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Engineering artificial cells to elucidate GPCR 5-HT1AR activity and plasma membrane compositional dependence

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Engineering artificial cells to elucidate GPCR 5-HT1AR activity and plasma membrane compositional dependence

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Engineering artificial cells to
elucidate GPCR 5-HT
1A
R
activity and plasma membrane
compositional dependence

Mary Gertrude L. Gutierrez

Doctor of Philosophy, Materials Science
in
The Faculty of the USC Graduate School
Mork Family Department of Chemical Engineering and Materials
Science

THE UNIVERSITY OF SOUTHERN CALIFORNIA

Principle Investigator: Prof. Noah Malmstadt, PhD

August 2016

1

Committee Members

Noah Malmstadt
Richard W. Roberts
Ralf Langen

2

Acknowledgements

Thank you to everyone who has supported and mentored me throughout my life and
especially through my three years of doctoral studies. When I first thought about pursuing a PhD
I remember naively saying that I would do it in three years. I’m not sure if at the time I was being
facetious, egotistic, or completely serious but this certainly was not an achievement that I could
have completed on my own. There are many who have inspired me and guided me throughout it
all, and I am eternally humbled and grateful. To my mom and dad, thank you for always believing
in me and supporting my sometimes naïve and idealistic ambitions. Thank you to my family,
especially my sisters and my nieces and nephews, for always inspiring me to want to be better in
all aspects of life. Thank you to Greg, for being there through the challenges of graduate school
life and providing me with all the love and support that made even the most frustrating days that
much better. To my USC family, thank you for all of the advice, guidance, and memories that we
shared together. And lastly to my advisor, Noah Malmstadt, thank you for all of your wisdom and
guidance. Thank you for your never ending support and providing me with opportunities that have
allowed me to grow in to an independent scientist. I hope that I can be as well versed an expert as
you are as I continue as a PhD scientist.

3

1. Preface
In unraveling the highly integrated network of cellular processes at the cell plasma
membrane, scientists have traditionally taken two approaches. The first is a top down approach
where cultured cell lines are probed and engineered to amplify certain functions and components
of interest. The second is a bottom up approach that allows the selection of specific materials for
mimicking certain features of the cell and can focus on single processes for investigation. While
the first approach is intrinsically physiological, investigation results are often convoluted and
inconclusive. For example, depending on the cell line used, binding affinities of drug molecules
widely differ. This illuminates the advantages of the second approach, for while more simplistic,
allows for more control over the biomimetic system. These bottom up approaches of cellular
engineering have uncovered biophysical properties of the cell plasma membrane. Here, we explore
the reverse engineering of simplistic biomimetic artificial cells and investigate the compositional
effects and dependence that plasma membrane lipids have on the human serotonin receptor, 5-
HT1AR. In pursuit of gaining fundamental knowledge and an academic understanding of the
specific and nonspecific interactions of lipids and membrane proteins, we employ techniques, such
as but not limited to, biophysics, chemical engineering, biology, chemistry and materials science,
and fabricate model membranes which we refer to as giant unilamellar protein-vesicles (GUPs)
that are incorporated with G protein coupled receptor (GPCR) 5-HT1AR and its associated G
proteins.
This report begins with an introduction to the importance of artificial membranes.
Properties of giant unilamellar vesicles (GUVs) are discussed and traditional methods of
fabrication are reviewed. Furthermore, protein incorporation into bilayer vesicles is reviewed, and
a discussion as to the current state of the field with regards to lipid and GPCRs interactions is
4

presented. In the following three chapters, the development, optimization and evaluation of GPCR
phase behavior, functionality and stability in GUPs is reported. In chapter 3, investigations that
successfully incorporate the human serotonin receptor into GUPs are presented with observations
on the phase partitioning of 5-HT1AR in liquid ordered and liquid disordered phase separating
vesicles. The following chapter further develops this platform and evaluates the activity of 5-
HT1AR in GUPs and elucidates the plasma membrane compositional dependence of 5-HT1A
receptor-catalyzed nucleotide exchange. Modulating cholesterol, sphingomyelin, and nonlamellar
lipids, we report a change in the rate of 5-HT1AR activity that is dependent on membrane
components. Chapter 4 extends this work into polymeric vesicles and an evaluation of 5-HT1AR
functionality and extended stability through cycles of lyophilization in diblock copolymer bilayer
vesicles is further discussed. Final remarks include a discussion of the extension of this work to
other GPCRs, namely the adenosine 2a receptor (A2aR) and an outlook for future works is
presented.

5

Table of Contents
1. Preface......................................................................................................................................... 3
2. Background ............................................................................................................................... 24
2.1 The cell plasma membrane ................................................................................................. 24
2.2 Bottom up approaches for mimicking cellular membranes ................................................ 25
2.2.1 Giant Unilamellar Vesicles (GUVs) as Model Membranes ........................................ 25
2.2.2 Polymersomes .............................................................................................................. 27
2.2.3 Fabricating GUVs ........................................................................................................ 28
2.3 Protein Incorporation into GUVs ........................................................................................ 30
2.3.1 Lipid-directed hydrogel method of protein incorporation ........................................... 30
2.3.2 Protein investigations in GUV models ........................................................................ 32
2.4 G Protein Coupled Receptors.............................................................................................. 34
2.4.1 Significance.................................................................................................................. 34
2.4.2 The human serotonin receptor ..................................................................................... 35
2.4.3 Compositional dependence of GPCRs: The case from rhodopsin ............................... 36
3. Phase behavior of 5-HT1AR in phase separated GUPs ............................................................. 39
3.1 Motivation ........................................................................................................................... 39
3.2 Methods and materials ........................................................................................................ 39
3.2.1 Materials ...................................................................................................................... 39
3.2.2 Fabrication of vesicles and protein incorporation ........................................................ 40
3.2.3 Antibody labeling......................................................................................................... 41
3.2.4 Antibody binding assay................................................................................................ 41
3.2.5 Antagonist binding assay ............................................................................................. 42
3.2.6 Microscopy .................................................................................................................. 42
3.2.7 Image processing ......................................................................................................... 43
3.3 Results and discussion ........................................................................................................ 43
3.3.1 Images of vesicle yield from agarose hydration method ............................................. 43
3.3.2 Antibody binding ......................................................................................................... 44
3.3.3 Phase separation and identification of liquid ordered phase ........................................ 45
3.3.4 Phase separation at varying cholesterol concentrations ............................................... 49
3.3.5 Ligand binding ............................................................................................................. 50
3.3.6 Antibody binding in DOPC systems ............................................................................ 51
6

3.4 Conclusions ......................................................................................................................... 52
4. Plasma membrane compositional dependence of 5-HT1A receptor-catalyzed nucleotide
exchange ....................................................................................................................................... 53
4.1 Motivation ........................................................................................................................... 53
4.2 Methods and materials ........................................................................................................ 55
4.2.1 Materials ...................................................................................................................... 55
4.2.2 Fabrication of vesicles and protein incorporation ........................................................ 56
4.2.3 5-HT1AR activity assay ................................................................................................ 57
4.2.4 Antibody labeling......................................................................................................... 58
4.2.5 Antibody label fluorescence quenching to determine protein orientation ................... 58
4.2.6 Fluorescence anisotropy measurements ....................................................................... 59
4.2.7 Microscopy .................................................................................................................. 59
4.2.8 Image processing ......................................................................................................... 60
4.2.9 Data analysis ................................................................................................................ 60
4.3 Results and discussion ........................................................................................................ 61
4.3.1 Protein orientation of GPCR 5-HT1AR in GUPs .......................................................... 61
4.3.2 Effects of lipid order on receptor-catalyzed nucleotide exchange ............................... 65
4.3.3 Activity assay validation .............................................................................................. 70
4.3.4 Effects of cholesterol and cholesterol analogues on receptor-catalyzed nucleotide
exchange ............................................................................................................................... 74
4.3.5 Effects of elastic curvature stress on receptor-catalyzed activity ................................ 76
4.4 Conclusions ......................................................................................................................... 79
5. 5-HT1AR incorporated polymeric vesicles retain functional activity through cycles of
dehydration and rehydration ......................................................................................................... 80
5.1 Motivation ........................................................................................................................... 80
5.2 Methods and materials ........................................................................................................ 81
5.2.1 Materials ...................................................................................................................... 81
5.2.2 Fabrication of polymer vesicles and protein incorporation ......................................... 82
5.2.3 5-HT1AR activity assay via microplate reader ............................................................. 83
5.2.4 pGUP lyophilization .................................................................................................... 83
............................................................................................................................................... 84
5.2.5 Antibody labeling......................................................................................................... 85
5.2.6 5-HT1A receptor and G protein identification via antibody binding ............................ 85
5.2.7 Antibody quenching to determine protein orientation ................................................. 86
7

5.2.8 Microscopy .................................................................................................................. 86
5.2.9 Image processing ......................................................................................................... 87
5.2.10 Data analysis .............................................................................................................. 87
5.3 Results and discussion ........................................................................................................ 88
5.3.1 Biased GPCR orientation in diblock copolymer pGUPs ............................................. 89
5.3.2. GPCR catalyzed nucleotide exchange activity in pGUPs........................................... 94
5.3.3 Lyophilization of pGUPs ............................................................................................. 97
5.4 Conclusions ....................................................................................................................... 103
6. Conclusions and future outlook .............................................................................................. 104
7. References ............................................................................................................................... 106

8

List of Figures

Figure 1. Examples of model membranes. Liposomes are ~100 nm and are often used for drug
delivery systems. GUVs are between 10-1000 μm and are typical biomimetic cellular models. The
bilayer of these membranes are made of phospholipids, with a typical lipid being 1-palmitoyl-2-
oleoyl-sn-glycero-phosphocholine (POPC). POPC exhibits a bilayer that is ~ 4 nm thick. Adapted
from Walde et al., 2010.
10
............................................................................................................. 25
Figure 2. Polymer membrane bilayers. Diblock copolymers, triblock copolymers, and multiblock
copolymers are shown. They can exhibit an I-shaped conformation or a U-shaped conformation in
bilayers as depicted above. Adapted from LoPresti et al., 2009.
15
............................................... 27
Figure 3. A) Gentle hydration requires the formation of a dried lipid film which is rehydrated with
an aqueous solution B) Electroformation requires the presence of an AC electric field during
rehydration of lipid films on conductive ITO glass. C) Gentle hydration (A) and electroformation
(B) can be used with liposome films instead of lipid films. D) Micropipet jetting is the formation
of GUVs from planar bilayers by sending a high energy fluid pulses through the planar bilayer. E)
Microfluidic devices uses double emulsion water-oil-water (w-o-w) droplets. Adapted from Walde
et al., 2010.
10
................................................................................................................................. 29
Figure 4. Lipid-directed hydrogel method of protein incorporation into bilayers.
43
A) Detergent
solubilized membrane proteins are mixed with molten low-melting temperature agarose. This is
spread onto a glass coverslip and allowed to gel. The detergent concentration is below the CMC.
B) A thin film of lipids is formed on top of the protein-agarose hydrogel. C) The system is swollen
with a physiologically relevant buffer and GUVs are formed with incorporated protein. ........... 31
Figure 5. Tissue-like packing of GUVs obtained using lipid-directed hydrogel method of protein
incorporation into bilayers. Scale bar is 25 μm. ........................................................................... 31
Figure 6. Confocal micrographs of protein incorporated GUPs with 1:3:1 POPC:BSM:Chol lipid
composition. A phase-separating lipid composition was used to ease visualization. Left images
show fluorescence from lipid label ATTO-488-DPPE, center show antibody-tagged Gα protein
9

subunit, and right show GUPs before and after fluorescent antagonist binding to 5-HT1A receptor.
10 µm scale bars apply to all panels in a row. .............................................................................. 33
Figure 7. GPCR structure and function. A) The X-ray crystal structure of GPCR Rhodopsin
showing 7 α-helical trans-membrane domains. Image taken from Palczewski et al., 2000.
60
B) A
schematic showing agonist activated GPCR catalysis of a G protein. ......................................... 34
Figure 8. 5-HT1AR agonists. (Left) D-lysergic acid diethylamide (LSD). (Right) 5-
hydroxytryptamine (serotonin, 5-HT). Both structures are tryptophan based molecules that
increase the activity of 5-HT1AR when bound. ............................................................................. 35
Figure 9. Bilayer hydrophobic thickness dependence of GPCR rhodopsin. Depending on the
hydrophobic matching between the bilayer and GPCR, the equilibrium state of rhodopsin can be
shifted towards the M-I inactive state or towards the M-II active state. Adapted from Soubias et
al., 2015.
85
..................................................................................................................................... 37
Figure 10. GUPs formed from protein-agarose thin film. Scale bars are 10 µm. Top images show
fluorescence from 491 nm excitation showing ATTO-488 labeled lipid and bottom images show
excitation at 561 nm with no visible fluorescence. The histogram shows the typical vesicle size
distribution for POPC lipid compositions (n=87). Mean GUP radius is 12.1 µm with a standard
error of 1.22 µm and a range of 1.1 µm to 47.1 µm. .................................................................... 44
Figure 11. GUP intensity increases during antibody binding. Left micrograph shows GUP at 491
nm immediately after exposure to antibody. Middle micrograph shows GUP at 561 nm
immediately after exposure to antibody. After one hour of incubation and prior to washing with
200 mM glucose in PBS (pH 7.4) increased intensity is observed at the surface of the GUP (right
micrograph). Scale bar is 5 µm, all images are confocal slices. The plot shows intensity of GUP
at 561 nm excitation. Maximum intensity peaks correspond to the exterior of the vesicle and
indicate successful antibody binding. ........................................................................................... 44
Figure 12. Increased intensity at exterior GUP surface. Confocal slice of GUPs at 491 nm (left)
and 561 nm (right) excitation with corresponding intensity plots. This image shows a large GUP
with smaller GUPs encapsulated inside it. As indicated in the right micrograph, binding only
occurs between the proteins on the surface of the outer GUP and the antibody. This is consistent
10

with the expected membrane impermeability of a high-molecular-weight protein. Scale bar is 5
µm. ................................................................................................................................................ 45
Figure 13. Phase separating vesicles with and without incorporated 5-HT1AR. Lipid label
fluorescence is on the top; 5-HT1AR antibody fluorescence is on the bottom. A) Phase separation
observed on GUV (1:1:3 POPC:Chol:BSM) without protein and incubated with antibody for one
hour. No signal is detected at 561 nm indicating that antibody does not bind to the lipid membrane.
B) Protein-incorporated GUP 1:1:3 POPC:Chol:BSM displaying phase separation prior to
antibody binding. (Confocal slice). C) GUP 1:1:3 POPC:Chol:BSM displaying phase separation
after one hour incubation with antibody. Signals from 491 nm and 561 nm excitation indicate
successful specific binding of antibody to 5-HT1AR. All scale bars are 5 µm. ............................ 46
Figure 14. Quantification of sizes of bright domains identifies them as liquid disordered. A) Shows
a ternary plot of POPC:Chol:BSM indicating the four compositions used in investigations with
representative images of their morphologies. B) An example of vesicle micrograph of the 1:1:3
composition as taken under 491 nm (top) and then made binary in ImageJ (bottom). Scale bar is 5
µm. C) Variability plot of ratio of bright pixels to total vesicle size versus concentrations of liquid
ordered-preferring lipids (%Chol + %BSM) and liquid disordered-preferring lipids (%POPC).
GUV on the y-axis refers to bilayer vesicles in general, both with and without protein incorporated.
....................................................................................................................................................... 47
Figure 15. Qualitative comparison of phase separated vesicles labeled with ATTO-488-DPPE
(Top) and labeled with rhodamine-DPPE (Bottom). Left set of micrographs correspond to
POPC:Chol:BSM lipid compositions at 1:1:3 and 2:1:2. Right micrographs correspond to
DOPC:Chol:BSM/DPPC compositions as listed. The micrograph for POPC:Chol:BSM 2:1:2
labeled with ATTO-488-DPPE is a confocal slice. All other images are Z-stack projections.
Rhodamine-DPPE has been previously reported to partition into the liquid disordered region of
synthetic lipid bilayers
6
and qualitative comparison confirms that ATTO-488-DPPE also partitions
into the liquid disordered region of phase-separated vesicles. ..................................................... 48
Figure 17. POPC-based GUP compositions after antibody binding. The pairs of images on the left
show controls without 5-HT1AR. 491 nm excitation is in the left image of each pair; 561 nm
excitation is on the right. No antibody binding is apparent. The triptychs of images on the right
11

show GUPs with 5-HT1AR after incubation with antibody. In each triptych, excitation at 491 nm
is on the left, 561 nm is in the center, and an overlay is on the right. Overlaid images indicate the
preferential segregation of 5-HT1AR into the liquid disordered phase (bright region) across a range
of cholesterol concentrations. The image 491 nm 2:1:2 POPC:Chol:BSM protein-free control is a
confocal slice, all other images are Z-projections. Scale bars are 5 µm. ...................................... 49
Figure 16. Antibody labeling results on GUPs at varying lipid compositions. A) GUVs showing
protein phase segregation via antibody binding. Excitation at 491 nm (left) and 561 nm (middle)
are overlaid in the right image. Top is 1:1:3 and bottom is 2:1:2 POPC:Chol:BSM. This shows
preferential segregation of 5-HT1AR in the liquid disordered (bright) phase. .............................. 49
Figure 18. Ligand binding results on protein incorporated GUPs with varying lipid compositions.
GUPs at 491 nm (left) and 640 nm (middle) excitation show 5-HT1AR segregation into the liquid
disordered phase after successful antagonist binding. Top is 1:1:3 POPC:Chol:BSM and bottom is
2:1:2. GPCR preferentially segregates to liquid disordered phase. All scale bars are 5 μm. ....... 50
Figure 19. POPC-based GUPs after incubation with the fluorescent 5-HT1AR ligand. The pairs of
images on the left show controls without 5-HT1AR. Micrographs are shown at 491 nm (left) and
640 nm (right) excitation. No ligand binding is apparent. The triptychs of images on the right show
GUPs with 5-HT1AR. In each triptych, excitation at 491 nm is on the left, 640 nm is in the center,
and an overlay is on the right. Scale bars are 5 µm. ..................................................................... 51
Figure 20. DOPC-based GUPs after antibody binding. The pairs of images on the left show
controls without 5-HT1AR. Micrographs are shown at 491 nm (left) and 561 nm (right) excitation.
No apparent non-specific antibody binding occurs. The triptychs of images on the right show GUPs
with 5-HT1AR. In each triptych, excitation at 491 nm is on the left, 561 nm is in the center, and an
overlay is on the right. Scale bars are 5 µm. ................................................................................ 52
Figure 21. Schematic of protein activity assay. GUPs are formed via hydrogel rehydration method.
Incubation with agonist unquenches encapsulated BODIPY-GTPγS fluorescence via G protein
binding. Fluorescence increase due to BODIPY-GTPγS binding to G protein is tracked over time
(see inset micrographs). ................................................................................................................ 54
12

Figure 22. QSY7 does not cross GUP membranes. GUPs encapsulating 400 nm liposomes were
imaged and quenched using QSY7. After incubation with QSY7 for ten minutes at room
temperature GUPs show an intensity decrease of 53.2% ± 2.7% while encapsulated liposomes do
not show any significant decrease in intensity (< 3% difference). QSY7 does not cross the bilayer
and effectively quenches fluorophore on the exterior of GUPs. Micrographs are Z-stack standard
deviation projections using confocal microscopy. Scale bar is 5 μm. .......................................... 61
Figure 23. Fluorescence quenching image analysis. All vesicles are made of 60%:40% BSM:Chol
(0% POPC). The control vesicles are made without protein. The leftmost micrographs show the 5-
HT1A receptor with rhodamine-antibody tagged on its cytosolic face. The center micrographs show
GUPs with the G proteins tagged with rhodamine-antibody. The rightmost micrographs show
vesicles with ATTO-488-DPPE fluorescent lipid. The top row shows vesicles prior to incubation
with QSY7 and bottom row shows GUPs after QSY7 quenching. The bottom plots show the
fluorescence intensities across the same line segment on before and after images. Fluorescence
intensity analysis was performed using values from the plots. The receptor retains 90% of its
intensity while the G protein and lipid tag, ATTO-488-DPPE, retain ~50% of their intensities. This
indicates a biased orientation of the receptor upon incorporation into GUPs. Scale bar is 10 μm.
....................................................................................................................................................... 62
Figure 24. GPCR orientation determination. 100% POPC and 60%:40%BSM:Chol GUPs were
formed with labeled antibodies to either the cytoplasmic domain of the GPCR or the Gα protein
subunit. Control vesicles were formed without protein and labeled fluorescently with ATTO-488-
DPPE. (A) Quenching of cytosolic-bound-rhodamine-antibody tagged 5-HT1AR results in
retention of ~90% of fluorescence. This indicates that incorporated receptors are oriented with the
N-terminus extracellular and C-terminus interior. Controls show 50% retained intensity. Analysis
of variance (ANOVA) indicates no significance between the two GUP samples, F(1, 14)=0.17,
P>0.69, as also confirmed with post-hoc Tukey-Kramer analysis. (B) G proteins tagged with
rhodamine-labeled antibody show an unbiased distribution between the inner and outer bilayer
leaflets. There is no significant difference between the GUPs and control, F(2,27)=0.64 P>0.53.
....................................................................................................................................................... 63
13

