Project Title: Effects of Overexpression of PSTPIP1 in Dictyostelium discoideum
Student Applicant: Erin Brannick, Biological Research Major, Loras College, Dubuque, IA
Faculty Mentor: Dr. Kate Cooper, Assistant Professor of Biology, Loras College, Dubuque, IA
Project Description
This project will delve into the functions of a family of proteins involved in cell migration in
Dictyostelium discoideum, a soil-living, eukaryotic organism. These cells grow independently, but can
migrate and form multicellular structures in adverse conditions. Because their genome contains many
genes that are homologous to higher eukaryotes (such as humans), Dictyostelium have become a model
organism for the scientific research community. They are most often used to study cell motility, but are also
useful in looking at phagocytosis, cell differentiation, and other similar processes. They are relatively
inexpensive and easy to grow and observe in a laboratory setting (Chisholm et al., 2006).
Because Dictyostelium are very useful organisms for understanding the process of cell migration,
some aspects of this process have been extensively researched already. It is understood that cells use
various proteins from their own cytosol to shape their membranes, and in cooperation with the
cytoskeleton, promote changes within the membrane that contribute to membrane trafficking, cell division,
and cell migration (Frost et al., 2009). One of the protein families shown to be involved in the process of cell
migration in Dictyostelium organisms is the PCH (pombe cdc15 homology) family. The PCH family may play
their role in cell migration in mammalian cells due to coordination of membrane and cytoskeletal
movements. Research has shown that the F-BAR domain is conserved in all PCH family proteins (Chitu and
Stanley, 2007). The conservation of this domain across numerous species and throughout evolution
indicates its importance in the function of the members of the PCH family (Tsujita et al., 2006). The F-BAR
domain functions by interacting with membrane phospholipids in a variety of mechanisms in order to
change curvature of the membrane of an organism. Changing the shape of the cell membrane and the
organization of the actin filaments induces cell movements (Chitu and Stanley, 2007). To date, six PCH
family genes encoding proteins with the F-BAR domain have been identified in Dictyostelium cells (Heath
and Insall 2008).
One of the PCH family proteins that potentially plays a role in cell migration is PSTPIP1 (proline
serine threonine phosphatase-interacting protein 1). This protein has not yet been studied in Dictyostelium
cells. In other cells, it has been found to form homodimers and generate filaments associated with cell
membranes, thus cell movement (Waite et al., 2009). Overall, previous research has been focused on the
PCH proteins and the effects they have on cell migration as a whole. The general mechanism of cell
migration is understood, however, the specific role of the each member of the PCH family is still unknown.
The goal of this research project is to examine the effects of the PSTPIP1 protein on cell migration in
Dictyostelium discoideum cells in order to increase the understanding of the mechanism of cell migration as
well as the specific, molecular role of the PCH family proteins in this process. It is important to understand
the mechanism of cell migration in model organisms as fully as possible, because these organisms’
functions are analogous to the same processes in human cells.
In order to investigate the role PSTPIP1 plays in cell migration in Dictyostelium cells, the cells must
be able to incorporate a foreign DNA vector into their own genome, the PSTPIP-specific overexpression
vector must be created, and experiments must be done to observe the effects of this overexpression. The
process of incorporating a foreign DNA vector has already been conducted yielding sufficient results. Going
forward, the goal of this project is to create a vector with a gene for fluorescently-tagged PSTPIP1 and
overexpress it by inserting it in Dictyostelium cells. The effects of this overexpression will be observed
through time-lapse videomicroscopy of chemotaxis. Cell speed and directionality will be investigated, as
well as the cellular localization of the PSTPIP1 protein during migration.
Methods & Means to be Utilized
Wildtype (KAX3 strain) Dictyostelium cells will be used (Dictybase stock center)
Calcium Phosphate Transfection (based on protocol in Chisholm et al., 2006)
In order for Dictyostelium cells to undergo transfection via calcium phosphate precipitation, they
are grown in HL5 media for three to four days. The day before the cells are going to be transformed, the
media should be switched from HL5 to 12.5mL Bis-Tris HL5 media. This media is replaced with 10mL fresh
Bis-Tris HL5 the day of the transformation in order to further reduce the phosphate.
The DNA that will be transformed into the cells also needs to be prepared. This is accomplished by
adding 0.24mL sterile water, 10µg DNA, 0.3mL 2x HBS, and 60µL 1.25M CaCl2. Once the DNA is prepared,
the Bis-Tris HL5 media should be removed from the cells, and the DNA suspension is added by pipetting
drops around the plate. The plates must sit for 30 minutes for the cells to take up the DNA vector. After this
period, 10mL fresh Bis-Tris HL5 is added and the plates are left alone for 4 to 8 hours to allow the cells to
adhere to the plate.
After the resting period, the Dictyostelium cells need to undergo a glycerol shock. This is done by
carefully removing the Bis-Tris HL5 media and gently adding 4mL of 18% glycerol in HBS. The glycerol is
exposed to the cells for exactly five minutes (within 10 seconds) and then gently taken off and replaced
with 10mL of HL5 media. The cells then need to sit overnight to develop phenotypic expression.
