cryoEM Studies of Muscle

肌肉的冷冻电镜研究

• 批准号: 10551733
• 负责人: KENNETH ALLEN TAYLOR
• 金额: $ 44.18万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10551733
• 关键词: Actins; Affect; Cardiac Myocytes; Chemicals; Cryo-electron tomography; Cryoelectron Microscopy; Defect; Development; Drosophila genus; Drosophila melanogaster; Electron Microscope; Elements; Excision; Filament; Freezing; Funding; Future; Genetic; Goals; Human; Hydration status; Image; In Situ; Invertebrates; Investigation; Learning; Link; Methodology; Methods; Microscope; Molecular; Muscle; Muscle Cells; Muscle function; Mutation; Myofibrils; Myopathy; Myosin ATPase; Nucleotides; Plastic Embedding; Property; Proteins; Research Project Grants; Resolution; Sarcomeres; Signaling Protein; Smooth Muscle Myocytes; Striated Muscles; Structure; System; Tail; Thick Filament; Thin Filament; Thinness; Three-Dimensional Image; Three-Dimensional Imaging; Vertebral column; Visualization; Work; crosslink; disease-causing mutation; image reconstruction; nanometer resolution; plasma protein Z

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.

项目概要 该研究项目的长期目标一直是并将继续是了解分子 现有的项目自 1983 年以来不断得到资助，并同时发展。 具有显微镜和电子 3D 图像重建方法的功能 最初专注于使用显微镜了解肌肉中的肌球蛋白-肌动蛋白相互作用。 化学固定、塑料嵌入和切片肌肉，现在建议利用该决议 冷冻电镜革命，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，然后返回冷冻活体原位成像肌动蛋白-肌球蛋白相互作用 横纹肌有四个主要成分：含有肌动蛋白的细丝、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 粗细丝到 Z 盘 这四个元素中最不为人所知的是粗细丝，即 Z 盘。 和连接细丝，其与粗细丝和 Z 盘的相互作用是人们最不了解的 因此，我们对 Z 盘和粗丝的研究可以为理解元素做出重大贡献。 我们的一项重大突破表明了肌球蛋白的卷曲螺旋尾部结构域。 可以以亚纳米分辨率甚至接近原子分辨率成像，该项目专注于 对多个物种的粗丝进行亚纳米分辨率成像，以检查其普遍性和 跨物种和肌肉类型的“弯曲分子结晶层”的结构保守。 该项目将利用果蝇（Drosophila melanogaster）来研究如何去除某些基因 成分蛋白影响肌球蛋白尾部与非肌球蛋白蛋白相互作用以增粗影响丝 我们将利用果蝇肌球蛋白尾部与已确定疾病相对应的突变。 导致人类横纹肌突变的 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，用于测定果蝇 Z 盘。 实验系统将有助于用各种信号蛋白装饰 Z 盘。 这些研究对肌原纤维内需要开发的成分的效用我们将通过以下方式进行调查。 使用 FIB/SEM 进行冷冻电子断层扫描，使 Lethocerus 和果蝇的冷冻水合肌原纤维变薄 使用各种核苷酸产生的状态，以观察肌球蛋白头如何与细丝原位相互作用。 最终，我们将从这些肌原纤维研究中学到的方法学知识应用到以下方面的研究中： 活体培养的平滑肌细胞和心肌细胞快速冷冻，通过 FIB/SEM 稀释以可视化主动相互作用 这项工作将为未来的结构研究开放所有结构。 存在于自然环境下的肌肉细胞中。