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; 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激活所必需的。这是人类明确说明的 疾病白细胞粘附缺乏-III。该疾病的患者在FERMT3中有突变，该基因 编码Kindlin-3，并患有威胁生命的出血和细菌感染。导致感染是因为 Kindlin-3缺乏白细胞在内 感染部位。中性粒细胞表达两个β2整合素αLβ2和αMβ2。我们制作并验证了人源 将人β2敲入小鼠β2基因座的小鼠。在这只小鼠中，β2整合素激活可以是 通过MAB 24和KIM127的结合监测，特定于人β2的激活表位。我们建议使用 流式细胞仪，通过定量动态足迹，超分辨率显微镜的活细胞成像。 超级风暴，最近在Ley实验室中开发，β2整合素中的浸润式显微镜人性化的显微镜 小鼠要解决三个特定目的：特定目标1是测试β2中Kindlin-3 pH结构域的作用 整合素激活。我们假设pH结构域是Kindlin-3募集到等离子体所必需的 膜。我们使用表达Kindlin-3的荧光融合蛋白的逆转录病毒翻译的中性粒细胞。 为了进行体内评估，我们用编码Kindlin- 3融合蛋白在体内实现原代小鼠中性粒细胞的表达。特定目标2是测试 Kindlin-3二聚体的重要性。二聚化将通过Förster共振能量转移评估 （FRET），既有总内反射（TIRF）显微镜（膜附近）和共聚焦显微镜（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。具体目的3是测试Kindlin-3如何与 talin-1和β2整合素。为了进行测试，我们利用现有的MCHERRY FP-TALIN-1转基因鼠标，因为 Talin-1对于逆转录病毒包装太大。 F（AB）由β2整合素激活报告基因KIM127和 MAB24将用于评估体外β2整合素构象，并使用人β2整合素敲击素 小鼠，在体内原发性嗜中性粒细胞中。项目2将通过执行活细胞与项目1密切合作 和上分辨率成像，以解决RAP-1结合对TALIN-1功能的作用。项目1调查人员 将协助项目2进行各种生化测定。提出的工作完成后，我们将 了解Kindlin-3和Talin-1如何配合以使β2整合素依赖性嗜中性粒细胞在流动下抑制。