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 过去的十年见证了冷冻电子显微镜（Cryo-EM）的显着改善 仪器，可以使用冷冻电子来对感兴趣的单元格的超微结构进行成像 断层扫描（冷冻-ET）。血小板通过形成凝块并停止出血在止血中起着至关重要的作用 血管损伤部位。静止的血小板的形状盘状。激活后血小板发生 戏剧性的形态变化，包括细胞骨架重排，膜受体激活和 质膜和颗粒释放中的重新分布。这些结构性变化与血小板有关 功能障碍，对包括血小板储存病变在内的出血疾病有影响， 血小板减少症和血栓形成。当前对血小板超微结构的理解主要来自 1950 - 1980年代的传输电子显微镜（TEM）研究。常规化学固定 已知TEM的生物样品在血小板细胞器中引起结构伪影 大分子。血小板的尺寸为3-5 µm，厚度为1-2 蜂窝冷冻-ET，我们在该项目中建议将尖端的低温ET方法应用于血小板并发展 有效的两个特定目标。在特定目标1中，我们将为3-开发多模式协议 人和鼠血小板的尺寸（3D）冷冻成像。包括整体在内的新成像方法 细胞冷冻-ET，相关光电子显微镜和无孔相位板的对比度增强的冷冻 ET将开发以可视化野生型小鼠，健康的人类供体和患者的血小板 异常颗粒。协议开发包括冷冻特性准备，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。 在特定的目标2中，我们将应用开发的冷冻ET协议来表征3D超微结构 血小板具有治疗意义。由于存储的短货架寿命有助于严重 可用于医院输血治疗的血小板短缺，制冷是一种潜在的 储存血小板的有前途的方法，以最大程度地减少细菌的生长并减少新陈代谢 贮存。但是，制冷会导致血小板的形态变化，并导致其快速清除 输血后。我们将使用冷藏的血小板作为开发冷冻-ET成像的模型系统 协议。新鲜和冷藏血小板中细胞超微结构的表征和比较将 进行，重点是微管和肌动蛋白细胞骨架的变化以及血小板的聚类 质膜上的受体。总体而言，在这个项目中，我们建议建立一个强大的尖端 血小板的冷冻成像协议，可以适应其他类型的细胞。可视化 空前结构分辨率的血小板还将详细比较健康和 患病的血小板，建立一个研究血小板生理学和病理生理学的生产平台。