partners is then
identified by mass spectrometry.9, 10
On the other hand, Co-IP is the immunoprecipitation of intact protein complexes. It is a method
that involves the use of an antibody targeting a known protein that is thought to be a member of a
larger protein complex (Figure 3.1B).7, 9 By binding this known protein to the antibody, it is
possible to extract the entire protein complex from the solution, allowing for the identification of
other, less characterized members of the complex by mass spectrometry.
AP-MS and Co-IP/MS offer several advantages such as high sensitivity, ability to identify
multiprotein complexes, and the potential to detect and quantify posttranslational modifications.
However, the main limitation is that weak or transient interactions are commonly disrupted and
lost during cell lysis and washing steps. In addition, a high-quality primary antibody is usually
necessary, and the occurrence of high background levels due to nonspecific protein binding during
the enrichment process is also a common occurrence.7, 9, 10
3.2.3. Chemical Crosslinking-Mass Spectrometry
To overcome the loss of weak or transient interactions in AP-MS, chemical crosslinking mass
spectrometry (XL-MS) may be utilized. XL-MS works by using a reagent that covalently

72
crosslinks nearby proteins (Figure 3-1C).7 Unnatural amino acids usually with photosensitive side
chains may also be incorporated into the POI to promote crosslinking to nearby proteins. After
digestion, cross-linked protein residues can then be identified by MS.7, 11, 12 Because crosslinking
also traps weak and transient interactors, XL-MS enables the use of more stringent conditions for
cell lysis and washing. A major challenge for XL-MS, however, is the processing of more complex
mass spectrometry data which require more complicated computational algorithms.7, 11, 12
While these methods demonstrate remarkable capabilities for studying biomolecular
interactions and are widely used, they also entail certain limitations. One such drawback is that a
biochemical fractionation step is usually required, which involves having the protein complexes
be efficiently extracted from cell/tissue while maintaining their integrity throughout the lysis,
purification and washing steps. It is therefore more difficult to characterize protein complexes
found in subcellular compartments with low solubility (for example, membranes, chromatin,
nuclear lamina, and cytoskeleton) because the harsh buffer conditions required to solubilize the
proteins located in these compartments often disrupt the protein-protein interactions.7, 13
3.3. PROXIMITY LABELING METHODS FOR PROTEIN-PROTEIN INTERACTIONS
Over the past decade, proximity labeling (PL) techniques have gained prominence because
they offer effective means to identify strong as well as weak and transient protein interactions.14
In general, PL is based on the fusion of a modifying enzyme to a targeting signal peptide or directly
to a POI (Figure 3.2). The modifying enzyme converts a cell-permeable substrate conjugated to an
affinity tag (often biotin) into a highly reactive intermediate which then diffuses and covalently
label nearby endogenous proteins with the extent of labeling being influenced by their spatial

73
Figure 3.2. Mechanism and evolution of the most common methods for enzyme-mediated
proximity labeling. (A) Schematic of a typical proximity labeling (PL) workflow. An enzyme (a
biotin ligase or a peroxidase) is fused to the protein of interest (bait) and targeted to a specific
subcellular location. Proximity labeling is achieved through the in situ enzymatic synthesis of
biotin-conjugated reactive intermediates, which subsequently diffuse away and react with nearby
proteins/nucleic acids (NAs). The nanometer-scale action radius of the intermediates (shown as a
red contour map) covers both proteins/NAs that tightly associate with the bait and those that
loosely interact in the same compartment, enabling PL to reach over multiple layers of protein–
protein/NA interactions. After cell lysis, biotinylated proteins are collected by affinity purification
and characterized by mass spectrometry. Biotinylated RNAs are analyzed by high-throughput
sequencing. (B) The mechanism and technology development timeline of PL. In the presence of
H2O2, APEX (green) converts biotin phenol to phenoxyl free radical, which reacts with the
adjacent tyrosine residues. In the presence of ATP, BioID (cyan) activates biotin into bio-AMP,
which reacts with lysine residues of neighboring proteins. The timeline describes a brief history of
major APEX- and BirA-mediated PL techniques. Methods highlighted in green, blue, and pink
refer to protein-centered, RNA-centered, and DNA-centered profiling, respectively. (Adapted from
Ref. 15).

74
proximity. The labeled protein complex or organelle can then be affinity purified and
characterized by mass spectrometry.
14, 16
In addition to studying protein-protein interactions, PL has found application in the proteomic
profiling of subcellular regions such as the mitochondrial matrix17 and intermembrane spaces
18
,
synaptic clefts19 and organelle contact sites.20, 21 PL has also been applied to study cell-cell
interactions22, 23 as well as signal transduction networks and pathways, including those ofGprotein-coupled receptors (GPCR)24, 25, mitogen-associated protein kinase (MAPK)26
, and Wnt
signaling pathway27. Lastly, PL can be used not only in a variety of cell types but also in different
organisms (bacteria28, protozoans29, fungi30, yeast31, worms32, viruses33, flies34, plants35, mouse22
and human36 tissue).
To date, two main classes of enzymes have been utilized for proximity-dependent
biotinylation: biotin ligases and peroxidases.
3.3.1. Biotin Ligase-Based Methods
The biotin ligase BirA is a conserved enzyme that facilitates the attachment of biotin to target
proteins. In the presence of ATP, BirA converts biotin into a reactive biotinoyl-5’-AMP (bio-AMP)
that is held within the BirA active site until it reacts specifically with lysine residues of the biotin
acceptor tag sequence in its substrate proteins, which are primarily restricted to a few carboxylases
in mammalian cells.37 Since the wild-type BirA has a very high specificity to its target sequence,
Choi-Rhee et al. mutated the active site of the E. coli BirA resulting in a R118G mutant (R118GBirA
or BirA*). This mutation not only significantly reduced BirA’s affinity for biotinoyl-5’-AMP, but
also allowed promiscuous biotinylation of surrounding molecules, i.e., biotinylation of lysine
residues without requiring specific amino acid sequence recognition.38, 39 Roux and coworkers
leveraged the promiscuity of BirA* to develop BioID, the first proximity labeling technique using

75
biotin ligase, and applied it to determine the proteins in proximity to the human lamin A.40
Subsequent BioID investigations into the architecture of the nuclear pore complex revealed that
BioID has an approximate labeling radius of 10nm.41
With a size of 35.1 kDa, BirA* is slightly larger than GFP and can sometimes prevent efficient
targeting of some fusion proteins. To overcome this problem, a smaller enzyme was developed
from the Aquifex aeolicus biotin ligase, named BioID2, which has R40G mutation.
42 While BioID2
works similarly to BioID, it was found that BioID2 it less disruptive due to its smaller size (26.4
kDa), and is capable of achieving high levels of biotinylation at lower biotin concentrations
(>3.2μM) than BioID, which requires a concentration of 50μM.42
Recently, three split versions of BioID were developed for protein-fragment complementation
assays.21, 43, 44 De Munter et al. split BirA* at E140/Q141 (split-BioID). The two inactive halves
of BirA* are separately fused to protein phosphatase PP1 (PP1) and to two known PP1 interactors,
nuclear inhibitor of PP1 (NIPP1) and RepoMan.43 The split-BioID approach was successful in
identifying the interactors of PP1, and also enabled a more comprehensive assessment of the
apparent interactors associated with the PP1-NIPP1 complex compared to those of the PP1-
RepoMan complex. The second version of split-BioID was developed by Schopp and coworkers
by splitting the BirA* at E256/G257 and used to study the miRNA silencing pathway.44 One
fragment was fused to Ago2 and the other to a specific component of the silencing pathway
complex. Their split-BioID technique applied to Ago2-TNRC6C and Ago2-Dicer revealed the
protein GIGYF2 as a regulator of miRNA-mediated translation repression.44 Finally, Kwak and
coworkers reported the third split-BioID method, termed ContactID, by splitting BirA* at
G78/G79.21 The split site was identified based on temperature factors (B factors), which are related
to the atomic flexibility at crystalline state, i.e., a high B factor for the split site on the flexible loop

76
of BirA* is required to preserve the structural integrity and biotinylation activity of the split-BioID
when reconstituted.21, 45
Split-BioID systems offer more spatiotemporal control and allow for lower level of nonspecific biotin labeling since the biotin ligase only becomes active when reconstituted. However,
BioID-based PL methods are constrained by their slow kinetics, which require biotin labeling of
18 to 24 hours.40, 42 To overcome this, Branon et al. reported two engineered biotin ligases,
TurboID and miniTurbo, which could biotinylate proteins at a significantly higher speed, providing
labeling times as short as 10 min without decreasing specificity.46 TurboID (35 kDa) and
miniTurbo (28 kDa) were produced from R118SBirA using a directed evolution. Each enzyme was
engineered from R118SBirA using a yeast display-based directed evolution method. TurboID was
found to be slightly more active than miniTurbo (1.5-2-fold) but it also has more background
labeling, i.e, it can utilized endogenous biotin and thus PL begins prior to incubation with
exogenous biotin.46 Split-TurboID has also been developed, and the split site at L73/G74 was
identified through a computational algorithm called SPELL (split protein reassembly by ligand or
light) which evaluates the energy profiles of candidate fragments against that of the full-length
protein, in combination to other parameters such as solvent accessibility, sequence conservation
and geometric constraints to identify and evaluate potential split sites.
20, 47 Both low-affinity (PPIdependent reconstitution) and high-affinity (PPI-independent reconstitution) versions of splitTurboID have been reported, and the split-TurboID labeling time of about 1 hour is still an
improvement compared to the split versions of BioID.20
Another recently engineered mutant called BASU (29 kDa) was derived from the Bacillus
subtilis BirA and demonstrated a >1000-fold faster kinetics (significant labeling possible for about
1 minute) and a >30-fold improvement on signal-to-noise compared to that of the E. coli BirA.48

77
The PL method called ChromID was developed by Villaseñor et al. who fused BASU to engineered
chromatin readers (eCRs). They then used it to identify proteins interacting with individual histone
marks H3K9me3, H3K4me3 and H3K27me3 in mouse embryonic stem cells.49
Finally, Kido and co-workers reported a novel BirA enzyme for PL, AirID (ancestral BirA for
proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme
reconstruction algorithm and metagenomic data.50 Results showed that AirID has higher
biotinylation activity toward interacting proteins both in vitro experiments and in cells, despite
exhibiting an 82% sequence similarity to BioID. AirID also has the potential to be used for longlasting experiments in living organisms, since it was shown to be less toxic for cells over time,
unlike BioID and TurboID.50
In general, PL methods based on biotin ligases have been widely used to study protein-protein
interactions because they are relative simple to implement, only requires incubation with
exogenous biotin, and it can be adapted to in vivo animal studies. However, since biotin is a
cofactor in many biological processes, care should be taken in designing biotin ligase PL
experiments particularly with TurboID and miniTurbo since their high biotinylation activity may
deplete endogenous biotin levels. Another drawback of biotin ligase PL methods is the relatively
long labeling time of least 10 min for efficient proximity labeling. As a consequence, other
proximity labeling methods based on peroxidase enzymes are usually preferred for experiments
requiring faster kinetics.
3.3.2. Peroxidase-Based Methods
Peroxidases represent another group of PL enzymes that can convert various substrates into
free radicals in the presence of hydrogen peroxide (H2O2). Horseradish peroxidase (HRP, 44 kDa)
is a well-known member of this group and was the first peroxidase to be used in PL studies. In the

78
presence of H2O2 it converts a substrate into a highly reactive radical that covalently tags
neighboring proteins on electron-rich amino acids, particularly tyrosine residues.51 Substrates
based from phenol derivatives produce phenoxyl radicals that are short-lived (<1 ms) and have a
small labeling radius (<20 nm).52, 53 Kotani et al. first reported that HRP can convert an aryl-azide
biotin reagent into free radical specials, called “enzyme-mediated activation of radical source” or
“EMARS” reaction54, which they then coupled to MS-based proteomics to identify protein
components of the cell membrane.55 Li and coworkers then introduced a variation of this HRPbased PL approach called selective proteomic proximity labeling using tyramide (SPPLAT), a
method that employs biotin-tyramide (biotin-phenol, BP) as the substrate for HRP in conjunction
with quantitative proteomics by stable isotope labeling through amino acids in cell culture
(SILAC).56 With the SPPLAT method, they provided the first PL proteomic profiling of B-cell
receptor clusters using the chicken B cell line DT40 as a model.56
One important disadvantage of HRP as a PL enzyme is that its structure is dependent upon two
Ca2+ ion-binding sites and four disulfide bond that are broken in cellular compartments with
reducing environments (e.g. cytosol and mitochondria), which inactivates the HRP.51 Because of
this, HRP-based PL is limited to oxidizing environments such as the ER lumen, the Golgi, or the
cell surface.19, 57
Another peroxidase used for PL is ascorbate peroxidase (APEX). Initially, APEX was
developed to catalyze the H2O2-dependent polymerization of 3,3’-diaminobenzidine (DAB) into a
localized precipitate to give high contrast upon OsO4 treatment and generating high resolution
electron microscopy images.58 APEX is a monomeric triple mutant (K14D/W41F/E112K) derived
from the dimeric pea or soybean ascorbate peroxidase (APX) and produced by structure-guided
rational design. Unlike HRP, APEX lacks disulfide and calcium-binding sites and thus it is active

79
in all cellular compartments, and has a smaller molecular weight (28 kDa).58 APEX was then
successfully employed in profiling the mitochondrial proteome in living cells.17, 18 The low
sensitivity of APEX was a major limitation to its utility for EM and proteomics applications,
notably because, when expressed at low levels, the APEX activity with DAB (for EM) and biotinphenol (for proteomics) becomes undetectable.59 Because of this, Lam et al. sought to improve the
sensitivity of APEX using yeast display-based directed evolution, and after three rounds of
evolution, an APEX2 version was generated, which contains the addition of A134P mutation into
the soybean-derived APEX. This second generation APEX2 was shown to have improved kinetics,
thermal stability, heme binding and resistance to high H2O2 concentrations.59
Similar to split-BioID and split-TurboID, split versions of HRP and APEX have also been
developed for protein-fragment complementation assays. Martell and colleagues developed the
split-HRP. Guided by the HRP structure, they rationally screened 17 HRP split sites located on
solvent exposed loop regions, determined that 213/214 was the best split site, and then utilized
directed evolution to further improve the complementation and labeling efficiency, yielding a final
split-HRP which has six mutations compared to HRP: T21I, P78S, R93G, N175S, N255D, and
L299R.60
They then fused the split-HRP fragments to the proteins neurexin (NRX) and neuroligin
(NLG) and enabled sensitive visualization of synapses between specific sets of neurons.60

Two versions of the split-APEX2 have been reported.61, 62 Xue and coworkers rationally
screened different split sites and concluded that sites 201/202 display the highest enzymatic
activity and lowest background signal. This split-APEX2 was then used to detect STIM1 and
Orial1 homodimers as well as the biotinylation sites on STIM1.61 Interestingly, another splitAPEX2 system (sAPEX2) was developed by Han et al. by first rationally determining that the site
200/201 was the best split site. The 1-200 fragment (called AP) was subjected to 4 generations of

80
directed evolution, resulting in 9 mutations and improved complementation with the 201-250
fragment (called EX) to produce a functional sAPEX2.62 Because the 200/201 cut site disrupted
the heme-binding site of APEX2, Han also recommended heme supplementation prior to biotinphenol labeling.62

Similarly, two more versions of APEX2 have been introduced.63, 64
The cysteine-free C32S
mutant of APEX2 (C32SAPEX2) was introduced by Huang and colleagues to improve the
expression in mammalian cells when fused to four SLC protein family members (SLC1A5,
SLC6A5, ALC6A14, and SLC7A1) compared to APEX2, potentially caused by mismatched
disulfide bond formation. They found that C32SAPEX2 exhibits comparable enzymatic activity to
APEX2, allows correct SLC fusion protein localization, and provides consistent levels of
biotinylation using biotin-phenol.63 On the other hand, Becker et al. found that APEX2 exhibits a
cytoplasmic-biased localization and identified a putative nuclear export signal (NES) at the
carboxy-terminus of APEX2 (NESAPEX2), structurally adjacent to the conserved heme binding
site.64 To remove this cytoplasmic-biased localization, they identified a unique separation-offunction mutant, APEX2-L242A, termed APEX3. They then tested the localization and
biotinylation functionality of APEX3 by fusing it to the nucleocytoplasmic shuttling
transcriptional factor, RELA.64
The peroxidase-dependent approaches, especially APEX2-based methods, have been
frequently used in PL studies because of their convenience and fast labeling kinetics compared to
biotin-ligase PL methods. A major drawback, however, is the use of hydrogen peroxide, which
can be toxic and thus limits the utility of peroxidase-based PL in in vivo studies.

81
3.4. PROXIMITY LABELING METHODS FOR PROTEIN-NUCLEIC ACID INTERACTIONS
3.4.1. DamID and Related Methods
DamID is one of the earliest PL methods and it was initially developed to map the DNA contact
sites of proteins.65 DamID works by fusing the E. coli DNA adenine methyltransferase (Dam) to a
protein of interest and is expressed at low levels inside living cells. Dam catalyzes the addition of
a methyl group to the N6 position of adenine in the sequence GATC. Endogenous methylation of
adenines is absent in most eukaryotes. Upon expression of the fusion protein in cultured cells or
in an intact organism, Dam will be targeted and recruited to specific loci that are the native binding
sites of the POI.66 Dam will then be able to methylate adenines in GATC sequences near the DNA
binding site of the POI. The methylated DNA can then be extracted from the cells, followed by
digestion with methylation sensitive restriction enzymes DpnI and DpnII. Protocols have been
developed to combine DamID with next-generation sequencing (NGS) in order to produce a map
(chromatin profile) of loci at which the POI has been in close proximity.67 DamID has been
successfully used to generate genome-wide maps of DNA-interacting proteins in different
organism such as Drosophila,
65 C. elegans,
68 yeast,69 plants,70 and mouse71 and human cell lines.72
To prevent mis-targeting of the Dam fusion protein and to avoid saturating methylation of
genomic DNA, Dam fusion proteins have to be expressed are very low levels. Traditional DamID
uses the “leaky” Drosophila hsp70 promoter in the absence of heat to allow low-level expression
of Dam.73 Better inducible systems have since been developed including the fusion of a
destabilization domain (DD), which causes a Dam-POI fusion to be rapidly targeted for
proteosomal degradation unless the protein is shielded by the synthetic small molecule Shield1.74,
75

82
For cell-specific Dam expression, targeted DamID or TaDa was reported.76 In this method, low
Dam levels are controlled using the expression of a bicistronic construct in which the Dam
sequence is placed downstream of a primary open reading frame (ORF). When the construct is
expressed, the primary ORF (may be a fluorescent protein reporter gene) is expressed at relatively
high levels, while ribosome re-initiation occurs very infrequently at the secondary ORF (Dam-POI
fusion), which results in very low levels of Dam translation.76 Dam-POI expression can be induced
only in cells of interest by using the Drosophila Gal4/UAS system, resulting in targeted
methylation in only the desired tissues.76
Compared to chromatin immunoprecipitation (ChIP), DamID does not require crosslinking.
However, several limitations of DamID-based methods include: sequence specificity of
methylation, potential disruption of the localization and binding of the POI and long labeling time
(∼24 h).77
3.4.2. APEX2- and BioID-based Methods
In addition to mapping protein-protein interactions, APEX2 PL has also been applied to
subcellular transcriptome mapping.78 Initial methods include APEX2 targeted to specific
subcellular compartments (nucleus or mitochondria) where it labeled RNA binding proteins
(RBPs). By combining APEX2 with either UV-crosslinking (Proximity-CLIP)79 or formaldehyde
crosslinking (APEX-RIP), biotinylated RBPs are enriched and co-purified with bound RNAs. The
crosslinked RNAs are then released and sequenced, while the biotinylated RBPs are identified by
mass spectrometry.
A method for RNA sequencing called APEX-Seq has also been developed where it was shown
that APEX2 can directly label RNAs. APEX2 was targeted to different subcellular regions
(nucleolus, nuclear lamina, nuclear pore, mitochondrial membrane, mitochondrial matrix) and

83
following RNA sequencing, APEX-Seq provided spatiotemporal information about the
organization of RNAs in live cells.80
This method has also been applied to study the organization
of translation initiation complex and repressive RNA granules.81 In addition, Zhou et al.
characterized the nucleic acid labeling capability of APEX2 and showed that guanosine residues
are the most prominently labeled nucleotide.82
APEX-RIP, Proximity-CLIP and APEX-Seq are protein-centric methods where the RNA
interaction partners of a specific protein bait can be identified by RNA sequencing. There are also
RNA-centric methods in which the protein partners of a specific RNA bait are identified.14, 83

RaPID (RNA–protein interaction detection) is an RNA-centric method that allows the
biotinylation of RBPs by tagging an RNA of interest with a BoxB aptamer to recruit a fusion
protein of the bacteriophage λN peptide and BASU. This method was used to study the interactions
between Zika virus RNA and host cell proteins.48 Similar RNA-centric approaches have been
reported by the fusion of MS2 coat protein (MCP) to BioID84 and to APEX285 to recruit these PL
enzymes to MS2-tagged RNAs. MCP-BioID and MCP-APEX2 identified proteins that interact
with exogenously expressed tagged RNA, which may not accurately reflect the interactome of
native transcripts.14
To target endogenous RNAs Han et al. targeted catalytically inactive Cas13d fused with
APEX2 and a double-stranded RNA-binding domain (dsRBD) (to enhance its binding affinity) to
human telomerase RNA. This allowed them to uncovered previously unknown interaction
between human telomerase RNA and the N6-methyladenosine (m6A) demethylase ALKBH5.
85
PL methods to study protein-DNA interactions are much more limited than PL methods for
protein-RNA studies. ChIP-seq and DamID are widely used to capture and sequence DNA regions
associated with a POI.
66, 86 An analogous method to APEX-RIP for identifying the genomic regions

84
proximal to POI is APEX-mediated chromatin labeling and purification (ALaP), where APEX2
biotinylate proteins nearby the POI bait, which are in turn crosslinked by formaldehyde to
neighboring DNA regions.
87 Subsequently, biotinylated protein–DNA fragment complexes are
enriched by streptavidin and analyzed by next-generation sequencing. ALaP-Seq has been shown
to have improved sensitivity but decreased specificity in comparison to traditional ChIP-seq.87
ALaP-Seq has been used to define the genomic contact sites of promyelocytic leukemia bodies
(PML), phase-separated nuclear structures that physically interact with chromatin.
87, 88
Finally, APEX-ID is a method reported by Tran and coworkers for mapping protein, RNA and
DNA interactions with the nuclear lamina.89 This method involved performing the APEX2 labeling
after paraformaldehyde fixation followed by extraction of the biotinylated preys (proteins, RNA
or DNA). They explored APEX-ID by fusing APEX2 to lamin B1, a nuclear lamina protein, and
identified (1) that a C-rich motif binding regulatory protein exhibits altered localization in laminnull cells; (2) that RNAs interacting with or proximal to the nuclear lamina have a long 3’ UTR
bias (also consistent with an observed bias toward longer 3’ UTRs in genes deregulated in laminnull cells); and (3) that short, H3K27me3-rich variable lamin associated domains (LADs) exist
throughout the cell cycle.89
3.4.3. Photoactivated PL methods
An alternative to direct biotinylation of RNA by APEX2 is chromophore-assisted proximity
labeling and sequencing (CAP-seq) that uses the blue light-activated singlet oxygen generator
miniSOG for proximity-dependent photo-oxidation of RNA nucleobases, particularly guanosine
bases.
90 The oxidized guanosines are subsequently crosslinked to propargyl amine (PA) probes that
are taken up by the cells. Following extraction, the modified RNA can then be labeled with biotin
through a click-chemistry reaction (between biotin-azide and the alkyne-conjugated RNA) and

85
enriched for RT-qPCR or RNA-seq.
90 CAP-seq has been tested in the mitochondrial matrix, ER
membrane (ERM), and the outer mitochondrial membrane (OMM) in HEK293T cells. The
temporal resolution of CAP-seq (~20 min) is lower than that of APEX-seq (1 min), but the results
of CAP-seq complement those of APEX-Seq data and the two approaches offer distinct
mechanisms for RNA labeling.14, 90 CAP-Seq has also been recently applied to study the spatial
organization of chromatin, notably by targeting miniSOG to subnuclear locations (nucleoplasm,
nuclear lamina and nucleolus).
91 Results have revealed that LADs cover 37.6 % of the genome,
and that these LADs overlap with heterochromatin hallmarks and are depleted with CpG islands.91
3.5.SUMMARY
Over the last decade, proximity labeling has emerged as an essential technique for investigating
known biomolecular interactions and identifying novel interactions. With innovations in method
development, PL proved to be suitable for studying interactions among protein, RNA, DNA and
even small molecules, and can be applied to various model organisms. While enzyme-based PL
are still the most widely used approach with biotin ligases and peroxidases, more studies are still
needed to further optimization those enzymesin terms of labeling kinetics and background labeling
(especially for BioID and TurboID). For protein–nucleic acid mapping, improving the efficiency
of RNA/DNA labeling by PL enzymes will be necessary, for instanceby using new
substrates/probes to enhance the sensitivity and the analysis of transcriptomes and genomes.
Finally, newer PL enzymes like miniSOG with its smaller size may be an alternative to systems
where the proper localization of the PL enzyme-fusion become disrupted . The newer PL enzymes
can also be complementary to the more traditional biotin ligase and peroxidase methods because
they differ in temrns of labeling mechanisms.

86
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CHAPTER 4: EXPLORING THE EMERIN INTERACTOME BY
APEX2-PROXIMITY PROTEOMICS
4.1. INTRODUCTION
Emerin is a conserved LEM domain protein that has been implicated in Emery-Dreifuss
Muscular Dystrophy (EDMD).1
It is an integral membrane domain that localizes primarily in the
INM, but it has been shown that it also localizes to the ONM and ER.2
It carries out multiple roles
within the nuclear lamina, contributes to the organization of nuclear structure, regulates
transcription, and influences chromatin architecture by binding and interacting with a number of
proteins including Lamin A/C, BAF, and actin.1, 3-7 These interactions have mostly been
determined by classical biochemical assays like affinity purification-MS, and yeast two-hybrid,
but these assays have the limitation of only being able to isolate high-affinity molecular
interactions under non-physiological conditions or in vitro, and they also have shortcomings when
it comes to organelle and/or subcellular protein localization and identification.8, 9 The past decade
has seen the growth of proximity labeling (PL) methods combined with mass spectrometry-based
proteomics as an alternative approach to detect and identify protein-protein interactions. PL offers
the unique advantage of studying protein interactions within living cells and even in living
organisms.10, 11 Proximity labeling involves tagging the endogenous interacting partners of a
protein of interest (bait) by first fusing the bait protein with a promiscuous enzyme that produces
reactive species capable of labeling amino acids proximal to the bait protein. The tagged proteins
can then be enriched and identified by mass spectrometry. 10, 12, 13
In this work, we utilized proximity labeling by an engineered ascorbate peroxidase APEX2 to
determine the proximity and interacting partners of emerin and those of EDMD-causing mutants

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P183H, Q133H and Δ95-99. We designed our PL experiments such that we are able to detect
proximity proteome at both the N- and C-terminus of emerin, on both sides of the ER and nuclear
membranes where emerin traffics before accumulating at the INM. Furthermore, we used
splitAPEX2 to detect proteins that specifically interact with emerin oligomers.
4.2. RESULTS AND DISCUSSION
4.2.1. Generating Stable V5-tagged APEX2-expressing Dermal Fibroblasts
Several studies have reported the consequences of emerin overexpression on its localization
and function. For example, Lee and co-workers observed that emerin overexpression in HeLa cells
downregulated the transcription of a number of genes including those regulated by the Wnt and
STAT pathways.14 In another research by Markiewicz et al. revealed that the overexpression of
emerin impedes β‐catenin signaling by preventing the nuclear accumulation of β‐catenin.
Additionally, Shimojima and coworkers reported that nuclear invagination is significantly
increased by emerin overexpression.2, 15
As this study aimed to elucidate the interacting partners of emerin via APEX2-mediated
biotinylation and given the aforementioned effects of emerin overexpression, we sought to ensure
that the expression of the APEX2-emerin fusion protein remains close to that of endogenous
emerin levels, notably to minimize the risk of altered cellular localizations and functions or the
identification of false-positive proximity partners. To achieve endogenous expressions of emerin
fusions to APEX2, we first knockdown the endogenous emerin by transducing wild-type human
skin fibroblasts (HDF+/y) with an shRNA lentivirus targeting the 3’-UTR of the EMD gene. After
1 week of selection with puromycin, we were able to establish a stable emerin knockdown

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Figure 4.1. Generation of emerin-knockdown fibroblasts by lentiviral transduction with shRNA
targeting the 3’-UTR of the EMD gene. (A) Confocal florescnce images of HDF+/y transduced with
scramble shRNA and EMD shRNA clearly showed a marked decrease in endogenous emerin.
Scale bars: 50 µm. (B) Confirmation of the emerin knockdown efficiency by western blot. Data
were normalized to β-actin expression. The results presented are the mean ± SD of two biological
replicates (each with three technical replicates). Student’s t-test for statistical significance. **** p
< 0.0001.
fibroblast cell line (EMDkd) and confirmed a knockdown efficiency of 75.7% by
immunofluorescence and immunoblotting (Figure 4.1).
4.2.2. Rational design of APEX2 fusions to emerin
To identify proteins that selectively interact with the emerin using proximity labeling, we
fused the engineered ascorbate peroxidase APEX2 to emerin. The inserted APEX2 was designed
with an additional V5 epitope tag to facilitate the detection of APEX2 fusions in cells and to
differentiate APEX2 fusions to emerin from endogenous emerin (Figure 4.2).

