Actn4 Links Inactive Integrin α5 With Actin in Zebrafish Somites

Integrins are key plasma membrane proteins that mediate cell-ECM adhesion and communication, and they rely on a conformational change for their activation and bidirectional signaling. However, there are few in vivo studies of integrin activation. Here, we identify Integrin α5 (Itgα5)-associated proteins in the physiological setting of zebrafish somite morphogenesis. Using label-free mass spectrometry, we compared Itgα5-associated proteins in different integrin activation states. As expected, we found active Itgα5 enriched extracellular matrix (ECM) proteins. Surprisingly, inactive Itgα5 incapable of binding ligand recruits actin cytoskeletal proteins as efficiently as the active integrin. We validated Itgα5's linking to actin adaptors using Parallel Reaction Monitoring (PRM). We then focused on α-actinin 4 (Actn4), an actin cross-linker, which we find preferentially associates with inactive Itgα5. Along zebrafish somite boundaries, Itgα5 and Actn4 displayed on and off co-localization, and Actn4 showed a stronger correlation with wild-type and inactive Itgα5 compared with the active Itgα5. We also found that deleting the actin-binding domain (Actn4^ABDdel) resulted in cytoplasmic retention and loss of colocalization with Itgα5. These findings suggest that Itgα5 and Actn4 cooperate during somite boundary formation and that actin cytoskeleton reorganization facilitates their colocalization. Furthermore, we showed ligand-binding-deficient Itgα5 associated with Paxillin a (Pxna), a scaffold protein highly enriched at somite boundaries and strongly correlated with activated Itgα5. This study provides novel insights into in vivo integrin activation and integrin-actin interactions and broadens our understanding of integrin's role in tissue morphogenesis. Data are available via ProteomeXchange with identifiers PXD024942, PXD065495, PXD058516, PXD058550, and PXD058747.