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: 9181427
• 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 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9181427
• 关键词: ANGPTL2 gene; Actins; Adhesions; Alpha Cell; Area; Biochemical; Biological Assay; Bundling; Cell Adhesion; Cell Line; Cell membrane; Cell physiology; Cell-Cell Adhesion; Cell-Free System; Cells; Cellular biology; Color; Complex; Crystallization; Data; Elongation Factor; Event; Filament; Filopodia; Fingers; Fluorescence Microscopy; Focal Adhesions; Genes; In Vitro; Knock-out; Lipid Bilayers; Mammalian Cell; Mediating; Membrane; Microfilaments; Microscopy; Modeling; Modification; Molecular; Myosin ATPase; Nature; Neoplasm Metastasis; Nutrient; Pathologic; Play; Population; Process; Proteins; Reagent; Role; Site; Small Interfering RNA; Source; Stress Fibers; Structure; System; Testing; Time; Transport Vesicles; Vesicle Transport Pathway; Virus; Virus Diseases; Work; base; cell growth regulation; cell motility; cofilin; directional cell; fascin; inhibitor/antagonist; live cell microscopy; novel; profilin; public health relevance; reconstitution; tool; transmission process; vasodilator-stimulated phosphoprotein

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.

描述（由申请人提供）：丝状虫是质膜的基于肌动蛋白的手指样突起，用于细胞中的多个功能，包括运动，细胞 - 细胞粘附，细胞 - 肌间肌粘附和病毒感染。对于许多此类过程，所涉及的丝状虫具有高度动态，组装和拆卸时间为几分钟的时间。至少需要三个基于肌动蛋白的活性才能进行丝状组装：细丝成核活性，细丝伸长活性和细丝捆绑活性。丝状组件的两个竞争模型在成核和伸长活性的性质上有所不同。在 “尖端成核”模型，诸如formins之类的蛋白质既充当成核和伸长因子。在“收敛伸长”模型中，ARP2/3复合物是成核因子，formins随后充当伸长因子，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是一个有效的丝端装配因子，我们建议对收敛伸长模型进行新的扩展 - FMNL3可以将任何现有的丝（ARP2/3依赖性或ARP2/3-独立依赖性）重塑为丝状丝虫，前提是它们含量含量。此外，我们表明FMNL3在细胞 - 细胞粘附中起作用。在此提案中，我们研究了哺乳动物细胞中的FMNL3，重点是以下目的。 AIM 1使用基于细胞的测定来定义FMNL3介导的丝状组件的机制。我们使用活细胞显微镜，抑制剂治疗和siRNA来测试FMNL3重新模型应力纤维/局灶性粘附相关的肌动蛋白丝的能力。 AIM 2使用无细胞系统使用纯化的蛋白（FMNL3，ARP2/3复合物，上限蛋白，Profilin，fascin）在受支持的脂质双层对支持的脂质双层的丝状组件中重构。使用该系统，我们将以受控的方式测试装配原理，并研究其他分子（VASP，Cofilin）的贡献。 AIM 3研究了细胞细胞粘附中的FMNL3功能，重点是FMNL3从细胞内存储位点到质膜的转运，并在此过程中激活在质膜上激活。总体而言，该项目将提供有关丝状组件的根本新机械信息，并在细胞细胞粘附中提供肌动蛋白动力学与早期事件之间的新分子连接。