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Figure 4.2. Schematic of V5-tagged APEX2 fusion constructs and principles of APEX2-mediated
proximity labeling. The APEX2 biotinylation zone is expected to be <20 nm.
We designed two APEX2 fusions to wild-type emerin – one at the N-terminus of emerin
(APEX2-EMD) and another at the C-terminus (EMD-APEX2) (Figure 4.2). Emerin is a
transmembrane protein that primarily localizes at the inner nuclear membrane (INM), but before
it accumulates at this membrane it also travels throughout the ER membrane and the outer nuclear
membrane (ONM). As such, the N-terminus of emerin is exposed to the cytoplasm during its ER
and ONM travel and to the nucleoplasm once it localizes at the INM. Similarly, the C-terminus of
emerin is exposed to the ER lumen and to the perinuclear space on its journey toward the INM.
Our N-terminal APEX2-EMD fusion orients APEX2 towards the cytoplasm and the nucleoplasm.

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Figure 4.3. The V5-tagged APEX2 fusion constructs used in this work.
We also sought to explore how three EDMD-inducing mutations of emerin (P183H, Q133H,
Δ95-99) might influence its interactions with protein partners, again using both N- and C-terminus
APEX2 fusions. Finally, we also developed means to study the proteomic interactome specific to
emerin oligomers. To achieve this, we utilized the split-APEX2 technology.16 This method
involves the division of APEX2 into two non-functional fragments, AP and EX, which only exhibit
peroxidase activity when they are brought together during molecular interactions. We fused each

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component to emerin (AP-V5-EMD and EX-Flag-EMD), designating these constructs as
splitAPEX2-EMD and EMD-splitAPEX2 (Figure 4.3), and co-expressed them in cells. Upon the
assembly of emerin dimers and higher order oligomers, the AP and EX fragments come into
sufficient proximity to facilitate the reconstitution of peroxidase activity. Using split-GFP imaging,
we already showed that split-protein fusions to emerin re-assemble only at sites of emerin
oligomerization and splitAPEX2-emerin fusions are expected to behave similarly.17
Considering that emerin faces a variety of cellular compartments along its trafficking route
toward the INM (cytoplasm, ER membrane, ER lumen, ONM, nucleoplasm and INM), we also
establish adequate spatial controls for our emerin proximity labeling studies, notably to deconvolve
compartment bystanders from specific emerin partners. This was achieved by directing APEX2 to
compartments where emerin is exposed and by fusing APEX2 to another protein that shares the
same trafficking route and final localization but does not interact with the emerin itself. Those
controls significantly mitigate the influence of non-specific bystanders on emerin’s proximity
proteomes, which facilitates the identification of proteins that are genuinely associated with
APEX2-emerin or emerin-APEX2, including both known and novel interaction partners.18 To this
end, we chose several spatial controls. The first one involve the use of a truncated lamin B receptor
(LBR1TM, aa: 1-238, only the N-terminus lamin binding domain and the first transmembrane
segment). LBR1TM does not bind emerin but diffuses in the same endomembranes as emerin (ER
and ONM) before accumulating at the INM.
19-21 APEX2 fusions to LBR1TM (both N-term. and
C-term.), serve as robust controls to account for the non-specific “en passant” biotinylation of
proteins embedded in those membranes due to emerin lateral diffusion. Other spatial controls
include APEX2-NES (cytoplasm), APEX2-NLS (nucleoplasm) and APEX2-KDEL (ER lumen)
fusions.

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4.2.3. APEX2 fused to emerin localizes predominantly to the INM and biotinylates endogenous
proteins
To characterize the functionality of APEX2-emerin and emerin-APEX2 fusion, we examined
their localization in HDFs. We first rescued emerin expression by transducing the EMDkd
fibroblasts with lentivirus for the expression of both N- and C-term. APEX2 fusions to wild-type
emerin (WT) or the three emerin mutants mentioned previously (P183H, Q133H, Δ95-99). After
10 days of selection with blasticidin, we were able to establish stable cells lines expressing all
those APEX2 constructs. For the splitAPEX2, we performed a double transduction, first with APemerin (selection by blasticidin) then with EX-emerin (selection by hygromycin) also in EMDkd
fibroblasts, while for spatial controls (APEX2-LBR1TM, LBR1TM-APEX2, APEX2-NES,
APEX2-NLS, and APEX2-KDEL) HDF+/y cells were used. As shown in Figure 4.4 (see also Figure
4.13.), each APEX2 construct is properly localized, as evidenced by the anti-V5 stain, and all
maintain a high level of biotin labeling efficiency (streptavidin staining).
Subsequently, we tested the biotin labeling efficiency of emerin-APEX2 fusions by western
blotting. Cells stably expressing the APEX2 constructs were incubated with biotin-phenol, and the
biotinylation reaction was initiated by the addition of H2O2. After 1 minute, the reaction was
stopped, and the cells were harvested. The clarified whole cell lysates were probed with
streptavidin-HRP and imaged by electrochemiluminescence. As shown in Figure 4.5, multiple
proteins were biotinylated in APEX2-EMD (and mutants) with similar biotinylated pattern
observed in the APEX2-LBR1TM, indicating that LBR1TM is an adequate and important spatial
control. In addition, we probed the expression level of both endogenous emerin and emerin fusion
to APEX2 in all cell lines to define the total emerin expression levels, and compared it to that of
control non-knocked down HDF+/y
. As shown in Figures 4.5. and 4.6., the combined expression of

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endogenous and APEX2 fusion to emerin sin all stable cell lines is non-significantly different from
that observed in HDF+/y. Combined with the proper cellular localization of those fusions (Figure
4.4), it indicates that APEX2 emerin fusions likely traffic and behave similarly to that of
endogenous emerin in cells.
From those western blot assays, we can also see that the extent of biotinylation induced by
APEX2 is largely above the signal expected for endogenously biotinylated proteins, to an extend
that endogenously biotinylated proteins are not visible (Figures 4.5. and 4.6.). It is also notable
that the biotinylation patterns for EMD-APEX2 are entirely distinct from those of APEX2-EMD
(Figures 4.5. and 4.6), which suggest that the interacting emerin partners to be detected will likely
differ depending on whether APEX2 is fused to the N- or C-terminus of emerin, as originally
expected.

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Figure 4.4. Characterization of biotinylation by confocal fluorescence imaging of human dermal
fibroblasts stably expressing APEX2. Cells were treated with 0.5 mM biotin-phenol (BP) for 30
min and the biotinylation reaction was triggered by the addition of 1mM H2O2. Cells were fixed
and stained with an anti-V5 antibody (green) to visualize APEX2 localization and StreptavidinAlexa Fluor 594 (red) to visualize biotinylated proteins. All cell lines show correct localization of
each fusion and effective biotinylation Scale bars, 50 μm.

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Figure 4.5. Characterization of biotinylation activity by western blotting for wild-type and mutated
N-terminal APEX2-emerin fusions and controls. (A) HDFs stably expressing APEX2-EMD
constructs, APEX2-LBR1TM, APEX2-NLS and APEX2-NES were treated with 0.5 mM biotinphenol (BP) for 30 min and 1mM H2O2 for 1 minute. This was followed by cell lysis, and clarified
whole-cell lysates were analyzed by streptavidin-HRP blotting. (B) Quantitation of emerin levels
on N-terminus constructs by western blot probed by anti-emerin antibody. Normalization is done
with respect to β-actin expression levels. All statistical comparisons to HDF EMD+/y with a student
t-test: **p<0.01, ns: non-significant.

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Figure 4.6. Characterization of biotinylation activity by western blotting for wild-type and mutated
C-terminal emerin-APEX2 fusions and controls. (A) HDFs stably expressing EMD-APEX2
constructs, LBR1TM-APEX2, APEX2-KDEL and APEX2-NES were treated with 0.5 mM biotinphenol (BP) for 30 min and 1mM H2O2 for 1 minute. This was followed by cell lysis, and clarified
whole-cell lysates were analyzed by streptavidin-HRP blotting. (B) Quantitation of emerin levels
on C-terminus constructs by western blot probed by anti-emerin antibody. Normalization is done
with respect to β-actin expression levels. All statistical comparisons to HDF EMD+/y with a student
t-test: *** p<0.001, ns: non-significant.
4.2.4. Quantitative Proteomic Profiling of Emerin Interacting Partners by APEX2-mediated
Biotinylation
Once we had optimized our biotinylation experiments, we prepared samples for proteomic
analysis. We cultured each APEX2-expressing cells in 150 mm plates and labeled them with biotin-

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phenol. Each APEX2-expressing cell line has two replicates. After labeling, cells were harvested
and lysed, and the biotinylated proteins from each sample were enriched using streptavidin beads.
Prior to enrichment, about 2.5% of the whole-cell lysate from each sample was removed for quality
control analysis by Western blotting to ensure that APEX2 fusion were properly expressed, and
that labeling occurred. After streptavidin bead enrichment, an additional 5% of the beads were
removed and biotinylated proteins were eluted for quality control analysis by silver staining to
ensure biotinylated proteins were indeed enriched.
The remaining streptavidin beads were then shipped to Harvard ThermoFisher Center for
Multiplexed Proteomics, for sample processing and quantitative mass spectrometry-based
proteomics. On-bead tryptic digestion was performed on each sample, followed by labeling with
a unique isobaric tandem mass tag (TMT) using TMTpro-18plex.22 This allowed the duplicate
samples from each group to be pooled and analyzed in a single mass spectrometry (MS) run to
compare and quantify relative protein abundance (Figure 4.7).
Raw MS files were then analyzed for data processing, including search, peptides identification,
protein assignment, TMT quantification and data normalization using the Fragpipe computational
proteomics platform, which includes the MSFragger proteomic search engine, Philosopher toolkit
for downstream post-processing of MSFragger search results, and TMT-Integrator for TMT
isobaric labeling-based quantification.23, 24 The Human reference proteome database
(UP000005640) was used for protein identification with false discovery rates of 1%. Downstream
statistical analysis, comparison and data visualization was then performed using Fragpipe-Analyst,
including experimental reproducibility (principal component analyses, sample correlations and
coefficients of variation), differential expression (DE) analyses at the protein level and
unsupervised clustering.25 For DE analysis, protein linear models combined with empirical

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Figure 4.7. Principle and experimental workflow of TMT-based MS proteomics. After on-bead
digestion, the tryptic peptides are labeled by a different Tandem Mass Tag (TMT). The samples
are pooled together and injected into the mass spectrometer for identification and quantitation.
Bayesian statistics from Limma were used to testfor statistically significant differences in
abundance between conditions.26 Both p-values and adjusted p-values (using the BenjaminiHochberg method) were computed.
4.2.4.1. Proteome From APEX2-EMD
In Figure 4.8A, principal component analysis (PCA) of the TMT-18-plex run presented in Fig.
4.7, reveals high reproducibility in detected protein variance between replicates (n=2) and
expected dissimilarities between spatial control samples (WT, APEX2-NLS, APEX2-NES) and
nuclear envelope associated samples (APEX2-emerinWT, all mutants, split-APEX2-emerinWT and
APEX2-LBR1TM). This observation suggests a high level of data quality in terms of
quantification, characterized by minimal technical variability. Importantly, samples from APEX2-

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Figure 4.8. (A) PCA plot and (B) correlation matrix plot to assess the quality of APEX2-EMD
samples sent for MS-based proteomics.
EMDWT and fusion to mutated emerin are clustered together with APEX2-LBR1TM, indicating
that both emerin and LBR1TM share similar cellular localization, as expected, and that LBR1TM
is a proper spatial control to account for “en passant” non-specific biotinylation of proteins due to
emerin membrane diffusion. The sample correlation heatmap (Figure 4.8B) aligned with
distributions from PCA plots, and indicates that samples with APEX2 fusions to EMD (including
mutants) and APEX2-LBR1TM formed their own distinct clusters. Interestingly, the splitAPEX2-
EMD samples cluster closer to the APEX2-NLS samples and are separated from other emerin
samples. While this might appear surprising at first, this observation is fully consistent with our
previous findings that oligomerized emerin is only found at the INM.17 It indicates that APEX2-
emerinWT and all emerin mutant samples likely contain detected proteins additionally located in
the ER membrane and the ONM.

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Figure 4.9. Schematic showing the filtering criteria for selection of proteins enriched in APEX2-
EMDWT samples relative to APEX2-LBR1TM.
We then contrasted biotinylated protein abundances between APEX2-EMD and APEX2-
LBR1TM using DE analyses based on Limma which were visualized on volcano plots. Additional
statistical analyses were performed on GraphPad PRISM based the Fragpipe-Analyst output files.
Overall, 4033 proteins with at least 1 peptide were isolated and identified during our screen
(Figure 4.9). For stringency and to limit the identification of false positives, we performed triple
filtration. Since emerin is a nuclear membrane protein, the APEX2-emerin fusion can have the
APEX2 localized facing either the nucleoplasm or the cytoplasm. To filter out the potential
partners in the inner nuclear membrane/nucleoplasmic side, the proteins with abundance ratio
(log2[APEX2-EMDWT/NLS]) > 0 were selected after removing those with cytoplasmic

109
localizations (log2[APEX2-EMDWT/NES] < 0), resulting in 506 proteins (NES-corrected).
Conversely, potential partners at the cytoplasmic side were selected by taking the proteins with
abundance ratio log2[APEX2-EMDWT/NES] > 0, removing those with nuclear localization
(log2[APEX2-EMDWT/NLS] < 0, resulting in 282 proteins (NLS-corrected). The NES- and NLScorrected proteins were combined (removing duplicates) leading to 546 enriched proteins. A third
filtration criterion was implemented by selecting the 2085 proteins enriched when APEX2-
EMDWT
-expressing cells are compared to untransfected wild-type fibroblasts (log2[APEX2-
EMDWT/FibroWT] > 0). The proteins found in both [APEX2-EMDWT/(NES-/NLS-corrected)] and
[APEX2-EMDWT/FibroWT] were then eliminated since these are non-specific proteins as a result
of background signal from the untransfected cells.27 This led to a reduction to 489 “EMDWT
-
enriched” proteins. The same selection protocol was performed for LBR1TM resulting to 349
“LBR1TM-enriched” proteins. The two protein lists were then combined (removing duplicates),
giving 636 proteins. For stringency and to limit the identification of false positives, we only
retained proteins having a minimum assignment of 2 peptides and discarded 1-peptiders. The final
list of 442 proteins were then subjected to DE analyses on Fragpipe-Analyst using the original
Fragpipe output results.
To identify true emerin interacting partners, we selected the proteins for which the ratio of
normalized abundance for the APEX2-EMDWT and APEX2-LBR1TM is equivalent or greater than
1.25-fold (enrichment log2[APEX2-EMDWT/APEX2-LBR1TM] > 0.322) with an adjusted
probability of false detection lower than p=0.05 (-log adj. p value <1.310). The rationale behind
this criterion is that, in our APEX2-EMDWT sample, any identified protein enriched over the
APEX2-LBR1TM spatial control likely represent true proximity partners of emerin positioned at

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or near the ER membrane, ONM and INM. Based on this rationale, the group of identified proteins
was reduced to 76 proteins that more likely to interact with emerin than LBR1TM (Figure 10A).
Figure 4.10. Proximity labeling proteomics results of APEX2-EMDWT. (A) Volcano plot and list
of all high confidence proteins identified where the APEX2-EMDWT/APEX2-LBR1TM ratio was
greater than 0.322. Highlighted in maroon/red are the previously known emerin interacting
partners. (B-C) Gene ontology analyses for cellular component (B) and biological processes (C)
of proteins from (A) that met our selection criteria.

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It is worth noting that we have identified and enriched previously known emerin-interacting
partners including lamin A/C,28 emerin,29, 30 Lap2β (TMPO_P42167),31 lamin B1,8
lamin B231 and
MAN1 (LEMD3)31 (Figure 10A).
To identify classes of proteins involved in different cellular processes, the 76 proteins were
further analyzed on ShinyGO v0.81 using the 4033 identified proteins as background (Figure 10BC).32 Gene ontology (GO) analysis showed that we have enriched proteins that are localized in the
inner nuclear membrane, as well as proteins we have enriched proteins that are mostly involved in
Figure 4.11. Venn diagrams showing the similarity of enriched proteins for the APEX2-EMD
compared to the emerin mutants.

112
protein and lipid transport. This is not surprising since Tran and coworkers have previously shed
light into the transport of emerin into the nuclear envelope and identified the endoplasmic
reticulum (ER)-Golgi intermediate compartment (ERGIC) as the compartment into which emerin
is sequestered before VAPB-dependent retrograde transport to the nuclear envelope.33
We then performed the same filtration steps and analyses for the APEX2-EMD mutants, and
identified 68, 74, and 82 enriched proteins for the P183H, Q133H and Δ95-99 emerin mutants,
respectively. Venn diagrams showing the overlap of enriched proteins compared to the wild-type
emerin were generated using using InteractiVenn (Figure 11).
34 Furthermore, GO analyses were
also done (Figure 12). While transport proteins from the ER and Golgi are still enriched for the
three emerin mutants, it is worth noting that it is only for the P183H mutant that the nuclear lamina
proteins lamin B1 and B2 were not enriched. While B-type lamins mainly interact with lamin B
receptor (LBR), it has been previously shown that emerin also interacts with B-type lamins.21, 35,
36 The loss of lamin B1 and B2 in the enriched proteins for the P183H mutant suggests that the
binding site for B-type lamins on emerin might be on the region around the residue 183, and further
studies is needed for confirmation. Additionally, the loss of B-type lamin interaction might also
contribute to the enhanced binding of the P183H mutant toward lamin A/C.37 A list of the enriched
proteins is given in Appendices 1 and 2.

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Figure 4.12. Gene ontology analysis for the cellular components (B) and biological processes (C)
of the enriched proteins for the APEX2-EMD P183H, Q133H and Δ95-99 mutants.
4.2.4.2. EMD-APEX2
Similarly, the C-terminus EMD-APEX2 samples were analyzed in Fragpipe and FragpipeAnalyst. The PCA plot and correlation heatmap shown in Figure 4.13 shows good clustering within
all EMD-APEX2 samples and LBR1TM-APEX2.

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3407 proteins with at least 1 peptide were isolated and identified during our screen (Figure
4.14). In this case, we performed double filtration. To filter out the potential partners from the
emerin C-terminus to cover the interactors of emerin due to its localization at the ER membrane,
the proteins with abundance ratio (log2[EMDWT
-APEX2/KDEL]) > 0, resulting in 410 proteins.
The second filtration criterion was implemented similarly for the non-specific proteins in the
untransfected wild-type fibroblasts (log2[APEX2-EMDWT/FibroWT] > 0, 1521 proteins), and
subtraction resulted to 353 “EMDWT
-enriched” proteins. On the other hand, similar protocol done
on the LBR1TM-APEX2 brought about 154 “LBR1TM-enriched” proteins. The final list of 305
proteins (peptides ≥ 2) were then subjected to DE analyses on Fragpipe-Analyst using the original
Fragpipe output results. DE analysis based on Limma of the EMD-APEX2 (log2[EMDWT
-APEX2/
LBR1TM- APEX2] > 0.322) and -log adj. p value < 1.310). Our data detected 163 proteins that
Figure 4.13. (A) PCA plot and (B) correlation matrix plot to assess the quality of EMD-APEX2
samples sent for MS-based proteomics.

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Figure 4.14. Schematic showing the filtering criteria for selection of proteins enriched in EMDWT
-
APEX2 samples relative to LBR1TM-APEX2.
are more likely interacting with emerin than LBR1TM, and represents the proteins interacting with
emerin at its C-terminus (perinuclear side). GO analysis on ShinyGo v0.81 using the 3407
identified proteins as background suggests that we have we have enriched proteins that are mostly
involved in cell adhesion, highlighting emerin’s role in migration and force transmission (Figure
4.15). In fact, Nastaly et al. showed that EMD knock-down significantly perturbed cell migratory
properties, and that cells lacking EMD exhibited increased velocity and persistence and impaired
chemotaxis efficiency.38 They also noted cells with EMD deficiency had smaller focal adhesions,
and accordingly, they transmitted less force to their substrate, which could explain the observed
increase in speed.38

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Figure 4.15. Proximity labeling proteomics results of EMDWT
-APEX2. (A) Volcano plot and list
of all high confidence proteins identified where the APEX2-EMDWT/APEX2-LBR1TM ratio was
greater than 0.322. (B-C) Gene ontology analyses for cellular component (B) and biological
processes (C) of proteins from (A) that met our selection criteria.

117
Of the emerin mutants studied, the EMD(Δ95-99) mutant exhibits the most significant
reduction in the abundance of enriched proteins with only 22 enriched proteins (Figure 4.16-17).
This particular mutant has been shown to have disrupted lamin A binding, slower mobility and
reduced self-association.17, 37 While certain enriched proteins remain implicated in cell adhesion
processes, further investigation is necessary to ascertain the effects of this mutant, particularly
since one of this deletion mutant involves Tyr99, a phosphorylation site, where it was shown that
emerin phosphorylation at Tyr99 is activated in cells and functions as a key mechanosensitive
signal in response to lowered matrix stiffness.39 A list of all the enriched proteins is given in
Appendices 1 and 2.
Figure 4.16. Venn diagrams showing the similarity of enriched proteins for the EMD-APEX2
compared to the emerin mutants.

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Figure 4.17. Gene ontology analysis for the cellular components (B) and biological processes (C)
of the enriched proteins for the P183H, Q133H and Δ95-99 mutants of EMD-APEX2.
4.2.5. Potential of splitAPEX2 for Profiling the Interactors of Emerin Oligomers
Studies have demonstrated that emerin can self-associate, with oligomerization being a key
factor in its interaction with the nucleoskeleton. Additionally, the impairment of this
oligomerization is associated with the functional loss seen in EDMD-causing emerin mutants.17,
30, 40

119
In this case, we aimed to explore the potential of utilizing splitAPEX2 to identify the specific
binding partners associated with emerin oligomers. Fernandez et al. previously co-expressed wildtype emerin fusions to self-complementary split-GFP fragments to irreversibly induce the
formation of complemented emerin-GFP-emerin species, and studied their lateral mobility by
complementation activated light microscopy (CALM).17, 41, 42 Their results showed that emerinGFP-emerin species localize almost exclusively at the nuclear envelope. Taking inspiration from
their results, we fused the AP and EX fragments to wild-type emerin and expressed them in EMDkd
fibroblasts. Figures 4.18 and 4.19 showed that in both the N- and C-terminus emerin fusions of the
splitAPEX2, proper emerin localization is still observed, and that sufficient biotinylation activity
is still observed. The reconstitution of the peroxidase activity of the splitAPEX2 is yet another
proof of emerin self-association.
Figure 4.18. Confocal microscopy imaging on HDFs expressing splitAPEX2-EMD and EMDsplitAPEX2. Proper localization was observed from all emerin fusions and biotinylation activity
was restored upon contact of the AP and EX fragments due to emerin self-association.

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Figure 4.19. Characterization of biotinylation activity by western blotting for the splitAPEX2-
emerin fusion constructs. HDFs stably expressing (A) splitAPEX2-EMD or (2) EMD-splitAPEX2
along with the other APEX2 constructs were treated with 0.5 mM biotin-phenol (BP) for 30 min
and 1mM H2O2 for 1 minute. This was followed by cell lysis, and clarified whole-cell lysates were
analyzed by streptavidin-HRP blotting.
To optimize conditions of biotinylation, we performed western blots, and showed that both
splitAPEX2-emerin follows the correct biotinylation patterns expected from the full length

121
Figure 4.20. Proximity labeling proteomics results of splitAPEX2-EMD. (A) Volcano plot and list
of all high confidence proteins identified where the splitAPEX2-EMD/APEX2-LBR1TM ratio
was greater than 0.322. Highlighted in maroon/red are the previously known emerin interacting
partners. (B-C) Gene ontology analyses for cellular component (B) and biological processes (C)
of proteins from (A) that met our selection criteria.

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APEX2-emerin fusions. It should be noted, however, that the EX fragment has been observed to
be prone to degradation16, which is why we chose to have a higher expression level of the EX
fragment, especially in the splitAPEX2-EMD. The biotinylation efficiency of the splitAPEX2-
EMD fusions, however, was not seemingly affected by the EX fragment instability.
Following the same selection criteria as the full length APEX-EMD samples, our results
showed that there are 98 enriched proteins in the splitAPEX2-EMD sample (Figure 4.20A).
Interestingly, these proteins have been mapped to nuclear proteins and are involved in mRNA
processing, splicing, DNA damage and repair and recombination (Figure 4.20 B-C). Surprisingly,
APEX2-EMD and splitAPEX2-EMD only shared four common enriched proteins (EMD, MAN1
(LEMD3), LEMD2, TOR1AIP1). Several studies might still be needed to optimize not just the
biotinylation conditions but also finding a proper spatial control for the splitAPEX2-emerin.
Our results showed that the splitAPEX2-emerin platform is a promising technique especially
in studying the interactions of emerin (dimers or oligomers) with proteins related to DNA and RNA
processes, and the effects of emerin self-association, especially for the Q133H and Δ95-99
mutants which were previously shown to have diminished capacity to self-assemble.17, 40
4.2.6. Comparison with our work with previous emerin proximity labeling
So far, there have been four proximity labeling studies using emerin as a bait protein – each
with different PL enzyme, controls, labeling strategies and even cell lines. Go et al. utilized BioID
on HEK293T cells31, Moser et al. used BioID on U2OS cells43, Cheng et al. utilized TurboID on
mouse embryonic fibroblasts (MEFs)44, and Muller et al. used a modified APEX2 assay called
RAPIDS (Rapamycin- and APEX-dependent identification of proteins by SILAC) on HeLa cells45
.

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In all the previous studies, they utilized the N-terminal fusion of the PL enzyme on emerin,
while our work entails proximity mapping from both termini of emerin. In any case, we were still
hoping that we would have significant similarities with the previous results. As illustrated in Figure
4.21, the enriched proteins exhibit variability across experiments. This variability suggests that the
results obtained from proximity labeling experiments using the same bait protein will differ
depending on both the experimental technique utilized and the type of cell studied.
Figure 4.21. Venn diagram comparing the list of enriched proteins for emerin proximity mapping
in our work and previously published studies.

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4.2.7. Search for the Missing BAF
Barrier-to-autointegration factor is one of the most prominent interacting partners of emerin.
Barrier-to-autointegration factor (BAF) is a DNA-bridging protein, highly conserved in
metazoans. BAF binds directly to LEM (LAP2, emerin, MAN1) domain nuclear membrane
proteins, including LAP2 and emerin.46, 47 While several evidence for BAF-emerin interaction has
already been provided by both in vitro assays and in cells by FRAP, FLIP and FRET analysis,4, 48
BAF has never been shown as an emerin interacting partner from proximity labeling experiments.
One reason might be because APEX2 biotinylates tyrosine residues on the surface of a proximal
protein. BAF is a small protein at 10 kDa that only has one tyrosine residue.46 This already limits
the potential of the biotin-phenoxyl radical to be able to find the lone tyrosine residue on BAF. In
addition to that, BAF is a DNA-bridging protein that is often in complex with lamin A/C and/or
emerin. This further limits chance of BAF to be biotinylated from proximity labeling experiments.
To mitigate this problem of detecting BAF-emerin interaction by proximity labeling, we
propose the use of the splitAPEX2 system by fusing on one fragment to emerin, and the other
fragment to BAF. For initial studies, we utilized emerin-null human skeletal myoblasts from a male
EDMD patient, and rescued emerin expression by transduction with lentivirus for the expression
of EX-Flag-EMD. After hygromycin selection, we knockdown the endogenous BAF levels by
transduction with shRNA targeting the 3’-UTR of BANF1 gene. Following puromycin selection,
we then rescued BAF expression by transduction with lentivirus for the expression of BAF-V5-
AP and selection with blasticidin.
We used the EX-Flag-EMD fusion to make the EX fragment face the nucleoplasm, making the
splitAPEX2 ready for reconstitution with BAF-V5-AP is interacting with or in very close
proximity to EX-Flag-EMD. Our initial results in Figure 4.22 show that both EX-Flag-EMD and

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Figure 4.22. Utilizing splitAPEX2 to detect the interaction of emerin with barrier-toautointegration factor in emerin-null human skeletal myoblasts rescued with EX-Flag-EMD, then
treated with BAF shRNA following by transduction with BAF-V5-AP.
BAF-V5-AP are properly localized, and that APEX2 reconstitutes upon close contact of emerin
and BAF. We therefore propose to use this split-APEX-BAF-EMD system for mapping the
interacting partners of the BAF-emerin dimer. In addition, we can also use this system for mapping
genomic domains interacting with BAF and emerin.

