INTRODUCTION

Clathrin-mediated endocytosis is the process by which extracellular macromolecules are

selectively internalized into a cell by the use of vesicles.
1
In clathrin-mediated endocytosis,
clathrin-coated pits form around the extracellular macromolecules that are to be internalized, and
these pits become vesicles as they separate from the cell membrane, which is triggered by
GTPases.
2
This process is essential to many different cell processes, such as signaling and cell
trafficking. There are a number of different proteins that facilitate the process of clathrin-
mediated endocytosis.
3
PACSIN2 is a protein that has been identified to play an important role
in clathrin-mediated endocytosis, specifically in zebrafish (Danio rerio).
4

PACSIN2 is a PCH protein, specifically a member of the F-BAR protein domain.
3
The
PCH family of proteins was originally thought of as a group of proteins that helped with
regulating cytokinesis and changes in actin structure. The PCH proteins are found in many
eukaryotes and all have a similar basic structure. This group of proteins is known to interact
with many other proteins and help control their organization and their subcellular dispersal.
PCH proteins have even been found to regulate the activity of other proteins. Some processes
that these proteins are known to be involved with include cell morphology, the mechanical and
functional integrity of organelles, cell motility, configuration of the actin cytoskeleton, and
protein trafficking. The PCH proteins are involved in these processes by their interactions of the
surface of the cell membrane, which allows them to be active in endocytosis and exocytosis.
5

Recent studies have shown that the F-BAR domain of PCH proteins allows them to bind to cell
membranes and cause deformations to them, which can result in changes in the curvature of the
membrane or tubulation of the membrane.
6
The PCH proteins induce invaginations by binding
to phospholipids on the cell membrane, which is how they specifically play a role in endocytosis.


Different proteins produce distinct shaped invaginations. For example, F-BAR domain proteins
are known to create a rather shallow crescent-shaped indentation.
2
In a study by Shih Lin et al.
(2012), it was found that the deformation of the plasma membrane by PACSIN2 in mammals
was reduced when full-length PACSIN2 was used to create invaginations compared to using
PACSIN1. Here, the tubular invaginations characteristic of PACSIN2 were still generated, but
the ability to create vesicles was impaired in comparison to the activity of PACSIN1.
3
Also, in a
study by de Kreuk et al. (2012), they found that PACSIN2 regulated EGF receptor surface
expression in HeLa cells and that it is important for ligand-independent trafficking of the EGF
receptor.
7

In previous studies, PACSIN2 has been shown to interact with other proteins during
endocytosis. In a study by Lam and Hordijk (2013), they found that PACSIN2 interacted with
Rac1 in membrane ruffles, where PACSIN2 seemed to be recruited to places where Rac1 was
working in order to induce internalization in those areas.
8
Interactions between PACSIN2 and
Rac1 were also shown in HeLa cells and mouse embryonic fibroblast cells in a study by de
Kruek et al. (2011). Here they found that PACSIN2 interacts with Rac1 specifically at its C-
terminus.
9
Edeling et al. (2009) performed a study which looked at how PACSIN affects
embryonic notochord development in zebrafish, but they mostly focused on PACSIN3 in this
study. However, they did mention that the endocytic components in cell movements during the
formation of the notochord in zebrafish are not well understood, although they did find that
PACSIN3 plays a key role in proper formation of the notochord.
4
The interactions of PACSIN2
with proteins in the cells of humans and other mammals has been studied by a number of people,
however, the interactions of PACSIN2 with other proteins in zebrafish are poorly understood.


Because this is a subject that has not been extensively researched, the purpose of this study was
to explore the interactions of PACSIN2 with other proteins in zebrafish cells.
METHODS
PCR for making GST-PACSIN2
Two different samples of GST-PACSIN2 were prepared, one using Paq 5000, and another using
the P fusion enzyme. While keeping them on ice, 1L of PACSIN2 DNA with a dilution of 1:50
was added to two eppendorf tubes. Then 2L of PACSIN2 Forward BamH1 primer and 2L of
PACSIN2 Reverse EcoR1 primer each having a concentration of 25 pmol/L were added to each
of the tubes, followed by the addition of 5L of 10x deoxynucleotide triphosphates (dNTPs) to
each tube. Then 5L of 10x buffer, 34L of double distilled water, and 1L of Paq 5000 were
added to the first tube, yielding a total volume of 50L. To the second tube, 10L of 5x buffer,
29L of double distilled water, and 1L of Pfusion enzyme were added in order to bring the final
volume to 50L. The samples were then put into the PCR machine set to cycle at 55 C Ior 45
seconds and 72 C for 2 minutes.
Gel Electrophoresis
In order to prepare a 0.8% agarose gel, 0.4g of DNA grade agarose was added to 50mL of 1x
TAE (Tris-acetate-EDTA) buffer, which was microwaved until the agarose dissolved. 50L of
EtBr were added to the mixture. The gel was poured and once the gel hardened, samples were
loaded and run at 100V for 30 minutes. A 1 Kb Plus DNA Ladder was used as reference for the
samples.
Gel Extraction of PCR Product GST-PACSIN2
Gel extraction was performed using the Qiagen protocol. GST-PACSIN2 was cut out of the
agarose gel using a razor blade and placed in an eppendorf tube. The gel weighed 187mg, and


