In our original application (funded from May 1, 1998-April 30, 2002), we proposed to identify genes required for early embryonic cell divisions in C. elegans, focusing on the isolation and investigation of conditional, embryonic-lethal mutants. In this funding period, we have identified 1600 conditional mutants, including several with defects in cytokineis that are the focus of this proposal. We have identified several intriguing classes of cytokinesis mutants, some required positively and others required for novel negative forms of regulation not previously documented. In our new Specific Aims, we propose to investigate the regulation and role of the Nedd8/Rub1p ubiquitin-like protein conjugation pathway, which negatively regulating cortical microfilament contractility outside the cleavage furrow during cytokinesis. We also propose to characterize the phenotypes of several new cytokinesis mutants we have identified, and to use positional cloning to determine the molecular identities of the mutated genes. These include one mutant with defects in furrow assembly and progression, one mutant with a late defect in cytokinesis, and two mutants with ectopic cleavage furrows either during or before mitosis. Finally, we propose to continue our screens for conditional, embryonic-lethal mutants with defects in cell division, and we also propose a novel screen that takes advantage of RNAi and our conditional mutants to identify more comprehensively genes that participate in cytokinesis and other processes. These genetic and molecular studies should provide new insight into the regulation and function of the microfilament and microtubule cytoskeleton, which mediate essential and conserved cellular processes, and are often associated with human disease.
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