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Positive and negative regulation of the cytokinesis contractility controller

胞质分裂收缩性控制器的正向和负向调节

基本信息

批准号：
9610830
负责人：
Priyanka Kothari
金额：
$ 4.45万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-02 至 2020-07-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9610830
关键词：
Actins Acute Affect Affinity Behavior Binding Biochemical Biochemical Pathway Biological Process Biology Cardiac Myosins Cell Shape Cell division Cell physiology Cells Chemicals Complex Contractile Proteins Crosslinker Cytokinesis Cytoskeletal Proteins Data Development Developmental Process Dictyostelium Dimerization Disease Dominant-Negative Mutation Embryonic Development Environment Enzymes Excision Feedback Filament Fluorescence Genetic Goals Hepatocyte Human Biology Image Immunoprecipitation In Vitro Interphase Light Lung Malignant neoplasm of pancreas Mass Spectrum Analysis Measures Mechanics Mediating Mitotic spindle Modeling Molecular Morphogenesis Myoblasts Myosin ATPase Myosin Type II Neoplasm Metastasis Oxidoreductase Post-Translational Protein Processing Process Production Proteins Regulation Resolution Role Scaffolding Protein Shapes Signal Pathway Signal Transduction Signaling Protein Site Spectrum Analysis Stress System Testing Tissues Work biophysical analysis cancer cell cell motility cortexillin I crosslink daughter cell genetic regulatory protein genetic selection human disease in vivo insight loss of function mechanical behavior mechanical force methylmalonate mutant non-muscle myosin overexpression propionyl-coenzyme A recruit response scaffold sensor single molecule

项目摘要

PROJECT SUMMARY Every biological process, ranging from cell migration to embryogenesis and tissue morphogenesis, relies on a cell’s ability to adapt to changing mechanical environments. While we understand many biochemical signaling pathways involved, the mechanisms that are integrated to govern a cell’s response to mechanical forces remain a mystery. Deciphering these interactions will shed light on the mechanical changes that drive both normal and disease state processes. To reveal how the cell responds to various forces, the Robinson lab studies Dictyostelium cytokinesis, a model shape change process by which one cell divides to form two daughter cells. The lab has discovered that cytokinesis is driven by an integrated control system composed of proteins that modulate their behavior in response to both mechanical and biochemical signals. Although we know many of the players involved in the cytokinetic control system, their biochemical interactions that allow force propagation through the cortical network are still unknown. My goal is to characterize the regulatory mechanisms that characterize these interactions, which will be critical to elucidate the mechanisms of a cell’s response to its mechanical environment. To identify the direct interactions that govern a cell’s mechanical response, we performed immunoprecipitation followed by mass spectrometry on two key nodes of the cytokinetic control system, the scaffolding protein IQGAP2 and the actin crosslinker cortexillin I. This approach led to the discovery of potential binding partners of these nodes. Using a combination of Fluorescence Cross- Correlation Spectroscopy (FCCS) and Single Molecule Pulldown (SiMPull), we have discovered a potential mechanism of inhibition by a negative regulator of the system, IQGAP1. To further understand how IQGAP1 mediates inhibition, I will purify key cytoskeletal proteins and use quantitative biochemical approaches to measure binding affinities and implement a chemically-inducible dimerization system to assess the inhibitory activity of IQGAP1. In addition, I will use super-resolution imaging during both interphase and cytokinesis to characterize alterations in complexes formed by these key cytoskeletal proteins that allow force transduction through the network. Moreover, I will determine the cellular role of methylmalonate semialdehyde dehydrogenase (mmsdh), which catalyzes the production of propionyl-coA. Mmsdh was identified as an interactor of cortexillin I, but was also previously identified in a genetic selection in our lab. It is possible that proteins may modified by propionylation, an underappreciated post-translational modification, which may facilitate positive regulation of the cytokinetic control system. Through a combination of genetics, mass spectrometry, and biophysical analyses, I will elucidate the cellular function of mmsdh. The work proposed here will decipher the molecular mechanisms of positive and negative regulation of the contractile network. This information will be critical for understanding the cell’s ability to sense and respond to mechanical forces, yielding insight into both normal developmental processes, as well as disease state progression.

项目摘要每一个生物过程，从细胞迁移到胚胎发生和组织形态发生，都依赖于一个细胞适应不断变化的机械环境的能力。虽然我们了解许多生物化学信号参与的途径，整合的机制来管理细胞对机械力的反应仍然是个谜破译这些相互作用将揭示驱动这两种机制的机械变化正常和疾病状态过程。为了揭示细胞对各种力的反应，罗宾逊实验室研究Dictyosteoblasts胞质分裂，一个细胞分裂形成两个细胞的模型形状变化过程该实验室已经发现，胞质分裂是由一个综合控制系统组成，调节它们的行为以响应机械和生物化学信号的蛋白质。虽然我们我知道许多参与细胞动力学控制系统的参与者，他们的生化相互作用，通过皮层网络的力传播仍然是未知的。我的目标是描述这些相互作用的特征机制，这将是至关重要的阐明细胞的机制，对机械环境的反应。为了确定控制细胞机械结构的直接相互作用，响应，我们进行了免疫沉淀，然后在两个关键节点上进行质谱分析。细胞动力学控制系统，支架蛋白IQGAP 2和肌动蛋白交联剂皮质素I。发现了这些节点的潜在结合伴侣。相关光谱（FCCS）和单分子下拉（SiMPull），我们发现了一个潜在的由系统的负调节剂IQGAP 1抑制的机制。为了进一步了解IQGAP 1 介导抑制，我将纯化关键的细胞骨架蛋白，并使用定量生化方法，测量结合亲和力，并实施化学诱导的二聚化系统，以评估抑制 IQGAP 1的活性。此外，我将在间期和胞质分裂期间使用超分辨率成像，表征由这些关键细胞骨架蛋白形成的复合物的改变，通过网络。此外，我将确定甲基丙二酸半醛的细胞作用在一些实施方案中，所述酶是脱氢酶（mmsdh），其催化丙酰-辅酶A的产生。 MMSDH被确定为 Cortexillin I的相互作用子，但先前也在我们实验室的遗传选择中鉴定出。有可能蛋白质可以通过丙酰化修饰，丙酰化是一种未被充分认识的翻译后修饰，促进细胞动力学控制系统的正调节。通过基因、质量光谱和生物物理分析，我将阐明MMSDH的细胞功能。工作提出的这里将破译收缩网络的正向和负向调节的分子机制。这些信息对于理解细胞感知和响应机械力的能力至关重要，产生对正常发育过程以及疾病状态进展的洞察。