To study the molecular mechanisms underlying collective epithelial migration, we used the human bronchial epithelial cell line, 16HBE [33
]. Scratching a 16HBE monolayer induces collective migration of cells into the space created. Cell streams, or flows can be seen within
the migrating epithelium, resulting in cells changing their position with respect to each other, a common characteristic of epithelial collective migration [34
Rho GTPases, particularly Rho, Rac and Cdc42, regulate the actin cytoskeleton and cell polarity in epithelial tissues. Through effector proteins such a Rho-kinase, WASP and mDia, they promote actin polymerization and actin-myosin filament contraction, and through effectors such Par6 and IQGAP, they mediate the establishment of apical-basal polarity and cell-cell junction formation [19
]. They are, therefore, important regulators of epithelial morphogenesis and migration. A key feature of these signaling pathways lies in the ability of upstream activators (GEFs) and inactivators (GAPs) to regulate Rho GTPases in a spatially restricted way. To explore their contribution to collective cell migration, we screened an siRNA library targeting the 67 known Rho family GAPs, in 16HBE cells and identified myosin-IXA, an actin binding, RhoGAP domain containing protein, as an important player in collective migration.
Depletion of myosin-IXA by siRNA resulted in uncoordinated, random migration in scratched monolayers and in islands, through the inability of cells to maintain cell-cell contacts. A large number of proteins are recruited to adherens junctions and tight junctions at cell-cell contact sites to control their assembly, organization, maintenance and association with actin filaments, including numerous actin-based motors, such as myosin-II, -VII, -VI and -X. Myosins regulate cell-cell contacts by remodeling the actin cytoskeleton with their actin cross-linking activity, or by motor function creating tension between actin filaments, or by delivering cargo components. A myosin-IXA knockout mouse revealed a crucial role for this protein in the differentiation of ependymal cells, where it is highly expressed, with defects seen in morphology, gene expression and junction formation, supporting our results on the importance of myosin-IXA function at cell-cell contacts [29
Myosins are a large family of actin binding motors, structurally diverse but sharing a conserved motor domain that binds to actin filaments and hydrolyzes ATP leading to translocation along the filament [36
]. Myosins bind selectively to actin filaments with different composition and spatial arrangement [37
]. Endogenous myosin-IXA and EGFP-myosin-IXA localize in puncta at cell-cell contact sites, along the circumferential, contractile actin bundle [29
]. The circumferential localization suggests that myosin-IXA harbors recognition motifs for contractile actin filaments, perhaps in the neck-tail region, similar to myosin-IIA and -IIB [38
Rho signaling is important during cell-cell contact formation and we recently described a role for the Rho target PRK2 in cell-cell junction maturation in 16HBE cells [39
]. Localized activation of RhoA at the cell-cell contact zone, visualized by FRET, has a distinct spatiotemporal pattern upon cell-cell interactions suggesting tight regulation by both GEFs and GAPs [21
]. Here we present evidence that the RhoGAP activity of myosin-IXA facilitates the assembly of cell-cell contacts and that this is particularly important during collective cell migration. First, we found that the contractile phenotype induced by depletion of myosin-IXA can be partially reversed by inhibiting Rho-kinase. Second, the phenotype observed after overexpression of myosin-IXA is abolished by an inactivating mutation in the RhoGAP catalytic site. Third, low level expression of the constitutively active RhoGAP domain partially rescues the cell scattering phenotype induced by myosin-IXA depletion. Fourth, RhoA activity at nascent cell-cell contacts, detected by FRET ratio imaging, is significantly increased in myosin-IXA-depleted cells. These data suggested that the GAP domain of myosin-IXA acts as a localized, negative regulator of Rho to control contractile forces during the early stages of cell-cell contact formation.
The interaction of lamellipodia from two colliding cells stimulates local clustering of E-cadherin and the recruitment of actin filaments, referred to as junctional actin. Junctional actin can be seen at very early stages of cell-cell interactions in both control and myosin-IXA-depleted colliding 16HBE cells, but in the absence of myosin-IXA this did not reorganize into radial actin filaments. As a consequence, junctions were not stabilized and cells moved away from each other. These observations indicate that myosin-IXA is essential during the assembly of radial actin filaments ().
We conclude that myosin-IXA is required during the assembly of radial actin filaments, seen within a few minutes after cell-cell contact. In migrating 16HBE cells, these appear to be essential for subsequent stabilization of contact sites and depletion of myosin-IXA leads to cell scattering. Maturation of cell-cell junctions is accompanied by Rho-dependent conversion of radial actin filaments into tangential acto-myosin filaments. We speculate that myosin-IXA is required locally to downregulate Rho activity during early stages of junction formation, as well as during the dynamic reorganization of cell-cell contacts seen during collective migration.