this number was multiplied by three to determine that 561L of Buffer QG should be added to
the tube. This mixture was incubated at 50 C for three minutes in order to dissolve the gel.
187L of isopropanol was added and 750L of this mixture were added to a spin column in a
collection tube and centrifuged at 1300rpm. The liquid that entered the collection tube was
discarded and 750L of Buffer PE were added followed by centrifugation. A new eppendorf
tube was placed under the spin column and 30L of Buffer EB was added and centrifuged. The
liquid that flowed through the spin column was kept, yielding a total volume of approximately
28L of product.
Digest of PACSIN2 and pGEX 4T-1 with BamH1
In order to cut PACSIN2 with BamHI, 4L of 10x buffer, 4L of 10x BSA, 1L of BamH1, and
3L of double distilled water were added to 28L of PACSIN2 while kept on ice. Next, the
vector pGEX 4T-1, which was prepared by Dr. Cooper, was cut with BamH1. This was done by
adding 3L of 10x buffer, 3L of 10x BSA, 1L of BamH1, and 13L of double distilled water
to 10L of pGEX 4T-1, while on ice. The samples were then placed in the incubator Ior
approximately one hour at 37 C.
Purification of PACSIN2 cut with BamH1
In order to purify the product from the digest with BamH1, 200L of Buffer PB were added to
the 40L of digest product. This was placed in a spin tube column and centrifuged at 1300rpm
for one minute. The liquid that flowed through the column was discarded and 750L of Buffer
PE was added to the tube followed by centrifugation. The flow-through liquid was discarded and
the spin column was placed in a new eppendorf tube. Then 30L of Buffer EB was added to the
spin column, which was centrifuged and the liquid that flowed through the column was kept,
resulting in approximately 28L of product.


Digest of PACSIN2 and pGEX 4T-1 with EcoR1
In order to cut the protein and vector with EcoR1, 4L of 10x buffer, 4L of 10x BSA, 1L of
EcoR1, and 3L of double distilled water were added to 28L of PACSIN2 and 28L of pGEX
4T-1, while kept on ice, resulting in two samples with a total volume of 40L. Both samples
were put in the incubator Ior approximately one hour at 37 C.
Purification of PACSIN2 and pGEX 4T-1 cut with BamH1 and EcoR1
40L of PACSIN2 and 40L of pGEX 4T-1 were placed into two separate quick spin columns.
200L of Buffer PB were added to each column and centrifuged at 1300rpm for 1 minute. The
waste in each collection tube was discarded. 750L of Buffer PE was added to each spin column
and centrifuged. Again, the waste was discarded. A new tube was placed under each of the spin
columns and 50L of Buffer EB was added to each spin column followed by centrifugation. The
flow-through from each spin column was kept as the final products from this procedure.
Pouring Plates
Plates were created to grow bacteria that would grow PACSIN2. 250ml of LB broth was made
by combining 2.5g of Tryptone, 1.25g of yeast extract, 2.5g of NaCl, and a little less than 250mL
of double distilled water. Once the mixture was dissolved, 3.75g of agar was added. The LB
broth was autoclaved for twenty minutes and allowed to cool for thirty minutes. Then 500L of
ampicillin was added to the broth and the plates were poured.
Ligation
A control and experimental sample were made to try to properly insert PACSIN2 into the vector.
The experimental sample was made by combining 1L of the vector pGEX 4T-1 with 16L of
the insert PACSIN2, 2L of T4 DNA Ligase 10x Buffer, and 1L of T4 DNA Ligase. The
control was made by combining 1L of the vector pGEX 4T-1 with 2L of T4 DNA Ligase 10x


