University of Southern California Dissertations and Theses

Temperature-mediated induction of caveolin-mediated endocytosis via elastin-like polypeptides

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Temperature-mediated induction of caveolin-mediated endocytosis via elastin-like polypeptides

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Content Temperature-mediated Induction of Caveolin-mediated
Endocytosis via Elastin-like Polypeptides

by
Yue Wang

A Thesis Presented to the
Faculty of the Graduate School
University of Southern California

In Partial Fulfillment of the Requirements for the Degree
Master of Science in
Pharmaceutical Sciences

May 2019

1
Acknowledgement:
To Dr. Andrew MacKay for being my PI and guiding me through my project.
To David Tyrpak for being my student mentor and training me through my project.
To Anh Truong, Zhe Li, Hugo Avila, Christina (Runzhong) Fu and Gao Huo for giving me great
help with my project.
To USC School of Pharmacy, RO1 funding from National Institutes of Health, the Ming Hsieh
Institute, etc for making my work possible.

2
Table of contents:

1. Introduction: ............................................................................................................................. 4
2. Materials and Methods ............................................................................................................. 9
2.1 Plasmid constructs .............................................................................................................. 9
2.2 Cell culture and transfection............................................................................................ 12
2.3 SDS-PAGE and immunoblot ........................................................................................... 12
2.4 Live cell imaging ............................................................................................................... 14
2.5 Immunofluorescence and confocal microscopy.............................................................. 16
2.6 Flow cytometry .................................................................................................................. 19
3. Results ...................................................................................................................................... 21
3.1 Generation and characterization of temperature-responsive CAV1-ELP fusion
proteins..................................................................................................................................... 21
3.2 Measurement of the Tt of CAV1-ELP fusion proteins ................................................... 26
3.3 Validation of the effect of CAV1-ELP assembly on CAVME by confocal microscopy
................................................................................................................................................... 31
3.4 Validation the effect of CAV1-ELPs aggregation on CAVME by flow cytometry ..... 34
4. Discussion................................................................................................................................. 37
5. Conclusion ............................................................................................................................... 39
Reference: .................................................................................................................................... 40

3
Abstract:
Endocytosis has an important role in cellular functions, including nutrient uptake,
signaling, antigen presentation, growth and differentiation, etc. To better understand endocytosis,
scientists have developed a variety of chemical and genetic endocytic manipulators. However,
most of these tools are irreversible or lack specificity. In addition, few technologies have been
found to specifically manipulate caveolin-mediated endocytosis (CAVME). To better study
caveolin-mediated endocytosis, we use Elastin-like polypeptides (ELPs) as a thermally responsive
tool to rapidly and specifically manipulate cellular processes, including CAVME. We hypothesize
that ELPs fused to a mainly functional protein involved in CAVME will modulate the pathway.
To test this hypothesis, ELPs were fused to Caveolin 1 (CAV1), a key functional protein associated
with CAVME. Before temperature stimulation, the CAV1-ELP fusion is soluble in aqueous
solution and CAVME stays in “off” status. Temperature increase can activate the self-assembly
of CAV1-ELP fusion proteins that switches CAVME to “on” status. These microdomains can
react to thermally stimulation within a few minutes, do not need exogenous stimulation, and are
specific for CAVME pathway. This thesis manuscript involves cloning a new CAV1-ELP
construct, determining its self-assembly temperature using a live cell imaging technique, and using
confocal microscopy to determine the effect of CAV1-ELP self-assembly on CAVME. This
manuscript demonstrates that assembly of CAV1-ELP through slight temperature modulation can
activate Caveolin-mediated endocytic pathway.

4
1. Introduction:

Before the existence of a potent tool to study endocytosis, it was simply defined by the
material a cell internalizes. It is classified as phagocytosis and pinocytosis. Phagocytosis refers to
large particles that are taken in. It is also described as cell eating. Pinocytosis refers to extracellular
medium that is taken in. It is also described as cell drinking. However, the advent of electronic
microscopy (EM) has provided a better understanding of subcellular anatomy. The clathrin-
mediated endocytosis was discovered when people studied the uptake of low density lipoprotein
(LDL)
[1]
. It was found that LDLs are recruited to receptors at the cell surface. Afterwards the
complex is wrapped by intercellular clathrin-coated pits, and then clathrin-coated vesicles (CCVs)
is formed
[2,3]
. Therefore, clathrin-mediated endocytosis (CMS) is also called receptor-mediated
endocytosis. However, contradictory additional non-clathrin endocytic pathways were soon
discovered, including caveolin-mediated endocytosis. It is now known that endocytosis is complex
and involves multiple pathways. Now a variety of endocytosis pathways have been discovered
including Clathrin-mediated endocytosis, Caveolin-mediated endocytosis, Flotillin-mediated
endocytosis.
Clathrin-mediated endocytosis (CME) is a key process in vesicular trafficking. It mediates
the cellular uptake of many extracellular ligands, receptors, and pathogens. Many researches have
been done on studying this cellular pathway. Clathrin is the key functional protein involved in
CME. After ligand stimulation, the Clathrin-light chain (CLC) and Clathrin-heavy chain (CHC)
assemble together to form a basket-like triskelion around the budding vesicle. Then the basket-like
triskelion forms clathrin-coated vesicles (CCVs) and further transports different cargos and ligands
to early endosomes.
5
After the discovery of CME, researchers subsequently discovered that there was a class of
clathrin-independent endocytosis. Then caveolin-mediated endocytosis is discovered. Caveolae
are small invaginations which shape like flasks
[4]
in cell membrane and are full of caveolins
[5]
. It
is essential in the formation of caveolae
[6,7]
. Three proteins are included in this family, which are
caveolin-1(CAV1), caveolin-2 (CAV2) and caveolin-3 (CAV3). Caveolin-1 and caveolin-2 are
expressed in numerous kinds of cells, except lymphocytes and neuroblastoma cells
[8,9]
. Caveolin-
3 is mainly expressed in skeletal and cardiac muscle and astrocytes
[10]
. Although the thorough
mechanism has not been fully understood, two features might be important in this mechanism.
Firstly, caveolin-1 binds to cholesterol and sphingolipids
[11,12]
. The ability to bind cholesterol
[13]

