Novel mechanisms regulating formins and cell polarity

描述（由申请人提供）：本研究的目的是确定肌动蛋白细胞骨架的组装动力学和结构是如何被形成蛋白控制的，以及它们在体内的活性调节机制。我们正在出芽酵母中研究这个问题，其中形成蛋白Bni1和Bnr1组装肌动蛋白“电缆”，在极化细胞生长中起重要作用。我们的实验室最近发现了四种由极性因子Bud6、Bud14、Smy1和Hof1介导的双胍调节新模式。值得注意的是，这些蛋白都与Bnr1的formmin同源FH2结构域结合，但在体外对Bnr1活性和体内不同的Bnr1依赖性突变肌动蛋白电缆表型有不同的影响。此外，我们还发现了Bud6和Bud14的新的体内配体，它们与Bud6和Bud14一起调节bnr1介导的肌动蛋白电缆组装。拟议的研究将定义这些蛋白质的细胞功能和机制，以及它们的综合作用如何协调肌动蛋白电缆的适当组装，使其具有特定的长度、结构和动态，以适应其功能。这项工作将提供一个更深入的了解分子活动和相互作用的基础上的细胞极性和形态发生。该项目采用多学科方法，结合遗传学、活细胞成像、生物化学和新型多波长单分子TIRF体外显微镜。目的：(1)阐明Bud6在调节Bnr1介导的肌动蛋白索组装中的具体作用和机制；(2)验证Bud14和Smy1提供维持肌动蛋白电缆长度、动力学和结构所需的不同形式的时间调节模式的假设；(3)确定多种fh2结合调节因子在体内和体外如何协调作用。

DESCRIPTION (provided by applicant): The goal of this research is to determine how the assembly dynamics and architecture of the actin cytoskeleton are controlled by formins and the mechanisms regulating their activities in vivo. We are studying this question in budding yeast, where the formins Bni1 and Bnr1 assemble actin 'cables' that play an essential role in polarized cell growth. Our lab recently discovered four novel modes of formin regulation mediated by the polarity factors Bud6, Bud14, Smy1, and Hof1. Remarkably, each of these proteins binds to the formin-homology FH2 domain of Bnr1, but has distinct effects on Bnr1 activity in vitro and distinct Bnr1-dependent mutant actin cable phenotypes in vivo. Further, we have identified novel in vivo ligands of Bud6 and Bud14 that function with them in regulating Bnr1-mediated actin cable assembly. The proposed research will define the cellular functions and mechanisms of these proteins, and how their combined effects coordinate the proper assembly of actin cables with a characteristic length, architecture, and dynamics that is tailored to their function. This work will provide a deeper understanding of the molecular activities and interactions that underlie cell polarity and morphogenesis. The project uses a multi-disciplinary approach, combining genetics, live-cell imaging, biochemistry, and novel multi-wavelength single molecule TIRF in vitro microscopy. The Aims are to: (1) Elucidate the specific roles and mechanisms of Bud6 in regulating Bnr1- mediated actin cable assembly; (2) Test the hypothesis that Bud14 and Smy1 provide distinct modes of formin temporal regulation required for maintaining actin cable length, dynamics, and architecture; and (3) Determine how the functions of multiple FH2-binding regulators are coordinated in vitro and in vivo.