Vinculin regulates directionality and cell polarity in two- and three-dimensional matrix and three-dimensional microtrack migration

During metastasis, cells can use proteolytic activity to form tube-like "micro- tracks" within the extracellular matrix (ECM). Using these microtracks, cells can migrate un- impeded through the stroma. To investigate the molecular mechanisms of microtrack migra- tion, we developed an in vitro three-dimensional (3D) micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Because focal adhesions are the pri- mary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the re- peated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knock- down disrupts cell polarity in microtracks. Vinculin also directs migration on two-dimensional (2D) substrates and in 3D uniform collagen matrices, as indicated by reduced speed, shorter net displacement, and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for focal adhesion kinase (FAK) activation in three dimensions, as vinculin knock- down results in reduced FAK activation in both 3D uniform collagen matrices and micro- tracks but not on 2D substrates, and, accordingly, FAK inhibition halts cell migration in 3D microtracks. Together these data indicate that vinculin plays a key role in polarization during migration.