Specification of actomyosin function in the cell

细胞内肌动球蛋白功能的规范

• 批准号: 8235439
• 负责人: Matthew Lord
• 金额: $ 28.51万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8235439
• 关键词: ATP phosphohydrolase; Actin-Binding Protein; Actinin; Actins; Actomyosin; Aneuploidy; Architecture; Biochemical; Biological Assay; Bundling; C-terminal; Cell Proliferation; Cells; Centrosome; Characteristics; Chromosomes; Complex; Cytokinesis; Cytoskeleton; Defect; Electron Microscopy; Endocytic Vesicle; Endocytosis; Ensure; Filament; Fimbrin; Fission Yeast; Fluorescence; Fluorescence Microscopy; Genetic; Human; In Vitro; Intracellular Transport; Knock-out; Laser Scanning Confocal Microscopy; Length; Light; Malignant Neoplasms; Mediating; Microfilaments; Modeling; Molecular; Motor; Motor Activity; Myosin ATPase; Myosin Type I; Myosin Type II; Myosin Type V; Neoplasm Metastasis; Physiological; Play; Population; Protein Isoforms; Recruitment Activity; Role; SM 22 muscle protein; Site; Sorting - Cell Movement; Specific qualifier value; Stress Fibers; Structure; Tail; Testing; Time; Tropomyosin; Yeasts; analytical ultracentrifugation; base; cancer cell; cell motility; cell transformation; constriction; crosslink; in vivo; insight; interest; novel; reconstitution; self assembly; tumor

DESCRIPTION (provided by applicant): The proposal tackles a fundamental question in the cytoskeleton field: How do different actin structures in the cell recruit the appropriate actin-binding proteins that adapt them for their given roles? Presently, we are focused on how myosin-I is directed to sites of endocytosis (actin patches), how myosin-II functions at contractile rings during cytokinesis, and how myosin-V is specified for intracellular transport along actin cables. Fission yeast is currently our model of choice owing to its tractable genetics, well-defined actin architecture, and our ability to assay myosin-I, -II, and -V function using both in vivo and in vitro approaches. In the long-term we aim to understand mechanisms specifying myosin motor function in human cells. We hypothesize that the composition of the actin track plays a crucial role in directing myosin motors to their appropriate actin structures. Aim 1 will employ a combination of time-lapse epi-fluorescence microscopy and biochemical assays to assess the contribution of actin filament cross-linkers (fimbrin and transgelin) in controlling tropomyosin (Tm) and myosin-I function at actin patches. Aim 2 will study the role of Tm, a-actinin, and the myosin-II tail in actomyosin ring function. In vitro myosin-bead assays will be used to define the mechanism by which Tm promotes myosin-II motility during ring assembly. Time-lapse epi- fluorescence and laser-scanning confocal microscopy will be used along with in vitro studies to examine the role of actin filament cross-linking by a-actinin during ring constriction and remodeling. The ability of the tail to direct the self-assembly of myosin-II into ensembles (that favor ring assembly) will also be tested using analytical ultracentrifugation and electron microscopy. Aim 3 seeks to understand how the actin track regulates myosin-V transport along cables. Myosin-bead assays and in vivo tracking of Myo52p motility by total internal reflection fluorescence (TIRF) microscopy will be employed to determine how Tm and parallel filament bundling regulate myosin-V motor function and intracellular motility. Our studies focus on the role of highly conserved actin-binding proteins and will have implications for actin function in human cancers. Changes in actin structure facilitates cell transformation, as actomyosin stress fibers make way for a more dynamic network facilitating cell motility and metastasis. Cell proliferation relies on actomyosin rings to power division. The >40 isoforms of Tm found in human cells are believed to play a key role in specifying different actomyosin structures. However, this idea has been difficult to test because it is not yet known which Tm isoforms regulate which specific myosin isoforms in such complex, non-muscle cells. Our use of fission yeast (with 1 Tm isoform and 5 myosins total) will overcome this complexity and provide new and novel insights into the specification of actomyosin structures. PUBLIC HEALTH RELEVANCE: Changes in specific Tm isoform levels correlate with changes in actin organization favoring metastasis or proliferation of cancer cells. Defective cytokinesis leads to unequal division of chromosomes (aneuploidy) and supernumerary centrosomes, characteristics of many human tumors. Our studies on actomyosin contractile rings and other actin structures in yeast will shed light on fundamental molecular mechanisms governing actin re-organization and function in cancer cells.

描述（由申请人提供）：该提案解决了细胞骨架领域中的一个基本问题：细胞中的不同肌动蛋白结构如何募集适当的肌动蛋白结合蛋白，以使其适应其给定角色？目前，我们专注于肌动蛋白I如何指向内吞作用部位（肌动蛋白斑块），肌球蛋白-II在细胞因子过程中如何在收缩环上起作用，以及如何指定肌动蛋白V沿肌动蛋白电缆的细胞内转运。裂变酵母目前是我们的首选模型，这是由于其可进行的遗传学，定义明确的肌动蛋白结构以及我们使用体内和体外方法测定肌球蛋白I，-II和-V功能的能力。从长远来看，我们旨在了解指定人类细胞中肌球蛋白运动功能的机制。 我们假设肌动蛋白轨道的组成在将肌球蛋白电机引导到其适当的肌动蛋白结构中起着至关重要的作用。 AIM 1将采用延时荧光显微镜和生化测定的结合，以评估肌动蛋白丝交联链（FIMBRIN和TRANSGELIN）在控制肌动蛋白斑块对肌动蛋白（TM）和肌动蛋白-I功能中的贡献。 AIM 2将研究TM，A-肌动蛋白和肌球蛋白-II尾巴在肌动蛋白环功能中的作用。体外肌球蛋白珠测定法将用于定义TM在环组装过程中促进肌球蛋白-II运动的机制。延时表演荧光和激光扫描共聚焦显微镜将与体外研究一起使用，以检查肌动蛋白丝丝在环收缩和重塑过程中通过A-肌动蛋白交联的作用。尾巴将肌球蛋白-II自组装引导到合奏（该偏爱环组件）的能力也将使用分析超速离心和电子显微镜进行测试。 AIM 3试图了解肌动蛋白轨道如何调节沿电缆的肌球蛋白V传输。将使用总内反射荧光（TIRF）显微镜对Myo52p运动的肌球蛋白珠测定和体内跟踪，以确定如何调节肌球蛋白V运动功能和细胞内运动。 我们的研究着重于高度保守的肌动蛋白结合蛋白的作用，并将对人类癌症中的肌动蛋白功能产生影响。肌动蛋白结构的变化促进了细胞转化，因为肌动球蛋白应力纤维为更动态的网络促进细胞运动和转移而铺平了道路。细胞增殖依赖于肌动蛋白环用于电力部。据信，在人类细胞中发现的TM的40种同工型在指定不同的肌球蛋白结构中起关键作用。但是，这个想法很难测试，因为尚不清楚哪种TM同工型调节这种复杂的非肌肉细胞中哪种特定的肌球蛋白同工型。我们使用裂变酵母（具有1 TM同工型和5种肌球蛋白）将克服这种复杂性，并为肌动蛋白结构的规范提供新的新颖见解。 公共卫生相关性：特定TM同工型水平的变化与有利于转移或癌细胞增殖的肌动蛋白组织的变化相关。缺陷的细胞因子会导致染色体（非整倍性）和超鼻中心体的不平等分裂，许多人类肿瘤的特征。我们对酵母中肌动球蛋白收缩环和其他肌动蛋白结构的研究将阐明癌细胞中肌动蛋白重组和功能的基本分子机制。