is essential for the vesicle biogenesis. Secondly, the caveolin-1 monomer tends to oligomerize with
itself or caveolin-2 to form higher order complexes
[14]
, which can drive the formation of caveolae.
Caveolin is firstly generated in the endoplasmic reticulum (ER)
[15]
, where it is a low molecular
weight oligomer. Then it is transported to Golgi, where it undergoes further oligomerization
[16]
.
After that it is then transported to cell membrane. It has a hairpin-shaped structure. Its scaffolding
domain and intramembrane domain are inserted in cell membrane while both of the N- and C-
terminus are faced to cytoplasm
[17]
. Caveolae is enriched in ganglioside GM1 molecule
[18]
, and
recently it has been found out that GM1 molecule is essential for caveolae maintenance and
caveolin trafficking. On the plasma membrane caveolae forms a lipid raft invagination that is
enriched in sphingolipid, specifically sphingomyelin and the glycosphingolipids ganglioside GD3,
GM1 and GM3
[19]
.
Caveolae plays an essential part in cellular signaling pathways. It acts as a platform to
contain and regulate different signaling proteins, including platelet-derived growth factor (PDGF),
epidermal growth factor receptor (EGFR), and the small GTPase H-Ras
[20]
.
6
Endocytosis regulates many processes, including ligand uptake, signal transduction, etc.
The disruption of endocytosis can cause diseases
[21,22]
. Some reports have shown that caveolin is
highly related to many types of tumors, and its role is divergent. It has been pointed out that
Caveolin-1 functions as a cancer suppressor
[23-25]
. Some studies proved that the human caveolin-
1 gene is located at the long arm of human chromosome 7 (7q31.1)
[26,27].
And the (C-A)n
microsatellite repeat marker D7S522 is located at the same position and is often deleted under
various cancer conditions
[28-32]
. However, some studies came up with the opposite idea that CAV-
1 may prompt cancer cell growth. In these studies, CAV-1 is highly expressed in prostate cancer
[33,34]
. In another case, gastrointestinal cancer, caveolin-1 seems to play a more complicated part.
Some study showed that CAV1 RNA and protein are expressed much less in human colon tumor
cell lines
[35]
, while another study showed that an increased expression of CAV1 is found in the
same cell lines
[36]
. Considering previous researching results, CAV distributed in different tissues
might have different contribution in development and progression of cancer. Another role caveolin
plays in promotion of cancer is that it is partly responsible for angiogenesis, which dramatically
facilitates tumor growth by providing plenty of nutrition cancer cells need to grow. Therefore,
being able to specifically manipulate CAVME has great value in illuminating its role in cancer
development.
Besides cancer, it has been found out that caveolin is also involved in other diseases like
atherosclerosis and restenosis
[37]
. One pathogenesis of atherosclerosis and restenosis might be due
to vascular smooth muscle cells (VSMC) proliferate and migrate to blood vessel intima
[38-40]
. It
has been found out that caveolin is responsible for suppression of VSMC proliferation
[41,42]
. Some
researchers have demonstrated that caveolin expression reduction in atherosclerosis and restenosis
cases. Therefore, it suggests that caveolin expression is in a reverse relationship with
7
atherosclerosis and restenosis. Furthermore, Hypertrophic cardiomyopathy (HCM) was found
related to abnormality in caveolin expression. HCM is a disease caused by cardiomyocyte
hypertrophy and fibrosis
[43]
, whose characteristic is a dramatically increased left ventricular mass.
Some researchers have shown that cav-1 knocked out mice tend to have trouble with progressive
cardiac hypertrophy. Additionally, Cav-1 knocked out mice showed dramatically much higher
pulmonary artery pressure
[44]
. Pulmonary hypertension could also be a result of pulmonary
fibrosis.
Besides, abnormality in caveolin is also involved in diabetes progression. It has been found
out that caveolin interacts with insulin receptor. A derivative peptide of caveolin can activate
the activity of insulin receptor kinase
[45]
. Therefore, the abnormality in caveolin is directly related
to abnormal blood sugar regulation.
Taking these facts into consideration, there is still a lot to do to have a comprehensive
understanding of caveolae and caveolin in the roles in trafficking, diseases pathogenesis.
In this thesis, I try to build a toolbox to understand and manipulate Caveolin-mediated
endocytosis. The idea is partly inspired from the previous finding in our lab---using the same
toolbox to understand and manipulate Clathrin-mediated endocytosis. The tool we use to achieve
our goal is called Elastin-like Polypeptides (ELPs). Elastin-like-polypeptides (ELPs) are thermal-
responsive and biocompatible polypeptide derived from human tropoelastin. ELPs are composed
of a pentameric repetitive sequence (VPGXG)n, where X is the guest residue which can be any
amino acid except proline and n is the sequence repeating number. In abbreviation, V96 means the
guest amino acid is Valine and the pentapeptide repeating number is 96. ELPs remain highly
soluble below the transition temperature (Tt). When heated above Tt, they undergo self-assemble
and become insoluble. This change is reversible when temperature falls below Tt. Modulation of
8
Tt can be achieved by changing the guest residue, sequence repeating number, and other factors,
like ELP concentration and ionic strength of solution. The transition temperature (Tt) is in
reversely relationship to the number of pentameric repeats. The larger the number of repeats, the
lower the Tt. As for the guest amino acid, a hydrophobic amino acid depresses the Tt, while a
hydrophilic amino acid elevates the Tt. Due to the special characteristics of ELPs, we propose it
to be utilized as a “switch on-off” way to manipulate caveolin-mediated endocytic pathway.
Our research group have previously shown that ELPs can be used as a tool to manipulate
Clathrin-mediated endocytosis (CMS). The idea was fusing an ELP construct to clathrin light
chain, a key effector protein in CMS. When ELP is heated above Tt, it will self-assemble and
sequester all the related proteins in CMS into a microdomain, and thus bring CMS to a halt.
For CLC-ELP design, there is a myc tag inserted between ELP and CLC for further
immunofluorescent detection. There are two reasons why CLC is chosen to fuse ELPs: 1. CLC is
essential to form the basket-like triskelion and to internalize cargos
[46]
. 2. Some study has shown
that attaching green fluorescent protein to CLC does not affect CLC’s normal function
[47]
.
Therefore, attaching ELP to CLC end won’t affect its function either. The conclusion of the study
is that by using ELPs as the tool, the sequestration of clathrin light chain can stop the clathrin-
mediated cell trafficking pathway.
Inspired by this result, we want to apply the similar idea to CAVME, and our hypothesis
is that ELP fused to CAV1 can activate this pathway. Before temperature increase, CAV1-ELP is
soluble in the cell membrane, and CAVME remains “OFF” due to the lack of association. Once
induced to phase separate, ELPs self-assemble and bring CAV1 monomers together to form higher
order complexity, and thus activate CAVME.
9
2. Materials and Methods

