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

项目摘要/总结 细胞利用一系列具有多种和互补特性的肌动蛋白结合蛋白来 组装、维护和拆卸一系列不同的 F-肌动蛋白网络，以促进不同的功能 基本功能包括运动、极化和分裂。为了发挥其功能，每一个 网络具有独特的架构，由其细丝的数量、长度和连接性定义， 这是通过肌动蛋白丝组装、重塑和重构的持续动态平衡来维持的 拆解。一个基本的挑战是理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。我们在这里 专注于细胞皮层，这是一个由肌动蛋白丝、交联剂和肌球蛋白马达组成的动态网络， 就在质膜下方，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整体如何发挥作用 协调一致地同时调节肌动蛋白丝网络结构的组装和动态 人们对活细胞皮层知之甚少。单细胞线虫胚胎提供了独特的强大功能 有机会在一个大单元中解决这些问题，该单元可以直接获得高分辨率 显微镜，具有强大的遗传学、转基因、CRISPR 和 RNAi。线虫皮质主要是 由线性丝和小丝束组成的 F-肌动蛋白网络组成，这些丝束由 福尔明 CYK-1 介导的 Profilin-肌动蛋白聚合，并由肌动蛋白丝交联剂修饰 塑蛋白 PLST-1、苯胺 ANI-1 和非肌肉肌球蛋白 II NMY-2 的微丝。相反，肌切蛋白 UNC-60A 和加帽蛋白 CAP-1/CAP-2 似乎是细丝分解和 分别是灯丝长度。 我们正在研究线虫皮质 F-肌动蛋白网络架构是如何形成的以及 通过依赖于形式的细丝组装、细丝的动态整合来维持 交联、细丝封端和依赖肌动蛋白丝切蛋白的细丝分解，同时网络 经历持续的肌球蛋白驱动的变形和流动。我们正在结合互补 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 实验室的专业知识（重组和生物物理分析）解决了两个问题 有关线虫 F-肌动蛋白皮质的结构和动力学的主要问题。首先，我们将 表征福尔明介导的肌动蛋白的基本动力学、调节和反馈控制 灯丝和束组装（目标 1）。二是考察基本面动态、调控情况 肌动蛋白丝切蛋白介导的肌动蛋白丝网络的分解和反馈控制 （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).