Specification of actomyosin function in the cell

细胞内肌动球蛋白功能的规范

• 批准号: 8413608
• 负责人: Matthew Lord
• 金额: $ 28.7万
• 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/8413608
• 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.

描述（由申请人提供）：该提案解决了细胞骨架领域的一个基本问题：细胞中不同的肌动蛋白结构如何招募适当的肌动蛋白结合蛋白以适应其给定的作用？目前，我们的重点是肌球蛋白-I 如何定向到内吞作用位点（肌动蛋白斑块）、肌球蛋白-II 在胞质分裂过程中如何在收缩环上发挥作用，以及肌球蛋白-V 如何沿着肌动蛋白电缆进行细胞内运输。裂殖酵母目前是我们选择的模型，因为它具有易处理的遗传学、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，我们的目标是了解人类细胞中指定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马达引导至适当的肌动蛋白结构方面起着至关重要的作用。目标 1 将结合使用延时落射荧光显微镜和生化检测来评估肌动蛋白丝交联剂（纤毛蛋白和转胶蛋白）在控制肌动蛋白斑块上的原肌球蛋白 (Tm) 和肌球蛋白-I 功能方面的贡献。目标 2 将研究 Tm、α-肌动蛋白和肌球蛋白-II 尾部在肌动球蛋白环功能中的作用。体外肌球蛋白珠测定将用于确定 Tm 在环组装过程中促进肌球蛋白-II 运动的机制。延时落射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α-肌动蛋白在环收缩和重塑过程中肌动蛋白丝交联的作用。尾部引导肌球蛋白-II 自组装成整体（有利于环组装）的能力也将使用分析超速离心和电子显微镜进行测试。目标 3 旨在了解肌动蛋白轨道如何调节肌球蛋白-V 沿电缆的运输。肌球蛋白珠测定和通过全内反射荧光 (TIRF) 显微镜对 Myo52p 运动的体内跟踪将用于确定 Tm 和平行丝束如何调节肌球蛋白-V 运动功能和细胞内运动。 我们的研究重点是高度保守的肌动蛋白结合蛋白的作用，并将对人类癌症中肌动蛋白的功能产生影响。肌动蛋白结构的变化促进细胞转化，因为肌动球蛋白应力纤维为更动态的网络让路，促进细胞运动和转移。细胞增殖依赖于肌动球蛋白环来驱动分裂。在人类细胞中发现的超过 40 种 Tm 亚型被认为在指定不同的肌动球蛋白结构中发挥着关键作用。然而，这个想法很难检验，因为尚不清楚哪种 Tm 同工型在这种复杂的非肌肉细胞中调节哪些特定的肌球蛋白同工型。我们使用裂殖酵母（总共有 1 个 Tm 亚型和 5 个肌球蛋白）将克服这种复杂性，并为肌动球蛋白结构的规范提供新的见解。