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4.3. CONCLUSIONS
In this study, we used APEX2-mediated proximity labeling to identify interacting partners of
emerin. PL offers several advantages compared to traditional biochemical assays, particularly in
its ability to identify interacting species within the environment of a living cell. One possible
disadvantage of proximity labeling-based methods, however, is that they may identify proteins that
are within a distance of 10-20 nm (the labeling radius), regardless of whether they interact or not,
suggesting that these non-interacting proteins are just in proximity of the bait protein at the time
of biotinylation. To circumvent this, we used a ratiometric enrichment approach by comparing
APEX2-EMD with several spatial controls including APEX2 fused to the truncated LBR1TM, a
related INM but not interacting protein, APEX2-NLS and APEX2-KDEL. This approach led us to
discover interacting partners of emerin that are mostly involved in protein and lipid transport,
confirming the previous observation about emerin shuttling to the nuclear envelope.
In addition, we utilized the splitAPEX2 technology to uncover the proteins that are selectively
interacting with emerin oligomers. This was done by fusing the inactive AP and EX fragments to
emerin. Upon self-association, the AP and EX fragments are brought close together such that the
peroxidase activity is restored. Furthermore, we splitAPEX2 to confirm the interaction of emerin
and BAF, a DNA-bridging protein that is a well-known interacting partner of emerin, but has never
been confirmed by any proximity labeling experiment.
4.4. MATERIALS AND METHODS
4.4.1. Plasmid cDNA constructs
The following plasmids were received from Addgene: pcDNA3 APEX2-NES (#49386), NLSMCP-AP_pLX304 (#120918), EX-HA-FRB-Cb5_pLX304 (item #120915), scramble shRNA

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(item #1864), EGFP-BAF (item #101772), LBR EGFP (item #128150), pSFFV_sfCherry2(1-10)
(item #82603), psPAX2 (#12260) and pCMV-VSV-G (#8454). Human wild-type emerin cDNA
was kindly provided by Dr Juliet Ellis, University College London, UK.
4.4.2. Design and cloning of APEX2 constructs
V5-tagged APEX2 constructs were generated by standard PCR techniques using Phusion PCR
polymerase. The Flag tag in the original pCDNA3-APEX2-NES was replaced with V5-tag by
including the DNA sequence for V5-tag in the PCR primers. The splitAPEX2 constructs were
made by overlap-extension PCR following the protocol of Behle.49
4.4.3. Cell culture
Wildtype and APEX2-expressing human skeletal fibroblasts cells were maintained in DMEM
(HyClone™ Cytiva) supplemented with 10% fetal bovine serum and penicillin/streptomycin
(growth medium, GM). Cells were cultured in a humidified incubator at 37 ℃ under 5% CO2.
Immortalized Emd-/y and Emd+/y human skeletal myoblasts were received from the MyoLine
Platform of the Institute of Myology, Paris, France. Emd-/y myoblasts are derived from a gluteus
maximus biopsy of a 9-year-old male patient carrying the frameshifted mutation EMD
c.651_655dup. Emd+/y myoblasts (Control) are derived from a paravertebral muscle biopsy of an
healthy male individual of the closest possible matching age (16-year-old, reference: AB1190,
MyoLine Platform).
The myoblasts were cultured in the myoblast proliferation medium (MPM) consisting of
Medium-199 (HyClone™ Cytiva) and low-glucose DMEM (Corning) in a 1:4 ratio supplemented
with 20% fetal bovine serum (Life Technologies), 0.40 mg/ml gentamicin (VWR), 5 ng/ml human
epithelial growth factor (PeproTech), 5 mg/ml insulin (Lonza), 0.5 ng/ml human basic fibroblast

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growth factor (PeproTech), 50 mg/ml fetuin (Sigma-Aldrich), 0.2 mM dexamethasone (Gold
Biotechnology). Cells were cultured in a humidified incubator at 37 ℃ under 5% CO2.
4.4.4. Lentiviral Production
To generate lentiviruses, HEK 293T cells plated in a T25 flask (Falcon) were transfected at
∼80% confluency with 0.5 mL of serum-free DMEM containing the lentiviral plasmid encoding
the APEX2 fusion proteins, the lentiviral packaging plasmids psPAX2 (Addgene #12260) and
pCMV-VSV-G (Addgene #8454) at 2:1.5:1 mass ratio, respectively, and 12 µL X-tremeGENE HP
(transfection protocol was followed according to manufacturer’s instructions). The next day, the
media was changed to 5 mL DMEM containing 10% FBS and 1% bovine serum albumin (Gold
Biotechnology). After 24 hours, the medium containing lentivirus particles was harvested and
stored at 4 °C, and replaced with another 5 mL DMEM containing 10% FBS and 1% bovine serum
albumin. A second harvest was performed after another 24 hours and combined with the previous
harvest, filtered through a 0.45 μm filter, aliquoted and stored at -80 °C until further use.
4.4.5. shRNA knockdown of emerin
EMD shRNA targeting the 3’-UTR of EMD and BAF shRNA targeting the 3’-UTR of BANF1
were separately subcloned into the lentiviral vector pLKO.1 scramble shRNA (Addgene). The
lentiviruses were as mentioned before. Cells were then infected at ~60% confluency with lentivirus
with the growth medium supplemented with 10 μg/mL polybrene and 1 mg/mL Synperonic F108.
50
After 2 days, the medium was replaced with growth medium supplemented with 2 μg/mL
puromycin in and antibiotic selection was done for 7 days. The selected cells were allowed to
recover and they are split into T-150 flasks, maintained in growth medium supplemented with
0.5 μg/mL puromycin then frozen as stocks.

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4.4.6. Generation of stable cell lines
EMDkd fibroblasts were then infected at ~60% confluency with lentivirus for the APEX2-
emerin fusion with the growth medium supplemented with 10 μg/mL polybrene and 1 mg/mL
Synperonic F108.50 After two days, antibiotic selection was started by replacing the medium with
a fresh growth medium supplemented with 10 μg/mL blasticidin and antibiotic selection was done
for 10 days. The selected cells were allowed to recover and they are split into T-150 flasks,
maintained in growth medium supplemented with 5 μg/mL blasticidin then frozen as stocks. For
the splitAPEX2-emerin, a double lentiviral infection was performs for the expression of AP and
EX emerin fusion. Double antibiotic selection was then performed with 10 μg/mL blasticidin and
200 μg/mL hygromycin. Selection was done for 14 days. The selected cells were maintained in
3 μg/mL blasticidin and 50 μg/mL hygromycin.
4.4.7. Proximity labeling using APEX2 followed by fluorescence imaging
APEX2-expressing cells were plated on fibronectin-coated glass coverslips (#1, Electron
Microscopy Sciences). After 2 days, the media was replaced with fresh GM (for fibroblasts) or
MPM (for myoblasts) supplemented with 6 µM hemin-Cl (Sigma-Aldrich) for 1.5 h. Cells were
then incubated with 0.5 mM biotin-phenol (APEXBio) (diluted in GM or MPM) for 30 min at 37
°C, followed by addition of 1 mM H2O2 (diluted in 1xPBS) for 1 min. The biotinylation reaction
was immediately quenched by removing the media and washing 3x with the quencher solution (10
mM sodium azide, 10 mM sodium ascorbate and 5 mM Trolox in PBS) followed by 1xPBS. Cells
were fixed with 4% paraformaldehyde in PBS for 15 min, rinsed 3x with 1xPBS. Subsequently,
the cells cells were permeabilized with 0.1% (v/v) Triton X100 for 10 minutes, rinsed 3x with
1xPBS, then blocked with 3% BSA in 1xPBS (blocking buffer) for 1 h at RT. The cells were
incubated in primary rabbit anti-V5 antibody (1:1000, Cell Signaling) and/or mouse anti-FLAG

130
(1:1000, Sigma-Aldrich) was used for 1 h at RT. The cells were washed three times with 1xPBS
followed by incubation with secondary antibodies (goat anti-rabbit Alexa Fluor 488, goat antimouse Alexa Fluor 647, Life Technologies) at a dilution of 1:1000 in 1xPBS for 1 h at RT.
Following 3x 1xPBS rinse, the cells were mounted using Fluoromount G with DAPI (Electron
Microscopy Sciences).
Microscopy images were acquired by confocal imaging on an Zeiss780 Upright Confocal
Laser Scanning Microscope, or on an inverted Eclipse Ti-E microscope (Nikon Instruments Inc.,
Melville, NY, USA), as described in Chapter 2. All image analyses were performed using FIJI
software (version 1.54, National Institutes of Health, Bethesda, MD, USA).
4.4.8. Proximity labeling using APEX2 followed by western blotting
For gels and Western blots experiments, cells expressing the APEX2 fusion constructs were
plated, and labeled with biotin-phenol as described above, and subsequently scraped and pelleted
by centrifugation at 3000 rpm for 1 min. The pellet was lysed by resuspending in RIPA lysis buffer
(50 mM Tris pH 8, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, Ix
protease inhibitor cocktail (APEXBio), and 1 mM PMSF) by gentle pipetting and incubating for
20 min on ice. Lysates were clarified by centrifugation at 13000g for 10 min at 4°C. Protein
concentration in clarified lysate was estimated with Bio-Rad DC Protein Assay prior to separation
on a mPAGE 4-12% Bis-Tris gel (Sigma-Aldrich). Silver-stained gels were generated using Pierce
Silver Stain Kit (ThermoFisher). For all Western blots, 15 µg proteins from whole cell lysates were
separated on mPAGE 4-12% Bis-Tris SDS-PAGE gels and were transferred to nitrocellulose
membrane (overnight, total of 1000 mA-h), and then stained by Ponceau S (5 min in 0.1% (w/v)
Ponceau S in 5% acetic acid/water). The blots were then blocked in 3% (w/v) BSA (ThermoFisher)
in TBS-T (Tris-buffered saline, 0.1% Tween 20) for at least 1 h at RT. Blots were then stained with

131
primary antibodies in 3% BSA (w/v) in TBS-T for 1 hr at RT or overnight at 4°C, washed four
times with TBS-T for 5 min each, then stained with HRP-conjugated secondary antibodies or
Pierce High-Sensitivity Streptavidin-HRP (1:20000) in 3% BSA (w/v) in TBS-T for 1 hr at RT.
The blots were washed four times with TBS-T for 5 min each prior to developing with
SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoScientific) and imaging on
a Bio-Rad ChemiDoc System.
4.4.9. Proximity labeling using APEX2 for mass spectrometry-based proteomic analysis
APEX-expressing cells were grown in 15 cm plates per proteomic sample. The same protocol
for APEX2 labeling as mentioned above, accounting for the larger volume of the growth medium.
Cell pellets were collected and lysed in 1 mL RIPA lysis buffer as described above, and clarified
by centrifugation at 13,000 rpm for 10 min at 4°C. Protein concentration was estimated as before
and 15 µg protein was run on SDS-PAGE gel for streptavidin blotting.
For enrichment of biotinylated material, 200 µL streptavidin-coated magnetic beads (Pierce)
were washed twice with RIPA buffer, then incubated with clarified lysates containing
approximately 1.5 mg protein for each sample with rotation overnight at 4°C, after which 5% of
beads were removed for quality control analysis of enrichment. The rest were washed three times
in 1xPBS, resuspended in 1xPBS, then stored at -80°C.
For the 5% beads, they were subsequently washed twice with 1 mL of RIPA lysis buffer, once
with 1 mL of 1 M KCl, once with 1 mL of 0.1 M Na2CO3, once with 1 mL of 2 M urea in 10 mM
Tris-HCl (pH 8.0), and twice with 1 mL RIPA lysis buffer. The biotinylated proteins were eluted
by boiling the beads in 100 µL of 3x protein loading buffer supplemented with 20 mM DTT and 2
mM biotin. After magnetic sepatation, the eluate is divided into two and run on 2 SDS PAGE gels.
One gel was stained using Pierce Silver Stain Kit, the other was used for streptavidin blotting.

132
These are the quality control steps before the samples are shipped on dry ice to the Harvard
ThermoFisher Center for Multiplexed Proteomics for on-bead digestion and MS analysis.
4.4.10. On Bead Digestion and Labeling
Protein-coated beads were washed three times with PBS. The beads were suspended in 200
mM EPPS pH 8.5 and digested for 6 hours with 1 µg trypsin. Acetonitrile was added to each
sample to achieve a final concentration of ~33%. Each sample was labelled with ~62.5 µg of
TMTPro reagents (ThermoFisher Scientific). Following confirmation of satisfactory labelling
(>97%), excess TMT was quenched by addition of hydroxylamine to a final concentration of 0.3%.
The full volume from each sample was pooled and acetonitrile was removed by vacuum
centrifugation. The samples were acidified with formic acid and desalted by StageTip eluted into
autosampler inserts (Thermo Scientific), dried in a speedvac and reconstituted with 5%
Acetonitrile, 5% formic acid for LC-MS/MS analysis.
4.4.11. Liquid chromatography and tandem mass spectrometry.
Mass spectrometric data were collected on an Orbitrap Eclipse mass spectrometer coupled to
a Proxeon NanoLC-1000 UHPLC (Thermo Fisher Scientific). The 100 µm capillary column was
packed in-house with 35 cm of Accucore 150 resin (2.6 μm, 150Å; ThermoFisher Scientific). Data
were acquired for 180 min per run. A FAIMS device was enabled during data collection and
compensation voltages were set at -40V, -60V, and -80V [PMID: 30672687]. MS1 scans were
collected in the Orbitrap (resolution – 60,000; scan range – 400-1600 Th; automatic gain control
(AGC) – standard; maximum ion injection time – automatic). MS2 scans were collected in the
Orbitrap following higher-energy collision dissociation (HCD; resolution – 50,000; AGC – 250%;
normalized collision energy – 36; isolation window – 0.5 Th; maximum ion injection time – 100
ms.

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4.4.12. MS Data Analysis
The .raw MS files were first converted into .mzML files using MSConvert from the
ProteoWizard software package.51 Fragpipe v.22 was used for MS data processing. Fragpipe is
comprehensive computational platform for analyzing mass spectrometry-based proteomics data
that incorporates the proteomic search engine MSFragger.23, 25 Tandem MS spectra were searched
against SwissProt human protein database using the following search parameters: MS1 and MS2
tolerance were set to 10 ppm and 20 ppm, respectively; trypsin was set at the enzyme, and 2
allowed missed cleavages was set; carbamidomethylation of cysteines and TMT labeling of lysine
and N-termini of peptides were considered static modifications; while oxidation of methionine and
N-terminal acetylation were considered variable. Proteins and peptides that passed ≤1% false
discovery rate threshold were retrained for subsequent analysis. Quantitation was performed using
Philosopher and with TMT reporter ion MS2 with mass tolerance of 20 ppm, virtual reference, and
the normalized intensities (median centering) were used for DE analysis.
4.4.13. Differential Expression Analysis by Fragpipe-Analyst
Output files from Fragpipe were loaded into Fragpipe-Analyst.25 Specifically, the parameters
are: (1) Data type: TMT; (2) median-normalized protein abundance file from MSFragger; (3)
experiment annotation file; (4) Min percentage of non-missing values globally: 100; (5) Min
percentage of non-missing values in at least one condition: 0; (6) DE Adjusted p-value cutoff: 0.05;
(7) DE Log2 fold change cutoff: 0.322; (8) No normalization; (9) No imputation; (10) BenjaminHochberg FDR correction. PCA plots, correlation plots, volcano plots are output files from
Fragpipe-Analyst. In addition, output files (as .txt) were exported for subsequent analysis.

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4.4.14. Statistical Analysis
Detection of the enriched proteins using the ratiometric approach was done on GraphPad
PRISM v.10 using the log2 (APEX-EMD/control) values and adjusted p-values. Functional
Annotation Analysis was done using ShinyGO, while Venn diagrams were generated by
InteractiVenn.32, 34
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(36) Clements, L.; Manilal, S.; Love, D. R.; Morris, G. E. Direct interaction between emerin and
lamin A. Biochem Biophys Res Commun 2000, 267 (3), 709-714. DOI:
10.1006/bbrc.1999.2023 From NLM Medline.
(37) Lee, K. K.; Haraguchi, T.; Lee, R. S.; Koujin, T.; Hiraoka, Y.; Wilson, K. L. Distinct functional
domains in emerin bind lamin A and DNA-bridging protein BAF. J Cell Sci 2001, 114 (Pt
24), 4567-4573. DOI: 10.1242/jcs.114.24.4567.
(38) Nastaly, P.; Purushothaman, D.; Marchesi, S.; Poli, A.; Lendenmann, T.; Kidiyoor, G. R.;
Beznoussenko, G. V.; Lavore, S.; Romano, O. M.; Poulikakos, D.; et al. Role of the nuclear
membrane protein Emerin in front-rear polarity of the nucleus. Nat Commun 2020, 11 (1),
2122. DOI: 10.1038/s41467-020-15910-9.
(39) Pradhan, R.; Ranade, D.; Sengupta, K. Emerin modulates spatial organization of chromosome
territories in cells on softer matrices. Nucleic Acids Res 2018, 46 (11), 5561-5586. DOI:
10.1093/nar/gky288.
(40) Herrada, I.; Samson, C.; Velours, C.; Renault, L.; Ostlund, C.; Chervy, P.; Puchkov, D.;
Worman, H. J.; Buendia, B.; Zinn-Justin, S. Muscular Dystrophy Mutations Impair the
Nuclear Envelope Emerin Self-assembly Properties. ACS Chem Biol 2015, 10 (12), 2733-
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(41) Bautista, M.; Fernandez, A.; Pinaud, F. A Micropatterning Strategy to Study Nuclear
Mechanotransduction in Cells. Micromachines (Basel) 2019, 10 (12). DOI:
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(42) Pinaud, F.; Dahan, M. Targeting and imaging single biomolecules in living cells by
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(43) Moser, B.; Basilio, J.; Gotzmann, J.; Brachner, A.; Foisner, R. Comparative Interactome
Analysis of Emerin, MAN1 and LEM2 Reveals a Unique Role for LEM2 in Nucleotide
Excision Repair. Cells 2020, 9 (2). DOI: 10.3390/cells9020463.
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Branon, T. C.; Ting, A. Y.; Loose, E.; Yates, J. R., 3rd; et al. Shared and Distinctive
Neighborhoods of Emerin and Lamin B Receptor Revealed by Proximity Labeling and
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(45) Muller, M.; James, C.; Lenz, C.; Urlaub, H.; Kehlenbach, R. H. Probing the Environment of
Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling. Cells
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NLM Medline.

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CHAPTER 5: BENCHMARKING APEX2 FOR MAPPING EMERINASSOCIATED DOMAINS
5.1. INTRODUCTION
The diverse array of roles that emerin fulfills within cells can be attributed primarily to its
extensive list of binding partners. In addition to its roles in mechanotransduction (through the
interactions with the LINC complex)1, 2 and in maintaining the nuclear architecture (through
binding with the nuclear lamina (NL) and the nuclear actomyosin network),3-7
emerin also share
with other LEM domain proteins a vital role in transcriptional regulation and in tethering the
chromatin to the nuclear envelope (NE) as it binds to HDAC3, GCL, Lmo7, β-catenin, BAF, and
Btf.8-14
In the interphase nucleus, hundreds of large genomic regions (termed lamina-associated
domains, LADs) are in close contact with the nuclear lamina.15 The genes in LADs are generally
inactive, indicating that LADs form a transcriptionally repressive chromatin compartment.
15 Given
that emerin is proposed to help in anchoring chromatin to the NL through its interactions with both
NL proteins and chromatin components like BAF, several studies have been reported to map the
specific genomic regions that emerin interacts with.16-18 These studies are mostly based on the
DamID technology in which emerin is fused to the E. coli DNA adenine methyltransferase.19 Upon
expression of emerin-Dam fusion in cells, the DNA regions that contain GATC motif and are in
close contact with emerin are methylated by the Dam protein.19 Emerin-DamID results showed
nearly identical results to that of Dam-LaminB1 in which that majority of LADs contains
chromatin modifications (decrease in H3K4me2 and increase in H3K27me3 and H3K9me2)
consistent with chromatin compaction and transcriptional repression.17

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Since the engineered ascorbate peroxidase APEX2 has been shown to directly label nucleic
acids20-22, we sought to adopt this method as an alternative approach to map the genomic regions
associated with emerin. Based on our successful development of APEX2 and split-APEX2 fusions
at the N-term. or C-term. of emerin for quantitative studies of emerin monomer and oligomer
proteomic neighborhoods in human dermal fibroblasts, myoblasts and muscle myotube cells, we
hoped to similarly determine the genomic neighborhoods specific to those two types of emerin
nanoscale organizations at the nuclear envelope. Indeed, the use of a common APEX2 labeling
approach offers a unique way to seamlessly correlate the functional re-organization of emerin as a
function of force with local changes in both proteomic and genomic environments at surface of a
nucleus, thus bridging the gap between nuclear mechano-proteomics and nuclear mechanogenomics, with nanometer resolution.
To do so, we took advantage of the work by Zhou et al. who previously synthesized and
screened a series of aromatic compounds and identified biotin-conjugated arylamines as novel
probes for APEX2 (Figure 5.1).23 These arylamine probes have significantly higher reactivity
towards nucleic acids than biotin-phenol (BP). For instance, biotin-aniline (Btn-An) proved to be
very efficient for labeling both RNA and DNA while biotin-naphthalene (Btn-Nap) and biotin-4-
hydroxybenzamide (Btn-4HB) react with DNA but not RNA. Because of its significant reactivity
towards RNA, Zhou and coworkers selected Btn-An and APEX2 labeling for the transcriptomic
profiling of the mitochondrial matrix, and they were able to capture all 13 mitochondrial messenger
RNAs and none of the cytoplasmic RNAs.23 Confirming the efficient reaction of Btn-An with
RNA, this substrate was also use together with HRP to detect glycan-conjugated RNAs
(glycoRNAs) localized on the cell surface and interacting with Siglec receptors.24

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As we were interested in labeling DNA rather than RNA, we initially considering using BtnNap or Btn-4HB, which, based on the screen by Zhou et al., are both very efficient at labeling
DNA (Fig. 5.1.). Yet, Btn-Nap retains significant reactivity toward proteins, while Btn-4-HB does
not. Although Btn-4-HB appeared to be excellent candidate for highly specific DNA labeling in
our assays, the compound is not commercially available. We thus decided to employ Btn-An as an
alternative APEX2 substrate since it has low reactivity to proteins, significantly better reactivity
toward DNA compared to BP, and is commercially available. This chapter summarizes the
optimization of protocols for the APEX2-mediated biotinylation and extraction of genomic DNA
(gDNA) in the proximity to emerin.
Figure 5.1. Screening of biotinylated arylamines for protein, DNA and RNA labeling. (Adapted
from Ref. 23)

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5.2. RESULTS AND DISCUSSION
5.2.1 Live cell biotinylation using BtAn
For the initial optimization of BtAn labeling, we used emerin-null human skeletal myoblasts
from an EDMD patient. We rescued emerin expression by lentiviral transduction of either APEX2-
emerin or emerin-APEX2, with a subsequent blasticidin treatment to select for stably expressing
cells. We then modified a few steps in the biotinylation and cell staining protocol previously
described in Chapter 4 for myoblasts expressing APEX2 fusions to emerin. In this modified
protocol (Protocol 1) cells were incubated with BtAn, biotinylation was triggered for 1 min with
H2O2, the reaction was quenched and cells were fixed with 4% paraformaldehyde (PFA) for 15
min. From our previous experience with BP labeling and other imaging assays, multiple rinses
with 1xPBS is sufficient to remove excess PFA and PFA inactivation/quenching is not usually
required. However, since BtAn is an arylamine and may potentially react and be crosslinked by
PFA, we added a PFA quenching step using tris-glycine buffer. Cells were then permeabilized with
Triton X-100 and treated with RNaseA to remove RNA and increase immunostaining specificity
to biotinylated DNA, since BtAn normally labels RNA more efficiently than DNA. Fluorescence
microscopy imaging results showed that there is high background in streptavidin staining after
BtAn labeling even for cells that do not strongly express APEX2-EMD (Figure 5.2, top panel).
Since APEX2-EMD is mostly localized in the INM we expected that biotinylation pattern to be
primarily at the nucleus, as observed for labeling with BP (Figure 5.2, lower panel). This is clearly
not the case for BtAn labeling in live myoblasts and the biotinylation pattern is weak and
distributed over the entire cell as shown by streptavidin staining. The radicals produced from BP
(biotin-phenoxyl radicals) and from alkyne-phenol (alkyne-phenoxyl radicals) have been shown
to not be able to cross membranes.25, 26 While no study has yet been performed on biotin-aminyl

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Figure 5.2. Labeling on live APEX2-expressing human skeletal myoblasts (Protocol 1).
radicals, we can assume that they have similar non-membrane crossing property. This suggested
that the biotinylation patterns observed likely stem from non-specific effects. Indeed, even control
myoblasts which do not express APEX2 showed very high streptavidin staining (Figure 5.2,
middle panel). These results suggest that residual BtAn remains within the cells even after multiple
1xPBS rinse, which can then react and crosslink with PFA during fixation.

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RNA and increase the chance to immunostain biotinylated DNA since BtAn was shown to more
efficiently label RNA than DNA. Fluorescence microscopy imaging results showed that there is
very high background in the streptavidin staining for BtAn labeled cells (Figure 5.2, upper panel).
APEX2-EMD is mostly localized in the INM so we expect that biotinylation will also be mostly
localized in the nucleus as in BP labeling (Figure 5.2, lower panel). This is clearly not the case for
the BtAn labeling of live myoblasts since the streptavidin signals across the entire cell is the same.
The reactive biotin-phenoxyl (from BP) and alkyne-phenoxyl (from alkyne-phenol) radicals have
been shown to not be able to cross membranes.25, 26 While no study has yet been performed on
biotin-aminyl radicals, we can assume that they have the same non-membrane crossing property
as biotin-phenoxyl and alkyne-phenoxyl radicals. Surprisingly, even the control myoblasts which
are not expressing APEX2 showed very high streptavidin signals (Figure 5.2, middle panel). These
results indicate that residual BtAn remains within the cells even after multiple 1xPBS rinse which
can then react and crosslink with PFA during fixation.
5.2.2 HRP-Mediated Biotinylation of Plasmid DNA
Because in our initial cell staining experiments, we did not observe biotinylation patterns
specific to the location of APEX2-EMD, we wanted to assess if this lack of specificity stems from
excessive streptavidin labeling backgrounds or is simply due to a very low labeling efficiency of
DNA. We thus decided to test both BP and BtAn labeling of plasmid DNA in vitro. In this case,
we used HRP instead of APEX2 since purified recombinant HRP is more readily available than
APEX2. HRP and APEX2 belong to the same peroxidase class and share the same substrates for
proximity labeling experiments.27, 28 20 μg plasmid DNA were labeled with 0.5 mM BP or Btn-

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Figure 5.3. Comparison of HRP-mediated labeling efficiency toward plasmid DNA. (A)
Representative dot blot image. 20 μg plasmid DNA were labeled with 0.5 mM BP or Btn-An, HRP,
and 1 mM H2O2 for different time points at RT. Following plasmid DNA recovery, 1 μg plasmid
DNA was dot blotted onto the membrane; (B) Quantitative analysis of biotinylation intensity.
Student’t t-test was used to test for significance, ****p < 0.0001.
An, HRP and 1 mM H2O2 for different time points at RT. The labeled plasmid DNA was then
recovered and purified, and 1 μg plasmid DNA was dot blotted onto a nitrocellulose membrane.
Quantitative analysis of the biotinylation signals clearly show that BtAn is a superior substrate for
DNA labeling over BP (Figure 5.3). As such, the apparent lack of specific biotinylation pattern at
the nucleus likely stems from excessive non-specific backgrounds that hide signals at the nuclear
envelope.
5.2.3 Optimization of Biotinylation on Fixed Cells
Having established that BtAn is a suitable substrate for DNA labeling, we attempted to enhance
its efficacy as a substrate for labeling genomic DNA (gDNA) in APEX2-EMD expressing cells.
This time, cell fixation by PFA, PFA quenching and Triton X-100 permeabilization were all done
before BtAn labeling (Protocol 2). This approach offers the advantage of: (1) preserving/freezing
the close association (if any) of genomic domains with APEX2-emerin; (2) removing excess PFA
by tris/glycine quenching before BtAn labeling to limit the possibility that BtAn get crosslinked

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Figure 5.4. Labeling on fixed APEX2-expressing human skeletal myoblasts following Protocol 2.
Labeling time is extended to 5 minutes. Background streptavidin staining on control myoblasts are
reduced but still significant compared to live cell labeling.
Figure 5.5. Labeling on fixed APEX2-expressing human skin fibroblasts following Protocol 2.
Labeling time is 1 minute. Similar background streptavidin staining is observed in non-APEX2
expressing fibroblasts, similar to control myoblasts.

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to PFA; and (3) shortening BtAn incubation time before H2O2 addition since cell are fully
permeabilized by TritonX-100 treatment before labeling.
As shown in Figure 5.4., implementing Protocol 2 in fixed cells significantly improved BtAn
labeling in terms of the background. Indeed, biotinylation patterns are localized specifically at the
nuclear envelope, as initially expected, and non-specific streptavidin staining is significantly
decreased in control myoblasts, even if reaction times are extended to 5 minutes instead of 1
minute. This is an important improvement compared to Protocol 1 in live cells.
To confirm this improvement, we also implemented Protocol 2 in fibroblasts expressing APEX2-
EMD (described in the APEX2-mediated proteomic profiling of Chapter 4). We observed
biotinylation patterns that were specific to the nuclear envelope as in myoblasts (Figure 5.5),
although streptavidin staining along the nuclear periphery was reduced because the APEX2
activation time was decreased to 1 minute, five time less than in myoblasts.
To further improve DNA labeling specificity and reduce background signals, Protocol 2 was
further modified, by performing the BtAn labeling step immediately after PFA fixation/quenching
and before TritonX-100 permeabilization. Although this additional modification of the protocol
entailed having to increase the incubation of BtAn on cells to 30 min in order to ensure adequate
diffusion across non-permeabilized cell compartments (Protocol 3), this last approach ended up
being the best protocol so far, with non-specific background signals being almost eliminated even
after extending the biotinylation reaction to 5 minutes (Figure 5.6).

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Figure 5.6. Labeling on fixed APEX2-expressing human skeletal myoblasts following Protocol 3.
Perfoming the Triton-X100 permeabilization after BtAn labeling significantly reduced background
staining on control myoblasts even for extended periods of biotinylation reaction.

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Figure 5.7. Assay to determine the biotin levels in the gDNA sample. (A) Chemical basis of the
fluorescent biotin assay; (B) biocytin standard curve. (Figure 5.6A adapted from Ref. 29)
5.2.4 Quantitation of Biotinylation Levels in the Genomic DNA of Labeled Cells
DNA biotinylated by APEX2-EMD likely represents gDNA domains that are interacting with
or are in close contact with EMD at the INM. In order to (potentially) identify these emerinassociated domains, the gDNA needs to be extracted following BtAn labeling. As of this date, we
have slightly modified the original Protocol 1 (live cell labeling) and Protocol 2 (fixed cell
labeling) used for immunostaining after BP/BtAn labeling to extend them to emerin-associated
gDNA extraction in dermal fibroblasts. Unfortunately, we have not had the time to extend those
extraction of human myoblast cells, nor to perform streptavidin bead enrichment of the
biotinylated gDNA and send samples for sequencing. However, to make sure that we successfully

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biotinylated fibroblasts gDNA, we used a Pierce™ Fluorescence Biotin Quantitation Kit to assess
the biotinylation levels in our extracted gDNA.
The quantification method is based upon a fluorophore-tagged avidin that is weakly complexed
with HABA (4’-hydroxyazobenzene-2-carboxylic acid) (Figure 5.7A).30 HABA quenched the
fluorophores attached to avidin, but once mixed with the biotinylated gDNA, HABA is displaced
due to the much higher affinity of biotin to avidin, which allows avidin to fluoresce.31 The amount
of biotin is thus measured by comparing the fluorescence signal to a biocytin standard curve
(Figure 5.7C). From the biocytin standards, we have determined that the response is linear until
20 pmol biotin/10 µL sample.
We thus performed gDNA labeling in live and fixed cells using both BP and BtAn for wildtype, APEX2-EMD- and EMD-APEX2-expressing fibroblasts. The perinuclear space location of
the APEX2 fusion in EMD-APEX2 (C-term.) is such that the labeling efficiency of the gDNA from
EMD-APEX2-expressing fibroblasts is not expected to be significant, compared to APEX2-EMDexpressing fibroblasts where APEX2 is directly facing the nucleoplasm. Thus, in addition to
comparing BP vs. BtAn labeling efficiencies, EMD-APEX2 cells serve as an additional control to
ensure that gDNA is being specifically biotinylated.
We note that in most cases, we extract more gDNA from live cells than fixed cells. This is not
surprising because there are more preparative steps when handling fixed cells, including
trypsinization and repeated rinsing before cell collection, compared to live cells, where simply
scrapping off cell plates is needed to collect cells. We used trypsin to lift off the cells

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Figure 5.8. Biotin levels of the labeled gDNA samples. (A) calculated and (B) normalized biotin
levels. Student’t t-test was used to test for significance, *p < 0.05.
from the plate because scraping is not that efficient in removing them from the plate, most likely
because of the crosslinking extent due to PFA fixation.

