The Effects of Folic Acid Concentration on Cell Movement in
D. Discoideum
Emily Sotelo
ABSTRACT
Chemotaxis is an essential cell process for development in Dictyostelium discoideum. In their
vegetative state, D. Discoideum is chemically attracted to its bacterial food source via excretion
of folic acid from the bacteria. In this research, we examine the effects of folic acid
concentration gradients on cell movement. This process is analyzed utilizing an under-agarose
chemotaxis assay containing two concentrations of folic acid. The results imply that higher
concentrations of folic acid impact the velocity of the cells towards it. Additionally, the results
show that concentration of folic acid has no impact on directional persistence of the cells.
INTRODUCTION:
Dictyostelium discoideum is a eukaryotic cell belonging to the kingdom Protista. D. discoideum grows and
develops through various life cycles which are all reliant on chemotaxis, or movement caused by attraction to a
chemical signal. In its vegetative state, Dictyostelium live as a single celled organism and feed off of bacteria.
The cells are attracted to their food source
via secretions of folic acid (FA) and other
chemicals released by the bacterial cells.
When FA is limited, it triggers the cells to
become sensitized to cAMP, another
chemical signaling molecule, which
results in cell aggregation (Kesbeke et al.
1990). Cell aggregation begins the cells
social life cycle. Aggregation of the cells
leads to the formation of a multicellular
slug, which has the capability to move
around until it finds favorable conditions.
Once favorable conditions are reached, it will form a stalk and fruiting body containing spores that, when
disturbed, are released. If conditions remain favorable, the spores will germinate and the cells will once again
live as a unicellular organism. This alternative life cycle gives the cells at better chance at survival in these
unfavorable conditions. This life cycle makes D. Discoideum classified as a cellular slime mold, meaning it
spends most of its life as a unicellular organism but it can also survive as a multicellular amoeba. Alternatively,
Dictyostelium can reproduce sexually. However, sexual reproduction in these cells is rare (Williams 2010). A
depiction of this cells life cycles, all of which are dependent on chemotaxis, can be found in Figure 1.
Dictyostelium cells have specialized receptors present on the cells surface which cause slightly different
responses based on the chemoattractant available (Kesbeke et al1990). In the cell’s vegetative state, FA is
recognized by the cell through the specific receptor on the cell membrane, causing the activation of a specific
G-protein. This activation of the G-protein creates a cascade of signaling events which allows the cell to move
to where the FA is highly concentrated, ultimately allowing the cell to move toward its food supply.
D. Discoideum is often used in research as beneficial model organism because it has a relatively short and
simplistic life cycle. Furthermore, a number of its gene sequences closely resemble that of more complex
eukaryotic species which makes it a great model organism in a variety of research areas. Understanding cellular
processes in these cells such as chemotaxis can aid in a further understanding of this process in larger, more
complex eukaryotes which are less accessible (Fey et al. 2009).
The purpose of this research was to compare the speed and directionality of this model organism towards two
different concentrations of folic acid via an under-agarose chemotaxis assay. It was hypothesized that the cells
exposed to the higher concentrated FA gradient would move faster and more directly toward the source. This is
because the highly concentrated FA will diffuse from its source more rapidly and therefore more cells will be
able to detect the FA. If more FA is binding to more receptors on the cell, it is possible that the intracellular
signal is amplified, causing the movement of the cell to be faster. Additionally, it was hypothesized that the
cells will be more directionally persistent toward the source in the higher concentration of FA. Since FA would
be considered a favorable environment, the cells do not need to react to cAMP signals, which cause the cells to
aggregate. Aggregation could cause the cells to move less directionally towards the source, which would be
predicted in lower source concentrations of folic acid.
METHODS:
To prepare the agarose plates, a 1% agarose/SM mixture was microwaved, pipetted into petri dishes and
allowed to cool. A spatula was used to release any connections between the solidified agar and the plate. Three
troughs were cut into the gel utilizing a trough-cutting template. A wet razor blade was used to make the cuts
and a spatula was used to remove the strips of cut agarose. Next, approximately 200uL of 1mM and 10mM folic
acid was added to the outer wells. Dictyostelium cells were suspended in SM media at a concentration of 107
cells per mL, and 100uL of the cells was added to the middle well. The plate was then left undisturbed for 15
minutes followed by placement of a round glass coverslip atop of the middle well only. After approximately 1-2
hours, a time-lapse movie was taken for both sides of the wells via a light microscope. The cell speed and
directionality were measured via the image software MTrackJ.
