Filopodia assembly by FMNL3: biochemical mechanism and cellular function

描述(申请人提供)：丝状伪足是质膜上以肌动蛋白为基础的指状突起，在细胞中具有多种功能，包括运动、细胞-细胞黏附、细胞-基质黏附和病毒感染。对于这些过程中的许多过程，所涉及的丝状伪足是高度动态的，在几分钟的时间尺度上组装和拆卸。丝轴组装至少需要三种基于肌动蛋白的活性：细丝成核活性、细丝伸长活性和丝束活性。两种相互竞争的丝轴组装模型在成核和延伸活动的性质上不同。在……里面 在“尖端成核”模型中，福尔马林等蛋白质既是成核因素，又是延伸因素。在“收敛伸长”模型中，Arp2/3复合体是成核因子，福尔马林随后作为伸长因子，也有助于重新模拟由Arp2/3生成的分支肌动蛋白网络。我们的结果表明，Forin FMNL3是一种有效的丝足组装因子，我们提出了一个新的收敛伸长模型的扩展-FMNL3可以将任何现有的细丝(Arp2/3依赖或Arp2/3不依赖)重塑为丝足，只要它们与质膜相邻。此外，我们还发现FMNL3在细胞间的黏附中起作用。在这个建议中，我们研究了哺乳动物细胞中的FMNL3，主要集中在以下目标。目的1使用基于细胞的分析来确定FMNL3介导的丝状尿路组装的机制。我们使用活细胞显微镜、抑制剂处理和siRNA来测试FMNL3将应力纤维/焦点黏附相关肌动蛋白细丝重新建模为丝足的能力。 目的2利用无细胞系统，利用纯化的蛋白质(FMNL3、Arp2/3复合体、封闭蛋白、Profilin、Fasin)在支撑的脂质双层上重建丝状集合体。有了这个系统，我们将以受控的方式测试组装原理，并研究其他分子(Vasp、Cofilin)的贡献。目的3研究FMNL3在细胞间黏附中的作用，重点是FMNL3的S从细胞内储藏部位向质膜的迁移，以及在此过程中质膜的激活。总体而言，该项目将从根本上提供有关丝状孔组装的新的机制信息，以及提供肌动蛋白动力学和细胞-细胞黏附早期事件之间的新的分子联系。

DESCRIPTION (provided by applicant): 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.