Mechanism of kindlin-3-dependent integrin activation

kindlin-3依赖性整合素激活机制

• 批准号: 10229369
• 负责人: Klaus F. Ley
• 金额: $ 54.93万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-05 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10229369
• 关键词: Address; Affinity; Bacterial Infections; Binding; Binding Sites; Biochemical; Biological Assay; Blood Platelets; Bone Marrow; Bone Marrow Transplantation; Cell Adhesion; Cell membrane; Cell surface; Cells; Chimeric Proteins; Confocal Microscopy; Crystallization; Cytoplasmic Tail; Cytosol; Data; Dimerization; Disease; Dissociation; Epitopes; Extracellular Domain; Flow Cytometry; Fluorescence Resonance Energy Transfer; G-protein Beta gamma; Genes; Genetic; Grain; HL-60 Cells; Hematopoietic stem cells; Hemorrhage; Hemostatic function; Human; IL8 gene; Image; In Vitro; Individual; Infection; Inflammation; Integrins; Knock-in Mouse; Knock-out; Label; Leukocyte Adhesion Deficiency; Leukocytes; Life; Ligands; Light; Macrophage-1 Antigen; Mediating; Membrane; Microfluidics; Microscopy; Modeling; Molecular; Molecular Conformation; Monitor; Monoclonal Antibodies; Mus; Mutation; Names; Optics; P-selectin ligand protein; PH Domain; Patients; Proteins; Reporter; Reporting; Research Personnel; Resolution; Role; Selectins; Signal Pathway; Site; Structure; System; Talin; Testing; Thrombosis; Time; Tissues; Transgenic Mice; Vascular Endothelium; Visualization; Work; base; chemokine; chemokine receptor; dimer; experimental study; gain of function; human disease; humanized mouse; in vivo; in vivo evaluation; intravital microscopy; live cell imaging; nanometer; neutrophil; promoter; quantitative imaging; reconstitution; reconstruction; recruit; retroviral transduction; sharpin; shear stress; stem cells; tool; vector

Description This is project 2 of the PPG “Cellular Mechanisms of Inflammation, Hemostasis, and Thrombosis”. Kindlin-3 is known to be required for integrin activation in platelets and leukocytes. This is starkly illustrated by the human disease Leukocyte Adhesion Deficiency-III. Patients with this disease have mutations in FERMT3, the gene encoding kindlin-3, and suffer from life-threatening bleeding and bacterial infections. Infections result because kindlin-3-deficient leukocytes including neutrophils cannot adhere to the vessel wall and thus fail to recruit to sites of infection. Neutrophils express two β2 integrins, αLβ2 and αMβ2. We made and validated a humanized mouse in which human β2 was knocked into the mouse β2 locus. In this mouse, β2 integrin activation can be monitored by binding of mAbs 24 and KIM127, specific for activation epitopes in human β2. We propose to use flow cytometry, live cell imaging by quantitative dynamic footprinting, superresolution microscopy by SuperSTORM, recently developed in the Ley lab, and intravital microscopy in the β2 integrin humanized mouse to address three specific aims: Specific Aim 1 is to test the role of the kindlin-3 PH domain in β2 integrin activation. We hypothesize that the PH domain is required for kindlin-3 recruitment to the plasma membrane. We use retrovirally transduced neutrophilic cells expressing fluorescent fusion proteins of kindlin-3. For in vivo assessment, we transduce kindlin-3-deficient hematopoitic stem cells with vectors encoding kindlin- 3 fusion proteins to achieve expression in primary mouse neutrophils in vivo. Specific Aim 2 is to test the importance of kindlin-3 dimerization. Dimerization will be assessed by Förster Resonance Energy Transfer (FRET), both in total internal reflection (TIRF) microscopy (near the membrane) and by confocal microscopy (in the cytosol). We hypothesize that the PH domain also may contribute to dimerization, similar to the dimerization of sharpin, which is studied in project 4. Specific Aim 3 is to test how kindlin-3 interacts with talin-1 and β2 integrins. To test this, we make use of the existing mCherry FP-talin-1 transgenic mouse, since talin-1 is too large for retroviral packaging. F(ab) made from the β2 integrin activation reporters KIM127 and mAb24 will be used to assess β2 integrin conformations in vitro and, using the human β2 integrin knockin mouse, in primary neutrophils in vivo. Project 2 will closely collaborate with project 1 by conducting live cell and superresolution imaging to address the role of Rap-1 binding for talin-1 function. Project 1 investigators will assist project 2 with various biochemical assays. When the proposed work is completed, we will understand how kindlin-3 and talin-1 cooperate to enable β2 integrin-dependent neutrophil arrest under flow.

描述 这是 PPG“炎症、止血和血栓形成的细胞机制”的项目 2。 Kindlin-3 是 已知是血小板和白细胞中整合素激活所必需的。人类就清楚地说明了这一点 疾病白细胞粘附缺陷-III。患有这种疾病的患者的 FERMT3 基因发生突变 编码kindlin-3，并遭受危及生命的出血和细菌感染。造成感染的原因是 kindlin-3 缺陷型白细胞（包括中性粒细胞）无法粘附在血管壁上，因此无法募集 感染部位。中性粒细胞表达两种 β2 整合素：αLβ2 和 αMβ2。我们制作并验证了人性化 小鼠，其中人β2被敲入小鼠β2基因座。在该小鼠中，β2整合素激活可以是 通过 mAb 24 和 KIM127 的结合进行监测，对人 β2 的激活表位具有特异性。我们建议使用 流式细胞术、定量动态足迹法活细胞成像、超分辨率显微镜 Ley 实验室最近开发的 SuperSTORM，以及 β2 整合素人源化的活体显微镜检查 小鼠来解决三个具体目标： 具体目标 1 是测试 kindlin-3 PH 结构域在 β2 中的作用 整合素激活。我们假设 PH 结构域是 kindlin-3 招募到血浆所必需的 膜。我们使用逆转录病毒转导的表达 kindlin-3 荧光融合蛋白的中性粒细胞。 为了进行体内评估，我们用编码 kindlin-3 的载体转导 kindlin-3 缺陷型造血干细胞。 3个融合蛋白在原代小鼠中性粒细胞中实现体内表达。具体目标 2 是测试 kindlin-3 二聚化的重要性。二聚化将通过福斯特共振能量转移进行评估 （FRET），无论是在全内反射（TIRF）显微镜（靠近膜）还是通过共聚焦显微镜（在 细胞质）。我们假设 PH 结构域也可能有助于二聚化，类似于 Sharpin 的二聚化，在项目 4 中进行了研究。具体目标 3 是测试 kindlin-3 如何与 talin-1 和 β2 整合素。为了测试这一点，我们利用现有的 mCherry FP-talin-1 转基因小鼠，因为 talin-1 对于逆转录病毒包装而言太大​​。 F(ab) 由 β2 整合素激活报告基因 KIM127 制成， mAb24 将用于体外评估 β2 整合素构象，并使用人 β2 整合素敲入 小鼠体内原代中性粒细胞。项目2将与项目1密切合作，开展活细胞研究 和超分辨率成像来解决 Rap-1 结合对 talin-1 功能的作用。项目1研究人员 将协助项目 2 进行各种生化检测。当建议的工作完成后，我们将 了解 kindlin-3 和 talin-1 如何合作，使 β2 整合素依赖性中性粒细胞在血流下停滞。