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.

项目总结 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，使得使用冷冻电子成像感兴趣的细胞的超微结构成为可能 体层摄影(冷冻-ET)。血小板通过形成血栓和止血在止血中起着至关重要的作用。 血管损伤的部位。静息状态的血小板呈盘状。在激活时，血小板会经历 显著的形态变化，包括细胞骨架重排，膜受体激活和 在质膜中重新分布，颗粒释放。这些结构变化与血小板有关 功能障碍，并对包括血小板储存损伤在内的出血障碍有影响， 血小板减少和血栓形成。目前对血小板超微结构的了解主要来源于 1950年-1980年S的透射电子显微镜研究。常规化学固定 已知用于透射电子显微镜的生物样本会导致血小板细胞器中的结构伪影和 大分子。直径3~5微米、厚度1~2微米的血小板大小有利于全血 细胞冷冻，我们在这个项目中建议将尖端的冷冻方法应用到血小板上，并开发 两个特定目标的有效工作流程。在具体目标1中，我们将制定多式联运协议，用于3- 人和小鼠血小板的三维(3D)冷冻成像。包括全息成像在内的新成像方法 细胞低温、相关光电子显微镜和无孔位相板对比度增强低温。 ET将被开发成可视化来自野生型小鼠、健康的人类捐赠者和患有癌症的患者的血小板 异常颗粒。方案开发包括低温样品制备、图像采集、图像 分析，重点是对血小板细胞器和大分子的可视化和量化。 在特定的目标2中，我们将应用开发的CRYO-ET方案来表征3D超微结构 具有治疗意义的血小板。由于储存的血小板保质期短，导致严重的 由于医院可供输血治疗的血小板短缺，冷藏是一种潜在的 一种有希望的保存血小板的方法，以最大限度地减少细菌生长和降低新陈代谢 储藏室。然而，冷藏会引起血小板的形态变化，并导致其快速清除 输液后。我们将使用冷冻的血小板作为发展冷冻成像的模型系统。 协议。新鲜和冷藏血小板细胞超微结构的表征与比较 重点是微管和肌动蛋白细胞骨架的变化，以及血小板的聚集 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、尖端的 适用于血小板的冷冻成像协议，可适用于其他类型的细胞。可视化 具有前所未有的结构分辨率的血小板也将能够详细比较健康和 疾病的血小板，为研究血小板生理学和病理生理学建立了一个生产性的平台。