Developmental control of spindle positioning in embryos.

这项工作的长期目标是阐明控制地位的机制 在有丝分裂纺锤体的发育过程中。主轴定位对于一个数字来说是必不可少的 发育过程，包括不对称分裂，极化的细胞 分裂产生不同命运的女儿。拟议的项目解决了 不对称分裂过程中纺锤体定位的分子机制 秀丽线虫胚胎。在线虫的单细胞胚胎中，let-99，a DEP结构域包含DEPDC1家族的蛋白，定位于不对称的 PAR蛋白的皮质条带模式。LET-99反过来又限制了皮质的定位 G蛋白信号的正调控因子GPR和LIN-5，作用于细胞的某些区域 细胞皮质。G蛋白信号是作用于星体的皮层牵引力所必需的 微管定位纺锤体，GPR和LIN-5与 微管运动动力蛋白。PAR蛋白、GPR和LIN-5的同源物是重要的 在几个不同的生物体中用于极性和主轴定位。然而，GPR如何 而LIN-5调节定位主轴的力对于任何系统都是未知的。再往前走 细菌产生GPR和LIN-5不对称的分子机制 雅致仍有待阐明。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力学基础的模型 微管结合蛋白和微管马达的动态和定位 由LET-99和GPR/LIN-5定位定义的皮质力域。实时成像 GFP标记的记者将被用于检查皮质微管动力学和 动力蛋白及其调节因子在皮质和微管加端的定位。 微管正端结合蛋白CLASP家族参与调控的假说 微管动力学有助于纺锤体定向，然后将纺锤体拴在一起 也可以通过现场成像和遗传分析相结合的方法进行调查。目标是 目的2是确定let-99与G)亚基的结合如何影响G蛋白 GPR定位在大脑皮层被抑制的途径。定量化分析 将使用免疫定位模式和双突变分析来确定 该途径的组成部分受LET-99的调节。生化方法将是 用于确定let-99是否影响G)活性或其与其他途径的联系 组件。由于通路组件的保守性，这些结果 研究将与许多系统中的不对称分裂有关。

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.