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.

项目摘要/摘要 丝状霉菌是质膜的基于肌动蛋白的手指样蛋白，用于多个 细胞的功能，包括运动，细胞 - 细胞粘合剂，细胞 - 肌间粘附和病毒感染。为了 其中许多过程中，所涉及的丝状疾病是高度动态的，组装和拆卸的 时间尺度为几分钟。至少需要三个基于肌动蛋白的活动才能进行丝状大会： 细丝核活动，细丝外延活动和细丝束活动。两个竞争 成核和伸长活性性质的丝源组装差异的模型。在“提示”中 成核”模型，诸如formins之类的蛋白质既充当成核和伸长因子。 “融合伸长”模型，ARP2/3复合物是成核因子，随后具有formins 充当伸长因素，也有助于重新建模分支的ARP2/3产生的肌动蛋白网络。我们的 结果表明，Formin fmnl3是潜在的丝虫装配因子，我们提出了一种新颖的 扩展到收敛伸长模型 - FMNL3可以重塑任何现有的细丝 （ARP2/3依赖性或ARP2/3独立于丝状），但只要它们是质膜即可。在 此外，我们表明FMNL3作用于细胞 - 细胞粘附。在此提案中，我们研究了FMNL3 哺乳动物细胞，重点是以下目标。 AIM 1使用基于单元的测定来定义 FMNL3介导的丝状组件的机理。我们使用活细胞显微镜，抑制剂治疗 和siRNA测试FMNL3重新建模应力纤维/局灶性粘合剂肌动蛋白的能力 丝状细丝。 AIM 2使用无细胞的系统来重建支持的丝状组件 使用纯化蛋白质（FMNL3，ARP2/3复合物，封盖蛋白，叶状蛋白，Fascin）的脂质双层。和 该系统，我们将以受控的方式测试集会原则，并调查 其他分子（VASP，cofilin）。 AIM 3研究了细胞细胞粘附中的FMNL3功能，重点是 从细胞内储存位点到质膜的FMNL3 s，在等离子体处激活 在此过程中的膜。总体而言，该项目将提供从根本上提供新颖的机理 有关丝状组件的信息，以及在肌动蛋白之间提供新颖的分子连接 细胞细胞粘附中的动力学和早期事件。