Effect

of knocking out specific genes on the number of

nuclei present in Dictyostelium discoideum cells

Ella Dyett





12/14/15

Abstract:
Dictyostelium discoideum (D. discoideum) cells are very similar to mammalian cells
in their protein sequences, which allows them to mimic human cells in experiments.
Dicty and mammalian cells are also similar in their development process. The
mitosis process is crucial to the cell development of D. discoideum because it is what
allows a cell to replicate. This experiment worked with 2 different genes (tklA,
pldB). These two genes are essential in the development process but we havent
been able to exactly figure out what they do. It demonstrated how these genes

affected the process because over-expressing them or knocking them out amplified
or completely took away the affect that protein had on the development. Therefore,
comparing the D. discoideum strains to the human developmental process showed
what effect the protein had. Studying these proteins was important because learning
more about the development of D. discoideum cells in general could allow for more
discoveries relating to mutations such as cancer and why they occur. D. discoideum
cells can be used in studies of cancer cells because their developmental process is
very similar to that of cancer cells. These cells are similar because they start off as
single cells but come together as an aggregate to survive once they are starved. In
this experiment, multiple nuclei were found when working with the following six
strains of D. discoideum: (TklAKO,TklAOE, PldBKO, PLDB0E, TklAKOpldBOE and
Ax2). Ax2 is used as a control cell because it is a wild type, meaning it has no
mutations unlike the other strains that are being used. The other strains are either
with the tkla or pldb taken out or more put in. Specifically, the number of nuclei per
cell was studied to figure out if compared to the Ax2, there was any significant
difference in the number of nuclei. The cells were grown on gel plates and were put
into shaking cultures with media, which contains bacteria for them to eat. After
there was a they were counted, then spun down in the centrifuge. During the spin
down the media was replaced with PBM (a detergent substance) to start the
starvation process. These cells were prepared on slides, then imaged through a
high-powered microscope and analyzed using the program Image J. After analysis
was done, three significant results between Ax2 and tklAKO were found (0.0006,
0.0477 and 0.002) and 2 significant differences between tklAKO and tklAOE (0.0212
and 0.0413). This means that the tklaKO cells most likely had a problem in the
mitosis process.

Introduction:
Dictyostelium discoideum (D. discoideium) cells are a slime mold that has been used
for many years as a model for biological cells (Annesley, 2009). D. discoideum cells
naturally live in soil and move by molding their photoplasm into fingerlike
structures (Annesley, 2009). They are particularly useful to study because their
protein sequence is very similar to Metezoa indicating they developed later than
originally thought. The complexity of its protein sequence is evidence that they
developed later than yeast because yeast have a less developed sequence (Annesley,
2009). D. discoideum have 24 classes of protein kinase that are not found in yeast
(Goldberg, 2006). This is important because if the sequence is more like the animal
sequence than yeast, it can be used as a model for human cells. (Eichinger, 2005)
During its developmental cycle, it takes on a variety of unicellular and multicellular
forms, thus allowing for the study of many different types of organisms (Goldberg,
2006). The part of the developmental process of D. discoideum that these specific
proteins are involved in is not well known in this field of research. The difference
between over expression (OE) and knocking out (KO) a protein is in overexpression
you are manipulating the cell to make more of the protein. In the knocking out
process, you are taking out the gene that makes this specific protein. The purpose of
this research is to see how changing the expression of the different proteins affects
the development of the cell.

Typically, there is only one nucleus per cell of D. discoideum. However, It was
observed that some strains with knocked out or overexpressed proteins had more
than one nucleus indicating a problem in the mitosis process. When specific proteins
were overexpressed or knocked out completely, it showed that these proteins
played a crucial role in this specific part of the developmental process, mitosis. More
than one nucleus per cell indicated that the cell was not going through the process of
splitting into two cells, or mitosis, which should happen after the second nucleus
forms (Zhou, 2008). The significance of this is that if it is known exactly what these
proteins do in the cell process, the findings could be used to discover more about
human cells and disease. This is true because of the similarities D. discoideum and
the human cell have and these proteins could be used in possible cures and
solutions to many diseases, such as cancer, that are mutations of the cell (Huber,
2014). It was predicted that the D. discoideum cell strains would have a nuclei per
cell count higher than one, except for the wild type Ax2. This would indicate that the
tklAKO cells were not splitting properly.

