The Role of Flightin in Thick Filament Assembly and Flight Muscle Mechanics

Flightin 在粗丝组装和飞行肌肉力学中的作用

• 批准号: 0090768
• 负责人: Jim Vigoreaux
• 金额: $ 24万
• 依托单位: University of Vermont & State Agricultural College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0090768&HistoricalAwards=false
• 关键词: Role; Flightin; Thick; Filament; Assembly

Striated muscle is a remarkable tissue. Within the cytoplasm of each muscle cell is a highly organized cytoskeletal lattice where actin and myosin are assembled in perfectly ordered, interdigitating arrays of thin and thick filaments. The architecture of the sarcomere is extraordinarily well designed to generate force for work and locomotion, as evidenced by its strong evolutionary conservation across animals of many phyla. The way in which myosin motors assemble into bipolar filaments of specific and uniform length, a requirement for proper muscle function, has long been a topic of great interest but few concrete answers have been found. An important goal of this research is to address this fundamental question in muscle biology through theinvestigation of the functional properties of Drosophila flightin. Previous work from this lab has shown that Drosophila indirect flight muscle (IFM) thick filaments assemble to abnormally long lengths in the absence of flightin. This project will combine in vivo and in vitro approaches to establish the mechanism by which flightin participates in thick filament length determination during IFM development. A central thesis of this work is that flightin is a myosin binding protein. The first aim will be to test the hypothesis that flightin binds to two sites in the myosin rod region. Recombinant fragments representing distinct parts of the myosin heavy chain protein will be tested for flightin binding using four different biochemical assays. The second aim will be to explore the role of flightin phosphorylation in thick filament assembly. Competing hypotheses, that phosphorylation is or is not required for proper assembly, will be tested using transgenic flies that express phosphorylation-site mutant flightin. Electron microscopy will be used to assess the effect of mutant flightin on sarcomere and thick filament length. These studies will be accompanied by experiments in vitro that will test the effect of flightin on myosin polymerization.The combination of classical and molecular genetics with biochemistry, cell structure and physiology/biophysics will result in a coherent, integrated view of the functional roles of flightin and provide valuable insight into the mechanism of thick filament assembly and the mechanism of stretch activation.

横纹肌是一种了不起的组织。在每个肌肉细胞的细胞质中是一个高度组织的细胞骨架晶格，肌动蛋白和肌球蛋白以完美有序的交错排列的细丝和粗丝组装在一起。肌节的结构设计得非常好，可以为工作和运动产生力量，这一点从它在许多门动物中强大的进化保守性就可见一斑。肌球蛋白马达如何组装成特定和统一长度的双极细丝，这是肌肉功能正常所必需的，长期以来一直是人们非常感兴趣的话题，但几乎没有找到具体的答案。本研究的一个重要目标是通过研究果蝇flightin的功能特性来解决肌肉生物学中的这一基本问题。该实验室之前的工作表明，在没有飞行蛋白的情况下，果蝇间接飞行肌肉(IFM)的粗丝可以聚集成异常长的长度。这个项目将结合体内和体外的方法来建立flightin在IFM发育过程中参与粗丝长度确定的机制。这项工作的一个中心论点是，flightin是一种肌球蛋白结合蛋白。第一个目标是验证Flightin与肌球蛋白杆区两个位置结合的假设。代表肌球蛋白重链蛋白不同部分的重组片段将使用四种不同的生化分析方法进行飞行蛋白结合测试。第二个目标将是探索Flightin磷酸化在粗丝组装中的作用。相互竞争的假设，即磷酸化是或不是正确组装所必需的，将使用表达磷酸化位点突变的flightin的转基因果蝇进行测试。电子显微镜将被用来评估突变的flightin对肌节和粗丝长度的影响。这些研究将伴随着体外实验，以测试flightin对肌球蛋白聚合的影响。将经典遗传学和分子遗传学与生物化学、细胞结构和生理学/生物物理学相结合，将导致对flightin功能作用的一致、完整的看法，并为粗丝组装和拉伸激活机制提供有价值的见解。