LAUSR

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-III. Kindlin-3 binds the β2 integrin cytoplasmic tail at a site distinct from talin-1, but the molecular mechanism by which kindlin-3 activates β2 integrins 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 found that kindlin-3 was recruited to the plasma membrane in response to IL-8 before induction of the H+ β2 integrin conformation. Intravital imaging revealed that EGFP-kindlin-3-reconstituted kindlin-3 knockout neutrophils arrest in vivo in response to CXCL1. EGFP-kindlin-3 in primary mouse neutrophils was also recruited to the plasma membrane prior to arrest. Upon arrest, we found small clusters of high affinity β2 integrin molecules within large areas of membrane-proximal kindlin-3-FP. Deletion of kindlin-3 or its pleckstrin homology (PH) in neutrophil-like HL-60 cells completely abolished H+ β2 integrin induction. IL-8 also triggered recruitment of the isolated kindlin-3 PH domain to the plasma membrane prior to arrest. In conclusion, we show that the kindlin-3 PH domain is both necessary and sufficient for recruitment to the plasma membrane, where full-length kindlin-3 is indispensable for the induction of high affinity β2 integrin.