Mechanisms of Formin-Mediated Actin Filament Assembly in Fission Yeast

裂殖酵母中福尔明介导的肌动蛋白丝组装机制

基本信息

批准号：
7318798
负责人：
David R Kovar
金额：
$ 25.91万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2012-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The focus of our research plan is to determine the fundamental biochemical mechanisms that govern how cells differentially coordinate actin filament assembly for a wide range of tasks. It has been recently established that formins promote actin assembly that drives diverse cellular processes such as division, polarization and migration. The hallmark features of formin proteins are two internal formin homology 1 and 2 domains (FH1FH2) that are flanked by regulatory domains. FH1FH2 domains cooperate to assemble actin filaments by a conceptually novel, but poorly understood, mechanism. Eukaryotic cells contain multiple formin isoforms utilized for different processes, but the mechanistic basis for functional specificity is not clear. Differential regulation, activation of each formin isoform at the right time and place, is probably required but it has not been tested. Additionally, I discovered that the rate of actin assembly varies significantly between diverse formins, suggesting that different cellular processes require different actin filament elongation rates. The unicellular fission yeast Schizosaccharomyces pombe is a superb model system for investigating formin biology because it is amenable to a wide-range of experimental strategies and because it contains three formins that are each required for a specific cellular process. I am initially focusing on the fundamentally important fission yeast cytokinesis formin Cdc12p. I plan to utilize a combination of genetics, cell biology and in vitro real-time observation of the assembly of individual actin filaments by evanescent wave fluorescence microscopy to address my hypothesis that both differential regulation and the specific actin filament elongation rate are functionally important. I will initially address three specific aims. Aim I: to elucidate the molecular parameters that determine the specific elongation rate of Cdc12p. Aim II: to determine the consequences for cytokinesis of altering the elongation rate of Cdc12p. Aim III: to elucidate the mechanism(s) of regulation of Cdc12p. Relevance: Formins assemble the actin filaments necessary for basic cellular processes such as the contractile ring during cell division and filopodial protrusions at the leading edge of motile cells. Cell division and motility are required for normal mammalian development, but then become unregulated in malignant tumors. By utilizing a combination of experimental approaches to study fission yeast formins rapid progress can be made, and given the similarity of formin biology between animal and fission yeast cells these studies will lay the groundwork for establishing their general mechanisms in animals.

描述（由申请人提供）：我们的研究计划的重点是确定基本的生化机制，这些机制控制细胞如何差异协调肌动蛋白丝组装的各种任务。最近已经确定，formins促进肌动蛋白组装，从而驱动各种细胞过程，例如分裂，极化和迁移。甲蛋白蛋白的标志性特征是两个内部formin同源性1和2域（FH1FH2），它们侧面是调节域。 FH1FH2领域合作通过概念上的新颖但知之甚少的机制来组装肌动蛋白丝。真核细胞包含用于不同过程的多种形式的同工型，但是功能特异性的机理基础尚不清楚。差异调节，在正确的时间和地点激活每个形式的同工型，但尚未进行测试。此外，我发现肌动蛋白组装的速率在不同的formins之间差异很大，这表明不同的细胞过程需要不同的肌动蛋白丝伸长率。单细胞裂变酵母菌型酸糖疗法是一种用于研究formin生物学的出色模型系统，因为它可以适合大量的实验策略，并且因为它包含三个formins，每个层都需要特定的细胞过程。我最初关注的是根本重要的裂变酵母菌细胞动力学formin cdc12p。我计划利用遗传学，细胞生物学和通过evaneScent波荧光显微镜组装单个肌动蛋白丝的体外实时观察，以解决我的假设，即差异调节和特定的肌动蛋白丝伸长率在功能上都很重要。我最初将解决三个具体目标。目的I：阐明确定CDC12P特异性伸长率的分子参数。 AIM II：确定改变Cdc12p伸长率的细胞因子的后果。 AIM III：阐明CDC12P调节的机制。相关性：formins组装基本细胞过程所必需的肌动蛋白丝，例如细胞分裂期间的收缩环和运动细胞前缘的丝状突起。正常的哺乳动物发育需要细胞分裂和运动性，但随后在恶性肿瘤中不受监管。通过利用实验方法的组合来研究裂变酵母造型的快速进步，并且鉴于动物和裂变酵母细胞之间形成生物学的相似性，这些研究将为建立动物的一般机制奠定基础。