Cytokinesis staging mechanisms

项目摘要 真核 适当 开始 女儿 癌变 过程 胞质 细胞分裂需要依赖关系，在这种关系中，晚期事件只有在早期事件发生后才开始。 完成例如，在胞质分裂过程中， 以精确和一致的顺序来完成架构重组， 细胞细胞机械分裂的失败会严重影响基因组的稳定性， 以及其他重要的人类疾病协调细胞动力学的机制 是不完全理解的，也是一个强烈的研究兴趣的主题。值得注意的是， 并且目前已知的控制它的调节剂是高度保守的。在芽殖酵母中工作，我们 监管 已经发现了一个系统，当早期的细胞分裂被延迟或有缺陷时， 让纠正机制有时间发挥作用。这种“检查站”途径，称为“执行 胞质分裂顺序”（ECO），通过稳定一个多基序蛋白，阻断一个胞质分裂特异性分泌 Ndr/Lats蛋白激酶Cbk 1的功能，Cbk 1是高度保守的“Ndr-河马”信号传导的关键组分 系统 Hippo信号通路是细胞形态发生、增殖和分化的重要调节因子。 真核细胞; Ndr/Lats激酶是其最下游的信号传导组分。我们之前的研究 为理解这些系统做出了重要的分析。我们是最早描述 河马信号系统在体内的特定功能，定义了独特的Ndr激酶磷酸化基序， 报道了第一个典型的底物对接的AGC家族蛋白激酶，并贡献了第一个， 只有Ndr/Lats激酶的晶体结构与Mob共激活剂结合。 该项目旨在确定我们发现的检查点机制如何阻止晚分泌 当该过程的早期阶段有缺陷时，胞质分裂蛋白质。它将包括探索在体内 与胞质分裂调节剂的对接相互作用，胞质分裂期间分泌组织的评估，以及 评价Ndr-hippo调节胞质分裂关键效应蛋白的重要性。我们还将探索 假设人类Ndr-hippo通路参与晚期胞质分裂的控制，使用两种不同的 接近。一方面，我们将确定人类Ndr激酶是否参与对接体内 磷酸化靶标，使用高度优化的噬菌体展示方法来鉴定可能的肽基序 与Ndr激酶- Mob共激活剂复合物相互作用。我们将使用一种新的配体足迹法， 开发用于绘制肽基序关联。在另一种方法中，我们将确定人类Ndr-hippo 通过构建仅表达类似物敏感性Ndr激酶的细胞系，这 将允许在经历正常和缺陷的细胞中快速和可逆地抑制这些激酶 胞质分裂

PROJECT SUMMARY Eukaryotic appropriately commence daughter carcinogenesis processes cytokinesis cell division requires dependency relationships in which late events start only after early ones are completed. During cytokinesis, for example, distinct large-scale morphological processes in precise and consistent order to accomplish architectural reorganization that produces two cells. Failure of mechanical division of cells can drastically affect genome stability, contributing to and other important human maladies. mechanisms that coordinate cytokinetic are incompletely understood, and a subject of intense research interest. Notably, machinery of and regulators currently known to control it are highly conserved. Working in budding yeast, we Regulatory have discovered a system that blocks late events of cytokinesis when early ones are delayed or defective, allowing time for corrective mechanisms to function. This “checkpoint” pathway, termed “Enforcement of Cytokinesis Order” (ECO), works by stabilizing a multi-motif protein that blocks a cytokinesis-specific secretion function of the Ndr/Lats protein kinase Cbk1, a key component of a highly conserved “Ndr-hippo” signaling system. Hippo signaling pathways are crucial regulators of morphogenesis, proliferation, and differentiation of eukaryotic cells; Ndr/Lats kinases are their downstream-most signaling components. Our prior research contributed crucial analysis for understanding of these systems. We were was among the first to describe specific functions of a hippo signaling system in vivo, defined the distinctive Ndr kinase phosphorylation motif, reported the first canonical substrate docking by an AGC-family protein kinase, and contributed the first and only crystal structure of an Ndr/Lats kinase bound to a Mob co-activator. This project seeks to determine how the checkpoint mechanism we discovered blocks secretion of late cytokinesis proteins when early stages of the process are defective. It will include exploration of in vivo docking interactions with cytokinesis regulators, assessment of secretion organization during cytokinesis, and evaluation of the importance of Ndr-hippo regulation of key effector proteins of cytokinesis. We will also explore the hypothesis that human Ndr-hippo pathways participate in control of late cytokinesis, using two different approaches. In one, we will determine if human Ndr kinases engage in docking interactions with in vivo phosphorylation targets, using a highly optimized phage display approach to identify possible peptide motifs that interact with Ndr kinase – Mob co-activator complexes. We will use a new ligand footprinting method we developed to map peptide motif associations. In another approach, we will determine if human Ndr-hippo signaling functions in cytokinesis by constructing cell lines expressing only analog sensitive Ndr kinases. This will allow rapid and reversible inhibition of these kinases in cells undergoing both normal and defective cytokinesis.