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In both live and fixed cells, the level of biotinylation is higher with BtAn than with BP as
substrate (Figure 5.8A). Biotin levels are higher in APEX2-EMD compared to EMD-APEX2, as
expected. It is also worth noting that significant levels of biotin are detected in wild-type
fibroblasts. This may be due to insufficient 1xPBS rinsing after labeling and a resulting excess of
BP or BtAn that persist during subsequent gDNA extraction and purification steps. Effectively, if
the biotin levels are normalized to those of wild-type fibroblasts for each condition, then it becomes
clearer that BtAn labeling on fixed APEX2-expressing cells is significantly higher (~3-fold
increase in detected biotin over fixed wild-type fibroblasts, and ~2-fold increase of live APEX2-
EMD).
5.3. CONCLUSIONS
Biotin-aniline has been reported to have significant labeling efficiencies toward RNA and
moderate labeling efficiencies toward DNA. Here, we optimized protocols for APEX2-mediated
biotinylation of DNA with BtAn and applied them to imaging of emerin-associated gDNA domains
in cells, to extract gDNA associated with those domains and to quantifies their levels of
biotinylation. While we determined that Protocol 3 is currently the best to reduce background
streptavidin signals, we unfortunately did not get the chance to implement it together with BtAn
labeling for gDNA extraction in skin fibroblast and human muscle cells. However, using a
modified Protocol 2, we did show that BtAn labeling on fixed cells produces high levels of gDNA
biotinylation. We also demonstrated that an APEX2-EMD fusion facing the nucleoplasm allows
for a specific biotinylation gDNA at the INM as compared to an EMD-APEX2 fusion facing the
perinuclear space.

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In future work, experiments involving the enrichment, isolation and subsequent sequencing of
biotinylated gDNA will likely allow for the identification of genome-wide emerin-associated
chromatin domains. Expanding those approaches to APEX2 and split-APEX2 fusions to EDMDinducing emerin mutations in cells subjected to varying mechanical stress on their nucleus, will
help define the genomic neighborhoods specific to emerin monomers and oligomers at the INM,
will offer means to explore the involvement of emerin-associated chromatin domains in nuclear
mechano-genomics and will significantly advance our understanding of EDMD as a disease.
5.4. MATERIALS AND METHODS
5.4.1. Cell Culture
Wildtype and APEX2-expressing human dermal fibroblasts cells were maintained in DMEM
(HyClone™ Cytiva) supplemented with 10% fetal bovine serum and penicillin/streptomycin
(growth medium, GM), as described in Chapters 2 and 4. Cells were cultured in a humidified
incubator at 37 ℃ under 5% CO2.
Immortalized Emd-/y and Emd+/y human skeletal myoblasts were received from the MyoLine
Platform of the Institute of Myology, Paris, France. Emd-/y myoblasts are derived from a gluteus
maximus biopsy of a 9-year-old male patient carrying the frameshifted mutation EMD
c.651_655dup. Emd+/y myoblasts (Control) are derived from a paravertebral muscle biopsy of an
healthy male individual of the closest possible matching age (16-year-old, reference: AB1190,
MyoLine Platform). Absence of emerin expression in the Emd-/y myoblasts was verified by
immunostaining and immunoblotting as shown in Chapter 4. Emd-/y myoblasts were infected with
lentivirus for expression of APEX2-EMD and stably expressing cells were selected following the
protocol given in Chapter 4.

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The myoblasts were cultured in the myoblast proliferation medium (MPM) consisting of
Medium-199 (HyClone™ Cytiva) and low-glucose DMEM (Corning) in a 1:4 ratio supplemented
with 20% fetal bovine serum (Life Technologies), 0.40 mg/ml gentamicin (VWR), 5 ng/ml human
epithelial growth factor (PeproTech), 5 mg/ml insulin (Lonza), 0.5 ng/ml human basic fibroblast
growth factor (PeproTech), 50 mg/ml fetuin (Sigma-Aldrich), 0.2 mM dexamethasone (Gold
Biotechnology). Cells were cultured in a humidified incubator at 37 ℃ under 5% CO2.
5.4.2. PROTOCOL 1: APEX2-mediated biotinylation in living cells
Cells were plated on fibronectin-coated glass coverslips (#1, Electron Microscopy Sciences) 2
days before APEX2 labeling. The media was replaced with GM (for fibroblasts) or MPM (for
myoblasts) supplemented with 6 µM hemin-Cl (Sigma-Aldrich) for 1.5 h. Cells were then
incubated with 0.5 mM biotin-phenol (BP) or biotin-aniline (BtAn) (diluted in GM or MPM) for
30 min at 37 °C, followed by addition of 1 mM H2O2 (diluted in 1xPBS) for 1 min. The
biotinylation reaction was immediately quenched by removing the media and washing 3x with the
quencher solution (10 mM sodium azide, 10 mM sodium ascorbate and 5 mM Trolox in PBS)
followed by 1xPBS. Cells were fixed with 4% paraformaldehyde in PBS for 15 min, rinsed 3x
with 1xPBS, and the excess PFA was quenched with 1x TG buffer (25 mM Tris, 192 mM glycine,
Bioland Scientific) for 15 min. Cells were permeabilized with 0.1% Triton X-100 for 10 min then
rinsed 3x with 1xPBS. To ensure the removal of RNA, the cells were incubated with 1 µL 10
mg/mL RNaseA (Qiagen) in PBS for 2 hours at 37 °C. The cells were rinsed with 1xPBS 3x before
proceeding to immunostaining protocol.
5.4.3. PROTOCOL 2: APEX2-mediated biotinylation in fixed cells
Cells were plated on fibronectin-coated glass coverslips (#1, Electron Microscopy Sciences) 2
days before APEX2 labeling. The media was replaced with GM (for fibroblasts) or MPM (for

155
myoblasts) supplemented with 6 µM hemin-Cl (Sigma-Aldrich) for 1.5 hr. Cells were fixed with
4% paraformaldehyde in PBS for 15 min, rinsed 3x with 1xPBS, and the excess PFA was quenched
with 1x TG buffer (25 mM Tris, 192 mM glycine, Bioland Scientific) for 15 min. Cells were
permeabilized with 0.1% Triton X-100 for 10 min then rinsed 3x with 1xPBS. Cells were then
incubated with 0.5 mM BP or BtAn (diluted in 1xPBS) for 30 min at 37 °C, followed by addition
of 1 mM H2O2 (diluted in 1xPBS) for 1 min. The biotinylation reaction was immediately quenched
by removing the media and washing 3x with the quencher solution followed by 1xPBS. Cells were
incubated with 1 µL 10 mg/mL RNaseA (Qiagen) in PBS for 2 hours at 37 °C.
5.4.4. PROTOCOL 3: APEX2-mediated biotinylation in fixed cells
Cells were plated on fibronectin-coated glass coverslips (#1, Electron Microscopy Sciences) 2
days before APEX2 labeling. The media was replaced with GM (for fibroblasts) or MPM (for
myoblasts) supplemented with 6 µM hemin-Cl (Sigma-Aldrich) for 1.5 hr. Cells were fixed with
4% paraformaldehyde in PBS for 15 min, rinsed 3x with 1xPBS, and the excess PFA was quenched
with 1x TG buffer (25 mM Tris, 192 mM glycine, Bioland Scientific) for 15 min. Cells were then
incubated with 0.5 mM BP or BtAn (diluted in 1xPBS) for 30 min at 37 °C, followed by addition
of 1 mM H2O2 (diluted in 1xPBS) for 1 min. The biotinylation reaction was immediately quenched
by removing the media and washing 3x with the quencher solution followed by 1xPBS. Cells were
permeabilized with 0.1% Triton X-100 for 10 min then rinsed 3x with 1xPBS. Cells were incubated
with 1 µL 10 mg/mL RNaseA (Qiagen) in PBS for 2 hours at 37 °C.
5.4.5. Immunofluorescence
Following RNAse incubation, cells were blocked in 3% BSA in 1xPBS (blocking buffer) for
1 h at RT. Cells were then stained with rabbit anti-V5 antibody (1:1000, Cell Signaling
Technology) for 1 h at RT. After washing 3×5 min with 1xPBS, staining with a goat anti rabbit-

156
Alexa Fluor 488 (1:500, Life Technologies) and Streptavidin-Alexa Fluor 594 (1:1000,
ThermoFisher, Life Technologies) secondary antibodies was done for 1 h at RT and washed 3×5
min with PBS. The coverslips were then DAPI-Fluoromount G (Electron Microscopy Sciences),
sealed with clear nail polish and stored at -20 °C until imaging.
5.4.6. Microscopy imaging
Microscopy images were acquired by confocal imaging on an Zeiss780 Upright Confocal
Laser Scanning Microscope, or on an inverted Eclipse Ti-E microscope (Nikon Instruments Inc.,
Melville, NY, USA), as described in Chapter 2. All image analyses were performed using FIJI
software (version 1.54, National Institutes of Health, Bethesda, MD, USA).
5.4.7. HRP-mediated labeling of plasmid DNA with biotin probes
Plasmid DNA was extracted from overnight culture of Stbl3™ Chemically Competent E. coli
(Invitrogen) using EndoFree MaxiPrep Kit (Qiagen) following the manufacturer’s protocol. 20 μg
plasmid DNA was incubated with 1 μL goat anti-mouse IgG (H+L)-HRP (Invitrogen) and 0.5 mM
BP or BtAn in nuclease-free water. The biotinylation reaction was triggered by mixing with 1 mM
H2O2 (30s, 1 min, 3 min, 5 min and 10 min) and stopped with quencher solution. For the negative
control, the plasmid DNA was incubated with BP or BtAn and HRP without were incubated
without H2O2 for 10 min. The plasmid DNA was then recovered and purified using NucleoSpin
Gel and PCR Clean-up (Macherey-Nagel) following manufacturer’s protocol. The concentration
of recovered DNA was determined by Nanodrop.
5.4.8. Dot blot characterization of DNA labeling with biotin probes
The DNA concentration for all recovered/labeled plasmid DNA was normalized by dilution
with nuclease-free water. Equal volume for all samples (total of 1 μg DNA) was dot-blotted into
nitrocellulose membrane. After air-drying, the DNA was crosslinked by baking the membrane at

157
80 °C for 2 h. The membrane was rinsed with 1x TBST twice, blocked with 3% BSA in 1xTBST
for 1 h, and incubated with Pierce™ High Sensitivity Streptavidin-HRP (1:15000,
ThermoScientific) in 3% BSA in 1xTBST for 1 h at RT. The membrane was washed with TBST
for 3×5 min followed by incubation in SuperSignal™ West Pico PLUS Chemiluminescent
Substrate (ThermoScientific) for 5 min. The membrane was imaged on ChemiDoc MP imaging
system (Bio-Rad). For quantitative analysis, data from 4 replicates of dot blot experiments were
averaged.
Dot blot images were analyzed using FIJI following the “horizontal lanes” protocol provided
in https://imagej.net/ij/docs/examples/dot-blot/ (accessed July 2024). Statistical analysis was
performed using GraphPad PRISM v.10 software.
5.4.9. APEX2-mdiated Biotinylation in Live Cells for genomic DNA (gDNA) extraction
Wild-type and APEX2-expressing human dermal fibroblasts were seeded in 100 mm plates.
After reaching ~90% confluence, the media was replaced with GM supplemented with 6 µM
hemin-Cl (Sigma-Aldrich) for 1.5 hr. The media was replaced with GM (for fibroblasts) or MPM
(for myoblasts) supplemented with 6 µM hemin-Cl (Sigma-Aldrich) for 1.5 h. Cells were then
incubated with 0.5 mM biotin-phenol (BP) or biotin-aniline (BtAn) (diluted in GM or MPM) for
30 min at 37 °C, followed by addition of 1 mM H2O2 (diluted in 1xPBS) for 1 min. The
biotinylation reaction was immediately quenched by removing the media and washing 3x with the
quencher solution (10 mM sodium azide, 10 mM sodium ascorbate and 5 mM Trolox in PBS)
followed by 1xPBS. The cells were scraped from the plate with 1 mL 1xPBS and collected by
centrifugation. The 1xPBS was removed by aspiration, then 125 µL 1xPBS was added and the
cells were transferred to a microcentrifuge tube.

158
5.4.10. APEX2-mediated Biotinylation in Fixed Cells for gDNA extraction
Labeling was performed using PROTOCOL 2 with some modifications. Briefly, wild-type and
APEX2-expressing human dermal fibroblasts were seeded in 100 mm plates. After reaching ~90%
confluence, the media was replaced with GM supplemented with 6 µM hemin-Cl (Sigma-Aldrich)
for 1.5 hr. Cells were fixed with 4% paraformaldehyde in PBS for 15 min, rinsed 3x with 1xPBS,
and the excess PFA was quenched with 1x TG buffer (25 mM Tris, 192 mM glycine, Bioland
Scientific) for 15 min. Cells were permeabilized with 0.1% Triton X-100 for 10 min then rinsed
3x with 1xPBS. Cells were then incubated with 0.5 mM BP or BtAn (diluted in 1xPBS) for 30 min
at 37 °C, followed by addition of 1 mM H2O2 (diluted in 1xPBS) for 5 min. The biotinylation
reaction was immediately quenched by removing the media and washing 3x with the quencher
solution followed by 1xPBS. 1 mL of trypsin was added, and the cells were incubated at 37 °C for
3 min. 1 mL of warm GM was then added to neutralize the trypsin and the cells were collected by
centrifugation. The trypsin/GM mixture was removed by aspiration, and the cell pellet was rinsed
with 1xPBS, triturated and collected once more by centrifugation. The 1xPBS was removed by
aspiration, then 125 µL 1xPBS was added and the cells were transferred to a microcentrifuge tube.
5.4.11. Genomic DNA extraction
1 µL 10 mg/mL RNaseA (Qiagen) and 1 µL 20 mg/mL Proteinase K (Sigma-Alridch) was
added to the collected cells from live and fixed cell labeling. The samples were incubated in a
shaking incubator at 37 °C overnight. The gDNA was then extracted using EasyPrepTM Genomic
DNA Kit (Bioland Scientific) following manufacturer’s protocol for cell samples. DNA
concentration was determined by Nanodrop.
5.4.12. Quantitative Analysis of Biotinylation in Labeled Genomic DNA

159
To detect the level of biotinylation in labeled gDNA, Pierce™ Fluorescence Biotin
Quantitation Kit was used following manufacturer’s protocol. 10 µL of gDNA sample (done in
duplicates) was used. The fluorescence was measured using Fluoroskan Ascent FL Fluoresence
Microplate Reader (Thermo Labsystems) with Ex485nm/Em527nm filters and 100 ms integration
time.
Calculations for biotinylation levels were done on Microsoft Excel, and statistical analysis
was performed using GraphPad PRISM v.10 software. Statistical significance was determined with
the Student’s t-test with a significance level of p < 0.05.
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APPENDIX 1:
SUMMARY OF ENRICHED PROTEINS FROM PROTEOMICS EXPERIMENTS
Table 1. Summary of Enriched Proteins from the APEX2-EMD Proteomic Experiments.
V5-APEX2-
EMDwt
V5-APEX2-
EMD(P183H)
V5-APEX2-
EMD(Q133H)
V5-APEX2-
EMD(Δ95-99)
splitAPEX2-
EMDwt
ZFPL1 ZFPL1 ZFPL1 ZFPL1 EMD
TRIP11 TRIP11 TRIP11 TRIP11 RACGAP1
GOLGA3 ACBD3 GOLGA3 GOLGA3 L3MBTL2
ACBD3 GOLGA3 EMD ACBD3 TPX2
YIPF4 YIPF4 ACBD3 YIPF4 TAF5
EMD EMD GOLGB1 EMD PMS1
SLC30A5 SLC30A5 SLC30A5 GOLGB1 NFATC2IP
GOLGB1 GOLGB1 YIPF4 SLC30A5 RCOR3
USO1 KCT2 USO1 KCT2 SMCHD1
KCT2 USO1 GOLGA2 USO1 EHMT1
GOLGA2 GOLGA2 KCT2 ZDHHC17 BAP18
ZDHHC21 TM9SF3 VPS13B OSBP EIF2AK2
OSBP OSBP ZDHHC17 GOLGA2 BLM
ZDHHC17 ZDHHC17 COG1 VPS13B SMARCD3
VPS13B VPS13B GOPC VPS54 FOXK1
TM9SF3 GOSR1 OSBP ZDHHC21 APOBEC3B
GOPC VPS54 GOSR1 ATP7A TMPO_P42166
COG1 ZDHHC21 SLC35E1 TM9SF3 BEND3
GOLGA4 COG1 TM9SF3 GOPC ZC3H13
GOSR1 GOPC GOLGA4 MAN1B1 MTA3
ARL1 RAB6A TMF1 TMF1 CMAS
RAB6A COG7 ATP7A ARL1 NFIC
ATP2C1 CUX1 ZDHHC21 GOSR1 PMS2
TMF1 FHIP2B ATP2C1 GOLGA4 KIF23
COG7 SLC35E1 FHIP2B RAB6A RALY
CUX1 MAN1B1 COG7 CUX1 GTF3C5
MON2 GOLGA4 VPS54 PDXDC1 ZCCHC8
VPS54 PDXDC1 PDXDC1 ARFGEF1 RBMXL1
MAN1B1 TMF1 CUX1 SLC35E1 KIFC1
SACM1L TMEM87A RIC1 SACM1L HNRNPC
ARFGEF1 ARL1 ARFGEF1 COG1 TOR4A
RIC1 CPD LEMD3 FHIP2B UHRF2
VTI1B ATP2C1 TMPO_P42166 COG7 RBM12B
CPD ARFGEF1 STX5 ATP6V0A2 FLYWCH1
ARF3 RIC1 MAN1B1 MON2 KIF18B
PDXDC1 SACM1L GBF1 CPD AKAP8L
ARFIP2 MON2 AKAP9 RIC1 NCAPD3
SCYL3 ARF3 YKT6 ATP2C1 ADNP2
SLC35E1 TMPO_P42166 MON2 TMEM87A BRWD3
FHIP2B YKT6 RAB6A YKT6 MSH2

192
TMEM87A ARFIP2 ARFIP2 ARFIP2 TRIM24
YKT6 NBEAL1 ATP6V0A2 STX5 ATF7IP
NBEAL1 ATP7A CPD GBF1 BARD1
ATP6V0A2 USP32 SEC23A ARF3 ZMYM3
GCC2 TMPO_P42167 OSBPL11 GCC2 BAZ1B
GBF1 ATAD2 ARL1 VTI1B ARID2
PIR ATP6V0A2 SACM1L AKAP9 KDM1B
USP32 SLC35B2 USP32 PI4K2A TAF15
COPB2 GBF1 SEC24C NBEAL1 SRSF10
ATP7A COPB2 LMNA OSBPL11 WRNIP1
PRRC1 COPA NBEAL1 USP32 BRCA2
COPA LEMD3 ARF3 ARMH3 TRIM33
TMPO_P42167 OSBPL11 TOR1AIP1 LEMD3 MSL1
ARF4 SCYL3 TMPO_P42167 COPB2 UPP1
OSBPL9 PI4K2A LMAN2 TMPO_P42166 ATAD2
LMNA PIR SCYL3 FHIP2A MAD1L1
LEMD3 TOR1AIP1 BET1 PIR TMPO_P42167
FHIP2A LMAN2 SEC23IP TMPO_P42167 SMARCD1
DHX32 SEC23A VTI1B COPA SMARCC2
OSBPL11 SEC23IP PIR LMAN2 WDHD1
SEC23A FLT4 HSPE1 SEC31A SMARCA5
AP4B1 LMNA COPB2 SEC23IP USP1
LMAN2 SLC27A1 DHX32 OSBPL9 FANCI
TOR1AIP1 AP4B1 PI4K2A PRRC1 SGO2
FLT4 KRT8 MYL9 BET1 ZNF618
SLC35B2 KRT75 LMNB1 SEC23A UBR5
SEC23IP LEMD2 SLC35B2 SCYL3 PBRM1
JARID2 PIGG LMNB2 TBC1D23 TLK2
PCM1 LEMD2 CEP131 SAFB
CEP131 PCM1 TOR1AIP1 DNMT3A
LEMD2 PRR14L LMNA ATR
LMNB1 FLT4 MTMR4 BRD7
ATP6V1C1 NCSTN CRACR2A XPC
SLC27A1 SLC27A1 FADS1 CENPF
LMNB2 AP4B1 KIF20B
NES ATP6V1C1 CDYL
PCM1 POLD2
LMNB1 TRA2B
SLC27A1 ZMYM2
LEMD2 TSPYL2
COG8 SMC1A
PRR14L ASXL2
BAZ1A
MMS22L
TPR
SMC5
COIL
LEMD3
KHDRBS3

193
DCAF1
EPC1
LEMD2
TOR1AIP1
TP53BP1
PHIP
IDH2
L3MBTL3
POU2F1
Table 2. Summary of Enriched Proteins from the EMD-APEX2 Proteomic Experiments.
EMDwtAPEX2-V5
EMD(P183H)-
APEX2-V5
EMD(Q133H)-
APEX2-V5
EMD(Δ95-99)-
APEX2-V5
EMDwtsplitAPEX2
GOLIM4 GOLIM4 GOLIM4 OCLN EMD
TFRC OCLN GALNT2 MMP14 NUCB2
OCLN TFRC GLG1 MPZL1 NUCB1
MMP14 MMP14 TFRC EPHA2 DCAKD
PTPRK NUCB1 MGAT2 PTK7 IDH2
MGAT2 GLG1 NUCB1 PTPRK TMED4
PTK7 MGAT2 TM9SF4 ITGA2 NAXE
NUCB1 GALNT2 HS2ST1 ALCAM PON2
ITGA2 NUCB2 FUT8 IGSF8 DOCK1
GLG1 HS2ST1 OCLN CTNNB1 TLCD4
CADM1 FUT8 NUCB2 DSG2 PNPLA6
MPZL1 PTPRK MAN1A2 IGSF3 HELZ2
EPHA2 TM9SF2 GALNT16 BCAM TMED10
BCAM IGFBP7 TM9SF2 DAG1 HLA-B
NUCB2 MAN1A2 QSOX2 CADM1 HSPA1A
DAG1 ITGA2 MMP14 CD44 TMPO_P42166
GALNT2 PTK7 MAN1B1 ITGAV TAP2
CDH2 GALNT16 TM9SF3 ELFN1 C1RL
TM9SF2 TM9SF4 CPD CXADR MPPE1
DSG2 EPHA2 THBS1 IGFBP7 TRIM59
ALCAM BCAM ST3GAL2 HMOX1 ALG10
FUT8 CDH2 C1GALT1C1 PTPRG IGFBP7
HS2ST1 QSOX2 SDF4 SLC27A1
IGSF8 DAG1 GALNT7 NES
CTNNB1 CADM1 NDST1 KRT2
ELFN1 SDF4 GOLM1 NEMP1
IGSF3 MAN1B1 KIAA0319L VIM
IGFBP7 CTNNB1 SEMA3C
CCN1 MPZL1 TGOLN2
GALNT16 DSG2 B4GALNT1
CD44 CPD GPR107
MRC2 IGSF8 C1GALT1
ITGAV TM9SF3 PTPRK

194
QSOX2 CCN1 PTK7
MAN1A2 ST3GAL2 COL1A1
TM9SF4 C1GALT1C1 MRC2
CXADR ALCAM TMED4
MAN1B1 IGSF3 LFNG
CPD CD44 XYLT2
TM9SF3 SEMA3C TPST1
EPHB2 HMOX1 EPHA2
C1GALT1C1 TMED4 TPST2
SDF4 KIAA0319L CDH2
KIAA0319L MAN2A2 MAN2A1
ST3GAL2 EMD MAN2A2
SEMA3C MRC2 GALNT17
FLRT2 NDST1 HAPLN1
NDST1 ELFN1 MPZL1
PLXNB2 GALNT7 CCN1
ITGA5 LAMA1 ITGB1
PTPRG CXADR CTNNB1
HMOX1 FLRT2 FAM3C
DCBLD2 GPR107 CHST14
LRRC4C C1GALT1 ITGA2
EMD EPHB2 FAM20B
GPR107 GOLM1 CD44
GALNT7 TGOLN2 DAG1
ANTXR1 GALNT17 CSGALNACT2
TMED4 PLXNB2 IGSF3
GOLM1 B4GALNT1 ALCAM
FAT1 TPST1 TMED2
CSPG4 ITGA5 DSG2
GALNT17 TPST2 TMED10
ATP1B1 ITGAV COLGALT2
MAN2A2 ANTXR1 EMD
B4GALNT1 LOXL4 BPNT2
LOXL4 IDH2 CHSY1
CEMIP2 SULT1A1 IGSF8
NOTCH2 PCDHB5 ITGA5
TPST1 TMED10 IGFBP7
INSR COLGALT2 SMIM19
PCDHB5 CEMIP2 ANTXR1
FXR1 PTPRG BCAM
TGOLN2 MAN2A1 LRPAP1
EPHA5 CSGALNACT2 POGLUT3
TPST2 XYLT2 CADM1
ITGA6 FAT1 CHPF2
XYLT2 SMIM19 ITGB2
SULT1A1 TMED2 HMOX1
CD109 FKBP10 PCDHB5
COLGALT2 C1RL CHPF
TENM3 FXR1 ELFN1

195
DDR2 FAM3C CEMIP2
C1GALT1 CHST14 EPHB2
PTPRS SLC39A10 FKBP10
NCAM1 LRRC4C IDH2
CSGALNACT2 NOTCH2 SULT1A1
TMED2 ELAVL1 FAT1
C1RL DCBLD2 POMGNT1
ELAVL1 CSPG4 SEZ6L2
IDH2 FAM20B CNPY3
SMIM19 CNPY3 PCYOX1L
HAPLN1 CHPF2 LMAN2
SLC39A10 PTPRS PLXNB2
PTPRF NCAM1 GPX7
LRRC8A CHSY1 B4GALT7
CHPF2 SEZ6L2 WLS
ITGB5 PCDHB11 DCAKD
FKBP10 CCN2 B4GALT1
MAN2A1 SEMA3A NAXE
SLC12A7 NAXE MMP2
CHST14 POMGNT1 TGM2
L1CAM ITGB5 PON2
DCAKD PHGDH ERP29
BTN2A1 GPX7 ELAVL1
SEZ6L2 HAPLN1 GLT8D1
FAM3C WLS GAPDH
NPTXR DCAKD TMED7
TMED10 SCD5 LOXL4
WLS SLC12A7 CXADR
CHSY1 CD109 DCBLD2
ITM2B LRPAP1 OAT
NFASC NPTXR RFT1
ATP1A1 L1CAM SEMA3A
NRP1 GAPDH ADAM10
PCDHB11 LMAN2 ITM2B
AMIGO2 RFT1 TMED1
PCDHB8 UXS1 COL11A1
CCN2 TENM3 GXYLT1
EGFR XXYLT1 LEMD2
LRPAP1 OAT TLCD4
TOP2B B4GALT7 B4GALT3
NAXE MMP2 MPPE1
CD47 GLT8D1 CYB5B
LOXL1 PTPRF ITGB5
SCD5 NRP1 CCN2
ADGRL1 HLA
-
B CLGN
ITGB2 TLCD4 SLC12A7
FLT4 ADAM10 TMED9
POMGNT1 LOXL1 EGFR
LRRC4B BTN2A1 APP

196
SEMA3A COL6A1 ERGIC2
HLA-B EGFR ERGIC3
PROCR ITGB2 HTATIP2
MMP2 ITM2B SLC39A10
TENM2 NFASC ANGPTL2
FAM20B HNRNPD KCTD7
TLCD4 RPS14 LRIG2
RPS14 FLT4 YIPF4
APP SLC27A1 FKBP7
MIA2 CD47 POMK
RFT1 ANGPTL2
ANGPTL2 POGZ
ADAM10 ERGIC3
ECM1 HNRNPDL
RAB1A HNRNPAB
SGCE SORT1
B4GALT7 ITM2C
ACAT2 YIPF4
MCAM LRIG2
POGZ ADGRL1
PTGFRN FKBP7
PHF3 HEG1
GLT8D1
PLD3
ATP7A
ERGIC3
ITM2C
HNRNPAB
SORT1
LPL
LRIG2
SLC27A1