Figure 25. Vesicle growth from agarose lipid film. The top micrographs show example images of
the agarose lipid film at the indicated time points during the hydration and vesicle swelling process.
Vesicles form as small vesicles that coalesce over time to form giant unilamellar vesicles. A 100%
POPC lipid mixture was used with 0.2%mol ATTO-488-DPPE as the fluorescent dye.
Micrographs are epifluorescent images. Scale bar is 20 µm. Systematic analysis of the film using
ImageJ particle analyzer is plotted below the micrographs. As time progresses the number of
vesicles decrease while the mean radius of the vesicle population increases, indicating vesicle
fusion as a means of vesicle formation using the agarose hydration approach.
41
......................... 64
Figure 26. Receptor-catalyzed nucleotide exchange rates in ternary and binary GUPs.
Fluorescence intensity over time of an average of five observations with identical experimental
conditions is shown as single individual curves. The shaded area around the points indicates the
standard error mean of averaged samples. Control samples are the average of all observations of
the listed compositions of GUPs tracked in the absence of agonist and are indicated in pink. (A)
GPCR activity rate in GUPs with increasing amounts of ordering components, BSM and Chol, in
ternary GUPs of POPC:BSM:Chol. Going from 100% POPC to 60%:40% BSM:Chol (0% POPC)
shows an increased rate of receptor-catalyzed nucleotide exchange. (B) GPCR activity rate in
binary GUPs of POPC:Chol. Increasing the amount of Chol in these systems increases the rate of
receptor-catalyzed nucleotide exchange. (C) Plot of rates from (A) and (B) by Chol and BSM
composition. Standard error bars are not included as they are smaller than the markers. See Table
1 for standard error data. Ternary GUPs indicated by circles in blue show faster protein rates than
binary GUPs indicted by squares in green .................................................................................... 67
Figure 27. Control fluorescence intensity curves for all compositions investigated. These data
represent protein activity with no agonist added; each curve represents a single sample. Each plot
is indicated by the lipid system and the basal rate for that system as determined by a single
exponential fit to the average of all curves shown in the plot. The control curves presented in the
main text represent this average. ................................................................................................... 68
Figure 28. Size distribution of GUPs of POPC:BSM:Chol. GUPs with rhodamine labeled antibody
tagged serotonin receptor were imaged using epifluorescence and size distribution analysis was
14

performed using ImageJ particle analyzer. Table shows average radius in µm for each of the
different compositions of GUPs, followed by respective histograms........................................... 70
Figure 29. Nonspecific BODIPY-GTPγS fluorescence with GUVs. GUVs without protein were
formed of 100% POPC and 0% POPC lipid compositions and incubated with (+Ag) and without
agonist (Ctl). GUVs show less than 5% fluorescence intensity increase while GUPs of the same
composition show over 75% fluorescence intensity increase. BODIPY-GTPγS interaction with
GUVs yields insignificant fluorescence........................................................................................ 71
Figure 30. Protein thermal stability investigated by pre-incubating membrane fragments. 5-HT1AR
membrane fragments were pre-incubated at 37 ºC for 0, 6, 18, and 24 hr and then incorporated in
GUPs for the 12-hour activity assay. GUPs were made of 0% POPC or 100% POPC. Percent
intensity increase was tracked over time and plotted. Plots are an average of 6 replicates and
shaded areas around the points of the curve are the standard error of the mean. 0, 6, and 18 hr pre-
incubated protein samples retain activity and fluorescence intensity increase. 24 hr pre-incubated
GUPs did not display significant fluorescence intensity increase over time. ............................... 72
Figure 31. Increasing agonist concentration. GUPs of 100% POPC and 0% POPC were formed
and subjected to activity assay with increasing amounts of 8-OH-DPAT, 0 M, 150 fM, 150 pM,
and 150 nM. A) Increasing the amount of agonist in the assay displays increasing rates of intensity
increase. B) Activity rates corresponding to curves in A. ............................................................ 73
Figure 32. Receptor-catalyzed nucleotide exchange rate of GUPs made with Chol analogs. (A).
Binary POPC:Ergosterol GUPs were assessed for receptor-catalyzed nucleotide exchange activity.
As mole percent of ergostolerol is increased, exchange rate also increases. The chemical structure
of ergostolerol is placed within the plot with its differences from Chol highlighted. (B) Binary
POPC:Epicholesterol GUPs also show an increase in receptor-catalyzed exchange rate with
increasing amounts of epicholesterol. Epicholesterol is a diastereomer of Chol with the hydroxyl
group on the alpha face of Chol as shown in the chemical structure within the plot. Control curves
are the average of all individual observations for the listed compositions of GUPs without agonist
incubation. ..................................................................................................................................... 75
15

Figure 33. GUP formation of binary POPC:DOPE compositions. While DOPE is a nonlamellar
forming lipid, GUPs are successfully formed in binary mixtures of POPC:DOPE with DOPE at
10%, 25%, 50%. In comparison with forming vesicles without proteins, GUPs of POPC:DOPE
are larger in radius. DOPE was run at 100 mol% using our agarose hydration method as a control.
No vesicles or bilayer structures were formed. The table shows relative average radii for different
POPC:DOPE compositions. These binary compositions are smaller than 100% POPC GUPs. . 77
Figure 34. 5-HT1A receptor-catalyzed nucleotide exchange rate of GUPs made of POPC:DOPE.
The overall rates are well above other compositions investigated, ranging from 9.38-22.67 (x 10
-
3
) min
-1
, see Table 1. However, as DOPE mole percent increases beyond 7.5% the protein
functional rate decreases. .............................................................................................................. 78
Figure 35. Schematic of pGUP formation, lyophilization, and functional assay. Step 1. Films of
agarose and protein are deposited on to a coverslip and a thin film of diblock copolymer PBd(650)-
PEO(400) is added. The system is swollen with 200 mM sucrose in PBS (pH 7.4) and BODIPY-
GTPγS. Step 2. Upon pGUP formation, vesicles are lyophilized. Step 3. After dehydrated storage
pGUPs are rehydrated with Milli Q water. Step 4. Rehydrated pGUPs are assayed for agonist
induced functionality. ................................................................................................................... 80
Figure 36. Schematic of experiments presented. The top schematic shows protein incorporation
into polymer bilayer membranes using hydrogel approach. A thin film of protein-agarose is made
on a coverslip. A thin film of polymer is made on top. The system is rehydrated with
physiologically relevant buffers and pGUPs are formed with GPCR incorporated in the polymer
bilayers. The lower left schematic shows protein orientation determination set-up. Antibodies for
either receptor or G protein are conjugated to NHS-rhodamine. The conjugated antibody is run
through a 40 kD spin column to remove excess rhodamine. Receptor or G protein is incubated and
tagged with respective rhodamine labeled antibody. The rhodamine-antibody-tagged receptor or
protein is used in the hydrogel matrix to form pGUPs. After formation, pGUPs are incubated with
QSY7 to determine orientation by quenching rhodamine on the exterior of pGUPs. The lower
middle schematic shows the steps in protein activity assessment. After formation of pGUPs using
the presented method, pGUPs are incubated with agonist and fluorescence unquenching due to
BODIPY-GTPγS binding to G protein is tracked over time. In the lower right schematic pGUP
16

lyophilization. After pGUP formation, pGUPs are flash frozen in liquid nitrogen and then
subjected to overnight vacuum at 0.5 torr to complete lyophilization. Lyophilized pGUPs can be
stored at -20 C and can be rehydrated using water to regain spherical shape and protein activity.
....................................................................................................................................................... 84
Figure 37. Top set: Confocal micrographs of pGUPs prior to lyophilization. The left micrograph
shows the polymer bilayer tagged with ATTO-488-DPPE. The right micrograph shows that
rhodamine antibody-tagged 5-HT1AR is evenly distributed throughout the polymer bilayer. Bottom
set: The left image shows a pGUP sample after lyophilization. Upon rehydration, pGUPs can still
be detected as shown in the right micrograph. All scale bars represent 5 µm. ............................. 88
Figure 38. pGUP polymer make-up confirmation. In the micrographs above, either polymer or
ATTO-488-DPPE was casted as a thin film on top of a hydrogel matrix without protein, with 5-
HT1AR, or with rhodamine-antibody tagged 5-HT1AR. The hydrogel is low-melting-temperature
agarose. To image the polymer without lipid dye ATTO-488-DPPE, micrographs were taken using
DIC. All other micrographs were collected using epifluorescence at the indicated wavelengths. All
scale bars are 25 μm and the scale bar in the first micrograph of a set of triptych images goes for
all images in that set. In the left set of polymer images, vesicles are formed on all three hydrogels.
When protein is included in the hydrogel, vesicles tend to float off of the hydrogel, which explains
the lack of vesicle density on the hydrogel surface. pGUPs can be found floating in the solution
above (not shown). In the right set of ATTO-488-DPPE images, vesicles are not observed to form
on three hydrogels investigated. This confirms that pGUPs formed using the hydrogel assisted
method of protein incorporation are indeed made of polymer. .................................................... 89
Figure 39. Nonspecific interaction of antibodies and pGUPs. The micrographs presented show
polymersomes before (left) and after (right) incubation with rhodamine-labeled antibody. Vesicles
were made without protein (pGUVs), with and without ATTO-488-DPPE, and with unlabeled
protein incorporated (pGUPs). Vesicles were incubated with either rhodamine-anti-5-HT1AR (Rh-
anti-receptor) or rhodamine-anti-G protein (Rh-anti-G protein). Images were taken using phase
contrast for vesicles without lipid label, at 491 nm for ATTO-488-DPPE, and at 561 nm for Rh-
antibodies. pGUVs without protein (top set) show no intrinsic fluorescence at 491 nm and 561 nm.
After incubation with antibody, antibody does not cross the membrane, and no fluorescence
17

accumulation is seen at the vesicle surface. pGUVs tagged with ATTO-488-DPPE show
fluorescence at 491 nm (middle set), and also show no fluorescence accumulation at vesicles
surface. In pGUPs with unlabeled protein (bottom set), and without lipid label, incubation with
Rh-anti-receptor does not show binding of the antibody to the exterior of the pGUP, or the N-
terminus of the receptor. The antibody does not cross the membrane and cannot reach the C-
terminus of the receptor. Furthermore when the same pGUPs are incubated with Rh-anti-G protein,
some fluorescence intensity accumulates on the surface the pGUPs, since G proteins are distributed
on both leaflets of the bilayer. All scale bars are 10 µm. ............................................................. 91
Figure 40. QSY7 quenches fluorescence of ruptured pGUPs with Rh-anti-receptor. pGUPs were
prepared with Rh-anti-receptor. pGUPs were settled and observed using confocal microscopy at
561 nm excitation. Scale bar is 5 µm. In the two top microcraphs, pGUPs were imaged prior to
and after incubation with QSY7 for ten minutes. The fluorescence intensity of the pGUP does not
change. pGUPs were ruptured. Micrographs are select images at the specified time points. The
intensity of all images are plotted as line segments spanning the top left to the bottom right. The
colors on the plot correspond to the colors of the time point text indicated. Select time points are
shown vesicle rupture, vesicle fragments and quenching of the fluorescence. ............................ 92
Figure 41. Fluorescence assessment of antibody-tagged pGUPs for determination of protein
orientation. pGUPs were made with either Rh-anti-receptor, Rh-anti-G protein or ATTO-488-
DPPE. The left micrograph pair shows excitation at 561 nm and shows the rhodamine tagged
receptor, the middle micrograph pair shows excitation at 561 nm showing the rhodamine tagged
G protein, and the right micrographs show excitation at 491 nm which shows fluorescence of
ATTO-488-DPPE. The top micrographs of each pair show the vesicles before quenching with
QSY7 and the bottom micrographs show vesicles after quenching. The plots show the intensity as
tracked by the colored line segments across each vesicle and show the change before and after
quenching. The receptor shows little change in intensity before and after quenching with QSY7
indicating a biased orientation of the receptor. The G protein and the ATTO-488-DPPE show
~50% decrease in fluorescence intensity indicating that these components are distributed on the
inner and outer leaflet of the bilayer. All scale bars are 10 µm. ................................................... 92
18

Figure 42. Retained fluorescence intensity of quenched monoclonal antibody-tagged pGUPs.
pGUPs were formed with monoclonal rhodamine antibody-tagged receptor or G protein and
subsequently quenched. Retained fluorescence intensity indicates the population of receptor in the
correct orientation and G Protein in the inner leaflet of the vesicles. Over 90% of the receptor
population is incorporated in the correct orientation while G proteins exists across both leaflets.
Control pGUPs were made with ATTO-488-DPPE throughout both bilayers; this lipid tag is
quenched ~50%. ............................................................................................................................ 93
Figure 43. pGUP formation using the agarose swelling method for protein incorporation. The
micrographs depicted are DIC time-lapse images as indicated above each image in seconds. Time
0 s represents the hydrogel and polymer film immediately prior to rehydration with 200 mM
sucrose in PBS (pH 7.4). Vesicle formation begins almost immediately and pGUPs start as
nanometer size vesicles and continue to grow and form micrometer sized vesicles. Coalescence of
the smaller vesicles into large vesicles is captured in the decrease in number of vesicles from 60 s
to 180 s to 300 s. The plot below the images shows the pGUP count and average radius in μm over
a 5-minute period. pGUP count initially spikes and decreases while radius increases and plateaus
over time, indicating the coalescence of vesicles during pGUP formation. ................................. 94
Figure 44. BODIPY-GTPγS does not display nonspecific binding to pGUVs. pGUVs without
protein were formed as described and omitting the protein from the hydrogel. pGUVs encapsulate
BODIPY-GTPγS and were incubated with and without agonist (8-OH-DPAT) and fluorescence
was tracked over time. As shown in the plot, pGUPs do not show significant fluorescence intensity
increases over time indicating that BODIPY-GTPγS has no unqunenching interaction with the
polymersome membrane. .............................................................................................................. 95
Figure 45. 5-HT1AR in pGUPS display physiological response to increasing antagonist
concentration while keeping agonist concentration constant. Fluorescence unquenching due to the
irreversible binding of BODIPY-GTPγS to G proteins was tracked for 12 hours for pGUPs formed
with increasing amount of antagonist spiperone and constant amount of agonist (+Ag). Increasing
the amount of antagonist spiperone in the system decrease the protein functional rate (See Table
4, for best-fit rates). The inset shows control curves for the pGUPs that were incubated without
agonist. 5-HT1AR basal activity is captured in the control, no agonist, pGUPs. .......................... 95
19

Figure 46. Curves of protein functional rate with varying antagonist. The points indicate the
average of 6 pGUP sample observations for each of the listed antagonist, methiothepine maleate,
NAN-190, and WAY 100635, and the shaded area around the points are the standard error mean.
The inset shows the control pGUPs that were not incubated with agonist and represents protein
basal activity in the specified pGUP samples. Each of the agonist induce samples (Ag+) and
corresponding control (Ctl) samples are plotted as a set below. The quantitative rates of both Ctl
and Ag+ rates can be found on Table 4. ....................................................................................... 96
Figure 47. Comparison of pGUPs before and after lyophilization. A) The left micrograph shows
a typical yield of pGUPs on the hydrogel before harvesting. The right micrograph shows a patch
of pGUPs that were lyophilized on the hydrogel for viewing purposes. pGUPs are smaller and
display a rough perimeter due to dehydration from lyophilization. B) This set of images shows
pGUPs after they have been harvested from the hydrogel and settled. The left micrograph shows
pGUP controls that were not lyophilized. The right micrograph shows typical pGUPs after
lyophilization and subsequent rehydration with Milli Q water. C) Histograms of size distributions
of pGUPs before lyophilization and after lyophilization. Prior to lyophilization the mean average
radius of a typical pGUP population is 6.03±0.24 μm. After lyophilization the mean average radius
of a typical pGUP population is 5.94±0.24 μm. ........................................................................... 98
Figure 48. Rates of non-lyophilized pGUPs after storage at 5˚ C 24 hr and 120 hr. While pGUPs
show an increase in fluorescence intensity over time, the percent increase reported for lyophilized
samples decreases indicating that the populations of pGUPs with protein activity is diminishing
and that lyohilization is needed for extended storage. The shaded areas around the points represent
the standard error mean of 6 samples using the identical experimental set-up. ......................... 100
Figure 49. Results of pGUPs without spiperone. pGUPs were formed without the antagonist,
spiperone, in the rehydration buffer and were lyophilized for 0 hour (0 hr, non lyophilized), 24
hours or 120 hours then assayed for protein functionality. For all time periods, the protein displays
inherent functionality and agonist induced activity, however, the rate of protein function for the
lyophilized samples are attenuated compared to the 0 hr non lyophilized control. The control
curves in the in-set are pGUP samples of the indicated lyophilization time that were not incubated
20

with agonist, and therefore indicate protein basal activity. Each of the agonist induce samples
(Ag+) and corresponding control (Ctl) samples are plotted as a set below ................................ 100
Figure 50. Functional rates of 5-HT1AR in polymersomes (pGUPs) versus various solutions.
Controls (Ctl), no agonist pGUPs, are plotted alongside agonist-exposed samples (+Ag). The
percent intensity increase of the samples indicates the population of functional protein. In DI water
and PBS, the 5-HT1AR displays no fluorescence activity. In 200 mM sucrose in PBS (pH 7.4) 5-
HT1AR displays weaker fluorescence intensity increase compared to pGUPs. Furthermore there is
no difference in rate between the Ctl and +Ag protein in 200 mM sucrose in PBS. .................. 101
Figure 51. Normalized rate of agonist and control protein diluted in 200 mM sucrose in PBS and
lyophilized for 24 hr. As presented in Figure 50, 5-HT1AR membrane fragments diluted in sucrose
rehydration buffer shows protein function, however, they do not show agonist dependence as
protein exposure to 8-OH-DPAT does not show an increase in protein functional rate. The curves
are averages of the data points presented. The rates as determined by a single exponential filling
of the control protein is 7.23±0.12 (x10
-3
min
-1
) and the rate of the agonist-incubated protein is
7.18±0.12 (x10
-3
min
-1
) ............................................................................................................... 102

21

List of Tables

Table 1. Summary of rates from all compositions. Lipid concentrations are given in mol%. ..... 69
Table 2. 5-HT1AR thermal stability. 24 hr time point displayed no measurable protein activity,
see Figure 28. ............................................................................................................................... 73
Table 3. Bilayer ordering results as determined by fluorescence anisotropy measurements for all
lipid compositions. Lipid concentrations are given in mol%. ...................................................... 76
Table 4. Physiological 5-HT1AR in pGUP response to changes in antagonist species and
concentration. ................................................................................................................................ 96
Table 5. Protein rates and percent increase in fluorescence intensity of pGUP controls (non-
lyophilized) and after lyophilization (24 hr and 120 hr). +Ag indicates added agonist; Ctl
samples represent basal activity (without agonist). Percent intensity is indicative of active 5-
HT1AR population. ........................................................................................................................ 98
Table 6. Rates of pGUPs formed and lyophilized without antagonist, spiperone. .................... 101

22

List of abbreviations
5-HT1AR Human serotonin receptor, 5-hydroxytryptamine subclass 1A receptor
7TM Seven-transmembrane (domain)
8-OH-DPAT 8-Hydroxy-2-(dipropylamino)tetralin hydrobromide
A2aR Adenosine 2A receptor
AAS Atomic absorption spectroscopy
AChR Human acetylcholine receptor
BSA Bovine serum albumin
BSM Porcine brain sphingomyelin
CHCl3 Chloroform
Chol Cholesterol
CMC Critical micelle concentration
COPD Chronic obstructive pulmonary disease
CXCR4 Chemokine C-X-C motif receptor 4
DMSO Dimethyl sulfoxide
DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
DPH Diphenylhexatriene
DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DPPE 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
DRD2 Dopamine receptor D2
DRM Detergent resistant membrane
FRET Förster resonance energy transfer
GDP Guanosine diphosphate
23

GPCR G protein coupled receptor
GTP Guanosine triphosphate
GUPs Giant unilamellar protein-vesicles
GUVs Giant unilamellar vesicles
HII Lipid hexagonal phase
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
hr Hour
Kd Dissociation constant
Ld Liquid disorderd phase
l-l Liquid-liquid
Lo Liquid ordered phase
LSD D-lysergic acid diethylamide
MeOH Methanol
MI Meta-I inactive state of rhodopsin
MII Meta-II activate state of rhodopsin
MOPS 3-morpholinopropane-1-sulfonic acid
NaOH Sodium hydroxide
NHS N-hydroxysuccinimide
PBS Phosphate buffered saline
PE Phosphoethanolamine
POPC 1-palmitoyl-3-oleoyl-sn-glycero-phosphocholine
ROS Rod outer segment
SBFI Sodium-binding benzofuran isopthalate
UV-vis Ultra violet-visible
w-o-w Water-oil-water, referring to double emulsion droplets
24

2. Background
2.1 The cell plasma membrane
The cell plasma membrane is the gateway to the cytosol and it is specific to the exterior-
most membrane that surrounds living cells. Eukaryotic cells, however, possess organelles
surrounded by other bilayer membranes which are essential to their compartmentalization. The
term plasma membrane is thus reserved for the membrane barrier between the interior and exterior
of the cell and components that make up the plasma membrane include a phospholipid bilayer and
membrane proteins. It is responsible for important cellular functions which include but are not
limited to drug uptake, cell-cell communication, transport and signaling.
1, 2, 3
The dynamic
processes of the plasma cell membrane are embedded in the phospholipid bilayer whose lipid
composition can vary between cell type and a person’s disease state.
4
Various phospholipids both
saturated and unsaturated, sphingolipids and sterols along with membrane proteins,
transmembrane and peripheral, make up the cell membrane.
1
The effects of plasma membrane
lipid composition on cellular function and protein activity is an area of research that not only
transcends a desire for fundamental knowledge of lipid-protein interactions but has the potential
to promote the understanding of membrane protein related diseases and enhance drug targeting.
5

Lipid-protein interactions are investigated using many approaches; bottom up approaches utilize
biomimetic bilayers that provide a simplistic foundation to elucidate such fundamental
interactions.

