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Methods and protocols for detecting the intracellular assembly of elastin-like polypeptides

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Methods and protocols for detecting the intracellular assembly of elastin-like polypeptides

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Methods and protocols for detecting the intracellular
assembly of elastin-like polypeptides

by

Yaocun Li

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

August 2020

Copyright 2020 Yaocun Li

ii

ACKNOWLEDGMENTS

First of all, I would like to thank my advisor, Dr. J. Andrew MacKay, who gave me work in
this fantastic laboratory where I learned many new practical technologies. Without the continuous
support and encouragement for my research from his group, I could not complete this thesis.

I would like to thank Dr. Zhipeng Lu and Dr. Ian S. Haworth for their careful review of my
thesis and their useful suggestions for my thesis writing.

I would also like to thank all the members of the laboratory. They gave me great support
during the laboratory work, especially David Tyrpak and Hugo Avila, for their guidance and
technical help.

Finally, I would like to express my most enormous gratitude to my parents. Without their
support, I could not study and obtain a degree at the University of Southern California.

iii

TABLE OF CONTENTS
ACKNOWLEDGMENTS ............................................................................................................ ii
List of Tables .................................................................................................................................. v
List of Figures ............................................................................................................................... vi
Abbreviations .............................................................................................................................. vii
Abstract ....................................................................................................................................... viii
1. Introduction ............................................................................................................................... 1
1.1 Biological Study .................................................................................................................... 5
1.2 Therapeutic peptide drug delivery ......................................................................................... 7
2. Materials .................................................................................................................................... 9
2.1 Reagents ................................................................................................................................ 9
3. Methods and Protocols ............................................................................................................11
3.1 Passage Cells ........................................................................................................................11
3.2 Transfection ......................................................................................................................... 12
3.3 Live-cell Imaging and Temperature Control during Imaging ............................................. 13
4. Results ...................................................................................................................................... 15
4.1 Characterization of the ELP-fusion proteins ....................................................................... 15
4.1.1 ELP constructs show thermal sensitivity after co-transfection ..................................... 15
4.1.2 Determination the Transition Temperature of the ELP-Clathrin Proteins .................... 17
4.2 Reversibility of these switch proteins ................................................................................. 22
5. Discussion................................................................................................................................. 23
6. Conclusion ............................................................................................................................... 25

iv

References .................................................................................................................................... 26

v

List of Tables
Table 1. Results of analysis of variance (anova)………………………………………………….19
Table 2. Results of Turkey Post-Hoc Test………………………………………………………...19
Table3. Results of one way anova………………………………………………………………..21

vi

List of Figures
Figure 1. The basic structure of the ELPs………………………………………………………….1
Figure 2. ELP fusion proteins designed to control the clathrin-mediated endocytosis…………….3
Figure 3. Module for the function of the Dynamin during the clathrin-mediated endocytosis…….6
Figure 4. ELPs’ self-assmble…………………………………………………………………….16
Figure 5. Clusters form during temperature increase…………………………………………….17
Figure 6. The transition temperature of each cell………………………………………………...18
Figure 7. Differences in mean levels of transfection type………………………………………..20
Figure 8. Reversibility of ELP’s activity…………………………………………………………22

vii

Abbreviations
ELP Elastin-like Polypeptides
Transition temperature Tt
Val Valine
Pro Proline
Gly Glycine
CLC Clathrin Light Chain
CHC Clathrin Heavy Chain
CME Clathrin-mediated Endocytosis
EPR Enhanced Permeability and Retention effect
DMEM Dulbecco's Modified Eagle Medium
FBS Fetal Bovine Serum

viii

Abstract
It is crucial to explore the role of proteins in various cellular processes. A common way to
study the function of proteins is to reduce the activity of a specific protein (gene deletion, siRNA,
small molecule inhibitor) to study the effect on the cell. Dr. MacKay’s team has developed a new
method to do this using proteins that are sensitive to temperature change-- Elastin-like
polypeptides (ELPs). A small increase in temperature will stimulate the assembly of ELPs inside
of cells. If ELPs are connected to a target protein, the resulting ELP fusion protein is soluble at
low temperature. When the temperature increased, the ELP fusion protein can be induced to
assemble into organelle-sized microdomains, thus close the cell pathway mediated by this protein.
This method's characteristic is that these organelle-sized microdomains can be thermally activated
and inactivated in a few minutes, which is reversible and does not need exogenous chemical
stimulation. This temperature triggered cell switching system provides a new platform for time
manipulation of transport mechanisms in healthy and disease cell models and has applications in
manipulating other intracellular pathways.

