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基结构的组装和动力学。哺乳动物拥有至少14种不同的基于肌动蛋白的结构，这些结构由细胞质肌动蛋白单体组成。因此，必须有严格的机制来控制每个结构的装配时间和地点。对于许多基于肌动蛋白的结构，其组装机制尚不清楚。Formin蛋白是肌动蛋白组装因子，哺乳动物的15种Formin异构体提供了在许多细胞环境中调节肌动蛋白组装的明确潜力。在之前的资助期间，我的实验室阐明了哺乳动物formins的许多生化特性。通过比较，我们发现所有哺乳动物的FH2结构域对肌动蛋白有三个核心作用：1)成核加速；2)伸长率调节；3)封顶蛋白抑制。此外，我们发现一些FH2结构域捆绑在细丝上。捆绑双胍FRL2是细胞中丝状足的有效诱导剂，提示捆绑活性在细胞中的作用。我们还发现另一种双胍，INF2，具有显著的加速肌动蛋白聚合和解聚的能力。在细胞中，INF2与内质网（ER）紧密结合，部分由c端法尼化介导。INF2解聚活性的破坏导致内质网塌陷，并在塌陷的内质网周围积累大量的肌动蛋白丝。INF2也与微管（MTs）紧密结合，可能在某些细胞环境中提供MTs和肌动蛋白之间的联系。在本提案中，我将使用生物化学/细胞结合的方法来解决formin介导的细胞结构的组装，使用INF2和FRL2作为模型。在Aim 1中，我确定了INF2和FRL2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Aim 2中，我阐明了这些生化活动的调控机制。在Aim 3中，我使用RNAi技术，以及在Aim 1中发现的突变体，来解决INF2和FRL2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形成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构这一过程时，就能达到这种程度的理解。这个提议启动了重构过程。

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.