2.1 Plasmid constructs

ELP expression vectors were synthesized using recursive directional ligation in Pet25b(+) plasmid.
Recursive directional ligation by Plasmid Reconstruction (PRe-RDL) can rapidly clone highly
repetitive polypeptides. Any desirable sequence and any length is achievable by this method
[48]
.
The CAV1 WT gene was obtained from Addgene (Cambridge, MA) and modified by inserting
DNA oligonucleotides with HindIII and EcoRV. Then DNA sequences encoding ELPs were
cloned into the downstream of CAV1-myc after digesting the ELP plasmid with MsII/ HindIII and
modified CAV1 plasmid with Hind III/EcoRV. The final opening frames of the fusion proteins
were verified by DNA sequencing (Retrogen, San Diego, CA). Then CAV1-ELP plasmids were
amplified in E coli. for further use.
In detail, my job was to build CAV1-V72 DNA plasmid. All the restriction enzymes are purchased
from New England Biolabs Inc. (NEB) Firstly, V72 is isolated from the plasmid by using
restriction enzymes. There are two experiment groups which contains two different V72 DNA
amount. The ingredients are shown in the table (Table 1) below. Next two tubes of samples were
loaded on the gel, and the DNA was run at 100V for 1h. The length of V72 DNA is 1128bp, the
desirable band was cut from the gel. The results and reference band are shown in figure 6.
Table 1. Ingredients of restriction digestion to isolate V72 DNA
V72 DNA 3μg (20.5μl) V72 DNA 2μg (13.7μl)
Cutsmart 10X 3μl Cutsmart 10X 3μl
10
HindIII-HF 3μl HindIII-HF 3μl
MsII 3μl MsII 3μl
H2O 0.5μl H2O 7.3μl
Total volume 30μl Total volume 30μl

Secondly, CAV1 DNA is isolated from the plasmid. There are 4 experiment groups which contains
4 different CAV1 colonies. The ingredients are shown in the table (Table 2) below. After
incubation at 37°C for 30min, enzymes are inactivated at 80°C for another 20 min. Then CAV1
DNA is dephosphorylated by using antarctic phosphatase, of which the ingredients are shown in
Table 3. The 4 samples were run on the gel at 100V for 40 min. The length of CAV1 DNA is
5388bp, cut the desirable band from the gel. The results and reference band are shown in Figure
7.
Table 2. Ingredients of restriction digestion to isolate CAV1 DNA
Group1
Total 20μl
Group2 Total 20
μl
Group3 Total 20
μl
Group4 Total 20
μl
CAV1
DNA
1.0 μg (3.88
μl)
CAV1
DNA
1.0 μ g
(3.80 μ
l)
CAV1
DNA
1.0 μ g
(3.69 μ
l)
CAV1
DNA
1.0 μ g
(3.40 μ
l)
11
Cut smart
buffer
10X
2μl
Cut
smart
buffer
10X
2μl
Cut
smart
buffer
10X
2μl
Cut
smart
buffer
10X
2μl
Hind III
2μl
Hind III
2μl
Hind III
2μl
Hind III
2μl
EcoRV
2μl
EcoRV
2μl
EcoRV
2μl
EcoRV
2μl
H2O
10.12μl
H2O
10.20μl
H2O
10.31μl
H2O
10.6μl

Table 3. Ingredients of restriction digestion to dephosphorylate CAV1 DNA
CAV1 DNA complex 20μl
Antarctic phosphatase 1μl
10X Anp Buffer 2.5μl
H2O 1.5μl
Total 25μl

Thirdly, the CAV1-V72 plasmid is built by using T4 ligase. The ingredients are shown in Table 4.
4 ratios of CAV1:V72 are chose to ligate the two parts, which are (CAV1:V72) 1:3; 1:5; 1:7; 1:9.
12
Then the 4 reaction complexes are incubated at 25°C for 2h. Afterwards, add 20μl ligation liquid
into Top10 component cells, put them on ice for 20 min, water bath at 42°C for 90 secs, then add
250μl medium S.O.C to cell tube, shake at 225rpm at 37°C for 1h; spin down at 3000rpm for
5min, then discard supernatant, resuspend the bacteria, plant on plate, incubate at 37°C overnight.
It turns out that the 1:9 ratio has the most colonies. After mini-prep, sequencing result shows the
desirable sequence, and restriction digestion result also shows the right molecular weight (Figure
8).
2.2 Cell culture and transfection

HEK 293T cells (ATCC, Manassas, VA) were maintained in DMEM (Thermo Fisher Scientific,
Waltham, MA) supplemented with 10% FBS (Corning, NY) in a humidified incubator with 5%
CO2 at 37 ℃. For CAV1-ELP protein expression, cells were transfected with Lipofectamine 3000
(Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s instructions. After
transfection, cells were cultured at 30 ℃ for 48 hours prior to assay.
2.3 SDS-PAGE and immunoblot

HEK 293T cells were plated in six-well plate and transfected the following day with CAV1-ELPs.
Table 4 shows the ingredients for transfection.
Table 4. Transfection ingredients for western blot
Tube 1 Tube 2
DNA Opti-MEM Lipofectamine3000 Opti-MEM DNA P3000 Reagent
13
CAV1-A96
125μl 5.625μl 125μl 7.15μl 5μl
CAV1-V96
125μl 5.625μl 125μl 5.25μl 5μl
CAV1-V72
125μl 5.625μl 125μl 4.13μl 5μl
CAV1-WT
125μl 5.625μl 125μl 5.28μl 5μl
Control

