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- 2. 1 Abstract Dictyostelium discoideum cells have a complex system of signalling networks that are activated by chemoattractants. These chemoattractant chemicals induce a response of the Dictyostelium cells to move closer to the chemical signal that the cell received, a phenomenon known as positive chemotaxis. Dictyostelium cells exhibit this by expressing CAR receptors on their surface that recognize the signal, cyclic AMP, that is sent out when the Dictyostelium cells are entering starvation mode due to a lack of bacterial food sources. When cells receive this signal, they go through a process called aggregation and form tall fruiting bodies with protruding spores in the hope of increasing its odds to disperse spores to locations that have more stable food sources. However, if a cell possesses a mutation in the cAMP signaling pathway or in the CAR receptors that receive cAMP, the Dictyostelium cells cannot make the starvation stress signal and therefore will not aggregate and eventually die of starvation. This study aimed at testing how mutations in genes for signals or signal receptors affect developmental pathways in two Dictyostelium discoideum strains, wild type Dictyostelium DNA (AX3) and a mutant (RI9). This process was carried out by culturing AX3 and RI9 DNA onto separate plates and then after a week plating AX3 DNA on 5 treatments: EGTA, caffeine, pH 4.4, 6.39, and 9.57. The results lead us to extracting and isolating AX3 and RI9 DNA and amplifying it using PCR. Once the amplification was complete, a gel electrophoresis was run to determine gene expression of AX3 and RI9 in ACA, cARA and cAR4. The results show that there is a loss of gene expression in RI9 (mutant strain) for the receptor, cARA. This means that the receptor cannot sense the cAMP signal and therefore cannot aggregate. However, the results do not confirm that the receptor is the only reason why the cells did not aggregate as there could be other factors involved. Yet, the
- 3. 2 gel electrophoresis results did show a band in the ACA column in both RI9 and AX3 DNA, meaning that there was not a mutation in creating the cAMP signal. Introduction Mutations can happen at any time or in any way, and can be harmful or unharmful depending on the genes involved. For example, mutations may be unharmful in the case of people with red hair. On the other hand, mutations can be harmful and cause death due to the mutation causing something to be amiss. Dictyostelium discoideum is an organism that helps us study mutations in the signaling pathway of the amoeba. Dictyostelium is a fungus referred to as a slime mold, but it feeds on bacteria and can be found on forest floors or under leaves and logs (Meima and Schaap 1999). Dictyostelium cells have developed numerous effective signaling networks that are activated by chemoattractants (FrancaKoh 2006). Chemotaxis is simple cell migration by chemical cues and is critical for biological processes (Willard and Devreotes 2006). Dictyostelium uses cyclic adenosine monophosphate (cAMP), as an extracellular chemoattractant. (Willard and Devreotes 2006). When food sources get scarce, some D. discoideum amoeba start to send out pulses of cAMP, a chemoattractant, that will induce surrounding cells to move towards them and also begin to secrete the cAMP signal themselves (Meima and Schaap 1999). They are motile like an amoebae but when food and nutrient resources are scarce, the cells secrete a signaling molecule, cAMP, that alerts other nearby D. discoideum cells to congregate and begin a relocation process to search for new food. Dictyostelium cells sense and receive the signal because when they are starving, Dictyostelium cells express cAMP receptors on their surface and migrate in response to waves of the
- 4. 3 chemoattractant, cAMP (Willard and Devreotes 2006). Once the cells receive the signal, they migrate toward the source of the cAMP causing the cells to aggregate. The aggregate group is known as a slug, which can move as a multicellular organismal unit. Once it moves to a new location, it forms a fruiting body as a starvation stress response (Brenner and Thoms 1984). Cyclic AMP also acts as an intercellular messenger to control stalk and cell maturation, germination, and spore production. Some cells anchor while others climb the stalk and form a spore (Meima and Schaap 1999). The fruiting body releases the spores that can be carried in the wind or transported on a passing