Mechanisms of Formin-Mediated Actin Filament Assembly - Renewal 01 - Resubmission

项目总结 细胞同时组装不同功能的肌动蛋白细胞骨架细丝，具有不同的 驱动极化、内吞、运动等基本过程的体系结构和动力学 和组织。多个基于肌动蛋白细丝的结构必须在单个拥挤的 维持细胞质的组织和功能特异性。多种肌动蛋白结合蛋白 不同的性质调节肌动蛋白的组装和组织。细胞仍准备组装肌动蛋白 在正确的时间和地点对信号作出反应的细丝，因为它们维持着大量的肌动蛋白 结合到小肌动蛋白结合蛋白Profilin的单体。Profilin可防止不必要的自发反应 肌动蛋白通过抑制核化而组装，因此需要一些因子来快速刺激肌动蛋白在 正确的时间和地点来响应信号。大多数新的肌动蛋白细丝是由一个不断扩大的清单产生的 成核和延长因素建立不同的肌动蛋白细丝结构所需的 不同的过程。福尔明利用一种概念上新颖的机制来刺激快速组装 伸长的长而直的细丝，通常由肌球蛋白马达拉动以产生收缩力量， 或提供极化轨道和/或支架，以建立/维持细胞极性和丝状基座 分机。Ena/Vasp还促进了长直细丝的伸长 迁移细胞的前沿。相反，Arp2/3复合体启动短分支的组装 提供推动力的细丝。一个尚未解决的主要问题是确定多样性 成核因子单独或联合作用将Profilin-肌动蛋白组装成功能不同的肌动蛋白 在拥挤的共同细胞质内的花丝。 我们的假设是，肌动蛋白单体是限制性的，因此通过 成核因子在维持不同肌动蛋白细丝之间的平衡方面起着至关重要的作用 在拥挤的细胞质内的结构。Profilin不仅促进Form in和EnA/Vasp介导的肌动蛋白 细丝伸长，但也允许福尔马林成功地与过量的Arp2/3复合体竞争 肌动蛋白单体。我们的研究目标是确定Profilin的基本细胞角色(S)，并遵循 在我们之前的拨款中取得的进展，以研究福尔马林 利用Profilin-肌动蛋白快速延长肌动蛋白细丝(目标I)。我们还在向高影响力领域扩张 研究福尔马林如何与Arp2/3复合体竞争Profilin-肌动蛋白推动不同 过程(AIM II)，以及Formin如何与Ena/Vasp竞争和/或合作组装肌动蛋白 丝状基座的花丝(AIM III)。

PROJECT SUMMARY Cells simultaneously assemble functionally diverse actin cytoskeleton filaments with distinct architectures and dynamics to drive fundamental processes such as polarization, endocytosis, motility and division. Multiple actin filament-based structures must self-organize within a single crowded cytoplasm to maintain organization and functional specificity. Numerous actin-binding proteins with different properties regulate actin assembly and organization. Cells remain poised to assemble actin filaments at the right time and place in response to signals because they maintain a large pool of actin monomers bound to the small actin-binding protein profilin. Profilin prevents unwanted spontaneous actin assembly by inhibiting nucleation, so factors are required to rapidly stimulate actin assembly at the right time and place in response to signals. Most new actin filaments are produced by an expanding list of nucleation and elongation factors that establish diverse actin filament architectures required for different processes. Formin utilizes a conceptually novel mechanism to stimulate the assembly of rapidly elongating long-straight filaments that are often pulled by myosin motors to generate contractile forces, or to provide polarized tracks and/or scaffolds that establish/maintain cell polarity and filopodia extension. Ena/VASP also promotes the elongation of long-straight filaments for extending filopodia at the leading edge of migrating cells. Conversely, Arp2/3 complex initiates the assembly of short-branched filaments that provide pushing forces. A major unresolved question is to determine how diverse nucleation factors work alone and in combination to assemble profilin-actin into functionally diverse actin filaments within a crowded common cytoplasm. Our hypothesis is that actin monomers are limiting, and therefore competition for profilin-actin by nucleation factors plays a critical role in maintaining a balance between different actin filament-based structures within a crowded cytoplasm. Profilin not only facilitates formin- and Ena/VASP-mediated actin filament elongation, but also allows formin to successfully compete with an excess of Arp2/3 complex for actin monomers. Our research goals are to determine the essential cellular role(s) of profilin, and follow up on progress made in our previous grant to investigate the precise mechanisms by which formins utilize profilin-actin for rapid actin filament elongation (Aim I). We are also expanding into the high impact area of investigating how formin competes with Arp2/3 complex for profilin-actin to drive different processes (Aim II), and how formin competes and/or cooperates with Ena/VASP to assemble actin filaments for filopodia (Aim III).