Dynamic control of actin network architecture in early C. elegans embryos

早期秀丽隐杆线虫胚胎中肌动蛋白网络结构的动态控制

• 批准号: 10630246
• 负责人: David R Kovar
• 金额: $ 47.35万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-04 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630246
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Address; Architecture; Biological Assay; Caenorhabditis elegans; Cell Shape; Cell division; Cell membrane; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Complex; Coupling; Crosslinker; Cytokinesis; Disease; Embryo; Equilibrium; F-Actin; Feedback; Filament; Generations; Genes; Genetic; Goals; Health; In Vitro; Length; Mechanics; Mediating; Microfilaments; Microscopy; Molecular; Morphogenesis; Motor; Myosin ATPase; Myosin Type II; Polymers; Property; Proteins; Quantitative Microscopy; RNA Interference; Regulation; Resolution; Shapes; System; Tissues; Total Internal Reflection Fluorescent; Work; anillin; biophysical analysis; cell cortex; cell motility; cofactor; cofilin; crosslink; experience; in vivo; molecular imaging; monomer; network architecture; non-muscle myosin; particle; plastin; polarized cell; polymerization; profilin; reconstitution; single molecule; tool

Project Abstract/Summary Cells utilize an array of actin binding proteins with diverse and complementary properties to assemble, maintain and disassemble a range of distinct F-actin networks to facilitate different fundamental functions including motility, polarization and division. To serve its function, each of these networks has a unique architecture defined by the number, lengths and connectivity of its filaments, which is maintained by a continuous dynamical balance of actin filament assembly, remodeling and disassembly. A fundamental challenge is to understand how functional network architectures are formed and maintained by the continuous coupling of architecture and assembly dynamics. Here we are focusing on the cell cortex, a dynamic network of actin filaments, crosslinkers and myosin motors, lying just beneath the plasma membrane, that undergoes rapid deformation and flow to drive cell movement, polarization, division and tissue morphogenesis. How ensembles of actin regulatory factors work in concert to simultaneously regulate actin filament network architecture assembly and dynamics at the cortex of living cells is poorly understood. The one cell C. elegans embryo provides a uniquely powerful opportunity to address these questions in a single large cell, that is directly accessible to high resolution microscopy, with powerful genetics, transgenics, CRISPR and RNAi. The C. elegans cortex is primarily composed of an F-actin network of linear filaments and small filament bundles that are assembled by formin CYK-1-mediated polymerization of profilin-actin, and decorated by actin filament crosslinkers plastin PLST-1, anillin ANI-1, and by mini-filaments of non-muscle myosin II NMY-2. Conversely, cofilin UNC-60A and capping protein CAP-1/CAP-2 appear to be key regulators of filament disassembly and filament length, respectively. We are addressing how the C. elegans cortical F-actin network architecture is formed and maintained through the dynamic integration of formin-dependent filament assembly, filament crosslinking, filament capping, and cofilin-dependent filament disassembly, all while the network experiences continuous myosin-driven deformation and flow. We are combining the complementary state-of-the-art in vivo expertise (quantitative single molecule imaging and particle analysis) and in vitro expertise (reconstitution and biophysical analysis) of the Ed Munro and David Kovar lab's to address two major questions concerning the architecture and dynamics of the C. elegans F-actin cortex. First, we will characterize the fundamental dynamics, regulation and feedback control of formin-mediated actin filament and bundle assembly (Aim 1). Second, we will investigate the fundamental dynamics, regulation and feedback control of cofilin-mediated actin filament disassembly of the formin actin filament networks (Aim 2).

项目摘要/摘要 细胞利用具有多种和互补特性的一系列肌动蛋白结合蛋白 组装，维护和拆卸一系列不同的F-肌动蛋白网络，以促进不同 基本功能，包括运动，极化和分裂。为了发挥其功能，每一个 网络具有独特的体系结构，其数量，长度和连接性的定义， 通过肌动蛋白丝组件，重塑和 拆卸。一个根本的挑战是了解如何形成功能网络架构 并通过建筑和组装动力学的连续耦合来维护。我们在这里 专注于细胞皮层，肌动蛋白丝，交联和肌球蛋白电动机的动态网络， 就在质膜下方，该质膜经历了快速变形和流动以驱动细胞运动， 极化，分裂和组织形态发生。肌动蛋白调节因素的合奏如何在 共同调节肌动蛋白丝网络架构组件和动态的音乐会 活细胞的皮质知之甚少。一个细胞C.秀丽隐杆线虫胚胎提供了独特的功能 在单个大单元中解决这些问题的机会，高分辨率可以直接访问 显微镜，具有强大的遗传学，转基因，CRISPR和RNAi。秀丽隐杆线虫皮层主要是 由线性细丝和小细丝束组成的F-肌动蛋白网络组成 formin cyk-1介导的profilin-actin的聚合，并由肌动蛋白丝交联链装饰 Plastin PLST-1，Anillin ani-1，以及非肌肉肌球蛋白II NMY-2的迷你丝。相反，Cofilin UNC-60A和上限蛋白CAP-1/CAP-2似乎是细丝拆卸和 细丝长度分别。 我们正在解决秀丽隐杆线虫皮质F-肌动蛋白网络架构如何形成，并 通过依赖形灯丝组件的动态整合，丝丝保持 网络时 经历连续肌球蛋白驱动的变形和流动。我们正在结合补充 最先进的体内专业知识（定量单分子成像和颗粒分析）和体外 Ed Munro和David Kovar Lab的专业知识（重建和生物物理分析）旨在解决两个 有关秀丽隐杆线虫F-肌动蛋白皮质的建筑和动态的主要问题。首先，我们会的 表征对甲型介导的肌动蛋白的基本动力，调节和反馈控制 细丝和束组件（AIM 1）。其次，我们将调查基本动态，调节 以及对formin肌动蛋白细丝网络拆卸的cofilin介导的肌动蛋白丝网络的反馈控制 （目标2）。