animal to a more desireable location of increased food resources and nutrients. However, mutants will disrupt this pathway by either preventing cAMP from being sent, or preventing cAMP from being received. ACA(adenylyl cyclase) makes cAMP from ATP and is responsible for gene expression (Shu et al. 2011). However, if there is a mutation in ACA, then we may be unable to detect that cAMP is being made from ATP at all, which therefore prevents cells from aggregating. There also could be a mutation in the receptor genes. The receptors we examine in this experiment are cARA and cARD. cARA mutants fail to bind or sense cAMP signaling and therefore will eventually die because they are unable to receive the signal to aggregate (Sun and Devreotes 1991). This concept is central to our study, as we will test how mutations in genes for signals or signal receptors affect developmental pathways in wild type DNA strain (AX3) and a mutant DNA strain (RI9). We also aim to compare the wild type strain of D. discoideum to the effects that various chemicals and pH levels have on the Dictyostelium aggregation, developmental patterns, and spore formation. Overall, we predict that the mutant RI9 strain will have severe development issues and a decrease in
- 5. 4 aggregation and fruiting body formation due to it’s cAMP mutation due to the fact that these stress signals are initiated by secretion of cAMP. Methods Culturing Dictyostelium discoideum on a bacterial food source Klebsiella aerogenes was used as a bacterial food source for the two Dictyostelium discoideum strains. Each strain was tested to see if there were apparent differences in the stress response and fruiting body development due to the presence or absence of the strain’s cAMP pathway. Using the K. aerogenes bacteria stock solution, 100 µl was pipetted and spread on two MS agar plates using aseptic techniques. 100 µl of each D. discoideum wild type (AX3) strain and mutant type (RI9) strain was pipetted onto the center of each bacterial lawn on the plate using aseptic techniques. The plates were then incubated at room temperature with ambient light and growth and development was observed over the next 72 hours. Effects of chemicals and pH on Dictyostelium discoideum development DNA extracted from the AX3 (wild type) strain was tested on five different treatments of various compounds and pH levels that were thought to have an effect on signaling cAMP. The tested treatments were MS agar plates that contained one each of EGTA, caffeine, and plates with a pH of 4.40, 6.39, and 9.57. A volume of 25 µl of Klebsiella aerogenes bacteria was spread on each 35 mm MS agar plate, followed by the addition of 25 µl of the AX3 Dictyostelium discoideum strain DNA onto each plate. Penetrating light was blocked out with aluminum foil, and the plates were incubated at room temperature for seven days. After this time period,
- 6. 5 observations were recorded and photos of each plate were taken with a Nikon SM22T Pixera Pro 150ES camera. Isolation of Dictyostelium discoideum genomic DNA The following protocol was used to extract PCRready genomic DNA from Dictyostelium strains AX3 and RI9: 125 µl of QuickExtract Solution was added to each of the two microcentrifuge tubes. A sterile metal loop was used to scrape up some fruiting bodies from the AX3 plate (bacterial cells were avoided). The tip of the metal loop was put into the QuickExtract solution and swirled to ensure the cells were transferred into the solution. The above procedure was done for the RI9 strain as well. Both microcentrifuge tubes were vortexed for 15 seconds and transferred to a 65°C water bath to incubate for 30 minutes. Then, they were vortexed for 15 seconds and incubated in 98°C for 8 minutes. Finally, they were vortexed for 15 more seconds and put back in 98°C incubation for 8 minutes. A volume of 3 ml of each tube was used for the PCR processing. The remaining DNA was stored at 20°C. PCR Amplification The following from Table 1 was added to eight PuReTaq ReadyToGo PCR tubes (GE Healthcare, Piscataway NJ) along with a bead containing Taq polymerase, buffer, Mg2+ , and dNTPs. PCR amplification of the DNA was performed by setting the program to incubate the reactions at 95°C for 5 minutes to activate the Taq polymerase, followed by 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute to allow the Taq polymerase to replicate the DNA fragments. The program ended with a 4°C hold until the samples were stored and prepared for gel electrophoresis.
