Cytokinesis and the Cytoskeleton in C. elegans Embryos

描述(申请人提供)：在我们最初的申请(资助时间为1998年5月1日至2002年4月30日)中，我们建议鉴定线虫早期胚胎细胞分裂所需的基因，重点是分离和研究条件突变、胚胎致死突变。在这一资助期间，我们已经确定了1600个条件突变，其中包括几个细胞分裂素缺陷的突变，这是这项提案的重点。我们已经确定了几类有趣的细胞质分裂突变体，其中一些是积极的，另一些是以前没有文献记载的新的负面调控形式所必需的。在我们的新的特定目标中，我们建议研究Nedd8/Rub1p泛素样蛋白结合途径的调节和作用，该途径在细胞质分裂过程中负向调节卵裂沟外的皮质微丝收缩。我们还建议对已鉴定的几个新的胞质分裂突变体进行表型鉴定，并使用定位克隆来确定突变基因的分子特性。这些突变包括一个具有皱纹组装和进展缺陷的突变，一个具有晚期细胞质分裂缺陷的突变，以及两个在有丝分裂期间或之前具有异位分裂皱纹的突变。最后，我们建议继续筛选具有细胞分裂缺陷的条件、胚胎-致命突变，并提出一种新的筛选方法，利用RNAi和我们的条件突变来更全面地识别参与胞质分裂和其他过程的基因。这些遗传学和分子研究将为微丝和微管细胞骨架的调节和功能提供新的见解，它们介导基本和保守的细胞过程，通常与人类疾病有关。

DESCRIPTION (provided by applicant): 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.