Filopodia assembly by FMNL3: biochemical mechanism and cellular function

FMNL3 的丝状伪足组装：生化机制和细胞功能

• 批准号: 8669584
• 负责人: HENRY N HIGGS
• 金额: $ 31.19万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8669584
• 关键词: Actins; Adhesions; Area; Biochemical; Biological Assay; Bundling; Cell Adhesion; Cell Line; Cell membrane; Cell physiology; Cell-Cell Adhesion; Cell-Free System; Cells; Cellular biology; Color; Complex; Data; Elongation Factor; Event; Filament; Filopodia; Fingers; Fluorescence Microscopy; Focal Adhesions; Genes; In Vitro; Knock-out; Life; Lipid Bilayers; Mammalian Cell; Mediating; Membrane; Microfilaments; Microscopy; Modeling; Modification; Molecular; Myosin ATPase; Nature; Neoplasm Metastasis; Nutrient; Play; Population; Process; Proteins; Reagent; Role; Site; Small Interfering RNA; Source; Stress Fibers; Structure; System; Testing; Time; Transport Vesicles; Virus; Virus Diseases; Work; base; cell growth regulation; cell motility; cofilin; directional cell; ena protein; fascin; inhibitor/antagonist; novel; profilin; public health relevance; reconstitution; tool; transmission process; vasodilator-stimulated phosphoprotein

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.

项目概要/摘要 丝状伪足是质膜上基于肌动蛋白的指状突起，用于多种 细胞中的功能包括运动、细胞-细胞粘附、细胞-基质粘附和病毒感染。为了 在许多这些过程中，所涉及的丝状伪足是高度动态的，在 分钟的时间尺度。丝状伪足组装至少需要三种基于肌动蛋白的活动： 细丝成核活性、细丝伸长活性和细丝成束活性。两人竞争 丝状伪足组装模型的不同之处在于成核和伸长活动的性质。在“提示 在“成核”模型中，福尔明等蛋白质既充当成核因子又充当延伸因子。 “收敛伸长”模型，Arp2/3复合物是成核因子，随后是formins 作为延伸因子，也有助于重新建模分支的 Arp2/3 生成的肌动蛋白网络。我们的 结果表明，formin FMNL3 是一种有效的丝状伪足组装因子，我们提出了一种新的 扩展至收敛伸长模型 - FMNL3 可以重塑任何现有的细丝 （Arp2/3 依赖或 Arp2/3 独立）到丝状伪足，前提是它们紧邻质膜。在 此外，我们还发现 FMNL3 在细胞间粘附中发挥作用。在本提案中，我们研究了 FMNL3 哺乳动物细胞，重点关注以下目标。目标 1 使用基于细胞的测定来定义 FMNL3介导的丝状伪足组装机制。我们使用活细胞显微镜、抑制剂治疗 和 siRNA 测试 FMNL3 重塑应力纤维/粘着斑相关肌动蛋白的能力 丝状成丝状伪足。目标 2 使用无细胞系统在支持的细胞上重建丝状伪足组件 使用纯化蛋白（FMNL3、Arp2/3 复合物、加帽蛋白、profilin、肌成束蛋白）构建脂质双层。和 在这个系统中，我们将以受控的方式测试装配原则并调查 其他分子（VASP、cofilin）。目标 3 研究 FMNL3 在细胞间粘附中的功能，重点关注 FMNL3 从细胞内储存位点转移到质膜，并在质膜处激活 在此过程中形成膜。总体而言，该项目将提供全新的机制 有关丝状伪足组装的信息，以及提供肌动蛋白之间的新颖分子连接 细胞-细胞粘附的动力学和早期事件。