The Role of PakD in the Adhesion of Dictyostelium

discoideum to a Substrate
Raphael Santore
The Packer Collegiate Institute
November 21, 2013
Abstract:
Regulation of the cells actin cytoskeleton is important for a number of cell processes,
including adhesion. Cancer cells adhere to each other to form tumors, and these cells can
metastasize and cancer can spread through the body. Dictyostelium discoideum cells were
researched in this lab as a model for the adhesive behavior of cancer cells in order to determine
the effect of different genes on adhesion. In this paper, we describe the role of PakD, a protein
that is involved in the regulation of the cells cytoskeleton, on the rate of de-adhesion to a
substrate. Cells were collected, starved, and allowed to adhere to a flask. De-adhesion was
initiated by applying shear stress to the cells. Cells that lost adhesion were then counted at
specific time points to find the percentage of cells that had de-adhered from their flask. We
found that while PakD did not have major role on total de-adhesion, it did slightly affect the
initial rates of de-adhesion. It was also found that starving the cells for different amounts of time
only affected the adhesion of the cells very slightly.
Acknowledgements:
My Research was conducted at Hunter College. Research was conducted beginning In April
2013. Research was done full time over the summer, and four hours a week during the school
year. First and foremost, I would like to thank Dr. Derrick Brazill for mentoring me, and
"
allowing me to work in his lab to conduct my research. I would also like to thank the other
students in the lab that occasionally assisted me with my research. I would also like to both of
my science research teachers at school. Finally, I would like to thank my parents for helping me
grasp many complicated topics, supporting me and encouraging me to continue my research.
Table of Contents:
Introduction ~ Pg. 3
Materials and Methods ~ Pg. 5
Results ~ Pg. 7
Discussion ~ Pg. 11
References ~ Pg. 13
List of Tables & Figures
Table 1: Percent Adhesion to Flask After 2-Hour Starvation Pg. 7
Table 2: Percent Adhesion to Flask After 3-Hour Starvation Pg. 8
Table 3: Rates of De-adhesion From 0-15 Minutes (Percentage Per Hour Pg. 10
Table 4: Rates of De-adhesion From 0-10 minutes (Percentage Per Hour) Pg. 11
Figure 1: The Life Cycle of Dictyostelium discoideum Pg. 3
Figure 1 shows the life cycle of Dictyostelium discoideum cells
Figure 2: Percent Adhesion Vs. Time after 2-Hour Starvation Pg. 7
Figure 2 shows a graph of the percentage of cells that were adhered to the flask at each
time point after being starved for 2 hours.
Figure 3: Percent Adhesion Vs. Time after 3-Hour Starvation Pg. 8
Figure 3 shows a graph of the percentage of cells that were adhered to the flask at each
time point after being starved for 3 hours.
#
Figure 4: Average Rates of De-adhesion Between 0 and 15 Minutes Pg. 9
Figure 4 shows the average slope of the line for each cell line represented between 0-15
minutes on the percent adhesion vs. time graph.

Figure 5: Average Rates of De-adhesion Between 0 and 10 Minutes Pg. 10
Figure 5 shows the average slope of the lines for each cell line between 0 and 10 minutes.
Introduction:
Dictyostelium discoideum is an amoeba that is often used as a model to study and
understand cell cohesion and adhesion in eukaryotic cells. Dictyostelium discoideum is a
unicellular amoeba that is more commonly known as a slime mold. The wild type Dictyostelium
discoideum cells are known as Ax2 (Pribic, 2011). These amoeba are used to study cell cohesion
and adhesion because they can adhere to each other and different substrates depending on the
amount of food they have. When Dictyostelium discoideum cells begin to starve, they secrete
chemical signals that cause them to aggregate (Serezani, 2008). When these cells aggregate, they
clump together and form a structure called the mound. The mound eventually develops into what
is known as the fruiting body. The fruiting body releases spores that then release the unicellular
amoeba again once food is present.

