Mechanisms of septin-actin cytoskeletal crosstalk

septin-肌动蛋白细胞骨架串扰的机制

• 批准号: 10677181
• 负责人: Joseph O Magliozzi
• 金额: $ 7.11万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677181
• 关键词: Actins; Acute; Address; Affect; Affinity; Architecture; Binding; Biological Assay; Biological Process; Bundling; C-terminal; Cardiovascular Diseases; Cell physiology; Cells; Collaborations; Complex; Cytokinesis; Cytoskeleton; Data; F-Actin; Filament; Foundations; Future; Genetic; Goals; Hereditary Neuralgic Amyotrophy; Higher Order Chromatin Structure; Human; Image; Immune System Diseases; In Vitro; Intracellular Transport; Kidney Diseases; Knowledge; Label; Length; Ligands; Link; Malignant Neoplasms; Mediating; Microfilaments; Mitotic; Modeling; Molecular; Morphogenesis; Mutagenesis; Mutation; Myosin Light Chains; Neck; Organism; Polymers; Property; Proteins; Regulation; Role; Saccharomyces cerevisiae; Saccharomycetales; Secretory Vesicles; Sedimentation process; Shapes; Site; Structure; System; Testing; Total Internal Reflection Fluorescent; Training; Vesicle; Work; Yeasts; autosome; cell growth; copolymer; deafness; design; experimental study; genetic regulatory protein; human disease; imaging approach; in vivo; in vivo evaluation; inhibitor; insight; live cell imaging; model organism; molecular imaging; mutant; nervous system disorder; novel; polarized cell; reconstitution; recruit; scaffold; single molecule; superresolution microscopy

PROJECT SUMMARY This proposal addresses how the cellular functions of two major cytoskeletal polymer systems, septins and actin, are coordinated and influence each other. To address this question, I will use the budding yeast S. cerevisiae, where septins were first discovered, and where I am readily able to combine a ‘bottom up’ in vitro reconstitution and single molecule imaging approach with a ‘top down’ genetics and live imaging approach. The proposal builds off of recent discoveries made in the Goode lab, which reveal that septins are organized at the yeast bud neck into 8-10 evenly-spaced bars, or “pillars”, which co-align with F-actin cables used for intracellular transport. While work in a number of model organisms has closely linked the in vivo functions of septins and actin, we still have only a limited understanding of the molecular mechanisms underlying this septin-actin crosstalk. The goal of this proposal is to define these mechanisms. S. cerevisiae express 5 different septin proteins, which co-polymerize into filaments and are further organized into higher order structures. My preliminary data show that one of the septins (Shs1) mediates direct binding to F-actin in vitro, and that loss of SHS1 disrupts actin cable architecture and function in vivo. In Aim1, I will use targeted mutagenesis to generate new shs1 separation-of-function mutants disrupting F-actin binding. I will then use these mutants to investigate how Shs1-mediated F-actin binding contributes to the alignment of actin cables with septin pillars in vivo, and intracellular transport of secretory vesicles. In Aim2, I will reconstitute purified septin oligomers and filaments decorated with actin- nucleating formins (Bnr1) and formin-regulatory proteins (e.g., Gin4, Bud6, and the Mlc1-Iqg1-Hof1 complex), and define their effects on F-actin assembly and organization by TIRF microscopy and single molecule imaging. These experiments will test several important hypotheses, including: (1) whether septins and Gin4 activate full- length Bnr1 from autoinhibition to promote actin assembly; (2) whether Iqg1 has regulatory effects on F-actin and Bnr1, like its human counterpart IQGAP1 (based on a recent study from the Goode lab; Hoeprich et al., 2022); and (3) whether septin oligomers/filaments themselves (via Shs1) directly influence F-actin bundling and dynamics. In parallel to these in vitro experiments, I will acutely deplete the same proteins in vivo (using degron tags) to determine how each contributes to the assembly and alignment of actin cables at the bud neck. My preliminary data already point to an exciting new role for Iqg1 in controlling actin cable formation during polarized cell growth. Together, the in vitro and in vivo work outlined in this proposal will: (i) clarify how septins, formins, and formin-regulatory proteins work in concert to shape actin networks, (ii) define new subunit-specific roles for septins in actin regulation, (iii) lay a strong foundation for launching my own lab focused on septin-actin crosstalk, and (iv) provide new leads that will allow me to extend this work in the future (on my own and via collaboration) into other systems.

项目总结 这项提案涉及两个主要的细胞骨架聚合物系统--Septins和Actin--的细胞功能。 是相互协调、相互影响的。为了解决这个问题，我将使用萌芽酵母酿酒酵母， 在那里，第一次发现了分隔素，在那里，我很容易地结合了一种‘自下而上’的体外重建 和单分子成像方法与‘自上而下’的遗传学和活体成像方法。提案建立了 根据古德实验室的最新发现，这些发现表明，隔质是在酵母芽颈组织起来的 形成8-10个均匀间隔的条状或“柱状”，它们与用于细胞内运输的F-肌动蛋白缆线共同对准。而当 在一些模式生物中的工作已经将体内的隔膜蛋白和肌动蛋白的功能紧密联系在一起，我们仍然有 对这种Septin-肌动蛋白串扰的分子机制了解有限。这样做的目的是 提案就是定义这些机制。酿酒酵母表达5种不同的Septin蛋白，它们共同聚合 形成细丝，并进一步组织成更高顺序的结构。我的初步数据显示，其中一个 Septins(Shs1)在体外介导与F-肌动蛋白的直接结合，而SHS1的缺失破坏了肌动蛋白的电缆结构 并在体内发挥作用。在Aim1中，我将使用定向突变来生成新的shs1功能分离 破坏F-肌动蛋白结合的突变体。然后我将使用这些突变体来研究Shs1介导的F-肌动蛋白是如何 结合有助于肌动蛋白电缆与体内的间隔素柱对齐，以及细胞内转运 分泌性小泡。在AIM2中，我将重建纯化的Septin寡聚体和用肌动蛋白装饰的细丝- 核化形成蛋白(Bnr1)和形成蛋白调节蛋白(例如Gin4、Bud6和MLc1-Iqg1-Hof1复合体)， 并通过TIRF显微镜和单分子成像确定它们对F-肌动蛋白组装和组织的影响。 这些实验将检验几个重要的假设，包括：(1)Septins和Gin4是否完全激活- (2)Iqg1是否对F-肌动蛋白有调节作用 和Bnr1，与其人类对应的IQGAP1一样(基于Goode实验室最近的一项研究；Hoeprich等人， 2022)；以及(3)Septin低聚体/细丝本身(通过Shs1)是否直接影响F-肌动蛋白捆绑和 动力学。在这些体外实验的同时，我将在体内剧烈地耗尽相同的蛋白质(使用degron 标签)，以确定每个标签对芽颈的肌动蛋白电缆的组装和对齐有何作用。我的 初步数据已经表明，Iqg1在控制极化过程中肌动蛋白电缆的形成方面发挥了令人兴奋的新作用 细胞生长。综上所述，这项提案中概述的体外和体内工作将：(I)阐明Septins，Form in， 和形成蛋白调节蛋白协同作用形成肌动蛋白网络，(Ii)定义新的亚基特定的作用 肌动蛋白调控中的Septin，(Iii)为我自己推出专注于Septin-Actin串扰的实验室奠定了坚实的基础， 以及(Iv)提供新的线索，使我能够在未来扩展这项工作(我自己和通过合作) 进入其他系统。