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

横纹肌是一种了不起的组织。在每个肌肉细胞的细胞质中都有一个高度组织的细胞骨架晶格，其中肌动蛋白和肌球蛋白以完全有序的、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，可以产生工作和运动的力量，这在许多门动物中都有很强的进化保守性。肌凝蛋白马达组装成特定且均匀长度的双极细丝的方式，是正常肌肉功能的要求，长期以来一直是一个非常有趣的话题，但很少找到具体的答案。本研究的一个重要目标是通过研究果蝇飞行的功能特性来解决肌肉生物学中的这个基本问题。该实验室之前的研究表明，果蝇的间接飞行肌（IFM）粗丝在没有飞行的情况下会聚集到异常长的长度。本项目将结合体内和体外方法，建立飞行蛋白参与IFM发育过程中粗丝长度确定的机制。这项工作的中心论点是飞行蛋白是一种肌凝蛋白结合蛋白。第一个目标将是测试飞行蛋白与肌凝蛋白棒区域的两个位点结合的假设。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来测试飞行蛋白的结合。第二个目标将是探索飞行蛋白磷酸化在粗丝组装中的作用。相互竞争的假设，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飞行蛋白的转基因果蝇进行测试。电子显微镜将用于评估突变体飞行蛋白对肌节和粗丝长度的影响。这些研究将伴随着体外实验，以测试飞行蛋白对肌球蛋白聚合的影响。经典遗传学和分子遗传学与生物化学、细胞结构和生理/生物物理学的结合将导致对飞行蛋白功能作用的连贯、综合的看法，并为厚丝组装机制和拉伸激活机制提供有价值的见解。

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.