197
APPENDIX 2:
LIST OF ENRICHED PROTEINS FROM PROTEOMIC EXPERIMENTS
Table 1. Enriched Proteins for APEX2-EMDWT
.
Protein
ID Entry Name Gene
Leng
th
Organis
m Protein Description
Q9H3P
7
GCP60_HU
MAN ACBD3 528
Homo
sapiens Golgi resident protein GCP60
Q9Y6B
7
AP4B1_HU
MAN AP4B1 739
Homo
sapiens AP-4 complex subunit beta-1
P61204
ARF3_HUM
AN ARF3 181
Homo
sapiens ADP-ribosylation factor 3
P18085
ARF4_HUM
AN ARF4 180
Homo
sapiens ADP-ribosylation factor 4
Q9Y6D
6
BIG1_HUM
AN
ARFGE
F1 1849
Homo
sapiens
Brefeldin A-inhibited guanine nucleotide-exchange
protein 1
P53365
ARFP2_HU
MAN ARFIP2 341
Homo
sapiens Arfaptin-2
P40616
ARL1_HUM
AN ARL1 181
Homo
sapiens ADP-ribosylation factor-like protein 1
P98194
AT2C1_HU
MAN
ATP2C
1 919
Homo
sapiens Calcium-transporting ATPase type 2C member 1
Q9Y48
7
VPP2_HUM
AN
ATP6V
0A2 856
Homo
sapiens V-type proton ATPase 116 kDa subunit a 2
P21283
VATC1_HU
MAN
ATP6V
1C1 382
Homo
sapiens V-type proton ATPase subunit C 1
Q04656
ATP7A_HU
MAN ATP7A 1500
Homo
sapiens Copper-transporting ATPase 1
Q9UPN
4
CP131_HUM
AN CEP131 1083
Homo
sapiens Centrosomal protein of 131 kDa
Q8WT
W3
COG1_HUM
AN COG1 980
Homo
sapiens Conserved oligomeric Golgi complex subunit 1
P83436
COG7_HUM
AN COG7 770
Homo
sapiens Conserved oligomeric Golgi complex subunit 7
P53621
COPA_HUM
AN COPA 1224
Homo
sapiens Coatomer subunit alpha
P35606
COPB2_HU
MAN COPB2 906
Homo
sapiens Coatomer subunit beta'
O75976
CBPD_HUM
AN CPD 1380
Homo
sapiens Carboxypeptidase D
Q13948
CASP_HUM
AN CUX1 678
Homo
sapiens Protein CASP
Q7L7V
1
DHX32_HU
MAN DHX32 743
Homo
sapiens
Putative pre-mRNA-splicing factor ATP-dependent
RNA helicase DHX32
P50402
EMD_HUM
AN EMD 254
Homo
sapiens Emerin
Q5W0
V3
FHI2A_HU
MAN FHIP2A 765
Homo
sapiens FHF complex subunit HOOK interacting protein 2A
Q86V8
7
FHI2B_HU
MAN FHIP2B 743
Homo
sapiens FHF complex subunit HOOK-interacting protein 2B
P35916
VGFR3_HU
MAN FLT4 1363
Homo
sapiens Vascular endothelial growth factor receptor 3

198
Q92538
GBF1_HUM
AN GBF1 1860
Homo
sapiens
Golgi-specific brefeldin A-resistance guanine
nucleotide exchange factor 1
Q8IWJ
2
GCC2_HUM
AN GCC2 1684
Homo
sapiens GRIP and coiled-coil domain-containing protein 2
Q08379
GOGA2_HU
MAN
GOLG
A2 1002
Homo
sapiens Golgin subfamily A member 2
Q08378
GOGA3_HU
MAN
GOLG
A3 1498
Homo
sapiens Golgin subfamily A member 3
Q13439
GOGA4_HU
MAN
GOLG
A4 2230
Homo
sapiens Golgin subfamily A member 4
Q14789
GOGB1_HU
MAN
GOLGB
1 3259
Homo
sapiens Golgin subfamily B member 1
Q9HD2
6
GOPC_HUM
AN GOPC 462
Homo
sapiens
Golgi-associated PDZ and coiled-coil motifcontaining protein
O95249
GOSR1_HU
MAN GOSR1 250
Homo
sapiens Golgi SNAP receptor complex member 1
Q92833
JARD2_HU
MAN JARID2 1246
Homo
sapiens Protein Jumonji
Q8NC5
4
KCT2_HUM
AN KCT2 265
Homo
sapiens Keratinocyte-associated transmembrane protein 2
Q8NC5
6
LEMD2_HU
MAN LEMD2 503
Homo
sapiens LEM domain-containing protein 2
Q9Y2U
8
MAN1_HU
MAN LEMD3 911
Homo
sapiens Inner nuclear membrane protein Man1
Q12907
LMAN2_HU
MAN LMAN2 356
Homo
sapiens Vesicular integral-membrane protein VIP36
P02545
LMNA_HU
MAN LMNA 664
Homo
sapiens Prelamin-A/C
P20700
LMNB1_HU
MAN LMNB1 586
Homo
sapiens Lamin-B1
Q03252
LMNB2_HU
MAN LMNB2 620
Homo
sapiens Lamin-B2
Q9UK
M7
MA1B1_HU
MAN
MAN1
B1 699
Homo
sapiens
Endoplasmic reticulum mannosyl-oligosaccharide
1,2-alpha-mannosidase
Q7Z3U
7
MON2_HU
MAN MON2 1717
Homo
sapiens Protein MON2 homolog
Q6ZS3
0
NBEL1_HU
MAN
NBEAL
1 2694
Homo
sapiens Neurobeachin-like protein 1
P48681
NEST_HUM
AN NES 1621
Homo
sapiens Nestin
P22059
OSBP1_HU
MAN OSBP 807
Homo
sapiens Oxysterol-binding protein 1
Q9BX
B4
OSB11_HU
MAN
OSBPL
11 747
Homo
sapiens Oxysterol-binding protein-related protein 11
Q96SU
4
OSBL9_HU
MAN
OSBPL
9 736
Homo
sapiens Oxysterol-binding protein-related protein 9
Q15154
PCM1_HUM
AN PCM1 2024
Homo
sapiens Pericentriolar material 1 protein
Q6P99
6
PDXD1_HU
MAN
PDXDC
1 788
Homo
sapiens
Pyridoxal-dependent decarboxylase domaincontaining protein 1
O00625
PIR_HUMA
N PIR 290
Homo
sapiens Pirin
Q96M2
7
PRRC1_HU
MAN PRRC1 445
Homo
sapiens Protein PRRC1

199
P20340
RAB6A_HU
MAN RAB6A 208
Homo
sapiens Ras-related protein Rab-6A
Q4AD
V7
RIC1_HUM
AN RIC1 1423
Homo
sapiens Guanine nucleotide exchange factor subunit RIC1
Q9NTJ
5
SAC1_HUM
AN
SACM1
L 587
Homo
sapiens Phosphatidylinositol-3-phosphatase SAC1
Q8IZE
3
PACE1_HU
MAN SCYL3 742
Homo
sapiens
Protein-associating with the carboxyl-terminal
domain of ezrin
Q15436
SC23A_HU
MAN
SEC23
A 765
Homo
sapiens Protein transport protein Sec23A
Q9Y6Y
8
S23IP_HUM
AN
SEC23I
P 1000
Homo
sapiens SEC23-interacting protein
Q6PCB
7
S27A1_HU
MAN
SLC27
A1 646
Homo
sapiens Long-chain fatty acid transport protein 1
Q8TA
D4
ZNT5_HUM
AN
SLC30
A5 765
Homo
sapiens Proton-coupled zinc antiporter SLC30A5
Q8TB6
1
S35B2_HUM
AN
SLC35
B2 432
Homo
sapiens Adenosine 3'-phospho 5'-phosphosulfate transporter 1
Q96K3
7
S35E1_HUM
AN
SLC35E
1 410
Homo
sapiens Solute carrier family 35 member E1
Q9HD4
5
TM9S3_HU
MAN
TM9SF
3 589
Homo
sapiens Transmembrane 9 superfamily member 3
Q8NB
N3
TM87A_HU
MAN
TMEM
87A 555
Homo
sapiens Transmembrane protein 87A
P82094
TMF1_HUM
AN TMF1 1093
Homo
sapiens TATA element modulatory factor
P42167
LAP2B_HU
MAN TMPO 454
Homo
sapiens
Lamina-associated polypeptide 2, isoforms
beta/gamma
Q5JTV
8
TOIP1_HUM
AN
TOR1A
IP1 583
Homo
sapiens Torsin-1A-interacting protein 1
Q15643
TRIPB_HU
MAN TRIP11 1979
Homo
sapiens Thyroid receptor-interacting protein 11
O60763
USO1_HUM
AN USO1 962
Homo
sapiens General vesicular transport factor p115
Q8NFA
0
UBP32_HU
MAN USP32 1604
Homo
sapiens Ubiquitin carboxyl-terminal hydrolase 32
Q7Z7G
8
VP13B_HU
MAN
VPS13
B 4022
Homo
sapiens Intermembrane lipid transfer protein VPS13B
Q9P1Q
0
VPS54_HU
MAN VPS54 977
Homo
sapiens Vacuolar protein sorting-associated protein 54
Q9UE
U0
VTI1B_HU
MAN VTI1B 232
Homo
sapiens
Vesicle transport through interaction with t-SNAREs
homolog 1B
Q9BSR
8
YIPF4_HUM
AN YIPF4 244
Homo
sapiens Protein YIPF4
O15498
YKT6_HUM
AN YKT6 198
Homo
sapiens Synaptobrevin homolog YKT6
Q8IUH
5
ZDH17_HU
MAN
ZDHHC
17 632
Homo
sapiens Palmitoyltransferase ZDHHC17
Q8IVQ
6
ZDH21_HU
MAN
ZDHHC
21 265
Homo
sapiens Palmitoyltransferase ZDHHC21
O95159
ZFPL1_HU
MAN ZFPL1 310
Homo
sapiens Zinc finger protein-like 1

200
Table 2. Enriched Proteins for APEX2-EMD(P183H).
Protein
ID Entry Name Gene
Leng
th
Organis
m Protein Description
Q9H3P
7
GCP60_HU
MAN ACBD3 528
Homo
sapiens Golgi resident protein GCP60
Q9Y6B
7
AP4B1_HU
MAN AP4B1 739
Homo
sapiens AP-4 complex subunit beta-1
P61204
ARF3_HUM
AN ARF3 181
Homo
sapiens ADP-ribosylation factor 3
Q9Y6D
6
BIG1_HUM
AN
ARFGE
F1 1849
Homo
sapiens
Brefeldin A-inhibited guanine nucleotide-exchange
protein 1
P53365
ARFP2_HU
MAN ARFIP2 341
Homo
sapiens Arfaptin-2
P40616
ARL1_HUM
AN ARL1 181
Homo
sapiens ADP-ribosylation factor-like protein 1
Q6PL1
8
ATAD2_HU
MAN ATAD2 1390
Homo
sapiens ATPase family AAA domain-containing protein 2
P98194
AT2C1_HU
MAN
ATP2C
1 919
Homo
sapiens Calcium-transporting ATPase type 2C member 1
Q9Y48
7
VPP2_HUM
AN
ATP6V
0A2 856
Homo
sapiens V-type proton ATPase 116 kDa subunit a 2
Q04656
ATP7A_HU
MAN ATP7A 1500
Homo
sapiens Copper-transporting ATPase 1
Q8WT
W3
COG1_HUM
AN COG1 980
Homo
sapiens Conserved oligomeric Golgi complex subunit 1
P83436
COG7_HUM
AN COG7 770
Homo
sapiens Conserved oligomeric Golgi complex subunit 7
P53621
COPA_HUM
AN COPA 1224
Homo
sapiens Coatomer subunit alpha
P35606
COPB2_HU
MAN COPB2 906
Homo
sapiens Coatomer subunit beta'
O75976
CBPD_HUM
AN CPD 1380
Homo
sapiens Carboxypeptidase D
Q13948
CASP_HUM
AN CUX1 678
Homo
sapiens Protein CASP
P50402
EMD_HUM
AN EMD 254
Homo
sapiens Emerin
Q86V8
7
FHI2B_HU
MAN FHIP2B 743
Homo
sapiens FHF complex subunit HOOK-interacting protein 2B
P35916
VGFR3_HU
MAN FLT4 1363
Homo
sapiens Vascular endothelial growth factor receptor 3
Q92538
GBF1_HUM
AN GBF1 1860
Homo
sapiens
Golgi-specific brefeldin A-resistance guanine
nucleotide exchange factor 1
Q08379
GOGA2_HU
MAN
GOLG
A2 1002
Homo
sapiens Golgin subfamily A member 2
Q08378
GOGA3_HU
MAN
GOLG
A3 1498
Homo
sapiens Golgin subfamily A member 3
Q13439
GOGA4_HU
MAN
GOLG
A4 2230
Homo
sapiens Golgin subfamily A member 4
Q14789
GOGB1_HU
MAN
GOLGB
1 3259
Homo
sapiens Golgin subfamily B member 1
Q9HD2
6
GOPC_HUM
AN GOPC 462
Homo
sapiens
Golgi-associated PDZ and coiled-coil motifcontaining protein

201
O95249
GOSR1_HU
MAN GOSR1 250
Homo
sapiens Golgi SNAP receptor complex member 1
Q8NC5
4
KCT2_HUM
AN KCT2 265
Homo
sapiens Keratinocyte-associated transmembrane protein 2
O95678
K2C75_HU
MAN KRT75 551
Homo
sapiens Keratin, type II cytoskeletal 75
P05787
K2C8_HUM
AN KRT8 483
Homo
sapiens Keratin, type II cytoskeletal 8
Q8NC5
6
LEMD2_HU
MAN LEMD2 503
Homo
sapiens LEM domain-containing protein 2
Q9Y2U
8
MAN1_HU
MAN LEMD3 911
Homo
sapiens Inner nuclear membrane protein Man1
Q12907
LMAN2_HU
MAN LMAN2 356
Homo
sapiens Vesicular integral-membrane protein VIP36
P02545
LMNA_HU
MAN LMNA 664
Homo
sapiens Prelamin-A/C
Q9UK
M7
MA1B1_HU
MAN
MAN1
B1 699
Homo
sapiens
Endoplasmic reticulum mannosyl-oligosaccharide
1,2-alpha-mannosidase
Q7Z3U
7
MON2_HU
MAN MON2 1717
Homo
sapiens Protein MON2 homolog
Q6ZS3
0
NBEL1_HU
MAN
NBEAL
1 2694
Homo
sapiens Neurobeachin-like protein 1
P22059
OSBP1_HU
MAN OSBP 807
Homo
sapiens Oxysterol-binding protein 1
Q9BX
B4
OSB11_HU
MAN
OSBPL
11 747
Homo
sapiens Oxysterol-binding protein-related protein 11
Q6P99
6
PDXD1_HU
MAN
PDXDC
1 788
Homo
sapiens
Pyridoxal-dependent decarboxylase domaincontaining protein 1
Q9BTU
6
P4K2A_HU
MAN PI4K2A 479
Homo
sapiens Phosphatidylinositol 4-kinase type 2-alpha
Q5H8A
4
PIGG_HUM
AN PIGG 983
Homo
sapiens GPI ethanolamine phosphate transferase 2
O00625
PIR_HUMA
N PIR 290
Homo
sapiens Pirin
P20340
RAB6A_HU
MAN RAB6A 208
Homo
sapiens Ras-related protein Rab-6A
Q4AD
V7
RIC1_HUM
AN RIC1 1423
Homo
sapiens Guanine nucleotide exchange factor subunit RIC1
Q9NTJ
5
SAC1_HUM
AN
SACM1
L 587
Homo
sapiens Phosphatidylinositol-3-phosphatase SAC1
Q8IZE
3
PACE1_HU
MAN SCYL3 742
Homo
sapiens
Protein-associating with the carboxyl-terminal
domain of ezrin
Q15436
SC23A_HU
MAN
SEC23
A 765
Homo
sapiens Protein transport protein Sec23A
Q9Y6Y
8
S23IP_HUM
AN
SEC23I
P 1000
Homo
sapiens SEC23-interacting protein
Q6PCB
7
S27A1_HU
MAN
SLC27
A1 646
Homo
sapiens Long-chain fatty acid transport protein 1
Q8TA
D4
ZNT5_HUM
AN
SLC30
A5 765
Homo
sapiens Proton-coupled zinc antiporter SLC30A5
Q8TB6
1
S35B2_HUM
AN
SLC35
B2 432
Homo
sapiens Adenosine 3'-phospho 5'-phosphosulfate transporter 1
Q96K3
7
S35E1_HUM
AN
SLC35E
1 410
Homo
sapiens Solute carrier family 35 member E1

202
Q9HD4
5
TM9S3_HU
MAN
TM9SF
3 589
Homo
sapiens Transmembrane 9 superfamily member 3
Q8NB
N3
TM87A_HU
MAN
TMEM
87A 555
Homo
sapiens Transmembrane protein 87A
P82094
TMF1_HUM
AN TMF1 1093
Homo
sapiens TATA element modulatory factor
P42166
LAP2A_HU
MAN TMPO 694
Homo
sapiens Lamina-associated polypeptide 2, isoform alpha
P42167
LAP2B_HU
MAN TMPO 454
Homo
sapiens
Lamina-associated polypeptide 2, isoforms
beta/gamma
Q5JTV
8
TOIP1_HUM
AN
TOR1A
IP1 583
Homo
sapiens Torsin-1A-interacting protein 1
Q15643
TRIPB_HU
MAN TRIP11 1979
Homo
sapiens Thyroid receptor-interacting protein 11
O60763
USO1_HUM
AN USO1 962
Homo
sapiens General vesicular transport factor p115
Q8NFA
0
UBP32_HU
MAN USP32 1604
Homo
sapiens Ubiquitin carboxyl-terminal hydrolase 32
Q7Z7G
8
VP13B_HU
MAN
VPS13
B 4022
Homo
sapiens Intermembrane lipid transfer protein VPS13B
Q9P1Q
0
VPS54_HU
MAN VPS54 977
Homo
sapiens Vacuolar protein sorting-associated protein 54
Q9BSR
8
YIPF4_HUM
AN YIPF4 244
Homo
sapiens Protein YIPF4
O15498
YKT6_HUM
AN YKT6 198
Homo
sapiens Synaptobrevin homolog YKT6
Q8IUH
5
ZDH17_HU
MAN
ZDHHC
17 632
Homo
sapiens Palmitoyltransferase ZDHHC17
Q8IVQ
6
ZDH21_HU
MAN
ZDHHC
21 265
Homo
sapiens Palmitoyltransferase ZDHHC21
O95159
ZFPL1_HU
MAN ZFPL1 310
Homo
sapiens Zinc finger protein-like 1
Table 3. Enriched Proteins for APEX2-EMD(Q133H).
Protein
ID Entry Name Gene
Leng
th
Organis
m Protein Description
Q9H3P
7
GCP60_HU
MAN ACBD3 528
Homo
sapiens Golgi resident protein GCP60
Q99996
AKAP9_HU
MAN AKAP9 3907
Homo
sapiens A-kinase anchor protein 9
P61204
ARF3_HUM
AN ARF3 181
Homo
sapiens ADP-ribosylation factor 3
Q9Y6D
6
BIG1_HUM
AN
ARFGE
F1 1849
Homo
sapiens
Brefeldin A-inhibited guanine nucleotide-exchange
protein 1
P53365
ARFP2_HU
MAN ARFIP2 341
Homo
sapiens Arfaptin-2
P40616
ARL1_HUM
AN ARL1 181
Homo
sapiens ADP-ribosylation factor-like protein 1
P98194
AT2C1_HU
MAN
ATP2C
1 919
Homo
sapiens Calcium-transporting ATPase type 2C member 1
Q9Y48
7
VPP2_HUM
AN
ATP6V
0A2 856
Homo
sapiens V-type proton ATPase 116 kDa subunit a 2

203
Q04656
ATP7A_HU
MAN ATP7A 1500
Homo
sapiens Copper-transporting ATPase 1
O15155
BET1_HUM
AN BET1 118
Homo
sapiens BET1 homolog
Q8WT
W3
COG1_HUM
AN COG1 980
Homo
sapiens Conserved oligomeric Golgi complex subunit 1
P83436
COG7_HUM
AN COG7 770
Homo
sapiens Conserved oligomeric Golgi complex subunit 7
P35606
COPB2_HU
MAN COPB2 906
Homo
sapiens Coatomer subunit beta'
O75976
CBPD_HUM
AN CPD 1380
Homo
sapiens Carboxypeptidase D
Q13948
CASP_HUM
AN CUX1 678
Homo
sapiens Protein CASP
Q7L7V
1
DHX32_HU
MAN DHX32 743
Homo
sapiens
Putative pre-mRNA-splicing factor ATP-dependent
RNA helicase DHX32
P50402
EMD_HUM
AN EMD 254
Homo
sapiens Emerin
Q86V8
7
FHI2B_HU
MAN FHIP2B 743
Homo
sapiens FHF complex subunit HOOK-interacting protein 2B
P35916
VGFR3_HU
MAN FLT4 1363
Homo
sapiens Vascular endothelial growth factor receptor 3
Q92538
GBF1_HUM
AN GBF1 1860
Homo
sapiens
Golgi-specific brefeldin A-resistance guanine
nucleotide exchange factor 1
Q08379
GOGA2_HU
MAN
GOLG
A2 1002
Homo
sapiens Golgin subfamily A member 2
Q08378
GOGA3_HU
MAN
GOLG
A3 1498
Homo
sapiens Golgin subfamily A member 3
Q13439
GOGA4_HU
MAN
GOLG
A4 2230
Homo
sapiens Golgin subfamily A member 4
Q14789
GOGB1_HU
MAN
GOLGB
1 3259
Homo
sapiens Golgin subfamily B member 1
Q9HD2
6
GOPC_HUM
AN GOPC 462
Homo
sapiens
Golgi-associated PDZ and coiled-coil motifcontaining protein
O95249
GOSR1_HU
MAN GOSR1 250
Homo
sapiens Golgi SNAP receptor complex member 1
P61604
CH10_HUM
AN HSPE1 102
Homo
sapiens 10 kDa heat shock protein, mitochondrial
Q8NC5
4
KCT2_HUM
AN KCT2 265
Homo
sapiens Keratinocyte-associated transmembrane protein 2
Q8NC5
6
LEMD2_HU
MAN LEMD2 503
Homo
sapiens LEM domain-containing protein 2
Q9Y2U
8
MAN1_HU
MAN LEMD3 911
Homo
sapiens Inner nuclear membrane protein Man1
Q12907
LMAN2_HU
MAN LMAN2 356
Homo
sapiens Vesicular integral-membrane protein VIP36
P02545
LMNA_HU
MAN LMNA 664
Homo
sapiens Prelamin-A/C
P20700
LMNB1_HU
MAN LMNB1 586
Homo
sapiens Lamin-B1
Q03252
LMNB2_HU
MAN LMNB2 620
Homo
sapiens Lamin-B2
Q9UK
M7
MA1B1_HU
MAN
MAN1
B1 699
Homo
sapiens
Endoplasmic reticulum mannosyl-oligosaccharide
1,2-alpha-mannosidase

204
Q7Z3U
7
MON2_HU
MAN MON2 1717
Homo
sapiens Protein MON2 homolog
P24844
MYL9_HUM
AN MYL9 172
Homo
sapiens Myosin regulatory light polypeptide 9
Q6ZS3
0
NBEL1_HU
MAN
NBEAL
1 2694
Homo
sapiens Neurobeachin-like protein 1
Q92542
NICA_HUM
AN NCSTN 709
Homo
sapiens Nicastrin
P22059
OSBP1_HU
MAN OSBP 807
Homo
sapiens Oxysterol-binding protein 1
Q9BX
B4
OSB11_HU
MAN
OSBPL
11 747
Homo
sapiens Oxysterol-binding protein-related protein 11
Q15154
PCM1_HUM
AN PCM1 2024
Homo
sapiens Pericentriolar material 1 protein
Q6P99
6
PDXD1_HU
MAN
PDXDC
1 788
Homo
sapiens
Pyridoxal-dependent decarboxylase domaincontaining protein 1
Q9BTU
6
P4K2A_HU
MAN PI4K2A 479
Homo
sapiens Phosphatidylinositol 4-kinase type 2-alpha
O00625
PIR_HUMA
N PIR 290
Homo
sapiens Pirin
Q5TH
K1
PR14L_HU
MAN
PRR14
L 2151
Homo
sapiens Protein PRR14L
P20340
RAB6A_HU
MAN RAB6A 208
Homo
sapiens Ras-related protein Rab-6A
Q4AD
V7
RIC1_HUM
AN RIC1 1423
Homo
sapiens Guanine nucleotide exchange factor subunit RIC1
Q9NTJ
5
SAC1_HUM
AN
SACM1
L 587
Homo
sapiens Phosphatidylinositol-3-phosphatase SAC1
Q8IZE
3
PACE1_HU
MAN SCYL3 742
Homo
sapiens
Protein-associating with the carboxyl-terminal
domain of ezrin
Q15436
SC23A_HU
MAN
SEC23
A 765
Homo
sapiens Protein transport protein Sec23A
Q9Y6Y
8
S23IP_HUM
AN
SEC23I
P 1000
Homo
sapiens SEC23-interacting protein
P53992
SC24C_HU
MAN
SEC24
C 1094
Homo
sapiens Protein transport protein Sec24C
Q6PCB
7
S27A1_HU
MAN
SLC27
A1 646
Homo
sapiens Long-chain fatty acid transport protein 1
Q8TA
D4
ZNT5_HUM
AN
SLC30
A5 765
Homo
sapiens Proton-coupled zinc antiporter SLC30A5
Q8TB6
1
S35B2_HUM
AN
SLC35
B2 432
Homo
sapiens Adenosine 3'-phospho 5'-phosphosulfate transporter 1
Q96K3
7
S35E1_HUM
AN
SLC35E
1 410
Homo
sapiens Solute carrier family 35 member E1
Q13190
STX5_HUM
AN STX5 355
Homo
sapiens Syntaxin-5
Q9HD4
5
TM9S3_HU
MAN
TM9SF
3 589
Homo
sapiens Transmembrane 9 superfamily member 3
P82094
TMF1_HUM
AN TMF1 1093
Homo
sapiens TATA element modulatory factor
P42166
LAP2A_HU
MAN TMPO 694
Homo
sapiens Lamina-associated polypeptide 2, isoform alpha
P42167
LAP2B_HU
MAN TMPO 454
Homo
sapiens
Lamina-associated polypeptide 2, isoforms
beta/gamma

205
Table 4. Enriched Proteins for APEX2-EMD(Δ95-99).
Protein
ID Entry Name Gene
Leng
th
Organis
m Protein Description
Q9H3P
7
GCP60_HU
MAN ACBD3 528
Homo
sapiens Golgi resident protein GCP60
Q99996
AKAP9_HU
MAN AKAP9 3907
Homo
sapiens A-kinase anchor protein 9
Q9Y6B
7
AP4B1_HU
MAN AP4B1 739
Homo
sapiens AP-4 complex subunit beta-1
P61204
ARF3_HUM
AN ARF3 181
Homo
sapiens ADP-ribosylation factor 3
Q9Y6D
6
BIG1_HUM
AN
ARFGE
F1 1849
Homo
sapiens
Brefeldin A-inhibited guanine nucleotide-exchange
protein 1
P53365
ARFP2_HU
MAN ARFIP2 341
Homo
sapiens Arfaptin-2
P40616
ARL1_HUM
AN ARL1 181
Homo
sapiens ADP-ribosylation factor-like protein 1
Q5T2E
6
ARMD3_HU
MAN
ARMH
3 689
Homo
sapiens Armadillo-like helical domain-containing protein 3
P98194
AT2C1_HU
MAN
ATP2C
1 919
Homo
sapiens Calcium-transporting ATPase type 2C member 1
Q9Y48
7
VPP2_HUM
AN
ATP6V
0A2 856
Homo
sapiens V-type proton ATPase 116 kDa subunit a 2
P21283
VATC1_HU
MAN
ATP6V
1C1 382
Homo
sapiens V-type proton ATPase subunit C 1
Q04656
ATP7A_HU
MAN ATP7A 1500
Homo
sapiens Copper-transporting ATPase 1
Q5JTV
8
TOIP1_HUM
AN
TOR1A
IP1 583
Homo
sapiens Torsin-1A-interacting protein 1
Q15643
TRIPB_HU
MAN TRIP11 1979
Homo
sapiens Thyroid receptor-interacting protein 11
O60763
USO1_HUM
AN USO1 962
Homo
sapiens General vesicular transport factor p115
Q8NFA
0
UBP32_HU
MAN USP32 1604
Homo
sapiens Ubiquitin carboxyl-terminal hydrolase 32
Q7Z7G
8
VP13B_HU
MAN
VPS13
B 4022
Homo
sapiens Intermembrane lipid transfer protein VPS13B
Q9P1Q
0
VPS54_HU
MAN VPS54 977
Homo
sapiens Vacuolar protein sorting-associated protein 54
Q9UE
U0
VTI1B_HU
MAN VTI1B 232
Homo
sapiens
Vesicle transport through interaction with t-SNAREs
homolog 1B
Q9BSR
8
YIPF4_HUM
AN YIPF4 244
Homo
sapiens Protein YIPF4
O15498
YKT6_HUM
AN YKT6 198
Homo
sapiens Synaptobrevin homolog YKT6
Q8IUH
5
ZDH17_HU
MAN
ZDHHC
17 632
Homo
sapiens Palmitoyltransferase ZDHHC17
Q8IVQ
6
ZDH21_HU
MAN
ZDHHC
21 265
Homo
sapiens Palmitoyltransferase ZDHHC21
O95159
ZFPL1_HU
MAN ZFPL1 310
Homo
sapiens Zinc finger protein-like 1