RESULTS:
Figure 1. Under-agarose chemotaxis assay of D. discoideum. An under-agarose chemotaxis assay was used to
analyze cell movement in D. discoideum in the presence of two differing concentrations of folic acid. Cells
were pipetted into the center well of a 1% agarose gel plate. Folic acid was added at 1mM and 10mM to the
outer-wells of the plate. (A) Cell movement was detected through a time-lapse movie using a light microscope.
A picture of the cells was taken every minute for a total of ten minutes. (B) The average cell speed of arbitrarily
selected cells (n=10) was measured using the image tracking software ImageJ and mTrackJ. A two-tailed paired
samples t-test resulted in a p-value of 1.23x10-5. (C) The average directional persistence of the n=10 cells was
also calculated. This directionality was determined using the total y-displacement divided by the total distance
traveled by the cell. A two-tailed paired samples t-test resulted in a p-value of 0.17.
Chemotaxis of vegetative D. discoideum was analyzed using an under-agarose chemotaxis assay with folic acid
as the chemoattractant (Figure 1). The purpose of this was to see if differing folic acid concentrations would
impact the average cell speed or directional persistence of the cells. The video montage displays the cell
movement at time 0, 5, and 10 minutes. The top sequence of pictures shows the cell movement of the cells
exposed to the 10mM FA while the bottom pictures show the cell movement of those exposed to 1mM FA. This
movement was further analyzed using the image tracking software ImageJ and mTrackJ. A comparison of
average cell speed of n=10 cells resulted in a p-value of 1.23x10-5. This indicated that there was a significant
difference in average cell speed between the cells exposed to 1mM FA and those exposed to 10mM FA. A
comparison of average directional persistence resulted in a p-value of 0.17. These results indicate that folic acid
did not create a significant difference in directionality between the two cell groups.
DISCUSSION:
Chemotaxis plays an important role during the life cycle of D. discoideum. In its vegetative state, chemotaxis is
used to locate its bacterial food source through the release of chemicals such as folic acid by the bacteria. It was
known that Dictyostelium contain a finite number of specific receptors for folic acid and that the cells respond
and move toward the folic acid via intracellular signaling pathways. The present study addresses the question of
whether or not folic acid concentration has an impact on this cell movement. The analysis of average cell
velocity in D. discoideum indicated that higher concentrations of FA correlates with a higher average cell speed
when compared to those exposed to lower concentrations. This increased cell speed may be associated with the
multiple folic acid receptors on the cell surface (Kesbeke et al 1990). Since more folic acid is present in the
higher concentrated end, it is possible that more folic acid would bind to the surface of the cells, causing a
quicker and potentially more amplified signal for the cell to move. On the contrary, when the average
directional persistence was compared, the results showed that folic acid concentration had no impact on the
directionality of the cell. Although individual cells showed some forward cell movement, others simply circled
around one location. This result is potentially caused by the multitude of signals the cells can respond to. For
example, Dictyostelium, even in the presence of folic acid, can still respond to the presence of cAMP in the
environment which is released by other cells (Kesbeke et al 1990). The response to these signals would cause
the cell to move toward whichever cell it is getting the signal from. Since there was a multitude of cells to
respond to, the cell would not move directionally toward any one location. To better the results, it may be
necessary to use multiple concentrations of FA to obtain a better analysis and conclusion as to how an
increasing or decreasing concentration gradient impacts cell movement in Dictyostelium Discoideum.
WORKS CITED:
Brown, D., Strassmann, J.E. The life cycles of Dictyostelium discoideum. CC Creative Commons Attribution –
Share Alike 3.0.
Fey, P., Gaudet, P., Curk, T., Zupan, B., Just, E. M., Basu, S., Merchant, S. N., Bushmanova, Y. A., Shaulsky,
G., Kibbe, W. A., Chisholm, R. L. (2009). dictyBase - a Dictyostelium bioinformatics resource
update. Nucleic Acids Res. 37, (Database issue): D515-19.
Kesbeke, F., PJM van Haastert, RJW De Wit, and BE Snaar-Jagalska. 1990. Chemotaxis to cyclic AMP and
folic acid is mediated by different G proteins in Dictyostelium discoideum. J. Cell Sci. 96: 669-673.
Williams, J.G. Dictyostelium Finds New Roles To Model. Genetics Society of America. 2010