Finding the Chemotactic Index, Directionality, and

Velocity in PkcA Inhibited Cells in Dictyostelium

discoideum

By Isabella Jansen
Hunter College with Sean Singh
April 5, 2017
Abstract:

In order to understand the signaling pathways of Protein Kinase C (PKC) in

Dictyostelium discoideum, the chemotactic index, directionality, and velocity of cells in

which Protein Kinase C-alpha (PkcA) has been overexpressed or knocked out, will be

tested. PkcA is thought to play a role in the motility of Dictyostelium. By learning about

the signalling pathways of PkcA, we will be able to learn more about this proteins

function in motility, and then apply that knowledge to cancer research. Dictyostelium is

an excellent model because of their complex life cycle, which is known to use similar

proteins as cancer. This experiment will be done by taking videos of cells where PkcA

has been either overexpressed or knocked out, and track their movements using

ImageJ, an image analysis program. The data collected by this program will indicate

how the different cells respond to Cyclic Adenosine Monophosphate (cAMP). To date,

errors in the data collecting program have been impeding progress for this project,

changes to the program will allow for accurate analysis of call motility in the near future.

Introduction:

In humans, Protein Kinase C (PKC) is known to play a role in the motility of cells,

such as during cancer metastasis. Current research focuses on the signaling pathways

of PKC; as well as ways to inhibit, or activate its activity. Dictyostelium discoideum

(Dicty) possesses a version of PKC, PkcA, which exhibits similar functions to the

human-version of the protein. Dictyostelium is a simpler model to research cellular

signaling pathways because of its unique life cycle and ease of both culturing and
genetics.1 During starvation, the cells go through many phases (Figure 1), which are

affected and disturbed when PkcA is disrupted.3,4 Dicty cells go through many phases in

their life cycle that require PkcA to function. Dicty starts as a multicellular organism but

when it is starved it aggregates to form and unicellular organism. It then goes through a

slug phase and culmination until it finally becomes a fruiting body in which spore cells

are at the tip. It is possible to pinpoint how PkcA disrupts cell motility by examining its

effects on the cell's response to a chemoattractant, cAMP, which normally serves as a

signal to aggregate during development1. This is done by analyzing the cells

chemotactic index which is a measure of the cells capability to sense and move

towards cAMP during starvation.5 Additional analysis includes measures of the cells

velocity and directionality, which are the speed of movement, and the cells capability to

move in a straight line, respectively. In order for an organism to have a good

directionality it needs to move in a straight line, either towards or away from the

chemoattractant.

So far, it is unknown how Dicty cells are supposed to behave when observing

their velocity, chemotactic index, and directionality when their levels of PkcA are

changed.6 Understanding how the Dicty behave and react when PkcA is changed will

give insight on how PkcA works in the organism. Understanding PkcA is vital to other

fields of research, such as cancer.7 When knocking out or overexpressing PkcA there

may be a change in what their capabilities are to do. Based on how the change of

protein presence affects the abilities of the dicty will give further insight on how the

signalling pathways of PkcA work which can then apply to cancer research.
Project Description:

The objective of this experiment is to learn about how PKC signaling

works and how inhibiting it or overexpressing it affects the function of Dicty cells. This

work is significant due to its potential applications to cancer research.8 If it is found that

by blocking PKC, the Dicty cells are stopped from aggregating, then it is possible that if

a PKC-like protein is inhibited in cancer cells, it will stop the cancer from spreading.

Research Strategy:

An extensive amount of videos are taken on each cell strain of Dicty. The

chemoattractant are placed at the top of the petri dish. The cells are placed in petri

dishes and filmed using a microscope. An image per minute is taken, for 20 minutes.

These videos will be then analyzed using a computer program called ImageJ. One

organism is then selected and a point was plotted as it moved along its trek. The

computer program then took these points and collected data. This is repeated in each

video for each individual cell that was capable of being viewed. Cells that are not

capable of being viewed are cells that are attached to or stuck to other cells which

would have skewed the data. Once each cell that is capable of being plotted is done, we

would move onto another cell line. The cell lines included are Ax2, PkcAOE, and PkcA-.

Ax2 w functions as the control group because they are not changed whatsoever, which

will provide an idea of what normal Dicty cells look like with chemotactic index,

directionality, and velocity. PkcOE means that PKC is being over expressed in the cells,
PkcA- means that PKC is not being expressed within the cells. This data was then

moved into an Excel sheet that can make graphs with the data.

So far, a few challenges have occurred while collecting the data due to the way

the program process the data. These challenges made it very difficult to work with the

data so it was decided to start afresh and change the way the program processed the

data in order to make the data easier to work with.

Figure 1: Dicty life cycle. Dicty cells start unicellular but become a multicellular organism

after aggregation.2
Figure 2: ImageJ processes the data and it is then moved into Excel to further make

graphs with the data.

Acknowledgements:

I would like to thank Dr. Brazil for allowing me to work at his lab, Sean Singh for

mentoring me for the last 2 years, and Zully Santiago for also mentoring me in the lab. I

give many thanks to all the Science Research teachers at packer for all the advice and

guidance. I would also like to acknowledge Hunter College and The Packer Collegiate

Institute for the opportunity.

References:

1) Loomis, William F. "Cell Signaling during Development of Dictyostelium." Science

Direct. Elsevier,n.d. Web. 15 Aug.
2016.<http://dx.doi.org/10.1016/j.ydbio.2014.04.001>.
2) "File:Dicty Life Cycle H01.svg." - Wikimedia Commons. Accessed March 02, 2016.
https://commons.wikimedia.org/wiki/File:Dicty_Life_Cycle_H01.svg.
3) Cooper, G. (n.d.). Actin, Myosin, and Cell Movement. Retrieved December 16, 2015,
from http://www.ncbi.nlm.nih.gov/books/NBK9961/
4) Mohamed, W., et al., Absence of catalytic domain in a putative protein kinase C
(PkcA) suppresses tip dominance in Dictyostelium discoideum. Dev. Biol.
(2015), http://dx.doi.org/10.1016/j.ydbio.2015.05.021
5) Fukujin, F., Nakajima, A., Shimada, N., & Sawai, S. (2016). Self-organization of
chemoattractant waves in Dictyostelium depends on F-actin and cellsubstrate
adhesion. Journal of the Royal Society Interface, 13(119), 20160233.
http://doi.org/10.1098/rsif.2016.0233
6) "Dictyostelium Discoideum Chemotaxis: Threshold for Directed Motion."
Dictyostelium Discoideum Chemotaxis: Threshold for Directed Motion. Science
Direct, n.d. Web. 13 Dec. 2016.
<http://www.sciencedirect.com/science/article/pii/S0171933506000264>.
7) Loomis, William F. "Cell Signaling during Development of Dictyostelium." Science
Direct. Elsevier, n.d. Web. 15 Aug. 2016.
<http://dx.doi.org/10.1016/j.ydbio.2014.04.001>.
8) Ibid