206
O15155
BET1_HUM
AN BET1 118
Homo
sapiens BET1 homolog
Q9UPN
4
CP131_HUM
AN CEP131 1083
Homo
sapiens Centrosomal protein of 131 kDa
Q8WT
W3
COG1_HUM
AN COG1 980
Homo
sapiens Conserved oligomeric Golgi complex subunit 1
P83436
COG7_HUM
AN COG7 770
Homo
sapiens Conserved oligomeric Golgi complex subunit 7
Q96M
W5
COG8_HUM
AN COG8 612
Homo
sapiens Conserved oligomeric Golgi complex subunit 8
P53621
COPA_HUM
AN COPA 1224
Homo
sapiens Coatomer subunit alpha
P35606
COPB2_HU
MAN COPB2 906
Homo
sapiens Coatomer subunit beta'
O75976
CBPD_HUM
AN CPD 1380
Homo
sapiens Carboxypeptidase D
Q9BS
W2
EFC4B_HU
MAN
CRACR
2A 731
Homo
sapiens
EF-hand calcium-binding domain-containing protein
4B
Q13948
CASP_HUM
AN CUX1 678
Homo
sapiens Protein CASP
P50402
EMD_HUM
AN EMD 254
Homo
sapiens Emerin
O60427
FADS1_HU
MAN FADS1 444
Homo
sapiens Acyl-CoA (8-3)-desaturase
Q5W0
V3
FHI2A_HU
MAN FHIP2A 765
Homo
sapiens FHF complex subunit HOOK interacting protein 2A
Q86V8
7
FHI2B_HUM
AN FHIP2B 743
Homo
sapiens FHF complex subunit HOOK-interacting protein 2B
Q92538
GBF1_HUM
AN GBF1 1860
Homo
sapiens
Golgi-specific brefeldin A-resistance guanine
nucleotide exchange factor 1
Q8IWJ
2
GCC2_HUM
AN GCC2 1684
Homo
sapiens GRIP and coiled-coil domain-containing protein 2
Q08379
GOGA2_HU
MAN
GOLG
A2 1002
Homo
sapiens Golgin subfamily A member 2
Q08378
GOGA3_HU
MAN
GOLG
A3 1498
Homo
sapiens Golgin subfamily A member 3
Q13439
GOGA4_HU
MAN
GOLG
A4 2230
Homo
sapiens Golgin subfamily A member 4
Q14789
GOGB1_HU
MAN
GOLGB
1 3259
Homo
sapiens Golgin subfamily B member 1
Q9HD2
6
GOPC_HUM
AN GOPC 462
Homo
sapiens
Golgi-associated PDZ and coiled-coil motifcontaining protein
O95249
GOSR1_HU
MAN GOSR1 250
Homo
sapiens Golgi SNAP receptor complex member 1
Q8NC5
4
KCT2_HUM
AN KCT2 265
Homo
sapiens Keratinocyte-associated transmembrane protein 2
Q8NC5
6
LEMD2_HU
MAN LEMD2 503
Homo
sapiens LEM domain-containing protein 2
Q9Y2U
8
MAN1_HU
MAN LEMD3 911
Homo
sapiens Inner nuclear membrane protein Man1
Q12907
LMAN2_HU
MAN LMAN2 356
Homo
sapiens Vesicular integral-membrane protein VIP36
P02545
LMNA_HU
MAN LMNA 664
Homo
sapiens Prelamin-A/C

207
P20700
LMNB1_HU
MAN LMNB1 586
Homo
sapiens Lamin-B1
Q9UK
M7
MA1B1_HU
MAN
MAN1
B1 699
Homo
sapiens
Endoplasmic reticulum mannosyl-oligosaccharide
1,2-alpha-mannosidase
Q7Z3U
7
MON2_HU
MAN MON2 1717
Homo
sapiens Protein MON2 homolog
Q9NY
A4
MTMR4_HU
MAN
MTMR
4 1195
Homo
sapiens
Phosphatidylinositol-3,5-bisphosphate 3-phosphatase
MTMR4
Q6ZS3
0
NBEL1_HU
MAN
NBEAL
1 2694
Homo
sapiens Neurobeachin-like protein 1
P22059
OSBP1_HU
MAN OSBP 807
Homo
sapiens Oxysterol-binding protein 1
Q9BX
B4
OSB11_HU
MAN
OSBPL
11 747
Homo
sapiens Oxysterol-binding protein-related protein 11
Q96SU
4
OSBL9_HU
MAN
OSBPL
9 736
Homo
sapiens Oxysterol-binding protein-related protein 9
Q15154
PCM1_HUM
AN PCM1 2024
Homo
sapiens Pericentriolar material 1 protein
Q6P99
6
PDXD1_HU
MAN
PDXDC
1 788
Homo
sapiens
Pyridoxal-dependent decarboxylase domaincontaining protein 1
Q9BTU
6
P4K2A_HU
MAN PI4K2A 479
Homo
sapiens Phosphatidylinositol 4-kinase type 2-alpha
O00625
PIR_HUMA
N PIR 290
Homo
sapiens Pirin
Q5TH
K1
PR14L_HU
MAN
PRR14
L 2151
Homo
sapiens Protein PRR14L
Q96M2
7
PRRC1_HU
MAN PRRC1 445
Homo
sapiens Protein PRRC1
P20340
RAB6A_HU
MAN RAB6A 208
Homo
sapiens Ras-related protein Rab-6A
Q4AD
V7
RIC1_HUM
AN RIC1 1423
Homo
sapiens Guanine nucleotide exchange factor subunit RIC1
Q9NTJ
5
SAC1_HUM
AN
SACM1
L 587
Homo
sapiens Phosphatidylinositol-3-phosphatase SAC1
Q8IZE
3
PACE1_HU
MAN SCYL3 742
Homo
sapiens
Protein-associating with the carboxyl-terminal
domain of ezrin
Q15436
SC23A_HU
MAN
SEC23
A 765
Homo
sapiens Protein transport protein Sec23A
Q9Y6Y
8
S23IP_HUM
AN
SEC23I
P 1000
Homo
sapiens SEC23-interacting protein
O94979
SC31A_HU
MAN
SEC31
A 1220
Homo
sapiens Protein transport protein Sec31A
Q6PCB
7
S27A1_HUM
AN
SLC27
A1 646
Homo
sapiens Long-chain fatty acid transport protein 1
Q8TA
D4
ZNT5_HUM
AN
SLC30
A5 765
Homo
sapiens Proton-coupled zinc antiporter SLC30A5
Q96K3
7
S35E1_HUM
AN
SLC35E
1 410
Homo
sapiens Solute carrier family 35 member E1
Q13190
STX5_HUM
AN STX5 355
Homo
sapiens Syntaxin-5
Q9NU
Y8
TBC23_HU
MAN
TBC1D
23 699
Homo
sapiens TBC1 domain family member 23
Q9HD4
5
TM9S3_HU
MAN
TM9SF
3 589
Homo
sapiens Transmembrane 9 superfamily member 3

208
Q8NB
N3
TM87A_HU
MAN
TMEM
87A 555
Homo
sapiens Transmembrane protein 87A
P82094
TMF1_HUM
AN TMF1 1093
Homo
sapiens TATA element modulatory factor
P42166
LAP2A_HU
MAN TMPO 694
Homo
sapiens Lamina-associated polypeptide 2, isoform alpha
P42167
LAP2B_HU
MAN TMPO 454
Homo
sapiens
Lamina-associated polypeptide 2, isoforms
beta/gamma
Q5JTV
8
TOIP1_HUM
AN
TOR1A
IP1 583
Homo
sapiens Torsin-1A-interacting protein 1
Q15643
TRIPB_HU
MAN TRIP11 1979
Homo
sapiens Thyroid receptor-interacting protein 11
O60763
USO1_HUM
AN USO1 962
Homo
sapiens General vesicular transport factor p115
Q8NFA
0
UBP32_HU
MAN USP32 1604
Homo
sapiens Ubiquitin carboxyl-terminal hydrolase 32
Q7Z7G
8
VP13B_HU
MAN
VPS13
B 4022
Homo
sapiens Intermembrane lipid transfer protein VPS13B
Q9P1Q
0
VPS54_HU
MAN VPS54 977
Homo
sapiens Vacuolar protein sorting-associated protein 54
Q9UE
U0
VTI1B_HU
MAN VTI1B 232
Homo
sapiens
Vesicle transport through interaction with t-SNAREs
homolog 1B
Q9BSR
8
YIPF4_HUM
AN YIPF4 244
Homo
sapiens Protein YIPF4
O15498
YKT6_HUM
AN YKT6 198
Homo
sapiens Synaptobrevin homolog YKT6
Q8IUH
5
ZDH17_HU
MAN
ZDHHC
17 632
Homo
sapiens Palmitoyltransferase ZDHHC17
Q8IVQ
6
ZDH21_HU
MAN
ZDHHC
21 265
Homo
sapiens Palmitoyltransferase ZDHHC21
O95159
ZFPL1_HU
MAN ZFPL1 310
Homo
sapiens Zinc finger protein-like 1
Table 5. Enriched Proteins for splitAPEX2-EMD.
Protei
n ID
Entry
Name
Gene Len
gth
Organis
m
Protein Description
Q6IQ
32
ADNP2_H
UMAN
ADNP
2
113
1
Homo
sapiens
Activity-dependent neuroprotector homeobox protein 2
Q9UL
X6
AKP8L_H
UMAN
AKAP
8L
646 Homo
sapiens
A-kinase anchor protein 8-like
Q9UH
17
ABC3B_H
UMAN
APOB
EC3B
382 Homo
sapiens
DNA dC->dU-editing enzyme APOBEC-3B
Q68C
P9
ARID2_H
UMAN
ARID2 183
5
Homo
sapiens
AT-rich interactive domain-containing protein 2
Q76L
83
ASXL2_H
UMAN
ASXL2 143
5
Homo
sapiens
Putative Polycomb group protein ASXL2
Q6PL
18
ATAD2_H
UMAN
ATAD2 139
0
Homo
sapiens
ATPase family AAA domain-containing protein 2
Q6V
MQ6
MCAF1_H
UMAN
ATF7I
P
127
0
Homo
sapiens
Activating transcription factor 7-interacting protein 1
Q1353
5
ATR_HUM
AN
ATR 264
4
Homo
sapiens
Serine/threonine-protein kinase ATR

209
Q8IX
M2
BAP18_H
UMAN
BAP18 172 Homo
sapiens
Chromatin complexes subunit BAP18
Q9972
8
BARD1_H
UMAN
BARD
1
777 Homo
sapiens
BRCA1-associated RING domain protein 1
Q9NR
L2
BAZ1A_H
UMAN
BAZ1
A
155
6
Homo
sapiens
Bromodomain adjacent to zinc finger domain protein 1A
Q9UI
G0
BAZ1B_H
UMAN
BAZ1
B
148
3
Homo
sapiens
Tyrosine-protein kinase BAZ1B
Q5T5
X7
BEND3_H
UMAN
BEND
3
828 Homo
sapiens
BEN domain-containing protein 3
P5413
2
BLM_HU
MAN
BLM 141
7
Homo
sapiens
RecQ-like DNA helicase BLM
P5158
7
BRCA2_H
UMAN
BRCA
2
341
8
Homo
sapiens
Breast cancer type 2 susceptibility protein
Q9NP
I1
BRD7_HU
MAN
BRD7 651 Homo
sapiens
Bromodomain-containing protein 7
Q6RI4
5
BRWD3_H
UMAN
BRWD
3
180
2
Homo
sapiens
Bromodomain and WD repeat-containing protein 3
Q9Y2
32
CDYL_HU
MAN
CDYL 598 Homo
sapiens
Chromodomain Y-like protein
P4945
4
CENPF_H
UMAN
CENPF 311
4
Homo
sapiens
Centromere protein F
Q8NF
W8
NEUA_HU
MAN
CMAS 434 Homo
sapiens
N-acylneuraminate cytidylyltransferase
P3843
2
COIL_HU
MAN
COIL 576 Homo
sapiens
Coilin
Q9Y4
B6
DCAF1_H
UMAN
DCAF
1
150
7
Homo
sapiens
DDB1- and CUL4-associated factor 1
Q9Y6
K1
DNM3A_H
UMAN
DNMT
3A
912 Homo
sapiens
DNA (cytosine-5)-methyltransferase 3A
Q9H9
B1
EHMT1_H
UMAN
EHMT
1
129
8
Homo
sapiens
Histone-lysine N-methyltransferase EHMT1
P1952
5
E2AK2_H
UMAN
EIF2A
K2
551 Homo
sapiens
Interferon-induced, double-stranded RNA-activated protein
kinase
P5040
2
EMD_HU
MAN
EMD 254 Homo
sapiens
Emerin
Q9H2
F5
EPC1_HU
MAN
EPC1 836 Homo
sapiens
Enhancer of polycomb homolog 1
Q9NV
I1
FANCI_H
UMAN
FANCI 132
8
Homo
sapiens
Fanconi anemia group I protein
Q4VC
44
FWCH1_H
UMAN
FLYW
CH1
716 Homo
sapiens
FLYWCH-type zinc finger-containing protein 1
P8503
7
FOXK1_H
UMAN
FOXK
1
733 Homo
sapiens
Forkhead box protein K1
Q9Y5
Q8
TF3C5_HU
MAN
GTF3C
5
519 Homo
sapiens
General transcription factor 3C polypeptide 5
P0791
0
HNRPC_H
UMAN
HNRN
PC
306 Homo
sapiens
Heterogeneous nuclear ribonucleoproteins C1/C2
P4873
5
IDHP_HU
MAN
IDH2 452 Homo
sapiens
Isocitrate dehydrogenase [NADP], mitochondrial
Q8NB
78
KDM1B_H
UMAN
KDM1
B
822 Homo
sapiens
Lysine-specific histone demethylase 2
O7552
5
KHDR3_H
UMAN
KHDR
BS3
346 Homo
sapiens
KH domain-containing, RNA-binding, signal transductionassociated protein 3

210
Q86Y
91
KI18B_HU
MAN
KIF18
B
852 Homo
sapiens
Kinesin-like protein KIF18B
Q96Q
89
KI20B_HU
MAN
KIF20
B
182
0
Homo
sapiens
Kinesin-like protein KIF20B
Q0224
1
KIF23_HU
MAN
KIF23 960 Homo
sapiens
Kinesin-like protein KIF23
Q9B
W19
KIFC1_HU
MAN
KIFC1 673 Homo
sapiens
Kinesin-like protein KIFC1
Q969
R5
LMBL2_H
UMAN
L3MB
TL2
705 Homo
sapiens
Lethal(3)malignant brain tumor-like protein 2
Q96J
M7
LMBL3_H
UMAN
L3MB
TL3
780 Homo
sapiens
Lethal(3)malignant brain tumor-like protein 3
Q8NC
56
LEMD2_H
UMAN
LEMD
2
503 Homo
sapiens
LEM domain-containing protein 2
Q9Y2
U8
MAN1_HU
MAN
LEMD
3
911 Homo
sapiens
Inner nuclear membrane protein Man1
Q9Y6
D9
MD1L1_H
UMAN
MAD1
L1
718 Homo
sapiens
Mitotic spindle assembly checkpoint protein MAD1
Q6ZR
Q5
MMS22_H
UMAN
MMS2
2L
124
3
Homo
sapiens
Protein MMS22-like
P4324
6
MSH2_HU
MAN
MSH2 934 Homo
sapiens
DNA mismatch repair protein Msh2
Q68D
K7
MSL1_HU
MAN
MSL1 614 Homo
sapiens
Male-specific lethal 1 homolog
Q9BT
C8
MTA3_HU
MAN
MTA3 594 Homo
sapiens
Metastasis-associated protein MTA3
P4269
5
CNDD3_H
UMAN
NCAP
D3
149
8
Homo
sapiens
Condensin-2 complex subunit D3
Q8NC
F5
NF2IP_HU
MAN
NFATC
2IP
419 Homo
sapiens
NFATC2-interacting protein
P0865
1
NFIC_HU
MAN
NFIC 508 Homo
sapiens
Nuclear factor 1 C-type
Q86U
86
PB1_HUM
AN
PBRM
1
168
9
Homo
sapiens
Protein polybromo-1
Q8W
WQ0
PHIP_HU
MAN
PHIP 182
1
Homo
sapiens
PH-interacting protein
P5427
7
PMS1_HU
MAN
PMS1 932 Homo
sapiens
PMS1 protein homolog 1
P5427
8
PMS2_HU
MAN
PMS2 862 Homo
sapiens
Mismatch repair endonuclease PMS2
P4900
5
DPOD2_H
UMAN
POLD2 469 Homo
sapiens
DNA polymerase delta subunit 2
P1485
9
PO2F1_HU
MAN
POU2F
1
743 Homo
sapiens
POU domain, class 2, transcription factor 1
Q9H0
H5
RGAP1_H
UMAN
RACG
AP1
632 Homo
sapiens
Rac GTPase-activating protein 1
Q9UK
M9
RALY_HU
MAN
RALY 306 Homo
sapiens
RNA-binding protein Raly
Q8IX
T5
RB12B_H
UMAN
RBM1
2B
100
1
Homo
sapiens
RNA-binding protein 12B
Q96E
39
RMXL1_H
UMAN
RBMX
L1
390 Homo
sapiens
RNA binding motif protein, X-linked-like-1
Q9P2
K3
RCOR3_H
UMAN
RCOR
3
495 Homo
sapiens
REST corepressor 3

211
Q1542
4
SAFB1_H
UMAN
SAFB 915 Homo
sapiens
Scaffold attachment factor B1
Q562
F6
SGO2_HU
MAN
SGO2 126
5
Homo
sapiens
Shugoshin 2
O6026
4
SMCA5_H
UMAN
SMAR
CA5
105
2
Homo
sapiens
SWI/SNF-related matrix-associated actin-dependent
regulator of chromatin subfamily A member 5
Q8TA
Q2
SMRC2_H
UMAN
SMAR
CC2
121
4
Homo
sapiens
SWI/SNF complex subunit SMARCC2
Q96G
M5
SMRD1_H
UMAN
SMAR
CD1
515 Homo
sapiens
SWI/SNF-related matrix-associated actin-dependent
regulator of chromatin subfamily D member 1
Q6ST
E5
SMRD3_H
UMAN
SMAR
CD3
483 Homo
sapiens
SWI/SNF-related matrix-associated actin-dependent
regulator of chromatin subfamily D member 3
Q1468
3
SMC1A_H
UMAN
SMC1
A
123
3
Homo
sapiens
Structural maintenance of chromosomes protein 1A
Q8IY
18
SMC5_HU
MAN
SMC5 110
1
Homo
sapiens
Structural maintenance of chromosomes protein 5
A6NH
R9
SMHD1_H
UMAN
SMCH
D1
200
5
Homo
sapiens
Structural maintenance of chromosomes flexible hinge
domain-containing protein 1
O7549
4
SRS10_HU
MAN
SRSF1
0
262 Homo
sapiens
Serine/arginine-rich splicing factor 10
Q9280
4
RBP56_H
UMAN
TAF15 592 Homo
sapiens
TATA-binding protein-associated factor 2N
Q1554
2
TAF5_HU
MAN
TAF5 800 Homo
sapiens
Transcription initiation factor TFIID subunit 5
Q86U
E8
TLK2_HU
MAN
TLK2 772 Homo
sapiens
Serine/threonine-protein kinase tousled-like 2
P4216
6
LAP2A_H
UMAN
TMPO 694 Homo
sapiens
Lamina-associated polypeptide 2, isoform alpha
P4216
7
LAP2B_H
UMAN
TMPO 454 Homo
sapiens
Lamina-associated polypeptide 2, isoforms beta/gamma
Q5JT
V8
TOIP1_HU
MAN
TOR1
AIP1
583 Homo
sapiens
Torsin-1A-interacting protein 1
Q9NX
H8
TOR4A_H
UMAN
TOR4
A
423 Homo
sapiens
Torsin-4A
Q1288
8
TP53B_HU
MAN
TP53B
P1
197
2
Homo
sapiens
TP53-binding protein 1
P1227
0
TPR_HUM
AN
TPR 236
3
Homo
sapiens
Nucleoprotein TPR
Q9UL
W0
TPX2_HU
MAN
TPX2 747 Homo
sapiens
Targeting protein for Xklp2
P6299
5
TRA2B_H
UMAN
TRA2
B
288 Homo
sapiens
Transformer-2 protein homolog beta
O1516
4
TIF1A_HU
MAN
TRIM2
4
105
0
Homo
sapiens
Transcription intermediary factor 1-alpha
Q9UP
N9
TRI33_HU
MAN
TRIM3
3
112
7
Homo
sapiens
E3 ubiquitin-protein ligase TRIM33
Q9H2
G4
TSYL2_H
UMAN
TSPYL
2
693 Homo
sapiens
Testis-specific Y-encoded-like protein 2
O9507
1
UBR5_HU
MAN
UBR5 279
9
Homo
sapiens
E3 ubiquitin-protein ligase UBR5
Q96P
U4
UHRF2_H
UMAN
UHRF
2
802 Homo
sapiens
E3 ubiquitin-protein ligase UHRF2
Q1683
1
UPP1_HU
MAN
UPP1 310 Homo
sapiens
Uridine phosphorylase 1

212
O9478
2
UBP1_HU
MAN
USP1 785 Homo
sapiens
Ubiquitin carboxyl-terminal hydrolase 1
O7571
7
WDHD1_
HUMAN
WDHD
1
112
9
Homo
sapiens
WD repeat and HMG-box DNA-binding protein 1
Q96S
55
WRIP1_H
UMAN
WRNI
P1
665 Homo
sapiens
ATPase WRNIP1
Q0183
1
XPC_HUM
AN
XPC 940 Homo
sapiens
DNA repair protein complementing XP-C cells
Q5T2
00
ZC3HD_H
UMAN
ZC3H1
3
166
8
Homo
sapiens
Zinc finger CCCH domain-containing protein 13
Q6NZ
Y4
ZCHC8_H
UMAN
ZCCH
C8
707 Homo
sapiens
Zinc finger CCHC domain-containing protein 8
Q9UB
W7
ZMYM2_
HUMAN
ZMYM
2
137
7
Homo
sapiens
Zinc finger MYM-type protein 2
Q1420
2
ZMYM3_
HUMAN
ZMYM
3
137
0
Homo
sapiens
Zinc finger MYM-type protein 3
Q5T7
W0
ZN618_HU
MAN
ZNF61
8
954 Homo
sapiens
Zinc finger protein 618
Table 6. Enriched Proteins for EMDWT
-APEX2.
Protei
n ID Entry Name Gene
Len
gth
Organis
m Protein Description
Q9BW
D1
THIC_HUM
AN ACAT2 397
Homo
sapiens Acetyl-CoA acetyltransferase, cytosolic
O1467
2
ADA10_HU
MAN ADAM10 748
Homo
sapiens
Disintegrin and metalloproteinase domaincontaining protein 10
O9491
0
AGRL1_HU
MAN ADGRL1 1474
Homo
sapiens Adhesion G protein-coupled receptor L1
Q1374
0
CD166_HU
MAN ALCAM 583
Homo
sapiens CD166 antigen
Q86SJ
2
AMGO2_H
UMAN AMIGO2 522
Homo
sapiens Amphoterin-induced protein 2
Q9UK
U9
ANGL2_HU
MAN ANGPTL2 493
Homo
sapiens Angiopoietin-related protein 2
Q9H6
X2
ANTR1_HU
MAN ANTXR1 564
Homo
sapiens Anthrax toxin receptor 1
P05067
A4_HUMA
N APP 770
Homo
sapiens Amyloid-beta precursor protein
P05023
AT1A1_HU
MAN ATP1A1 1023
Homo
sapiens
Sodium/potassium-transporting ATPase subunit
alpha-1
P05026
AT1B1_HU
MAN ATP1B1 303
Homo
sapiens
Sodium/potassium-transporting ATPase subunit
beta-1
Q0465
6
ATP7A_HU
MAN ATP7A 1500
Homo
sapiens Copper-transporting ATPase 1
Q0097
3
B4GN1_HU
MAN
B4GALN
T1 533
Homo
sapiens Beta-1,4 N-acetylgalactosaminyltransferase 1
Q9UB
V7
B4GT7_HU
MAN B4GALT7 327
Homo
sapiens Beta-1,4-galactosyltransferase 7
P50895
BCAM_HU
MAN BCAM 628
Homo
sapiens Basal cell adhesion molecule

213
Q7KY
R7
BT2A1_HU
MAN BTN2A1 527
Homo
sapiens Butyrophilin subfamily 2 member A1
Q9NS0
0
C1GLT_HU
MAN C1GALT1 363
Homo
sapiens
Glycoprotein-N-acetylgalactosamine 3-betagalactosyltransferase 1
Q96EU
7
C1GLC_HU
MAN
C1GALT1
C1 318
Homo
sapiens C1GALT1-specific chaperone 1
Q9NZP
8
C1RL_HUM
AN C1RL 487
Homo
sapiens Complement C1r subcomponent-like protein
Q9BY6
7
CADM1_H
UMAN CADM1 442
Homo
sapiens Cell adhesion molecule 1
O0062
2
CCN1_HUM
AN CCN1 381
Homo
sapiens CCN family member 1
P29279
CCN2_HUM
AN CCN2 349
Homo
sapiens CCN family member 2
Q6YH
K3
CD109_HU
MAN CD109 1445
Homo
sapiens CD109 antigen
P16070
CD44_HUM
AN CD44 742
Homo
sapiens CD44 antigen
Q0872
2
CD47_HUM
AN CD47 323
Homo
sapiens Leukocyte surface antigen CD47
P19022
CADH2_HU
MAN CDH2 906
Homo
sapiens Cadherin-2
Q9UH
N6
CEIP2_HU
MAN CEMIP2 1383
Homo
sapiens Cell surface hyaluronidase CEMIP2
Q9P2E
5
CHPF2_HU
MAN CHPF2 772
Homo
sapiens Chondroitin sulfate glucuronyltransferase
Q8NC
H0
CHSTE_HU
MAN CHST14 376
Homo
sapiens Carbohydrate sulfotransferase 14
Q86X5
2
CHSS1_HU
MAN CHSY1 802
Homo
sapiens Chondroitin sulfate synthase 1
Q8IYK
4
GT252_HU
MAN
COLGAL
T2 626
Homo
sapiens Procollagen galactosyltransferase 2
O7597
6
CBPD_HU
MAN CPD 1380
Homo
sapiens Carboxypeptidase D
Q8N6
G5
CGAT2_HU
MAN
CSGALN
ACT2 542
Homo
sapiens
Chondroitin sulfate Nacetylgalactosaminyltransferase 2
Q6UV
K1
CSPG4_HU
MAN CSPG4 2322
Homo
sapiens Chondroitin sulfate proteoglycan 4
P35222
CTNB1_HU
MAN CTNNB1 781
Homo
sapiens Catenin beta-1
P78310
CXAR_HU
MAN CXADR 365
Homo
sapiens Coxsackievirus and adenovirus receptor
Q1411
8
DAG1_HU
MAN DAG1 895
Homo
sapiens Dystroglycan 1
Q8WV
C6
DCAKD_H
UMAN DCAKD 231
Homo
sapiens Dephospho-CoA kinase domain-containing protein
Q96PD
2
DCBD2_HU
MAN DCBLD2 775
Homo
sapiens
Discoidin, CUB and LCCL domain-containing
protein 2
Q1683
2
DDR2_HU
MAN DDR2 855
Homo
sapiens Discoidin domain-containing receptor 2
Q1412
6
DSG2_HUM
AN DSG2 1118
Homo
sapiens Desmoglein-2
Q1661
0
ECM1_HU
MAN ECM1 540
Homo
sapiens Extracellular matrix protein 1

214
P00533
EGFR_HUM
AN EGFR 1210
Homo
sapiens Epidermal growth factor receptor
Q1571
7
ELAV1_HU
MAN ELAVL1 326
Homo
sapiens ELAV-like protein 1
P0C7U
0
ELFN1_HU
MAN ELFN1 828
Homo
sapiens Protein ELFN1
P50402
EMD_HUM
AN EMD 254
Homo
sapiens Emerin
P29317
EPHA2_HU
MAN EPHA2 976
Homo
sapiens Ephrin type-A receptor 2
P54756
EPHA5_HU
MAN EPHA5 1037
Homo
sapiens Ephrin type-A receptor 5
P29323
EPHB2_HU
MAN EPHB2 1055
Homo
sapiens Ephrin type-B receptor 2
Q9Y28
2
ERGI3_HU
MAN ERGIC3 383
Homo
sapiens
Endoplasmic reticulum-Golgi intermediate
compartment protein 3
O7506
3
XYLK_HU
MAN FAM20B 409
Homo
sapiens Glycosaminoglycan xylosylkinase
Q9252
0
FAM3C_HU
MAN FAM3C 227
Homo
sapiens Protein FAM3C
Q1451
7
FAT1_HUM
AN FAT1 4588
Homo
sapiens Protocadherin Fat 1
Q96A
Y3
FKB10_HU
MAN FKBP10 582
Homo
sapiens Peptidyl-prolyl cis-trans isomerase FKBP10
O4315
5
FLRT2_HU
MAN FLRT2 660
Homo
sapiens Leucine-rich repeat transmembrane protein FLRT2
P35916
VGFR3_HU
MAN FLT4 1363
Homo
sapiens Vascular endothelial growth factor receptor 3
Q9BY
C5
FUT8_HUM
AN FUT8 575
Homo
sapiens Alpha-(1,6)-fucosyltransferase
P51114
FXR1_HUM
AN FXR1 621
Homo
sapiens RNA-binding protein FXR1
Q8N42
8
GLT16_HU
MAN GALNT16 558
Homo
sapiens Polypeptide N-acetylgalactosaminyltransferase 16
Q6IS24
GLT17_HU
MAN GALNT17 598
Homo
sapiens Polypeptide N-acetylgalactosaminyltransferase 17
Q1047
1
GALT2_HU
MAN GALNT2 571
Homo
sapiens Polypeptide N-acetylgalactosaminyltransferase 2
Q86SF
2
GALT7_HU
MAN GALNT7 657
Homo
sapiens N-acetylgalactosaminyltransferase 7
Q9289
6
GSLG1_HU
MAN GLG1 1179
Homo
sapiens Golgi apparatus protein 1
Q68CQ
7
GL8D1_HU
MAN GLT8D1 371
Homo
sapiens Glycosyltransferase 8 domain-containing protein 1
O0046
1
GOLI4_HU
MAN GOLIM4 696
Homo
sapiens Golgi integral membrane protein 4
Q8NBJ
4
GOLM1_HU
MAN GOLM1 401
Homo
sapiens Golgi membrane protein 1
Q5VW
38
GP107_HU
MAN GPR107 600
Homo
sapiens Protein GPR107
P10915
HPLN1_HU
MAN HAPLN1 354
Homo
sapiens Hyaluronan and proteoglycan link protein 1
P01889
HLAB_HU
MAN HLA-B 362
Homo
sapiens
HLA class I histocompatibility antigen, B alpha
chain

