Formin regulation by a novel three-component mechanism

项目摘要 这项建议的重点是调节福尔马林，一个保守的家族的肌动蛋白组装促进蛋白， 在其快速生长的带刺末端形成并加工拉长肌动蛋白细丝。具体来说，它使用肌动蛋白 以酿酒酵母中的电缆形成为模型。虽然福尔敏的结构和功能已经在一些 详细来说，相对较少的人知道福尔马林活性是如何在细胞内的空间和时间上受到控制的 产生特定形状和大小的肌动蛋白结构。最近的研究表明，哺乳动物的同源物 可以同时与肌动蛋白细丝的带刺末端结合，形成 ‘决策复合体’和催化彼此的解离来调节肌动蛋白的生长和长度1，2。这些令人兴奋 观察结果提出了新的问题，包括复杂的决策机制是否在 其他系统，以及额外的福尔明和/或CP的结合伙伴是否会影响机制。我的 初步数据显示，酵母CP(Cap1/2)和酵母形成素结合蛋白Bud14协同工作以 将福尔曼Bnr1从带刺的末端移开。因此，我的数据表明了一种新的三组分(Bud14-Bnr1- Cap1/2)决定复合体，用于调节细胞肌动蛋白结构的生长。在本提案中，我将在 体外单分子成像以实时可视化这一新的机制。此外，我会决定哪一位 Bud14的结构域和分子相互作用参与了这一机制。此外，我将使用酵母遗传学 和活细胞成像来测试体内的三组分机制，并确定它如何调节适当的 形成具有特定形状和长度的肌动蛋白缆索，该缆索的形状和长度最适合于分泌性囊泡的运输。最后，我 将使用实时成像来定义Bud14粒子在细胞中的动态运动，以及双分子荧光 互补(BIFC)实验研究Bud14和Cap1/2与Bnr1在蕾颈的活体相互作用。 总之，这项工作将为福尔马林如何在体内受到控制提供新的见解。这项工作将扩大我们的 了解控制细胞内肌动蛋白组装的基本、保守的机制，并提供新的 对与肌动蛋白调控缺陷相关的人类疾病状态的潜在基础的洞察。具体的 目的是：(1)使用单分子成像来定义Bud14和Cap1/2合作的机制 控制肌动蛋白细丝带刺末端的Forin活性；(2)检测Bud14-Bnr1-Cap1/2在体内的作用 肌动蛋白线缆长度和极化分泌的调节机制。

Project Summary This proposal focuses on the regulation of formins, a conserved family of actin assembly-promoting proteins that nucleate and processivly elongate actin filaments at their fast-growing barbed ends. Specifically, it uses actin cable formation in S. cerevisiae as a model. While formin structure and function have been defined in some detail, comparatively little is known about how formin activities are spatially and temporally controlled in cells to produce actin structures of a particular shape and size. Recent studies have shown that mammalian homologs of capping protein (CP) and formin can bind simultaneously to the barbed ends of actin filaments, forming ‘decision complexes’ and catalyzing each other’s dissociation to tune actin growth and length1,2. These exciting observations have raised new questions, including whether the decision complex mechanism is conserved in other systems, and whether additional binding partners of formins and/or CP can influence the mechanism. My preliminary data show that yeast CP (Cap1/2) and the yeast formin-binding protein Bud14 work in concert to displace the formin Bnr1 from barbed ends. Thus, my data suggest a novel three-component (Bud14-Bnr1- Cap1/2) decision complex used to regulate the growth of cellular actin structures. In this proposal, I will use in vitro single-molecule imaging to visualize this novel mechanism in real time. Further, I will determine which domains and molecular interactions of Bud14 contribute to the mechanism. In addition, I will use yeast genetics and live-cell imaging to test the three-component mechanism in vivo, and determine how it regulates the proper formation of actin cables with a characteristic shape and length optimized for secretory vesicle traffic. Finally, I will use live-imaging to define the dynamic movements of Bud14 particles in cells, and Bimolecular Fluorescence Complementation (BiFC) assays to study live interactions of Bud14 and Cap1/2 with Bnr1 at the bud neck. Together, this work will provide new insights into how formins are controlled in vivo. This work will expand our understanding of fundamental, conserved mechanisms controlling actin assembly in cells, and provide new insights into the underlying basis of human disease states associated with defects in actin regulation. The specific aims are: (1) Use single-molecule imaging to define the mechanism by which Bud14 and Cap1/2 collaborate to control formin activity at the barbed ends of actin filaments; (2) Test the in vivo role of the Bud14-Bnr1-Cap1/2 mechanism in regulating actin cable length and polarized secretion.