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Figure 1: The Life Cycle of Dictyostelium discoideum (Wikipedia, 2012)
Studying cell adhesion and cohesion in Dictyostelium discoideum cells is relevant
because it can be used as a model for cancer cells in humans. Once cancer cells begin to
multiply, they form tumors. A very serious problem for many cancer patients is that there is a
point in which some cancer cells no longer adhere tightly to the tumor. The cells metastasize, or
break off, from the tumor and spread throughout the body. Cancer can then spread to multiple
different parts of the body (Shimoyama, 1992). By studying Dictyostelium discoideum cells we
will be able to learn more about which genes affect and control the adhesion of cells. Then will
hopefully be able to apply that knowledge to homologous genes in humans and better understand
and treat cell metastasis (Brazill, 2012).
Many of the genes in Dictyostelium discoideum have homologs in humans so studying
these genes can give very important insights into the effect similar genes may have in human
cells. The specific gene worked with in this experiment was pakD. PakD is known to be involved
in the regulation of the protein actin, which is an important element of the cytoskeleton in the
cell. Actin is a key part of cell adhesion (Pribic, 2011). The PakD ortholog in humans is protein
kinase C, or PKC.
To study pakD, it was deleted in the wild-type AX2 cells to create !"#$
%
cells. The cells
were cultured in a liquid nutrient solution called HL-5. That solution contained essential
nutrients that the cells needed to grow. In order to starve Dictyostelium discoideum cells, they
were washed free of HL-5, and transferred into a solution called PBM, which is essentially a
buffered water solution that does not contain nutrients necessary to the cells growth. Under
these conditions Dictyostelium discoideum cells begin to starve, and adhere to each other and
their substrate.
%
The rate at which the cells fell off of the flask over time was measured after both !"#$
%

cells and Ax2 cells were starved. This data was collected to compare the !"#$
%
cell line to the
wild type. The rate at which the cells fell off the side of the flask is known as the rate of de-
adhesion.
The point of the research was to investigate the role of PakD on the cells adhesion to the
bottom of a flask. Because PakD is known to regulate actin fibers, which are involved in cell
adhesion, it was predicted that the cells with PakD deleted would not adhere to the flask as
tightly, and would fall off more easily. It was also predicted that longer starvation, would likely
increase adhesion.
Materials and Methods:
Cell Collection and Starving
In order to examine the adhesion or de-adhesion of the cells, a specific number of cells
had to be used. In order to obtain that number of cells, ten microliters of each cell line were
collected and the cells were counted in a hemacytometer under 400x magnification. The number
of cells counted in the 10 microliters was used to determine the concentrations of cells in cells
per milliliter. Knowing this concentration, the specific number of cells required, 1x10
7
, can be
taken out of the solution. Next, 1x10
7
cells were transferred into a 50-milliliter falcon tube and
centrifuged for 3 minutes at 3000 RPM to create a pellet of the cells at the bottom of the tube.
The supernatant (HL-5 media) was removed carefully, and the cells were re-suspended in 10 mL
of PBM. In order to eliminate any remaining nutrients, the cells were then centrifuged for 3
minutes at 3000 RPM and re-suspended in PBM two more times. The cells were then re-
suspended in 2 milliliters of PBM and counted again to find the final concentration of cells in the
solution. That concentration was then used to calculate how many milliliters of cell solution were
&
required to collect 4x10
6
cells. The calculated volume of solution was then transferred into a
flask, and supplemented to 4 mL with PBM, making the concentration 1x10
6
cells per milliliter.
The cells were then shaken at 120 RPM for 10 minutes to ensure that none of the cells adhere to
each other or the flask before we were ready to begin starving the cells. The cells were then
counted to determine the total number of cells in the trial. The flask was left sitting for either 2 or
3 hours so the cells could starve and adhere to the flask.
Determining Cell Concentrations at Different Time Points
After either the two or three-hour starvation period, the flask of cells was put onto the shaker at
60 RPM. The cells were on the shaker in order to create shear stress and cause the cells that were
not tightly adhered to the bottom of the flask to detach, so that only strongly adhered cells remain
adhered. 0.5 milliliters of the cell solution were removed from the flask at 0,5,10,20, and 40-
minute time points. The number of non-adhered cells at each time- point as then determined by
counting in a hemacytometer. This count gave us the number of cells that had detached from the
bottom of the flask back into the solution.
Calculations and Analysis:
All of the cell counts were put into an excel spreadsheet. For each trial, the cell counts for each
time point were subtracted from the initial number of cells. To find the percent of cells that had
adhered to the bottom of the flask, the difference between the initial and the respective time point
was divided by the initial number of cells. Those values were then plotted on an percent adhesion
vs. time graph. The decrease in percent adhesion over time observed in all trials indicated that
the cells were de-adhering from the flask. In order to determine how the rate of de-adhesion
changed over time, the slope of the percent adhesion vs. time graphs were calculated between
specific time intervals. The slope of the lines between 0-10 minutes, and 0-15 minutes were
'
calculated to determine over what time interval there was the most variation in adhesion from the
wild type. The error bars are based on the values of the standard error of the mean for each time
point. This was calculated by dividing the standard deviation by the square root of the number of
trials.
Results:
TABLE 1. Percent Adhesion to Flask After 2-Hour Starvation


