| We use the social amoeba Dictyostelium discoideum to study cytokinesis, the process of cell division. Three proteins were previously identified to be required for cytokinesis, but the precise stage of cytokinesis each protein affected was unknown. RacE is a member of the rho family of small GTPases. Clathrin is a member of the protein lattice involved in vesicle budding from membranes. Large volume sphere A (LvsA) is a novel protein that contains domains also found in proteins that mediate traffick and lipid signaling events. The focus of this project was to characterize the cytokinesis defects of the mutants for these three proteins. To directly address this question, a novel protocol for viewing cells live in suspension culture was developed. Other forms of microscopy were also extensively used, including immunofluorescence, confocal microscopy, and GFP fusion proteins. Molecular biology techniques such as cloning, PCR, and homologous recombination were also used. In addition, a collaboration was initiated for the biophysical measurement of cortical tension. As a result of these studies, we determined that racE mutants have extremely low levels of cortical tension that cause multiple ruptures in the cortex during division. RacE null cells assemble a contractile ring for division, but they cannot constrict it to completion. RacE may regulate organization of F-actin in the cortex. Clathrin null cells have defects assembling and constricting the contractile ring. Clathrin null cells develop abnormal dual constrictions that do not divide the cell, but cause the equator to bulge between them. LvsA-mutants form dual constrictions, similar to clathrin null cells. LvsA protein localizes to the contractile vacuole, and is important for the organization and function of this organelle. In addition, IvsA-cells do not properly localize the protein calmodulin to the contractile vacuole. This IvsA-calmodulin defect is also similar to the phenotype of clathrin null cells. This suggests that roles of LvsA and clathrin may overlap a common signalling pathway in cytokinesis. |