Methodology:
Six strains of D. discoideum: (TklAKO,TklAOE, PldBKO, PLDB0E, TklAKOpldBOE and
Ax2) were obtained from a gel plate in which, they were grown from a shaking
culture. The cells were later put into a shaking culture with media, which contained
bacteria that they ate for food. 1 ml of these cells were taken from the shaking
cultures using a pipette and were transferred to a special slide with 1mm boxes.
The number of cells per box was counted using a microscope (include type of
microscope). The cells were spun down using a centrifuge; once to remove the
media, and twice to clean the cells. The last two times, they were spun with 10
microliters of PBM a detergent solution that removed nutrients from the cells so it
was known exactly when they were starved. The appropriate amount of pbm was
added to change the concentration of the cell solution to 0.5x106/ml. The cells were
placed using a dropper in an 8 well slide. They were left for an hour. The cells were
fixed with 95% ethanol and left to soak for 10 minutes. Thee ethanol was poured
out and the cells were left to air dry for 5 minutes. The cells were stained with 0.4
ml of DAPI solution in each well. The cells were left to stain in the dark for 5
minutes, then the wells of the slide were removed from the slide and a coverslip was
mounted. The edges were sealed with clear nail polish. Pictures were taken per
strain of D. discoideum. The pictures were put into the program image J and split
into two, one picture of the full cells and one of just the nuclei. The pictures were put
into PowerPoint and the cells and the nuclei inside were counted.

Results:

Figure 1: This is the aggregate data of all the trials, comparing the average data, showing the number of
nuclei per cell compared to the percentage of total cells using the Ax2, tklAKO and tklAOE strains.

The aggregate data, an average of all the trials, was also collected for all the strains.
On average, the Ax2, pldBOE and tklAKOpldBOE strains had the highest percentage of
one nucleus per cell and the lowest percentages in both multinucleate columns.While
tklAKO had the highest percentage of 2 and 3+ nuclei and the smallest percentage of
cells with a single nucleus. However overall, there are more cells with only one nucleus.

Figure 2: These are the p-values for some of the different strains.

As shown in figure 2 all of the p-values are significant when comparing Ax2 to
tklAKO These values show if a jump or change in the data is due to actual differences
or if it can be attributed to scientific or human error. If the p-value is below 5% it is
considered significant and if it below 1% it is considered to be very significant. As

shown in figure 5 all of the p-values are significant when comparing Ax2 to tklAKO.

Discussion:
The percentages of D. discoideum that were multi-nucleate were relatively high but
that was what was expected from observing the cells before the experiment. The
significant difference found between tklAKO cells and Ax2/ tklAOE cells was likely
caused by a problem in the mitosis process of the tklAKO cells. This could signify
that tklAKO is essential in the development process, and more specifically mitosis.
Comparing Ax2 to tklAOE, there was no significant difference, meaning that the
number of nuclei per cell was most likely not caused by a mitosis problem. It is
possible that this result could change as more data is collected.
When comparing tklAKO to tklAOE only two of the results were considered of
significance, which further points to the conclusion that the tklAKO cells have a
problem in the mitosis process. This is relevant because this could be a major
connection between the tklA protein and the development process and, therefore, a
step towards figuring out what it actually does in this process. The only hypothesis
made was that the cells used in this experiment would have a nuclei count higher
than one so in that sense the hypothesis was met. However, since this was the first
time the experiment was attempted, it was unclear what the results should be.
This research advances the field of D. discoideum because if we learn more about
these 2 genes (tklA and pldB) and how they affect the cell we could possibly alter
them to create cures or treatments for diseases such as cancer. There was possible
error on many fronts, but the experimental design was the best-known way to
complete this research. Error that could have occurred was some cells could have
been lost in the spin down process with the changing out of fluid and in the ethanol
soaking process. They could be lost in both of these processes by coming loose and
going into the liquid that was being discarded. Ultimately, what is important about
this research is it can do so much for people who are really suffering, which is
important.




Acknowledgments:
I would like to thank Sean Singh, Derrick Brazill and the rest of the Brazill lab group
at Hunter College for all of their help. I would also like to thank my research
teachers, Mr. Holzinger and Ms. Schmitz.







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