Recruitment of kindlin-3 to plasma membrane through its PH domain precedes high affinity β2 integrin activation and neutrophil arrest

Abstract

Abstract Leukocytes including neutrophils must adhere to the vascular endothelium in order to respond to injury or infection. Integrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is required for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type III syndrome. However, the molecular mechanism by which kindlin-3 regulates β2 integrin activation is unknown. Here we measured the spatiotemporal dynamics of kindlin-3 and β2 integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting (qDF) microscopy and kindlin-3-fluorescent protein (FP) fusion proteins, we find that kindlin-3 is recruited to the plasma membrane prior to induction of the H+ β2 integrin conformation. Deletion of kindlin-3 or its lipid-binding pleckstrin homology (PH) domain completely abolished H+ β2 integrin induction. Intravital imaging of bone marrow chimeric mice revealed that EGFP-tagged kindlin-3-expressing neutrophils, but not control kindlin-3-deficient neutrophils, arrested in cremaster venules in vivo in response to CXCL1. Our data show that kindlin-3 is indispensable for the H+ β2 integrin conformation and crucial for integrin-mediated leukocyte adhesion during inflammation.