After incubation at 30℃ for 48 hours, cells were put on ice in the 4°C fridge for 45min. Whole
cell lysates were prepared by lysis buffer containing 30Mm CHAPS, 1% SDS and protease
inhibitor (Cell Signaling Technology, Danvers, MA) (pH=8), sonicated in ice water bath for 10min
and subsequently spun for 2 hours in the 4℃fridge. Before running the gel, the proteins sample
sat in room temperature for 1h instead of being heated at 95℃ for 5min due to the heat-sensitive
characteristic of CAV1-ELPs. 50µg of total protein was separated on a PAGEr EX 4-12% gradient
gel (Lonza, Morristown, NJ) and then transferred onto a nitrocellulose membrane using wet tank
transfer method. The membrane was first immunoblotted with primary antibody anti-myc (Cell
Signaling Technology, Danvers, MA), then incubated with secondary antibody anti-mouse IgG
(HRP-linked) (Cell Signaling Technology, Danvers, MA). Protein bands were visualized using
ProSignal Dura Chemiluminescent Substrate (Genesee Scientific, San Diego, CA) and imaged on
a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA).
14

2.4 Live cell imaging

Cells were plated on 35mm glass bottom dishes (MatTek Corporation, Ashland, MA). In the
following day, group 1 (dual group) cells were transfected with both CAV1-ELP and GFP-V60
while group 2 (single group) cells were only transfected with GFP-V60. The cell plating and cell
transfecting protocols in detail is stated in the following:
On Day 1, HEK 293T cells were passages into 4 35mm Mattek plates. Old DMEM medium was
sucked out from the flask. The flask was washed with 7 mL of PBS. After wash, 3 mL of trypsin
was added and the flask was incubated for about 3 minutes at 37
o
C. When cells are visually no
longer attached, pipette gun with 10 mL pipette was used to mix up and down. The flask was held
at an angle to wash any cells down. 7 mL of DMEM + FBS was added to deactivate trypsin. Then
10 mL of cells was transferred to falcon tube. The tube was centrifuged at 200 rcf for 6 minutes
while plates were prepared with poly D-lysine. Then cells were resuspended in 7 mL media. Cells
number was counted using cell counter and microscope. After removing Poly D-lysine, one million
cells were plated into each plate after calculating the cell density based on the result of cell
counting. Each plate was filled with media until the total volume is 2.5ml.
Day 2, Cells were transfected with CAV1-V72 and/or GFP-V60 DNAs. The ingredients are shown
in Table 5.
Table 5. Ingredients of HEK 293T cell transfection for live cell imaging
Tube 1 Lipofectamine 3000 Opti-MEM total
15
12.5μl 300μl 312.5μl
Tube 2
Lipofectamine 3000 Opti-MEM total
12.5μl 300μl 312.5μl
Tube 3
P3000 Reagent Opti-MEM GFP-V60 total
11μl 295μl 6.54 μl 312.5μl
Tube 4
P3000 Reagent Opti-MEM GFP-V60&CAV1-V72 total
11μl 295μl 3.27μl & 4.6 μl 321.5μl

Liquid in tube 3 was added into tube 1; liquid in tube 4 was added into tube 2. Tube 1 and tube 2
were vortexed and spun down. The two tubes were incubated at room temperature for 15min.
meanwhile, cells in 35mm mattek plate were washed with PBS and refilled with 2.5 ml optimum.
Then 250μl of transfection mix was added to each Mattek plate evenly. The plates were incubated
at 30°C for 48h.
Day 4, live cell imaging. Opti-MEM was sucked out from Mattek plates, washed with PBS. 2.5ml
live cell imaging solution was added to each plate. 3 drops of NucBlue live cell stain were added
to each plate. Mattek plates are plated into a temperature controlled microscope stage (Linkam
Scientific). Temperature increased from 8 ℃ to 45 ℃ slowly while the medium temperature in the
dish was monitored by a digital probe. The co-assembly of CAV1-ELP and GFP-V60 and the
assembly of GFP-V60 itself were imaged by a DIAPHOT epifluorescent microscope equipped
with a DS digital camera (Nikon Instruments, Melville, NY). Each cell had a pile of images took
16
during the time ramp. The number of puncta was counted by eye. The contour of each puncta was
circled manually and measured by the software. The standard deviation of pixel value was used to
measure the transition temperature. Firstly, cells were randomly selected in the view. After circling
their contours and add them to ROI manager, the “Analyze---Set Measurements” function was
chosen to calculate the standard deviation of each slide. Then the transition temperature was
calculated by an algorithm developed in our laboratory.
2.5 Immunofluorescence and confocal microscopy

Immunofluorescence is conducted to validate the effect of CAV1-ELP assembly on CAVME.
Detailed protocols are stated in the following:
On Day 1, HEK 293T cells were passages into 2 12-well plates. Old DMEM medium was sucked
out from the flask. The flask was washed with 7 mL of PBS. After wash, 3 mL of trypsin was
added and the flask was incubated for about 3 minutes at 37
o
C. When cells are visually no longer
attached, pipette gun with 10 mL pipette was used to mix up and down. The flask was held at an
angle to wash any cells down. 7 mL of DMEM + FBS was added to deactivate trypsin. Then 10
mL of cells was transferred to falcon tube. The tube was centrifuged at 200 rcf for 6 minutes while
plates were prepared with poly D-lysine. Then cells were resuspended in 7 mL media. Cells
number was counted using cell counter and microscope. After removing Poly D-lysine, 0.3 million
cells were plated into each plate after calculating the cell density based on the result of cell
counting. Each plate was filled with media until the total volume is 1ml.
On Day 2, cells are transfected with CAV1-A96, CAV1-V96, CAV1-V72 respectively. The
ingredients are shown in Table 6 below.

