Mechanisms of Formin-Mediated Actin Filament Assembly in Fission Yeast

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，这些机制决定细胞如何以不同的方式协调肌动蛋白丝的组装，以完成广泛的任务。最近已经证实，形成蛋白促进肌动蛋白组装，从而驱动多种细胞过程，如分裂、极化和迁移。双胍蛋白的标志性特征是两个内部双胍同源1和2结构域（FH1FH2），两侧是调控结构域。FH1FH2结构域通过一种概念新颖但尚不清楚的机制协同组装肌动蛋白丝。真核细胞含有多种formin亚型，用于不同的过程，但功能特异性的机制基础尚不清楚。不同的调控，在适当的时间和地点激活每种双胍异构体，可能是必需的，但尚未经过测试。此外，我发现不同形成蛋白之间的肌动蛋白组装率差异很大，这表明不同的细胞过程需要不同的肌动蛋白丝伸长率。单细胞分裂酵母Schizosaccharomyces pombe是研究formin生物学的一个极好的模型系统，因为它适用于广泛的实验策略，并且因为它包含三种formin，每种formin都是特定细胞过程所必需的。我首先关注的是至关重要的分裂酵母胞质双胍Cdc12p。我计划结合遗传学、细胞生物学和通过瞬变波荧光显微镜对单个肌动蛋白丝的组装进行体外实时观察，以解决我的假设，即差异调节和特定肌动蛋白丝伸长率在功能上都很重要。我将首先阐述三个具体目标。目的一：阐明决定Cdc12p比伸长率的分子参数。目的二：确定改变Cdc12p延伸率对细胞分裂的影响。目的三：阐明Cdc12p的调控机制。相关：形成蛋白聚集了基本细胞过程所必需的肌动蛋白丝，如细胞分裂时的收缩环和运动细胞前缘的丝状突起。细胞分裂和运动是正常哺乳动物发育所必需的，但在恶性肿瘤中却变得不受调节。通过结合实验方法研究分裂酵母形成蛋白可以取得快速进展，并且考虑到动物和分裂酵母细胞之间的formin生物学相似性，这些研究将为建立其在动物中的一般机制奠定基础。

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.