The Biochemical Basis for the Mechanics of Cytokinesis

细胞分裂机制的生化基础

• 批准号: 8972015
• 负责人: DOUGLAS N ROBINSON
• 金额: $ 31.12万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 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/8972015
• 关键词: 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; response; success; transmission process; viscoelasticity

DESCRIPTION (provided by applicant): Cytokinesis, the separation of a mother cell into two daughters, is an essential life process. Cytokinesis success is critical to the health and fidelityof 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 wil 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.

描述（由申请人提供）：将母细胞分为两个女儿的细胞因子是一个重要的生活过程。细胞因子的成功对于单细胞的健康和保真度，多细胞发育和预防疾病至关重要。在此提案中，我们建立在破译细胞因子力学和机械感应的分子基础的框架基础上。在AIM 1中，我们使用dictyostelium，研究肌球蛋白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是像机械化学反馈系统的结构，该机械反馈系统整合了有丝分裂主轴和机械应力的信号，以调整裂解沟时肌球蛋白II水平。使用光漂白后的荧光恢复，我们将分析有或没有应用机械应力以及野生型和选定突变体的关键蛋白的动力学。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。然后将相互作用的蛋白质列表与我们已经鉴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和心脏收缩系统功能有关。因此，我们有兴趣查看这些PTM是否有助于机械感觉反馈系统和细胞因子细胞形状的变化。我们还将使用纯化的蛋白质来确定IQGAP如何调节皮质辛基和可能的肌球蛋白II功能。在AIM 2中，我们将扩展微管-race-14-3-3-肌球蛋白II途径，我们发现。该途径控制全局/极性皮层力学，皮质张力和细胞因子形状变化。我们将利用遗传和生化方法来识别种族的相互作用者。我们还将确定14-3-3控制肌球蛋白II双相厚细丝组件的机制。然后，我们将确定人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相互作用的保守性质，以及在14-3-3和另一种与癌症相关的蛋白S100A4/MTS1之间共享的类似机制。总体而言，在此更新应用中提出的工作旨在发展对促进和调节细胞形状变化和控制皮质张力，肌球蛋白II II动力学和细胞因子的途径的力量传播的复杂理解。