25

2.2 Bottom up approaches for mimicking cellular membranes
2.2.1 Giant Unilamellar Vesicles (GUVs) as Model Membranes

Bottom up approaches to forming artificial cells focus on bilayer vesicles as model
membranes (Figure 1). The formation of liposomes, nanometer sized bilayer vesicles, was
introduced in the 1960’s and was a technique for understanding membrane biophysics. This has
been further extended to use as drug delivery systems.
6, 7
For example, AmbiSome
®
and DOXIL
®

are prescribed medical therapeutics that are liposome based drug delivery systems.
8
Methods for
forming liposome sized bilayer vesicles were mostly based on rehydration of lipid films followed
by extrusion or sonication. In 1986, Angelova and Dimitrov introduced the electroformation
method that facilitated the fabrication of micrometer sized giant unilamellar vesicles (GUVs).
9

Since then GUVs have been widely used to model the cellular plasma membrane.
10
As larger
Figure 1. Examples of model membranes. Liposomes are ~100 nm and are often used for drug delivery systems.
GUVs are between 10-1000 μm and are typical biomimetic cellular models. The bilayer of these membranes are
made of phospholipids, with a typical lipid being 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC). POPC
exhibits a bilayer that is ~ 4 nm thick. Adapted from Walde et al., 2010.
10

26

micron sized platforms GUVs have been exploited for probing physiochemical properties of
biological membranes and proteins can be incorporated into the bilayer to further understand
activity at the plasma membrane.
10

Due to their size, GUVs are easily accessible via microscopy and fluorescence based
techniques are typically employed to visualize phase separation, transport and other membrane
phenomena. The compositions of GUVs can consist of synthetic lipids, natural lipids, or cellular
lipids from blebbed membranes of live cells and the ratio of these components can be controlled
in bottom up approaches of membrane formation.
10-13
GUVs as membrane models offer the same
characteristics of the plasma membrane and are often utilized in modeling membrane fusion,
observing lipid oxidation at the plasma membrane, exploring permeability across the membrane,
and mimicking cellular compartmentalization.
14
Lipid phase behavior provides insight into the
complex nature of bilayer membranes with work focusing on phase coexistence in lipid bilayers.
15

Furthermore, the incorporation of proteins into fluorescently tagged GUVs allows for observations
on protein phase behavior and protein biophysical properties.
16
For example transport of
incorporated α-lysin can be tracked using fluorescence dyes as demonstrated by Pokorny et al. in
2006.
17
Encapsulation of in vitro transcription components allows for protein expression and
assembly within these bilayer vesicles expanding our understanding and ability to mimic cellular
compartmentalization and function.
While GUVs offer many advantages in probing membrane biophysics, they are less stable
than liposomes, and lipidic GUVs are less stable than polymersomes. In 1993, Mui et al. reported
on GUV sensitivity to osmotic gradients and showed that GUVs obey Laplace’s Law relating lysis
pressure, tension, and vesicle diameter.
18
Focusing on lysis, where maximal membrane tension is
exceeded, the vesicle radius and osmotic pressure difference between the interior and exterior of
27

the vesicle are inversely related.
18
To this extent GUVs are not typically stored for long periods of
time while liposomes can be lyophilized for extended storage and drug delivery purposes.
8, 19

2.2.2 Polymersomes
GUVs are not limited to having
bilayers solely made of membrane
lipids. The use of block copolymers to
form polymersomes was introduced in
1999 by Discher et al., and has provided
researchers with another platform to investigate membrane material properties.
20-22
Diblock
copolymers and triblock copolymers have been used to make vesicles that vary from 100s of
nanometers to 100s of micrometers in diameter (Figure 2).
23
Polymersomes are more stable and
less permeable than their lipidic counterparts and have been exploited and hybridized with cellular
components such as lipids and proteins for drug delivery and research.
21, 24, 25
For example, phase
separation has been modeled in lipid-polymer vesicles offering a platform for “windows” of lipid
bilayers to be observed in a polymeric framework.
26
The encapsulation of nanometer sized
polymersomes within GUVs further provides insight into the compartmentalization of living
cells.
27
In recent years, protein incorporation into varying lamellar phase polymeric vesicles has
been reported.
28, 29
Polymersomes and lipid based GUVs are platforms for understanding specific
and nonspecific chemical interactions of membrane components such that methods for fabricating
these bilayer vesicles have been engineered for decades.
Figure 2. Polymer membrane bilayers. Diblock copolymers,
triblock copolymers, and multiblock copolymers are shown.
They can exhibit an I-shaped conformation or a U-shaped
conformation in bilayers as depicted above. Adapted from
LoPresti et al., 2009.
15

28

2.2.3 Fabricating GUVs
The most traditional methods for GUV fabrication are gentle hydration and
electroformation (Figure 3).
10, 30
More recently micropipette jetting and microfluidic devices have
been used to make uniformly sized vesicles, and hydrogel assisted approaches have also gained
much popularity.
10, 30, 31
Gentle hydration is the approach of rehydrating a dried lipid film with
water or buffer which allows for the formation of bilayer vesicles due to the interactions of the
hydrophilic headgroup and hydrophobic tails of lipids.
32
This approach may be followed by
sonication or extrusion to form liposome sized vesicles.
6, 32
With the gentle hydration approach
however, vesicle size tends to be bimodal and unrestricted and this approach is also reported to
yield more defects than fabrication via electroformation.
33
The introduction of an electric field to
the system of gentle hydration as presented by Angelova (1986) to electroform GUVs is reported
to form more GUVs of micron size and is widely used in the field of membrane biophysics.
9
Using
the electroformation approach, a thin film of lipids is spread on a piece of ITO (indium-tin-oxide)-
coated glass and is rehydrated in the presence of an electric field; this causes the formation of
GUVs. In 1999, Angelova reported the kinetics of applying an AC field to fabricate GUVs and
concluded that an alternating electric field causing a change in direction and magnitude of the field
intensity could be a driving force for lipid movement and may assist in the formation of bilayers.
34

Pott et al. report a modification to this process where proteoliposomes are used instead of a lipid
film.
35
While electroformation is widely used to form GUVs, increasing GUV complexity with the
integration of membrane proteins is of concern as the exposure of proteins and protein precursors
to an electric field can denature proteins and damage biomolecules.
29

Micropipette jetting and
microfluidic devices are two other
approaches used for GUV engineering
(Figure 3). Micropipette jetting
requires the use of high frequency
fluid pulses from an inkjet device to
promote the formation a GUV from a
lipid bilayer.
36, 37
This approach
allows for size uniformity,
encapsulation of material, and control
over membrane asymmetry. When
stringent size control of GUVs is
desired microfluidic devices can also
be used. This approach exploits oil and
aqueous phases of liquids flowed through a miniature device to create water-in-oil droplets using
double emulsions.
38
Due to the amphiphilic nature of phospholipids, they can arrange themselves
accordingly around the double emulsion droplets. Subsequently each leaflet of the GUV bilayer
can be made in different steps. Typically the inner leaftlet is formed around an aqueous droplet in
oil, then a second layer of lipids is assembled on top to form a bilayer with an aqueous interior and
exterior, though oil may be present in between the bilayer.
38
However, while this approach offers
uniform and high throughput fabrication of GUVs, residual oil between bilayer leaflets is of major
concern.
Figure 3. A) Gentle hydration requires the formation of a dried
lipid film which is rehydrated with an aqueous solution B)
Electroformation requires the presence of an AC electric field
during rehydration of lipid films on conductive ITO glass. C)
Gentle hydration (A) and electroformation (B) can be used with
liposome films instead of lipid films. D) Micropipet jetting is the
formation of GUVs from planar bilayers by sending a high energy
fluid pulses through the planar bilayer. E) Microfluidic devices
uses double emulsion water-oil-water (w-o-w) droplets. Adapted
from Walde et al., 2010.
10

30

Though electroformation of GUVs is the method that is regularly used for bilayer vesicle
fabrication, hydrogel assisted formation of GUVs is a method that has gained increasing
popularity. This approach was reported by Horger et al. in 2009 and utilizes an agarose hydrogel
that quickly introduces water into the system to form GUVs.
31
Using this approach, a hydrogel
film is made on top of a substrate and a lipid film is formed on top of the hydrogel. These films
are rehydrated with physiological buffers such as phosphate buffered saline (PBS) and GUVs
readily form on top of the hydrogel due to water influx from the hydrogel film pushing against the
lipids which forms vesicles.
31
Continued formation of small vesicles from the hydrogel results in
crowding and coalescence of these smaller vesicles in to giant vesicles. This approach has been
broadened by Lopez-Mora et al. to include dextran hydrogels with varying amounts of cross-
linking to vary the size of GUVs formed.
39
Furthermore, this approach has been used to effectively
incorporate protein as initially presented by Hansen et al. in 2013.
40
The hydrogel-assisted method
of GUV formation also allows for rehydration using physiological ionic strength buffers and use
of lipids with net formal charges, two features that are problematic in electroformation.

2.3 Protein Incorporation into GUVs
2.3.1 Lipid-directed hydrogel method of protein incorporation
Incorporation of intrinsic membrane proteins into GUVs using the lipid-directed hydrogel
method demonstrated by Hansen et al. alleviates obstacles that previous GUV fabrication
approaches faced when attempting to incorporate proteins, namely high detergent concentration
and need for use of physiological ionic strength buffers.
40
Unlike other methods of GUV
fabrication that pose damage to proteins, this approach does not require an electric field, proteins
are gelled in an agarose hydrogel and prevents them from desiccation and damage, protein
31

precursor encapsulation is not
required, and furthermore the
typically high concentration of
detergent used to prepare
solubilized proteins is diluted
below the critical micelle
concentration (CMC) and
therefore does not negatively
affect the formation of a lipid
bilayer.
40
Agarose-assisted
hydration involves the drop
casting of an agarose film onto a
clean glass coverslip. For protein
incorporation, proteins are mixed
with molten agarose prior to thin
film coating. This protein-agarose is gelled and lipids are
added to form a thin film on top of the gelled protein-agarose.
Excess solvent is removed with a stream of N2 and the system
is rehydrated with a physiologically relevant sucrose buffer
(Figure 4). The amphiphilic nature of vesicle building blocks
provides a hydrophobic driving force for the self-assembly and
directed insertion of proteins into lipid vesicles. In their report,
Hansen et al. incorporated two types of transmembrane water
z
Figure 5. Tissue-like packing of
GUVs obtained using lipid-directed
hydrogel method of protein
incorporation into bilayers. Scale
bar is 25 μm.
Figure 4. Lipid-directed hydrogel method of protein incorporation
into bilayers.
43
A) Detergent solubilized membrane proteins are mixed
with molten low-melting temperature agarose. This is spread onto a
glass coverslip and allowed to gel. The detergent concentration is
below the CMC. B) A thin film of lipids is formed on top of the
protein-agarose hydrogel. C) The system is swollen with a
physiologically relevant buffer and GUVs are formed with
incorporated protein.
32

channel proteins, Aquaporin Z from E. coli and SOPIP2;1 from spinach.
40
Using this approach
they are able to assemble tissue-like packing of GUVs (Figure 5) and they observed the phase
behavior of the aquaporins and reported permeability measurements which indicated conserved
protein functionality.
40

2.3.2 Protein investigations in GUV models
Protein incorporated into model membranes allows for investigations that span
observations on functionality, ligand and antibody binding, and protein partition into different lipid
phases. Demonstration of protein functionality in GUVs is a method to determine the degree of
success of different protein incorporation approaches. In 2004, utilizing partially dehydrated
proteoliposomes and rehydrating them in an AC electric field, Girard et al. report the transport of
H
+
and Ca
2+
by incorporated bacteriorhodopsin, and Ca
2+
by –ATPase.
41
Despite their report
however, the electric field required by the method calls into question the integrity of the entire
protein population. Moreover, also using proteoliposomes as their precursors, Horger et al.
extended their application of hydrogel assisted GUV formation with human p-glycoprotein
(ABCB1) into giant vesicles and report on its active transport of rhodamine 123 across the
membrane.
42
While protein incorporation into liposome sized vesicles has been reported,
43, 44

reports on protein incorporation into micrometer sized vesicles without the use of an electric field
is relatively sparse.
Ligand- and antibody-binding has been are demonstrated in protein-incorporated GUVs
and are of high interests in particular for drug discovery with targets such as G protein-coupled
receptors (GPCRs). In 2013, Kang et al. reported antibody and ligand binding to the human
acetylcholine receptor (AChR).
45
Using a hydrogel stamping method to form GUVs, they
33

demonstrated binding of fluorescent neurotoxin α-bungarotoxin, selective for AChR.
46
In 2014,
May et al. incorporated the GPCR dopamine receptor D2 (DRD2) into polymeric vesicles using an
in vitro expression approach.
47
They too showed antibody and ligand binding on liposome sized
polymersomes that were dependent on the encapsulation of biomolecules for in vitro expression
of the GPCR.
47
Using the same approach de Hoog et al. incorporated the chemokine C-X-C motif
receptor 4 (CXCR4) into polymersomes and tracked its binding to antibodies via surface plasmon
resonance.
48
Ligand binding studies in model membrane system has implications in further
understanding diseases and protein activity. Much work is still needed and a body of work
regarding its optimization is presented in the following chapters.
In addition to observations of protein
function and protein binding activity, GUVs
offer a unique platform for modeling lipid raft
formation.
3
Lipid rafts are areas on the cell
plasma membrane where proteins,
sphingolipids, and cholesterol congregate to
assist in signaling pathways.
49
These rafts
have been indirectly identified in cells using
detergent resistant membrane techniques
(DRM) where cells are exposed to detergents
that force separation of membrane
components.
49
While DRMs have been
heavily used to associate proteins to lipid rafts, in 2005 Lichtenburg et al. advised that “finding a
molecule in DRM does not prove its localization in rafts (and vice versa)”.
50
They further go on to
Figure 6. Confocal micrographs of protein incorporated
GUPs with 1:3:1 POPC:BSM:Chol lipid composition. A
phase-separating lipid composition was used to ease
visualization. Left images show fluorescence from lipid
label ATTO-488-DPPE, center show antibody-tagged
G α protein subunit, and right show GUPs before and
after fluorescent antagonist binding to 5-HT 1A receptor.
10 µm scale bars apply to all panels in a row.
34

say that not all rafts are detergent resistant and that some DRM fractions have shown to be
unrelated to rafts.
50

Liquid ordered and liquid disordered phase separation in model membranes (Figure 6) is
a prominent biomimetic platform for the assessment of lipid phase behavior which avoids artifacts
like those associated with DRM induced separation.
51, 52
In GUVs, liquid-liquid phase separating
mixtures mimic lipid rafts where areas rich in sphingomyelin, cholesterol, and saturated lipids are
identified as the liquid ordered domain or lipid raft.
53
Phase-separating GUVs provide a platform
that directly visualizes different phases via fluorescence microscopy. Reconstitution of fluorescent
integral membrane proteins into GUVs thus provides opportunities for observing the phase
partitioning of proteins like α-hemolysin as demonstrated by Hansen et al. in their report on protein
incorporation into GUVs.
40

2.4 G Protein Coupled Receptors
2.4.1 Significance
An important family of proteins is the G protein
Coupled Receptors (GPCRs). This is the largest family of
membrane proteins that accounts for 2% of the human genome
and over 800 have been identified.
54
GPCRs are involved in
neuronal function and are the target of over ~40% of medical
therapies on the market.
55, 56
The two best-selling therapeutics
targeted at GPCRs in 2000 were Zyprexa
®
an antipsychotic
drug targeting serotonin and dopamine receptors, and Claritin
®

an anti-allergy therapeutic targeting the histamine H1
Figure 7. GPCR structure and
function. A) The X-ray crystal
structure of GPCR Rhodopsin
showing 7 α-helical trans-
membrane domains. Image taken
from Palczewski et al., 2000.
60
B) A
schematic showing agonist
activated GPCR catalysis of a G
protein.
35

receptor.
56
GPCRs are categorized into five classes based on their sequence homology and
structural similarities.
57
The largest class of GPCRs is class A, which includes serotonin,
dopamine, histamine, and rhodopsin.
57, 58

GPCRs are involved in signaling pathways and bind to a variety of signaling molecules
such as neurotransmitters.
54, 59
GPCRs are integral membrane proteins with seven transmembrane
α-helices.
60
They associate with heterotrimeric G proteins on the cytosolic side of membranes
(Figure 7). Upon binding an extracellular ligand, a GPCR can catalyze GDP/GTP exchange on
the G protein alpha subunit, eliciting an intracellular signaling cascade resulting in events such as
apoptosis or cell proliferation.
61
As clinically relevant proteins, GPCRs are heavily studied and
their reconstitution in lipid matrices is a critical necessity for structural and functional
investigations.
14

2.4.2 The human serotonin receptor
An important GPCR that is found
throughout the central nervous system is the 5-
hydroxytryptamine receptor subtype 1A, 5-
HT1AR, also known as the human serotonin
receptor.
62
Activation of 5-HT1AR can lead to the
inhibition of adenylyl cyclase, closing of calcium
channels and opening of potassium channels.
63

This receptor regulates mood, emotions and responses to stress and is implicated in diseases such
as schizophrenia, depression, and bipolar disorder.
64, 65
The implications of 5-HT1AR in psychiatric
disordered stems from the fact that D-lysergic acid diethylamide (LSD) is a compound that induces
Figure 8. 5-HT 1AR agonists. (Left) D-lysergic
acid diethylamide (LSD). (Right) 5-
hydroxytryptamine (serotonin, 5-HT). Both
structures are tryptophan based molecules that
increase the activity of 5-HT 1AR when bound.
36

psychotic behaviors in healthy individuals.
66
It is structurally similar to serotonin (Figure 8) and
is known to bind to 5-HT1AR in the raphe nuclei of the brain stem.
66
Alterations in 5-HT1AR have
also been reported in individuals with depression and in victims who have committed suicide.
65

Thus this receptor is particularly important for the therapeutic effects of antidepressant and
antipsychotic drugs, and lipid modulation of the receptor, specifically cholesterol modulation of
5-HT1AR, has implications in psychotic diseases.
67-69

2.4.3 Compositional dependence of GPCRs: The case from rhodopsin
Lipid compositional dependence of GPCRs is important because the composition of the
plasma membrane varies with age, diet, and disease state, making lipid bilayer modulation of
GPCRs function a possible mode of disease etiology.
5
Variation in plasma membrane composition
across tissues may lead widely distributed GPCRs to behave differently in different tissue types.
4

Membrane lipid composition is known to affect the behavior of integral membrane proteins
in general and GPCRs in particular.
70-75
In reviews by Lee (2004) and Brown (1994 and 2012), the
lipid compositional effects on proteins are conceptualized in terms of changes in bulk bilayer
properties and chemically nonspecific interactions of bilayers and proteins.
73-75
Changes in bulk
bilayer material properties such as curvature stress and line tension due to membrane composition
(experimentally testable via bilayer lipid substitutions) can shift the equilibrium between various
protein conformations.
There has been extensive study of the effect of various lipid bilayer properties on the GPCR
rhodopsin. Rhodopsin is a class A GPCR that is found in the rod outer segments (ROS) of eyes.
In vision rhodopsin detects photons and amplifies and converts the light signal into electrical
signals.
76
Hydrophobic mismatch, bilayer-induced protein oligomerization, curvature elastic
37

stress, lipid structure, lateral pressure profiles in lipid bilayers, and the effects of close range
(annular) and long range (non-annular) lipids on rhodopsin have all been reported to shift the
equilibrium between the inactive meta-I state (MI) and the active meta-II state (MII).
73-75, 77-80
In
particular, adding phosphoethanolamine lipids (PE) to lipid bilayers increases bilayer curvature
stress, and shifts the rhodopsin equilibrium from MI towards MII.
78, 79, 81
Cholesterol, which has
membrane-ordering effects, shifts the equilibrium towards MI.
79, 82, 83
Rhodopsin activity also
depends on bilayer thickness, increasing in bilayers that match the thickness of its transmembrane
domain (Figure 9).
84
Furthermore, rhodopsin-rhodopsin interactions at physiological lipid-to-
protein ratios of 70:1 favor the MI inactivate state due to oligomerization or superposition of lipid
domains surrounding the GPCR.
79

Based on evolutionary relationships (80% of GPCRs are class A—or “rhodopsin-like”—
receptors), there is reason to believe that other GPCRs may display similar dependencies.
71, 85, 86

In addition, cholesterol binding
may play a role in GPCR
function. Hanson et al. reported
a cholesterol binding site in the
adenosine 2A (A2aR) receptor;
they further identified 96
GPCRs with sequence
homology to this binding site.
87

While these results suggest lipid bilayer composition sensitivity of GPCRs, current knowledge
regarding the role that lipids play in the activity of GPCRs other than rhodopsin is scant. Thus,
Figure 9. Bilayer hydrophobic thickness dependence of GPCR
rhodopsin. Depending on the hydrophobic matching between the bilayer
and GPCR, the equilibrium state of rhodopsin can be shifted towards the
M-I inactive state or towards the M-II active state. Adapted from
Soubias et al., 2015.
85

38

there is need to develop a synthetic system that stringently controls plasma membrane composition
and effectively incorporates proteins to better understand GPCR activity and properties.
39

3. Phase behavior of 5-HT
1A
R in phase separated GUPs

This work has been published.
88

3.1 Motivation
While 5-HT1AR has been incorporated into the membranes of nanoscale liposomes, this
membrane format is inaccessible to fluorescence microscopy, making direct observations of phase
segregation impossible. Giant unilamellar vesicles (GUVs), 10-100 µm in diameter, are more
suitable for such direct observations. Here, we investigate and report the incorporation of 5-HT1AR
into giant unilamellar protein-vesicles (GUPs) and directly observe 5-HT1AR in liquid-liquid (l-l)
phase separated vesicles. Through fabrication of GUPs containing 5-HT1AR membrane fragments,
we confirm the protein’s incorporation and location in the membrane through primary antibody
labeling and ligand binding. We observe that 5-HT1AR preferentially segregates into the
cholesterol-poor liquid disordered region. Furthermore, varying concentrations of cholesterol and
brain sphingomyelin in the membrane does not affect the partitioning of 5-HT1AR.