0
Minutes
5
Minutes
10
Minutes
15
Minutes
20
Minutes
40
Minutes
Ax2

88.6 62.5 47.4 32.2 15.8 5.63
Ax2 With
Latrunculin
14.2 12.0 12.0 7.01 7.53 8.22
pakD
-


84.9 67.2 51.6 36.4 17.4 5.88

Table 1: Average percent of cells that adhered to the bottom of the flask, or the substrate after the
cells were starved for 2 hours. These values were calculated by using cell counts at each time
point and the total initial number of cells in the flask to determine what percentage of cells had
remained adhered to the side of the flask.

Figure 2: The average percentage of cells that were adhered to the flask at each time point after
being starved for 2 hours. This graph is a representation of the data shown in Table 1. The red
(
line shows Ax2 after two hours of being starved and the orange shows !"#$
%
after 2 hours of
being starved. The green line shows cells that were kept in a solution mixed with Latrunculin, a
substance that affects the actin cytoskeleton of the cells and causing them not to adhere. The wild
type (Ax2) and the !"#$
%
cells both show a decrease in adhesion to the flask over time.

TABLE 2. Percent Adhesion to Flask After 3-Hour Starvation


0
Minutes
5
Minutes
10
Minutes
15
Minutes
20
Minutes
40
Minutes
Ax2

90.6 64.8 47.8 35.2 19.9 7.02
Ax2 With
Latrunculin
14.2 12.0 12.0 7.03 7.53 8.22
!"#$
%


85.72 68.4 49.2 35.9 20.3 7.26

Table 2: The average percentage of cells that adhered to the side of the flask, or the substrate,
after the cells were starved for 3 hours. These values were calculated by using cell counts at each
time point and the total initial number of cells in the flask to determine what percentage of cells
had remained adhered to the side of the flask.

Figure 3: The average percentage of cells that were adhered to the flask at each time point after
being starved for 3 hours. This graph is a representation of the data shown in Table 1. The red
)
line shows Ax2 after three hours of being starved and the orange shows !"#$
%
after 2 hours of
being starved. The green line shows cells that were kept in a solution mixed with Latrunculin, a
substance that disrupts the actin cytoskeleton of the cells and causing them not to adhere. The
wild type (Ax2) and the !"#$
%
cells both show a decrease in adhesion to the flask over time.


Figure 4: The average slope of the line for each cell line represented between 0-15 minutes on
the percent adhesion vs. time graph. The slope of the line signifies the rate at which the cells de-
adhere from the side of the flasks. It should be noted that these are the absolute values of each
slope.




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TABLE 3. Rates of De-adhesion From 0-15 Minutes (Percentage Per Hour)
Ax2 2 Hour Ax2
3
Hour
Ax2 With
Latrunculin
!"#$
%
2
Hour
!"#$
%
3
Hour
3.68 3.67 0.435 3.22 3.11
Table 3: The rates of de-adhesion for each cell line over 15 minutes in percentage per minute.


Figure 5: The rate of de-adhesion of the cells in percentage of cells adhered per minute. The
slopes of the lines are greater than those in over 15 minutes, showing that the average rate of de-
adhesion over a 10 minute interval is greater.



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TABLE 4. Rates of De-adhesion From 0-10 minutes (Percentage Per Hour)
Ax2 2 Hour Ax2
3
Hour
Ax2 With
Latrunculin
!"#$
%
2
Hour
!"#$
%
3
Hour
4.12 4.2 0.22 3.33 3.39

