Mechanisms of Formin-Mediated Actin Filament Assembly in Fission Yeast

描述(申请人提供)：我们研究计划的重点是确定控制细胞如何在广泛的任务中不同地协调肌动蛋白细丝组装的基本生化机制。最近证实，福尔马林促进肌动蛋白的组装，从而驱动不同的细胞过程，如分裂、极化和迁移。Forin蛋白的显著特征是两个内部Forin同源1和2结构域(FH1FH2)，两侧是调节结构域。FH1FH2结构域通过一种概念上新颖但知之甚少的机制协作组装肌动蛋白细丝。真核细胞含有多种用于不同过程的福尔敏异构体，但功能特异性的机制基础尚不清楚。不同的调节，即在正确的时间和地点激活每一种福尔曼异构体，可能是必需的，但它还没有被测试。此外，我发现肌动蛋白的组装速度在不同的福尔马林之间有很大的差异，这表明不同的细胞过程需要不同的肌动蛋白细丝延长率。单细胞裂解酵母菌是研究福尔马林生物学的一个极好的模型系统，因为它可以适应广泛的实验策略，而且它包含三种福尔马林，每一种福尔马林都是特定细胞过程所必需的。我首先关注的是根本上重要的分裂酵母胞质分裂形成蛋白CDc12p。我计划利用遗传学、细胞生物学和通过消逝波荧光显微镜对单个肌动蛋白细丝组装的体外实时观察相结合的方法来解决我的假设，即差异调节和特定的肌动蛋白细丝延长率在功能上都很重要。我将首先谈到三个具体目标。目的I：阐明决定CdC12P比延伸率的分子参数。目的II：确定改变CdC12P的延伸率对胞质分裂的影响。目的III：阐明CDC12P的调控机制(S)。相关性：Formins组装基本细胞过程所需的肌动蛋白细丝，如细胞分裂时的收缩环和活动细胞前缘的丝状突起。细胞分裂和运动是哺乳动物正常发育所必需的，但在恶性肿瘤中则变得不受调控。利用多种实验方法相结合的方法来研究裂解酵母福尔马林可以取得快速的进展，鉴于动物和裂变型酵母细胞福尔曼生物学上的相似性，这些研究将为在动物中建立其一般机制奠定基础。

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.