Cell Cycle Regulation of Membrane Trafficking

膜运输的细胞周期调控

• 批准号: 10607265
• 负责人: Joshua Nathaniel Bembenek
• 金额: $ 23.9万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-15 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607265
• 关键词: Cell; Cycle; Regulation; Membrane; Trafficking

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 trigger 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. This proposal describes research aimed at understanding how separase promotes exocytosis in collaboration with other closely related cell cycle regulators that have well characterized roles in chromosome segregation. The dynamic localization of separase is regulated during cell division and only localizes to vesicles during anaphase. Proposed research 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 separation of function alleles of separase. The functions of PPH-5 and HSP-90 will be characterized to determine whether they directly regulate separase during the meiotic divisions. 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. Finally, biochemical and genetic approaches will be employed to identify substrates or targets of separase on vesicles to define the mechanism by which it promotes exocytosis. 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.

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。细胞如何在胞质分裂期间触发膜运输还不清楚。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。该提案旨在了解分离酶在胞吐中的新作用 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。 这项建议描述了旨在了解分离酶如何促进协同胞吐作用的研究 与其他密切相关的细胞周期调节因子在染色体分离中具有明确的作用。 分离酶的动态定位在细胞分裂过程中受到调节， 后期拟议的研究将调查染色体分离调节剂如何控制分离酶 活性和定位于囊泡。不可降解的securin的过表达将被用来确定如何 这种抑制性伴侣蛋白控制分离酶的胞吐功能。PPH-5磷酸酶突变和 其激活因子HSP-90被鉴定为分离酶功能等位基因分离的抑制因子。的功能 将表征PPH-5和HSP-90的特性，以确定它们是否直接调节分离酶， 减数分裂分离酶的磷酸化位点将被定位，磷酸化突变体将被研究 以确定它们如何影响分离酶功能。PPH-5将被测试，以确定它是否直接 体外使分离酶脱磷酸化。最后，将采用生物化学和遗传学方法来鉴定 底物或目标的分离酶的囊泡，以确定其促进胞吐作用的机制。这些 将使用遗传上易处理的C.线虫胚胎这项工作将提供新的见解 细胞如何在胞质分裂期间协调染色体分离与胞吐作用的基本过程， 这与理解正常发育和诸如不育和癌症的疾病有关。