Comparative molecular physiology of mammalian formins

描述（由申请人提供）：我的实验室研究哺乳动物细胞中肌动蛋白结构的组装和动力学。哺乳动物拥有至少14个不同的肌动蛋白为基础的结构，从一个共同的池细胞质肌动蛋白单体组装。因此，必须存在严格的机制来控制每个结构的组装时间和地点。对于许多肌动蛋白为基础的结构，组装机制知之甚少。形成蛋白是肌动蛋白装配因子，15种哺乳动物肌动蛋白同工型在许多细胞环境中提供了明确的调节肌动蛋白装配的潜力。在前一个资助期内，我的实验室阐明了哺乳动物formin的许多生化特性。使用比较方法，我们发现研究的所有哺乳动物formin的FH 2结构域对肌动蛋白具有三种核心活性：1）成核加速; 2）延伸速率调节; 3）加帽蛋白抑制。此外，我们发现一些FH 2结构域成束纤维。成束酶FRL 2是细胞中丝状伪足的有效诱导物，表明成束活性的细胞作用。我们还发现，另一个酶，INF 2，具有显着的能力，加速肌动蛋白聚合和解聚。在细胞中，INF 2与内质网（ER）紧密结合，部分由C-末端法尼基化介导。INF 2解聚活性的破坏导致ER塌陷和塌陷ER周围的多产肌动蛋白丝积累。INF 2也与微管（MT）紧密结合，可能在某些细胞环境中提供MT和肌动蛋白之间的联系。在这个建议中，我将使用一个组合的生化/细胞的方法来解决组装的formin介导的细胞结构，使用INF 2和FRL 2作为模型。在目的1中，我确定了INF 2和FRL 2负责特定生化活动的区域：肌动蛋白解聚，肌动蛋白捆绑，MT结合和膜结合。在目的2，我阐明这些生化活动的调节机制。在目标3中，我使用RNAi技术，沿着目标1中鉴定的突变体，以解决INF 2和FRL 2功能背后的细胞机制。这些目标对于我在机械水平上理解这些formin的细胞功能的长期目标至关重要。我相信，当我们能够在无细胞系统中重建这一过程时，就会达到这种理解水平。该提案启动了重组过程。 公共卫生相关性：肌动蛋白丝是哺乳动物细胞的重要组成部分，在几乎所有的生理阶段发挥作用。本研究探讨哺乳动物细胞控制肌动蛋白丝组装的基本分子机制。我们的发现为治疗涉及这些机制故障的病理提供了新的和令人兴奋的机会。

DESCRIPTION (provided by applicant): My laboratory studies assembly and dynamics of actin-based structures in mammalian cells. Mammals possess at least 14 distinct actin-based structures, which assemble from a common pool of cytoplasmic actin monomers. Hence, rigorous mechanisms must exist to control the time and place of assembly for each structure. For many actin-based structures, assembly mechanisms are poorly understood. Formin proteins are actin assembly factors, and the 15 mammalian formin isoforms provide clear potential to regulate actin assembly in many cellular contexts. In the preceding grant period, my laboratory elucidated many of the biochemical properties of mammalian formins. Using a comparative approach, we found that the FH2 domains of all mammalian formins studied had three core activities on actin: 1) nucleation acceleration; 2) elongation rate regulation; and 3) capping protein inhibition. In addition, we found that some FH2 domains bundled filaments. The bundling formin, FRL2, is a potent inducer of filopodia in cells, suggesting a cellular role for bundling activity. We also find that another formin, INF2, has the remarkable ability to accelerate both actin polymerization and depolymerization. In cells, INF2 is tightly bound to the endoplasmic reticulum (ER), mediated in part by C-terminal farnesylation. Disruption of INF2's depolymerization activity causes ER collapse and prolific actin filament accumulation around the collapsed ER. INF2 also binds tightly to microtubules (MTs), possibly providing a link between MTs and actin in some cellular contexts. In this proposal, I will use a combined biochemical/cellular approach to address assembly of formin-mediated cellular structures, using INF2 and FRL2 as models. In Aim 1, I identify regions of INF2 and FRL2 responsible for specific biochemical activities: actin depolymerization, actin bundling, MT binding, and membrane binding. In Aim 2, I elucidate regulatory mechanisms for these biochemical activities. In Aim 3, I use RNAi techniques, along with mutants identified in Aim 1, to address the cellular mechanisms behind INF2 and FRL2 function. These aims are vital to my long-term goal of understanding the cellular functions of these formins at a mechanistic level. I believe that this level of understanding will be reached when we can reconstitute the process in a cell-free system. This proposal initiates the reconstitution process. PUBLIC HEALTH RELEVANCE: Actin filaments are vital components of mammalian cells, playing roles in almost all phases of physiology. This research investigates fundamental molecular mechanisms by which mammalian cells control assembly of actin filaments. Our findings provide new and exciting opportunities for therapies against pathologies involving malfunction of these mechanisms.