Cryo-ET structural studies of platelets

血小板的冷冻电子断层扫描结构研究

• 批准号: 9920191
• 负责人: Renhao Li
• 金额: $ 19.5万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920191
• 关键词: 3-Dimensional; Actins; Address; Agonist; Algorithms; Biological; Biological Models; Blood Coagulation Disorders; Blood Platelet Disorders; Blood Platelets; Caliber; Cell membrane; Cells; Coagulation Process; Cryo-electron tomography; Cryoelectron Microscopy; Cytoplasmic Granules; Cytoskeleton; Development; Disease; Electron Microscopy; Electrons; F-Actin; Filopodia; Freezing; Functional disorder; Future; Growth; Hemorrhage; Hemostatic function; Hospitals; Human; Hydration status; Image; Image Analysis; Ions; Label; Length; Lesion; Life; Light; Link; Membrane; Membrane Glycoproteins; Metabolism; Methods; Microtubules; Monitor; Morphologic artifacts; Morphology; Mus; Organelles; Pathway interactions; Patients; Phase; Physiology; Platelet Activation; Play; Preparation; Protocols documentation; Receptor Activation; Refrigeration; Resolution; Rest; Role; Sampling; Shapes; Site; Specimen; Structure; Structure-Activity Relationship; Surface; Techniques; Technology; Therapeutic; Thick; Thrombocytopenia; Thrombosis; Time; Transfusion; Transmission Electron Microscopy; Visualization; Wild Type Mouse; cell type; chemical fixation; contrast enhanced; detector; human disease; human imaging; image processing; imaging modality; instrumentation; interest; macromolecule; microscopic imaging; multimodality; nanometer; oligomycin sensitivity-conferring protein; particle; platelet storage lesion; protocol development; receptor; reconstruction; tomography; vascular injury

PROJECT SUMMARY The past decade has witnessed dramatic improvements to cryo-electron microscopy (cryo-EM) instrumentation, making it possible to image the ultrastructure of a cell of interest using cryo-electron tomography (cryo-ET). Platelets play a vital role in hemostasis by forming a clot and stopping bleeding at the site of vascular injury. Resting platelets are discoid in shape. Upon activation platelets undergo dramatic morphological changes, including cytoskeletal rearrangement, membrane receptor activation and redistribution in the plasma membrane, and granule release. These structural changes are linked to platelet dysfunction and have implications for bleeding disorders including platelet storage lesions, thrombocytopenia, and thrombosis. Current understanding of platelet ultrastructure is derived mainly from transmission electron microscopy (TEM) studies in the 1950-1980’s. Conventional chemical fixation of biological samples for TEM is known to cause structural artifacts in platelet organelles and macromolecules. The sizes of platelets, 3-5 µm in diameter and 1-2 in thickness, is conducive to whole cellular cryo-ET, we propose in this project to apply cutting-edge cryo-ET methods to platelets and develop effective workflows in two Specific Aims. In Specific Aim 1, we will develop multimodal protocols for 3- dimensional (3D) cryo-ET imaging of human and murine platelets. New imaging methods including whole cellular cryo-ET, correlative light electron microscopy, and hole-free phase-plate contrast enhanced cryo- ET will be developed to visualize platelets from wild-type mice, healthy human donors, and patients with abnormal granules. Protocol development includes cryo-specimen preparation, image acquisition, image analysis with an emphasis on visualization and quantification of platelet organelles and macromolecules. In Specific Aim 2, we will apply the developed cryo-ET protocols to characterize the 3D ultrastructure of platelets with therapeutic implications. As short shelf-life of stored platelets contributes to the severe shortage of platelets available for transfusion treatment in the hospital, refrigeration is a potentially promising method to store platelets in order to minimize bacterial growth and reduce metabolism during storage. However, refrigeration causes morphological changes of platelets and leads to their fast clearance after transfusion. We will use refrigerated platelets as a model system for developing the cryo-ET imaging protocols. Characterization and comparison of cellular ultrastructure in fresh and refrigerated platelets will be carried out, with a focus on changes in microtubules and the actin cytoskeleton, and clustering of platelet receptors on the plasma membrane. Overall, in this project we propose to establish a robust, cutting-edge cryo-ET imaging protocol for platelets, which can be adapted to other types of cells. Visualization of platelets at unprecedented structural resolution will also enable detailed comparison of healthy and diseased platelets, establishing a productive platform for studying platelet physiology and pathophysiology.

项目摘要 在过去的十年里，低温电子显微镜（cryo-EM）取得了巨大的进步 仪器，使得有可能使用冷冻电子成像感兴趣的细胞的超微结构 断层扫描（cryo-ET）。血小板在止血中起着至关重要的作用，通过形成凝块并在一定程度上止血。 血管损伤的部位静息血小板呈盘状。血小板活化后， 戏剧性的形态学变化，包括细胞骨架重排，膜受体活化和 在质膜中的再分布和颗粒释放。这些结构变化与血小板 功能障碍并暗示出血性疾病包括血小板储存损伤， 血小板减少症和血栓形成。目前对血小板超微结构的认识主要来自于 透射电子显微镜（TEM）研究在1950-1980年代。常规化学固定 已知用于TEM的生物样品在血小板细胞器中引起结构伪影， 大分子血小板的大小，直径为3-5微米，厚度为1-2微米，有利于整体 细胞冷冻ET，我们在这个项目中建议将尖端的冷冻ET方法应用于血小板， 在两个具体目标的有效工作流程。在具体目标1中，我们将为3- 人和鼠血小板的三维（3D）冷冻ET成像。新的成像方法，包括整体 细胞冷冻ET，相关光电子显微镜，和无孔相位板对比增强冷冻， ET将被开发用于可视化来自野生型小鼠、健康人类供体和患有血小板减少症的患者的血小板。 异常颗粒方案制定包括冷冻标本制备、图像采集、图像处理、 血小板细胞器和大分子的可视化和定量分析。 在具体目标2中，我们将应用开发的cryo-ET方案来表征 具有治疗意义的血小板。由于储存血小板的保质期短， 由于医院缺乏可用于输血治疗的血小板，冷藏是一种潜在的 储存血小板以最大限度地减少细菌生长和减少代谢的有前途的方法， 存储.然而，冷藏引起血小板的形态学变化，并导致其快速清除 输血后我们将使用冷冻血小板作为开发冷冻ET成像的模型系统 协议.新鲜和冷藏血小板细胞超微结构的特征和比较 进行，重点是微管和肌动蛋白细胞骨架的变化，以及血小板聚集 细胞膜上的受体。总的来说，在这个项目中，我们建议建立一个强大的，先进的 用于血小板的冷冻ET成像方案，其可适用于其他类型的细胞。可视化 血小板在前所未有的结构分辨率也将使详细比较健康和 疾病血小板，建立一个生产平台，研究血小板生理和病理生理。