What is D. discoideum ?

1. The Effect of PKC Inhibitors and Activators on Cell Aggregation of Dictyostelium discoideumVanessa Hardy1, Paola Giraldo2, and Derrick Brazill21Edward R. Murrow High School Brooklyn, New York 2Hunter College/CUNY New York, New York

2. 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

3. CMF cAMP CMF-R cAR1 Pkc PldB Guanylyl Cyclase AdenylylCyclase Motility Gene Expression CMF and cAMP Signal Pathways

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

5. 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 106 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.

6. 1 2 3 4 5 6 Ax2 +PBM Ax2 +DMSO Ax2 + Inh 1 or 2 Ax2 + Act 1 or 2 …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 2-6. • 1ml 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 1st 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.

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

8. 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. Results Expected Results Observed Results

9. 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.

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