17
Table 6. Ingredients of cell transfection for immunofluorescence
Lipid Mix:
Lipofectamine3000 Opti-MEM Total *3
22.5μl 427.5μl 450μl
DNA Mixes:
CAV1-V72 (656.3ng/ μl)
P3000 Reagent Opti-MEM Total *1
11μl 14.4μl 425μl 450μl
CAV1-V96 (476ng/ μl)
P3000 Reagent Opti-MEM Total *1
15.10μl 14.4μl 420.5μl 450μl
CAV1-A96 (349.5ng/ μl)
P3000 Reagent Opti-MEM Total *1
20.6μl 14.4μl
415x
450μl

Three lipofectamine 3000 mixes were made in three 1.5 mL Eppendorf tubes. Tubes were
vortexed, briefly centrifuged, and incubated 5 minutes at room temperature in the hood. Tubes
were sprayed with 70% alcohol before placing back into hood. While the lipofectamine 3000 mixes
were being incubated, DNA mixes were made, vortexed and briefly centrifuged. Tubes were
sprayed with 70% alcohol before placing back into hood. Corresponding DNA mixes tubes were
added to lipofectamine 3000 tubes. Mixed tubes were vortexed, briefly centrifuged, and incubated
for 15 minutes at room temperature in the hood. Meanwhile, two 12-well plates were prepared by:
18
aspirating old DMEM/FBS, washing with PBS, refilling with 900 uL optimem to each well of the
two 12-well plates. After incubation of DNA/lipofectamine 3000 mixes, 100μl of each mix was
added to the corresponding wells. 12-well plates were incubated at 30°C for 48 hours.
On Day 4, two 12-well plates were taken out of incubator, put on ice in the 4°C fridge for 40
min. Meanwhile CXTB (attached to AF555) dilution was made:21μl CXTB(1000X) + 20.979ml
media. The dilution tube was plated on ice. The media was aspirated and the plates were washed
with cold DPBS. Four wells in the 24 wells are set to be the control group---without CXTB. 1ml
DMEM media was added to those 4 wells. 1ml CXTB dilution was added to the rest 20 wells. The
plates were put in dark 4°C environment for 10 min. The plates were shaken gently every 3 min.
The plates were taken out and the liquid was aspirated. The plates were rinsed with DPBS 3
times.1ml media was added to each well. One plate was picked ramdomly to receive 4°C
treatment and the other to receive 37°C treatment. The 4°C treatment plate was put in the 4°
C fridge (covered with foil) and the 37°C treatment plate was put on the 37°C heatplate (covered
with foil as well) for 45min. Meanwhile 26ml 4% paraformaldehyde (16% stock diluted in PBS),
50ml 50mM NH4Cl (in PBS), 26ml 0.1% Triton-X-100 (10% Triton-X-100 diluted in PBS), and
53ml 1% BSA solution (in PBS) were made. The plated were fixed in 4% paraformaldehyde in
PBS for 15 minutes. Each well was rinsed with 1ml 50mM NH4Cl (in PBS) for 5 min. then with
1ml PBS, 5min*3. Each well was permeabilized with 1ml 0.1% or Triton X-100 for 10 minutes.
Each well was rinsed with 1ml PBS, 5min*3. The glass cover slip was incubated with the primary
Ab (1:1000 dilution) at 4°C overnight.
On Day 5, the two 12-well plates were washed with PBS 5 min*5 times. Chicken anti-mouse
AF647 (we ran out of goat anti-mouse AF488) secondary antibody (1:500 dilution in 1%BSA)
19
was added and plated were incubated for 1h in room temperature with gently shaking. 1ml DAPI
dilution (1:1000 in PBS) was added to per well of the two 12-well plates and incubated at room
temperature for 5min with gently shaking. Plates were washed with PBS 5min*5 times with gently
shaking. The slides were sealed using mounting media.
2.6 Flow cytometry

Flow cytometry is also used to validate the effect of CAV1-ELP assembly on CAVME. On Day
1, HEK 293 T cells are plated in two 6-well plates. Each well receives 2ml DMEM-Dulbecco’s
modified eagle medium (Thermo Fisher Scientific, Waltham, MA) and 0.5ml cell suspension
(approximately 1 million cells for each plate). There is no Poly-D-Lysine added to the wells before
cells are plated because cells are better to get off the plates easily when being collected for flow
cytometry. On Day 2, Cells are transfected as Figure 1 shows. Two plates receive the same
transfection protocol. For each plate, two wells are transfected with CAV1-V96; two wells are
transfected with CAV1-A96; two wells receive no transfection.

20
Figure 1. Flow cytometry transfecting instruction

Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA) is used to facilitate the
transfection. Dilute Lipofectamine 3000 Reagent in Opti-MEM reduced serum medium (Thermo
Fisher Scientific, Waltham, MA). Each well receives 3.75 μl Lipofectamine 3000 Reagent;
Prepare master mix of DNA by diluting DNA in Opti-MEM medium, then add P3000 Reagent.
Each well receives 2.5 μg DNA, and 5μl P3000 Reagent. Diluted DNA was added to each tube
of diluted Lipofectamine 3000 Reagent (1:1 ratio). DNA-lipid complex was added to each well.
After that, cells are incubated in 30°C for 48h. When cells are incubated for 24h, opti-MEM was
sucked out and replaced with same amount of DMEM media. During this procedure so far, all the
washing uses PBS that contains calcium and magnesium. On Day 4, the two 6-well plates were
taken out from the incubator, and put on ice in the 4°C fridge for 50 min. One plate was randomly
chosen, and replaced with 37°C pre-warmed DMEM media. Then this plate is put on a hot plate
which is set to 37°C for 40min. The medium in the other plate is replaced with 4°C DMEM
media. Then this plate is put on ice in 4°C fridge for 40min. After temperature treatment, wash
cells with 4°C PBS that does not contain calcium and magnesium. Cholera Toxin B- AF647
antibody was diluted 1000 times in 4°C DMEM media (18µL to 18ml). For cells that are
transfected with CAV1-V96 or CAV1-A96, each well receives 2ml mix. For cells that receives no
transfection, each well receives same amount of 4°C DMEM media. Two plates were wrapped
with foil and put on ice in 4°C fridge for 10min. After 10min incubation, cells were gotten off
the plate by pipetting gently, and cell suspension was collected in 1.5ml Eppendorf tube. Cells
21
were spun down at 0.3rcf for 6min. Supernatant were sucked out gently and cells were suspended
with 1ml PBS (without calcium and magnesium). This step was repeated twice. Then Cholera
Toxin (CXTB)-AF647 labeled CAV1-myc-V96 or CAV1-myc-A96 transfected 293T cells were
washed and resuspended in PBS and then stained by LIVE/DEAD Fixable Aqua (L34957,
Invitrogen) for 20 min on ice, followed by fixation with 4% paraformaldehyde for 20 min at RT.
Residual paraformaldehyde were completely removed by 2X washing with permeabilization wash
buffer (421001, BioLegend). Cells were subsequently subject to anti-myc-AF488 antibody
(2279S, Cell Signaling Technology) staining after 1:50 dilution in intracellular staining perm wash
buffer for 30 min on ice. Finally, cells were washed two more times with permeabilization wash
buffer and resuspended in PBS and recorded by flow cytometer (BD LSRFORTESSA X-20) and
analyzed by FlowJo software, version 10. The first gate was set on live cells based on LIVE/DEAD
Fixable Aqua staining. With non-transfected cells as the control, the second gate was set to myc-
positive population, representing successfully transfected population. Eventually, CXTB signal
was compared among groups only in transfected cell population.
3. Results

