cryoEM Studies of Muscle

项目摘要 这个研究项目的长期目标一直是，并将继续是对分子的理解 肌肉功能的机制。现有的项目自1983年以来一直在资助，并同步发展。 既有显微镜的能力，又有从电子中重建三维图像的方法 显微镜。最初专注于了解肌肉中肌球蛋白-肌动蛋白的原位相互作用 通过化学固定、塑料嵌入和肌肉切片，它现在提出利用分辨率 以最高分辨率研究肌肉主要结构元素的低温电子显微镜的革命 使用分离的组分，然后在冷冻活体中原位成像肌动蛋白-肌球蛋白相互作用 肌肉细胞。横纹肌有四个主要成分：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形圆盘和用于连接的连接丝 将粗细的细丝连接到Z盘。这四种元素中最不为人所知的是粗丝，即Z形圆盘 以及连接丝，它与粗丝和Z盘的相互作用最不为人所知 元素。因此，我们对Z圆盘和粗丝的研究可以对理解 所有这三个元素。在我们的一项重大突破表明肌球蛋白的卷曲尾部结构域之后 可以亚纳米分辨率成像，甚至接近原子分辨率，该项目最初专注于 几个物种的粗丝的亚纳米分辨率成像，以检查共性和 跨物种和肌肉类型的“弯曲分子晶层”的结构保守性。这个 该项目将利用果蝇，黑腹果蝇，来研究如何遗传移除某些 组成蛋白影响肌球蛋白尾部如何与非肌球蛋白相互作用，从而影响粗丝 属性。我们将利用果蝇肌球蛋白尾巴中与既定疾病相对应的突变 导致人类横纹肌发生突变。Z-Disk将使用在我们的 实验室从无脊椎动物中分离Z盘用于果蝇Z盘的测定。这个 实验系统将有助于用各种信号蛋白修饰Z盘。归根结底， 这些成分研究的实用性需要在肌原纤维内开发。我们将通过以下方式进行调查 冷冻电子断层扫描冷冻水合果蝇和石斑鱼的肌原纤维变薄 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学到的方法论应用到 活培养的血管和心肌细胞快速冷冻，通过FIB/SEM稀释以观察活跃的相互作用 在粗丝和细丝之间。这项工作将向未来的结构研究开放所有的结构 在其自然环境中存在于肌肉细胞中。

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.