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

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

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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.

项目摘要细胞因子（将母细胞分为两个女儿）是一个必不可少的生活过程。细胞因子成功对于单细胞，多细胞发育和疾病预防的健康和保真度至关重要。在此提案中，我们建立在破译细胞因子分子基础的框架上力学和机械感应。在AIM 1中，使用dictyostelium，我们研究了肌球蛋白II-皮质辛I-I-IQGAP2- 驱动蛋白-6途径（赤道机械敏感途径）。该蛋白质网络的结构像机械化学反馈系统将有丝分裂主轴和机械应力的信号集成到在裂解沟中调整肌球蛋白II水平。在光漂白后使用荧光恢复，我们将分析有或没有应用机械应力的关键蛋白的动力学以及野生类型的动力学和选定突变体。由此，我们将解密蛋白质如何相互依赖于机械敏感的积累。我们将使用下拉，然后使用LC-MS来识别相互作用的蛋白质。相互作用蛋白的列表将然后将其与我们已经确定的遗传相互作用蛋白的列表进行比较。初步数据确定参与翻译后修饰（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有关，并在心脏收缩系统功能。因此，我们有兴趣看看这些PTM是否有助于机械增强反馈系统和细胞因子细胞形状变化。我们还将使用纯化的蛋白质确定IQGAP如何调节Cortexillin和Myosin II功能。在AIM 2中，我们将扩展我们发现的Microtule-Race-Race-14-3-3-肌球蛋白II途径。该途径控制全球/极地皮质力学，皮质张力和细胞因子形状变化。我们将利用遗传和生化识别种族交互者的方法。我们还将确定14-3-3控制的机制肌球蛋白II双极厚细丝组件。然后，我们将确定人类14-3-3蛋白如何调节人肌球蛋白II厚细丝组件。初步数据指向14-3-3-肌球蛋白II的保守性质 14-3-3与另一种与癌症相关的蛋白之间共享的相互作用和可能的类似机制 S100A4/MTS1。总体而言，此续签应用中的拟议工作旨在发展复杂的了解促进和调节细胞形状变化的力传播以及控制皮质张力，肌球蛋白II动力学和细胞因子。