Spatiotemporal Control of Dynein Function by Num1

描述（由申请人提供）：有丝分裂纺锤体定位和核迁移对于确定子细胞的大小、位置和发育命运以及确保遗传物质的忠实分离至关重要。主轴定位是由负端定向马达，动力蛋白的活动介导的。动力蛋白是多种基本细胞过程所必需的，包括细胞分裂过程中的染色体分离和纺锤体定位。这些过程的失败会导致非整倍体，这是人类癌症的一个标志。为了正确定位有丝分裂纺锤体，动力蛋白的功能在空间和时间上都受到严格的调节。在后期，动力蛋白瞄准并固定在细胞皮层，在那里它的运动活动介导纺锤体定位。动力蛋白活性的调节机制尚不清楚，然而，在酵母中，已经确定了一个皮质锚点- Num1。目的/具体目的：我的目标是充分表征动力蛋白的时空调节。为了回答这个基本问题，我将研究动力蛋白的皮质锚点Num1对其的调节。我将在后期检查Num1传输，并确定此过程所需的组件。我将确定Num1的寡聚状态，并在体外鉴定与Num1相互作用的动力蛋白亚基。最后，我将确定负责寡聚化和动力蛋白相互作用的Num1结构域。了解动力蛋白与其皮质锚点之间相互作用的确切性质，以及锚点的调节，将促进我们对纺锤体定位的基本生物学过程和相关人类疾病的理解。

DESCRIPTION (provided by applicant): Mitotic spindle positioning and nuclear migration are critical for determining the size, position and developmental fate of daughter cells, and ensuring faithful segregation of the genetic material. Spindle positioning is mediated by the activity of the minus end-directed motor, dynein. Dynein is required for a variety of fundamental cellular processes, including chromosome segregation and spindle orientation during cell division. Failures of these processes can lead to aneuploidy, which is a hallmark of human cancer. To correctly position the mitotic spindle, dynein function is tightly regulated both spatially and temporally. During anaphase, dynein is targeted to, and anchored at the cell cortex, where its motor activity mediates spindle positioning. The mechanism by which dynein activity is regulated is not known, however, in yeast, a cortical anchor - Num1 - has been identified. OBJECTIVE/SPECIFIC AIMS: My goal is to fully characterize the spatial and temporal regulation of dynein. To answer this fundamental question, I will examine the regulation of dynein by its cortical anchor, Num1. I will examine Num1 transit during anaphase and identify the components required for this process. I will determine oligomeric state of Num1 and identify the subunit of dynein that interacts with Num1 in vitro. Finally, I will determine the Num1 domain responsible for oligomerization and dynein interaction. Understanding the exact nature of the interaction between dynein and its cortical anchor, as well as the regulation of that anchor, will progress our understanding of the basic biological process of spindle positioning, and related human disease.