The Biochemical Basis for the Mechanics of Cytokinesis

细胞分裂机制的生化基础

基本信息

批准号：
8628296
负责人：
DOUGLAS N ROBINSON
金额：
$ 32.71万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8628296
关键词：
14-3-3 Proteins Acetylation Actins Binding Biochemical Biological Assay Breast Cancer Cell Buffers Cannibalism Cardiac Cell Proliferation Cell Shape Cell division Cells Cellular Mechanotransduction Cellular Morphology Chemicals Columnar Epithelium Complement Complex Contractile System Cytokinesis Data Daughter Development Dictyostelium Enzymes Feedback Fluorescence Recovery After Photobleaching Genetic Genetic Suppression Genome Grant Health Human Imaging Techniques Kinesin Learning Life Liquid substance Maintenance Malignant Neoplasms Maps Mechanical Stress Mechanics Methods Microtubules Mitosis Mitotic spindle Modeling Molecular Mothers Myosin Type II Nature Nonmuscle Myosin Type IIA Normal Cell Organism Output Pathway interactions Post-Translational Protein Processing Process Production Property Proteins Race Research Shapes Signal Transduction Site Structure Surface Tension System Testing Thick Filament Time Work base biophysical techniques cancer cell cancer therapy cellular development cortexillin I disorder prevention egg interest mutant non-muscle myosin oocyte maturation physical process public health relevance response success transmission process viscoelasticity

项目摘要

Project Summary Cytokinesis, the separation of a mother cell into two daughters, is an essential life process. Cytokinesis success is critical to the health and fidelity of single cells, multi-cellular development, and disease prevention. In this proposal, we build upon our framework for deciphering the molecular underpinnings of cytokinesis mechanics and mechanosensing. Using Dictyostelium, in Aim 1, we study the Myosin II-Cortexillin I-IQGAP2- Kinesin-6 pathway (the equatorial mechanosensitive pathway). This network of proteins is structured like a mechanochemical feedback system that integrates signals from the mitotic spindle and mechanical stress to tune the myosin II levels at the cleavage furrow. Using fluorescence recovery after photobleaching, we will analyze the dynamics of key proteins with and without applied mechanical stress and in wild type and selected mutants. From this, we will decipher how proteins depend on each other for mechanosensitive accumulation. We will use pull-downs followed by LC-MS to identify interacting proteins. The list of interacting proteins will then be compared to the lists of genetic interacting proteins we have already identified. Preliminary data identify important enzymes involved in post-translational modifications (PTMs), such as propionylation and acetylation. Acetylation of myosin II and other proteins has been implicated in mammalian mitosis and in cardiac contractile system function. Thus, we are interested to see if these PTMs contribute to the mechanosensory feedback system and cytokinesis cell shape change. We will also use purified proteins to determine how IQGAPs modulate cortexillin and possibly myosin II function. In Aim 2, we will expand the Microtubule-RacE-14-3-3-Myosin II pathway, which we discovered. This pathway controls the global/polar cortex mechanics, cortical tension and cytokinesis shape change. We will draw upon genetic and biochemical methods to identify interactors of racE. We will also determine the mechanism by which 14-3-3 controls myosin II bipolar thick filament assembly. We will then determine how human 14-3-3 proteins modulate human myosin II thick filament assembly. Preliminary data points toward the conserved nature of 14-3-3-myosin II interactions and a possible similar mechanism shared between 14-3-3 and another cancer-related protein S100A4/Mts1. Overall, proposed work in this renewal application strives to develop a sophisticated understanding of the force transmission that promotes and regulates cell shape change and the pathways that control cortical tension, myosin II dynamics, and cytokinesis.

项目概要细胞分裂，即母细胞分裂成两个子细胞，是一个重要的生命过程。胞质分裂成功对于单细胞、多细胞发育和疾病预防的健康和保真度至关重要。在这个提案中，我们建立在破译胞质分裂分子基础的框架之上力学和机械传感。在目标 1 中，我们使用盘基网柄菌研究肌球蛋白 II-Cortexillin I-IQGAP2- Kinesin-6 通路（赤道机械敏感通路）。这个蛋白质网络的结构就像一个机械化学反馈系统，整合有丝分裂纺锤体和机械应力的信号调整卵裂沟处的肌球蛋白 II 水平。利用光漂白后的荧光恢复，我们将分析有和没有施加机械应力以及野生型和选定的关键蛋白质的动态突变体。由此，我们将破译蛋白质如何相互依赖来进行机械敏感积累。我们将使用 Pull-downs，然后使用 LC-MS 来识别相互作用的蛋白质。相互作用蛋白质的列表将然后与我们已经确定的遗传相互作用蛋白列表进行比较。初步数据识别参与翻译后修饰 (PTM) 的重要酶，例如丙酰化和乙酰化。肌球蛋白 II 和其他蛋白质的乙酰化与哺乳动物有丝分裂和心脏收缩系统功能。因此，我们有兴趣了解这些 PTM 是否有助于机械感觉反馈系统和胞质分裂细胞形状的变化。我们还将使用纯化的蛋白质确定 IQGAP 如何调节皮质西林和可能的肌球蛋白 II 功能。在目标 2 中，我们将扩展我们发现的微管-RacE-14-3-3-肌球蛋白 II 通路。该通路控制全球/极地皮质力学、皮质张力和胞质分裂形状的变化。我们将利用遗传和生化识别racE相互作用者的方法。我们还将确定 14-3-3 控制的机制肌球蛋白 II 双极粗丝组件。然后我们将确定人类 14-3-3 蛋白如何调节人类肌球蛋白 II 粗丝组件。初步数据表明 14-3-3-肌球蛋白 II 的保守性 14-3-3 和另一种癌症相关蛋白之间的相互作用以及可能存在的相似机制 S100A4/Mts1。总体而言，该更新应用程序中拟议的工作致力于开发一个复杂的了解促进和调节细胞形状变化的力传递及其途径控制皮质张力、肌球蛋白 II 动力学和胞质分裂。