Structural Role of Flightin in Thick Filaments of Drosophila Flight Muscle

智能运动是生物体的基本属性之一。在大多数动物中，运动是由横纹肌的收缩提供动力的，之所以这样称呼是因为它们高度规则和重复的蛋白质细丝组织，在显微镜下被视为条纹。所有横纹肌都含有粗丝，其主要成分是肌球蛋白，这是一种进化上保守的运动蛋白，负责产生肌肉收缩的力量。来自不同肌肉类型的粗丝具有由非肌球蛋白粗丝蛋白赋予的不同结构和机械特性。因此，这些蛋白质在确定肌肉类型之间的功能差异方面发挥着重要作用。这项研究将解决flightin的功能，flightin是一种新的粗丝蛋白，对果蝇（Drosophila melanogaster）飞行肌肉的结构完整性和机械稳定性至关重要。具体而言，本项目旨在阐明flightin和肌球蛋白之间的分子相互作用，通过体外生物化学研究和体内遗传学方法相结合。这项工作的一个关键假设是，一个保守的氨基酸区域flightin（约52个氨基酸）确定了一个新的肌球蛋白结合域，是广泛存在于两个主要群体的无脊椎动物，昆虫和甲壳类动物。为了阐明flightin功能的结构基础，一个详细的三维模型的厚丝从果蝇飞行肌肉将产生断层扫描从电子显微镜获得的图像。结合分子标记技术和比较有和没有flightin的细丝结构将导致在厚细丝中的flightin的识别和定位。这些研究将提供第一个更高分辨率的三维结构的粗丝从一个模型遗传生物，并将提供基础的结构分析过多的果蝇飞行肌肉突变体与已知的遗传，生化和机械缺陷。此外，该研究的影响将扩展到进化生物学和生物遗传学领域，这些领域旨在了解以昆虫为代表的生命形式的多样性，可以说昆虫是最成功的动物群体。更广泛的影响该研究将在教育领域以及为果蝇和肌肉研究界生产生物试剂方面产生影响。以前的NSF资助这个研究小组一直在培训本科生和研究生，包括来自代表性不足的群体和弱势背景的成员，该项目旨在将研究与继续教育机会相结合。从这项研究中产生的知识将找到它的方式，以先进的本科和研究生专业课程，评论文章和书籍章节打算为研究界，和文章和故事的非科学观众。结果将通过我们的网站，同行评审期刊上的出版物以及将在科学会议上展示其工作的本科生和研究生广泛传播。所有试剂（DNA克隆、突变株和转基因株）将免费提供给其他研究者。

Intellectual MeritMovement is one of the fundamental properties of living organisms. In most animals, movement is powered by contraction of striated muscles, so called because of their highly regular and repetitive organization of protein filaments that are seen as striations under a microscope. All striated muscles contain thick filaments whose main component is myosin, the evolutionarily conserved motor protein responsible for generating the force for muscle contraction. Thick filaments from different muscle types have distinct structural and mechanical properties that are imparted by non-myosin thick filament proteins. Thus, these proteins play a significant role in defining functional differences among muscle types. This research will address the functions of flightin, a novel thick filament protein that is essential for the structural integrity and mechanical stability of the flight muscle in the fruit fly, Drosophila melanogaster. Specifically, this project aims to elucidate the molecular interaction between flightin and myosin through a combination of in vitro biochemical studies and in vivo genetic approaches. A key hypothesis of this work is that a conserved amino acid region in flightin (of approximately 52 amino acids) identifies a new myosin binding domain that is widespread among two major groups of invertebrates, the insects and the crustaceans. To elucidate the structural basis of flightin function, a detailed three dimensional model of the thick filament from the fruit fly flight muscle will be generated by tomography from images obtained with an electron microscope. A combination of molecular labeling techniques and comparison of filament structures with and without flightin will result in the identification and localization of flightin in the thick filament. These studies will provide the first higher resolution three-dimensional structure of thick filaments from a model genetic organism and will provide the foundation for the structural analysis of a plethora of Drosophila melanogaster flight muscle mutants with known genetic, biochemical, and mechanical defects. Additionally, the impact of this research will extend to areas of evolutionary biology and phylogenetics that seek to understand the diversification of life forms as exemplified by the insects, arguably the most successful group of animals.Broader impactsThis research will have impacts in the areas of education and the generation of biological reagents for the Drosophila and muscle research communities. Previous NSF funding to this research group has been instrumental in training undergraduate and graduate students, including members from underrepresented groups and disadvantaged backgrounds, and this project is designed to integrate research with continuing educational opportunities. Knowledge generated from this research will find its way to advanced undergraduate and graduate specialty courses, review articles and book chapters intended for the research community, and articles and stories for the non-scientific audience. Results will be broadly disseminated through our website, publications in peer-reviewed journals, and by undergraduate and graduate students who will present their work at scientific meetings. All reagents (DNA clones, mutant and transgenic strains) will be freely available to other investigators.