3.2 Methods and materials
3.2.1 Materials
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), porcine brain sphingomyelin,
(BSM), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-
sn-glycero-3-phosphocholine (DPPC) and biotin-1,2-dipalmitoyl-sn-glycero-3-
phosphoethanolamine (DPPE) were acquired from Avanti Polar Lipids (USA) and used without
further purification. ATTO-488-labeled DPPE was used as a fluorescent tag without further
purification (ATTO-TEC, Germany). All reagents such as but not limited to agarose (Type IX, gel
40

point 8-17˚C), phosphate buffered saline (PBS), sodium hydroxide (NaOH), sucrose, and glucose
were of analytical grade (Sigma Aldrich, USA). Membrane fragments containing 5-HT1AR (Perkin
Elmer, USA), 5-HT1AR antibodies (Thermo Fisher, USA), and 5HT1A-633-AN2, a fluorescent
derivative of the well-known 5-HT1AR antagonist NAN-190 (Sigma Aldrich, USA), were used
without further purification. Sykes-Moore chambers (Bellco, USA) and standard 25 mm no. 1
glass coverslips (ChemGlass, USA) were used throughout. 18.2 MΩ  cm Milli-Q water was used
in all experiments (EMD Millipore, USA). Protein desalting micro spin columns (Thermo
Scientific, USA) and NHS-rhodamine (Thermo Scientific, USA) were used as per the
manufacturers’ instructions.

3.2.2 Fabrication of vesicles and protein incorporation
25 mm no. 1 coverslips were cleaned via sonication in concentrated NaOH for 30 minutes
at 35°C. Subsequent washing with water was performed to remove excess NaOH. Coverslips were
further plasma treated in a PDC-32G benchtop plasma cleaner (Harrick Plasma, USA) for 15
minutes. Coverslips were held in Sykes-Moore chambers for vesicle formation.
GUPs were formed using the agarose hydration method as reported by Hansen et al., 2013
40

and adapted from methods reported by Horger et al., 2009.
31
Briefly, a 1:19 w/v mixture of
membrane fragment suspension and agarose (2% w/v) was drop cast onto coverslips and a thin
film was made. For control GUV fabrication, protein was omitted from the thin film and only 2%
w/v agarose was used. The film was allowed to gel at room temperature. Lipid solutions of 5 mg/ml
in CHCl3 containing 0.2% mol biotin-DPPE and 0.4% mol ATTO-488-DPPE were added drop
wise to the protein-agarose film. Solvent was evaporated using a stream of N2. Lipids were
41

rehydrated with 200 mM sucrose in PBS (pH 7.4) above the gel-liquid transition temperature of
all of the lipids (~45°C) for 20 minutes (Figure 10).
For individual imaging, vesicles were harvested from the coverslip and diluted in 3x 200
mM glucose in PBS (pH 7.4). Vesicles were allowed to settle in glucose for 15 minutes at 45°C
and then anchored to coverslips functionalized with bovine serum albumin (BSA)-biotin.
Functionalized coverslips were freshly cleaned and treated with BSA-biotin (1% w/v) for 1 hour
at room temperature, leading to physiosorption of BSA onto the glass surface. Coverslips were
subsequently washed with Milli-Q water and exposed to avidin (1 mg/ml) immediately prior to
anchoring individual vesicles. Antibody binding and antagonist binding was performed at 37°C
and all imaging was performed at 30°C.
39, 89

3.2.3 Antibody labeling
5-HT1AR antibodies were equilibrated to room temperature and conjugated to NHS-
rhodamine in DMSO at 10x molar excess. Sodium bicarbonate was added as per manufacturer’s
instructions to raise the pH to 8.0. The solution was allowed to react for one hour at room
temperature. After one hour antibodies were desalted using spin columns according the
manufacturer’s instructions. Labeled antibody UV-vis absorbance was read on a NanoDrop ND-
1000 (Thermo Fisher, USA). Antibody concentration was determined to be 7.7 mg/mL and
labeling efficiency was calculated to be 0.83.

3.2.4 Antibody binding assay
Vesicles previously prepared for individual imaging were exposed to 1:1000 rhodamine-
labeled 5-HT1AR antibodies at 37°C for 1 hour. GUVs and GUPs were imaged before and after to
42

track the increased intensity at vesicle surfaces as a result of successful antibody binding. Samples
were subsequently washed with 200 mM glucose in PBS to remove excess fluorescent antibody.
GUVs and GUPs were imaged at 491 nm and 561 nm excitation corresponding to 523 nm and 575
nm emission respectively.

3.2.5 Antagonist binding assay
GUVs and GUPs anchored to a glass coverslips of observation chambers were exposed to
1 mM of 5HT1A-633-AN2, a fluorescent derivative of the 5-HT1AR antagonist NAN-190. The
concentration is 1-log unit above the Kd per product specification. Samples were incubated at 37°C
for 10 minutes.
64
GUVs and GUPs were washed with 200 mM glucose in PBS to remove excess
fluorophore and were imaged to observe antagonist binding at 491 nm and 640 nm excitation
corresponding to 523 nm and 650 nm emission respectively.

3.2.6 Microscopy
Imaging was done on a TI-Eclipse inverted microscope (Nikon, Japan) equipped with a
spinning-disc CSUX confocal head (Yokogawa, Japan) and a 16-bit Cascade II 512 EMCCD
camera (Photometrics, USA). Excitation of fluorophores was done using 50 mW solid-state lasers
at 491 nm, 561 nm, and 640 nm (Coherent Inc., Germany) at 200 ms exposure time. All images
were taken using a Plan-Apo 60x NA1.43 oil immersion Nikon objective. Z-stack images were
separated by 0.5 µm steps. Temperature control during imaging was performed using a heating-
cooling stage with a stability and accuracy of 0.1°C (Bioscience Tools, USA).

43

3.2.7 Image processing
All images were processed and analyzed using ImageJ. Standard deviation projections of
Z-stacks were produced using standard ImageJ stack tools. Fluorescent micrographs of GUVs and
GUPs using 491 nm excitation are shown using the ImageJ green lookup table, micrographs using
561 nm excitation are shown using the ImageJ red lookup table and all images using 640 nm
excitation are shown using the ImageJ blue lookup table. All images are presented without any
further processing adjustments or corrections and (with the exception of control images that show
no objects) are scaled from minimum to maximum intensity. Images in which no objects could be
discerned were adjusted to match intensity histogram of the rhodamine-band image in Figure 13
(Mean intensity: 1387.3, Standard Deviation: 3249.4, Minimum: 34.7, Maximum: 21711.7). The
adjustment eliminated the artifactual apparent amplification of background noise that occurs if
min-max scaling is applied to blank images. Images are presented as Z-stack projections unless
otherwise specified. All other analysis was done using JMP.

3.3 Results and discussion
3.3.1 Images of vesicle yield from agarose hydration method
GUPs and GUVs (vesicles without protein) were made of POPC, BSM, and Chol: a simple
tertiary mixture known to phase separate at certain compositions and temperatures.
53
POPC is
found in the outer leaflet of the plasma membrane
90
and is a major component of lipids extracted
from biological sources. Vesicles were labeled with 0.2 mol% ATTO-488-DPPE to facilitate
fluorescent observation of vesicles. Bilayer vesicles were swollen from a lipid film cast over a thin
layer of hydrogel in which 5-HT1AR membrane fragments were suspended in molten low-melting-
44

temperature agarose (Figure 10). Protein-free
control GUVs were fabricated by omitting the
membrane fragments from the agarose.

3.3.2 Antibody binding
For the assessment of antibody binding,
samples were incubated at physiological
temperature (37°C) with 5-HT1AR antibodies
labeled with rhodamine for one hour. Antibody
binding could be observed prior to observing
phase separation. GUVs and GUPs were
imaged prior to antibody exposure at both 491
nm and 561 nm excitation. After exposure to
1:1000 NHS-rhodamine conjugated 5-HT1AR
polyclonal antibody for one
hour at 37°C, GUPs were
imaged and increased intensity
at the edges (exterior) of GUPs
was observed at 561 nm
excitation. See Figure 11 and
12. To further confirm
antibody binding to 5-HT1AR
at the surface of GUPs, large
Figure 10. GUPs formed from protein-agarose thin
film. Scale bars are 10 µm. Top images show
fluorescence from 491 nm excitation showing ATTO-
488 labeled lipid and bottom images show excitation
at 561 nm with no visible fluorescence. The histogram
shows the typical vesicle size distribution for POPC
lipid compositions (n=87). Mean GUP radius is 12.1
µm with a standard error of 1.22 µm and a range of
1.1 µm to 47.1 µm.
Figure 11. GUP intensity increases during antibody binding. Left
micrograph shows GUP at 491 nm immediately after exposure to
antibody. Middle micrograph shows GUP at 561 nm immediately after
exposure to antibody. After one hour of incubation and prior to washing
with 200 mM glucose in PBS (pH 7.4) increased intensity is observed at
the surface of the GUP (right micrograph). Scale bar is 5 µm, all images
are confocal slices. The plot shows intensity of GUP at 561 nm
excitation. Maximum intensity peaks correspond to the exterior of the
vesicle and indicate successful antibody binding.
45

GUPs containing small GUPs were imaged. Fluorescence intensity was only observed on the
exterior of the larger GUPs as depicted in Figure 12. 5-HT1AR antibodies have high molecular
weight and are not expected to permeate across the membrane. The analysis in Figure 12 confirms
this.

3.3.3 Phase separation and identification of liquid ordered phase
Figure 13A shows a phase separated GUV without protein after incubation with labeled
antibody; no antibody is seen to associate with the membrane. Figure 13B shows a protein-
incorporated phase separated GUP prior to antibody incubation. After exposing GUPs to antibody
for one hour, binding of the labeled antibody to 5-HT1AR is clearly observed (Figure 13C). The
protein co-segregates with the ATTO-488-DPPE fluorescent lipid tag.
Partitioning of ATTO-488-DPPE into the liquid disordered phase was confirmed by
measuring domain size as a function of composition — dark domains occupy less vesicle surface
area as the concentration of ordered phase-preferring lipids (BSM and Chol) is decreased (Figure
14). These dark regions can be identified as liquid ordered based on previous work showing that
this lipid system exhibits liquid-liquid (l-l) coexistence at the temperatures studied here.
53
The
Figure 12. Increased intensity at
exterior GUP surface. Confocal
slice of GUPs at 491 nm (left)
and 561 nm (right) excitation
with corresponding intensity
plots. This image shows a large
GUP with smaller GUPs
encapsulated inside it. As
indicated in the right
micrograph, binding only occurs
between the proteins on the
surface of the outer GUP and the
antibody. This is consistent with
the expected membrane
impermeability of a high-
molecular-weight protein. Scale
bar is 5 µm.
46

ratios of bright pixels to total
GUV size (in pixels) in
micrographs were plotted as
a function of lipid
composition (Figure 14).
Furthermore, the ATTO-
488-DPPE lipid label
segregates in a manner
identical to that of
rhodamine-labeled DPPE,
which has been previously
shown to segregate
preferentially to liquid disordered domains (Figure 15).
91

To quantify the regions of bright and dark in phase separated vesicles, representative
images were thresholded to binary and the number of white pixels was compared to the number of
black pixels each vesicle. 1:1:3, 2:1:2, 5:1:4, and 3:6:11 POPC:Chol:BSM compositions (n = 4 for
each composition) were used in order to span the phase separation envelope as previously
described by Veatch et al. (see Figure 14B).
53
Figure 14B shows an example of an image made
binary and Figure 14C shows a plot of the ratio of bright pixels to total vesicle area versus the
composition in terms of liquid ordered-preferring (cholesterol and BSM) and disordered-preferring
(POPC) lipids. The plot clearly shows that as the concentration of cholesterol and BSM is
decreased in the system, the ratio of bright pixels to dark pixels increases. Since this system has
previously been shown to exhibit l-l phase coexistence at the temperature we studied here, the
Figure 13. Phase separating vesicles with and without incorporated 5-HT 1AR.
Lipid label fluorescence is on the top; 5-HT 1AR antibody fluorescence is on
the bottom. A) Phase separation observed on GUV (1:1:3 POPC:Chol:BSM)
without protein and incubated with antibody for one hour. No signal is
detected at 561 nm indicating that antibody does not bind to the lipid
membrane. B) Protein-incorporated GUP 1:1:3 POPC:Chol:BSM displaying
phase separation prior to antibody binding. (Confocal slice). C) GUP 1:1:3
POPC:Chol:BSM displaying phase separation after one hour incubation with
antibody. Signals from 491 nm and 561 nm excitation indicate successful
specific binding of antibody to 5-HT 1AR. All scale bars are 5 µm.
47

Figure 14. Quantification of sizes of bright domains identifies them as liquid disordered. A) Shows a ternary
plot of POPC:Chol:BSM indicating the four compositions used in investigations with representative images of
their morphologies. B) An example of vesicle micrograph of the 1:1:3 composition as taken under 491 nm (top)
and then made binary in ImageJ (bottom). Scale bar is 5 µm. C) Variability plot of ratio of bright pixels to total
vesicle size versus concentrations of liquid ordered-preferring lipids (%Chol + %BSM) and liquid disordered-
preferring lipids (%POPC). GUV on the y-axis refers to bilayer vesicles in general, both with and without protein
incorporated.
48

bright region can be identified as liquid disordered phase while the dark region is liquid ordered
phase.
Figure 15 shows images of phase-separated vesicles tagged with rhodamine-DPPE
alongside phase separated vesicles of the same composition tagged with ATTO-488-DPPE.
Rhodamine-DPPE has been previously reported to partition into the liquid disordered phase.
91
Therefore qualitative comparison of the morphologies of the vesicles makes it apparent that
ATTO-488-DPPE partitions into the liquid disordered phase of vesicles. This further confirms the
segregation of 5-HT1AR into the liquid disordered phase or bright region of phase separated
vesicles as presented in the next section (Figure 16).

Figure 15. Qualitative comparison of phase separated vesicles labeled with ATTO-488-DPPE (Top) and labeled
with rhodamine-DPPE (Bottom). Left set of micrographs correspond to POPC:Chol:BSM lipid compositions at
1:1:3 and 2:1:2. Right micrographs correspond to DOPC:Chol:BSM/DPPC compositions as listed. The
micrograph for POPC:Chol:BSM 2:1:2 labeled with ATTO-488-DPPE is a confocal slice. All other images are
Z-stack projections. Rhodamine-DPPE has been previously reported to partition into the liquid disordered region
of synthetic lipid bilayers
6
and qualitative comparison confirms that ATTO-488-DPPE also partitions into the
liquid disordered region of phase-separated vesicles.
49

3.3.4 Phase separation at varying cholesterol concentrations
Figure 176. Antibody labeling results
on GUPs at varying lipid
compositions. A) GUVs showing
protein phase segregation via antibody
binding. Excitation at 491 nm (left)
and 561 nm (middle) are overlaid in
the right image. Top is 1:1:3 and
bottom is 2:1:2 POPC:Chol:BSM.
This shows preferential segregation of
5-HT 1AR in the liquid disordered
(bright) phase.

Figure 167. POPC-based GUP compositions after antibody binding. The pairs of images on the left show controls
without 5-HT 1AR. 491 nm excitation is in the left image of each pair; 561 nm excitation is on the right. No antibody
binding is apparent. The triptychs of images on the right show GUPs with 5-HT 1AR after incubation with antibody.
In each triptych, excitation at 491 nm is on the left, 561 nm is in the center, and an overlay is on the right. Overlaid
images indicate the preferential segregation of 5-HT 1AR into the liquid disordered phase (bright region) across a
range of cholesterol concentrations. The image 491 nm 2:1:2 POPC:Chol:BSM protein-free control is a confocal
slice, all other images are Z-projections. Scale bars are 5 µm.
50

5-HT1AR segregates to the liquid disordered phase over a range of compositions spanning
the immiscible region of the POPC:Chol:BSM phase diagram.
53
Figure 16 shows two
compositions of POPC:Chol:BSM (1:1:3 and 2:1:2) yielding phase-separated vesicles with protein
incorporated following antibody binding. 5-HT1AR preferentially segregates into liquid disordered
phase regardless of sphingomyelin presence, contradicting previous DRM-based reports.
92, 93
This
preference for the disordered phase remains the case as cholesterol concentration is varied across
the immiscible region of the phase diagram as shown in Figure 17 which also includes control
images.

3.3.5 Ligand binding
To confirm the proper folding of the protein, a fluorescent antagonist, a NAN-190
derivative, was used to identify 5-HT1AR in bilayer vesicles by means of a ligand binding assay
(Figure 18). The ligand only associates to GUPs and therefore only when protein is present
(negative control in Figure 19). This binding indicates that the protein is properly folded with an
available binding site. Furthermore, as shown in Figure 18 and 19, overlap of fluorescence from
Figure 18. Ligand binding results on
protein incorporated GUPs with
varying lipid compositions. GUPs at
491 nm (left) and 640 nm (middle)
excitation show 5-HT 1AR segregation
into the liquid disordered phase after
successful antagonist binding. Top is
1:1:3 POPC:Chol:BSM and bottom is
2:1:2. GPCR preferentially
segregates to liquid disordered phase.
All scale bars are 5 μm.

51

the lipid and the ligand is observed, indicating that upon binding of the antagonist, 5-HT1AR
remains in the liquid disordered phase.

3.3.6 Antibody binding in DOPC systems
Previous reports suggest that functionality of 5-HT1AR is dependent on sphingomyelin.
94,
95
To test the role of sphingomyelin in GPCR phase behavior, GUPs made from varying
concentrations of BSM together with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol were also investigated. The
DOPC/DPPC-based system has been shown to separate into micron-scale liquid domains over a
wide range of temperatures and is not subject to photooxidation-based artifacts that have been
noted in POPC-based systems.
33, 96, 97
We prepared phase separated vesicles in which BSM was
replaced in part or completely with DPPC. Replacing POPC with DOPC in this system facilitates
phase separation with DPPC (which we used as a saturated lipid to substitute for BSM) and
Figure 19. POPC-based GUPs after incubation with the fluorescent 5-HT 1AR ligand. The pairs of images on
the left show controls without 5-HT 1AR. Micrographs are shown at 491 nm (left) and 640 nm (right) excitation.
No ligand binding is apparent. The triptychs of images on the right show GUPs with 5-HT 1AR. In each triptych,
excitation at 491 nm is on the left, 640 nm is in the center, and an overlay is on the right. Scale bars are 5 µm.
52

eliminates potential photooxidation artifacts. Ternary compositions of 3:2:5 DOPC:Chol:DPPC
and 3:2:5 DOPC:Chol:BSM and a quaternary composition of 3:2.5:2.5:2 DOPC:Chol:DPPC:BSM
were investigated (Figure 20). In all compositions, 5-HT1AR is observed to partition into the liquid
disordered phase suggesting that sphingomyelin has no particular effect on partitioning.

