The Role of Mammalian Diaphanous Formin 1 in Leading Edge Cell Protrusion

哺乳动物透明 Formin 1 在前缘细胞突出中的作用

• 批准号: 10679354
• 负责人: Andrea Ramirez
• 金额: $ 4.77万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679354
• 关键词: Actins; Adhesions; Binding; Biosensor; Cell physiology; Cells; Cellular Structures; Cellular biology; Cytoskeleton; Data; Detection; Development; Disease; Event; F-Actin; Family; Fluorescence Resonance Energy Transfer; Focal Adhesions; Goals; Guanine Nucleotide Exchange Factors; Immune; Invaded; Knowledge; Malignant Neoplasms; Mechanics; Mediating; Membrane; Microfilaments; Molecular Conformation; Molecular Mechanisms of Action; Monomeric GTP-Binding Proteins; Pathway interactions; Play; Polymers; Protein Isoforms; Protein Tyrosine Kinase; Proteins; Regulation; Reporting; Role; SH3 Domains; Signal Pathway; Signal Transduction; Site; Structure; Testing; Time; Vav guanine-nucleotide exchange factor; Visualization; cell motility; citrate carrier; directional cell; genetic manipulation; improved; member; migration; mutant; novel therapeutic intervention; optogenetics; paralogous gene; polymerization; protein expression; recruit; red fluorescent protein; rho; rho GTP-Binding Proteins; spatiotemporal; tool; wound healing

ABSTRACT The actin cytoskeleton is regulated by members of the p21 Rho family of small GTPases by propagating signals to downstream effectors including the mammalian diaphanous-related formin 1, mDia1. The formin mDia1 directly modulates the actin cytoskeleton by nucleating actin and assembling linear actin filaments. In addition to the enzymatic activity, mDia1 can recruit proteins containing SH3 domains such as non-receptor tyrosine kinase, Src. There is a limited understanding of the mDia1-specific, RhoGTPase-mediated functions that contribute to cellular protrusive structures, specifically at the leading edge. To protrude, cells require polymerization of actin filaments to mechanically extend the leading edge. The newly extended protrusion can become stabilized by formation of adhesion sites or retracted by disassembly of adhesions. In this proposal we seek to delineate RhoGTPase paralogs that regulate mDia1 and its subsequent downstream functions in leading edge cell protrusions. We hypothesize that the RhoGTPases RhoC and/or RhoA are activating mDia1 at the leading edge to recruit Src to sites of adhesion and to polymerize actin filaments, respectively. To gain understanding into the RhoGTPase regulation of mDia1, in Aim 1 we will develop Förster Resonance Energy Transfer (FRET) based biosensor tools capable of reporting the activity status of mDia1 in living cells. We will focus on the development of Near-Infra red (NIR) fluorescent protein biosensor of mDia1 to allow simultaneous detection of cyan-yellow FRET RhoGTPase biosensors or to use the blue-green optogenetic tools targeting RhoGTPases and their upstream regulators, in single living cells. We will use this approach to determine the RhoC and RhoA regulation of mDia1 at the leading edge. For Aim 2, we will uncover the downstream function of mDia1 by recruitment of Src to sites of adhesion and how this impacts adhesion turnover during protrusive events. Additionally, we will elucidate the spatiotemporal dynamics of mDia1 in mediating linear actin assembly at the leading edge protrusions. We will discern the mDia1-specific, localized activities and functions using our newly developed mDia1 biosensor in WT and RhoGTPase perturbed cells. Our approach will allow us to delineate the spatiotemporal dynamics of mDia1 and the respective RhoGTPase paralogs regulating them in cells during leading edge protrusions.

抽象的 肌动蛋白细胞骨架受P21 Rho小型GTPases家族的调节 向下游效应的信号，包括哺乳动物diaphanous相关的formin 1，MDIA1。形式 MDIA1通过成核肌动蛋白并组装线性肌动蛋白丝直接调节肌动蛋白细胞骨架。在 除酶活性外，MDIA1可以募集含有SH3结构域的蛋白质，例如非受体 酪氨酸激酶，SRC。对MDIA1特异性的RhoGTPase介导的功能有限的理解有限 这有助于细胞突出结构，特别是在前缘。伸直，细胞需要 肌动蛋白丝的聚合可以机械扩展前缘。新扩展的突出可以 通过形成粘合剂位点的形成或通过粘合剂拆卸而稳定。在这个建议中，我们 寻求描述调节MDIA1及其随后的下游功能的Rhogtpase car） 前缘细胞的突起。我们假设Rhogtpases Rhoc和/或RhoA正在激活MDIA1 在招募SRC到粘合剂位置并分别将肌动蛋白丝的位置招募。获得 了解MDIA1的RHOGTPase调节，在AIM 1中，我们将发展Förster共振能量 基于转移（FRET）的生物传感器工具，能够报告活细胞中MDIA1的活性状态。我们将 专注于MDIA1的近距离红色（NIR）荧光蛋白生物传感器的发展以允许简单 检测青色黄色的fret rhogtpase生物传感器或使用蓝绿色光遗传学工具靶向 Rhogtpases及其上游调节剂，在单个活细胞中。我们将使用这种方法来确定 RHOC和RHOA在前沿的MDIA1调节。对于AIM 2，我们将发现下游功能 通过将SRC募集到粘合剂位点，以及这如何影响粘合剂的粘合剂转移，使MDIA1的摄入量 事件。此外，我们将阐明MDIA1的空间时间动力学介导线性肌动蛋白组装 在前缘突起。我们将使用我们 在WT和Rhogtpase扰动细胞中新开发的MDIA1生物传感器。我们的方法将使我们能够 描述MDIA1的时空动力学和相对的Rhogtpase旁系同源物调节它们 前缘突起期间的细胞。