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是必不可少的，包括不对称分裂，即极化细胞分裂产生不同命运的后代。本项目研究了模式生物秀丽线虫在不对称分裂过程中，保守的PAR极性蛋白和下游中间体LET-99调节纺锤体位置的机制。关于let-99函数的几个假设将被检验。在目标1中，PAR蛋白将let-99定位于皮质带的遗传途径将通过检测let-99在突变背景中的定位来确定。此外，对于皮质和不对称定位是必要的和充分的let-99蛋白的区域将使用转基因来识别。在目标2中，将通过改变let-99分布的突变的延时视频显微镜来检验皮质let-99带控制纺锤体定位的假设。目的3验证LET-99拮抗G蛋白信号通路的假说，G蛋白信号通路在纺锤体定位中起作用。将研究遗传交互作用，并将使用免疫共沉淀和双杂交分析来分析let-99和G蛋白途径组件之间的可能联系。在目标4中，将检验SPN-2基因与LET-99共同作用以拮抗G蛋白信号的假设。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体的发育都很重要，PAR蛋白在许多生物体中都是保守的，包括人类。因此，从这项工作中获得的见解将具有广泛的相关性，并可能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.