1

1. Introduction
Many fundamental cellular processes are mediated by protein-protein interactions.
Accordingly, control of protein-protein interactions may elucidate primary mechanisms in cell
biology.
[1]
Over the past few years, a series of tools have been designed to control the activity of
the target proteins to study specific cellular pathways.
[2]
These induction systems respond to
endogenous proteins, exogenous small molecules, and environmental sensors such as pH
[3]
, light
[4]
,
heat
[5]
, and glucose
[6]
. Unfortunately, these methods often take a long time to induce and may also
produce off-target effects. An ideal method should be rapid and reversible. To achieve this goal,
we explored biopolymers named Elastin-like polypeptides.
Elastin-like polypeptides (ELPs) are biosynthesized polymers. The basic structure of the
ELPs is shown below. (Figure 1.)

Figure1. The basic structure of the ELPs.

V P G X
G
n

2

The sequence of the ELP monomeric unit is [Val-Pro-Gly-X-Gly]n. The "X" represents
variable amino acid, and the "n" represents the repeat number of this single monomeric unit.
Different amino acids and the repeat number influence the general properties of the ELPs, such as
transition temperature (Tt), which is the most critical feature of the construct. At the transition
temperature (Tt), ELPs undergo a reversible phase change within a very narrow temperature range
(2-3
o
C). Below this critical temperature, ELPs are highly soluble, solvated, and disordered. Once
the temperature rises to the Tt, ELPs gather rapidly, forming a micro-meter size cluster. The whole
process can be completed in several minutes and is reversible.
This Tt of the specific ELP is affected by many factors, like the sequence of the polypeptide,
molecular weight, concentration, ionic strength, and acidity and alkalinity of the solution.
[7]
If the
residues are more hydrophobic, this will reduce the Tt of the ELP. On the contrary, if the residues
are more hydrophilic, it will increase the Tt of the ELP.
[8]
Also, studies have shown that
increasing molecular weight without changing the overall hydrotherapy significantly reduces
Tt.
[9]
Additionally, substituents containing groups susceptible to ionization can adjust Tt by
changing the pH value of the solution.
[10]
However, it should be noted that the first glycine in the
ELP sequence cannot be replaced by any other amino acid except alanine, because it will hinder
the formation of beta-transformation.
[11]

3

Figure 2. ELP fusion proteins designed to control the clathrin-mediated endocytosis. a) The gene construct of two
different ELP fusion proteins. b) The Schematic of clathrin triskelia with and without the ELP molecule. c) Below the
critical temperature, the ELP fusions remain soluble, and the whole-cell pathway would not be blocked. d) After
increase the temperature to the critical temperature (transition temperature), the ELP would form to the micrometer-
level clusters and interrupt the interaction between the clathrin light chain and the clathrin heavy chain. Thus, block
the pathways of endocytosis.

4

To determine whether temperature-mediated assembly of ELPs can control a cellular pathway,
we used ELPs to control clathrin-mediated endocytosis. After the stimulation of the bind between
receptors and ligands on the cell's membrane, the clathrin light chain (CLC) and the clathrin heavy
chain (CHC) moved from the cytoplasm to the cell membrane and form basket-like triskelion to
help the formation of the vesicles for cargo endocytosis. In this experiment, we connect the ELP
molecule to the clathrin light chain (CLC) for the following reasons: i) the clathrin light chain is
vital for helping to form the triskelion.
[12]
ii) The function of the CLC would not be interrupted by
adding green fluorescent protein as a marker.
[13]
The formation of the triskelion needs the
interaction between the clathrin light chain and the clathrin heavy chain.
[14]
(Figure 2.)

