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如何由formins控制以及调节其体内活性的机制。我们正在芽殖酵母中研究这个问题，在芽殖酵母中，形成蛋白Bni 1和Bnr 1组装肌动蛋白“电缆”，在极化细胞生长中发挥重要作用。我们的实验室最近发现了由极性因子Bud 6，Bud 14，Smy 1和Hof 1介导的四种新的Hof 1调节模式。值得注意的是，这些蛋白质中的每一个都与Bnr 1的formin同源FH 2结构域结合，但对Bnr 1体外活性和体内不同的Bnr 1依赖性突变体肌动蛋白电缆表型具有不同的影响。此外，我们已经确定了新的体内配体的Bud 6和Bud 14的功能与他们在调节Bnr 1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，以及它们的组合效应如何协调肌动蛋白电缆的正确组装，并具有为其功能量身定制的特征长度，结构和动力学。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。其目的是：（1）阐明Bud 6在调节Bnr 1介导的肌动蛋白缆索组装中的特定作用和机制;（2）检验Bud 14和Smy 1提供维持肌动蛋白缆索长度、动力学和结构所需的不同时间调节模式的假设;（3）确定多种FH 2结合调节剂的功能如何在体外和体内协调。

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.