3.1 Generation and characterization of temperature-responsive CAV1-ELP fusion proteins

The results of V72 DNA and CAV1 DNA isolation are shown respectively in Figure 2 and Figure
3. The result of restriction digestion of newly-built CAV1-V72 plasmid is shown in Figure 4.
22

Figure 2. The figure shows the desirable band of V72 DNA after restriction digestion. There are
two experiment groups which contains two different V72 DNA amount. HindIII and MsII were
used for restriction digestion. The desirable band at 1128bp was cut for ligation.
23

Figure 3. The figure shows the desirable band of CAV1 DNA after restriction digestion. There are
4 experiment groups which contains 4 different CAV1 colonies. HindIII and EcoRV are used for
restriction digestion. The desirable band at 5324bp was cut for ligation.

24

Figure 4. Restriction digestion result shows the right molecular weights of CAV1 and V72. After
ligation, restriction digestion was conducted to double check the desirable sequence. In single
digested group, only HindIII enzyme was used to digest the newly-built plasmid, and in double
digested group, HindIII and NotI were used to digested the newly-built plasmid.
A library of CAV1-ELP constructs including CAV1-A96, CAV1-V96, CAV1-V72 and CAV1-
WT were designed and cloned. CAV1-V72 construct was cloned by me; CAV1-A96 was cloned
by David Tyrpak; CAV1-V96 and CAV1-WT were cloned by Anh Truong. An ELP is a
pentameric repeat of a five amino acid sequence (Val-Pro-Gly-X-Gly)n, where X and n are a guest
residue and the repeating number of the sequence respectively. Due to the characteristic of ELP,
different transition temperatures of different sequences can be achieved by changing the guest
residue and the repeating number. Basically, the construct of CAV1-A96 serves as a negative
control since Alanine is very hydrophilic and CAV1-A96 still remains soluble in aqueous solution
when it is heat above 80 ℃. Therefore, it is assumed as temperature insensitive. CAV1-WT is a
25
construct without being attached an ELP, therefore it also freely distributed on the lipid raft and
should not undergo phase separation during thermal stimulation. CAV1-V72 and CAV1-V96 can
undergo phase change and self-assemble when heated above transition temperature, and their
respective transition temperature were measured using a live cell imaging method developed in
our laboratory (detail in the next section). The molecular weights of the four constructs were
estimated based on their amino acid sequence, and were confirmed by western blot.

Figure 5. Design and characterization of CAV1-ELPs. a. Genes encoding four ELPs (A96, V96,
V72, WT) were attached to the C-terminus of CAV1 with a myc tag for immune-detection of
protein expression. b. Molecular weights of each CAV1-ELP construct were estimated based on
their amino acid sequence, and were confirmed by subsequent western blot. c. CA1-ELPs plasmids
were transfected into HEK 293T cells. CAV1-ELP proteins were detected by anti-myc primary
antibody. GAPDH served as a control to indicate the protein amount. Lane 1. No transfection 2.
CAV1-WT 3. CAV1-V72 4. CAV1-V96 5. CAV1-A96.
26

3.2 Measurement of the Tt of CAV1-ELP fusion proteins

To precisely measure the transition temperature of CAV1-ELP constructs, our group have
developed a technique to make CAV1-ELP visible and its Tt measurable. Normally, CAV1-ELPs
are invisible under fluorescence microscope. However, there is a special characteristic (Figure 6)
of ELPs we can utilize, which is that similar ELPs can co-assemble. This means when a cell is
transfected with two ELPs constructs, assembly of the construct with a lower Tt induces anothers
assembly with a higher Tt. Due to this characteristic, GFP-V60 plasmid is transfected along with
CAV1-V72 (dual transfection). In this case, CAV1-ELP self-assembly will be visible because of
GFP-V60 self-assembly. What’s more, dual transfected cells display larger but less numerous
puncta, compared to the control group---single transfected cells (GFP-V60 only), which makes it
easy for us to identify dual vs. single transfected cells (Figure 7). After imaging cells, transition
temperature was analyzed with ImageJ software. During the time ramp, as temperature increases,
puncta gradually form within the cell and the standard deviation of image pixel value increases as
well (Figure 8). When temperature reaches Tt, the standard deviation of pixel value will
dramatically jump. The threshold is set up as the 4 standard deviations away from the average
level, which is assumed as its transition temperature (Figure 9). This method and data analysis
were also done by David Tyrpak. The results of each CAV1-ELP’s transition temperature is
shown in Figure 10. Tt of CAV1-V96 was measured by Hugo Avila by doing the same protocol as
described previously. It turned out that CAV1-V96 has a transition temperature about 33℃ while
the transition temperature of CAV1-V72 is higher, which is about 37℃. Each time a CAV1-ELP
construct’s Tt was measured, the Tt of GFP-V60 was measured as well. Therefore, the Tt of GFP-
V60 was measured two time separately, and the results are pretty close to each other, which means
27
this method is pretty reliable and consistent. It turned out that the Tt of GFP-V60 was about 40℃.
There is a significance difference of Tt between dual and single transfected cells (Figure 11).

