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，管理细胞如何差异协调肌动蛋白丝组装广泛的任务。最近已经确定，formins促进肌动蛋白组装，驱动不同的细胞过程，如分裂，极化和迁移。FH 1蛋白的标志性特征是两个内部的FH 1同源性1和2结构域（FH 1 FH 2），其侧翼是调节结构域。FH 1FH 2域合作组装肌动蛋白丝的概念新颖，但知之甚少，机制。真核细胞含有多种用于不同过程的β-淀粉样蛋白异构体，但功能特异性的机制基础尚不清楚。可能需要差异调节，即在正确的时间和地点激活每一种亚型，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丝伸长速率。单细胞裂殖酵母裂殖酵母是一个极好的模型系统，用于研究裂殖生物学，因为它适合于广泛的实验策略，因为它包含三个formin，每个formin都是特定的细胞过程所需的。我最初专注于根本重要的裂变酵母胞质分裂Cdc 12 p。我计划利用遗传学，细胞生物学和在体外实时观察单个肌动蛋白丝的装配倏逝波荧光显微镜来解决我的假设，即差异调节和特定的肌动蛋白丝伸长速率在功能上是重要的。首先，我将讨论三个具体目标。目的一：阐明决定Cdc 12 p特异性延伸率的分子参数。目的II：确定改变Cdc 12 p的延伸率对胞质分裂的影响。目的III：阐明Cdc 12 p的调控机制。相关性：形成蛋白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伪足突起。细胞分裂和运动是正常哺乳动物发育所必需的，但在恶性肿瘤中则变得不受调节。通过利用实验方法的组合来研究裂殖酵母形成蛋白，可以取得快速进展，并且鉴于动物和裂殖酵母细胞之间的生物学相似性，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.