THICK FILAMENT MYOSIN AND C-PROTEIN ARRANGEMENT

肌球蛋白和辅助蛋白如C- 鱼类、鸟类和哺乳动物的粗丝蛋白质 骨骼肌仍然没有被清楚地阐明。 最近的工作 我们的实验室对青蛙粗丝结构的研究， 已经证明脊椎动物肌肉的结构 可以通过电子显微镜的组合进行分析， 光学衍射分析和计算机图像分析。 此外，已经表明来自菲奇的粗长丝， 鸟类和哺乳动物的肌肉可以用类似的 有序排列的横桥;从而使这些细丝 适用于通过相同类型的技术进行分析， 被证明对青蛙的粗丝有用。 具体目标是 拟议的研究是扩展用于 青蛙粗丝的结构的测定 鱼类、鸟类和哺乳动物骨骼肌的粗丝。 这些研究的主要目标是确定 肌球蛋白和辅助蛋白在肌纤维中的排列 这些脊椎动物类的纤维。 要完成的研究 这些目标将包括：1）使用消极的组合 染色、铂阴影、快速冷冻深蚀刻低温研究， 和光学衍射分析，以阐明主要的 纤维的超微结构特征，并评估 与结构相比， 从X射线衍射图中预期的参数 肌丝的计算机图像分析 最好的电子显微镜图像，以确定 肌球蛋白头的旋转对称性和排列， 产生结构的三维重建， 细丝; 3）使用免疫标记研究来确定 辅助蛋白如C蛋白和 X-蛋白，并将此信息与 在三维空间中观察到的细丝结构 重建。 这些研究将提供基本的科学 关于肌肉结构的信息，因此将提供一个 更好地理解病理条件 可能会影响这个组织的功能

The arrangement of myosin and accessory protein such as C- protein in the thick filaments of fish, avian, and mammalian skeletal muscles is still not clearly elucidated. Recent work in our laboratory on the structure of frog thick filaments however, has demonstrated that the structure of vertebrate muscles may be amenable to analysis by a combination of electron microscopy, optical diffraction analysis, and computer image analysis. Additionally, it has been shown that thick filaments from fich, avian, and mammalian muscle can be isolated with a similarly well ordered array of crossbridges; thus making these filaments suitable for analysis by the same type of techniques which have proven useful for the frog thick filaments. The specific aim of the proposed research is to extend the techniques used for the frog thick filaments to the determination of the structure of the thick filaments of fish, avian, and mammalian skeletal muscles. The major objective of theses studies will be the determination of the arrangement of the myosin and accessory proteins in the filaments of these vertebrate classes. The studies to accomplish these aims will include: 1) the use of a combination of negative staining, platinum shadowing, rapid-freeze deep-etch cryo studies, and optical diffraction analysis to elucidate the major ultrastructural features of the filaments and to assess the preservation of the filaments as compared to the structural parameters expected from X-ray diffraction patterns of living muscle; 2) the use of computer image analysis of the filament images in the best electron micrographs to determine the rotational symmetry and arrangement of the myosin heads, and to produce a three-dimensional reconstruction of the structure of the filament; 3) the use of immunolabelling studies to determine the probable location of accessory proteins such as C-protein and X-protein on the filaments, and to correlate this information with the structure of the filament as seen in the three dimensional reconstructions. These studies will provide basic scientific information about the structure of muscle, and will thus provide a better foundation for understanding how pathological conditions may affect the functioning of this tissue.