Developmental control of spindle positioning in embryos

描述（由申请人提供）：有丝分裂纺锤体的正确定位对许多发育过程至关重要，包括不对称分裂，其中极化细胞分裂产生具有不同命运的子细胞。该项目研究了保守的PAR极性蛋白和下游中间体LET-99在模式生物秀丽隐杆线虫不对称分裂过程中调节纺锤体位置的机制。关于LET-99功能的几个假设将被测试。在Aim 1中，PAR蛋白在皮质带中定位LET-99的遗传途径将通过检查突变背景下LET-99的定位来确定。此外，LET-99蛋白的皮质和不对称定位所必需和充分的区域将使用转基因来鉴定。在Aim 2中，皮层LET-99带控制纺锤体定位的假设将通过LET-99分布改变的突变体的延时视频显微镜进行验证。目的3将验证LET-99拮抗纺锤体定位中的G蛋白信号通路的假设。将检查遗传相互作用，并使用共免疫沉淀法和双杂交法分析LET-99与G蛋白途径组分之间可能存在的关联。在Aim 4中，我们将测试spn-2基因与LET-99一起拮抗G蛋白信号传导的假设。从长远来看，这些研究将导致对协调极性和纺锤体取向的分子机制的深入理解。不对称分裂对所有多细胞生物的发育都很重要，PAR蛋白在包括人类在内的许多生物中都是保守的。因此，从这项工作中获得的见解将具有广泛的相关性，也有助于理解导致癌症或其他疾病的发育异常。

DESCRIPTION (provided by applicant): Proper positioning of the mitotic spindle is essential for a number of developmental processes, including asymmetric divisions in which a polarized cell divides to produce daughters with different fates. This project addresses the mechanisms by which the conserved PAR polarity proteins and a downstream intermediate, LET-99, regulate spindle position during asymmetric divisions in the model organism Caenorhabditis elegans. Several hypotheses about LET-99 function will be tested. In Aim 1, the genetic pathway by which the PAR proteins localize LET-99 in a cortical band will be determined by examining LET-99 localization in mutant backgrounds. In addition, the regions of the LET-99 protein that are necessary and sufficient for cortical and asymmetric localization will be identified using transgenes. In Aim 2, the hypothesis that the cortical LET-99 band controls spindle positioning will be tested by time-lapse video microscopy of mutants with altered LET-99 distributions. Aim 3 will test the hypothesis that LET-99 antagonizes the G protein signaling pathway that functions in spindle positioning. Genetic interactions will be examined and possible associations between LET-99 and components of the G protein pathway will be assayed using co-immunoprecipitations and two-hybrid assays. In Aim 4, the hypothesis that the spn-2 gene acts with LET-99 to antagonize G protein signaling will be tested. In the long term, these studies will lead to an in-depth understanding of the molecular mechanisms that coordinate polarity and spindle orientation. Asymmetric divisions are important to the development of all multcellular organisms, and the PAR proteins are conserved in many organisms, including humans. Thus, insights gained from this work will be of wide relevance and could also aid in the understanding of developmental abnormalities that lead to cancer or other disorders.