215
P09601
HMOX1_H
UMAN HMOX1 288
Homo
sapiens Heme oxygenase 1
Q9972
9
ROAA_HU
MAN
HNRNPA
B 332
Homo
sapiens Heterogeneous nuclear ribonucleoprotein A/B
Q7LG
A3
HS2ST_HU
MAN HS2ST1 356
Homo
sapiens Heparan sulfate 2-O-sulfotransferase 1
P48735
IDHP_HUM
AN IDH2 452
Homo
sapiens Isocitrate dehydrogenase [NADP], mitochondrial
Q1627
0
IBP7_HUM
AN IGFBP7 282
Homo
sapiens Insulin-like growth factor-binding protein 7
O7505
4
IGSF3_HU
MAN IGSF3 1194
Homo
sapiens Immunoglobulin superfamily member 3
Q969P
0
IGSF8_HU
MAN IGSF8 613
Homo
sapiens Immunoglobulin superfamily member 8
P06213
INSR_HUM
AN INSR 1382
Homo
sapiens Insulin receptor
P17301
ITA2_HUM
AN ITGA2 1181
Homo
sapiens Integrin alpha-2
P08648
ITA5_HUM
AN ITGA5 1049
Homo
sapiens Integrin alpha-5
P23229
ITA6_HUM
AN ITGA6 1130
Homo
sapiens Integrin alpha-6
P06756
ITAV_HUM
AN ITGAV 1048
Homo
sapiens Integrin alpha-V
P05107
ITB2_HUM
AN ITGB2 769
Homo
sapiens Integrin beta-2
P18084
ITB5_HUM
AN ITGB5 799
Homo
sapiens Integrin beta-5
Q9Y28
7
ITM2B_HU
MAN ITM2B 266
Homo
sapiens Integral membrane protein 2B
Q9NQ
X7
ITM2C_HU
MAN ITM2C 267
Homo
sapiens Integral membrane protein 2C
Q8IZA
0
K319L_HU
MAN
KIAA0319
L 1049
Homo
sapiens Dyslexia-associated protein KIAA0319-like protein
P32004
L1CAM_HU
MAN L1CAM 1257
Homo
sapiens Neural cell adhesion molecule L1
Q0839
7
LOXL1_HU
MAN LOXL1 574
Homo
sapiens Lysyl oxidase homolog 1
Q96JB
6
LOXL4_HU
MAN LOXL4 756
Homo
sapiens Lysyl oxidase homolog 4
P06858
LIPL_HUM
AN LPL 475
Homo
sapiens Lipoprotein lipase
O9489
8
LRIG2_HU
MAN LRIG2 1065
Homo
sapiens
Leucine-rich repeats and immunoglobulin-like
domains protein 2
P30533
AMRP_HU
MAN LRPAP1 357
Homo
sapiens Alpha-2-macroglobulin receptor-associated protein
Q9NT9
9
LRC4B_HU
MAN LRRC4B 713
Homo
sapiens Leucine-rich repeat-containing protein 4B
Q9HCJ
2
LRC4C_HU
MAN LRRC4C 640
Homo
sapiens Leucine-rich repeat-containing protein 4C
Q8IWT
6
LRC8A_HU
MAN LRRC8A 810
Homo
sapiens Volume-regulated anion channel subunit LRRC8A
O6047
6
MA1A2_HU
MAN MAN1A2 641
Homo
sapiens
Mannosyl-oligosaccharide 1,2-alpha-mannosidase
IB

216
Q9UK
M7
MA1B1_HU
MAN MAN1B1 699
Homo
sapiens
Endoplasmic reticulum mannosyl-oligosaccharide
1,2-alpha-mannosidase
Q1670
6
MA2A1_HU
MAN MAN2A1 1144
Homo
sapiens Alpha-mannosidase 2
P49641
MA2A2_HU
MAN MAN2A2 1150
Homo
sapiens Alpha-mannosidase 2x
P43121
MUC18_HU
MAN MCAM 646
Homo
sapiens Cell surface glycoprotein MUC18
Q1046
9
MGAT2_HU
MAN MGAT2 447
Homo
sapiens
Alpha-1,6-mannosyl-glycoprotein 2-beta-Nacetylglucosaminyltransferase
Q96PC
5
MIA2_HUM
AN MIA2 1412
Homo
sapiens Melanoma inhibitory activity protein 2
P50281
MMP14_HU
MAN MMP14 582
Homo
sapiens Matrix metalloproteinase-14
P08253
MMP2_HU
MAN MMP2 660
Homo
sapiens 72 kDa type IV collagenase
O9529
7
MPZL1_HU
MAN MPZL1 269
Homo
sapiens Myelin protein zero-like protein 1
Q9UB
G0
MRC2_HU
MAN MRC2 1479
Homo
sapiens C-type mannose receptor 2
Q8NC
W5
NNRE_HU
MAN NAXE 288
Homo
sapiens NAD(P)H-hydrate epimerase
P13591
NCAM1_H
UMAN NCAM1 858
Homo
sapiens Neural cell adhesion molecule 1
P52848
NDST1_HU
MAN NDST1 882
Homo
sapiens
Bifunctional heparan sulfate N-deacetylase/Nsulfotransferase 1
O9485
6
NFASC_HU
MAN NFASC 1347
Homo
sapiens Neurofascin
Q0472
1
NOTC2_HU
MAN NOTCH2 2471
Homo
sapiens Neurogenic locus notch homolog protein 2
O9550
2
NPTXR_HU
MAN NPTXR 500
Homo
sapiens Neuronal pentraxin receptor
O1478
6
NRP1_HUM
AN NRP1 923
Homo
sapiens Neuropilin-1
Q0281
8
NUCB1_HU
MAN NUCB1 461
Homo
sapiens Nucleobindin-1
P80303
NUCB2_HU
MAN NUCB2 420
Homo
sapiens Nucleobindin-2
Q1662
5
OCLN_HU
MAN OCLN 522
Homo
sapiens Occludin
Q9Y5F
2
PCDBB_HU
MAN PCDHB11 797
Homo
sapiens Protocadherin beta-11
Q9Y5E
4
PCDB5_HU
MAN PCDHB5 795
Homo
sapiens Protocadherin beta-5
Q9UN
66
PCDB8_HU
MAN PCDHB8 801
Homo
sapiens Protocadherin beta-8
Q9257
6
PHF3_HUM
AN PHF3 2039
Homo
sapiens PHD finger protein 3
Q8IV0
8
PLD3_HUM
AN PLD3 490
Homo
sapiens 5'-3' exonuclease PLD3
O1503
1
PLXB2_HU
MAN PLXNB2 1838
Homo
sapiens Plexin-B2
Q7Z3K
3
POGZ_HU
MAN POGZ 1410
Homo
sapiens Pogo transposable element with ZNF domain

217
Q8WZ
A1
PMGT1_HU
MAN
POMGNT
1 660
Homo
sapiens
Protein O-linked-mannose beta-1,2-Nacetylglucosaminyltransferase 1
Q9UN
N8
EPCR_HUM
AN PROCR 238
Homo
sapiens Endothelial protein C receptor
Q9P2B
2
FPRP_HUM
AN PTGFRN 879
Homo
sapiens Prostaglandin F2 receptor negative regulator
Q1330
8
PTK7_HUM
AN PTK7 1070
Homo
sapiens Inactive tyrosine-protein kinase 7
P10586
PTPRF_HU
MAN PTPRF 1907
Homo
sapiens Receptor-type tyrosine-protein phosphatase F
P23470
PTPRG_HU
MAN PTPRG 1445
Homo
sapiens Receptor-type tyrosine-protein phosphatase gamma
Q1526
2
PTPRK_HU
MAN PTPRK 1439
Homo
sapiens Receptor-type tyrosine-protein phosphatase kappa
Q1333
2
PTPRS_HU
MAN PTPRS 1948
Homo
sapiens Receptor-type tyrosine-protein phosphatase S
Q6ZRP
7
QSOX2_HU
MAN QSOX2 698
Homo
sapiens Sulfhydryl oxidase 2
P62820
RAB1A_HU
MAN RAB1A 205
Homo
sapiens Ras-related protein Rab-1A
Q96A
A3
RFT1_HUM
AN RFT1 541
Homo
sapiens
Man(5)GlcNAc(2)-PP-dolichol translocation protein
RFT1
P62263
RS14_HUM
AN RPS14 151
Homo
sapiens Small ribosomal subunit protein uS11
Q86SK
9
SCD5_HUM
AN SCD5 330
Homo
sapiens Stearoyl-CoA desaturase 5
Q9BR
K5
CAB45_HU
MAN SDF4 362
Homo
sapiens 45 kDa calcium-binding protein
Q1456
3
SEM3A_HU
MAN SEMA3A 771
Homo
sapiens Semaphorin-3A
Q9998
5
SEM3C_HU
MAN SEMA3C 751
Homo
sapiens Semaphorin-3C
Q6UX
D5
SE6L2_HU
MAN SEZ6L2 910
Homo
sapiens Seizure 6-like protein 2
O4355
6
SGCE_HUM
AN SGCE 437
Homo
sapiens Epsilon-sarcoglycan
Q9Y66
6
S12A7_HU
MAN SLC12A7 1083
Homo
sapiens Solute carrier family 12 member 7
Q6PCB
7
S27A1_HU
MAN SLC27A1 646
Homo
sapiens Long-chain fatty acid transport protein 1
Q9ULF
5
S39AA_HU
MAN
SLC39A1
0 831
Homo
sapiens Zinc transporter ZIP10
Q96E1
6
SMI19_HU
MAN SMIM19 107
Homo
sapiens Small integral membrane protein 19
Q9952
3
SORT_HUM
AN SORT1 831
Homo
sapiens Sortilin
Q1684
2
SIA4B_HU
MAN ST3GAL2 350
Homo
sapiens
CMP-N-acetylneuraminate-beta-galactosamidealpha-2,3-sialyltransferase 2
P50225
ST1A1_HU
MAN SULT1A1 295
Homo
sapiens Sulfotransferase 1A1
Q9NT6
8
TEN2_HUM
AN TENM2 2774
Homo
sapiens Teneurin-2
Q9P27
3
TEN3_HUM
AN TENM3 2699
Homo
sapiens Teneurin-3

218
P02786
TFR1_HUM
AN TFRC 760
Homo
sapiens Transferrin receptor protein 1
O4349
3
TGON2_HU
MAN TGOLN2 437
Homo
sapiens Trans-Golgi network integral membrane protein 2
Q96M
V1
TLCD4_HU
MAN TLCD4 263
Homo
sapiens TLC domain-containing protein 4
Q9980
5
TM9S2_HU
MAN TM9SF2 663
Homo
sapiens Transmembrane 9 superfamily member 2
Q9HD
45
TM9S3_HU
MAN TM9SF3 589
Homo
sapiens Transmembrane 9 superfamily member 3
Q9254
4
TM9S4_HU
MAN TM9SF4 642
Homo
sapiens Transmembrane 9 superfamily member 4
P49755
TMEDA_H
UMAN TMED10 219
Homo
sapiens
Transmembrane emp24 domain-containing protein
10
Q1536
3
TMED2_HU
MAN TMED2 201
Homo
sapiens Transmembrane emp24 domain-containing protein 2
Q7Z7H
5
TMED4_HU
MAN TMED4 227
Homo
sapiens Transmembrane emp24 domain-containing protein 4
Q0288
0
TOP2B_HU
MAN TOP2B 1626
Homo
sapiens DNA topoisomerase 2-beta
O6050
7
TPST1_HU
MAN TPST1 370
Homo
sapiens Protein-tyrosine sulfotransferase 1
O6070
4
TPST2_HU
MAN TPST2 377
Homo
sapiens Protein-tyrosine sulfotransferase 2
Q5T9L
3
WLS_HUM
AN WLS 541
Homo
sapiens Protein wntless homolog
Q9H1B
5
XYLT2_HU
MAN XYLT2 865
Homo
sapiens Xylosyltransferase 2
Table 7. Enriched Proteins for EMD(P183H)-APEX2.
Protei
n ID
Entry
Name
Gene Len
gth
Organis
m
Protein Description
O1467
2
ADA10_H
UMAN
ADAM10 748 Homo
sapiens
Disintegrin and metalloproteinase domaincontaining protein 10
O9491
0
AGRL1_H
UMAN
ADGRL1 147
4
Homo
sapiens
Adhesion G protein-coupled receptor L1
Q1374
0
CD166_HU
MAN
ALCAM 583 Homo
sapiens
CD166 antigen
Q9UK
U9
ANGL2_H
UMAN
ANGPTL
2
493 Homo
sapiens
Angiopoietin-related protein 2
Q9H6
X2
ANTR1_H
UMAN
ANTXR1 564 Homo
sapiens
Anthrax toxin receptor 1
Q0097
3
B4GN1_H
UMAN
B4GALN
T1
533 Homo
sapiens
Beta-1,4 N-acetylgalactosaminyltransferase 1
Q9UB
V7
B4GT7_H
UMAN
B4GALT
7
327 Homo
sapiens
Beta-1,4-galactosyltransferase 7

219
P5089
5
BCAM_HU
MAN
BCAM 628 Homo
sapiens
Basal cell adhesion molecule
Q7KY
R7
BT2A1_H
UMAN
BTN2A1 527 Homo
sapiens
Butyrophilin subfamily 2 member A1
Q9NS
00
C1GLT_H
UMAN
C1GALT
1
363 Homo
sapiens
Glycoprotein-N-acetylgalactosamine 3-betagalactosyltransferase 1
Q96E
U7
C1GLC_H
UMAN
C1GALT
1C1
318 Homo
sapiens
C1GALT1-specific chaperone 1
Q9NZ
P8
C1RL_HU
MAN
C1RL 487 Homo
sapiens
Complement C1r subcomponent-like protein
Q9BY
67
CADM1_H
UMAN
CADM1 442 Homo
sapiens
Cell adhesion molecule 1
O0062
2
CCN1_HU
MAN
CCN1 381 Homo
sapiens
CCN family member 1
P2927
9
CCN2_HU
MAN
CCN2 349 Homo
sapiens
CCN family member 2
Q6YH
K3
CD109_HU
MAN
CD109 144
5
Homo
sapiens
CD109 antigen
P1607
0
CD44_HU
MAN
CD44 742 Homo
sapiens
CD44 antigen
Q0872
2
CD47_HU
MAN
CD47 323 Homo
sapiens
Leukocyte surface antigen CD47
P1902
2
CADH2_H
UMAN
CDH2 906 Homo
sapiens
Cadherin-2
Q9UH
N6
CEIP2_HU
MAN
CEMIP2 138
3
Homo
sapiens
Cell surface hyaluronidase CEMIP2
Q9P2
E5
CHPF2_H
UMAN
CHPF2 772 Homo
sapiens
Chondroitin sulfate glucuronyltransferase
Q8NC
H0
CHSTE_H
UMAN
CHST14 376 Homo
sapiens
Carbohydrate sulfotransferase 14
Q86X
52
CHSS1_H
UMAN
CHSY1 802 Homo
sapiens
Chondroitin sulfate synthase 1
Q9BT
09
CNPY3_H
UMAN
CNPY3 278 Homo
sapiens
Protein canopy homolog 3
P1210
9
CO6A1_H
UMAN
COL6A1 102
8
Homo
sapiens
Collagen alpha-1(VI) chain
Q8IY
K4
GT252_HU
MAN
COLGAL
T2
626 Homo
sapiens
Procollagen galactosyltransferase 2
O7597
6
CBPD_HU
MAN
CPD 138
0
Homo
sapiens
Carboxypeptidase D
Q8N6
G5
CGAT2_H
UMAN
CSGALN
ACT2
542 Homo
sapiens
Chondroitin sulfate Nacetylgalactosaminyltransferase 2
Q6UV
K1
CSPG4_H
UMAN
CSPG4 232
2
Homo
sapiens
Chondroitin sulfate proteoglycan 4
P3522
2
CTNB1_H
UMAN
CTNNB1 781 Homo
sapiens
Catenin beta-1
P7831
0
CXAR_HU
MAN
CXADR 365 Homo
sapiens
Coxsackievirus and adenovirus receptor
Q1411
8
DAG1_HU
MAN
DAG1 895 Homo
sapiens
Dystroglycan 1

220
Q8W
VC6
DCAKD_H
UMAN
DCAKD 231 Homo
sapiens
Dephospho-CoA kinase domain-containing
protein
Q96P
D2
DCBD2_H
UMAN
DCBLD2 775 Homo
sapiens
Discoidin, CUB and LCCL domain-containing
protein 2
Q1412
6
DSG2_HU
MAN
DSG2 1118 Homo
sapiens
Desmoglein-2
P0053
3
EGFR_HU
MAN
EGFR 121
0
Homo
sapiens
Epidermal growth factor receptor
Q1571
7
ELAV1_H
UMAN
ELAVL1 326 Homo
sapiens
ELAV-like protein 1
P0C7
U0
ELFN1_H
UMAN
ELFN1 828 Homo
sapiens
Protein ELFN1
P5040
2
EMD_HU
MAN
EMD 254 Homo
sapiens
Emerin
P2931
7
EPHA2_H
UMAN
EPHA2 976 Homo
sapiens
Ephrin type-A receptor 2
P2932
3
EPHB2_H
UMAN
EPHB2 105
5
Homo
sapiens
Ephrin type-B receptor 2
Q9Y2
82
ERGI3_HU
MAN
ERGIC3 383 Homo
sapiens
Endoplasmic reticulum-Golgi intermediate
compartment protein 3
O7506
3
XYLK_HU
MAN
FAM20B 409 Homo
sapiens
Glycosaminoglycan xylosylkinase
Q9252
0
FAM3C_H
UMAN
FAM3C 227 Homo
sapiens
Protein FAM3C
Q1451
7
FAT1_HU
MAN
FAT1 458
8
Homo
sapiens
Protocadherin Fat 1
Q96A
Y3
FKB10_HU
MAN
FKBP10 582 Homo
sapiens
Peptidyl-prolyl cis-trans isomerase FKBP10
Q9Y6
80
FKBP7_H
UMAN
FKBP7 222 Homo
sapiens
Peptidyl-prolyl cis-trans isomerase FKBP7
O4315
5
FLRT2_HU
MAN
FLRT2 660 Homo
sapiens
Leucine-rich repeat transmembrane protein
FLRT2
P3591
6
VGFR3_H
UMAN
FLT4 136
3
Homo
sapiens
Vascular endothelial growth factor receptor 3
Q9BY
C5
FUT8_HU
MAN
FUT8 575 Homo
sapiens
Alpha-(1,6)-fucosyltransferase
P5111
4
FXR1_HU
MAN
FXR1 621 Homo
sapiens
RNA-binding protein FXR1
Q8N4
28
GLT16_HU
MAN
GALNT1
6
558 Homo
sapiens
Polypeptide N-acetylgalactosaminyltransferase
16
Q6IS2
4
GLT17_HU
MAN
GALNT1
7
598 Homo
sapiens
Polypeptide N-acetylgalactosaminyltransferase
17
Q1047
1
GALT2_H
UMAN
GALNT2 571 Homo
sapiens
Polypeptide N-acetylgalactosaminyltransferase
2
Q86SF
2
GALT7_H
UMAN
GALNT7 657 Homo
sapiens
N-acetylgalactosaminyltransferase 7
P0440
6
G3P_HUM
AN
GAPDH 335 Homo
sapiens
Glyceraldehyde-3-phosphate dehydrogenase
Q9289
6
GSLG1_H
UMAN
GLG1 117
9
Homo
sapiens
Golgi apparatus protein 1

221
Q68C
Q7
GL8D1_H
UMAN
GLT8D1 371 Homo
sapiens
Glycosyltransferase 8 domain-containing
protein 1
O0046
1
GOLI4_HU
MAN
GOLIM4 696 Homo
sapiens
Golgi integral membrane protein 4
Q8NB
J4
GOLM1_H
UMAN
GOLM1 401 Homo
sapiens
Golgi membrane protein 1
Q5V
W38
GP107_HU
MAN
GPR107 600 Homo
sapiens
Protein GPR107
Q96S
L4
GPX7_HU
MAN
GPX7 187 Homo
sapiens
Glutathione peroxidase 7
P1091
5
HPLN1_H
UMAN
HAPLN1 354 Homo
sapiens
Hyaluronan and proteoglycan link protein 1
Q9UL
I3
HEG1_HU
MAN
HEG1 138
1
Homo
sapiens
Protein HEG homolog 1
P0188
9
HLAB_HU
MAN
HLA-B 362 Homo
sapiens
HLA class I histocompatibility antigen, B alpha
chain
P0960
1
HMOX1_H
UMAN
HMOX1 288 Homo
sapiens
Heme oxygenase 1
Q9972
9
ROAA_HU
MAN
HNRNPA
B
332 Homo
sapiens
Heterogeneous nuclear ribonucleoprotein A/B
Q1410
3
HNRPD_H
UMAN
HNRNP
D
355 Homo
sapiens
Heterogeneous nuclear ribonucleoprotein D0
O1497
9
HNRDL_H
UMAN
HNRNP
DL
420 Homo
sapiens
Heterogeneous nuclear ribonucleoprotein Dlike
Q7LG
A3
HS2ST_HU
MAN
HS2ST1 356 Homo
sapiens
Heparan sulfate 2-O-sulfotransferase 1
P4873
5
IDHP_HU
MAN
IDH2 452 Homo
sapiens
Isocitrate dehydrogenase [NADP],
mitochondrial
Q1627
0
IBP7_HUM
AN
IGFBP7 282 Homo
sapiens
Insulin-like growth factor-binding protein 7
O7505
4
IGSF3_HU
MAN
IGSF3 119
4
Homo
sapiens
Immunoglobulin superfamily member 3
Q969P
0
IGSF8_HU
MAN
IGSF8 613 Homo
sapiens
Immunoglobulin superfamily member 8
P1730
1
ITA2_HUM
AN
ITGA2 118
1
Homo
sapiens
Integrin alpha-2
P0864
8
ITA5_HUM
AN
ITGA5 104
9
Homo
sapiens
Integrin alpha-5
P0675
6
ITAV_HU
MAN
ITGAV 104
8
Homo
sapiens
Integrin alpha-V
P0510
7
ITB2_HU
MAN
ITGB2 769 Homo
sapiens
Integrin beta-2
P1808
4
ITB5_HU
MAN
ITGB5 799 Homo
sapiens
Integrin beta-5
Q9Y2
87
ITM2B_H
UMAN
ITM2B 266 Homo
sapiens
Integral membrane protein 2B
Q9NQ
X7
ITM2C_H
UMAN
ITM2C 267 Homo
sapiens
Integral membrane protein 2C
Q8IZ
A0
K319L_HU
MAN
KIAA031
9L
104
9
Homo
sapiens
Dyslexia-associated protein KIAA0319-like
protein

222
P3200
4
L1CAM_H
UMAN
L1CAM 125
7
Homo
sapiens
Neural cell adhesion molecule L1
P2539
1
LAMA1_H
UMAN
LAMA1 307
5
Homo
sapiens
Laminin subunit alpha-1
Q1290
7
LMAN2_H
UMAN
LMAN2 356 Homo
sapiens
Vesicular integral-membrane protein VIP36
Q0839
7
LOXL1_H
UMAN
LOXL1 574 Homo
sapiens
Lysyl oxidase homolog 1
Q96JB
6
LOXL4_H
UMAN
LOXL4 756 Homo
sapiens
Lysyl oxidase homolog 4
O9489
8
LRIG2_HU
MAN
LRIG2 106
5
Homo
sapiens
Leucine-rich repeats and immunoglobulin-like
domains protein 2
P3053
3
AMRP_HU
MAN
LRPAP1 357 Homo
sapiens
Alpha-2-macroglobulin receptor-associated
protein
Q9HC
J2
LRC4C_H
UMAN
LRRC4C 640 Homo
sapiens
Leucine-rich repeat-containing protein 4C
O6047
6
MA1A2_H
UMAN
MAN1A2 641 Homo
sapiens
Mannosyl-oligosaccharide 1,2-alphamannosidase IB
Q9UK
M7
MA1B1_H
UMAN
MAN1B1 699 Homo
sapiens
Endoplasmic reticulum mannosyloligosaccharide 1,2-alpha-mannosidase
Q1670
6
MA2A1_H
UMAN
MAN2A1 114
4
Homo
sapiens
Alpha-mannosidase 2
P4964
1
MA2A2_H
UMAN
MAN2A2 115
0
Homo
sapiens
Alpha-mannosidase 2x
Q1046
9
MGAT2_H
UMAN
MGAT2 447 Homo
sapiens
Alpha-1,6-mannosyl-glycoprotein 2-beta-Nacetylglucosaminyltransferase
P5028
1
MMP14_H
UMAN
MMP14 582 Homo
sapiens
Matrix metalloproteinase-14
P0825
3
MMP2_HU
MAN
MMP2 660 Homo
sapiens
72 kDa type IV collagenase
O9529
7
MPZL1_H
UMAN
MPZL1 269 Homo
sapiens
Myelin protein zero-like protein 1
Q9UB
G0
MRC2_HU
MAN
MRC2 147
9
Homo
sapiens
C-type mannose receptor 2
Q8NC
W5
NNRE_HU
MAN
NAXE 288 Homo
sapiens
NAD(P)H-hydrate epimerase
P1359
1
NCAM1_H
UMAN
NCAM1 858 Homo
sapiens
Neural cell adhesion molecule 1
P5284
8
NDST1_H
UMAN
NDST1 882 Homo
sapiens
Bifunctional heparan sulfate N-deacetylase/Nsulfotransferase 1
O9485
6
NFASC_H
UMAN
NFASC 134
7
Homo
sapiens
Neurofascin
Q0472
1
NOTC2_H
UMAN
NOTCH2 247
1
Homo
sapiens
Neurogenic locus notch homolog protein 2
O9550
2
NPTXR_H
UMAN
NPTXR 500 Homo
sapiens
Neuronal pentraxin receptor
O1478
6
NRP1_HU
MAN
NRP1 923 Homo
sapiens
Neuropilin-1
Q0281
8
NUCB1_H
UMAN
NUCB1 461 Homo
sapiens
Nucleobindin-1

223
P8030
3
NUCB2_H
UMAN
NUCB2 420 Homo
sapiens
Nucleobindin-2
P0418
1
OAT_HUM
AN
OAT 439 Homo
sapiens
Ornithine aminotransferase, mitochondrial
Q1662
5
OCLN_HU
MAN
OCLN 522 Homo
sapiens
Occludin
Q9Y5
F2
PCDBB_H
UMAN
PCDHB1
1
797 Homo
sapiens
Protocadherin beta-11
Q9Y5
E4
PCDB5_H
UMAN
PCDHB5 795 Homo
sapiens
Protocadherin beta-5
O4317
5
SERA_HU
MAN
PHGDH 533 Homo
sapiens
D-3-phosphoglycerate dehydrogenase
O1503
1
PLXB2_H
UMAN
PLXNB2 183
8
Homo
sapiens
Plexin-B2
Q7Z3
K3
POGZ_HU
MAN
POGZ 141
0
Homo
sapiens
Pogo transposable element with ZNF domain
Q8WZ
A1
PMGT1_H
UMAN
POMGN
T1
660 Homo
sapiens
Protein O-linked-mannose beta-1,2-Nacetylglucosaminyltransferase 1
Q1330
8
PTK7_HU
MAN
PTK7 107
0
Homo
sapiens
Inactive tyrosine-protein kinase 7
P1058
6
PTPRF_HU
MAN
PTPRF 190
7
Homo
sapiens
Receptor-type tyrosine-protein phosphatase F
P2347
0
PTPRG_H
UMAN
PTPRG 144
5
Homo
sapiens
Receptor-type tyrosine-protein phosphatase
gamma
Q1526
2
PTPRK_H
UMAN
PTPRK 143
9
Homo
sapiens
Receptor-type tyrosine-protein phosphatase
kappa
Q1333
2
PTPRS_HU
MAN
PTPRS 194
8
Homo
sapiens
Receptor-type tyrosine-protein phosphatase S
Q6ZR
P7
QSOX2_H
UMAN
QSOX2 698 Homo
sapiens
Sulfhydryl oxidase 2
Q96A
A3
RFT1_HU
MAN
RFT1 541 Homo
sapiens
Man(5)GlcNAc(2)-PP-dolichol translocation
protein RFT1
P6226
3
RS14_HU
MAN
RPS14 151 Homo
sapiens
Small ribosomal subunit protein uS11
Q86S
K9
SCD5_HU
MAN
SCD5 330 Homo
sapiens
Stearoyl-CoA desaturase 5
Q9BR
K5
CAB45_H
UMAN
SDF4 362 Homo
sapiens
45 kDa calcium-binding protein
Q1456
3
SEM3A_H
UMAN
SEMA3A 771 Homo
sapiens
Semaphorin-3A
Q9998
5
SEM3C_H
UMAN
SEMA3C 751 Homo
sapiens
Semaphorin-3C
Q6UX
D5
SE6L2_HU
MAN
SEZ6L2 910 Homo
sapiens
Seizure 6-like protein 2
Q9Y6
66
S12A7_HU
MAN
SLC12A7 108
3
Homo
sapiens
Solute carrier family 12 member 7
Q6PC
B7
S27A1_HU
MAN
SLC27A1 646 Homo
sapiens
Long-chain fatty acid transport protein 1
Q9UL
F5
S39AA_H
UMAN
SLC39A1
0
831 Homo
sapiens
Zinc transporter ZIP10

