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.

项目概要 过去十年冷冻电子显微镜 (cryo-EM) 取得了惊人的进步 仪器，使得使用冷冻电子对感兴趣的细胞的超微结构成像成为可能 断层扫描（冷冻电子断层扫描）中，血小板通过形成凝块和止血而在止血中发挥着至关重要的作用。 血管损伤部位的血小板呈盘状。 显着的形态变化，包括细胞骨架重排、膜受体激活和 质膜中的重新分布和颗粒释放与血小板有关。 功能障碍并对出血性疾病有影响，包括血小板储存病变， 目前对血小板超微结构的认识主要来源于血小板减少症和血栓形成。 1950-1980 年代的透射电子显微镜 (TEM) 研究。 已知用于 TEM 的生物样品会导致血小板细胞器和 血小板的大小，直径3-5μm，厚度1-2μm，有利于整体。 细胞冷冻电子断层扫描，我们在这个项目中建议将尖端的冷冻电子断层扫描方法应用于血小板并开发 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- 开发多模式协议。 人类和小鼠血小板的三维 (3D) 冷冻 ET 成像，包括整个血小板的新成像方法。 细胞冷冻 ET、相关光学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、健康人类捐赠者和患有糖尿病的患者的血小板 异常颗粒的方案开发包括冷冻样本制备、图像采集、图像。 分析重点是血小板细胞器和大分子的可视化和定量。 在具体目标 2 中，我们将应用开发的冷冻 ET 协议来表征 3D 超微结构 具有治疗意义的血小板，因为储存的血小板保质期短会导致严重的并发症。 医院可用于输血治疗的血小板短缺，冷藏是一个潜在的解决方案 储存血小板的有前途的方法，以最大限度地减少细菌生长并减少新陈代谢 然而，冷藏会导致血小板形态发生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。 新鲜和冷藏血小板的细胞超微结构的表征和比较。 进行，重点关注微管和肌动蛋白细胞骨架的变化以及血小板的聚集 总的来说，在这个项目中，我们建议建立一个强大的、尖端的受体。 用于血小板的冷冻 ET 成像协议，可适用于其他类型的细胞可视化。 血小板以前所未有的结构分辨率也将能够详细比较健康和 患病血小板，为研究血小板生理学和病理生理学建立了一个富有成效的平台。