5

1.1 Biological Study
And an effective way to study the function of a specific protein is to reduce or knockdown its
activity. Endocytosis is an essential physiological process of cells, and it is an important way for
cells to absorb substances. The formation of endocytic vesicles requires the recruitment of various
proteins from the cytosol, which coordinates the inward bending of the plasma membrane to form
a deep inverted bud and then promote its division. One protein directly involved in a fission
reaction is GTPase dynamin, a founding member of the GTPase family, which plays different roles
in the whole-cell membrane remodeling event. The role of dynamic proteins in endocytosis has
been studied for more than 20 years.
[15][16][17][18]
A large amount of evidence shows that Dynamin
assembles into helical polymers in the neck of the budding vesicles. The conformational changes
depending on GTP hydrolysis promote the splitting of underlying tubular membranes to form free
inner circulation vesicles.
[19]
The most well studied endocytic pathway is clathrin-mediated endocytosis (CME), which
relies on clathrin proteins to endocytose and ferry cargo. Notably, clathrin proteins have been used
as ELP fusion proteins.
[20]
Five stages of CME have been identified by using ultrastructural and
cellular biological observations through the formation of clathrin-coated vesicles:
[11]
i) Nucleation.
ii) Cargo Selection. iii) Clathrin coat assembly. iv) Vesicle scission and v) Uncoating and clathrin
component recycling. Since the cargo is selected and bound by AP2 or cargo-specific adaptor
protein, clathrin coating must be assembled to help transfer the cargo. Triskelia move to the plasma
membrane through AP2 and auxiliary adaptor protein. In the absence of clathrin, AP2 is absorbed
into the plasma membrane and interaction with nucleation form a puncta structure. However, this

6

kind of structure cannot mature, and cargo cannot be successfully transferred.
After the formation of the clathrin-mediated vesicle, the mechanochemical enzyme
Dynamin scissions the vesicle from the plasma membrane. (Figure 3.)
[21][22]
BAR domain-
containing proteins help absorb the clathrin-coated vesicle.
[23][24][25]
Then this kind of protein helps
mediate the scission by performing the GTP hydrolysis-dependent conformational
change.
[26][27][28][29]
After the internalization of the cargo and the scission of the vesicle, free clathrin
is mechanically returned to the cytoplasm where it is reabsorbed and reused in another round of
vesicle formation.

Figure 3. Module for the function of the Dynamin during the clathrin-mediated endocytosis

7

1.2 Therapeutic peptide drug delivery
In addition to their use as tools for biological research, ELPs are also very useful in
therapeutic peptide drug delivery, especially for cancer treatment.
[30]
. Tumor tissue has
considerable differences with normal tissue in regards to anatomical and structural characteristics.
Tumor blood vessels are unevenly distributed and often lack adequate lymphatic drainage, which
can lead to uneven blood flow, slow blood flow, and abnormal hemodynamics in tumor tissues.
Therefore, compared with normal tissues, macromolecules and soluble polymer-carriers can
preferentially permeate and accumulate in tumors in a phenomenon called the EPR (Enhanced
Permeability and Retention effect). Also, the literature shows that there is a thermal effect around
tumor cells called hyperthermia, which results in a slight increase in the temperature around the
tumor cells. Therefore, if the Tt of an ELP is between the temperature of normal tissues and the
tumor cells, then the ELP will self-assemble around the tumor cells. Based on this principle, if the
ELP is linked to the drug for cancer treatment, the fusion protein can self-assemble near the tumor
cells, and concentrate around the tumor cells better through the EPR effect, which can achieve a
better therapeutic effect on tumors. Studies of implanting human tumors into nude mice clearly
showed that hyperthermia of tumors resulted in increased accumulation of ELP polypeptide.
[31]
To
better explain the accumulation of ELP caused by this thermal effect. The researchers also
measured the gathering of the same ELP in cancer cells with hyperthermia and without
hyperthermia. The results showed that the accumulation of ELP in heat tumors was twice as much
as that of the same polypeptide without hyperthermia, and the accumulation of heat-responsive
polypeptide in heat tumors was caused by the reversal and subsequent aggregation of the heat-

8

responsive polypeptide, not by the non-specific physiological polypeptide.

1.3. Aim of the Study
The purpose of this study was to determine the transition temperature of ELP fusion proteins
inside of live cells. Based on the previous study of the intracellular ELPs,
[27],
two polymers were
selected for fusion with CLC: i) A96-CLC, ii) V96-CLC. Unfortunately, there is nothing
fluorescent on the ELPs-CLCs, and we cannot see when they self-assemble without fixing/killing
the cells and staining the ELPs with fluorescent antibodies. However, ELPs self-assemble with
each other, even when they are attached to different proteins. Accordingly, by co-transfecting cells
with an ELP-CLC and another fluorescent ELP fusion protein, the Tt of the ELP-CLC proteins can
be visualized through co-assembly with the fluorescent ELP fusion protein.
In this experiment, we choose GFP-V60 for the co-transfection ELP and observe intracellular
assembly. Three kinds of samples were created: i) Single transfection of GFP-V60 only, ii) Co-
transfection of A96-CLC plus GFP-V60, and iii) co-transfection of V96-CLC plus GFP-V60. Then,
we created a method allowing us to obtain the images of detecting the intracellular assembly in the
live cells.

