Novel mechanisms regulating formins and cell polarity

描述(申请人提供)：这项研究的目标是确定肌动蛋白细胞骨架的组装动力学和结构是如何由福尔马林控制的，以及在体内调节它们活动的机制。我们正在萌芽酵母中研究这个问题，在那里，Forins Bni1和Bnr1组装肌动蛋白“电缆”，在极化细胞生长中发挥关键作用。我们实验室最近发现了由极性因子Bud6、Bud14、Smy1和Hof1介导的四种新的福尔敏调控模式。值得注意的是，这些蛋白中的每一个都与Bnr1的形成蛋白同源FH2结构域结合，但在体外对Bnr1的活性和在体内对Bnr1依赖的突变肌动蛋白电缆表型有不同的影响。此外，我们还发现了新的体内配体Bud6和Bud14，它们与它们一起调节Bnr1介导的肌动蛋白电缆组装。这项拟议的研究将确定这些蛋白质的细胞功能和机制，以及它们的联合作用如何协调肌动蛋白电缆的适当组装，这些电缆具有根据其功能量身定做的特有长度、结构和动力学。这项工作将提供对细胞极性和形态发生基础的分子活动和相互作用的更深层次的理解。该项目采用了多学科方法，结合了遗传学、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Bud6在调节Bnr1介导的肌动蛋白电缆组装中的具体作用和机制；(2)检验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时间调控模式的假设；以及(3)确定多个FH2结合调控因子的功能在体外和体内是如何协调的。

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.