THICK FILAMENT MYOSIN AND C-PROTEIN ARRANGEMENT

肌球蛋白和辅助蛋白如C-蛋白的排列 鱼、鸟和哺乳动物粗丝中的蛋白质 骨骼肌仍未被清楚地阐明。最近的工作在 然而，我们实验室对青蛙粗丝的结构进行了研究， 已经证明脊椎动物肌肉的结构可能是 可以通过结合电子显微镜进行分析， 光学衍射分析和计算机图像分析。 此外，已经表明，来自FICH的粗丝， 鸟类和哺乳动物的肌肉可以用类似的方法分离出来 排列整齐的交叉桥；因此使这些细丝 适用于具有相同类型的分析技术 证明对青蛙的粗丝很有用。的具体目的 拟议的研究是为了扩展用于 青蛙粗丝的结构测定 鱼、鸟和哺乳动物骨骼肌的粗丝。 这些研究的主要目标将是确定 肌球蛋白及其附属蛋白在人体内的排列 这些脊椎动物的细丝。要完成的研究 这些目标将包括：1)使用否定词的组合 染色，铂阴影，快速冷冻深低温蚀刻研究， 和光学衍射分析来阐明主要的 细丝的超微结构特征，并评估 与结构相比较的细丝的保存 生物X射线衍射图的预期参数 2)利用计算机图像分析技术对细丝进行分析 最好的电子显微照片中的图像来确定 肌球蛋白头部的旋转对称性和排列，以及 制作一个三维重建的结构 细丝；3)使用免疫标记研究 C-蛋白和C-蛋白等辅助蛋白的可能定位 细丝上的X蛋白，并将这一信息与 在三维空间中看到的细丝的结构 重建。这些研究将提供基本的科学依据 有关肌肉结构的信息，因此将提供 更好地了解病理情况 可能会影响这种组织的功能。

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.