Filopodia assembly by FMNL3: biochemical mechanism and cellular function

项目摘要/摘要 丝状伪足是质膜上基于肌动蛋白的指状突起，用于 在细胞中的功能包括运动、细胞-细胞黏附、细胞-基质黏附和病毒感染。为 这些过程中的许多，所涉及的丝状伪足都是高度动态的，在 以分钟为单位的时间尺度。丝状尿管组装至少需要三种基于肌动蛋白的活动： 灯丝成核活性、灯丝伸长活性和灯丝束活性。两个竞争对手 丝状径向组装的模型在成核和延伸活动的性质上有所不同。在“提示”中 成核“模型中，蛋白质如福尔马林既是成核因子又是伸长因子。 “收敛伸长”模型，Arp2/3复合体是成核因素，随后是福尔马林 作为延伸因子，也有助于对Arp2/3生成的分支肌动蛋白网络进行重新建模。我们的 结果表明，Forin FMNL3是一种有效的丝状装配因子，我们提出了一种新的 对收敛伸长模型的扩展-FMNL3可以重塑任何现有的细丝 (Arp2/3依赖或Arp2/3不依赖)丝状孢子，前提是它们与质膜相邻。在……里面 此外，我们还发现FMNL3在细胞间的黏附中起作用。在本提案中，我们研究了FMNL3 哺乳动物细胞，专注于以下目的。AIM 1使用基于细胞的分析来定义 FMNL3介导的丝状尿路组装机制。我们使用活细胞显微镜，抑制剂治疗 和siRNA，以测试FMNL3重塑应力纤维/焦点黏附相关肌动蛋白的能力 花丝变成花丝基座。AIM 2使用无细胞系统在支撑体上重建丝状基座组件 使用纯化的蛋白质(FMNL3、Arp2/3复合体、封闭蛋白、Profilin、Fasin)进行脂双层。使用 这个系统，我们将以受控的方式测试组装原理，并调查 其他分子(Vasp、Cofilin)。目的3研究FMNL3在细胞-细胞黏附中的作用，重点是 S将FMNL3从细胞内的储存部位转移到质膜上，并在血浆中激活 膜在这一过程中。总体而言，该项目将提供从根本上新颖的机制 有关丝状轴组装的信息，以及提供肌动蛋白之间的新的分子连接 细胞-细胞黏附的动力学和早期事件。

Project Summary/Abstract Filopodia are actin-based finger-like protrusions from the plasma membrane, and are used for multiple functions in cells including motility, cell-cell adhesion, cell-substratum adhesion, and viral infection. For many of these processes, the filopodia involved are highly dynamic, assembling and disassembling on a time scale of minutes. At least three actin-based activities are required for filopodial assembly: filament nucleation activity, filament elongation activity, and filament bundling activity. Two competing models for filopodial assembly differ in the nature of the nucleation and elongation activities. In the "tip nucleation" model, proteins such as formins act as both nucleation and elongation factors. In the "convergent elongation" model, Arp2/3 complex is the nucleation factor, with formins subsequently acting as elongation factors that also help re-model the branched Arp2/3-generated actin network. Our results show that the formin FMNL3 is a potent filopodial assembly factor, and we propose a novel extension to the convergent elongation model - that FMNL3 can remodel any existing filaments (Arp2/3-dependent or Arp2/3-independent) to filopodia, provided they abut the plasma membrane. In addition, we show that FMNL3 acts in cell-cell adhesion. In this proposal, we study FMNL3 in mammalian cells, focusing on the following aims. Aim 1 uses cell-based assays to define the mechanism of FMNL3-mediated filopodial assembly. We use live-cell microscopy, inhibitor treatments and siRNA to test the ability of FMNL3 to re-model stress fiber/focal adhesion-associated actin filaments into filopodia. Aim 2 uses a cell-free system to reconstitute filopodial assembly on supported lipid bilayers using purified proteins (FMNL3, Arp2/3 complex, capping protein, profilin, fascin). With this system, we will test assembly principles in a controlled manner and investigate the contributions of other molecules (VASP, cofilin). Aim 3 investigates FMNL3 function in cell-cell adhesion, focusing on FMNL3¿s transit from intracellular storage sites to the plasma membrane, and activation at the plasma membrane during this process. Overall, this project will provide fundamentally novel mechanistic information on filopodial assembly, as well as providing novel molecular connections between actin dynamics and early events in cell-cell adhesion.