Mechanism of kindlin-3-dependent integrin activation

描述 这是PPG项目2“炎症、止血和血栓形成的细胞机制”。Kindlin-3是 已知是激活血小板和白细胞中整合素所必需的。这一点由人类鲜明地说明了 疾病白细胞黏附缺陷-III。患有这种疾病的患者有FERMT3基因突变 编码kindlin-3，并患有危及生命的出血和细菌感染。感染是因为 缺乏Kindlin-3的白细胞，包括中性粒细胞，不能附着在血管壁上，因此无法招募到 感染部位。中性粒细胞表达两种β-2整合素，αLβ2和αMβ2。 人β2基因被敲入小鼠β2基因座的小鼠。在这只小鼠中，β2整合素的激活可以 通过mAb24和KIM127的结合来监测，人β2中的激活表位是特异的。 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实验室最近开发的超级风暴，以及人源化的β2整合素的活体显微镜 小鼠解决三个特定目的：特定目的1是测试Kindlin-3PH结构域在β2中的作用 整合素激活。我们假设PH域是Kindlin-3重新募集到血浆中所必需的 薄膜。我们使用逆转录病毒转导的中性粒细胞表达Kindlin-3的荧光融合蛋白。 为了体内评估，我们用编码kindlin-3的载体转导kindlin-3缺陷的造血干细胞。 3融合蛋白在原代小鼠中性粒细胞体内表达。具体目标2是测试 Kindlin-3二聚化的重要性。二聚化反应的Förster共振能量转移 (FRET)，全内反射(TIRF)显微镜(膜附近)和共聚焦显微镜(In 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，这在项目4中研究。具体目标3是测试kindlin-3如何与 Talin-1和β-2整合素。为了测试这一点，我们使用了现有的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了。F(Ab)由β2整合素激活报告基因KIM127和 MAb24将用于评估β2整合素的体外构象，并利用人β2整合素敲打 小鼠，体内存在原代中性粒细胞。项目2将通过进行活细胞与项目1密切合作 以及超分辨率成像，以解决Rap-1结合对talin-1功能的作用。项目1调查员 将协助项目2进行各种生化分析。当建议的工作完成后，我们会 了解Kindlin-3和talin-1如何协同使β2整合素依赖的中性粒细胞在FLOW状态下停止。

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.