Regulation of formins and cell polarity in yeast

描述(由申请人提供)：我们的长期目标是对肌动蛋白细胞骨架如何指导细胞极性和细胞形态发生有一个高度机械性的理解。所有活细胞都有为其独特的生理功能量身定做并至关重要的内部和外部结构。此外，细胞结构可以响应于各种提示而快速改变。这些事件背后的机制仍然知之甚少，对细胞生物学家来说是一个重大的挑战。最近，一个被称为Forins的保守蛋白质家族已经成为细胞中肌动蛋白组装和重塑的关键调节因子，通常直接作用于Rho GTP酶的下游。Formins是一种大的多结构域蛋白质，在多种生物体的细胞极性、细胞分裂、细胞迁移、内吞作用和细胞黏附中发挥重要作用。Formins通过一种新的机制直接使肌动蛋白组装成核，并保持连续附着在细丝的生长末端，保护末端不受覆盖蛋白的影响，同时引导新的肌动蛋白亚基的插入。虽然在过去的五年里，在阐明Forin蛋白的结构、机制和功能方面取得了快速的进展，但对于Forin活性是如何在细胞内的空间和时间上进行调节的，人们知之甚少。在这项提案中，我们将使用萌芽酵母酿酒酵母作为模式生物来解决这个问题。哺乳动物有15个不同的Forin基因，而酿酒酵母只有两个(Bni1和Bnr1)，因此提供了一个分析Forin调控的简化模型。酵母还允许我们采取多学科的方法，结合遗传学、生物化学和活细胞成像。Bni1和Bnr1具有不同的定位和动力学，并组装两组不同的肌动蛋白缆线。这些电缆作为靶向分泌和极化细胞生长所需的极化轨道。我们将确定其中一种福尔马林(Bnr1)在体内是如何调节的，这将为细胞极性和细胞形态发生的分子基础提供关键的机械见解。该提案的具体目的是：(1)如何将Bnr1锚定在芽颈，从自身抑制状态激活/释放，然后从肌动蛋白细丝末端回收用于新一轮肌动蛋白组装？(2)Bnr1-调节器(Bud14)的活性和细胞功能是如何由其体内结合伙伴(Kel1和Kel2)控制的？确定这些事件的分子基础不仅对于了解正常的人类细胞生物学和生理学至关重要，而且对于确定编码形态发生决定因素的基因突变如何导致癌症、出生缺陷和神经退行性疾病等疾病状态也至关重要。

DESCRIPTION (provided by applicant): Our long-term goal is to gain a highly mechanistic understanding of how the actin cytoskeleton directs cell polarity and cell morphogenesis. All living cells have internal and external structures tailored to and critical for their distinctive physiological functions. Further, cell architecture can be changed rapidly in response to various cues. The mechanisms underlying these events remain poorly understood and represent a major challenge for cell biologists to define. Recently, a conserved family of proteins called formins has emerged as crucial regulators of actin assembly and remodeling in cells, often functioning directly downstream of Rho GTPases. Formins are large multi-domain proteins that play essential roles in cell polarity, cell division, cell migration, endocytosis, and cell adhesion in a wide range of organisms. Formins directly nucleate actin assembly by a novel mechanism and remain processively attached to the growing end of the filament, protecting the end from capping proteins while guiding insertion of new actin subunits. While the last five years have seen rapid progress in elucidating formin protein structure, mechanism and function, comparatively little is known about how formin activities are regulated spatially and temporally in cells. In this proposal, we will address this question using the budding yeast Saccharomyces cerevisiae as a model organism. Whereas mammals have 15 different formin genes, S. cerevisiae has only two (Bni1 and Bnr1), and hence offers a simplified model to dissect formin regulation. Yeast also allows us to take a multidisciplinary approach, combining genetics, biochemistry, and live cell imaging. Bni1 and Bnr1 have distinct localization and dynamics, and assemble two distinct sets of actin cables. These cables serve as polarized tracks required for targeted secretion and polarized cell growth. We will determine how one of these formins (Bnr1) is regulated in vivo, which will provide key mechanistic insights into the molecular basis of cell polarity and cell morphogenesis. The Specific Aims of the proposal are: (1) How is Bnr1 anchored at the bud neck, activated/released from an autoinhibited state, and then retrieved from actin filament ends for new rounds of actin assembly? (2) How are the activities and cellular functions of a novel Bnr1-regulator (Bud14) controlled by its in vivo binding partners (Kel1 and Kel2)? Defining the molecular basis of these events is critical not only for understanding normal human cell biology and physiology, but also for determining how mutations in the genes encoding morphogenetic determinants give rise to disease states including cancer, birth defects, and neurodegenerative disorders.