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: 10280989
• 负责人: David R Kovar
• 金额: $ 47.34万
• 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/10280989
• 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; Complex; Coupling; Crosslinker; Cytokinesis; Disease; Embryo; Equilibrium; F-Actin; Feedback; Filament; Generations; Genetic; Goals; Health; In Vitro; Length; Mechanics; Mediating; Microfilaments; Microscopy; Molecular; Morphogenesis; Motor; Myosin ATPase; Myosin Type II; Property; Proteins; Quantitative Microscopy; RNA Interference; Regulation; Resolution; Shapes; Structure; System; Tissues; Total Internal Reflection Fluorescent; Transgenic Organisms; Work; anillin; base; 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 细胞利用一系列具有不同和互补性质的肌动蛋白结合蛋白， 组装、维持和分解一系列不同的F-肌动蛋白网络，以促进不同的细胞功能。 基本功能包括运动、极化和分裂。为了发挥其功能，每一个 网络具有由其细丝的数量、长度和连通性定义的独特架构， 这是由肌动蛋白丝组装、重塑和 拆卸一个基本的挑战是了解功能网络架构是如何形成的 并通过架构和装配动力学的持续耦合来维持。我们到了 专注于细胞皮层，肌动蛋白丝，交联剂和肌球蛋白马达的动态网络， 就在质膜之下，经历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。肌动蛋白调节因子的集合如何在 音乐会，同时调节肌动蛋白丝网络结构组装和动力学在 对活细胞的皮层了解甚少。一个细胞C。线虫胚胎提供了一种独特的强大的 有机会在一个单一的大细胞解决这些问题，这是直接访问高分辨率 显微镜，强大的遗传学，转基因，CRISPR和RNAi。梭线虫皮层主要是 由线状纤维和小纤维束组成的F-肌动蛋白网络组成， CYK-1介导的profilin-actin聚合，并被肌动蛋白丝交联剂修饰 plastin PLST-1，anillin ANI-1，和非肌球蛋白II NMY-2的微丝。相反，cofilin CAP-60 A和帽蛋白CAP-1/CAP-2似乎是丝分解的关键调节因子， 长丝长度分别。 我们正在解决如何C。线虫皮层F-肌动蛋白网络结构形成， 维持通过动态整合的formin依赖的细丝组装，细丝 交联，细丝帽，和cofilin依赖的细丝拆卸，所有这些都是网络 经历连续肌球蛋白驱动的变形和流动。我们将互补的 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 专业知识（重建和生物物理分析）的艾德芒罗和大卫科伐实验室的解决两个 主要问题的架构和动力学的C。线虫F-肌动蛋白皮层。一是 表征形成蛋白介导的肌动蛋白的基本动力学、调节和反馈控制 灯丝和束组件（目标1）。其次，我们将研究基本的动态， 和反馈控制的cofilin介导的肌动蛋白丝解体的肌动蛋白丝网络 (Aim 2）。