Formin regulation by a novel three-component mechanism

通过新型三组分机制进行 Formin 调节

• 批准号: 9906494
• 负责人: Alison Catherine Wirshing
• 金额: $ 6.49万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906494
• 关键词: Actins; Address; Affect; Architecture; Back; Binding; Binding Proteins; Biological Assay; Biological Process; Bundling; Cardiovascular Diseases; Cell Cycle Stage; Cells; Characteristics; Complement; Complex; Cytoskeleton; Data; Defect; Dimensions; Dissociation; Family; Fiber; Filament; Fluorescence; Fluorescence Anisotropy; Genetic; Goals; Growth; Image; Immune System Diseases; In Vitro; Kidney Diseases; Kinetics; Label; Lead; Length; Malignant Neoplasms; Mediating; Microfilaments; Modeling; Molecular; Mothers; Movement; Mutation; Myosin ATPase; Neck; Organelles; Organism; Plus End of the Actin Filament; Polymers; Proteins; Regulation; Role; SH3 Domains; Saccharomyces cerevisiae; Secretory Vesicles; Shapes; Site; Structure; System; Testing; Time; Tube; Work; Yeasts; base; cell growth; cofilin; comparative; deafness; experimental study; human disease; in vivo; in vivo evaluation; insight; live cell imaging; molecular imaging; molecular modeling; mutant; novel; overexpression; particle; prevent; recruit; single molecule; stoichiometry; yeast genetics

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.

项目摘要 该提案的重点是造型的调节，造型是肌动蛋白组装蛋白的保守家族， 在快速生长的带刺末端的成核和过程拉长肌动蛋白丝。具体来说，它使用肌动蛋白 酿酒酵母中的电缆形成为模型。虽然在某些 细节，关于形式活动如何在细胞中如何在空间和临时控制到的细节知之甚少 产生特定形状和大小的肌动蛋白结构。最近的研究表明，哺乳动物同源物 封口蛋白（CP）和Formin的结合可以简单地与肌动蛋白丝的刺末端结合，形成 “决策综合体”，并催化彼此的分离为调节肌动蛋白的生长和长度1,2。这些令人兴奋 观察提出了新问题，包括决策复杂机制是否保存在 其他系统，以及造型和/或CP的其他结合伙伴是否会影响机制。我的 初步数据表明，酵母CP（CAP1/2）和酵母菌结合蛋白Bud14协同工作 从带刺的末端取代formin bnr1。这就是我的数据表明了一个新颖的三个组件（bud14-bnr1-- CAP1/2）用于调节细胞肌动蛋白结构生长的决策复合体。在此提案中，我将使用 体外单分子成像实时可视化这种新型机制。此外，我将确定哪个 Bud14的结构域和分子相互作用有助于该机制。此外，我将使用酵母遗传学 和活细胞成像以在体内测试三组分机制，并确定如何调节适当 肌动蛋白电缆的形成具有特征形状和针对秘密场地流量优化的长度。最后，我 将使用现场成像来定义细胞中Bud14颗粒的动态运动和双分子荧光 互补（BIFC）测定，研究Bud14和Cap1/2与BNR1在芽颈的实时相互作用。 这项工作将共同提供有关如何在体内控制formins的新见解。这项工作将扩大我们的 了解控制细胞中肌动蛋白组件的基本，组成的机制，并提供新的 对与肌动蛋白调节中缺陷相关的人类疾病状态的基础的见解。具体 目的是：（1）使用单分子成像来定义Bud14和Cap1/2合作的机制 在肌动蛋白丝的带状末端控制形孔活性； （2）测试Bud14-BNR1-CAP1/2的体内作用 控制肌动蛋白电缆长度和极化分泌的机制。