- 7. 6 Table 1. Amounts of each D. discoideum DNA strain and primer added to perform PCR amplification of the DNA. Tube 1 2 3 4 5 6 7 8 AX3 DNA 3 µl 3 µl 3 µl 3 µl RI9 DNA 3 µl 3 µl 3 µl Sterile Water 12 µl 12 µl 12 µl 12 µl 12 µl 12 µl 15 µl 22 µl ACA Forward Primer 5 µl 5 µl ACA Reverse Primer 5 µl 5 µl cARD Forward Primer 5 µl 5 µl 5 µl cARD Reverse Primer 5 µl 5 µl 5 µl cARA Forward Primer 5 µl 5 µl cARA Reverse Primer 5 µl 5 µl c Visualization of PCR via Gel Electrophoresis In order to visualize the DNA segments that were amplified in each of the strains and primer combinations, gel electrophoresis was used to separate fragments of DNA based on molecular mass. This process was carried out to determine where the mutant type D. discoideum strain was deficient in the cAMP signaling pathway. Each of the PCR products produced in Table 1 were mixed with 5 µl 6X GelRed loading dye, which binds to DNA and fluoresces when exposed to UV light. Also, an 11A DNA molecular weight ladder that was provided by the
- 8. 7 laboratory prep room was combined with 10 µl of the loading dye and ran with the mutant and wildtype D. discoideum DNA samples. A 1.2% wt/vol agarose gel containing 6X TAE (TrisAcetateEDTA) buffer was used and submerged in 1X TAE liquid buffer to run the analysis. Gel electrophoresis was ran at 120V for approximately one hour, until the bands were visibly separated. The resulting gel was observed under UV light with a UV transilluminator to see whether gene expression is causing RI9 signal or not in conditions of starvation (See Figure 2). Results The Dictyostelium discoideum AX3 DNA strain had varying responses to growing on different pH environments and chemicals. The SM agar plate that contained a pH 4.4 environment yielded the best growth and showed signs of higher levels of aggregation and larger stalks and sporangium size compared to Dictyostelium growing on a pH 6.39 and 9.57 environment, leading us to conclude that Dictyostelium discoideum grow best under acidic conditions (Figure 1A, B, and C). Secondly, the chemical analysis part of this test showed that caffeine completely inhibited stalk and fruiting body formation and showed little to no slug formation and aggregation (Figure 1E). Dictyostelium responded moderately to growth on EGTA, as we see complete aggregation, stalk, and sporangium formation, but it was clear that the stalk and sporangium size were still less than those growing in the pH 4.4 environment. DNA analysis using PCR and gel electrophoresis also yielded us data that confirmed our hypothesis. Due to the lack of a DNA band in lane seven, the mutant strain lacks the cARA gene (Lane 7, Figure 2) which is important in the aggregation stage of the developmental cycle.
- 9. 8 Because of this, the mutant RI9 was unable to produce fruiting bodies as a stress signal to relocate to alternate food sources when resources become scarce. Yet the mutant does contain the ACA gene (Lane 5, Figure 2). This means that the RI9 strain cells are able to take ATP and convert it to cAMP, which is the signaling molecule that it sends to other Dictyostelium cells to come together and aggregate. Cells that are well fed do not have cAMP receptors on their cell surface, and therefore, will not respond to the amplification of the cAMP signal if a nearby cell is starving. The cAMP is the initial signal that starts the cells aggregating, and its continued secretion is necessary for differentiation of cells. If a cell is unresponsive to cAMP, it will not act as part of the multicellular body and will therefore starve. Figure 1. Development of Dictyostelium discoideum aggregation on varying pH levels and chemical gradients after 7 days of growth. Figure A represents growth on a pH 4.4 environment, Figure B on a 6.39 environment, Figure C on a 9.57 environment, Figure D growth on EGTA, and Figure E on caffeine. Images were taken at a 3.6X magnification and scale bar represents 1mm.