9

2. Materials
2.1 Reagents
Cell Culture
• HEK293T Cells (#CRL-3216, ATCC, Manassas, V A)
• Dulbecco's Modified Eagle Medium (DMEM) (11995065, Thermo Fisher Scientific,
Waltham, MA)
• Fetal Bovine Serum (FBS) ((#35-011-CV , Corning, NY)
• Trypsin-EDTA (0.05%) (25300054, Thermo Fisher Scientific, Waltham, MA)
• T75 Cell Culture Flask (4616, Laguna Scientific, Laguna Niguel, CA)
• 1X dPBS (#25-508, Genesee, San Diego, CA)

Transfection
• Lipofectamine™ 3000 Transfection Reagent (L3000008, Life Technologies, Carlsbad,
CA)
• Opti-MEM™ (Reduced Serum Medium) (31985070, Thermo Fisher Scientific, Waltham,
MA)
• Poly-D-Lysine (P6407, Sigma-Aldrich, St. Louis, MO)
• 35mm glass bottom dishes (P35G-0-10-C, MatTek Corporation, Ashland, MA)

Live Cell Imaging

10

• Live Cell Imaging Solution (A14291DJ, Thermo Fisher Scientific, Waltham, MA)
• LSM 880 (Carl Zeiss, Oberkochen, Germany)
• LD LCI Plan Apochromat 25x/0.8 numerical aperture objective for
oil/water/glycerol/silicone immersion (420852-9871-000, Carl Zeiss, Oberkochen,
Germany)
• Stage attachment Z PIEZO WSB 500 (D) (432339-9000-000, Carl Zeiss, Oberkochen,
Germany)
• Stage insert Z PIEZO WSB 500 for Heating Inserts P S1 / Mxx S1 (D) (432339-9050-000,
Carl Zeiss, Oberkochen, Germany)
• Immersol W (444969-0000-000, Carl Zeiss, Oberkochen, Germany)
• Argon 488 laser line (Carl Zeiss, Oberkochen, Germany)

2.2 Image Analysis
• FIJI
• SIAL (a FIJI plugin for image randomization and blinding: https://sites.imagej.net/D-tear/)
• Template Matching (an optional FIJI plugin for stabilizing lateral drift in time lapse images:
http://sites.imagej.net/Template_Matching/)

11

3. Methods and Protocols
3.1 Passage Cells
HEK293T cells used for imaging were grown in a T75-flask in the incubator with 5% CO2 at
37
o
C. Cells were passaged onto 35mm glass bottom dishes (MatTek plates) using Trypsin-EDTA
(0.05%) before transfection.
The DPBS was used to remove the dead cells from the T75 flask. After washing, we used 3 mL of
Trypsin-EDTA and allowed it to incubate for 3 minutes at 37
o
C in the incubator. The cell media
was made by 7 mL of DMEM + 10% FBS to deactivate the Trypsin-EDTA. Then, 10 mL of cells
were transferred to a 15 mL falcon tube and centrifuged at 200 RFC for 10 minutes. During this
time, the MatTek plates were prepared with Poly-D-Lysin: We pipetted 2 mL of Poly-D-Lysine to
each plate and incubated for 5 min. When incubation is completed, Poly-D-Lysine was removed,
and 2 ml of cell media was added to each plate. (Plate is now prepared for cell plating). After
centrifugation, we aspirated the solution from the falcon tube and resuspended cells in 10 mL cell
media. Before adding cells to the MatTek plates, we counted cells in the plate using a cell counter
and microscope. Then, cells were transferred to plates for subsequent transfection incubated in the
incubator at 37
o
C for 24 h.

