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联蛋白活化所需的。这一点在人类身上得到了很好的说明 白细胞粘附缺陷病-III.患有这种疾病的患者有FERMT 3基因突变， 编码kindlin-3，并患有危及生命的出血和细菌感染。感染的原因是 包括嗜中性粒细胞在内的Kindlin-3缺陷型白细胞不能粘附于血管壁， 感染部位。中性粒细胞表达两种β2整合素αLβ2和αMβ2。我们制作并验证了一个人性化的 将人β2敲入小鼠β2基因座的小鼠。在这种小鼠中，β2整合素活化可以被抑制。 通过mAb 24和KIM 127的结合进行监测，特异于人β2中的活化表位。我们建议使用 流式细胞术，定量动态足迹法活细胞成像， 最近在Ley实验室开发的SuperSTORM和β2整合素人源化的活体显微镜 具体目标1是测试kindlin-3 PH结构域在β2 整合素活化。我们假设PH结构域是kindlin-3募集到血浆中所必需的 膜的我们使用逆转录病毒转导的表达kindlin-3的荧光融合蛋白的嗜中性细胞。 对于体内评估，我们用编码kindlin-3的载体转染kindlin-3缺陷型造血干细胞。 3融合蛋白，以实现在原代小鼠中性粒细胞中的体内表达。具体目标2是测试 Kindlin-3二聚化的重要性。将通过Förster共振能量转移评估二聚化 （FRET），无论是在全内反射（TIRF）显微镜（膜附近）和共聚焦显微镜（在 胞质溶胶）。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，这是在项目4中研究的。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来说太大。F（ab）由β2整联蛋白活化报告基因KIM 127和 mAb 24将用于体外评估β2整联蛋白构象，并使用人β2整联蛋白敲入 小鼠，体内原发性中性粒细胞。项目2将与项目1密切合作， 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调查员 将协助项目2进行各种生化分析。当拟议的工作完成后，我们会 了解kindlin-3和talin-1如何协同作用，使β2整合素依赖性中性粒细胞在血流下停滞。