(110) Alterations in Kindlin-3 recruitment to plasma membrane during neutrophil recruitment visualized by intravital imaging

Introduction/Rationale: Neutrophils respond rapidly to infection and are highly migratory cells. Migratory behavior is coordinated by integrins which bind to ligands to regulate crawling. Activation of integrins is tightly regulated by proteins including Kindlin-3 (K3). Lack of K3 leads to a failure of integrin activation and neutrophil migration resulting in a susceptibility of the host to life-threatening infections and death, a disease known as leukocyte adhesion deficiency 3 (LAD3). The mechanism by which K3 regulates integrin activation remains unclear. We hypothesize that K3 is recruited early to the membrane prior to integrin activation whereupon it then recruits other proteins to mediate integrin conformational changes.

Methods: We generated a knock-in mouse by fusing a fluorescent protein, mScarlet-I, to K3 (KI-K3), allowing tracking of KI-K3 subcellularly using live animal intravital imaging (IVM).

Results: We confirmed that all hemopoietic cells express KI-K3 and the transgenic mouse survives to adulthood and old age. However, this fusion induced allelic hypomorphicity as it reduced total KI-K3 mRNA and protein levels and caused compensatory neutrophilia. We studied neutrophil migration in the cremaster muscle and skin. Imaging revealed that KI-K3 is homogenously distributed during circulation, concentrated to the neutrophil-endothelial interface during luminal adherence, crawling and transmigration and polarized to the cell lamellipodia and uropod after diapedesis and during migration within tissues. We bred KI-K3 mice to mice expressing human β2-integrin (hITGB2) and quantified integrin activation using the antibody mAb24 which binds to the high-affinity conformation of β2-integrin. Activation of β2-integrin was reduced by half in hITGB2/KI-K3 mice as compared to hITGB2 mice alone.

Conclusion: Our real time in vivo observations demonstrate that localization of K3 within neutrophils is highly dynamic and reorganizes differentially during the recruitment cascade.