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 这个建议集中在formins的调节上，formins是一个保守的肌动蛋白组装促进蛋白家族， 在其快速生长的有倒钩的末端成核并过程性地拉长肌动蛋白丝。具体来说，它使用肌动蛋白 S.作为一种模式。虽然在一些文献中已经定义了微结构和功能， 详细地说，相对而言，我们对细胞中的细胞活动是如何在空间和时间上受到控制的知之甚少， 产生特定形状和大小的肌动蛋白结构。最近的研究表明， 加帽蛋白（CP）和肌动蛋白可以同时结合到肌动蛋白丝的倒刺末端，形成 “决定复合物”和催化彼此的解离，以调整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，包括决策复杂机制是否保守， 其他系统，以及是否额外的结合配偶体的formin和/或CP可以影响的机制。我 初步数据显示，酵母CP（Cap 1/2）和酵母formin结合蛋白Bud 14协同工作， 从带倒钩的端部置换Bnr 1。因此，我的数据表明，一种新的三组分（Bud 14-Bnr 1-Bnr 2）， Cap 1/2）决定复合物，用于调节细胞肌动蛋白结构的生长。在本提案中，我将使用 体外单分子成像，以可视化这种新的机制，在真实的时间。此外，我将确定 Bud 14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 形成具有特征形状和长度的肌动蛋白电缆，以优化分泌囊泡的运输。最后我 将使用实时成像和双分子荧光来定义细胞中Bud 14颗粒的动态运动 互补（BiFC）测定以研究Bud 14和Cap 1/2与Bnr 1在芽颈处的活相互作用。 总之，这项工作将提供新的见解如何在体内控制formin。这项工作将扩大我们的 了解基本的，保守的机制控制肌动蛋白组装在细胞中，并提供新的 深入了解与肌动蛋白调节缺陷相关的人类疾病状态的潜在基础。具体 目的是：（1）使用单分子成像来确定Bud 14和Cap 1/2协同作用的机制， 在肌动蛋白丝的有刺末端处的控制肌动蛋白活性;（2）测试Bud 14-Bnr 1-Cap 1/2的体内作用 调节肌动蛋白索长度和极化分泌的机制。