The CryoEM Motility Assay

CryoEM 活力测定

基本信息

批准号：
8464005
负责人：
KENNETH ALLEN TAYLOR
金额：
$ 15.71万
依托单位：
FLORIDA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8464005
关键词：
3-Dimensional ATP phosphohydrolase Actins Address Affinity Back Binding Biochemistry Biological Assay Biotin Carbon Collodion Crosslinker Cryoelectron Microscopy Cytoskeleton Defect Development Disease Electron Microscopy Equus caballus F-Actin Filament Film Fluorescence Freezing Gel Head Image Imagery In Vitro Individual Label Lasers Lead Light Microscope Lipids Liquid substance Methods Microfilaments Microtubules Molecular Conformation Molecular Motors Motion Motor Movement Muscle Myocardium Myopathy Myosin ATPase Myosin Type II Nature Phase Population Preparation Proteins Psyche structure Pyroxylin Radiation Recombinants Reporter Resolution Running Smooth Muscle Solutions Specimen Structure System Techniques Technology Therapeutic Thick Filament Thin Filament Three-Dimensional Imaging Tissues Water cell motility crosslink experience falls light microscopy monolayer novel reconstitution research study tomography

项目摘要

DESCRIPTION (provided by applicant): In vitro motility assays using fluorescently labeled proteins have helped define the function of individual molecular motors, particularly those that run on filament tracks in the cytoskeleton which comprise either F- actin or microtubules. While these experiments have defined many motor parameters, they do not image the motor molecule directly, rather they record movements of the fluorescent reporter. This proposal will build on the extensive experience obtained over almost 26 years of light microscopy on single molecular motors by in vitro motility assays by developing a cryoelectron microscopy (cryoEM) technique that will enable the direct visualization of active myosin motors themselves, not just their bound labels, as "freeze frames" of the ensemble action of a population of asynchronously functioning motors. This technique, dubbed "The CryoEM Motility Assay" will facilitate 3-D imaging of an ensemble of motors first visualized by fluorescent light microscopy and subsequently rapidly frozen for 3-D visualization using cryoEM. In some cases this may enable particular motor molecules to be imaged in 3-D at ~2 nm resolution. Many muscle diseases, particularly those of heart muscle, have been found to be caused by either defects in the function of myosin motors themselves, or defects in the thin filament proteins. Many diseases of non-muscle tissue are due to defects in cytoplasmic myosin motors. While biochemistry has often described the enzymatic defect, it has not been possible to image the active molecules to observe firsthand the altered structure or interaction. The CryoEM Motility Assay seeks to address this deficiency. The enhanced basic understanding of the structure of active motors may lead to therapeutic advances.

描述（由申请人提供）：使用荧光标记蛋白的体外运动测定有助于确定单个分子马达的功能，特别是那些在包含F-肌动蛋白或微管的细胞骨架中的细丝轨道上运行的分子马达。虽然这些实验定义了许多运动参数，但它们并没有直接对运动分子进行成像，而是记录了荧光报告基因的运动。该提案将建立在通过开发冷冻电子显微镜 (cryoEM) 技术，通过体外运动测定，在近 26 年的单分子马达光学显微镜方面获得了丰富的经验，该技术将能够直接可视化活性肌球蛋白马达本身，而不仅仅是其结合的标签，如“冻结帧” “一组异步运行的电机的整体动作。这项被称为“CryoEM Motility Assay”的技术将有助于对一组电机进行 3D 成像，首先通过荧光显微镜进行可视化，然后使用 CryoEM 快速冷冻以进行 3D 可视化。在某些情况下，这可能使特定的运动分子能够以约 2 nm 的分辨率进行 3D 成像。已发现许多肌肉疾病，特别是心肌疾病，是由肌球蛋白马达本身的功能缺陷或细丝蛋白的缺陷引起的。许多非肌肉组织的疾病是由于细胞质肌球蛋白马达的缺陷造成的。虽然生物化学经常描述酶缺陷，但不可能对活性分子进行成像以直接观察改变的结构或相互作用。 CryoEM 活力测定旨在解决这一缺陷。增强对主动运动结构的基本了解可能会带来治疗进展。