3.4 Conclusions
The GPCR 5-HT1AR membrane fragment-incorporated GUPs that we demonstrate here
allow for the first direct observation of the phase behavior of GPCRs in model membranes. Our
observations contradict conclusions from DRM-based studies and show that 5-HT1AR resides in
the liquid disordered phase of membranes. Sphingomyelin does not have an effect on the
preferential segregation of 5-HT1AR. This work provides a foundation for further investigations to
characterize GPCRs through microscopic observation of model membranes.
Figure 20. DOPC-based GUPs after antibody binding. The pairs of images on the left show controls without 5-
HT 1A R. Micrographs are shown at 491 nm (left) and 561 nm (right) excitation. No apparent non-specific antibody
binding occurs. The triptychs of images on the right show GUPs with 5-HT 1AR. In each triptych, excitation at
491 nm is on the left, 561 nm is in the center, and an overlay is on the right. Scale bars are 5 µm.
53

4. Plasma membrane compositional dependence of 5-HT
1A

receptor-catalyzed nucleotide exchange
This work has been published.
98

4.1 Motivation
Plasma membrane compositional dependence has been widely studied for the GPCR
rhodopsin. However, the extent to which bulk membrane properties affect other GPCRs is not well
understood. For example, the human serotonin receptor (5-HT1AR) is an important rhodopsin-like
GPCR that is involved in a number of psychological and stress-related diseases.
55
While much is
known about the implications of 5-HT1AR in psychiatric diseases and depression, the relationship
between 5-HT1AR and the plasma membrane has remained elusive. Understanding this relationship
could lead to further understanding of lipid-protein dependencies, disease etiology and would
demonstrate membrane lipid composition as a biochemical control parameter highlighting the
possibility that compositional changes related to aging, diet, or disease could impact cell signaling
functions.
5

Chattopadhyay and coworkers have extensively studied the cholesterol dependence of 5-
HT1AR. Working largely with systems based on depletion and replenishment of membrane
components in ligand binding and cell-based activity assays,
99
they have shown that cholesterol
depletion can inhibit downstream receptor activity of 5-HT1AR.
100-102
However, they have also
reported that depending on the cell type, depletion of the membrane ordering components
cholesterol and sphingomyelin can either increase or decrease agonist binding of 5-HT1AR.
103-106

54

There is clearly a strong effect of membrane
lipids on 5-HT1AR, though with conflicting
results largely depending on cell type, it is
essential to have a protein reconstitution system
in which the lipid environment can be stringently
controlled.
Here, we measure the lipid-dependent
activity of 5-HT1AR by reconstituting the protein
in GUPs with controlled composition using an
agarose hydration method (Figure 21). We
previously used this method to characterize 5-
HT1AR co-segregation with the liquid disordered
phase in liquid-liquid phase separating
membranes as discussed in the previous
chapter.
88
For this work, we encapsulate
BODIPY-GTPγS, a quenched fluorophore, into
GUPs with reconstituted 5-HT1AR and cognizant
G protein subunits. Incubating GUPs with an extracellular agonist, 8-hydroxy-2-
(dipropylamino)tetralin hydrobromide (8-OH-DPAT), triggers receptor activation and exchange
of G protein-bound-GDP for BODIPY-GTPγS. This irreversible exchange unquenches BODIPY-
GTPγS fluorescence (Figure 21) and receptor-catalyzed nucleotide exchange is measured. We use
this system to study the effects of lipid order, cholesterol concentration, and membrane curvature
stress on 5-HT1AR activity. We observed that increasing the concentration of membrane-ordering
Figure 21. Schematic of protein activity assay.
GUPs are formed via hydrogel rehydration
method. Incubation with agonist unquenches
encapsulated BODIPY-GTPγS fluorescence via G
protein binding. Fluorescence increase due to
BODIPY-GTPγS binding to G protein is tracked
over time (see inset micrographs).
55

components increases 5-HT1AR-catalyzed nucleotide exchange, and cholesterol analogs that have
a greater tendency to order the membrane than cholesterol itself results in greater increases in
receptor activity. Introduction of curvature stress from nonlamellar forming lipids also affects the
5-HT1AR activity as is the case of rhodopsin.

4.2 Methods and materials
4.2.1 Materials
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), brain sphingomyelin, (BSM),
cholesterol (Chol), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were acquired
from Avanti Polar Lipids (USA). The fluorescently tagged lipids ATTO-488-DPPE and ATTO-
550-DPPE were obtained from ATTO-TEC (Germany). Ergosterol was from Sigma Aldrich
(USA) and epicholesterol was obtained Steraloids (USA). Membrane fragments containing 5-
HT1AR were from Perkin Elmer (USA), the monoclonal antibody to 5-HT1AR was from EMD
Millipore (USA), and Gαi antibodies were from Thermo Fisher (USA). The agonist 8-Hydroxy-2-
(dipropylamino)tetralin hydrobromide (8-OH-DPAT) was from Sigma Aldrich, USA. The
antagonist spiperone was from Tocris (UK). Solutions of ligands were made to 10 mM in DMSO,
except for 8-OH-DPAT, which was prepared at 10 mM in 200 mM sucrose in PBS (pH 7.4).
GTPγS and QSY7 were obtained from Life Technologies (USA). Other chemicals including low
melting temperature agarose, phosphate buffered saline (PBS), methanol (MeOH), dimethyl
sulfoxide (DMSO), diphenylhexatriene (DPH), chloroform (CHCl3), sucrose, and glucose were of
analytical grade (Sigma Aldrich, USA). All chemicals and biomolecules were used as shipped
without further purification. Sykes-Moore chambers (Bellco, USA), standard 25 mm no. 1 glass
coverslips (ChemGlass, USA), and flat bottom 96-well plates (BD Biosciences, USA) were used
56

throughout all experiments. 18.2 MΩ  cm Milli-Q water was used in all experiments (EMD
Millipore). Protein desalting micro spin columns and NHS-Rhodamine (both from Thermo Fisher,
USA) were used as per manufacturer’s instructions.

4.2.2 Fabrication of vesicles and protein incorporation
25 mm no. 1 coverslips were cleaned via sonication in methanol for 30 minutes at 35°C.
Coverslips were dried and plasma treated in a PDC-32G benchtop plasma cleaner (Harrick Plasma,
USA) for 15 minutes. Coverslips were held in Sykes-Moore chambers for vesicle formation.
Protein-incorporated GUPs were formed using methods previously published.
88
Briefly, a
lipid film was made on freshly cleaned coverslips from lipid solutions of 3-4 mg/mL in CHCl3.
Solvent was evaporated using a stream of N2. A 1:1 v/v mixture of membrane fragment suspension
and agarose (3% w/v) was drop casted onto the same coverslips and a thin film was formed. Protein
concentration was estimated based on the reported protein concentrations as supplied in the
membrane fragments. Protein was added to the GUP formation system so as to achieve a final
receptor concentration of 0.2 nM in the GUP suspension. This corresponds to a synthetic lipid-to-
added-protein ratio of 100:1 and lipid-to-receptor ratio of 2x10
6
:1. For control giant unilamellar
vesicle (GUV) fabrication, protein was omitted from the hydrogel film and only 1.5% w/v agarose
was used. The film was allowed to gel and the system was hydrated with 200 mM sucrose in PBS
(pH 7.4) with 100 µM spiperone and BODIPY-γ-FL-GTP (5 µM). Lipid films were swollen for
30 minutes above the transition temperature of the lipids, ~40°C. Bilayer vesicles were harvested
from the coverslip and diluted in 3x of an isoosmotic glucose solution (200 mM glucose in PBS,
pH 7.4). GUPs were allowed to sediment in glucose for 30 minutes at room temperature.
57

Liposomes for encapsulation in quenching assay controls were made by extruding agarose-
formed giant vesicles. GUVs were made of POPC with 0.2 mol% ATTO-550-DPPE fluorescent
lipid and protein omitted from the agarose film. After swelling the vesicles were harvested and
extruded through a 400 nm filter with 12 passes.
Liposomes used for anisotropy measurements were made via sonication. Investigated
compositions were doped with 0.5 mol% DPH. 1 mg/mL of the lipid mixture was formed on the
side of a cell culture tube and rehydrated with water. Lipids were then sonicated for 1 hour at 37°C.

4.2.3 5-HT1AR activity assay
Prepared and settled GUPs were transferred to a flat bottom 96-well plate. 8-OH-DPAT
was added to each well to a final concentration of 150 nM immediately prior to reading
fluorescence. Control GUPs were run without the addition of the agonist. As additional controls
for the system, rehydration buffer with BODIPY-GTPγS, rehydration buffer with BODIPY-
GTPγS and agonist, and protein in membrane fragments as shipped diluted in rehydration buffer
with BODIPY-GTPγS and agonist were read. Protein in membrane fragments as shipped indicated
the intrinsic receptor-catalyzed nucleotide exchange of the membrane preparations with their
native lipid composition. Buffer with and without agonist were used for fluorescence correction
(i.e photobleaching or autofluoresence). Samples were read at physiological temperature 37°C
every 5 minutes for twelve hours (hr) to ensure complete activity assessment. Fluorescence reading
of BODIPY-GTPγS unquenching was done on a Biotek Synergy H4 Microplate Reader equipped
with a xenon flash lamp. Excitation was set to 485/20 nm and emission at 528/20 nm at a read
height of 7 mm.

58

4.2.4 Antibody labeling
5-HT1AR monoclonal and Gαi antibodies were equilibrated to room temperature and
conjugated to the NHS ester of rhodamine in DMSO at 10x molar excess. Sodium bicarbonate was
added as per manufacturer’s instructions to raise the solution pH to 8.0. The solution was allowed
to react for one hour at room temperature and overnight at 5°C. Rhodamine-labeled antibodies
were subsequently desalted using spin columns according to the manufacturer’s instructions.
Labeled antibody UV-vis absorbance was read on a NanoDrop ND-1000 (Thermo Fisher, USA).
Rhodamine labeled Gαi proteins antibody concentration was determined to be 22 µM and labeling
efficiency was calculated to be 1.15. Rhodamine labeled 5-HT1AR monoclonal antibody
concentration was 3.2 μM with 1.41 labeling efficiency.

4.2.5 Antibody label fluorescence quenching to determine protein orientation
5-HT1AR membrane fragments were incubated with rhodamine-labeled 5-HT1AR
monoclonal antibodies or rhodamine labeled Gαi monoclonal antibodies, 1:1000 dilution. The
labeled protein mixture was used in the agarose film for GUP formation. GUPs made of 0:3:2 and
1:0:0 POPC:BSM:Chol were fabricated as described above. GUPs were harvested, settled, and
placed in observation chambers. GUPs were imaged via epifluorescence prior to quenching.
QSY7, a quenching molecule, was then added to the observation chambers, 0.1 µM final
concentration, and incubated in the dark for 10 minutes. GUPs were imaged after incubation and
the amount of quenched fluorescence intensity was analyzed.

59

4.2.6 Fluorescence anisotropy measurements
Bilayer membranes were formed as liposomes. 1 mg of the desired lipid composition was
dried as a thin film in a cell culture tube, rehydrated with Milli Q water and sonicated as described
above. All lipid compositions were doped with 0.5 mol % DPH for membrane fluidity
measurements via fluorescence anisotropy. Fluorescence anisotropy measurements were made at
37°C on a QuantaMaster 4 spectroflourometer equipped with a xenon arc lamp (75 W). Readings
were performed using a slit width of 10 nm, excitation at 354 nm and emission at 435 nm. All
anisotropy readings were done for 60 seconds. Data from three separate samples were averaged
and are presented.

4.2.7 Microscopy
Imaging was done on a TI-Eclipse inverted microscope (Nikon, Japan) equipped with a
spinning-disc CSUX confocal head (Yokogawa, Japan) and a 16-bit Cascade II 512 EMCCD
camera (Photometrics, USA). Confocal excitation of fluorophores was done using 50 mW solid-
state lasers at 491 nm for BODIPY-GTPγS and ATTO-488-DPPE (emission filter centered at 525
nm), 561 nm for rhodamine (emission filter centered at 595 nm), and 640 nm for 5HT1A-633-
AN2 (emission filter centered at 660 nm, all lasers from Coherent Inc., Germany). All confocal
images were taken using a Plan-Apo 60x NA1.43 oil immersion Nikon objective. Z-stack images
were separated by 0.2 μm steps. Epifluorescence imaging was performed on the same microscope
with illumination from a 130 W mercury lamp (Intensilight, Nikon, Japan). Rhodamine and
ATTO-550-DPPE emission was excited using a green filter (528-552 nm bandpass, 540 nm cut-
on wavelength). Temperature control during imaging was performed using a heating-cooling stage
with a stability and accuracy of 0.1°C (Bioscience Tools, USA).
60

4.2.8 Image processing
All images were processed and analyzed using ImageJ. All confocal images are presented
as standard deviation projections of Z-stacks and were produced using standard ImageJ Stack
Tools. Particle analysis and measurements were performed using ImageJ Analyze Tools.
Fluorescent micrographs of vesicles using 491 nm excitation are shown using the ImageJ green
lookup table, micrographs using 561 nm excitation are shown using the Image J orange lookup
table, and images excited using 640 nm wavelength are shown using the ImageJ blue lookup table.
All images are presented without any further processing adjustments or corrections and are scaled
from minimum to maximum intensity.

4.2.9 Data analysis
Fluorescence microtiter results were collected and analyzed using JMP. About five
separate GUP sample preparations were averaged to obtain a single curve for each of the
compositions investigated. Standard error of the mean values for each data point were determined
and are plotted as shaded areas around the curves showing the average values (Figure 26-27, 29-
31, 32, and 34). Control curves are the average of all individual observations for the indicated
composition. They include samples representing all the variations in lipid compositions. Upon
obtaining the raw fluorescence intensities (arbitrary units, a.u), they were converted to percent
fluorescence intensity increase to account for variation in sample preparation. Furthermore, these
were normalized to 1 to aid in comparing the rates of specific data sets. The data from each lipid
composition were fit to a single exponential using the JMP mechanistic growth analysis and the
rates were obtained. Statistical analysis using ANOVA followed by post-hoc Tukey Kramer
pairwise comparison of means were done using JMP with a 95% confidence interval (α=0.05).
61

Fluorescence intensity analysis of GUPs prior to and after quenching was performed in
ImageJ. A line segment was drawn across a GUP and the intensity profiles across GUP diameter
were obtained. Intensities across the same line segment of unquenched and quenched GUPs were
averaged and the percent of retained fluorescence intensity was calculated.

4.3 Results and discussion
4.3.1 Protein orientation of GPCR 5-HT1AR in GUPs
Protein orientation was determined in GUPs formed in the presence of rhodamine-labeled
monoclonal antibody for either receptor (5-HT1AR antibody) or G protein (Gαi antibody). The 5-
HT1AR antibody used binds to the
cytosolic domain. A fluorescence
quencher (QSY7) was added to a
suspension of non-phase separating
solution of GUPs. QSY7 is a
commercially available molecule that
efficiently quenches the emission from a
broad range of fluorophores via Förster
resonance energy transfer (FRET) and
contact quenching.
107
This charged,
hydrophilic quencher accesses
fluorophores only on GUP exteriors, as
shown in Figure 22. In Figure 22, GUP
made of 15:3:2 POPC:BSM:Chol (75%
Figure 22. QSY7 does not cross GUP membranes. GUPs
encapsulating 400 nm liposomes were imaged and quenched
using QSY7. After incubation with QSY7 for ten minutes at
room temperature GUPs show an intensity decrease of
53.2% ± 2.7% while encapsulated liposomes do not show
any significant decrease in intensity (< 3% difference).
QSY7 does not cross the bilayer and effectively quenches
fluorophore on the exterior of GUPs. Micrographs are Z-
stack standard deviation projections using confocal
microscopy. Scale bar is 5 μm.
62

POPC) and tagged with ATTO-488-DPPE were formed to encapsulate 400 nm liposomes tagged
with ATTO-500-DPPE. Liposomes were fabricated in the same way as control GUPs without
protein then subsequently extruded through a 400 nm polycarbonate filter. Liposomes were mixed
with agarose during fabrication to allow for encapsulation in GUPs. GUPs were harvested and
settled to remove excess liposomes in the surrounding buffer. After incubation with QSY7 for ten
minutes at room temperature GUPs show an intensity decrease of 53.2% ± 2.7% while
encapsulated liposomes do not show any significant decrease in intensity (< 3% difference). Thus,
QSY7 does not cross the bilayer
membrane of our GUPs and
effectively quenches fluorophore
on the exterior of GUPs.
GUPs made of 100% 1-
palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC) and
60%:40% brain sphingomyelin
(BSM):cholesterol (Chol) (0%
POPC) were formed in the
presence of labeled antibodies and
exposed to QSY7 (0.1 mg/mL
final concentration) for 10
minutes at room temperature. To
determine the extent of
quenching, images of the 5-
Figure 23. Fluorescence quenching image analysis. All vesicles are
made of 60%:40% BSM:Chol (0% POPC). The control vesicles are
made without protein. The leftmost micrographs show the 5-HT 1A
receptor with rhodamine-antibody tagged on its cytosolic face. The
center micrographs show GUPs with the G proteins tagged with
rhodamine-antibody. The rightmost micrographs show vesicles with
ATTO-488-DPPE fluorescent lipid. The top row shows vesicles prior
to incubation with QSY7 and bottom row shows GUPs after QSY7
quenching. The bottom plots show the fluorescence intensities across
the same line segment on before and after images. Fluorescence
intensity analysis was performed using values from the plots. The
receptor retains 90% of its intensity while the G protein and lipid tag,
ATTO-488-DPPE, retain ~50% of their intensities. This indicates a
biased orientation of the receptor upon incorporation into GUPs. Scale
bar is 10 μm.

63

HT1AR with rhodamine-antibody
tagged on its cytosolic face were taken
before and after exposure to QSY7.
ATTO-488-DPPE lipid dye was imaged
before and after incubation in QSY7 on
both GUPs with tagged 5-HT1AR and
GUVs without protein; GUVs without
protein were used as controls. A line
segment was drawn across each vesicle
and the intensity profile of the line
segment across the vesicle before and
after incubation with QSY7 were
compared as shown in Figure 23.
Figure 24A shows that in both
lipid compositions, fluorescent labels
on 5-HT1AR antibodies retain over 90%
of their initial intensity. This indicates
that the reconstituted receptor is
incorporated in a biased and correct
orientation, with the cytoplasmic domain in the GUP interior. An analysis of variance (ANOVA)
on these compositions did not yield a significant variation among conditions, F(1, 14)=0.17,
P>0.69. A post-hoc Tukey-Kramer analysis of mean pairs of the two GUP compositions (α=0.05)
further rejects the null hypothesis that the means of the two GUP compositions are statistically
Figure 24. GPCR orientation determination. 100% POPC and
60%:40%BSM:Chol GUPs were formed with labeled
antibodies to either the cytoplasmic domain of the GPCR or the
G α protein subunit. Control vesicles were formed without
protein and labeled fluorescently with ATTO-488-DPPE. (A)
Quenching of cytosolic-bound-rhodamine-antibody tagged 5-
HT 1AR results in retention of ~90% of fluorescence. This
indicates that incorporated receptors are oriented with the N-
terminus extracellular and C-terminus interior. Controls show
50% retained intensity. Analysis of variance (ANOVA)
indicates no significance between the two GUP samples, F(1,
14)=0.17, P>0.69, as also confirmed with post-hoc Tukey-
Kramer analysis. (B) G proteins tagged with rhodamine-labeled
antibody show an unbiased distribution between the inner and
outer bilayer leaflets. There is no significant difference between
the GUPs and control, F(2,27)=0.64 P>0.53.
64

different. The control for the data set is a GUP formed with a fluorescently labeled lipid, ATTO-
488-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (ATTO-488-DPPE). The fluorescent
label is quenched on the exterior of the GUP but retained on the interior (~50% intensity retention,
see Figure 24).
A similar analysis was done with rhodamine tagged antibodies that bind to the Gαi subunit
that couples to 5-HT1AR (Figure 24B). In this case, only ~65% of the original intensity remains
after quenching with QSY7. Thus G protein subunits do not display a significant bias to either the
Figure 25. Vesicle growth from agarose lipid film. The top micrographs show example images of the agarose
lipid film at the indicated time points during the hydration and vesicle swelling process. Vesicles form as small
vesicles that coalesce over time to form giant unilamellar vesicles. A 100% POPC lipid mixture was used with
0.2%mol ATTO-488-DPPE as the fluorescent dye. Micrographs are epifluorescent images. Scale bar is 20 µm.
Systematic analysis of the film using ImageJ particle analyzer is plotted below the micrographs. As time
progresses the number of vesicles decrease while the mean radius of the vesicle population increases, indicating
vesicle fusion as a means of vesicle formation using the agarose hydration approach.
41

65

interior or exterior leaflet of our GUP bilayers. These measurements do not statistically differ from
the negative control data (see Figure 24B). The lack of preference towards bilayer leaflets
observed of the G proteins could be attributed to the fact that G proteins are anchored to the bilayer
via a palmitoylated lipid tails rather than being integral to the membrane.
The orientation of the receptor may be induced by membrane curvature.
108
In forming
GUPs using our method, GUPs initially form as small vesicles, likely of nanometer scale. Overtime
these smaller vesicles coalesce to form giant unilamellar vesicles (Figure 25).
31
At small vesicle
diameters, membrane curvature is high, facilitating curvature-induced orientation. The retention
of protein orientation in a biased and correct manner as facilitated by our method of GUP formation
offers a platform that decreases the effects of incorrect receptor orientation in GPCR
investigations.

