Functional Studies of Flightin, a Thick Filament Protein of Drosophila Flight Muscles

摘要本项目的目的是确定飞行蛋白的功能特性，飞行蛋白是一种与昆虫飞行肌粗丝相关的蛋白质。这种蛋白质很有趣，因为它可能与这种肌肉的一些特殊特性有关，即它的快速跳动。与典型的脊椎动物肌肉细胞的收缩和放松与细胞内钙浓度的上升和下降相协调的循环不同，昆虫的飞行肌肉在钙浓度保持高的情况下经历持续的快速收缩和放松循环。飞行蛋白被发现与昆虫细胞的肌凝蛋白厚丝紧密相关。Flightin可能只是稳定细胞中高度组织的纤维结构，或者可能在肌动蛋白和肌凝蛋白相互作用期间参与一种根本不同类型的收缩控制。在其他肌肉细胞类型中也可能发现Flightin或其相关基因，其中Flightin可能具有非常不同或相关的功能特性。该项目将解决三个具体问题，以了解果蝇（Drosophila melanogaster）的飞行功能，果蝇因其作为强大模型系统的许多优势而被选中。(1)缺乏飞行蛋白的突变飞行肌细胞在收缩时是否比正常细胞更不稳定？如果飞行蛋白在稳定肌动蛋白和肌凝蛋白丝的高度有序排列中起着基本的结构作用，这是可以预料的。(2)飞行蛋白与肌凝蛋白丝相互作用的确切方式是什么？详细的结合分析应该阐明飞行蛋白如何稳定丝结构或控制肌动蛋白-肌凝蛋白相互作用，因为许多肌动蛋白和肌凝蛋白结构的细节已经知道。(3)飞行蛋白分子的磷酸化在控制其活性中起什么作用？由于该分子可以存在于磷酸化和非磷酸化状态，类似于其他磷酸化蛋白的磷酸化c状态可以被酶控制，很可能这些不同的状态在功能上也是不同的。在flightin的发现中发挥了重要作用的首席研究员将纯化flightin以研究其结合特性、磷酸化位点和其他生化特性。他和他的联合首席研究员将整合各种其他技术和方法的结果，包括电子显微镜，分子和经典遗传学，以及生理学技术，为这些问题提供清晰连贯的答案。＊＊＊

9728868 Vigoreaux Lay Abstract The goal of this project is to determine the functional properties of flightin, a protein associated with the thick filaments of insect flight muscle. This protein is interesting because it may be involved in some of the special properties of this muscle, namely its very rapid beating. Unlike a typical vertebrate muscle cell which contracts and relaxes in a cycle coordinated with a rise and then fall in the concentration of intracellular calcium, insect flight muscle undergoes continuing rapid cycles of contraction and relaxation while the calcium concentration remains high. Flightin is found tightly associated with the myosin thick filaments of the insect cells. Flightin may simply stabilize the highly organized filament architecture in the cells or may be involved in a fundamentally different type of contraction control during the interaction of actin and myosin. Flightin or a relative may also be found in other muscle cell types where it may have very different or related functional properties. The project will address three specific questions to understand flightin function in the fruit fly, Drosophila melanogaster, chosen for its many advantages as a powerful model system. (1) Are mutant flight muscle cells that lack flightin less stable than normal cells when these cells contract? This would be expected if flightin plays a basic structural role to stabilize the highly ordered arrays of actin and myosin filament assemblies? (2) What is the precise way flightin interacts with the myosin filament? Detailed analysis of binding should elucidate how flightin might stabilize filament structure or control actin-myosin interaction since many details of the actin and myosin structures are already known. (3) What role does phosphorylation of the flightin molecule play in controlling its activity? Since the molecule can exist in phosphorylated and non-phosphorylated states, by analogy to other phosphorylated proteins whose state of phosphorylation c an be enzymatically controlled, it is very likely that these different states are also functionally different. The principal investigator, who was instrumental in the discovery of flightin, will purify flightin to study its binding properties, sites of phosphorylation, and other biochemical properties. He and his co-principal investigator will integrate results from a variety of other techniques and approaches, including electron microscopy, molecular and classical genetics, and physiological techniques, to provide clear and coherent answers to these questions. ***