The CryoEM Motility Assay

描述（由申请人提供）：使用荧光标记的蛋白质的体外运动性测定已经帮助定义了单个分子马达的功能，特别是那些在细胞骨架中的细丝轨道上运行的分子马达，所述细胞骨架包括F-肌动蛋白或微管.虽然这些实验已经定义了许多马达参数，但它们并不直接对马达分子成像，而是记录荧光报告分子的运动。该提案将建立在 通过开发冷冻电子显微镜（cryoEM）技术，在近26年的光学显微镜下通过体外运动试验对单分子马达进行检测，获得了丰富的经验，该技术将使活性肌球蛋白马达本身直接可视化，而不仅仅是其结合标签，作为异步功能马达群体的整体作用的“定格”。这项技术，被称为“CryoEM运动性测定”，将有助于三维成像的电机的合奏首先可视化的荧光显微镜，随后迅速冻结的三维可视化使用cryoEM。在某些情况下，这可以使得特定的马达分子能够以约2nm的分辨率以3D成像。许多肌肉疾病，特别是心肌疾病，已被发现是由肌球蛋白马达本身功能缺陷或细丝蛋白缺陷引起的。许多非肌肉组织的疾病是由于胞质肌球蛋白马达缺陷引起的。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运动性测定旨在解决这一缺陷。增强对主动马达结构的基本理解可能会导致治疗进展。 公共卫生相关性：许多肌肉疾病，特别是心肌疾病，已被发现是由肌球蛋白马达本身功能缺陷或细丝蛋白缺陷引起的。许多非肌肉组织的疾病是由于胞质肌球蛋白马达缺陷引起的.该项目将导致增强对主动马达结构的基本理解，这可能导致治疗进展。

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. PUBLIC HEALTH RELEVANCE: 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. This project will lead to enhanced basic understanding of the structure of active motors that may lead to therapeutic advances.