Figure 6. The assembly of an ELP construct with a lower Tt can induce the assembly of another
ELP construct with a higher Tt. Thus, transfecting the cell with both GFP-V60 and CAV1-ELP can
visualize ELP assembly.
28

Figure 7. Dual transfected and single transfected cell are distinguishable in aspects of puncta
number and puncta size. Dual transfected cells tend to form big but less numerous puncta while
single transfected cells tend to form small but numerous puncta.

29
Figure 8. The standard deviation of pixel value increases as temperature increases. When
temperature reaches Tt, the standard deviation of pixel value will dramatically increase. Cell 1 and
Cell 2 are the standard deviation-temperature profile of dual transfected cell while Cell 3 and Cell
4 the standard deviation-temperature profile of single transfected cell.

Figure 9. The threshold is set up as the point four standard deviations away from the average level.
The temperature at this point is defined as Tt. This threshold is set up by David Tyrpak.
30

Figure 10. The results of transition temperatures of CAV1-V72 and CAV1-V96. Transition
temperature of CAV1-V72 was measured by Yue Wang; transition temperature of CAV1-V96 was
messured by Hugo Avila. Both of the investigators shared the same protocol.
31

Figure 11. The significance difference of Tt between dual and single transfected cells. This figure
is made by David Tyrpak.
3.3 Validation of the effect of CAV1-ELP assembly on CAVME by confocal microscopy

To validate whether CAVME can be triggered by assembly of CAV1-ELPs, HEK 293T cells
transfected with CAV1-ELPs received two temperature treatments respectively (4 ℃and 37℃) for
40 minutes. Prior to assay, the GM1 molecule located on the extracellular side of caveolae was
pre-stained at 4 ℃ with AlexaFluor555 conjugated Cholera Toxin B. GM1 molecule is highly
concentrated in caveolae and can be internalized by CAVME. Cholera toxin B (CXTB)
specifically binds to GM1. After incubation, fixation and washing, CAV1-ELP was treated with
anti-myc primary antibody and Alexa Fluor-488 secondary antibody. From the results (Figure 12),
it is shown that at 4℃, CAV1-V72 and CXTB were freely distributed at cell surface. There was
32
no significant colocalization of CAV1-V72 and CXTB. However, at 37℃, CAV1-V72 transferred
from cell surface into cytoplasm and self-assembled into intense green fluorescent clusters.
Meanwhile, CXTB also formed clustered and these clusters significantly colocalized with CAV1-
V72 clusters. Similar results were observed for CAV1-V96. At 37℃, CAV1-V96 formed clusters
and significantly colocalized with CXTB while no significant colocalization was observed at 4℃
. For CAV1-A96, since A96 is physiological temperature insensitive and serves as a negative
control, even when temperature rose to 37℃, CAV1-A96 did not undergo phase separate nor self-
assemble. As a consequence, no significant colocalization was observed at 37℃ between CAV1-
A96 and CXTB. For CAV1-WT, since CAV1 was not fused to an ELP construct, CAVME cannot
be triggered by temperature stimulation. Therefore, no significant colocalization was observed at
37℃ between CAV1-A96 and CXTB. In summary, the results above showed that stimulated by
temperature increase, CAV1-V72 and CAV1-V96 undergo phase separate and self-assemble, thus
CAVME is activated and GM1 in internalized as a consequence.
33

Figure 12. CAV1-ELP assembly activates CAVME and initiates molecule internalization. HEK
293T cells were transfected with CAV1-ELPs. GM1 molecule was labeled by CXTB (red) and
CAV1-ELPs were treated by anti-myc antibody (green). Cells were labeled by CXTB prior to
temperature treatment (4 ℃ and 37 ℃). Afterwards, images were obtained by using confocal
34
microscopy. Green: AlexaFluor-488 conjugated anti-myc antibody; Red: AlexaFluor-555
conjugated CXTB; Blue: DAPI; Scale bar: 5µm.
3.4 Validation the effect of CAV1-ELPs aggregation on CAVME by flow cytometry

CAV1-ELPs transfected cell are labeled with AF647 conjugated Cholera Toxin Unit B. AF647
Fluorescent intensity are detected by flow cytometry. The results are showed in the following
several figures. For this part, cell preparation experiment was done by me; Flow cytometry and
analysis were performed by Hao Guo.

35
Figure 13. Fluorescent intensity overlay of cells transfected with CAV1-V96 4°C treatment
and 37°C treatment. The figure was made by Hao Guo.

Figure 14. Fluorescent intensity overlay of cells transfected with CAV1-A96 4°C treatment
and 37°C treatment. The figure was made by Hao Guo.
36
There are bigger CXTB signal increase from CAV1-V96 37°C to CAV1-V96 4°C. Details are
shown as below:
Table 7. CXTB signal difference of CAV1-V96 group and CAV1-A96 group. Data in this table
was obtained from Hao Guo.

