Structure-Function Analysis of Focal Adhesion Protein Vinculin in Neocortical Axon Growth and Development

Abstract

Axon growth and extension are dependent on the interaction of neurons with extracellular matrix (ECM). The interaction between cells and ECM is known as focal adhesion (FA). Vinculin is one the major proteins of FA complex and its interaction with actin and talin has been shown to be critical for FA formation in fibroblasts. But the role of vinculin in mammalian neuronal growth and development has not been studied at all. Here, we show that deletion of vinculin in mouse neocortical neurons attenuates axon growth both in vitro and in vivo. We also found that deletion of vinculin increases neuronal migration significantly during corticogenesis. To understand the mechanism by which vinculin regulates axon growth and migration of neurons, we generated several functional mutants of vinculin which involved the head, neck, and tail domains. Using these mutants, we found that constitutively active vinculin can enhance the growth of axons and increase neuronal migration rate. Vinculin-talin interaction has been shown to be indispensable for the formation of proper FA in fibroblast. Interestingly, we observed that abolishing the interaction between vinculin and talin did not affect either the axon growth or migration, which challenges the central dogma of requirement of vinculin-talin interaction for vinculin mediated functions. We observed that all the domains of vinculin are required for proper axonal growth but not for neuronal migration. Strikingly, we observed that the expression of only the tail domain of vinculin (Vcl-T) increased the branching of neurites, attenuated axon growth, increased cell soma area, and delayed neuronal migration in a dominant negative manner. Blocking Arp2/3 protein complex (another FA protein complex) abolished the excessive branching phenotype indicating that vinculin tail domain mediated branching required the activity of Arp2/3 protein complex. Interestingly, abolishing the interaction between the actin and Vcl-T did not show any of the abnormalities shown by Vcl-T. This indicated that in the absence of the head and neck domain of vinculin, the interaction of the tail domain with actin gets modified that causes changes in cell cytoskeleton behaviour and results into various morphological changes. Fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) analysis of neurons expressing Vcl-T indicated that the mobility of actin was increased in these cells which might be responsible for increased branching, delayed migration and other changes observed. Thus, our findings provide novel insights into the role of vinculin and its functional domains in regulating neuronal growth.