Neutrophil Microscopy and Quantitative Imaging Core B

中性粒细胞显微镜和定量成像 Core B

• 批准号: 10470239
• 负责人: Bill William Kiosses
• 金额: $ 30.06万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-16 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10470239
• 关键词: 3-Dimensional; Actins; Adhesions; Apolipoprotein E; Apoptosis; Apoptotic; Arterial Fatty Streak; Autoimmunity; Azurophilic Granule; Bacteria; Biological Assay; Biology; Cardiovascular Diseases; Cell Death; Cell membrane; Cells; Cellular biology; Cessation of life; Collaborations; Coronary Arteriosclerosis; Cytoplasmic Granules; Cytoskeleton; Development; Dimensions; Disease; Docking; Electron Microscopy; Ensure; Event; Exocytosis; Fibrosis; Fluorescence Microscopy; Gelatinases; Generations; Goals; High Fat Diet; Human; Hyperlipidemia; Image; Inflammasome; Inflammation; Inflammatory; Light; Longevity; MMP9 gene; Mediating; Microscopy; Mitochondria; Modeling; Molecular; Mouse Strains; Multivesicular Body; Mus; Neutrophil Activation; Organelles; Pathway interactions; Patients; Performance; Phagocytosis; Play; Process; Production; Productivity; Program Research Project Grants; Proteins; Quality Control; Quantitative Microscopy; Reactive Oxygen Species; Research; Resolution; Resources; Role; Sampling; Stimulus; Technology; Time; Vasculitis; Vesicle; Work; analytical tool; autoinflammatory; base; cost; data sharing; design; extracellular; fungus; in vivo; innovation; intravital microscopy; light microscopy; live cell microscopy; microscopic imaging; migration; mouse model; nanometer; neutrophil; novel strategies; progenitor; quantitative imaging; single molecule; trafficking; western diet

SUMMARY Neutrophils are the first line of defense against bacteria and fungi and induce inflammation. The study of the molecular mechanisms mediating neutrophil activation is therefore of fundamental importance. Visualizing these mechanisms requires unique, state-of-the-art technologies and approaches. To achieve this, Core B is designed to coordinate, design, develop and execute new technical approaches applied to the understanding of neutrophil biology in inflammation. Core B will work with all three Projects and will provide relevant imaging and microscopy assays in mice and human samples. The core utilizes available cutting-edge technology to implement new experimental approaches and to help elucidate the intricate cell biology processes proposed in research plans of Projects 1 (Dr. Hedrick), Project 2 (Dr. Catz) and Project 3 (Dr. Hoffman). To this end, we will have the dedicated effort of a Technology Core director (Dr. Kiosses) who will allocate specific time and efforts to support the goals of the PPG. Core B optimizes the workflows of neutrophil imaging, image quantification and data sharing for all projects. It provides stringent quality controls and ensures uniformity of performance of multiple key technologies applied to the study of neutrophils and precursors in the setting of coronary artery disease. Core B will implement unique live-cell approaches to study neutrophil vesicular trafficking, cytoskeleton remodeling and cell death pathways. Core B is technically and conceptually innovative because it utilizes, neutrophil-dedicated, novel approaches that include super-resolution microscopy and quantitative analyses of neutrophil dynamics to elucidate neutrophil functions and to elucidate their role in inflammation. In relationship with Project 1, Core B will identify neutrophils and their precursors in atherosclerotic plaques during NETosis and pyroptosis in conditions of inflammasome activation at various stages of plaque development in high-fat diet, both ex-vivo and in vivo using the ApoE-/- Western diet model of CAD mice. In association with Project 2, Core B will utilize single-molecule super-resolution microscopy and Total Internal Reflection Fluorescence Microscopy to study molecular mechanisms regulating vesicular trafficking and actin remodeling in neutrophil precursors. Core B will also utilize Correlated-Light Electron Microscopy (CLEM) to characterize the newly identified precursor-specific granules (PSGs). In collaboration with Project 3, Core B will examine three-dimensional localization and spatial changes in the mitochondrial network as the cells undergo apoptotic or necroptotic cell death in both neutrophils and their precursors from mouse models and human neutrophils from patients with CAD. In conclusion, Core B is uniquely designed to use the valuable technology available in La Jolla to the fullest extent possible to maximize neutrophil-dedicate research and productivity associated with the PPG.

概括 中性粒细胞是抵抗细菌和真菌并诱发炎症的第一道防线。该研究的 因此，介导中性粒细胞激活的分子机制至关重要。可视化这些 机制需要独特、最先进的技术和方法。为了实现这一点，设计了Core B 协调、设计、开发和执行应用于理解中性粒细胞的新技术方法 炎症中的生物学。核心 B 将与所有三个项目合作，并提供相关的成像和显微镜检查 在小鼠和人类样本中进行测定。核心利用现有的尖端技术来实现新的 实验方法并帮助阐明研究计划中提出的复杂的细胞生物学过程 项目 1（Hedrick 博士）、项目 2（Catz 博士）和项目 3（Hoffman 博士）。为此，我们将有 技术核心总监（Kiosses 博士）的奉献精神，他将分配特定的时间和精力来支持 PPG 的目标。 Core B 优化中性粒细胞成像、图像量化和数据的工作流程 为所有项目共享。它提供严格的质量控制并确保多个性能的一致性 应用于冠状动脉疾病背景下中性粒细胞和前体细胞研究的关键技术。 Core B 将采用独特的活细胞方法来研究中性粒细胞囊泡运输、细胞骨架 重塑和细胞死亡途径。 Core B 在技术和概念上都是创新的，因为它利用了： 中性粒细胞专用的新颖方法，包括超分辨率显微镜和定量分析 中性粒细胞动力学，以阐明中性粒细胞功能并阐明其在炎症中的作用。在关系中 通过项目 1，核心 B 将在 NETosis 期间识别动脉粥样硬化斑块中的中性粒细胞及其前体 高脂饮食中斑块形成的各个阶段的炎症小体激活条件下的细胞焦亡， 使用 CAD 小鼠的 ApoE-/- 西方饮食模型进行离体和体内实验。与项目 2 相关，核心 B将利用单分子超分辨率显微镜和全内反射荧光显微镜 研究调节中性粒细胞前体中囊泡运输和肌动蛋白重塑的分子机制。 Core B 还将利用相关光电子显微镜 (CLEM) 来表征新发现的 前体特异性颗粒（PSG）。与项目 3 合作，核心 B 将检查三维 当细胞发生凋亡或坏死性细胞凋亡时，线粒体网络的定位和空间变化 小鼠模型中的中性粒细胞及其前体以及患有以下疾病的患者中的人类中性粒细胞死亡 计算机辅助设计。总之，Core B 的独特设计旨在充分利用拉霍亚现有的宝贵技术 尽可能最大限度地提高与 PPG 相关的中性粒细胞专用研究和生产力。