The CryoEM Motility Assay

描述(申请人提供)：使用荧光标记蛋白质的体外运动性分析有助于确定单个分子马达的功能，特别是那些运行在细胞骨架中细丝轨迹上的分子马达，这些细胞骨架由F-肌动蛋白或微管组成。虽然这些实验定义了许多马达参数，但它们并不直接成像马达分子，而是记录荧光报告器的运动。这项提议将建立在 近26年来，通过开发低温电子显微镜(CryoEM)技术对单分子马达进行体外运动性分析，获得了丰富的经验，该技术将使活跃的肌球蛋白马达本身直接可视化，而不仅仅是它们的结合标签，作为一组异步功能马达整体动作的“定格框架”。这项被称为“低温电磁动力分析”的技术将有助于对马达整体进行三维成像，首先通过荧光显微镜进行可视化，然后快速冻结，以便使用低温电磁进行三维可视化。在某些情况下，这可能使特定的马达分子能够以~2纳米的分辨率在3-D中成像。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的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细胞质肌球蛋白马达的缺陷引起的。虽然生物化学经常描述酶的缺陷，但还不可能成像活性分子来第一手观察改变的结构或相互作用。低温EM动力分析试图解决这一缺陷。对主动运动结构的基本了解的加强可能会导致治疗的进展。

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.