Cell Cycle Regulation of Membrane Trafficking

项目摘要 细胞分裂需要一系列精心协调的基本事件，这些事件必须受到精确的调控。 细胞分裂的一个中心特征是将染色体准确地分离成子细胞。其他 细胞的隔间也必须小心地包装成子细胞，并与 染色体分离。膜运输途径是完成胞质分裂所必需的。 细胞分裂结束。细胞如何在胞质分裂过程中控制膜的运输还不是很清楚。大的 蛋白酶分离酶是染色体分离的中心角色，因为它在粘附素切割中发挥作用，这是 允许在后期开始进行染色体分离。染色体分离后，分离酶促进 后期发生的几件事。这项提议旨在了解分离酶在胞吐作用中的新作用。 胞质分裂所需的RAB-11小泡。分离酶也调节大皮质颗粒的胞吐作用 在减数分裂后期I阻断多精细胞，这是一个理想的背景下分析这一调控途径。 我们将使用生化和遗传方法来鉴定分离酶的底物或结合伙伴。 囊泡来定义它促进胞吐作用的机制。分离酶的动态定位是 在细胞分裂过程中被调节，而在细胞分裂后期，分离酶仅定位于小泡。我们将调查如何 染色体分离调节因子控制着分离酶的活性和对小泡的定位。过度表达 不可降解的Securin将被用来确定这种抑制性伴侣如何控制胞外功能 是分离的。PPH-5磷酸酶及其激活剂HSP-90的突变被鉴定为抑制 分离酶突变体的胚胎致死性。分离酶的磷酸化位点将被绘制和磷酸化 将对突变体进行研究，以确定它们如何影响分离酶功能。PPH-5将进行测试，以确定是否 在体外直接去磷酸化分离酶。PPH-5和HSP-90的功能特征如下 确定它们是否在减数分裂过程中直接调节分离酶。这些研究将是 使用了遗传上易驯化的线虫胚胎。这项工作将为细胞如何 在胞质分裂过程中协调染色体分离和胞吐的基本过程，这是 与了解正常发育和不孕不育和癌症等疾病有关。

Project Summary Cell division requires a carefully coordinated series of essential events that must be precisely regulated. A central feature of cell division is the accurate segregation of chromosomes into daughter cells. Other compartments of the cell must also be carefully packaged into daughter cells, and coordinated with chromosome segregation. Membrane trafficking pathways are essential for the completion of cytokinesis at the end of cell division. How cells control membrane trafficking during cytokinesis is not well understood. The large protease separase is a central player in chromosome segregation due to its role in cohesin cleavage, which allows chromosome separation at the onset of anaphase. After chromosome segregation, separase promotes several events during anaphase. This proposal aims to understand a novel role of separase in the exocytosis of RAB-11 vesicles required for cytokinesis. Separase also regulates exocytosis of large cortical granules during anaphase of meiosis I to block polyspermy, which is an ideal context to analyze this regulatory pathway. We will use biochemical and genetic approaches to identify substrates or binding partners of separase on vesicles to define the mechanism by which it promotes exocytosis. The dynamic localization of separase is regulated during cell division and separase only localizes to vesicles during anaphase. We will investigate how chromosome segregation regulators control separase activity and localization to vesicles. Overexpression of non-degradable securin will be used to determine how this inhibitory chaperone controls the exocytic function of separase. Mutations of the PPH-5 phosphatase and its activator HSP-90 were identified as suppressors of embryo lethality of separase mutants. Separase phosphorylation sites will be mapped and phosphorylation mutants will be studied to determine how they affect separase function. PPH-5 will be tested to determine if it directly dephosphorylates separase in vitro. The functions of PPH-5 and HSP-90 will be characterized to determine whether they directly regulate separase during the meiotic divisions. These studies will be performed using the genetically tractable C. elegans embryo. This work will provide new insight into how cells coordinate the essential process of chromosome segregation with exocytosis during cytokinesis, which is relevant to understanding normal development and diseases such as infertility and cancer.