Spatiotemporal Control of Dynein Function by Num1

描述(申请人提供)：有丝分裂纺锤体定位和核迁移对于决定子代细胞的大小、位置和发育命运以及确保遗传物质的忠实分离至关重要。纺锤体的定位是由负末端定向马达--动力蛋白的活动调节的。动力蛋白是各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向。这些过程的失败可能导致非整倍体，这是人类癌症的一个标志。为了正确定位有丝分裂纺锤体，动力蛋白功能在空间和时间上都受到严格的调控。在后期，动力蛋白以细胞皮质为靶点并锚定在那里，在那里它的运动活动调节纺锤体的定位。然而，在酵母中，已经确定了一个皮质锚-Num1-调控动力蛋白活性的机制尚不清楚。目标/特定目标：我的目标是全面描述动力蛋白的空间和时间调节。为了回答这个基本问题，我将研究动力蛋白的皮质锚Num1对其的调节。我将检查后期的1号中转，并确定这一过程所需的组件。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，我将确定负责齐聚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之间相互作用的确切性质，以及该锚的调节，将有助于我们理解纺锤体定位的基本生物学过程，以及相关的人类疾病。 与公共卫生相关：了解动力蛋白的空间和时间调节非常重要，因为细胞质动力蛋白的正确功能对于执行各种基本的细胞过程至关重要，包括染色体分离和纺锤体定位，这些过程的缺陷会导致非整倍体，这是人类癌症的标志。

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. PUBLIC HEALTH RELEVANCE: Understanding the spatial and temporal regulation of dynein is highly significant because proper functioning of cytoplasmic dynein is crucial for executing a variety of fundamental cellular processes, including chromosome segregation and spindle orientation, defects of which would lead to aneuploidy, which is a hallmark of human cancer.