Buffer, 1L of T4 DNA Ligase, and 16L of double distilled water. Both of these samples were
vortexed and centrifuged for approximately ten seconds and allowed to sit at room temperature.
Transformation
Cells were thawed and transferred to two new tubes. 10L oI the experimental ligation was
added to the Iirst tube and 10L oI the control ligation was added to the second tube with the
thawed cells. They were kept on ice Ior approximately 10 minutes and put into a water bath set
at 42 C Ior exactly 45 seconds. The samples were immediately put back on ice and 900L oI LB
broth were added to each tube. They were placed in the incubator at 37 C while shaking Ior one
hour. Plates that were prepared using the above procedure were taken out of the incubator and a
sterilized glass spreader was used to spread 100L of either the experimental or control sample
onto them. Then the plates were placed back in the incubator to allow the bacteria to grow.
Picking Colonies for Minipreps
In order to prepare for minipreps, colonies were grown in LB broth. This was done by adding
6L of ampicillin and 3mL of LB broth to a 15mL tube and touching a pipet tip to a colony that
grew on the experimental plate and putting it in the tube. The tube was then autoclaved.
Minipreps
Colonies were poured into separate eppendorf tubes and centrifuged for one minute at 1300rpm.
The liquid was dumped off of the pellets and 100L of Solution 1 was added to each tube. The
samples were vortexed in order to resuspend the pellet. 200L of Solution 2 was added to each
tube, and the samples were mixed by inverting the tubes. 150L of Solution 3 was added to each
tube and the tubes were inverted to mix the samples. The samples were then centrifuged at
1300rpm for ten minutes. 400L of isopropanol was added to new tubes and 400 L of sample
was added to each of these new tubes while avoiding the precipitate. The samples were


centrifuged at 1300rpm for ten minutes and as much isopropanol as possible was poured out of
the tubes. A large DNA pellet could be seen on the bottom of the tubes. 200L of double
distilled water was added to each tube and the pellets were redissolved by vortexing the samples.
20L of 3M NaOAc was added to each sample followed by 1mL of 100% EtOH. The samples
were placed in the centrifuge for 5 minutes at 1300rpm. The liquid was dumped out of the tubes
and the pellet was washed with 70% EtOH and. Then 40L of 40g/mL RNase in double
distilled water was added to each sample and the pellet was resuspended.
Digests of Minipreps
For the first digests, 5L of each miniprep was combined with 1L of 10x Multicore buffer, 1L
of 10x BSA, 0.5L of EcoR1, 0.5L of EcoRV, and 2L of double distilled water in a separate
tube. Then 5L of each miniprep was mixed with 1L oI 10x BuIIer D, 1L oI 10x BSA, 0.5L
oI EcoRV, 0.5L oI Bgl II, and 2L oI double distilled water. The tubes were placed in the
incubator Ior one hour at 37 C. The digests were then run on a gel using the above procedure Ior
gel electrophoresis to look for proper insertion of PACSIN2 into the vector.

RESULTS
The creation of GST-tagged PACSIN2 was important so it would be possible to detect PACSIN2
in later analyses of this project, such as the GST pull-down assay. In order to try to make GST-
PACSIN2, P fusion enzyme and Paq 5000 were used in order to see which method was more
successful. GST-PACSIN2 has approximately 2,223 base pairs, and looking at Figure 1, two
bands of various strengths can be seen around 2,000 bp (lane 2 and lane 4). Based on the size
and darkness of the band in lane 4 (Figure 1) it was decided that the P fusion enzyme worked
better to make GST-PACSIN2, and this sample was extracted from the gel for further analysis.



Figure 1: Gel Electrophoresis of GST-PACSIN2. GST-PACSIN2 has approximately 2223
base pairs. PCR was used to try to create GST-PACSIN2 by using either Paq 5000 or P fusion
enzyme. PACSIN2 DNA with a 1:50 dilution was exposed to PACSIN2 Forward BamH1 primer
and PACSIN2 Reverse EcoR1 primer with either Paq 5000 or P Iusion enzyme. The PCR
program was set to cycle at 55 C Ior 45 seconds and 72 C Ior 2 minutes. Gel electrophoresis was
conducted by adding 10L of the PCR product with Paq 5000 with 2L of dye to lane 2 and
10L of the PCR product with P fusion enzyme with 2L of dye to lane 4. A 1 Kb Plus DNA
Ladder was added to lane 1 to use as a reference tool.

DISCUSSION
The production of GST-PACSIN2 was successful using the P fusion enzyme in this experiment.
Here I will go on to discuss all of my findings and relate them to previous research.
REFERENCES
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