12

3.2 Transfection
Cells were transfected using Lipofectamine™ 3000 Kit. The transfection reagents and
plasmid DNA were diluted in Opti-MEM™ Reduced Serum Medium. We mimicked the protocol
for a single 6-well plate preparing lipofectamine mix for MatTek plates. For each 35 mm plate, the
transfection mix include 125 ul DNA mixture ( 2.5 ug total of plasmid DNA, P3000 Reagent at a
concentration of 2µg/µl of DNA and Opti-MEM up to 125µl) and 125 ul lipofectamine mixture
(5µl of Lipofectamine 3000 and 120µl of Opti-MEM). Before transfection, two different mixtures
were mixed before adding to the plates. Then this master mix was incubated at room temperature
for 15 minutes before adding to the plates. During the incubation, aspirate the cell media in the
MatTek plates and wash the plates with dPBS. Then refill the plates with 2ml of Opti-MEM™
Reduced Serum Medium. After incubation, add 250 ul master mix to each plate. Plates were then
placed in the incubator at 30
o
C for three days (72 hrs) for imaging.
For dual transfected cells (GFP-V60 + V96-CLC and GFP-V60 + A96-CLC), 1.25 ug of each
DNA was used for each plate to make the master mix. The rest of the protocol was the same as the
single transfected cell plates.

13

3.3 Live-cell Imaging and Temperature Control during Imaging
The live cell images were obtained with a LSM 880 confocal microscope equipped with an
airy scan detector and definite focus module. We used Immersol W instead of water as the
immersion media because it has the same refractive index of water but does not evaporate easily.
The 488-argon laser line was used to excite the GFP-V60, and all images were obtained using
airy scan fast mode with definite focus. The laser power was selected at 17%, and then the laser
light was passed through a main beam splitter (MBS). After exciting the cells, the emitted light
passed through a dual bandpass (495nm-550nm) and long pass (>= 570nm) filter before reaching
the detector. For the detector parameters, we chose 625 master gain and 1.00 digital gain. All
images were 16-bit. For automatic image acquisition, we used the "Time Series" module and
acquired images every 20 seconds. For each plate, 90 images were obtained.
The circulating temperature bath was used for temperature control during the imaging. The
cells in the 35mm dish were placed on ice and carried to the microscope room. Then it was placed
on a PeCon Heating Insert P Lab-TekTM S1stage attached to a circulating temperature bath. To
measure the temperature more precisely, we used a PeCon Control Sensor T S1 which was placed
directly into the live cell imaging solution.
When all the instruments are installed and set up, the temperature of the water bath should be
set to 60
o
C. Once the temperature of the water bath reached 60
o
C, it was maintained it at 60
o
C.
During this period, the images were acquired every 20 seconds. In 30 minutes, a total of 90 images
were captured.

14

Critical Points and Comments
The probe must be put directly into the solution to determine the temperature of the solution
as accurately as possible. Also, although the set temperature of the water bath was 60
o
C, the
temperature of the solution was less than 60
o
C. During the whole process of image capture, the
temperature range of the solution was about 20-40
o
C.
We were using Immersol W instead of DI water as the immersion solution. If water was used,
the water evaporated as the temperature increased and quality of the images decreased dramatically.
In addition, during the process of heating up the position of cells may change as the imaging dish
expands. Accordingly, the ‘definite focus’ mode was used at all times to ensure that the microscope
was properly focused.

15

4. Results
All image analysis was performed using FIJI. Before data analysis, it is recommended that
any data files be randomly named to minimize human interference, including identifying transition
temperature, drawing ROI, and selecting thresholds for subsequent microdomains analysis. At this
point, we developed a Java program to rename the data files to random three digit numbers.

4.1 Characterization of the ELP-fusion proteins
4.1.1 ELP constructs show thermal sensitivity after co-transfection
Using the confocal microscope, we evaluated the intracellular assembly of targeted ELP-
fusion proteins. In this experiment, we wanted to determine the transition temperature of the ELPs
inside the live cells. However, there is nothing fluorescent on the ELPs-CLCs, so we cannot see
when they self-assemble without fixing/killing the cells and staining the ELPs with fluorescent
antibodies. In addition, based on previous data, A96-CLC does not self-assemble at physiological
temperatures. However, after co-transfection with GFP-V60, A96-CLC started to self-assemble at
around 35
o
C (Figure 4.). This thermal sensitivity shows ELPs self-assemble, even when they are
attached to different proteins. So, by co-transfecting with GFP-V60, we can see when the ELPs-
CLC self-assemble because they will pull GFP-V60 proteins into the assembled coacervates. The
same thing happened to V96-CLC; by co-transfection with GFP-V60, V96-CLC shows a similar
thermal sensitivity compared to the A96-CLC, albeit at a different temperature.

