Comparative molecular physiology of mammalian formins

哺乳动物福明的比较分子生理学

基本信息

批准号：
7845997
负责人：
HENRY N HIGGS
金额：
$ 21.85万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-29 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7845997
关键词：
Actins Address Affinity Area Binding Biochemical Biological Assay Cell Culture System Cell division Cell physiology Cell-Free System Cells Chimeric Proteins Collaborations Collection Disease Equipment Filament Filopodia Fluorescence Funding Goals Human Human Resources Image Imagery Immune Immune System Diseases In Vitro Investigation Investments Laboratories Laboratory Study Length Life Malignant Neoplasms Mammals Measures Mediating Membrane Microfilaments Microscope Microscopy Microtubules Molecular Neurophysiology - biologic function Persons Physiology Population Process Property Protein Isoforms Proteins Pyrenes RNA Interference Recovery Resolution Role Skeleton Sum System Techniques Therapeutic Intervention Time Tubulin United States National Institutes of Health base cellular imaging charge coupled device camera comparative depolymerization fluorescence microscope fluorophore graduate student millisecond polymerization population based public health relevance research study response

项目摘要

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.

描述（由申请人提供）：本申请是对 NOT-OD-09-058 的回应，“NIH 宣布恢复法案资金可用于竞争性修订申请”。 My R01 研究哺乳动物福尔明蛋白的生化和细胞功能，福尔明蛋白是有效的肌动蛋白组装因子。哺乳动物拥有 15 种不同的亚型，但人们对其中许多亚型的细胞功能知之甚少。我关注两种哺乳动物福明，INF2 和 FRL2。 INF2 在两个方面是独特的。从生物化学角度来看，INF2 加速肌动蛋白聚合和解聚。在细胞中，INF2 定位于 ER 膜。 INF2 还与微管 (MT) 紧密结合。 FRL2 在体外使肌动蛋白丝成束，并在细胞中引起强大的丝状伪足组装。最初应用的目的是：1）剖析 INF2 和 FRL2 中的基序对肌动蛋白/MT 的介导作用； 2)确定INF2和FRL2的调控机制； 3)分析INF2和FRL2的细胞作用。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。我提议与该领域的专家合作，在目标 3 中进行活细胞显微镜检查。这一竞争性修订通过更详细地解决机制来扩展这些目标。这种扩张是通过在两个领域的投资实现的：1）购买设备，极大地提高我们的分析能力； 2）增加人员。所需的设备将通过以下方式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/绿图像。人员方面，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 INF2 解聚活性的机制及其对 MT 动力学的影响。在目标 2 中，我们将建立一个无细胞系统，用于 INF2 介导的肌动蛋白和 ER 膜上的 MT 动力学。在目标 3 中，我们将把 FRL2 动力学与活细胞中肌动蛋白的动力学相关联。公共健康相关性：肌动蛋白是一种构成细胞骨架的蛋白质，其聚合对于人类细胞中至少 15 个过程至关重要，包括涉及细胞分裂、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，我希望开发针对癌症和免疫疾病等疾病的治疗干预措施。