Roles of CBP7 and Rap Proteins in Dictyostelium Cell Migration and Development

Abstract

Calcium ions are involved in the regulation of diverse cellular processes. Fourteen genes encoding calcium binding proteins have been identified in Dictyostelium. CBP7, one of the 14 CBPs, is composed of 169 amino acids and contains four EF-hand motifs. Here, I investigated the roles of CBP7 in the development and cell migration of Dictyostelium cell and found that high levels of CBP7 exerted a negative effect on cells aggregation during development, possibly by inhibiting chemoattractant-directed cell migration. While cells lacking CBP7 exhibited normal development and chemotaxis similar that of wild-type cells, CBP7 overexpressing cells completely lost their chemotactic abilities to move toward increasing cAMP concentrations. This resulted in inhibition of cellular aggregation, a process required for forming multicellular organisms during development. Low levels of cytosolic free calcium were observed in CBP7 overexpressing cells, which was likely the underlying cause of their lack of chemotaxis. These results demonstrate that CBP7 plays an important role in cell spreading and cell-substrate adhesion. cbp7 null cells showed decreased cell size and cell-substrate adhesion. In addition, experiments using point-mutated CBP7 cells showed that all EF-hand domain of CBP7 was important for CBP7 to function in the developmental process. Yeast two-hybrid experiments using CBP7 as a bait showed that CBP7 specifically binds to activation RasG. These results suggest that CBP7 has important roles in the development process through all of CBP7 EF-hand domains and by binding to RasG. The present study contributes to further understanding the role of calcium signaling in regulation of cell migration and development and the relationship between the calcium signaling and Ras signaling pathways. Ras proteins are small, monomeric GTPases that act as crucial regulators of a number of cellular signaling pathways, including proliferation, cell migration, differentiation, and apoptosis. The functions of most of these Ras proteins in development and cell migration have not been studied yet. Therefore, I investigated the roles of RapB and RapC in cell migration and development. RapB and RapC has the highest homology (86.6%, 50.9 % amino acid identity) with Rap1, which is a key regulator in cell adhesion and cell migration. To investigate the functions of RapB, I prepared constitutively active form of GFP-RapB cells (GFP-RapB G31V cells) and dominantly negative form of GFP-RapB cells (GFP-RapBS36N cells). I found that GFP-RapBS36N cells displayed decreased cell size and weak cell-substrate adhesion than wild-type cells. These results demonstrate that RapB positive plays cell spreading and cell-substrate adhesion. In addition, I investigated the roles of RapB in the development and cell migration of Dictyostelium cells and found that GFP-RapB G31V cells exerted late development, possibly through strong cell-substrate adhesion appeared inhibition of chemoattractant-mediated cell migration in development. These results demonstrate that RapB negative plays in cell migration and development. To investigate the functions of RapC, I prepared rapC null cells by homologous recombination. Unexpectedly, RapC might have opposite functions to Rap1 and RapB. rapC null cells showed flattened and spread morphology and strong cell-substrate adhesion compared to wild-type cells. The results demonstrate that RapC plays an important negative role in cell spreading and cell-substrate adhesion as opposed to Rap1 and RapB. In addition, rapC null cells had a cytokinesis defect. These phenotypes of rapC null cells were rescued by overexpressing GFP-RapC, suggesting that RapC is required for cytokinesis. I investigated the roles of RapC in the development and cell migration of Dictyostelium cells and found that rapC null cells exerted a negative effect on cells aggregation and fruiting body formation during development, possibly through strong cell-substrate adhesion appeared inhibition of chemoattractant-mediated cell migration and electrical stimulation in development. These results demonstrate that RapC plays an important positive role in cell migration and development as opposed to Rap1 and RapB. The localization assay showed that RapB and RapC localized to the cell plasma membrane and preferentially at the leading edge in migrating cells. The present study contributes to our understanding of the molecular mechanism of Rap proteins that regulates cytoskeletal rearrangement and morphogenesis in Dictyostelium