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

项目摘要/摘要 细胞利用一系列具有不同和互补特性的肌动蛋白结合蛋白来 组装、维护和拆解一系列不同的F-肌动蛋白网络，以促进不同的 基本功能包括动力、极化和分裂。为了履行其职能，其中每一个 网络具有由其细丝的数量、长度和连接性定义的独特体系结构， 它由肌动蛋白细丝组装、重塑和 拆卸。一个根本的挑战是了解功能网络架构是如何形成的 并由体系结构和组装动态的持续耦合来维持。我们到了 关注细胞皮质，这是一个由肌动蛋白细丝、交联剂和肌球蛋白马达组成的动态网络， 就在质膜下面，它经历了快速的变形和流动来驱动细胞运动， 极化、分裂和组织形态发生。肌动蛋白调节因子在体内的作用 Concert同时规范肌动蛋白细丝网络体系结构组装和动力学 人们对活细胞的皮质知之甚少。单细胞线虫胚胎提供了一种独特的强大的 在高分辨率可直接访问的单个大单元中解决这些问题的机会 显微镜，具有强大的遗传学、转基因、CRISPR和RNAi。线虫的皮层主要是 由F-肌动蛋白网络组成，该网络由线状细丝和小丝束组成，由 Forin Cyk-1介导的Profilin-肌动蛋白聚合及肌动蛋白纤维交联剂修饰 纤溶酶原蛋白PLST-1、苯胺素ANI-1和非肌肉肌球蛋白II的微丝nMy-2。相反，合并 UNC-60A和帽蛋白CAP-1/CAP-2似乎是细丝分解和修复的关键调节因子 分别为细丝长度。 我们正在研究线虫皮质F-肌动蛋白网络结构是如何形成的，以及 通过依赖于形成素的细丝组件、细丝的动态集成来维持 交联化、细丝封顶和依赖于胶丝蛋白的细丝分解，所有这些都是在网络 经历持续的肌球蛋白驱动的变形和流动。我们正在将互补的 最先进的体内和体外技术(定量单分子成像和颗粒分析) Ed Munro和David Kovar实验室的专业知识(重建和生物物理分析)将解决两个问题 关于线虫F-肌动蛋白皮质的结构和动力学的主要问题。首先，我们将 福尔明介导肌动蛋白的基本动力学、调控和反馈控制 细丝和束状组件(目标1)。第二，我们将调查基本的动态，监管 并反馈控制cofilin介导的肌动蛋白微丝分解形成肌动蛋白微丝网络 (目标2)。

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).