Comparative molecular physiology of mammalian formins

描述（由申请人提供）：此申请是对NOT-OD-09-058的回应，“NIH宣布恢复法案基金可用于竞争性修订申请”。我的R01研究了哺乳动物双胍蛋白的生化和细胞功能，这是一种有效的肌动蛋白组装因子。哺乳动物拥有15种不同的异构体，对其中许多异构体的细胞功能了解甚少。我主要关注两种哺乳动物形成因子，INF2和FRL2。INF2在两个方面是独一无二的。生物化学上，INF2加速肌动蛋白的聚合和解聚。在细胞中，INF2定位于内质网膜。INF2也能紧密结合微管（mt）。FRL2在体外捆绑肌动蛋白丝，并在细胞中引起强大的丝状足组装。最初申请的目的是：1)解剖介导肌动蛋白/MTs的INF2和FRL2中的基序；2)确定INF2和FRL2的调控机制；3)分析INF2和FRL2在细胞中的作用。我建议使用传统的基于人群的生化技术来实现前两个目标。我提议在aim 3中与该领域的专家合作进行活细胞显微镜。这种竞争性修订通过在更详细的层面上解决机制问题来扩展这些目标。这种扩张是通过在两个领域的投资实现的：1)购买设备，大大提高了我们的分析能力；2)增加人员。所要求的设备将提高我的荧光显微镜：1)允许全内反射（TIRF）显微镜；2)允许在活细胞中几乎同时获取红/绿图像。人事方面，我建议增加一名研究生。目标扩展如下。在Aim 1中，我们将剖析INF2解聚活性的机制，以及它对MT动力学的影响。在Aim 2中，我们将建立一个无细胞系统，用于内质膜上INF2介导的肌动蛋白和MT动力学。在目标3中，我们将把FRL2动力学与活细胞中的肌动蛋白动力学联系起来。

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.