Mechanism of kindlin-3-dependent integrin activation

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.