Table 4: The rates of de-adhesion of each cell line over 10 minutes in percentage per minute.
Discussion:
Adhesion is a significant process in certain microorganisms such as Dictyostelium
discoideum. Adhesion is important to study because it gives insight into the understanding of cell
metastasis. The adhesion of cells is likely by some specific proteins. !"#$
%
seems not to have
much of an effect on the total de-adhesion of the cells to their substrate. For two hours of
starvation, over 20 and 40 minutes the cells have similar percent de adhesions. The wild type had
5.6 percent of the cells still adhered to the flask after 40 minutes, wand the !"#$
%
cells had 5.8
percent. The wild type had 17 percent adhered after 20 minutes and !"#$
%
had 15 percent
adhered. However there does seem to be some effect on the rates of de-adhesion prior to 20 and
40 minutes. The slopes of the lines in between time points on the percent adhesion vs. time graph
represent the rate of de-adhesion over time, or the rate at which the cells detach from the flask.
These rates are not the same over each time point. T he slope of the lines for Ax2 are slightly
more steep in the first 15 minutes at a rate of 3.6 percent per minute, while the !"#$
%
cells fall
off the side of the flask at a rate of 3.2 percent per minute. This shows a faster rate of de-
adhesion over that time interval. However because the cells have similar values after 20 minutes,
it can be inferred that the rate of adhesion in !"#$
%
over the next five minutes must have been
faster than that of the wild type. The rate of de-adhesion is even faster over the first 10 minutes
as displayed in figure 4. The rate of de-adhesion of the wild type is 4.12 percent per minute,
while the !"#$
%
is 3.3 percent per minute. This implies that the initial rate of de-adhesion is
,"
greater than the rate later on, signifying a decreased rate of de-adhesion over time. This
signifies that the PakD gene is likely involved in de-adhesion within the first 20 minutes of the
cells being shaken. If it were involved in later portions, the data would show different rates of
adhesion during later times because the PakD is a knockout gene and therefore would not be
performing its function. However because it has little effect on later rates of adhesion, it is likely
not regulating adhesion after the 20-minute time point. Because there is the greatest difference in
the de-adhesion of the cells in the first 20 minutes, the graphs of the slopes over later time points
are not as significant. The three-hour starvation does not seem to seem to have much effect on
the total adhesion. It does seem to cause the cells to de-adhere slightly, however the difference in
percentage adhered at each time point is fairly small showing that while it may have some effect,
the effect is not drastic. After being starved for 3 hours, only about 2 percent more of the cells
adhered to the side of the flask for both the wild type and the !"#$
%
cells. It also seems not to
affect the rates of adhesion very much, because in both figures 3 and 4 the values of the rates for
3 hour and 2 hour starvation are very close. For !"#$
%
Over 15 minutes the rate is 3.22 percent
per minute for 2-hour starvation versus 3.11 percent per minute for 3-hour starvation. In the wild
type cells it is 3.68 percent per minute for 2-hour starvation versus 3.67 percent per minute for
the 3-hour starvation. There is also little difference betw een the rates of de-adhesion for 2 and 3
hour starvation with the first 10 minutes. The percentage de-adhered per minute is 4.12 percent
for Ax2 cells starved for 2 hours rather than the 4.2 percent per minute of Ax2 cells starved for 3
hours. The difference for !"#$
%
2-hour starvation versus 3-hour starvation is a rate of 3.33
percent per minute and 3.39 percent per minute, respectively. In order to better determine if time
that the cells are starved for affects the adhesion of the cells to the substrate, the cells could be
starved for longer and shorter periods of time. If the cells were starved for 1 hour and 4 hours,
,#
there would be 2 more trials that could be analyzed to try and determine a whether or not there is
a relationship between time starved and the cells adhesion to the substrate.
While PakD did affect the adhesion of the cells to the substrate, it only affected it very
slightly. The time that the cells were starved also slightly effected the adhesion of the cells. In
the future, more research will be done using PakD cells to see how the gene may effect the cells
motility. It also will be helpful to collect data using separate genes when the cells have been
starved for different lengths than just two and three hours.

References:
Brazill, Derrick 21 November 2012. Personal communication.

Pribic, J., Garcia, R., Kong, M., & Brazill, D. (2011, April 29). Paxillin and Phospholipase
D Interact to Regulate Actin-Based Processes in Dictyostelium discoideum . Eukaryotic Cell,
10(7), 978-984. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/21531871

Serezani, C. H., Ballinger, M. N., Aronoff, D. M., & Peters-Golden, M. (2008,
March 6). Cyclic AMP Master Regulator of Innate Immune Cell Function.
American Journal of Respiratory Cell and Molecular Biology, 39, 127-132.
Retrieved from http://ajrcmb.atsjournals.org/content/39/2/127.abstract

Shimoyama, Yutaka, Akira Nagafuchi, and Shin Fujita. "Cadherin Dysfunction in a
Human Cancer Cell Line: Possible Involvement of Loss of !-Catenin Expression in
Reduced Cell-Cell Adhesiveness." The Journal of Cancer Research (1992): n. pag. The Journal
of Cancer Research. Web. <http://cancerres.aacrjournals.org/>.

[Life Cycle of Dictyostelium Discoideum] [Diagram]. March 3,12, . Retrieved from
http://en.wikipedia.org/wiki/Dictyostelium_discoideum






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