4.3.2 Effects of lipid order on receptor-catalyzed nucleotide exchange
Lipid order dependence of 5-HT1AR was investigated by varying ternary compositions of
GUPs made from POPC, BSM, and Chol. None of the compositions investigated were liquid-
liquid phase separating lipid mixtures.
53
Compositions studied span from pure POPC (less ordered)
to binary BSM:Chol (more ordered). Protein was added to the GUP formation system so as to
achieve a final receptor concentration of 0.2 nM in the GUP suspension. This corresponds to a
synthetic lipid-to-added-protein ratio of 100:1. GUPs are expected to contain about 20% of other
lipid and other proteins contributed from membrane fragment preparations. The same product lot
and concentration of membrane fragment preparation were used throughout all experiments,
meaning that the lipid and other protein composition in GUPs from the membrane fragments
remains a constant background composition. The composition-dependent effects observed in this
66

report can therefore be attributed to the synthetic lipids used in our GUP preparations. 5-HT1AR
GUPs were fabricated in the presence of the antagonist spiperone to limit basal activity. GUPs
were then exposed to excess agonist, 8-OH-DPAT to determine rates of receptor-catalyzed
nucleotide exchange.
For functional assays, non-phase separating GUPs were fabricated and transferred to 96-
well plates and fluorescence measurements were taken every five minutes for twelve hours (hr)
(Figure 26). Raw fluorescence intensity data was converted to percentages to account for
differences in GUP sample size and then normalized to facilitate rate comparisons. Each
fluorescence curve represents on average five separate GUP samples (Table 1). Percent intensity
increase of each sample at each time point was averaged for the indicated composition. Control
curves are averaged from all samples of a composition set without agonist, and represent basal
activity (Figure 27). Individual rates of control GUPs and GUPs exposed to agonist can be seen
in Table 1. As seen in Figure 26, increasing membrane-ordering components (BSM:Chol)
increases the measured rate of receptor-catalyzed nucleotide exchange of 5-HT1AR. Protein in
membrane fragments as shipped was diluted in 200 mM sucrose in 1x PBS with BODIPY- GTPγS,
exposed to 8-OH-DPAT and assayed using the same approach as the GUPs as a positive control
for intrinsic activity; these were not pre-exposed to spiperone and displayed a rate of5.29 (x 10
-3
)
min
-1
. GUPs made completely out of BSM:Chol have a rate constant over double that of pure
POPC GUPs (10.83 versus 4.26 (x 10
-3
) min
-1
). Thus it is observed that increase ordering
component BSM and Chol increases 5-HT1AR activity in our GUPs.

67

Figure 26. Receptor-catalyzed nucleotide
exchange rates in ternary and binary GUPs.
Fluorescence intensity over time of an average of
five observations with identical experimental
conditions is shown as single individual curves.
The shaded area around the points indicates the
standard error mean of averaged samples.
Control samples are the average of all
observations of the listed compositions of GUPs
tracked in the absence of agonist and are
indicated in pink. (A) GPCR activity rate in
GUPs with increasing amounts of ordering
components, BSM and Chol, in ternary GUPs of
POPC:BSM:Chol. Going from 100% POPC to
60%:40% BSM:Chol (0% POPC) shows an
increased rate of receptor-catalyzed nucleotide
exchange. (B) GPCR activity rate in binary
GUPs of POPC:Chol. Increasing the amount of
Chol in these systems increases the rate of
receptor-catalyzed nucleotide exchange. (C) Plot
of rates from (A) and (B) by Chol and BSM
composition. Standard error bars are not
included as they are smaller than the markers.
See Table 1 for standard error data. Ternary
GUPs indicated by circles in blue show faster
protein rates than binary GUPs indicted by
squares in green
68

Previous work by the Chattopadhyay group has shown that ligand binding of the 5-HT1A
serotonin receptor depends on cholesterol and sphingomyelin. They report that cleaving the
headgroup of sphingomyelin decreases ligand binding ability of the serotonin receptor.
103

Furthermore, they report that in solubilized membranes, depletion of cholesterol decreases ligand
binding ability while depletion of cholesterol in neuronal cells enhances ligand binding.
105, 106

These reports provide conflicting conclusions regarding the ligand binding ability of 5-HT1AR,
and further work on membrane ordering components on 5-HT1AR and other GPCRs is relatively
limited. Our results provide further insight into understanding the role of bulk bilayer membrane
properties on GPCR activity, specifically the rates of receptor-catalyzed nucleotide exchange.
These membrane properties could be the result of nonspecific interactions of membrane ordering
lipids or through cholesterol binding to the receptor.

Figure 27. Control fluorescence intensity curves for all compositions investigated. These data represent protein
activity with no agonist added; each curve represents a single sample. Each plot is indicated by the lipid system
and the basal rate for that system as determined by a single exponential fit to the average of all curves shown in
the plot. The control curves presented in the main text represent this average.

69

Table 1. Summary of rates from all compositions. Lipid concentrations are given in mol%.

POPC BSM Chol
Agonist
Rate
Std Error
Control
Rate
Std Error
(10
-3
min
-1
) (10
-3
min
-1
) (10
-3
min
-1
) (10
-3
min
-1
)
100 0 0 4.26 0.09 1.23 0.05
75 15 10 6.66 0.12 2.23 0.05
50 10 40 7.06 0.13 2.67 0.05
25 35 40 8.29 0.15 2.91 0.05
0 60 40 10.83 0.26 3.24 0.05
POPC Chol
90 10 4.91 0.11 1.97 0.10
75 25 5.18 0.15 2.94 0.05
50 50 8.06 0.22 3.09 0.05
POPC Ergosterol
90 10 10.19 0.15 3.59 0.07
75 25 12.82 0.23 3.88 0.06
50 50 17.26 0.34 6.22 0.87
POPC Epicholesterol
90 10 8.04 0.10 3.11 0.03
75 25 8.92 0.11 3.17 0.02
50 50 9.88 0.14 3.21 0.02
POPC DOPE
97.5 2.5 11.99 0.06 3.05 0.07
92.5 7.5 22.67 0.14 4.92 0.08
90 10 19.66 0.03 4.91 0.09
75 25 16.12 0.02 4.86 0.10
50 50 9.38 0.01 4.79 0.09
70

4.3.3 Activity assay validation
The measurement of receptor-catalyzed
nucleotide exchange rates for GUPs was done
through an activity assay as described in the
Methods and Materials section 4.2. Briefly,
GUPs were made using an agarose hydration
method by making a thin film of protein-
agarose, casting a thin film of the desired lipid
composition and rehydrating the system with
PBS with BODIPY- GTPγS and spiperone.
Through the rehydration process the BODIPY-
GTPγS is encapsulated in the GUPs. Spiperone
is added to limit intrinsic protein basal activity.
GUPs are settled in an isosmotic glucose
solution and then transferred to a 96-well plate.
Prior to reading, 8-OH-DPAT is added to sample
wells and the plate is read at 37°C every 5
minutes for 12 hr. Ternary POPC:Chol:BSM
GUP size distributions are shown in Figure 28.
In order to validate this experimental set up,
BODIPY-GTPγS autofluorescence, protein
thermal stability, and GUP response to varying
Figure 28. Size distribution of GUPs of
POPC:BSM:Chol. GUPs with rhodamine labeled
antibody tagged serotonin receptor were imaged
using epifluorescence and size distribution analysis
was performed using ImageJ particle analyzer. Table
shows average radius in µm for each of the different
compositions of GUPs, followed by respective
histograms.
71

amounts of agonists were
evaluated. Throughout the
following discussion, 100%
POPC refers to vesicles
fabricated from pure POPC
while 0% POPC refers to
vesicles fabricated from a 3:2
molar ratio of BSM:Chol.
To determine the degree
of non-specific interaction between BODIPY-GTPγS and lipids, GUVs of 100% POPC and 0%
POPC (60%BSM :40%Chol) were made without protein. These GUVs were made using the same
methods as GUP formation but omitting the protein from the agarose. GUVs contained BODIPY-
GTPγS and were subjected to fluorescence intensity microplate reading at 37 ºC for 12 hr with and
without the addition of agonist, 8-OH-DPAT. As shown in Figure 29, the percent intensity
increase is roughly 5% for both GUV compositions and for both control (Ctl) and agonist exposed
(+Ag) conditions. This amount of fluorescence intensity increase is significantly less than the
typical fluorescence increase observed in GUPs with and without agonist. GUPs display a
fluorescence intensity increase above 75% (normalized to 1 elsewhere in this work for ease of
comparison of rates).
To determine expected protein thermal stability over the 12-hour experimental course at
37 ºC, 5-HT1AR membrane preparations were subjected to pre-incubation at 37 ºC for 0, 6, 18, and
24 hr prior to being used for GUP formation and subsequent evaluation of receptor-catalyzed
nucleotide exchange via the activity assay. As seen in Figure 30 and Table 2,
Figure 29. Nonspecific BODIPY-GTPγS fluorescence with GUVs.
GUVs without protein were formed of 100% POPC and 0% POPC lipid
compositions and incubated with (+Ag) and without agonist (Ctl). GUVs
show less than 5% fluorescence intensity increase while GUPs of the
same composition show over 75% fluorescence intensity increase.
BODIPY-GTPγS interaction with GUVs yields insignificant
fluorescence.

72

0hr, 6hr, and 18hr time points, the rates of receptor activity in GUPs of 100% POPC and 0% POPC
were not statistically different. This indicates that the protein is stable up to 30 hr of incubation at
37 ºC. At 24 hr of pre-incubation however, the protein displays no significant fluorescence increase
and thus no rate is obtained from the activity assay. Thus, sometime between 30 hr (18 hr of pre-
incubation) and 36 hr (24 hr of pre-incubation) the system fails to show protein activity.

Figure 30. Protein thermal stability investigated by pre-incubating membrane fragments. 5-HT 1AR membrane
fragments were pre-incubated at 37 ºC for 0, 6, 18, and 24 hr and then incorporated in GUPs for the 12-hour
activity assay. GUPs were made of 0% POPC or 100% POPC. Percent intensity increase was tracked over time
and plotted. Plots are an average of 6 replicates and shaded areas around the points of the curve are the standard
error of the mean. 0, 6, and 18 hr pre-incubated protein samples retain activity and fluorescence intensity
increase. 24 hr pre-incubated GUPs did not display significant fluorescence intensity increase over time.
73

Table 2. 5-HT1AR thermal stability. 24 hr time point displayed no measurable protein activity, see
Figure 28.

0% POPC 100% POPC
Pre -
Incubation
time
+Ag Ctl +Ag Ctl
Rate +/- Std
Error
(10
-3
min
-1
)
Rate +/- Std
Error
(10
-3
min
-1
)
Rate +/- Std
Error
(10
-3
min
-1
)
Rate +/- Std
Error
(10
-3
min
-1
)
0 hr 10.83 +/- 0.26 3.24 +/- 0.05 4.26 +/- 0.09 1.23 +/-0.05
6 hr 10.47 +/- 0.10 4.41 +/- 0.04 4.96 +/- 0.54 1.49 +/- 0.49
18 hr 9.63 +/- 0.21 3.45 +/- 0.15 5.06 +/- 0.19 1.20 +/- 0.12
24 hr N/A N/A N/A N/A

Figure 31. Increasing agonist concentration. GUPs of 100% POPC and 0% POPC were formed and subjected
to activity assay with increasing amounts of 8-OH-DPAT, 0 M, 150 fM, 150 pM, and 150 nM. A) Increasing
the amount of agonist in the assay displays increasing rates of intensity increase. B) Activity rates
corresponding to curves in A.
74

To observe a pharmacological response to the addition of ligand, GUPs of 100% POPC
and 0% POPC were exposed to increasing amount of agonist. GUPs were formed and assayed as
previously described and 0 M, 150 fM, 150 pM, or 150 nM of agonist was added immediately
prior to fluorescence intensity reading for 12 hr. As expected, increasing amounts of agonist
resulted in increased rates of 5-HT1AR catalyzed nucleotide exchange (Figure 31). The rates do
not, however, display a logarithmic increase due to the presence of antagonist spiperone.

4.3.4 Effects of cholesterol and cholesterol analogues on receptor-catalyzed nucleotide
exchange
The independent effects of Chol on protein functionality were also investigated, using
binary POPC:Chol systems (Figure 26B and 260C, Table 1). At Chol concentrations of 0%, 10%,
25%, and 50%, we observed increasing rates of 5-HT1AR catalyzed nucleotide exchange. The rates
fitted from a single exponential curve are shown in Table 1 and fluorescence intensity curves are
depicted in Figure 26B. While increasing Chol concentration increased catalyzed exchange rates,
GUPs with both ordering components Chol and BSM had a higher rate than any binary POPC:Chol
GUP.
5-HT1AR Chol dependence has been previously observed,
106, 109
but it is unclear whether
the effects are due to direct cholesterol binding to the receptor or due to cholesterol-induced
changes in bulk membrane bilayer properties.
110, 111
Such properties may include ordering, or the
capacity of cholesterol to sequester ligands near the bilayer surface.
112
Cholesterol has been shown
to increase bilayer thickness with increasing concentration, and alterations to the energy of
membrane elastic deformations due to increased lateral area compressibility coefficient and elastic
75

bending modulus as also been observed.
113
To
determine if the effects of Chol are due to direct
binding or changes in membrane properties
(ordering, packing density, etc), the effects of
the Chol analogs epicholesterol and ergosterol
were examined. Ergosterol is the primary sterol
in fungal membranes and epicholesterol is a
Chol diastereomer;
114
their structures are shown
in Figure 32. The effects of ergosterol and
epicholesterol are shown in Figure 32A and
32B, respectively. As the amount of either sterol
increases, the measured rate of receptor-
catalyzed nucleotide exchange increases. This
suggests that changes in membrane properties
rather than direct Chol binding result in
increased receptor activity. A previous report
suggested cholesterol binding increases the
ligand binding ability of 5-HT1AR but does
report on receptor-catalyzed nucleotide
exchange of the serotonin receptor,
101
thus our
results provide initial insight into the effects of cholesterol and its analogues to receptor-catalyzed
exchange rates and activity.
Figure 32. Receptor-catalyzed nucleotide exchange
rate of GUPs made with Chol analogs. (A). Binary
POPC:Ergosterol GUPs were assessed for receptor-
catalyzed nucleotide exchange activity. As mole
percent of ergostolerol is increased, exchange rate
also increases. The chemical structure of ergostolerol
is placed within the plot with its differences from
Chol highlighted. (B) Binary POPC:Epicholesterol
GUPs also show an increase in receptor-catalyzed
exchange rate with increasing amounts of
epicholesterol. Epicholesterol is a diastereomer of
Chol with the hydroxyl group on the alpha face of
Chol as shown in the chemical structure within the
plot. Control curves are the average of all individual
observations for the listed compositions of GUPs
without agonist incubation.

76

Our results further show that ergosterol shows a greater effect than epicholesterol in
increasing rates of receptor exchange. Ergosterol is also known to induce more membrane ordering
than epicholesterol or Chol.
114
Thus, fluorescence anisotropy measurements of vesicles formed
without protein and doped with 0.5 mol% diphenylhexatriene (DPH) were performed to determine
the relative degrees of ordering in the bilayer.
115
Results shown in Table 3 indicate that lipid
ordering increases from Chol to epicholsterol to ergosterol. The idea that membrane order is the
key determinant of 5-HT1AR activity is borne out by the fact that the most highly ordered
compositions studied here (incorporating both Chol and BSM)
116
display the highest rates of
receptor-catalyzed nucleotide exchange.

4.3.5 Effects of elastic curvature stress on receptor-catalyzed activity
Elastic curvature stress as a result of nonlamellar phase forming lipids in bilayer
membranes have been shown to affect the MI-MII equilibrium of rhodopsin shifting it towards the
MII state.
80
To elucidate if elastic curvature stress as a membrane property affects 5-HT1AR
activity, GUPs made from POPC and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)
were assessed. POPC:DOPE GUPs with 2.5%, 7%, 10%, 25%, and 50% DOPE (Figure 33 and
Figure 34) were analyzed. We observed that adding DOPE increases the measured GPCR activity
rate overall, however the rate initially increases then decreases with increasing DOPE going from
% POPC Anisotropy Anisotropy Anisotropy Anisotropy Anisotropy
100 0.146 ± 0.008
90 0.168 ± 0.004 0.182 ± 0.003 0.171 ± 0.002 0.149 ± 0.003
75 0.184 ± 0.005 0.224 ± 0.001 0.215 ± 0.003 0.212 ± 0.004 0.143 ± 0.004
50 0.287 ± 0.012 0.248 ± 0.001 0.268 ± 0.002 0.241 ± 0.003 0.142 ± 0.001
25 0.271 ± 0.005
0 0.331 ± 0.006
POPC:Chol POPC:Ergosterol POPC:Epicholesterol POPC:DOPE POPC:Chol:BSM
Std Dev Std Dev Std Dev Std Dev Std Dev
Table 3. Bilayer ordering results as determined by fluorescence anisotropy measurements for all
lipid compositions. Lipid concentrations are given in mol%.
77

11.99 to 22.67 to 9.38 (x 10
-3
) min
-1
(Table 1). These rates are faster than rates observed in all
DOPE-free compositions reported. According to fluorescence anisotropy measurements (Table 3)
POPC:DOPE GUPs are no more ordered than pure POPC membranes, thus the effects of DOPE
are likely not due to membrane
ordering effects as seems to be
the case for Chol and the
cholesterol analogues studied
here.
The increased rate of 5-
HT1AR activity can be
considered in terms of elastic
curvature stress and bilayer
membrane pressure profile. Pure
DOPE is known to exist in the
curved hexagonal phase (HII) at
physiological temperatures.
73
In
compositions with at least 20%
lamellar forming lipids,
however, bilayers are formed
with DOPE and show increased
membrane frustration due to
negative curvature induced by
Figure 33. GUP formation of binary POPC:DOPE compositions.
While DOPE is a nonlamellar forming lipid, GUPs are successfully
formed in binary mixtures of POPC:DOPE with DOPE at 10%, 25%,
50%. In comparison with forming vesicles without proteins, GUPs
of POPC:DOPE are larger in radius. DOPE was run at 100 mol%
using our agarose hydration method as a control. No vesicles or
bilayer structures were formed. The table shows relative average
radii for different POPC:DOPE compositions. These binary
compositions are smaller than 100% POPC GUPs.
78

the cone shape of DOPE. Coupling of
the spontaneous curvature of PE lipid
bilayers to the conformational changes
of transmembrane proteins has also
been suggested to relieve membrane
stress, lowering the free energy of the
system.
73, 75, 78
As an annular lipid
immediately next to the protein, DOPE
can fill free volume between integral
proteins and bilayer components; it can
also assist in the hydrophobic matching of lipid to bilayer.
73
Lipids can stretch to match the
hydrophobic length of the protein and these lipids are depicted as having negative curvature which
suggests a possible location of DOPE in the bilayer.
71, 73, 78
Thus, the presence of DOPE in our
GUPs with a lipid to protein ratio of 100:1, and a peak rate of 5-HT1AR activity at 7.5-10 mol%
could be explained in terms of the release of elastic curvature stress due to favorable lipid and
protein interactions at these specific bilayer compositions.
Membrane curvature has been linked to membrane pressure profiles in molecular dynamic
simulations of bilayer membranes.
117
Thus, the effects of DOPE on the GPCR may also be
considered in terms of its modulation of the bilayer membrane pressure profile. In molecular
dynamic studies, simulated DOPE pressure profiles show an additional negative peak as compared
to DOPC.
117
This negative peak is in the headgroup region of the membrane and is indicative of
where the bilayer would contract to minimize free energy due to the cone shape of DOPE.
117

Because the DOPE headgroup is small it changes membrane pressure and hydration, which may
Figure 34. 5-HT 1A receptor-catalyzed nucleotide exchange rate
of GUPs made of POPC:DOPE. The overall rates are well above
other compositions investigated, ranging from 9.38-22.67 (x 10
-
3
) min
-1
, see Table 1. However, as DOPE mole percent increases
beyond 7.5% the protein functional rate decreases.
79

result in the modulation of GPCR function that we report here. Simulations with ergosterol in
bilayer membranes also display a change in membrane pressure profiles similar to that seen with
DOPE.
118
Thus, the shape of DOPE lipids and the propensity for ergosterol to order lipids have
similar effects on altering the membrane pressure profile; the mechanism by which this change
affects protein function would therefore be common to both compositional changes.

4.4 Conclusions
We report direct observations showing lipid compositional dependence of 5-HT1AR
activity in GUPs. Increasing the concentration of order-inducing lipid components (Chol,
ergosterol, epicholesterol, BSM) increases the receptor-catalyzed nucleotide exchange rate of 5-
HT1AR. Furthermore we see significant rate increases in the presence of DOPE, suggesting that
the elastic energy dependence widely reported for rhodopsin also holds true for 5-HT1AR. Changes
in membrane pressure due to DOPE and ergosterol may also modulate GPCR protein function.
Protein incorporated into model membranes using this approach allows for controlled
compositional changes in protein membrane environment and the ease of the experimental method
provides opportunities for evaluating the effects of various ligands and effectors on orphan GPCRs
and other proteins. Stringent control of lipid composition in model membranes may further
elucidate novel therapeutic approaches for diseases related to GPCR activity. This approach,
therefore, provides a platform for evaluating lipidic parameters effecting GPCR exchange and
protein activity that cannot be investigated using in vivo or traditional methods.
80

5. 5-HT
1A
R incorporated polymeric vesicles retain functional
activity through cycles of dehydration and rehydration

This work has been submitted for publication as of June 24, 2016.