The day after the transfection, the cells should be observed with a fluorescent microscope to
determine the degree of phenotypic expression. If successful, the transformants can be selected using
10mL fresh HL5 that
contains 5 to 10µg/mL G418
and 200µg/mL
streptomycin. Aspirate this
solution up and down to
remove the cells from the
plate, and place them into
microtiter well plates. The
original plate should be
saved and replenished with
fresh medium. The cells
Figure 1. Phenotypic Expression of GFP-Actin Binding Domain in Dictyostelium
should be fed twice a week
following Transfection. Wildtype Dictyostelium cells were transfected using the
for two to four weeks.
calcium phosphate precipitation protocol in Chisholm et al., 2006. The transfected
In order to optimize
plate (right) shows some cells with significant fluorescence compared to the control
this process in Dictyostelium,
plate (left). The cells were transfected with the pDXA-GFPABD120 plasmid (Knecht
several experiments were
et al., 1998) for 8 hours with 2xHBS pH 7.2. The cells exhibit some background
conducted to determine the
fluorescence, which is compounded by being on plastic dishes instead of glass (see
most successful conditions.
budget).
Results thus far have
revealed that the most transformants are obtained using cells at a concentration of 0.5x105cells/mL, 2x HBS
solution at pH 7.2, and a resting period after exposure to the DNA of eight hours (Figure 1). Transformants
were selected using 60mm plates, not microtiter well plates.
Creating the PSTPIP1 Overexpression Vector
The PSTPIP1 vector will be prepared by first obtaining RNA from Dictyostelium cells using an
RNAqueous® Kit by Life Technologies. This RNA will be converted to complementary DNA using the MMuLV reverse transcriptase enzyme from New England Biolabs. A polymerase chain reaction (PCR) will then
be conducted on that cDNA using the primers specific for the
Dictyostelium PSTPIP1 gene - dPSTPIP1. The DNA plus primer
L 1 2
solution will be run on an agarose DNA gel. The gel will be
investigated for the presence of the PSTPIP1 protein by noting
the size and location of the bands present and determining if
the results match the size of the PSTPIP1 protein. A previous
research student began this protocol and the results of his
experiment can be seen in Figure 2.
After the dPSTPIP1 gene has been isolated, it must be
incorporated into a vector in order to overexpress the gene in
Dictyostelium. This is done by treating the mRFPmars in pBsrH
plasmid in the cells with enzymes (BAMH1 and ECOR1) to
create ‘sticky ends’ for the vector. The vector will also be
treated
with these enzymes to create complementary
Figure 2. Dictyostelium PSTPIP1 Gene Copied
overhangs in order to insert itself into the plasmid. If inserted
from cDNA by PCR. PSTPIP1-specific primers
correctly, the cells should begin expressing red fluorescent
were used to run a PCR reaction on
protein attached to the dPSTPIP1.
Dictyostelium cDNA and the product run on a
gel (lanes 1 and 2). The band is the correct,
expected size for dPSTPIP1 as compared to the 1
kb plus DNA ladder (L).
Observing the Effects of Overexpression (based on protocol in
Chisholm et al., 2006)
The effects of overexpression of PSTPIP1 on cell
migration will be observed by performing an under-agarose
assay and noting the movements (folate chemotaxis) of the transformed Dictyostelium cells compared to
cells that are not over-expressing PSTPIP1. Changes in direction of the cell’s path, migration speed, and
efficiency will be examined.
SM media must be used because HL5 media contains traces of folate and will affect the migration
of the cells toward a folate source. A 1mM folic acid working solution (prepared from 50mM folic acid stock
solution) will be loaded onto the middle trough of an agarose gel plate with three troughs cut 39mm long,
2mm wide, and 5mm apart. Cells at a concentration of 5x106-1x107 cells/mL are loaded into the other two
troughs. The plate should sit flat for one hour before observing cell migration using a time lapse function on
a fluorescent microscope (Olympus IX-81) equipped with a digital camera (Olympus XM10) and Cellsens
software for image capture and analysis.
Student Activity & Responsibility
Ms. Brannick has already been working on this project for several months. She has learned how to
culture the cells, and independently investigated protocols to optimize transfection. She will work with Dr.
Cooper to learn PCR, DNA cloning techniques, time-lapse microscope, and data analysis using Image J. She
will then be responsible for repeating all experiments, compiling the data, and doing the statistical analysis.
She will work with Dr. Cooper at all steps to interpret data and plan experiments. The project likely will
require about 20 hours a week in the summer (10 weeks), as well as 5 hours a week for much of the school
year (2013-2014). Ms. Brannick will then be responsible for putting together a poster presentation of the
work, as well as collaborating with Dr. Cooper to prepare a manuscript for publication.
Desired Outcomes
The transfection of Dictyostelium cells through the calcium phosphate precipitation method has
already yielded cells expressing fluorescent protein. Previous student projects have shown the PSTPIP1
gene can be isolated from cDNA. By combining this knowledge, we plan to create the vector with
fluorescently-tagged dPSTPIP1, overexpress it in Dictyostelium cells, and analyze the effects of this
overexpression on cell migration as well as determining where this protein is expressed during cell
movements. Overall, we hope to gain insight into the role of a specific protein in cell migration that has not
previously been studied in this important model organism.
