The Effect of PKC Inhibitors and Activators on Cell Aggregation of Dictyostelium discoideum Vanessa Hardy 1, Paola Giraldo 2, and Derrick Brazill 2 1 Edward R. Murrow High School Brooklyn, New York 2 Hunter College/CUNY New York, New York

What is D. discoideum? Social amoeba that undergoes different developmental stages –Aggregates when there is lack of food The process of development involves chemotaxis, cell signaling, and cellular differentiation Used in lab research because of its similarity to mammalian cells to study signal transduction pathways Blanton and Grimson, Texas Tech University

CMF-R cAR1 cAMP PldB CMF Motility Gene Expression Guanylyl Cyclase Adenylyl Cyclase Pkc CMF and cAMP Signal Pathways

Objective To prove that Pkc inhibits PldB in D. discoideum’s CMF and cAMP signal transduction pathways, using Pkc inhibitors and activators.

Material and Methods Low Cell Density Assay Wild-type cells (Ax2) and pldB - cells (β12) are used in the experiment. Cell concentrations are calculated by counting Ax2 and β12 cells on a hemocytometer. 6.8 x 10 6 cells are placed in a Falcon tube and centrifuged at 1300rpm for 5 minutes. Medium is decanted and cells are resuspended and washed in 10ml of PBM. Supernatant is decanted and cells are resuspended again in 8ml PBM.

…Continued Three 24-well plates are set up to contain 400µl of PBM, DMSO, Inhibitor 1 or 2, Activator 1 or 2, and Rapamycin from column ml of cells are placed in six Eppendorf tubes, and 1µl of DMSO, Inhibitor 1or 2,Activator 1 or 2, and 2µl of Rapamycin are added respectively. 800µl of cells are taken from the Eppendorf tubes and placed in the 1 st well of each row in the 24-well plate. A serial dilution of 1:2 is made from well 1 to well 2, from well 2 to well 3 and so on. 400µl is taken from well 6 and tossed. Plates are placed in 21°C incubator and examined after 18hrs Ax2 +PBM Ax2 +DMSO Ax2 + Inh 1 or 2 Ax2 + Act 1 or 2

Stages of Aggregation Figure 1. Single Cells Figure 4. Aggregation Figure 3. Streaming Figure 2. Initial Streaming

Results Pkc inhibitor added to Ax2 cells would lead to no aggregation. Pkc activator added to Ax2 cells would lead to aggregation at low cell density. Pkc inhibitor and activator added to β12 cells would not affect cell aggregation. The results are inconsistent. Sometimes β12 cells did not aggregate at low cell density or did not aggregate earlier than the Ax2 cells as expected. There are some slight differences observed between the controls: PBM and DMSO. Rapamycin also allows the cells to aggregate at low cell density which means it acts more like an activator. Expected ResultsObserved Results

Conclusions and Future Experiments The hypothesis is not supported by the results in the experiment. The results are inconsistent and β12 cells didn’t aggregate at low cell density or aggregate earlier than the Ax2 cells as expected. We may need to test different concentration of inhibitors and activators in D. discoideum cells to determine the ideal concentration needed to observe differences. A future experiment is to establish whether pldB over- expressing cells will result in aggregation if Pkc inhibits PldB.

Acknowledgements Harlem Children Society/ Project Seed Dr. Sat Bhattacharya Dr. Derrick Brazill Paola Giraldo Yi Chen