Optogenetic interrogation of integrin αVβ3 function in endothelial cells

The integrin alpha V beta 3 is reported to promote angiogenesis in some model systems but not in others. Here, we used optogenetics to study the effects of alpha V beta 3 interaction with the intracellular adapter kindlin-2 ( Fermt2) on endothelial cell functions potentially relevant to angiogenesis. Because interaction of kindlin-2 with alpha V beta 3 requires the C-terminal three residues of the beta 3 cytoplasmic tail ( Arg-Gly-Thr; RGT), optogenetic probes LOVpep and ePDZ1 were fused to beta 3 Delta RGT-GFP and mCherry-kindlin-2, respectively, and expressed in beta 3 integrin-null microvascular endothelial cells. Exposure of the cells to 450 nm ( blue) light caused rapid and specific interaction of kindlin-2 with alpha V beta 3 as assessed by immunofluorescence and total internal reflection fluorescence ( TIRF) microscopy, and it led to increased endothelial cell migration, podosome formation and angiogenic sprouting. Analyses of kindlin-2 mutants indicated that interaction of kindlin-2 with other kindlin-2 binding partners, including c-Src, actin, integrin-linked kinase and phosphoinositides, were also likely necessary for these endothelial cell responses. Thus, kindlin-2 promotes alpha V beta 3-dependent angiogenic functions of endothelial cells through its simultaneous interactions with beta 3 integrin and several other binding partners. Optogenetic approaches should find further use in clarifying spatiotemporal aspects of vascular cell biology.