16

Figure 4. In this figure, V60 represents GFP-V60, A96 represents GFP-V60 + A96-CLC, and V96 represents GFP-
V60 + V96-CLC. If A96 is co-transfected with GFP-V60, the species will self-assemble during the heating process,
and they will form clusters in the cytoplasm.

17

4.1.2 Determination the Transition Temperature of the ELP-Clathrin Proteins
After randomly renaming the files, the transition temperature of each cell could be determined
by visual inspection. When the cluster begins to appear in the cytoplasm of the image, the
temperature of that frame was recorded. (Figure 5.) Repeat this step to count the transition
temperature of all cells. All images were analyzed by Fiji ImageJ software.

V96-CLC+GFP-V60
A96-CLC+GFP-V60
GFP-V60
Figure 5. Clusters form during
temperature increase.The red arrow in
the figure shows that the clusters
started to form when the temperature
increase. Record temperature when the
images appeared the clusters in the
cytoplasm

18

Figure 6 shows the distribution of the transition temperature of all cells. The plots in the
figure may indicate that the transition temperature of GFP-V60 + V96-CLC is lower than GFP-
V60. Also, the transition temperature of GFP-V60 + A96-CLC may be slightly lower than GFP-
V60. However, to confirm these conclusions, we need to make a statistical analysis of these data.

Figure 6. The transition temperature of each cell. The points were colored based on the date they were imaged.

19

The analysis of variance (anova) shows that the transfection type is a statistically significant
predictor of the transition temperature. (Table 1.)
Df Sum Sq Mean Sq F Value Pr (>F)
Transfection Type 2 47.83 23.916 3.318 0.049*
Residuals 32 230.63 7.207
Table 1. Results of analysis of variance (anova).

The results show that the p-value is 0.049, below 0.05. So the transfection type is a significant
predictor of the transition temperature. The Tukey Post-Hoc test is used to figure out which
transfection groups are statistically significantly different from each other. (Table 2.)

Transfection Type diff lwr upr P adj
V60 + A96 2.5098039 -0.6228912 5.6424991 0.1364197
V60 + V96 -2.2431373 -4.7304948 0.2442203 0.0836676
V96 + A96 0.2666667 -3.0318927 3.5652260 0.9784922
Table 2. Results of Tukey Post-Hoc test. In this table, V60 represents GFP-V60, A96 represents GFP-V60 + A96-
CLC, and V96 represents GFP-V60 + V96-CLC.

20

Unfortunately, none of the above p-values are significant. However, for the GFP-V60 vs. dual
transfection groups, the confidence intervals ("lwr," "upper" columns) are heavily lopsided, and
the point estimates for the difference in transfection temperature ("diff" column) are pretty
significant, nearly a 2.5-degree difference. To see this, we can plot the confidence intervals from
the tukey result. (Figure 7.)

Figure 7. Differences in mean levels of transfection type. In this figure, A96 represents GFP-V60 + A96-CLC, and
V96 represents GFP-V60 + V96-CLC.

A96/V60
V96/V60

21

These confidence intervals suggest that, in reality, there is a true biological difference. The P-
value isn't getting small enough because of the low sample sizes and high variability
We used one-way anova to test whether there is a significant difference in transition
temperature between GFP-V60 and GFP-V60 + V96-CLC. (Table 3.) This table shows that the p-
value is already below 0.05. And we can see the V96-CLC+GFP-V60 group has a transition
temperature of 2.24
o
C below GFP-V60.

Transfection
Type
Estimate Std. Error t value Pr(>|t|)
(Intercept) 36.1765 0.6106 59.243 <2e-16
V96 -2.2431 0.9493 -2.363 0.0256
Table 3. Results of one way anova. In this table, V96 represents GFP-V60 + V96-CLC. We dropped the A96-CLC
group since it has a low sample size, is partially confounded with date, and because it is so close to the V96-CLC
group. Including it in the model requires degrees of freedom, which impacts the adjusted P-value criteria, without
contributing any explanatory power. Because we only have two levels, we can directly check whether there is a
statistically significant difference between GFP-V60 ("intercept" column in table 3) and V96-CLC + GFP-V60.