There is more significant fluorescent signal increase from 37°C to 4°C in CAV-V96 compared
to CAV1-A96. That proves the initial hypothesis that CAV1-ELP phase change can activate
CAVME and internalize GM1 outside the cell membrane. CAV1-V96 has a transition temperature
at 33°C, therefore at 37°C, it undergoes phase change, activate CAVME and internalize GM1.
Therefore, there is a reduction of CXTB signal compared to 4°C where CAV1-ELP remains
soluble in cytoplasm. For CAV1-A96 is temperature insensitive, and it does not react to
Construct transfect and incubation
temperature
Higher CXTB percentage (signal intensity
above 8*10^2)
CAV1-V94 4°C
47.2
CAV1-V96 37°C
41.2
CAV1-A96 4°C
44.4
CAV1-A96 37°C
41.2
37
temperature change. Therefore, there is not much difference between CAV1-A96 4°C group and
CAV1-A96 37°C group.
4. Discussion

There is no doubt that endocytosis is essential in a variety of cellular processes and diseases.
However, the existing chemical methods to study endocytosis are not good enough. They either
lack of specificity or produce a significant time lag which allows cells develop compensation
mechanism. In this manuscript, a thermos-responsive protein polymer ELP was described as a tool
to study caveolin-mediated endocytosis. Manipulated by the same tool, it has been shown in
previous research that by fusing ELP to a key effector protein, clathrin light chain, the clathrin-
mediated endocytosis can be inhibited by temperature increase and this inhibition is reversible,
rapid and specific. Inspired by this idea, similar technique was used for CAVME manipulation.
When we tried to characterize the library of CAV1-ELP constructs by western blot, it was strange
that the temperature-sensitive two constructs CAV1-V72 and CAV1-V96 were always missing on
the blot. At first, the lysis buffer was changed RIPA to CHAPS. CHAPS detergent, (3-((3-
cholamidopropyl) dimethyllammonio)-1-propanesulfonate), is a zwitterionic detergent that has a
hydrophobic head and a hydrophilic head, which resembles the structure of membrane protein.
Therefore, it is specialized for membrane protein solubilization and good at protecting the native
state of proteins. After this change, the results were better, but it was still often that case that
CAV1-V72 and CAV1-V96 were missing on the blot. However, after one attempt that the transfer
method was changed from iBlot2 dry blotting system (1.5A 5min) to semi-wet method (20V
overnight), the two temperature-sensitive CAV1-ELP constructs appeared on the blot. The method
was validated by several repeating. In the end, the most possible speculation was that the large
38
amount of heat produced during the fierce and fast transferring process by iBlot2 dry blotting
system may have contributed to the aggregation of temperature-sensitive CAV1-ELP constructs.
Altenatively, caveolin is an integral membrane protein which could be heat-unstable and is easy
to aggregate. These factors contributed to the aggregation of CAV1-ELPs and may have caused
them not to appear. In contrast, transferring proteins from gel to blot by semi-wet overnight method
is mild and produces much less heat, which reduces dramatically the production of heat and
appears to allow transfer and detection of CAV1-ELPs on the blot.
The activation of CAVME was confirmed under confocal microscopy. At 37 ℃ ,
immunofluorescence suggested significant assembly of CAV1-V96 and CAV1-V72 in cells, and
they significantly colocalized with CXTB. In contrast, due to physiological temperature
insensitivity, there was no significant colocalization between CAV1-A96 and CXTB; Due to the
absence of ELP, CAV1-WT cannot be activated by temperature stimulation. All of the observation
above validated the activation of CAVME by CAV1-ELP self-assembly.
For the flow cytometry experiment, Cells attached to the plate was directly washed by PBS. Then
it turned out that too many cells were lost during the washing step and there was barely any cell
left on the plate after several times of washing. Therefore, we changed the way of washing cells.
After washing plates with PBS, cell suspensions were collected in Eppendorf tubes, then spn down
the tubes at 0.3rcf for 6min. After centrifuge, supernatant is taken out and fresh PBS is added to
the tube. Then the cells pellets were resuspended into suspension. By using this way to wash cells,
plenty of cells are left after several times of washing.

39
5. Conclusion

This thesis provides a novel way to manipulate Caveolin-mediated endocytosis. By using Elasin-
like polypeptides as a tool and fusing it with caveolin, the mainly functional protein in CAVME,
the aggregation of CAV1-ELP can activate CAVME. It has been tested that the activation of
CAVME can internalize GM1 molecule by different methods including immunofluorescence and
flow cytometry. For future direction, given the fact that caveolin is essential in many diseases’
development and progression, the ELP tool can be potentially applied in exploring the relationship
between caveolin expression abnormality and pathogenesis.

40
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Biomacromolecules 11, 944-952.

Abstract (if available)

Abstract Endocytosis has an important role in cellular functions, including nutrient uptake, signaling, antigen presentation, growth and differentiation, etc. To better understand endocytosis, scientists have developed a variety of chemical and genetic endocytic manipulators. However, most of these tools are irreversible or lack specificity. In addition, few technologies have been found to specifically manipulate caveolin-mediated endocytosis (CAVME). To better study caveolin-mediated endocytosis, we use Elastin-like polypeptides (ELPs) as a thermally responsive tool to rapidly and specifically manipulate cellular processes, including CAVME. We hypothesize that ELPs fused to a mainly functional protein involved in CAVME will modulate the pathway. To test this hypothesis, ELPs were fused to Caveolin 1 (CAV1), a key functional protein associated with CAVME. Before temperature stimulation, the CAV1-ELP fusion is soluble in aqueous solution and CAVME stays in “off” status. Temperature increase can activate the self-assembly of CAV1-ELP fusion proteins that switches CAVME to “on” status. These microdomains can react to thermally stimulation within a few minutes, do not need exogenous stimulation, and are specific for CAVME pathway. This thesis manuscript involves cloning a new CAV1-ELP construct, determining its self-assembly temperature using a live cell imaging technique, and using confocal microscopy to determine the effect of CAV1-ELP self-assembly on CAVME. This manuscript demonstrates that assembly of CAV1-ELP through slight temperature modulation can activate Caveolin-mediated endocytic pathway.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Wang, Yue (author)

Core Title Temperature-mediated induction of caveolin-mediated endocytosis via elastin-like polypeptides

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/24/2020

Defense Date 05/10/2019

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

Tag caveolin-mediated endocytosis,elastin-like polypeptide,HEK 293T cells,OAI-PMH Harvest,transition temperature

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor MacKay, J. Andrew (committee chair), Duncan, Roger (committee member), Zhang, Yong (committee member)

Creator Email wang586@usc.edu,yuew1118@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-148979

Unique identifier UC11662664

Identifier etd-WangYue-7284.pdf (filename),usctheses-c89-148979 (legacy record id)

Legacy Identifier etd-WangYue-7284.pdf

Dmrecord 148979

Document Type Thesis

Format application/pdf (imt)

Rights Wang, Yue

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA