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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化信号所涉及的途径，整合来控制细胞对机械力的反应的机制仍然是一个谜。破译这些相互作用将揭示驱动两者的机械变化正常和疾病状态过程。为了揭示细胞如何对各种力做出反应，罗宾逊实验室研究盘基网柄菌胞质分裂，这是一种模型形状变化过程，通过该过程一个细胞分裂形成两个细胞子细胞。实验室发现细胞分裂是由一个集成控制系统驱动的，该系统由以下部分组成：响应机械和生化信号来调节其行为的蛋白质。虽然我们了解参与细胞因子控制系统的许多参与者，他们的生化相互作用使得通过皮质网络的力传播仍然未知。我的目标是描述监管的特征表征这些相互作用的机制，这对于阐明细胞的机制至关重要对其机械环境的响应。识别控制细胞机械的直接相互作用响应，我们对两个关键节点进行了免疫沉淀，然后进行了质谱分析细胞因子控制系统、支架蛋白 IQGAP2 和肌动蛋白交联剂皮质西林 I。这种方法导致这些节点的潜在结合伙伴的发现。使用荧光交叉的组合相关光谱 (FCCS) 和单分子下拉 (SiMPull)，我们发现了一种潜在的系统负调节因子 IQGAP1 的抑制机制。进一步了解 IQGAP1 如何介导抑制，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二聚化系统来评估抑制 IQGAP1 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来表征这些关键细胞骨架蛋白形成的复合物的变化，这些蛋白允许力转导通过网络。此外，我将确定丙二酸甲酯半醛的细胞作用脱氢酶 (mmsdh)，催化丙酰辅酶 A 的产生。 Mmsdh 被确定为皮质西林 I 的相互作用者，但之前也在我们实验室的遗传选择中被鉴定出来。有可能蛋白质可以通过丙酰化进行修饰，丙酰化是一种未被充分重视的翻译后修饰，这可能促进细胞因子控制系统的正向调节。通过遗传学、质量的结合通过光谱测定和生物物理分析，我将阐明 mmsdh 的细胞功能。提议的工作这里将破译收缩网络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，深入了解正常发育过程以及疾病状态进展。