In addition to the scientific benefits of this project, working on this project will have many benefits
for Ms. Brannick as she will continue to gain hands-on experience on laboratory techniques, but even more
importantly, she will continue to gain experience in designing experiments and analyzing data, which will be
beneficial in her future career.
Budget & Justification
We are asking McElroy for a student stipend to compensate Ms. Brannick for some of her time
spent working on this project, important funds for the molecular biology reagents for the project to create
the fluorescently-tagged dPSTPIP1 (PCR enzymes, restriction enzymes), and culture dishes with glass
bottoms so that the cells can be imaged while migrating using fluorescence without the background glow
from the plastic dishes. All of these requested reagents are essential for the success of the proposed
project.
Request:
Amount requested ($):
Stipend for Erin Brannick
500
Molecular biology reagents and supplies:
Enzymes and reagents for cloning (PCR & restriction enzymes) 550
DNA purification kits (Qiagen, gel extraction, PCR clean-up)
550
Glass dishes:
35mm glass-bottomed dishes, uncoated (MatTek)
400
Total
$ 2000
Bibliography
Chisholm, R. L., Gaudet, P., Pilcher, K. E., Fey, P., et al. (2006). dictyBase, the Model Organism Database for
Dictyostelium discoideum. Nucleic Acids Res 34, D423-7. Various links available at:
http://dictybase.org/techniques/index.html.
Chitu, V., Stanley, E. R. (2007). Pombe Cdc15 Homology (PCH) Proteins: Coordinators of MembraneCytoskeletal Interactions. TRENDS in Cell Biology 17, 145-156.
Frost, A., Unger, V. M., and Camilli, P. (2009). The BAR Domain Superfamily: Membrane-Molding
Macromolecules. Journal of Cell Science 137, 191-196.
Heath, R. J. W., Insall, Robert H. (2008). Dictyostelium MEGAPs: F-Bar Domain Proteins that Regulate
Motility and Membrane Tubulation in Contractile Vacuoles. Journal of Cell Science 121, 1054-1064.
Knecht, D. A., Lee, E., Pang, K. M. (1998). Use of a Fusion Protein between GFP and an Actin-Binding Domain
to Visualize Transient Filamentous-Actin Structures. Curr Biol 8 (7): 405-308.
Tsujita, K., Suetsugu, S., Sasaki, N., Furutani, M., et al. (2006). Coordination Between the Actin Cytoskeleton
and Membrane Deformation by a Novel Membrane Tubulation Domain of PCH Proteins is Involved
in Endocytosis. Journal of Cell Biology 172, 269-279.
Waite, A. L., Schaner, P., Richards, N., Masters, S. L., et al. (2009). Pyrin Modulates the Intracellular
Distribution of PSTPIP1. PLoS ONE 4, 6147.
Biographical sketches:
Erin C. Brannick
I am currently a junior biological research major at Loras College (GPA: 4.0; Major GPA: 4.0). I am
the executive secretary of the Health Science Club and Loras Student Alumni Council organizations. I am
also a member of the Delta Epsilon Sigma National Honor Society. I have served as the supplemental
instructor for the introductory biology class for the past four semesters. I try to shed a positive light on
Loras College in all of my involvements and put my whole heart into all that I do.
Because I wanted to develop my passions for science and learning, I began working on
undergraduate research under the direction of Dr. Cooper at the start of my junior year. I have continued to
pursue research throughout the year, and have gained exceptional knowledge about the techniques
required for my project specifically, but also the demands of research in general. I hope to make significant
progress in gaining knowledge about the PSTPIP1 protein in Dictyostelium by graduation. Research has
already been a great opportunity for applying my biological knowledge, and I am grateful for the chance to
continue. After graduation, I plan to enroll in a dual degree PharmD/PhD program in order to conduct
medicinal research in the future.
Kate M. Cooper (Faculty Mentor)
As an Assistant Professor of Biology at Loras College I teach Introductory Biology, Cellular and
Molecular Biology, Immunology, and courses for non-majors on Darwin, the Science of Food, and the
Biology of Women. I am currently mentoring five students on cellular and molecular biology research
projects related to my interests in molecular biology and cell migration.
I graduated from Luther College (B.A., 2002), where I developed an interest in biology and
undergraduate research and received a McElroy Graduate Fellowship to help support me during my
doctoral studies in the Cellular and Molecular Biology program at the University of Wisconsin (Madison).
While there, I was funded through a National Institutes of Health (NIH) training grant, and was a Howard
Hughes Medical Institute (HHMI) Fellow in Scientific Teaching, taking classes and gaining hands-on
experience teaching and mentoring undergraduate students. I graduated with my Ph.D. in June 2008 and
joined the Loras biology faculty in August 2008. I received McElroy Student/Faculty Research Fund awards
with former students for 2009-2010, and again 2011-2012 to study this family of proteins and was part of
our institution’s grant from the Carver Foundation 2010-2011 which funded the microscopy equipment that
will be utilized in this project.