224
Q96E1
6
SMI19_HU
MAN
SMIM19 107 Homo
sapiens
Small integral membrane protein 19
Q9952
3
SORT_HU
MAN
SORT1 831 Homo
sapiens
Sortilin
Q1684
2
SIA4B_HU
MAN
ST3GAL
2
350 Homo
sapiens
CMP-N-acetylneuraminate-beta-galactosamidealpha-2,3-sialyltransferase 2
P5022
5
ST1A1_HU
MAN
SULT1A
1
295 Homo
sapiens
Sulfotransferase 1A1
Q9P27
3
TEN3_HU
MAN
TENM3 269
9
Homo
sapiens
Teneurin-3
P0278
6
TFR1_HU
MAN
TFRC 760 Homo
sapiens
Transferrin receptor protein 1
O4349
3
TGON2_H
UMAN
TGOLN2 437 Homo
sapiens
Trans-Golgi network integral membrane
protein 2
Q96M
V1
TLCD4_H
UMAN
TLCD4 263 Homo
sapiens
TLC domain-containing protein 4
Q9980
5
TM9S2_H
UMAN
TM9SF2 663 Homo
sapiens
Transmembrane 9 superfamily member 2
Q9HD
45
TM9S3_H
UMAN
TM9SF3 589 Homo
sapiens
Transmembrane 9 superfamily member 3
Q9254
4
TM9S4_H
UMAN
TM9SF4 642 Homo
sapiens
Transmembrane 9 superfamily member 4
P4975
5
TMEDA_H
UMAN
TMED10 219 Homo
sapiens
Transmembrane emp24 domain-containing
protein 10
Q1536
3
TMED2_H
UMAN
TMED2 201 Homo
sapiens
Transmembrane emp24 domain-containing
protein 2
Q7Z7
H5
TMED4_H
UMAN
TMED4 227 Homo
sapiens
Transmembrane emp24 domain-containing
protein 4
O6050
7
TPST1_HU
MAN
TPST1 370 Homo
sapiens
Protein-tyrosine sulfotransferase 1
O6070
4
TPST2_HU
MAN
TPST2 377 Homo
sapiens
Protein-tyrosine sulfotransferase 2
Q8NB
Z7
UXS1_HU
MAN
UXS1 420 Homo
sapiens
UDP-glucuronic acid decarboxylase 1
Q5T9
L3
WLS_HU
MAN
WLS 541 Homo
sapiens
Protein wntless homolog
Q8NB
I6
XXLT1_H
UMAN
XXYLT1 393 Homo
sapiens
Xyloside xylosyltransferase 1
Q9H1
B5
XYLT2_H
UMAN
XYLT2 865 Homo
sapiens
Xylosyltransferase 2
Q9BS
R8
YIPF4_HU
MAN
YIPF4 244 Homo
sapiens
Protein YIPF4
Table 8. Enriched Proteins for EMD(Q133H)-APEX2.
Protei
n ID
Entry
Name
Gene Len
gth
Organis
m
Protein Description

225
O1467
2
ADA10_H
UMAN
ADAM10 748 Homo
sapiens
Disintegrin and metalloproteinase domaincontaining protein 10
Q1374
0
CD166_HU
MAN
ALCAM 583 Homo
sapiens
CD166 antigen
Q9UK
U9
ANGL2_H
UMAN
ANGPTL
2
493 Homo
sapiens
Angiopoietin-related protein 2
Q9H6
X2
ANTR1_H
UMAN
ANTXR1 564 Homo
sapiens
Anthrax toxin receptor 1
P0506
7
A4_HUMA
N
APP 770 Homo
sapiens
Amyloid-beta precursor protein
Q0097
3
B4GN1_H
UMAN
B4GALN
T1
533 Homo
sapiens
Beta-1,4 N-acetylgalactosaminyltransferase 1
P1529
1
B4GT1_H
UMAN
B4GALT
1
398 Homo
sapiens
Beta-1,4-galactosyltransferase 1
O6051
2
B4GT3_H
UMAN
B4GALT
3
393 Homo
sapiens
Beta-1,4-galactosyltransferase 3
Q9UB
V7
B4GT7_H
UMAN
B4GALT
7
327 Homo
sapiens
Beta-1,4-galactosyltransferase 7
P5089
5
BCAM_HU
MAN
BCAM 628 Homo
sapiens
Basal cell adhesion molecule
Q9NX
62
IMPA3_HU
MAN
BPNT2 359 Homo
sapiens
Golgi-resident adenosine 3',5'-bisphosphate 3'-
phosphatase
Q9NS
00
C1GLT_H
UMAN
C1GALT
1
363 Homo
sapiens
Glycoprotein-N-acetylgalactosamine 3-betagalactosyltransferase 1
Q96E
U7
C1GLC_H
UMAN
C1GALT
1C1
318 Homo
sapiens
C1GALT1-specific chaperone 1
Q9BY
67
CADM1_H
UMAN
CADM1 442 Homo
sapiens
Cell adhesion molecule 1
O0062
2
CCN1_HU
MAN
CCN1 381 Homo
sapiens
CCN family member 1
P2927
9
CCN2_HU
MAN
CCN2 349 Homo
sapiens
CCN family member 2
P1607
0
CD44_HU
MAN
CD44 742 Homo
sapiens
CD44 antigen
P1902
2
CADH2_H
UMAN
CDH2 906 Homo
sapiens
Cadherin-2
Q9UH
N6
CEIP2_HU
MAN
CEMIP2 138
3
Homo
sapiens
Cell surface hyaluronidase CEMIP2
Q8IZ5
2
CHSS2_H
UMAN
CHPF 775 Homo
sapiens
Chondroitin sulfate synthase 2
Q9P2
E5
CHPF2_H
UMAN
CHPF2 772 Homo
sapiens
Chondroitin sulfate glucuronyltransferase
Q8NC
H0
CHSTE_H
UMAN
CHST14 376 Homo
sapiens
Carbohydrate sulfotransferase 14
Q86X
52
CHSS1_H
UMAN
CHSY1 802 Homo
sapiens
Chondroitin sulfate synthase 1
O1496
7
CLGN_HU
MAN
CLGN 610 Homo
sapiens
Calmegin
Q9BT
09
CNPY3_H
UMAN
CNPY3 278 Homo
sapiens
Protein canopy homolog 3

226
P1210
7
COBA1_H
UMAN
COL11A
1
180
6
Homo
sapiens
Collagen alpha-1(XI) chain
P0245
2
CO1A1_H
UMAN
COL1A1 146
4
Homo
sapiens
Collagen alpha-1(I) chain
Q8IY
K4
GT252_HU
MAN
COLGAL
T2
626 Homo
sapiens
Procollagen galactosyltransferase 2
O7597
6
CBPD_HU
MAN
CPD 138
0
Homo
sapiens
Carboxypeptidase D
Q8N6
G5
CGAT2_H
UMAN
CSGALN
ACT2
542 Homo
sapiens
Chondroitin sulfate Nacetylgalactosaminyltransferase 2
P3522
2
CTNB1_H
UMAN
CTNNB1 781 Homo
sapiens
Catenin beta-1
P7831
0
CXAR_HU
MAN
CXADR 365 Homo
sapiens
Coxsackievirus and adenovirus receptor
O4316
9
CYB5B_H
UMAN
CYB5B 150 Homo
sapiens
Cytochrome b5 type B
Q1411
8
DAG1_HU
MAN
DAG1 895 Homo
sapiens
Dystroglycan 1
Q8W
VC6
DCAKD_H
UMAN
DCAKD 231 Homo
sapiens
Dephospho-CoA kinase domain-containing
protein
Q96P
D2
DCBD2_H
UMAN
DCBLD2 775 Homo
sapiens
Discoidin, CUB and LCCL domain-containing
protein 2
Q1412
6
DSG2_HU
MAN
DSG2 1118 Homo
sapiens
Desmoglein-2
P0053
3
EGFR_HU
MAN
EGFR 121
0
Homo
sapiens
Epidermal growth factor receptor
Q1571
7
ELAV1_H
UMAN
ELAVL1 326 Homo
sapiens
ELAV-like protein 1
P0C7
U0
ELFN1_H
UMAN
ELFN1 828 Homo
sapiens
Protein ELFN1
P5040
2
EMD_HU
MAN
EMD 254 Homo
sapiens
Emerin
P2931
7
EPHA2_H
UMAN
EPHA2 976 Homo
sapiens
Ephrin type-A receptor 2
P2932
3
EPHB2_H
UMAN
EPHB2 105
5
Homo
sapiens
Ephrin type-B receptor 2
Q96R
Q1
ERGI2_HU
MAN
ERGIC2 377 Homo
sapiens
Endoplasmic reticulum-Golgi intermediate
compartment protein 2
Q9Y2
82
ERGI3_HU
MAN
ERGIC3 383 Homo
sapiens
Endoplasmic reticulum-Golgi intermediate
compartment protein 3
P3004
0
ERP29_HU
MAN
ERP29 261 Homo
sapiens
Endoplasmic reticulum resident protein 29
O7506
3
XYLK_HU
MAN
FAM20B 409 Homo
sapiens
Glycosaminoglycan xylosylkinase
Q9252
0
FAM3C_H
UMAN
FAM3C 227 Homo
sapiens
Protein FAM3C
Q1451
7
FAT1_HU
MAN
FAT1 458
8
Homo
sapiens
Protocadherin Fat 1
Q96A
Y3
FKB10_HU
MAN
FKBP10 582 Homo
sapiens
Peptidyl-prolyl cis-trans isomerase FKBP10

227
Q9Y6
80
FKBP7_H
UMAN
FKBP7 222 Homo
sapiens
Peptidyl-prolyl cis-trans isomerase FKBP7
Q9BY
C5
FUT8_HU
MAN
FUT8 575 Homo
sapiens
Alpha-(1,6)-fucosyltransferase
Q8N4
28
GLT16_HU
MAN
GALNT1
6
558 Homo
sapiens
Polypeptide N-acetylgalactosaminyltransferase
16
Q6IS2
4
GLT17_HU
MAN
GALNT1
7
598 Homo
sapiens
Polypeptide N-acetylgalactosaminyltransferase
17
Q1047
1
GALT2_H
UMAN
GALNT2 571 Homo
sapiens
Polypeptide N-acetylgalactosaminyltransferase
2
Q86SF
2
GALT7_H
UMAN
GALNT7 657 Homo
sapiens
N-acetylgalactosaminyltransferase 7
P0440
6
G3P_HUM
AN
GAPDH 335 Homo
sapiens
Glyceraldehyde-3-phosphate dehydrogenase
Q9289
6
GSLG1_H
UMAN
GLG1 117
9
Homo
sapiens
Golgi apparatus protein 1
Q68C
Q7
GL8D1_H
UMAN
GLT8D1 371 Homo
sapiens
Glycosyltransferase 8 domain-containing
protein 1
O0046
1
GOLI4_HU
MAN
GOLIM4 696 Homo
sapiens
Golgi integral membrane protein 4
Q8NB
J4
GOLM1_H
UMAN
GOLM1 401 Homo
sapiens
Golgi membrane protein 1
Q5V
W38
GP107_HU
MAN
GPR107 600 Homo
sapiens
Protein GPR107
Q96S
L4
GPX7_HU
MAN
GPX7 187 Homo
sapiens
Glutathione peroxidase 7
Q4G1
48
GXLT1_H
UMAN
GXYLT1 440 Homo
sapiens
Glucoside xylosyltransferase 1
P1091
5
HPLN1_H
UMAN
HAPLN1 354 Homo
sapiens
Hyaluronan and proteoglycan link protein 1
P0960
1
HMOX1_H
UMAN
HMOX1 288 Homo
sapiens
Heme oxygenase 1
Q7LG
A3
HS2ST_HU
MAN
HS2ST1 356 Homo
sapiens
Heparan sulfate 2-O-sulfotransferase 1
Q9BU
P3
HTAI2_HU
MAN
HTATIP2 242 Homo
sapiens
Oxidoreductase HTATIP2
P4873
5
IDHP_HU
MAN
IDH2 452 Homo
sapiens
Isocitrate dehydrogenase [NADP],
mitochondrial
Q1627
0
IBP7_HUM
AN
IGFBP7 282 Homo
sapiens
Insulin-like growth factor-binding protein 7
O7505
4
IGSF3_HU
MAN
IGSF3 119
4
Homo
sapiens
Immunoglobulin superfamily member 3
Q969P
0
IGSF8_HU
MAN
IGSF8 613 Homo
sapiens
Immunoglobulin superfamily member 8
P1730
1
ITA2_HUM
AN
ITGA2 118
1
Homo
sapiens
Integrin alpha-2
P0864
8
ITA5_HUM
AN
ITGA5 104
9
Homo
sapiens
Integrin alpha-5
P0555
6
ITB1_HU
MAN
ITGB1 798 Homo
sapiens
Integrin beta-1

228
P0510
7
ITB2_HU
MAN
ITGB2 769 Homo
sapiens
Integrin beta-2
P1808
4
ITB5_HU
MAN
ITGB5 799 Homo
sapiens
Integrin beta-5
Q9Y2
87
ITM2B_H
UMAN
ITM2B 266 Homo
sapiens
Integral membrane protein 2B
Q96M
P8
KCTD7_H
UMAN
KCTD7 289 Homo
sapiens
BTB/POZ domain-containing protein KCTD7
Q8IZ
A0
K319L_HU
MAN
KIAA031
9L
104
9
Homo
sapiens
Dyslexia-associated protein KIAA0319-like
protein
Q8NC
56
LEMD2_H
UMAN
LEMD2 503 Homo
sapiens
LEM domain-containing protein 2
Q8NE
S3
LFNG_HU
MAN
LFNG 379 Homo
sapiens
Beta-1,3-N-acetylglucosaminyltransferase
lunatic fringe
Q1290
7
LMAN2_H
UMAN
LMAN2 356 Homo
sapiens
Vesicular integral-membrane protein VIP36
Q96JB
6
LOXL4_H
UMAN
LOXL4 756 Homo
sapiens
Lysyl oxidase homolog 4
O9489
8
LRIG2_HU
MAN
LRIG2 106
5
Homo
sapiens
Leucine-rich repeats and immunoglobulin-like
domains protein 2
P3053
3
AMRP_HU
MAN
LRPAP1 357 Homo
sapiens
Alpha-2-macroglobulin receptor-associated
protein
O6047
6
MA1A2_H
UMAN
MAN1A2 641 Homo
sapiens
Mannosyl-oligosaccharide 1,2-alphamannosidase IB
Q9UK
M7
MA1B1_H
UMAN
MAN1B1 699 Homo
sapiens
Endoplasmic reticulum mannosyloligosaccharide 1,2-alpha-mannosidase
Q1670
6
MA2A1_H
UMAN
MAN2A1 114
4
Homo
sapiens
Alpha-mannosidase 2
P4964
1
MA2A2_H
UMAN
MAN2A2 115
0
Homo
sapiens
Alpha-mannosidase 2x
Q1046
9
MGAT2_H
UMAN
MGAT2 447 Homo
sapiens
Alpha-1,6-mannosyl-glycoprotein 2-beta-Nacetylglucosaminyltransferase
P5028
1
MMP14_H
UMAN
MMP14 582 Homo
sapiens
Matrix metalloproteinase-14
P0825
3
MMP2_HU
MAN
MMP2 660 Homo
sapiens
72 kDa type IV collagenase
Q53F3
9
MPPE1_H
UMAN
MPPE1 396 Homo
sapiens
Metallophosphoesterase 1
O9529
7
MPZL1_H
UMAN
MPZL1 269 Homo
sapiens
Myelin protein zero-like protein 1
Q9UB
G0
MRC2_HU
MAN
MRC2 147
9
Homo
sapiens
C-type mannose receptor 2
Q8NC
W5
NNRE_HU
MAN
NAXE 288 Homo
sapiens
NAD(P)H-hydrate epimerase
P5284
8
NDST1_H
UMAN
NDST1 882 Homo
sapiens
Bifunctional heparan sulfate N-deacetylase/Nsulfotransferase 1
Q0281
8
NUCB1_H
UMAN
NUCB1 461 Homo
sapiens
Nucleobindin-1
P8030
3
NUCB2_H
UMAN
NUCB2 420 Homo
sapiens
Nucleobindin-2

229
P0418
1
OAT_HUM
AN
OAT 439 Homo
sapiens
Ornithine aminotransferase, mitochondrial
Q1662
5
OCLN_HU
MAN
OCLN 522 Homo
sapiens
Occludin
Q9Y5
E4
PCDB5_H
UMAN
PCDHB5 795 Homo
sapiens
Protocadherin beta-5
Q8NB
M8
PCYXL_H
UMAN
PCYOX1
L
494 Homo
sapiens
Prenylcysteine oxidase-like
O1503
1
PLXB2_H
UMAN
PLXNB2 183
8
Homo
sapiens
Plexin-B2
Q7Z4
H8
PLGT3_H
UMAN
POGLUT
3
507 Homo
sapiens
Protein O-glucosyltransferase 3
Q8WZ
A1
PMGT1_H
UMAN
POMGN
T1
660 Homo
sapiens
Protein O-linked-mannose beta-1,2-Nacetylglucosaminyltransferase 1
Q9H5
K3
SG196_HU
MAN
POMK 350 Homo
sapiens
Protein O-mannose kinase
Q1516
5
PON2_HU
MAN
PON2 354 Homo
sapiens
Serum paraoxonase/arylesterase 2
Q1330
8
PTK7_HU
MAN
PTK7 107
0
Homo
sapiens
Inactive tyrosine-protein kinase 7
Q1526
2
PTPRK_H
UMAN
PTPRK 143
9
Homo
sapiens
Receptor-type tyrosine-protein phosphatase
kappa
Q6ZR
P7
QSOX2_H
UMAN
QSOX2 698 Homo
sapiens
Sulfhydryl oxidase 2
Q96A
A3
RFT1_HU
MAN
RFT1 541 Homo
sapiens
Man(5)GlcNAc(2)-PP-dolichol translocation
protein RFT1
Q9BR
K5
CAB45_H
UMAN
SDF4 362 Homo
sapiens
45 kDa calcium-binding protein
Q1456
3
SEM3A_H
UMAN
SEMA3A 771 Homo
sapiens
Semaphorin-3A
Q9998
5
SEM3C_H
UMAN
SEMA3C 751 Homo
sapiens
Semaphorin-3C
Q6UX
D5
SE6L2_HU
MAN
SEZ6L2 910 Homo
sapiens
Seizure 6-like protein 2
Q9Y6
66
S12A7_HU
MAN
SLC12A7 108
3
Homo
sapiens
Solute carrier family 12 member 7
Q9UL
F5
S39AA_H
UMAN
SLC39A1
0
831 Homo
sapiens
Zinc transporter ZIP10
Q96E1
6
SMI19_HU
MAN
SMIM19 107 Homo
sapiens
Small integral membrane protein 19
Q1684
2
SIA4B_HU
MAN
ST3GAL
2
350 Homo
sapiens
CMP-N-acetylneuraminate-beta-galactosamidealpha-2,3-sialyltransferase 2
P5022
5
ST1A1_HU
MAN
SULT1A
1
295 Homo
sapiens
Sulfotransferase 1A1
P0278
6
TFR1_HU
MAN
TFRC 760 Homo
sapiens
Transferrin receptor protein 1
P2198
0
TGM2_HU
MAN
TGM2 687 Homo
sapiens
Protein-glutamine gamma-glutamyltransferase
2
O4349
3
TGON2_H
UMAN
TGOLN2 437 Homo
sapiens
Trans-Golgi network integral membrane
protein 2

230
P0799
6
TSP1_HU
MAN
THBS1 117
0
Homo
sapiens
Thrombospondin-1
Q96M
V1
TLCD4_H
UMAN
TLCD4 263 Homo
sapiens
TLC domain-containing protein 4
Q9980
5
TM9S2_H
UMAN
TM9SF2 663 Homo
sapiens
Transmembrane 9 superfamily member 2
Q9HD
45
TM9S3_H
UMAN
TM9SF3 589 Homo
sapiens
Transmembrane 9 superfamily member 3
Q9254
4
TM9S4_H
UMAN
TM9SF4 642 Homo
sapiens
Transmembrane 9 superfamily member 4
Q1344
5
TMED1_H
UMAN
TMED1 227 Homo
sapiens
Transmembrane emp24 domain-containing
protein 1
P4975
5
TMEDA_H
UMAN
TMED10 219 Homo
sapiens
Transmembrane emp24 domain-containing
protein 10
Q1536
3
TMED2_H
UMAN
TMED2 201 Homo
sapiens
Transmembrane emp24 domain-containing
protein 2
Q7Z7
H5
TMED4_H
UMAN
TMED4 227 Homo
sapiens
Transmembrane emp24 domain-containing
protein 4
Q9Y3
B3
TMED7_H
UMAN
TMED7 224 Homo
sapiens
Transmembrane emp24 domain-containing
protein 7
Q9BV
K6
TMED9_H
UMAN
TMED9 235 Homo
sapiens
Transmembrane emp24 domain-containing
protein 9
O6050
7
TPST1_HU
MAN
TPST1 370 Homo
sapiens
Protein-tyrosine sulfotransferase 1
O6070
4
TPST2_HU
MAN
TPST2 377 Homo
sapiens
Protein-tyrosine sulfotransferase 2
Q5T9
L3
WLS_HU
MAN
WLS 541 Homo
sapiens
Protein wntless homolog
Q9H1
B5
XYLT2_H
UMAN
XYLT2 865 Homo
sapiens
Xylosyltransferase 2
Q9BS
R8
YIPF4_HU
MAN
YIPF4 244 Homo
sapiens
Protein YIPF4
Table 9. Enriched Proteins for EMD(Δ95-99)-APEX2.
Protein
ID
Entry Name Gene Leng
th
Organism Protein Description
Q13740 CD166_HUM
AN
ALCA
M
583 Homo
sapiens
CD166 antigen
P50895 BCAM_HUM
AN
BCAM 628 Homo
sapiens
Basal cell adhesion molecule
Q9BY67 CADM1_HU
MAN
CADM
1
442 Homo
sapiens
Cell adhesion molecule 1
P16070 CD44_HUMA
N
CD44 742 Homo
sapiens
CD44 antigen
P35222 CTNB1_HUM
AN
CTNN
B1
781 Homo
sapiens
Catenin beta-1

231
P78310 CXAR_HUM
AN
CXAD
R
365 Homo
sapiens
Coxsackievirus and adenovirus receptor
Q14118 DAG1_HUMA
N
DAG1 895 Homo
sapiens
Dystroglycan 1
Q14126 DSG2_HUMA
N
DSG2 1118 Homo
sapiens
Desmoglein-2
P0C7U0 ELFN1_HUM
AN
ELFN1 828 Homo
sapiens
Protein ELFN1
P29317 EPHA2_HUM
AN
EPHA2 976 Homo
sapiens
Ephrin type-A receptor 2
P09601 HMOX1_HU
MAN
HMOX
1
288 Homo
sapiens
Heme oxygenase 1
Q16270 IBP7_HUMA
N
IGFBP
7
282 Homo
sapiens
Insulin-like growth factor-binding
protein 7
O75054 IGSF3_HUMA
N
IGSF3 1194 Homo
sapiens
Immunoglobulin superfamily member 3
Q969P0 IGSF8_HUMA
N
IGSF8 613 Homo
sapiens
Immunoglobulin superfamily member 8
P17301 ITA2_HUMA
N
ITGA2 1181 Homo
sapiens
Integrin alpha-2
P06756 ITAV_HUMA
N
ITGAV 1048 Homo
sapiens
Integrin alpha-V
P50281 MMP14_HUM
AN
MMP1
4
582 Homo
sapiens
Matrix metalloproteinase-14
O95297 MPZL1_HUM
AN
MPZL
1
269 Homo
sapiens
Myelin protein zero-like protein 1
Q16625 OCLN_HUMA
N
OCLN 522 Homo
sapiens
Occludin
Q13308 PTK7_HUMA
N
PTK7 1070 Homo
sapiens
Inactive tyrosine-protein kinase 7
P23470 PTPRG_HUM
AN
PTPRG 1445 Homo
sapiens
Receptor-type tyrosine-protein
phosphatase gamma
Q15262 PTPRK_HUM
AN
PTPRK 1439 Homo
sapiens
Receptor-type tyrosine-protein
phosphatase kappa
Table 10. Enriched Proteins for EMD-splitAPEX2.
Protei
n ID
Entry
Name
Gene Len
gth
Organis
m
Protein Description
Q5BK
T4
AG10A_HU
MAN
ALG1
0
473 Homo
sapiens
Dol-P-Glc:Glc(2)Man(9)GlcNAc(2)-PP-Dol
alpha-1,2-glucosyltransferase
Q9NZ
P8
C1RL_HU
MAN
C1RL 487 Homo
sapiens
Complement C1r subcomponent-like protein
Q8WV
C6
DCAKD_H
UMAN
DCA
KD
231 Homo
sapiens
Dephospho-CoA kinase domain-containing
protein
Q1418
5
DOCK1_H
UMAN
DOC
K1
1865 Homo
sapiens
Dedicator of cytokinesis protein 1

232
P5040
2
EMD_HUM
AN
EMD 254 Homo
sapiens
Emerin
Q9BY
K8
HELZ2_HU
MAN
HELZ
2
2896 Homo
sapiens
3'-5' exoribonuclease HELZ2
P0188
9
HLAB_HU
MAN
HLAB
362 Homo
sapiens
HLA class I histocompatibility antigen, B alpha
chain
P0DM
V8
HS71A_HU
MAN
HSPA
1A
641 Homo
sapiens
Heat shock 70 kDa protein 1A
P4873
5
IDHP_HUM
AN
IDH2 452 Homo
sapiens
Isocitrate dehydrogenase [NADP], mitochondrial
Q1627
0
IBP7_HUM
AN
IGFB
P7
282 Homo
sapiens
Insulin-like growth factor-binding protein 7
P3590
8
K22E_HUM
AN
KRT2 639 Homo
sapiens
Keratin, type II cytoskeletal 2 epidermal
Q53F3
9
MPPE1_HU
MAN
MPPE
1
396 Homo
sapiens
Metallophosphoesterase 1
Q8NC
W5
NNRE_HU
MAN
NAX
E
288 Homo
sapiens
NAD(P)H-hydrate epimerase
O1452
4
NEMP1_H
UMAN
NEM
P1
444 Homo
sapiens
Nuclear envelope integral membrane protein 1
P4868
1
NEST_HU
MAN
NES 1621 Homo
sapiens
Nestin
Q0281
8
NUCB1_H
UMAN
NUC
B1
461 Homo
sapiens
Nucleobindin-1
P8030
3
NUCB2_H
UMAN
NUC
B2
420 Homo
sapiens
Nucleobindin-2
Q8IY1
7
PLPL6_HU
MAN
PNPL
A6
1375 Homo
sapiens
Patatin-like phospholipase domain-containing
protein 6
Q1516
5
PON2_HU
MAN
PON2 354 Homo
sapiens
Serum paraoxonase/arylesterase 2
Q6PC
B7
S27A1_HU
MAN
SLC2
7A1
646 Homo
sapiens
Long-chain fatty acid transport protein 1
Q0351
9
TAP2_HUM
AN
TAP2 686 Homo
sapiens
Antigen peptide transporter 2
Q96M
V1
TLCD4_HU
MAN
TLCD
4
263 Homo
sapiens
TLC domain-containing protein 4
P4975
5
TMEDA_H
UMAN
TME
D10
219 Homo
sapiens
Transmembrane emp24 domain-containing
protein 10
Q7Z7
H5
TMED4_H
UMAN
TME
D4
227 Homo
sapiens
Transmembrane emp24 domain-containing
protein 4
P4216
6
LAP2A_HU
MAN
TMP
O
694 Homo
sapiens
Lamina-associated polypeptide 2, isoform alpha
Q8IW
R1
TRI59_HU
MAN
TRIM
59
403 Homo
sapiens
Tripartite motif-containing protein 59
P0867
0
VIME_HU
MAN
VIM 466 Homo
sapiens
Vimentin

Abstract (if available)

Abstract Emerin is an integral membrane protein of the inner nuclear membrane. Mutations in the EMD gene can cause Emery-Dreifuss Muscular Dystrophy (EDMD), a disorder characterized by progressive skeletal muscle wasting, irregular heart rhythms and contractures of major tendons. Despite extensive research aimed at identifying the binding partners of emerin and understanding its structural organization at the nuclear envelope, how the interacting partners of emerin influence the nanoscale organization of emerin remains poorly defined.
First, we aimed to develop a simple platform based on cell micropatterning to study the role of emerin during nuclear mechanotransduction. By optimizing the microcontact printing of the extracellular matrix protein fibronectin on HMDS-modifed glass coverslips and micropatterning cells, we were able to imposing specific physical strains at the nuclear envelope to reveal some of the mechanotransducing functions of emerin in response to mechanical cues. By utilizing two-dimensional cell micropatterning on fibronectin substrates of varying widths, it is possible to efficiently impose incremental steady-state mechanical strains at the cell nuclear envelope and to reveal some of the mechanotransducing responses of emerin, as well as the effect of the nucleoskeletal proteins lamin A/C and actin.
Next, we aimed to define the nanoscale proteomic landscape of wild-type as well as EDMD-relevant mutants by proximity-dependent biotinylation using the engineered ascorbate peroxidase APEX2 combined with mass spectrometry-based proteomics. When activated by H2O2, APEX2 catalyzes the conversion of its substrate, biotin-phenol, into short-lived and highly reactive radicals that can form covalent bonds with electron-rich amino acids, including tyrosine, present in nearby endogenous proteins. By using APEX2 fusion to wild-type emerin and EDMD-causing mutants, we hypothesized that the emerin mutations result in altered interactions that could provide insight into the mechanism of EDMD. Moreover, inspired by prior research employing splitGFP to illustrate the self-association of emerin, we have adapted the splitAPEX2 platform to identify proteins that interact specifically with emerin oligomers.
Finally, recent reports of the capability of APEX2 to biotinylate nucleic acids has led us to explore its potential to map the genomic regions closely associating with emerin. By simply switching to another substrate, biotin-aniline, that has a higher biotinylation specifity towards nucleic acid, we have optimized protocols to confirm APEX2 biotinylation of the genomic DNA by fluorescence imaging and quantitation of biotin levels of extracted genomic DNA.

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Asset Metadata

Creator Bautista, Markville Bulosan (author)

Core Title Investigations on the muscular dystrophy protein emerin: from nuclear mechanotransduction to molecular interactions

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Degree Conferral Date 2024-12

Publication Date 01/13/2025

Defense Date 08/28/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag ascorbate peroxidase,cell micropatterning,emerin,muscular dystrophy,nuclear mechanotransduction,OAI-PMH Harvest,proximity labeling

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Pinaud, Fabien (committee chair), Chen, Lin (committee member), Qin, Peter (committee member)

Creator Email markvillebautista@gmail.com,mbbautis@usc.edu

Unique identifier UC11399FAEK

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Legacy Identifier etd-BautistaMa-13747

Document Type Dissertation

Format theses (aat)

Rights Bautista, Markville Bulosan

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Source 20250113-usctheses-batch-1234 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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