Developmental Control of Spindle Positioning in Embryos

描述（由申请人提供）：不对称分裂，其中极化细胞分裂产生具有不同命运的子代，有助于发育期间的细胞命运特化以及干细胞维持。该项目旨在探讨秀丽隐杆线虫胚胎不对称分裂过程中纺锤体定位和胞质分裂的分子机制。与其他系统一样，C.线虫单细胞胚胎依赖于一个保守的途径，其中PAR极性蛋白调节非经典G蛋白信号通路组分的分布。我们确定LET-99是DEPDC 1家族的一员，是这一途径中的新参与者。LET-99通过PAR-3和PAR-1蛋白定位在不对称的皮质带型中。LET-99反过来限制了G蛋白信号传导的正调节因子GPR和LIN-5在细胞皮质的某些区域的皮质定位。这些中间体的不对称定位是纺锤体定位的一个基本特征，因为GPR和LIN-5与微管运动动力蛋白的调节剂相关联，以产生移动纺锤体的不对称皮质拉力。一旦纺锤体被定位，它就会向皮层发出信号，以确定卵裂的平面。PAR蛋白如何促进纺锤体定位因子的不对称性，以及G蛋白通路如何与其他信号机制整合，仍有待阐明。目标1中提出的实验将定义PAR蛋白调节LET-99不对称性的分子机制。将使用体外激酶测定和体内转基因研究来测试PAR-1直接磷酸化LET-99以抑制其在后皮质定位的假设。PAR-3通过单独的机制抑制LET-99在前部的定位，这将使用实时成像和遗传分析的组合进行研究。还将测试LET-99相互作用蛋白在LET-99皮质锚定中的作用。目的2的目标是确定LET-99/G？通路如何与Rho家族GT3信号转导整合，以正确定位胞质分裂沟相对于纺锤体。结合生化相互作用试验的突变体定位模式的定量分析将用于确定这些途径如何相互作用。还将测试LET-99通过其部分RhoGAP结构域直接结合Rho GTP酶的假设。最后，目标3将检验LET-99的人类直系同源物DEPDC 1和DEPDC 1B与LET-99具有相似功能的假设。具体来说，我们将测试的假设，这些蛋白质与G或Rho和参与纺锤体运动或胞质分裂。由于途径组分的保守性，这些研究的结果将与许多系统中的不对称分裂相关，并将定义一类新蛋白质DEPDC 1家族的功能。

DESCRIPTION (provided by applicant): Asymmetric divisions, in which a polarized cell divides to produce daughters with different fates, contribute to cell fate specification during development as well as stem cell maintenance. The proposed project addresses the molecular mechanisms of spindle positioning and cytokinesis during asymmetric divisions in the Caenorhabditis elegans embryo. As in other systems, asymmetric division in the C. elegans one-cell embryo relies on a conserved pathway in which the PAR polarity proteins regulate the distribution of components of a non-canonical G protein signaling pathway. We identified LET-99, a member of the DEPDC1 family, as a new player in this pathway. LET-99 is localized in an asymmetric cortical band pattern by the PAR-3 and PAR-1 proteins. LET-99 in turn restricts the cortical localization of the positive regulators of G protein signaling, GPR and LIN-5, to certain regions of the cell cortex. The asymmetric localization of these intermediates is an essential feature of spindle positioning, because GPR and LIN-5 associate with regulators of the microtubule motor dynein to generate the asymmetric cortical pulling forces that move the spindle. Once the spindle is positioned, it signals back to the cortex to determine the plane of cleavage. How the PAR proteins promote asymmetry of spindle positioning factors, and how the G protein pathway is integrated with other signaling mechanisms, remains to be elucidated. The experiments proposed in Aim 1 will define the molecular mechanisms by which the PAR proteins regulate LET-99 asymmetry. The hypothesis that PAR-1 directly phosphorylates LET-99 to inhibit its localization at the posterior cortex will be tested using in vitro kinase assays followed by in vivo transgenic studies. PAR-3 inhibits LET-99 localization at the anterior via a separate mechanism, which will be investigated using a combination of live-imaging and genetic analysis. LET-99 interacting proteins will also be tested for a role in LET-99 cortical anchoring. The goal of Aim 2 is to determine how the LET-99/G¿ pathway is integrated with Rho-family GTPase signaling to properly position the cytokinesis furrow relative to the spindle. Quantitative analysis of localization patterns in mutants combined with biochemical interaction assays will be used to determine how these pathways interact. The hypothesis that LET-99 directly binds Rho GTPases via its partial RhoGAP domain will also be tested. Finally, Aim 3 will test the hypothesis that the human orthologs of LET-99, DEPDC1 and DEPDC1B, have a similar function to LET-99. Specifically we will test the hypothesis that these proteins associate with G¿ or Rho and are involved in spindle movements or cytokinesis. Because of the conservation of pathway components, the results of these studies will be relevant to asymmetric division in many systems and will define the function of a novel class of proteins, the DEPDC1 family.