- 10. 9 Figure 2. DNA analysis gel after PCR amplification using Dictyostelium discoideum wild type (AX3) and mutant (RI9) strains with various primers. Discussion The purpose of this study was to investigate mutated components of cAMP pathways in Dictyostelium in order to understand the importance of cAMP signaling in Dictyostelium development. We hypothesized the mutation in RI9’s cAMP signaling pathway would result in those cells being unable to reach the fruiting body development phase after the stationary phase. This results in the cells resorting to full starvation. We predicted that there was a mutation in the
- 11. 10 signaling pathway involved with ACA or one of the cAMP receptors. After completion of the experiment we can conclude that our hypothesis was supported. Analysis of the gel electrophoresis, proved that the mutant RI9 strain does have the ACA gene but it does not have the cARA gene. Since the cARA gene is not present in the RI9 strain, it therefore is not able to undergo chemotaxis or aggregation. These results align with a study showing that strains that do not have the cARA gene fail to undergo chemotaxis, aggregation into a multicellular unit, and begin cAMP signaling (Klein 1988). These results make sense when combined with the rest of our data for the mutant RI9 strain. Since the ACA gene is present, we know that cAMP is being synthesized from ATP. However, since cARA determines cellcell signaling of cAMP, we know that there is a signaling problem where RI9 does not receive cAMP and therefore aggregation and development cannot be complete (Kim 1997). Although the mutant RI9 strain was consistent with the findings of other experiments, our AX3 wild type strain was not. In our experiment, the gene for cARD was not found in our gel electrophoresis while in the majority of others it was present. This then would lead to aggregation as normal, while forming slugs and fruiting bodies that were abnormal in their multicellular development (Parent 1996). Normally however, the wild type, AX3 would show genes for ACA, cARA, and cARD which would lead to a full developmental life cycle. After completion of this experiment, questions were asked whether the RI9 strain would ever be able to grow. Research shows that it is possible that in high enough concentrations of cAMP, the mutant RI9 strain would still be able to complete the developmental cycle and produce fruiting bodies (Sun 1991). This hypothesis can lead us to future studies: testing the requirement of cAMP concentration for a cARA receptor deficient mutant to complete the
- 12. 11 developmental cycle. This research could lead us into important knowledge about the adaptability of mutant strains of Dictyostelium. Other studies could look into the importance of the cAMP pathway in the human body. Since these receptors are so important in Dictyostelium, one can only imagine what the outcome would be if a human cell was mutated that inhibited them. By experimenting with human cells, we could further the knowledge of how the human body performs. In conclusion, it was found that the mutant RI9 strain is deficient in its cARA gene. Without the cARA gene, cAMP is not received and therefore the developmental pathway is halted. This is an important finding because it allowed us to investigate mutated components of cAMP pathways in Dictyostelium discoideum in order to understand the importance of cAMP signaling in Dictyostelium development.
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- 14. 13 Sonnemann, J., Aichem, A., and Schlatterer, C. Dissection of the cAMP induced cytosolic response in Dictyostelium discoideum: the role of cAMP receptor subtypes and G protein subunits. FEBS Letters. 436(2):271276. Shu, S., Liu, X., Kriebel, P., Daniels, M., and Korn, E. (2011) Actin crosslinking proteins cortexillin I and II are required for cAMP signaling during Dictyostelium chemotaxis and development. Molecular Biology of the Cell. 23:390400 Sun, T. J., Devreotes, P. N. (1991).Gene targeting of the aggregation stage cAMP receptor cAR1 in Dictyostelium. Genes and Development. 5:572582. Wang, B. and A. Kuspa. (1997). Dictyostelium development in the absence of cAMP. Science. 277(5323):251254. Willard, S., and Devreotes, P. (2006). Signaling pathways mediating chemotaxis in the social amoeba, Dictyostelium discoideum. European Journal of Cell Biology. 85(9):897904.