5.1 Motivation
Since their discovery in 1999 polymersomes have been used as biomimetic platforms to
better understand physiological and material properties of cells.
20
Compared to their liposomal
counterparts, polymersomes display greater stability, decreased permeability, and have been
exploited and hybridized with cellular components such as lipids and proteins for drug delivery
and research.
21, 24

25
They have
been used as drug delivery
systems and offer robust
platforms for drug discovery and
cellular functional screening.
25, 27,
119
While the incorporation of
proteins into polymersomes has
been reported current efforts
remain limited.
28, 29, 47, 48
Current
approaches that incorporate
GPCRs into polymersomes are
limited by 1) the need for
encapsulation of expression
Figure 35. Schematic of pGUP formation, lyophilization, and
functional assay. Step 1. Films of agarose and protein are deposited on
to a coverslip and a thin film of diblock copolymer PBd(650)-PEO(400)
is added. The system is swollen with 200 mM sucrose in PBS (pH 7.4)
and BODIPY-GTPγS. Step 2. Upon pGUP formation, vesicles are
lyophilized. Step 3. After dehydrated storage pGUPs are rehydrated
with Milli Q water. Step 4. Rehydrated pGUPs are assayed for agonist
induced functionality.
81

components, 2) the lack of cognizant G protein subunits, and 3) liposomal sized vesicles of 100-
150 nm make them inaccessible to microscopy. To overcome these limitations we present a robust
platform for incorporation of GPCRs into diblock copolymer giant unilamellar polymersomes in
the micrometer range that allows for observations of GTP/GDP exchange on G proteins catalyzed
by the human serotonin receptor 5-HT1AR (Figure 35). We further exploit the stability of
polymersomes and show that following lyophilization and rehydration, polymeric giant
unilamellar protein-vesicles (pGUPs) with integrated GPCRs retain vesicle integrity and protein
function.

5.2 Methods and materials
5.2.1 Materials
Diblock copolymer poly(butadiene-b-ethyleneoxide) (PBd(650)-PEO(400)) was acquired
from Polymer Source (Canada) and used without further purification. ATTO-488-DPPE
fluorescence tag was used as indicated (ATTO-TEC, Germany). All reagents such as but not
limited to low melt-temperature agarose, phosphate buffered saline (PBS), dimethyl sulfoxide
(DMSO), chloroform (CHCl3), methanol (MeOH), sucrose, glucose, WAY 100635, methiothepin
maleate and agonist 8-Hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) were of
analytical grade from Sigma Aldrich, USA. Membrane fragments containing 5-HT1AR (Perkin
Elmer, USA), 5-HT1AR monoclonal antibodies (Millipore, USA) and Gαi protein antibodies
(Thermo Fisher, USA), antagonist spiperone (Tocris, UK) and NAN-190 (Tocris, UK) were used
without further purification. Sykes-Moore chambers (Bellco, USA), standard 25mm no. 1 glass
coverslips (ChemGlass, USA), and flat bottom 96-well plates (BD Biosciences, USA) were used
throughout all experiments. 18.2 MΩ  cm Milli-Q water was used in all experiments (EMD
82

Millipore, USA). Protein desalting micro spin columns (Thermo Scientific, USA) and NHS-
rhodamine (Thermo Scientific, USA) were used as per the manufacturer’s instructions. BODIPY-
GTPγS and QSY7 were obtained from Life Technologies, USA and used as directed by
manufacturer’s instructions. NHS-rhodamine was made in DMSO at 25 mg/ml. Solutions of
ligands were made to 10 mM in DMSO. 8-OH-DPAT was made at 10 mM in 200mM sucrose in
1xPBS (pH 7.4).

5.2.2 Fabrication of polymer vesicles and protein incorporation
25 mm no. 1 coverslips were cleaned via sonication in MeOH for 30 minutes at 35°C.
Coverslips were dried and then plasma treated in a PDC-32G benchtop plasma cleaner (Harrick
Plasma, USA) for 15 minutes. Coverslips were held in Sykes-Moore chambers for vesicle
formation. Protein-incorporated diblock copolymer giant unilamellar protein-vesicles (pGUPs)
were formed using methods similar to those previously published for lipid vesicles.
40, 88
Briefly, a
polymer film was made on freshly cleaned coverslips from a 5 mg/mL diblock copolymer solution
in CHCl3. Solvent was evaporated using a stream of N2 gas. A 1:1 v/v mixture of membrane
fragment suspension and low-melting temperature agarose (3% w/v) was drop casted onto the
same coverslips and a thin film was formed and allowed to gel. Protein concentration was
estimated based on the reported protein concentrations as supplied in the membrane fragments.
Protein was added to the GUP formation system so as to achieve a final receptor concentration of
0.2 nM in the GUP suspension. This corresponds to a synthetic lipid-to-added-protein ratio of
~280:1 and lipid-to-receptor ratio of ~2x10
6
:1. For control polymer giant unilamellar vesicle
(pGUV) fabrication without protein, protein was omitted from the hydrogel film and only 1.5%
w/v agarose was used. The system was hydrated with 200 mM sucrose in PBS (pH 7.4) with 100
83

μM spiperone and BODIPY-γ-FLGTP (70 nM final concentration). The films were swollen for 20
minutes at room temperature. Bilayer vesicles were harvested from the coverslip and diluted in 3x
of an isoosmotic glucose solution (200 mM glucose in PBS, pH 7.4). pGUPs were allowed to
sediment in glucose for 30 minutes at room temperature (Figure 36).

5.2.3 5-HT1AR activity assay via microplate reader
Prepared and settled pGUPs were transferred to a flat bottom 96-well plate. Agonist 8-OH-
DPAT was added to each well to a final concentration of 150 nM immediately prior to reading
fluorescence. Control pGUPs were run without the addition of the agonist. Rehydration buffer with
and without agonist and protein were read as positive and negative controls. Samples were read at
physiological temperature (37° C) every 5 minutes for twelve hr to ensure complete activity
assessment. Fluorescence reading of BODIPY-GTPγS unquenching was done on a Biotek Synergy
H4 Microplate Reader equipped with a xenon flash lamp. Excitation was set to 485/20 nm and
emission at 528/20 nm at a read height of 7 mm (Figure 36).

5.2.4 pGUP lyophilization
Protein incorporated vesicles were prepared and were not subjected to sedimentation.
Samples were transferred to microcentrifuge tubes with a hole pierced through the top to allow for
water evaporation during lyophilization. Samples were first flash-frozen in liquid nitrogen for 5
minutes. Immediately following, samples were placed under a vacuum at 0.5 torr overnight to
complete lyophilization. If needed, samples were stored with dessicant at -20°C (Figure 36).

84

Figure 36. Schematic of experiments presented. The top schematic shows protein incorporation into polymer
bilayer membranes using hydrogel approach. A thin film of protein-agarose is made on a coverslip. A thin film
of polymer is made on top. The system is rehydrated with physiologically relevant buffers and pGUPs are formed
with GPCR incorporated in the polymer bilayers. The lower left schematic shows protein orientation
determination set-up. Antibodies for either receptor or G protein are conjugated to NHS-rhodamine. The
conjugated antibody is run through a 40 kD spin column to remove excess rhodamine. Receptor or G protein is
incubated and tagged with respective rhodamine labeled antibody. The rhodamine-antibody-tagged receptor or
protein is used in the hydrogel matrix to form pGUPs. After formation, pGUPs are incubated with QSY7 to
determine orientation by quenching rhodamine on the exterior of pGUPs. The lower middle schematic shows the
steps in protein activity assessment. After formation of pGUPs using the presented method, pGUPs are incubated
with agonist and fluorescence unquenching due to BODIPY-GTPγS binding to G protein is tracked over time. In
the lower right schematic pGUP lyophilization. After pGUP formation, pGUPs are flash frozen in liquid nitrogen
and then subjected to overnight vacuum at 0.5 torr to complete lyophilization. Lyophilized pGUPs can be stored
at -20 C and can be rehydrated using water to regain spherical shape and protein activity.

85

5.2.5 Antibody labeling
5-HT1AR monoclonal and Gαi antibodies were equilibrated to room temperature and
conjugated to NHS rhodamine in aqueous buffer (PBS, pH 7.4, DMSO <5%) at 10x molar
excess of NHS-rhodamine. Sodium bicarbonate was added as per manufacturer’s instructions to
raise the solution pH to 8.0. The solution was allowed to react for one hour at room temperature
and then overnight at 5 °C. Rhodamine-labeled antibodies were subsequently desalted using spin
columns according the manufacturer’s instructions. Labeled antibody UV-vis absorbance was read
on a NanoDrop ND-1000 (Thermo Fisher, USA). Rhodamine-labeled Gαi antibody concentration
was determined to be 22 μM and labeling efficiency was calculated to be 1.15. Rhodamine labeled
5-HT1AR monoclonal antibody concentration was 3.2 μM with 1.41 labelling efficiency (Figure
36).

5.2.6 5-HT1A receptor and G protein identification via antibody binding
An aliquot of 5-HT1AR membrane fragment was incubated with 1:1000 labeled antibody,
either 5-HT1AR monoclonal or Gαi at 37° C for 1 hour. Labeled protein was used in the agarose
film for pGUP formation. pGUPs were formed as previously described with spiperone and
BODIPY-GTPγS omitted from the rehydration buffer. Polymer solutions included 0.2% ATTO-
4880-DPPE for ease of imaging. pGUPs were harvested, settled in an isoosmotic glucose solution
and transferred to observation chambers. pGUPs imaged at 491 nm and 561 nm excitation
corresponding to 523 nm and 575 nm emission respectively.

86

5.2.7 Antibody quenching to determine protein orientation
5-HT1AR membrane fragments were incubated with rhodamine-labeled anti-5-HT1AR
monoclonal antibodies or rhodamine-labeled Gαi monoclonal antibodies, 1:1000 dilution. The
labeled protein mixture was used in the agarose film for pGUP formation. For control vesicles,
labeled protein was omitted from the preparation and instead 0.2% ATTO-488-DPPE was used.
pGUPs were harvested, settled, and placed in observation chambers. pGUPs were imaged via
epifluorescent microscopy prior to quenching. QSY7, a membrane impermeable quenching
molecule, was then added to the observation chambers, 1:1000 dilution, and incubated in the dark
for 10 minutes. pGUPs were imaged before and after incubation and the amount of quenched
intensity was analyzed. Control data without tagged protein shows 50% quenching of ATTO-488-
DPPE fluorophore, indicative of even distribution of fluorescent dye in the vesicles (Figure 36).

5.2.8 Microscopy
Imaging was done on a TI-Eclipse inverted microscope (Nikon, Japan) equipped with a
spinning-disc CSUX confocal head (Yokogawa, Japan) and a 16-bit Cascade II 512 EMCCD
camera (Photometrics, USA). Confocal excitation of fluorophores was done using 50 mW solid-
state lasers at 491 nm for ATTO-488-DPPE and 561 nm for rhodamine (Coherent Inc., Germany).
All confocal images were taken using a Plan-Apo 60x NA1.43 oil immersion Nikon objective.
Epifluorescence imaging was performed on the same microscope with illumination from a 130 W
mercury lamp (Intensilight, Nikon, Japan). Rhodamine emission was excited using a green filter
(528-552 nm bandpass, 540 nm cut-on wavelength). Temperature control during imaging was
performed using a heating-cooling stage with a stability and accuracy of 0.1°C (Bioscience Tools,
USA).
87

Differential interference contrast (DIC) images were collected on an Axio Observer Z1
(Zeiss, Germany) inverted microscope using an EC Plan-Neofluar 40x objective and equipped
with a Hamamatsu CMOS camera (Hamamatsu, Japan). Illumination was provided by a halogen
lamp 12V 100W using a differential interference contrast prism with polarizer (Zeiss, Germany).

5.2.9 Image processing
All images were processed and analyzed using ImageJ. Particle analysis and measurements
were performed using ImageJ Analyze Tools. Fluorescent micrographs of vesicles using 491 nm
excitation are shown using the ImageJ green lookup table and micrographs using 561 nm excitation
are shown using the Image J orange hot lookup table. All images are presented without any further
processing adjustments or corrections and are scaled from minimum to maximum intensity.

5.2.10 Data analysis
Fluorescence microtiter results were collect and analyzed using JMP. Six separate
observations were fitted and averaged to obtain a single curve with standard error mean values.
Rates were fitted to the JMP Mechanistic Growth curve using a single exponential. Statistical
analysis using a Tukey-Kramer pairwise comparison of means were done using JMP with a 95%
confidence interval (α=0.05).

88

5.3 Results and discussion
We utilize the agarose hydration method that we have previously discussed in the previous
chapters for the incorporation of 5-HT1AR into giant unilamellar vesicles made of diblock
copolymer.
40, 88
We incorporate membrane preparations of 5-HT1AR with associated G proteins
into polymeric membranes made of polybutadiene-b-poly(ethylene oxide) (PBd(650)-PEO(400))
at a polymer to protein ratio of ~280:1. To
detect the functionality of 5-HT1AR in the
diblock copolymer bilayers, pGUPs were
formed to encapsulate BODIPY-GTPγS, a
quenched fluorophore. When an agonist binds
to 5-HT1AR on the pGUPs, G protein subunits
exchange bound GDP for BODIPY-GTPγS
and this exchange unquenches its
fluorescence. Using this system we detect
physiological responses of 5-HT1AR in the
presence of different antagonists and further
show retained protein function after
lyophilizing and rehydrating pGUPs. Figure
35 and 36 show schematics of experiments
presented in this work. Figure 37 shows
pGUP confocal images before and after lyophilization. The GPCRs, visualized using a rhodamine-
labeled anti-5-HT1AR antibody, are evenly distributed throughout the bilayer in both non-
lyophilized and lyophilized pGUPs (Figure 37).
Figure 37. Top set: Confocal micrographs of pGUPs
prior to lyophilization. The left micrograph shows the
polymer bilayer tagged with ATTO-488-DPPE. The
right micrograph shows that rhodamine antibody-
tagged 5-HT 1AR is evenly distributed throughout the
polymer bilayer. Bottom set: The left image shows a
pGUP sample after lyophilization. Upon rehydration,
pGUPs can still be detected as shown in the right
micrograph. All scale bars represent 5 µm.
89

5.3.1 Biased GPCR orientation in diblock copolymer pGUPs
The polymeric composition of pGUPs made using our proposed hydrogel assisted approach
was confirmed. Three types of hydrogels were prepared on clean glass coverslips: 1) low-melt
temperature agarose 2) low-melt temperature agarose with 5-HT1AR membrane preparations 3)
low-melt temperature agarose with rhodamine-labeled anti-5-HT1AR. On top of each of these
hydrogels either the diblock copolymer or 1 mg/ml solution of ATTO-488-DPPE was added. The
systems were individually rehydrated with 200 mM sucrose in PBS and observed using DIC for
the short polymer and epifluoresence for the fluorescent lipid. As seen in Figure 38, vesicles are
formed using only the polymer but not using the ATTO-488-DPPE lipid dye on all three types of
hydrogels. This confirms that the pGUPs presented with and without 0.2 mol% ATTO-488-DPPE
Figure 38. pGUP polymer make-up confirmation. In the micrographs above, either polymer or ATTO-488-DPPE
was casted as a thin film on top of a hydrogel matrix without protein, with 5-HT 1AR, or with rhodamine-antibody
tagged 5-HT 1AR. The hydrogel is low-melting-temperature agarose. To image the polymer without lipid dye
ATTO-488-DPPE, micrographs were taken using DIC. All other micrographs were collected using
epifluorescence at the indicated wavelengths. All scale bars are 25 μm and the scale bar in the first micrograph of
a set of triptych images goes for all images in that set. In the left set of polymer images, vesicles are formed on all
three hydrogels. When protein is included in the hydrogel, vesicles tend to float off of the hydrogel, which explains
the lack of vesicle density on the hydrogel surface. pGUPs can be found floating in the solution above (not shown).
In the right set of ATTO-488-DPPE images, vesicles are not observed to form on three hydrogels investigated.
This confirms that pGUPs formed using the hydrogel assisted method of protein incorporation are indeed made
of polymer.
90

are primarily made of diblock copolymer. Furthermore, using this approach we assume that the
diblock copolymer takes on an I-shape conformation leading to the formation of a bilayer vesicles
as depicted in Figure 25.
23

To determine the location of 5-HT1AR and its orientation in pGUPs, monoclonal
rhodamine-labelled anti-5-HT1AR or anti-G proteins were incubated with membrane preparations
of 5-HT1AR prior to incorporation into pGUPs and then subsequently quenched. The monoclonal
5-HT1AR antibody binds to the cytosolic face of the receptor. The incorporation of the antibody
during the preparation results in antibody bound to the receptor to be inside the pGUPs for the
correctly oriented GPCRs (C-terminus cytosolic and the N-terminus extracellular), and outside the
pGUPs for the incorrect orientation. QSY7, a commercially available promiscuous membrane
impermeable fluorescent quencher was used.
First, the nonspecific interactions of the antibodies with the diblock copolymer bilayer
membrane was investigated. Vesicles with, pGUPs, and without protein, pGUVs, were made with
and without lipid label, ATTO-488-DPPE. These vesicles were incubated with rhodamine labelled
anti-5-HT1AR or anti-G protein for 1 hour at 37 ˚C. Figure 39 shows the lack of nonspecific
interaction of the receptor and G protein antibodies to various polymeric vesicles. It can be seen,
that the rhodamine-labelled antibodies do not bind to the vesicles without protein (pGUVs) as
fluorescence intensity is not accumulated at vesicle surfaces. Therefore the antibodies do not
interact with the polymer membrane and fluorescence due to antibody binding is due to the
presence of 5-HT1AR or its associated G protein subunits. As a positive control, pGUP with Rh-
anti-receptor were incubated with QSY7 and then exposed to 2x volume amounts of Milli-Q water
to induce vesicle rupture/popping. Images were taken every second during exposure to water and
intensity was tracked. Select time points are shown in Figure 40 shows vesicle rupture, vesicle
91

Figure 39. Nonspecific interaction of antibodies and pGUPs. The micrographs presented show polymersomes
before (left) and after (right) incubation with rhodamine-labeled antibody. Vesicles were made without protein
(pGUVs), with and without ATTO-488-DPPE, and with unlabeled protein incorporated (pGUPs). Vesicles were
incubated with either rhodamine-anti-5-HT 1AR (Rh-anti-receptor) or rhodamine-anti-G protein (Rh-anti-G protein).
Images were taken using phase contrast for vesicles without lipid label, at 491 nm for ATTO-488-DPPE, and at
561 nm for Rh-antibodies. pGUVs without protein (top set) show no intrinsic fluorescence at 491 nm and 561 nm.
After incubation with antibody, antibody does not cross the membrane, and no fluorescence accumulation is seen
at the vesicle surface. pGUVs tagged with ATTO-488-DPPE show fluorescence at 491 nm (middle set), and also
show no fluorescence accumulation at vesicles surface. In pGUPs with unlabeled protein (bottom set), and without
lipid label, incubation with Rh-anti-receptor does not show binding of the antibody to the exterior of the pGUP, or
the N-terminus of the receptor. The antibody does not cross the membrane and cannot reach the C-terminus of the
receptor. Furthermore when the same pGUPs are incubated with Rh-anti-G protein, some fluorescence intensity
accumulates on the surface the pGUPs, since G proteins are distributed on both leaflets of the bilayer. All scale bars
are 10 µm.

92

Figure 40. QSY7 quenches fluorescence of
ruptured pGUPs with Rh-anti-receptor.
pGUPs were prepared with Rh-anti-
receptor. pGUPs were settled and observed
using confocal microscopy at 561 nm
excitation. Scale bar is 5 µm. In the two top
microcraphs, pGUPs were imaged prior to
and after incubation with QSY7 for ten
minutes. The fluorescence intensity of the
pGUP does not change. pGUPs were
ruptured. Micrographs are select images at
the specified time points. The intensity of
all images are plotted as line segments
spanning the top left to the bottom right.
The colors on the plot correspond to the
colors of the time point text indicated.
Select time points are shown vesicle
rupture, vesicle fragments and quenching
of the fluorescence.
Figure 41. Fluorescence assessment of antibody-tagged pGUPs for determination of protein orientation. pGUPs
were made with either Rh-anti-receptor, Rh-anti-G protein or ATTO-488-DPPE. The left micrograph pair shows
excitation at 561 nm and shows the rhodamine tagged receptor, the middle micrograph pair shows excitation at
561 nm showing the rhodamine tagged G protein, and the right micrographs show excitation at 491 nm which
shows fluorescence of ATTO-488-DPPE. The top micrographs of each pair show the vesicles before quenching
with QSY7 and the bottom micrographs show vesicles after quenching. The plots show the intensity as tracked
by the colored line segments across each vesicle and show the change before and after quenching. The receptor
shows little change in intensity before and after quenching with QSY7 indicating a biased orientation of the
receptor. The G protein and the ATTO-488-DPPE show ~50% decrease in fluorescence intensity indicating that
these components are distributed on the inner and outer leaflet of the bilayer. All scale bars are 10 µm.
93

fragments and quenching of the
fluorescence due to exposure to QSY7.
These vesicles were anchored to the
coverslip using BSA-Biotin modification.
This ensures proper tracking of vesicle
fragment fluorescence since they are
anchored to the coverslip and cannot
readily diffuse away.
To determine protein orientation,
pGUPs made with rhodamine-labelled
anti-5-HT1AR (Rh-anti-receptor) or
rhodamine-labelled anti-G protein (Rh-anti-G protein) were formed and then exposed to a
promiscuous membrane impermeable quencher, QSY7 as previously described for a lipid system
in section 4.3.1. Fluorescence intensity across line segments of vesicles before and after quenching
were analyzed and quantified (Figure 41). Results of retained fluorescence after quenching in
pGUPs are presented in Figure 42. Similarly to lipid GUP systems, less than 10% of the Rh-anti-
receptor fluorescence is quenched, indicating that 5-HT1AR displays a biased orientation with the
C-terminus cytosolic and the N-terminus extracellular and in the correct physiological orientation.
The peripheral G protein subunits are distributed in both polymer bilayer leaflets (~55%
fluorescence retention) without a bias towards either the inner or outer leaflet of the assumed
polymer bilayer as also observed in lipid GUPs. The G proteins are anchored to the membrane via
a single tether, lacking the complex structure needed for membrane orientation bias.
Figure 42. Retained fluorescence intensity of quenched
monoclonal antibody-tagged pGUPs. pGUPs were formed
with monoclonal rhodamine antibody-tagged receptor or G
protein and subsequently quenched. Retained fluorescence
intensity indicates the population of receptor in the correct
orientation and G Protein in the inner leaflet of the
vesicles. Over 90% of the receptor population is
incorporated in the correct orientation while G proteins
exists across both leaflets. Control pGUPs were made with
ATTO-488-DPPE throughout both bilayers; this lipid tag
is quenched ~50%.
94

The results indicate the formation of pGUPs using the agarose technique is likely the initial
formation of nanoscale liposomes, as discussed in section 4.3.1; the high curvature causes proteins
to orient themselves within the bilayer.
31, 108
These liposomes then coalesce into larger pGUPs
during the agarose rehydration process to form giant vesicles on the micrometer scale (Figure 43).