22

4.2 Reversibility of these switch proteins
To test the reversibility of this process of phase separation, we cooled the cells that have
formed clusters, and observed whether ELPs would become soluble again. After acquired images
from the cells, the clusters formed in the cells disappeared after cooling down. The results
demonstrate that one round of heating and cooling caused no loss of the ELPs' activity. (Figure 8.)

Figure 8. After cooling the cells formed clusters, ELPs became soluble again and evenly distributed in the
cytoplasm, which proves that this process is reversible.

23

5. Discussion
There is no doubt that protein interaction and assembly is essential in a variety of cellular
processes. However, the existing methods to detect protein function are not ideal.
[2]
They typically
produce a time lag that allows cells to develop compensation mechanisms, leading to inaccurate
results. Through this experiment, we have established a set of methods that can be used to
determine the transition temperature of ELPs. Also, for some ELPs without thermal properties
(A96-CLC), they can become thermal sensitive by transfection with other ELPs (GFP-V60), which
are thermally sensitive.
It was shown in the previous study that when cells are dually transfected, micro-
domain assembly occurs at a lower temperature in comparison to cells transfected with single ELP.
[32]
In this experiment, V96-CLC shows the same properties. The Tt of V96-CLC + GFP-V60 group
is 2.24
o
C below the GFP-V60 group. (Table 3.)
When we tried to characterize the library of A96-ELP constructs by live cell imaging, it is
strange that the transfection efficiency of A96 was significantly lower than GFP-V60 + V96-CLC
or GFP-V60 alone. But unfortunately, no matter how we changed the conditions of the transfection
step, the final transfection efficiency of A96-CLC + GFP-V60 is significantly lower than the other
two groups (V96-CLC + GFP-60 and GFP-V60 only).
In addition, it should be noted that the clusters size formed by V96-CLC + GFP-V60 and
GFP-V60 alone is very similar. Therefore, Tt can also be an important indicator to distinguish V96-
CLC + GFP-60 and GFP- V60 alone.

24

As in the previous experiment
[20]
,

the three ELPs in this experiment all showed reversibility.
When we cooled the cells that have formed clusters, theses clusters in the cells disappeared after
cooling down, which means one round of heating and cooling caused no loss of ELPs' activity.
(Figure 8.)

25

6. Conclusion
This experiment provides an effective way to detect the Tt of the elastin-like polypeptides.
Compared with the traditional methods, this method is not only efficient and reversible but may
also can prevent cells from producing compensation effects.
Given the fact that clathrin is essential for receptor-mediated endocytosis, future studies could
explore the relationship between ELP-CLC expression and assembly with cell signaling.

26

References
1. Ausländer S, Ausländer D, Müller M, Wieland M, Fussenegger M. Programmable single-
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Abstract (if available)

Abstract It is crucial to explore the role of proteins in various cellular processes. A common way to study the function of proteins is to reduce the activity of a specific protein (gene deletion, siRNA, small molecule inhibitor) to study the effect on the cell. Dr. MacKay’s team has developed a new method to do this using proteins that are sensitive to temperature change—Elastin-like polypeptides (ELPs). A small increase in temperature will stimulate the assembly of ELPs inside of cells. If ELPs are connected to a target protein, the resulting ELP fusion protein is soluble at low temperature. When the temperature increased, the ELP fusion protein can be induced to assemble into organelle-sized microdomains, thus close the cell pathway mediated by this protein. This method's characteristic is that these organelle-sized microdomains can be thermally activated and inactivated in a few minutes, which is reversible and does not need exogenous chemical stimulation. This temperature triggered cell switching system provides a new platform for time manipulation of transport mechanisms in healthy and disease cell models and has applications in manipulating other intracellular pathways.

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Asset Metadata

Creator Li, Yaocun (author)

Core Title Methods and protocols for detecting the intracellular assembly of elastin-like polypeptides

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 08/02/2020

Defense Date 07/31/2020

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

Tag clusters,elastin-like polypeptides,methods,OAI-PMH Harvest,transition temperature

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mackay, Andrew (committee chair), Ian, Haworth (committee member), Lu, Zhipeng (committee member)

Creator Email dk868@163.com,yaocunli@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-354792

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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...

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