cryoEM Studies of Muscle

项目摘要 这项研究项目的长期目标一直是并将继续是了解分子 肌肉功能的机制。现有的项目自1983年以来一直得到资助， 利用显微镜和从电子显微镜重建三维图像的方法的能力， 显微镜.最初专注于了解肌球蛋白-肌动蛋白的相互作用，在肌肉原位使用 化学固定，塑料包埋和切片肌肉，它现在建议利用分辨率 cryoEM的革命，以尽可能高的分辨率研究肌肉的主要结构元素 使用分离的成分，然后返回到成像肌动蛋白-肌球蛋白的相互作用，在原位冷冻活 肌肉细胞横纹肌有四种主要成分：含肌动蛋白的细丝，肌球蛋白， 包含粗丝、用于交联反平行细丝的Z盘和用于连接的连接丝 将粗纤维连接到Z盘上这四个元素中最不为人所知的是粗丝，Z盘 连接丝与粗丝和Z盘的相互作用是最不清楚的 元素因此，我们对Z盘和粗丝的研究可以为理解 这三个要素。在我们的一项重大突破表明肌球蛋白的卷曲尾域 可以在亚纳米分辨率下成像，甚至接近原子分辨率，该项目最初集中在 亚纳米分辨率成像的厚丝从几个物种，以检查的一般性和 跨物种和肌肉类型的“弯曲分子晶体层”的结构保守性。的 该项目将利用果蝇，黑腹果蝇，调查如何遗传删除某些 组分蛋白质影响肌球蛋白尾部如何与非肌球蛋白相互作用以影响粗丝 特性.我们将利用果蝇肌球蛋白尾部的突变， 导致人类横纹肌突变Z盘将使用我们开发的方法进行研究 实验室从无脊椎动物中分离Z盘，用于测定黑腹果蝇Z盘。的 实验系统将有助于用各种信号蛋白装饰Z盘。最终 这些关于组分的研究的实用性需要在肌原纤维内进行开发。我们将调查 冷冻电子断层扫描用FIB/SEM细化的白蛉和果蝇的冷冻水化肌原纤维， 使用各种核苷酸产生的状态，以观察肌球蛋白头部如何与细丝原位相互作用。 最终，我们将把我们从这些肌原纤维研究中所学到的方法应用到以下研究中： 快速冷冻的活培养平滑肌细胞和心肌细胞，通过FIB/SEM进行稀释，以观察活性相互作用 在粗丝和细丝之间。这项工作将开放给未来的结构调查所有的结构 存在于肌肉细胞中。

Project Summary The long term goal of this research project has been and continues to be an understanding of the molecular mechanism of muscle function. The existing project, funded continuously since 1983, has evolved in parallel with the capabilities of both microscopes and methods for 3-D image reconstruction from electron microscopes. Originally focused on understanding myosin-actin interactions in situ in muscle using chemically fixed, plastic embedded and sectioned muscle, it now proposes to take advantage of the resolution revolution in cryoEM to study the major structural elements of the muscle at the highest resolution possible using isolated components, followed by a return to imaging actin-myosin interactions in situ in frozen live muscle cells. Striated muscles have four major components: actin-containing thin filaments, myosin- containing thick filaments, a Z-disk to crosslink antiparallel thin filaments and a connecting filament to link the thick filaments to the Z-disk. The least understood of these four elements are the thick filament, the Z-disk and the connecting filament, whose interactions with the thick filament and Z-disk are its least understood elements. Thus, our study of Z-disk and thick filament can make major contributions to an understanding of all three elements. Following a major breakthrough of ours that showed that coiled-coil tail domain of myosin can be imaged at subnanometer resolution, even near atomic resolution, the project concentrates initially on subnanometer resolution imaging of thick filaments from several species, to examine the generality and structural conservation of the “curved molecular crystalline layers” across species and muscle types. The project will utilize the fruit fly, Drosophila melanogaster, to investigate how genetic removal of certain component proteins affects how the myosin tails interact with non-myosin proteins to affect thick filament properties. We will utilize mutations in the myosin tail of Drosophila that correspond to established disease causing mutations in human striated muscle. The Z-disk will be studied using methodology developed in our lab to isolate Z-disks from invertebrates applied to determination of the Drosophila melanogaster Z-disk. The experimental system will facilitate decoration of the Z-disks with various signaling proteins. Ultimately, the utility of these studies on components needs development within the myofibril. We will investigate by cryoelectron tomography frozen-hydrated myofibrils of Lethocerus and Drosophila thinned using FIB/SEM in states produced using various nucleotides to see how myosin heads interact with the thin filament in situ. Ultimately, we will apply what we have learned methodologically from these myofibril studies to studies of live cultured smooth and cardiac muscle cells fast frozen, thinned via FIB/SEM to visualize active interactions between thick and thin filaments. This work will open to future structural investigation all the structures present in a muscle cell within their natural context.