Comparative molecular physiology of mammalian formins

描述（由申请人提供）：本申请是对NOT-OD-09-058“NIH宣布恢复法案资金可用于竞争性修订申请”的回应。我的R 01研究哺乳动物肌动蛋白的生化和细胞功能，这是有力的肌动蛋白组装因子。哺乳动物拥有15种不同的formin，许多这些异构体的细胞功能知之甚少。我专注于两个哺乳动物formin，INF 2和FRL 2。INF 2在两个方面是独一无二的。在生物化学上，INF 2加速肌动蛋白聚合和解聚。在细胞中，INF 2定位于ER膜。INF 2也紧密结合微管（MT）。FRL 2在体外捆绑肌动蛋白丝，并在细胞中引起强大的丝状伪足组装。最初申请的目的是：1）剖析INF 2和FRL 2介导肌动蛋白/MT效应的基序; 2）确定INF 2和FRL 2的调控机制; 3）分析INF 2和FRL 2的细胞作用。我建议使用传统的基于群体的生物化学技术来实现前两个目标。我提议与该领域的一位专家合作，在aim 3中进行活细胞显微镜检查。这一竞争性修订通过在更详细的层面上解决机制来扩展这些目标。这一扩张是通过两个方面的投资实现的：1）购买设备，大大提高了我们的分析能力; 2）增加人员。所要求的设备将升级我的荧光显微镜：1）允许全内反射（TIRF）显微镜; 2）允许在活细胞中几乎同时采集红色/绿色图像。在人事方面，我建议增加一名研究生。目标的扩展如下。在目标1中，我们将剖析INF 2的解聚活性的机制，以及它对MT动力学的影响。目的二是建立一个无细胞系统，研究INF 2介导的肌动蛋白和MT在内质网膜上的动态变化。在目标3中，我们将FRL 2动力学与活细胞中肌动蛋白的动力学相关联。 公共卫生相关性：肌动蛋白是一种构成细胞骨架的蛋白质，其聚合对人类细胞中至少15个过程至关重要，包括参与细胞分裂，神经功能和免疫细胞功能的过程。我的实验室研究肌动蛋白聚合在特定过程中是如何控制的。通过了解这些过程，我希望开发出癌症和免疫性疾病等疾病的治疗干预措施。

DESCRIPTION (provided by applicant): This application is in response to NOT-OD-09-058, "NIH announces the availability of Recovery Act funds for competitive revision applications". My R01 investigates the biochemical and cellular functions of mammalian formin proteins, which are potent actin assembly factors. Mammals possess 15 distinct formins, and cellular function is poorly understood for many of these isoforms. I focus on two mammalian formins, INF2 and FRL2. INF2 is unique in two respects. Biochemically, INF2 accelerates both actin polymerization and depolymerization. In cells, INF2 localizes to ER membranes. INF2 also binds microtubules (MTs) tightly. FRL2 bundles actin filaments in vitro and causes robust filopodia assembly in cells. The aims of the original application were to: 1) dissect motifs in INF2 and FRL2 mediating effects on actin/MTs; 2) determine regulatory mechanisms for INF2 and FRL2; and 3) analyze the cellular roles of INF2 and FRL2. I proposed to conduct the first two aims using conventional population-based biochemical techniques. I proposed to conduct live cell microscopy in aim 3 in collaboration with an expert in the field. This competitive revision expands these aims by addressing mechanism at a much more detailed level. This expansion is made possible by investments in two areas: 1) equipment purchases that dramatically increase our analytical capability; and 2) addition of personnel. The requested equipment will up- grade my fluorescence microscope by: 1) allowing Total Internal Reflection (TIRF) microscopy; and 2) allowing near simultaneous red/green image acquisition in live cells. For personnel, I propose to add one graduate student. Expansion of aims will be as follows. In Aim 1, we will dissect the mechanism of INF2's depolymerization activity, as well as its effect on MT dynamics. In Aim 2, we will establish a cell-free system for INF2- mediated actin and MT dynamics on ER membrane. In Aim 3, we will correlate FRL2 dynamics with those of actin in live cells. PUBLIC HEALTH RELEVANCE: Actin is a protein that makes up the skeletons of cells, and whose polymerization is vital for at least 15 processes in human cells, including processes involved in cell division, neural function, and immune cell function. My laboratory studies how actin polymerization is controlled in specific processes. By understanding these processes, I hope to develop therapeutic interventions for diseases such as cancer and immunological diseases.