Real-time TIRF imaging reveals sequential recruitment of kindlin-3 and talin-1 during β2 integrin activation 2247812

Introduction Neutrophil recruitment involves a multi-step adhesion cascade crucial for innate immunity and depends on the activation of β2 integrins. The cytoplasmic adaptor proteins, kindlin-3 and talin-1 regulate the β2 integrin activation by binding to its β cytoplasmic tail, yet the sequence of their recruitment under physiological conditions remain poorly understood. Methods To address this, we generated double knock-in mice expressing fluorescently tagged mScarlet-kindlin-3 and eGFP-talin-1, which enabled real-time co-visualization of these adaptor proteins during β2 integrin activation. Results TIRF microscopy analysis of primary neutrophils revealed a novel two-stage recruitment sequence, wherein kindlin-3 was recruited first during rolling and within seconds of arrest, followed by talin-1 recruitment after firm adhesion. Gradually, the talin-1 signal intensity exceeded that of kindlin-3, suggesting a transition from kindlin-3 driven β2 integrin priming to talin-1 mediated cytoskeletal reinforcement and stabilization of adhesion sites. Conclusion In conclusion, this study refines current paradigms of β2 integrin activation by providing real-time evidence of unexpected sequential recruitment of kindlin-3 and talin-1 to the integrin cytoplasmic tail during the leukocyte adhesion cascade.