5.3.2. GPCR catalyzed nucleotide exchange activity in pGUPs
To observe protein function in the synthetic polymer bilayers, pGUPs were formed, settled
in glucose and transferred to a 96-microtiter plate. pGUPs were formed in the presence of
antagonist, spiperone (final concentration 14 µM unless otherwise stated), to reduce protein basal
activity and incubated with agonist 8-Hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-
DPAT) at 37°C for 12 hours (hr). Fluorescence unquenching of BODIPY-GTPγS due to G protein
binding was monitored every 5 minutes. Control pGUVs without protein do not display
nonspecific BODIPY-GTPγS unquenching, as shown in Figure 44. Tracked measured
fluorescence curves over time were first calculated as percent intensity increase to account for
Figure 43. pGUP formation using the agarose swelling
method for protein incorporation. The micrographs
depicted are DIC time-lapse images as indicated above
each image in seconds. Time 0 s represents the hydrogel
and polymer film immediately prior to rehydration with
200 mM sucrose in PBS (pH 7.4). Vesicle formation
begins almost immediately and pGUPs start as
nanometer size vesicles and continue to grow and form
micrometer sized vesicles. Coalescence of the smaller
vesicles into large vesicles is captured in the decrease in
number of vesicles from 60 s to 180 s to 300 s. The plot
below the images shows the pGUP count and average
radius in μm over a 5 -minute period. pGUP count
initially spikes and decreases while radius increases and
plateaus over time, indicating the coalescence of vesicles
during pGUP formation.

95

variation in sample size (i.e. pGUP population).
Each curve is an average of six independent
replicates of the same experimental protocol.
Fluorescence intensity increase curves were then
normalized for ease in viewing the differences in
receptor-catalyzed nucleotide exchange rate. Rates
are obtained from single exponential fitting on the
fluorescence intensity increase versus time curves.
Agonist activated fluorescence intensity of the
pGUPs displays a much faster rate than control
pGUPs not exposed to agonist indicating that 5-
HT1AR function is agonist
induced in the diblock
copolymer membranes
(Figure 45).
5-HT1AR in pGUPs
displays physiological
responses to varying
amounts of antagonist.
Reducing the final
concentration of spiperone
in pGUPs from 14 µM to 14
pM and keeping the
Figure 44. BODIPY-GTPγS does not display
nonspecific binding to pGUVs. pGUVs without
protein were formed as described and omitting the
protein from the hydrogel. pGUVs encapsulate
BODIPY-GTPγS and were incubated with and
without agonist (8-OH-DPAT) and fluorescence
was tracked over time. As shown in the plot,
pGUPs do not show significant fluorescence
intensity increases over time indicating that
BODIPY-GTPγS has no unqunenching
interaction with the polymersome membrane.

Figure 45. 5-HT 1AR in pGUPS display physiological response to increasing
antagonist concentration while keeping agonist concentration constant.
Fluorescence unquenching due to the irreversible binding of BODIPY-GTPγS
to G proteins was tracked for 12 hours for pGUPs formed with increasing
amount of antagonist spiperone and constant amount of agonist (+Ag).
Increasing the amount of antagonist spiperone in the system decrease the
protein functional rate (See Table 4, for best-fit rates). The inset shows control
curves for the pGUPs that were incubated without agonist. 5-HT 1AR basal
activity is captured in the control, no agonist, pGUPs.

96

Table 4. Physiological 5-HT1AR in pGUP response to changes in antagonist species and
concentration.
Antagonist
Agonist Induced
Rate ± Std Er
Control Rate
± Std Er
[x10
-3
min
-1
] [x10
-3
min
-1
]
14 pM Spiperone 10.2 ± 0.3 4.2 ± 0.1
14 nM Spiperone 9.6 ± 0.3 3.2 ± 0.1
14 µM Spiperone 7.7 ± 0.1 3.1 ± 0.1
14 µM Methiothepin 8.5 ± 0.2 2.8 ± 0.1
14 µM NAN-190 5.7 ± 0.1 3.6 ± 0.1
14 µM WAY 100635 5.9 ± 0.1 3.4 ± 0.1

Figure 46. Curves of protein functional rate with varying antagonist. The points indicate the average of 6 pGUP
sample observations for each of the listed antagonist, methiothepine maleate, NAN-190, and WAY 100635, and
the shaded area around the points are the standard error mean. The inset shows the control pGUPs that were not
incubated with agonist and represents protein basal activity in the specified pGUP samples. Each of the agonist
induce samples (Ag+) and corresponding control (Ctl) samples are plotted as a set below. The quantitative rates
of both Ctl and Ag+ rates can be found on Table 4.
97

concentration of added agonist 8-OH-DPAT constant, results in an expected increase in the
protein functional rate (Table 4 and Figure 45). The inset of Figure 45 shows control curves,
where pGUPs were formed and incubated without agonist. Rate fitted from single exponential
fittings of curve obtained from agonist incubated and control pGUPs are shown in Table 4.
Furthermore, other known 5-HT1AR antagonist, methiothepin maleate (methiothepin),
NAN-190, and WAY 100635, that are known to inhibit agonist binding more strongly than
spiperone also reduce the protein functional rate (Figure 46 and Table 4).
120-122
Thus pGUPs as
fabricated are sensitive to spiperone concentration and shows a reduction in rate due to other
anatagonists.

5.3.3 Lyophilization of pGUPs
A remarkable feature of the 5-HT1AR pGUPs is their stabilitythrough lyophilization and
rehydration. Lyophilization is a process also known as cryo-freezing or cryopreservation where
material is flash frozen and then subjected to a vacuum where the material is desiccated into a
powder for long term storage.
8
The lyophilization of membranes and proteins has implications in
drug delivery. Lyophilzation of nanometer sized vesicles has been optimized and lyoprotectants,
water replacement, and use of vitrification models has been implemented for extended shelf-life.
8,
123-125
This is particularly important for storage of liposomes containing thermosensitive medical
therapeutics.
8
While lyophilization is a desirable approach, freeze drying of proteins often renders
them nonfunctional and larger lipid vesicles (>5 µm) typically display fracturing upon
lyophilization.
8, 19

Since polymersomes are known for increased stability, we formed pGUPs as described
above and subjected them to flash freezing for five minutes in liquid nitrogen followed by
98

overnight vacuum at 0.5 torr and -35°C to completely lyophilize the samples. Lyophilized pGUPs
were kept frozen with desiccant at -20°C for extended storage. At 24 hr and 120 hr lyophilized
samples were rehydrated with deionized water (37°C) for 20 minutes. Rehydrated samples were

Table 5. Protein rates and percent increase in fluorescence intensity of pGUP controls (non-
lyophilized) and after lyophilization (24 hr and 120 hr). +Ag indicates added agonist; Ctl samples
represent basal activity (without agonist). Percent intensity is indicative of active 5-HT1AR
population.

Lyophilization
Time [hr]
Sample Rate x 10
-3
[min
-1
]
Increase in
Fluorescence Intensity
0 Ctl 2.4 ± 0.1 85% ± 11%
24 Ctl 3.0 ± 0.1 73% ± 8%
120 Ctl 2.5 ± 0.2 83% ± 12%
0 + Ag 7.7 ± 0.1 80% ± 10%
24 + Ag 7.3 ± 0.2 77% ± 7%
120 + Ag 6.5 ± 0.1 85% ± 9%
Figure 47. Comparison of pGUPs before and after lyophilization. A) The left micrograph shows a typical yield
of pGUPs on the hydrogel before harvesting. The right micrograph shows a patch of pGUPs that were lyophilized
on the hydrogel for viewing purposes. pGUPs are smaller and display a rough perimeter due to dehydration from
lyophilization. B) This set of images shows pGUPs after they have been harvested from the hydrogel and settled.
The left micrograph shows pGUP controls that were not lyophilized. The right micrograph shows typical pGUPs
after lyophilization and subsequent rehydration with Milli Q water. C) Histograms of size distributions of pGUPs
before lyophilization and after lyophilization. Prior to lyophilization the mean average radius of a typical pGUP
population is 6.03±0.24 μm. After lyophilization the mean average radius of a typical pGUP population is
5.94±0.24 μm.
99

observed using epifluorescence microscopy and analyzed via fluorescence microtiter plate assay
as previously described. 24 hr and 120 hr pGUPs were still vesicular and retained their size as can
be seen in Figure 47. The histograms in Figure 47 show pGUPs that are not lyophilized have a
mean radius of 6.03±0.24 μm. After lyophilization the mean average radius of the pGUP
population is 5.94±0.24 μm.
Furthermore 24 hr and 120 hr lyophilized pGUPs display agonist induced functional rates
comparable to that of pGUPs that were freshly prepared and assayed without further storage,
7.3±0.2, 65.±0.1 versus 7.7±0.1 (x 10
-3
min
-1
) respectively (Table 5) . A Tukey-Kramer pair wise
comparison of means (α=0.05) shows that the differences in rates are not statistically significant.
The protein retains agonist-induced activity and protein functionality suggesting that the polymer
stabilizes the GPCR. . The percent intensity increase of the pGUPs accounts for varying amounts
of pGUP population in individual microtiter wells and is also indicative of the population of
functional receptors. Table 5 also shows that the percent intensity increase does not vary
significantly across all samples and thus the functional protein populations in pGUPs do not
decrease upon lyophilization.
Furthermore, non-lyophilized pGUPs stored at 5˚ C for 24 hr and 120 hr display little
fluorescence intensity increase and diminishing percent fluorescence intensity increase, as seen in
Figure 48, which indicates a decrease in the population of active protein during storage without
lyophilization. These results indicate that our polymeric bilayers protect protein integrity during
lyophilization and extended dehydrated storage, and that lyophilization is necessary for extended
storage of functional proteins in pGUPs.
100

Figure 49. Results of pGUPs without spiperone. pGUPs were formed without the antagonist, spiperone, in the
rehydration buffer and were lyophilized for 0 hour (0 hr, non lyophilized), 24 hours or 120 hours then assayed for
protein functionality. For all time periods, the protein displays inherent functionality and agonist induced activity,
however, the rate of protein function for the lyophilized samples are attenuated compared to the 0 hr non
lyophilized control. The control curves in the in-set are pGUP samples of the indicated lyophilization time that
were not incubated with agonist, and therefore indicate protein basal activity. Each of the agonist induce samples
(Ag+) and corresponding control (Ctl) samples are plotted as a set below

Figure 48. Rates of non-lyophilized pGUPs after storage at 5˚ C 24 hr and 120 hr. While pGUPs show an increase
in fluorescence intensity over time, the percent increase reported for lyophilized samples decreases indicating that
the populations of pGUPs with protein activity is diminishing and that lyohilization is needed for extended storage.
The shaded areas around the points represent the standard error mean of 6 samples using the identical experimental
set-up.
101

Table 6. Rates of pGUPs formed and lyophilized without antagonist, spiperone.
Lyophilization
Sample
Rate
(hr) x 10
-3
[min
-1
]
0 Ctl 5.7±0.1
24 Ctl 3.0±0.1
120 Ctl 3.6±0.1
0 + Ag 16.0±0.4
24 + Ag 5.3±0.1
120 + Ag 6.8±0.2

To determine if antagonist binding also stabilizes 5-HT1AR in our polymeric vesicles,
pGUPs were formed without spiperone and subjected to lyophilization. Lyophilized pGUPs
without spiperone in the system displays some functional activity as shown in Figure 49. Best fit
rates determined from the curves in Figure 49 are quantified in Table 6. The 0 hr, non-lyophilized
agonist induced and control samples display a faster rate than the 24 hr and 120 hr lyophilized
samples. Protein retains agonist-induced activity and protein functionality promoted by polymer
stabilization. Decrease in rates
from 0 hr to 24 hr to 120 hr in
lyophilized samples without bound
antagonist shows that ligand
binding is also necessary to
promote and retain integrity of
protein function in pGUPs.
Proteins may be stabilized
by ligands and sugars, which can
aid in keeping their functional
integrity during lyophilization by
Figure 50. Functional rates of 5-HT 1AR in polymersomes (pGUPs)
versus various solutions. Controls (Ctl), no agonist pGUPs, are
plotted alongside agonist-exposed samples (+Ag). The percent
intensity increase of the samples indicates the population of
functional protein. In DI water and PBS, the 5-HT 1AR displays no
fluorescence activity. In 200 mM sucrose in PBS (pH 7.4) 5-HT 1AR
displays weaker fluorescence intensity increase compared to pGUPs.
Furthermore there is no difference in rate between the Ctl and +Ag
protein in 200 mM sucrose in PBS.
102

decreasing aggregation and providing H-bonding.
126-129
It has been previously shown that
increasing sucrose content increases the physical stability of proteins.
129
Furthermore, interactions
between buffer species and proteins can replace water molecules during lyophilzation which
further stabilizes the protein in a dehydrated state.
127
To determine if the stabilization in our
systems is due in part from concentrated sugar present in the buffer used for pGUP formation (200
mM sucrose in PBS), membrane fragments of 5-HT1AR were bound to spiperone and then diluted
in deionized water, PBS (pH 7.4), or 200 mM sucrose in PBS (pH 7.4) with concentrations similar
to our pGUP system. After 24 hr of lyophilization, samples were rehydrated and assessed for
protein function. 5-HT1AR membrane fragments diluted in Milli Q water (DI water) or PBS did
not retain its function (Figure 50). In 200 mM sucrose in PBS, the protein displays a similar
functional rate for both control and agonist treated samples. Despite displaying protein function,
the rates do not discriminate between control and agonist exposed samples suggesting that the
protein has lost its agonist binding ability as shown by the overlapping curves in Figure 51.
Furthermore the percent intensity increase of these samples were well below that of the pGUP
samples, indicating that only a small population of proteins retained some function (Figure 50).
Figure 51. Normalized rate of agonist
and control protein diluted in 200 mM
sucrose in PBS and lyophilized for 24
hr. As presented in Figure 50, 5-
HT 1AR membrane fragments diluted
in sucrose rehydration buffer shows
protein function, however, they do not
show agonist dependence as protein
exposure to 8-OH-DPAT does not
show an increase in protein functional
rate. The curves are averages of the
data points presented. The rates as
determined by a single exponential
filling of the control protein is
7.23±0.12 (x10
-3
min
-1
) and the rate of
the agonist-incubated protein is
7.18±0.12 (x10
-3
min
-1
)
103

Thus, while these results suggest that sugar stabilizes 5-HT1AR to some extent during
lyophilization, its protective ability is much lower than the overall stability and protection offered
by our pGUPs, which not only retain vesicle shape and size, but also retain protein functional
integrity.

5.4 Conclusions
Using an agarose rehydration technique we not only show successful incorporation of
GPCR 5-HT1AR into diblock copolymer bilayer vesicles in the form of pGUPs but also show
increased protein stability during lyophilization and extended dehydrated storage. Successfully
reconstituted 5-HT1AR in diblock copolymer pGUP vesicles on the micrometer scale exhibits
expected responses to different antagonists and at a variety of concentrations. Rehydration of
pGUPs after 24 hr and 120 hr of lyophilization retains vesicle size and consistent protein function
and offers increased stability as compared to buffered sugar solutions and pGUPs that are not
lyophilized. Thus we offer a simple platform to investigate protein function in polymer vesicles in
the form of pGUPs. Extension of this work to other types of GPCRs is currently being conducted.

104

6. Conclusions and future outlook
The isolated and tunable GPCR plasma membrane platform developed and discussed in
the previous chapters offers a novel approach to answering emerging questions about the role and
function of these druggable targets. Initial work providing observations on 5-HT1AR phase
behavior, fundamental work describing how 5-HT1AR interacts with lipids in the plasma
membrane, orientational bias of the receptor in bilayer membranes, and increased stability of the
human serotonin receptor in polymeric membranes are only the beginning of furthering our
knowledge about these important signaling drug targets. The hydrogel assisted approach for
fabricating membranes incorporated with GPCRs is a compartmentalized, oriented, and vesicular
system that not only allows for cell mimicking, but allows for isolated studies that focus on both
or either extracellular and intracellular characteristics of GPCRs. Extended stability and storage
through lyophilization offers potential for drug discovery efforts and polymeric membranes
provide flexibility for exploring the biophysics of GPCRs.
In collaboration with structural biology, this platform can elucidate pressing questions in
GPCR biology to further understand the nature and function of these proteins. In 2014, Katritch et
al. reported a sodium pocket in the seven-transmembrane (7TM) helical domain of the adenosine
2a receptor (A2aR).
130
The 7TM is a highly conserved domain found across all GPCRs and the Na
+

pocket is found when an antagonist is bound to the receptor; upon receptor activation, however,
this Na
+
pocket collapses.
130
The reports from this structural study suggests that GPCRs may have
functions as sodium transporters, though the direction of movement of the Na
+
released during
agonist activation is unknown. The location of the Na
+
can therefore only be investigated using
GUPs that isolate the two faces of the GPCR with known protein orientation.
105

A fluorescence based technique similar to the activity assays presented here, where Na
+

binds to a fluorescent indicator can be implemented within the GUP platform. GUP bilayers
incorporated with purified antagonist bound A2aR will be formed using the hydrogel assisted
approach. The protein buffer and rehydration buffer will be sodium-free, and may be composed of
HEPES or MOPS to maintain pH~7. Sodium-binding benzofuran isopthalate (SBFI) will be
present in the rehydration buffer and will be encapsulated and surround the GUPs. Using confocal
microscopy, GUPs will be incubated with an agonist and the release of the Na
+
from its binding
pocket can be tracked by increased fluorescence due to Na
+
binding to SBFI on the interior or
exterior of the GUP.
In the case that fluorescence signal-to-noise poses a challenge, the investigation can be
performed using atomic absorption spectroscopy (AAS). GUPs in sodium free buffer will be
incorporated with antagonist bound A2sR and then incubated with agonist. The agonist bound GUP
system will be dialyzed against Na
+
and then analyzed using AAS. Detection of Na
+
will indicate
the presence of Na
+
within the GUPs after agonist receptor activation. Analysis on non-dialyzed
samples will provide the amount of Na
+
that is released to the exterior of the GUP.
In furthering this work, extension of phase behavior and plasma lipid interactions to A2aR
is currently being pursued. A2aR is implicated in diseases such as chronic obstructive pulmonary
disease (COPD) and addiction.
131
As a medical target for respiratory diseases, understanding the
effects of the plasma membrane on this GPCR, and other GPCRs in general, has the potential to
revolution medical therapies and our understanding of the proteins associated with common
diseases. Using the GUP platform described throughout has the potential to revolutionize the
fundamental understanding of protein-lipid interactions and highlights lipid-targeted therapies as
practical means to fight disease.
106

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Abstract (if available)

Abstract G protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome and are the target of over 50% of therapeutics on the market. Understanding the role of GPCRs in behavior and disease has led to medical innovations to combat psychiatric and respiratory diseases. The human serotonin receptor, GPCR 5-HT1AR is implicated in schizophrenia and bipolar disorder. While many drugs have been designed to target the serotonin receptor to combat psychiatric and behavioral disorders, information regarding the biophysics of this protein in the plasma membrane is scant. In this work, the successful incorporation of 5-HT1AR in giant unilamellar protein-vesicles (GUPs) is demonstrated and GPCR partitioning into liquid ordered phases of phase separating GUPs is presented. The bilayer compositional dependence of 5-HT1AR activity is reported and extended storage and stability of the serotonin receptor in polymeric-GUPs (pGUPs) is also demonstrated. These observations and discussions further our understanding of GPCRs and offer novel approaches for drug design and discovery.

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Creator Gutierrez, Mary Gertrude L. (author)

Core Title Engineering artificial cells to elucidate GPCR 5-HT1AR activity and plasma membrane compositional dependence

School Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Materials Science

Publication Date 07/21/2016

Defense Date 05/02/2016

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

Tag bilayer,GPCR,OAI-PMH Harvest,plasma membrane,serotonin,vesicle

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Malmstadt, Noah (committee chair), Langen, Ralf (committee member), Roberts, Richard W. (committee member)

Creator Email gertrudegutierrez@gmail.com,mlgutier@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-270374

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