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，这些事件必须得到精确的调节。 细胞分裂的一个中心特征是染色体准确地分离成子细胞。其他 细胞的隔室也必须小心地包装成子细胞，并与 染色体分离细胞膜运输途径对于细胞分裂的完成是必不可少的， 细胞分裂的结束。细胞如何控制胞质分裂期间的膜运输还没有很好地理解。大 蛋白酶分离酶是染色体分离中的主要参与者，这是由于其在粘着蛋白切割中的作用， 使染色体在分裂后期开始时分离。染色体分离后，分离酶促进 后期的几个事件。该建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囊泡。分离酶也调节大皮质颗粒的胞吐作用 在减数分裂后期I，阻断多精受精，这是一个理想的背景下分析这一调控途径。 我们将使用生物化学和遗传学的方法来确定底物或分离酶的结合伴侣， 囊泡，以确定其促进胞吐作用的机制。分离酶的动态定位是 在细胞分裂过程中受到调节，分离酶仅在后期定位于囊泡。我们将调查如何 染色体分离调节剂控制分离酶活性和定位于囊泡。过表达 将使用不可降解的securin来确定这种抑制性伴侣如何控制胞吐功能 的分离。PPH-5磷酸酶和它的激活剂HSP-90的突变被鉴定为是 分离酶突变体的胚致死率。将绘制分离酶磷酸化位点，并将磷酸化 将研究突变体以确定它们如何影响分离酶功能。PPH-5将被测试，以确定它是否 在体外直接使分离酶脱磷酸。PPH-5和HSP-90的功能将被表征为 确定它们是否在减数分裂期间直接调节分离酶。这些研究报告将 使用遗传上易处理的C.线虫胚胎这项工作将为细胞如何 协调胞质分裂期间染色体分离的基本过程与胞吐作用，即 与了解正常发育和疾病，如不孕症和癌症有关。

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.