Developmental control of spindle positioning in embryos.

这项工作的长期目标是阐明控制位置的机制 有丝分裂纺锤体在发育过程中。主轴定位对于一些 包括不对称分裂，其中极化细胞 产生了不同命运的女儿拟议项目涉及 在不对称分裂过程中纺锤体定位的分子机制 秀丽隐杆线虫胚胎。在C.线虫单细胞胚胎，LET-99，a DEPDC 1家族的含DEP结构域的蛋白，定位于不对称的 皮质带型的PAR蛋白。LET-99反过来限制了皮质定位 G蛋白信号传导的正调节因子GPR和LIN-5，在细胞的某些区域， 细胞皮层G蛋白信号是皮质拉力作用于星体层所必需的。 微管定位纺锤体，GPR和LIN-5与微管的调节因子相关。 微管运动动力蛋白PAR蛋白、GPR和LIN-5的同源物是重要的 在几种不同的生物体中的极性和纺锤体定位。然而，如何GPR 和LIN-5调节力的位置主轴是未知的任何系统。进一步 C. GPR和LIN-5不对称性产生的分子机制。 elegans仍然有待阐明。目标1中提出的实验将有助于改进 通过确定微管如何产生力的机械基础模型 动力学和微管结合蛋白和马达的定位与 由LET-99和GPR/LIN-5定位定义的皮质力域。实时成像 的GFP标记的报告将被用来检查皮质微管动力学和 动力蛋白及其调节物在皮质和微管正端的定位。 微管加端结合蛋白的clasp家族调节 微管动力学，以促进纺锤体的方向，然后系绳纺锤体将 也可以使用实时成像和遗传分析相结合的方法进行研究。目标 目的2是确定LET-99与G）亚基的结合如何影响G蛋白 因此，在皮质抑制GPR定位。定量分析 免疫定位模式和双突变分析将用于确定 该途径的组分由LET-99调节。生物化学方法将是 用于确定LET-99是否影响G）活性或其与其它途径的关联 件.由于途径组分的保守性，这些结果 研究将与许多系统中的不对称划分相关。

The long-term goal of this work is to elucidate the mechanisms that control the position of the mitotic spindle during development. Spindle positioning is essential for a number of developmental processes, including asymmetric divisions in which a polarized cell divides to produce daughters with different fates. The proposed project addresses the molecular mechanisms of spindle positioning during asymmetric divisions in the Caenorhabditis elegans embryo. In the C. elegans one-cell embryo, LET-99, a DEP domain containing protein of the DEPDC1 family, is localized in an asymmetric cortical band pattern by the PAR 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. G protein signaling is required for cortical pulling forces that act on astral microtubules to position the spindle, and GPR and LIN-5 associate with regulators of the microtubule motor dynein. Homologs of the PAR proteins, GPR and LIN-5 are important for polarity and spindle positioning in several different organisms. However, how GPR and LIN-5 regulate forces that position spindles is not known for any system. Further the molecular mechanism by which asymmetries of GPR and LIN-5 are generated in C. elegans remain to be elucidated. The experiments proposed in Aim 1 will help refine models for the mechanistic basis of force generation by determining how microtubule dynamics and the localization of microtubule binding proteins and motors correlates with the cortical force domains defined by LET-99 and GPR/LIN-5 localization. Live-imaging of GFP-tagged reporters will be used to examine cortical-microtubule dynamics and the localization of dynein and its regulators both at the cortex and on microtubule plus-ends. The hypothesis that the clasp family of microtubule plus-end binding proteins regulates microtubule dynamics to facilitate spindle orientation and then to tether the spindle will also be investigated, using a combination of live-imaging and genetic analysis. The goal of Aim 2 is to determine how binding of LET-99 to G) subunits affects the G protein pathway such that GPR localization is inhibited at the cortex. Quantitative analysis of immunolocalization patterns and double mutant analysis will be used to determine which components of the pathway are regulated by LET-99. Biochemical approaches will be used to determine if LET-99 affects G) activity or its association with other pathway components. Because of the conservation of pathway components, the results of these studies will be relevant to asymmetric division in many systems.