Kindlin‐3 recruitment to the plasma membrane in neutrophils precedes high affinity integrin activation

Abstract

Integrin‐mediated adhesion of circulating leukocytes to the blood vessel wall starts by arrest from rolling. Activation of integrins by inside‐out signaling 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 critically required for leukocyte adhesion. Mutations of kindlin‐3 cause leukocyte adhesion deficiency type III (LAD‐III) syndrome, characterized by severe bleeding (a platelet adhesion defect) and recurring infections (a neutrophil adhesion defect). The clinical significance of kindlin‐3 has been extensively studied; however, the molecular mechanism by which kindlin‐3 regulates the β2 integrin activation is unknown. Kindlin‐3 can bind the β2 integrin cytoplasmic tail at a site distinct from the talin‐1 binding site. It remains unclear whether kindlin‐3 affects individual integrin conformation and/or integrin clustering. Here we monitored the spatiotemporal dynamics of kindlin‐3 and β2 integrin conformation changes during neutrophil rolling, arrest and spreading under flow (6 dyn/cm2) using high‐resolution quantitative dynamic footprinting (qDF) microscopy in combination with an integrin conformation‐specific antibody reporting assay and kindlin‐3 fusion reporters. We find that kindlin‐3 is recruited to the plasma membrane during neutrophil adhesion prior to induction of H+ β2 integrin conformation, indicating that kindlin‐3 is required for H+ integrin conformation. Indeed, deletion of kindlin‐3 in neutrophil‐like HL‐60 cells completely abolishes high affinity β2 integrin. Together, our data suggest that kindlin‐3 is permissive for the high affinity β2 integrin conformation.Support or Funding InformationThis work was supported by funding from the National Institutes of Health, USA (NIH, P01‐HL078784).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.