This irradiation procedure allows us to maintain spatial control of the secondary irradiated regions and the initial cluster geometry down to a 5-mm resolution, and thereby enables the precise control of the cellular microenvironment in collective migration. N-cadherin, a cell adhesion protein, was localized along the cell-cell contact sites on both the homogenous and nanopatterned surface. Cadherins are critical membrane proteins that form trans-homodimers between neighboring cells and hence play essential roles in maintaining cell-cell junctions during epithelial collective migration. In addition, we have demonstrated that a sufficient incubation time is required for the maturation of E-cadherinmediated cell-cell contacts, when MDCK cells become capable of showing collective migration characteristics. Taking these factors into consideration, we concluded that a 9-hour incubation is sufficient for cell-cell contacts to mature on either the homogenous substrate or the nanopatterned substrate. However, when the cells were released from their confinements by irradiating the surrounding regions, we observed dramatically different outcomes of cell migration on the two substrates. On the homogenous surface, the cellular cluster expanded in a radial fashion toward the previously idle areas and maintaining the cellcell contacts between the neighboring cells. The sustained cell-cell interactions during cell migration can also be clearly seen from the localization of N-cadherin at the sites of cell-cell contact. Most of the cells formed a pancakelike shape and showed directed migration from the center of the cluster to outward. These cellular behaviors are similar to the sheet-like collective motions of epithelial cells during the wound healing process of scratched confluent monolayers. On the other hand, on the nanopatterned substrate, the cells gradually became disconnected and migrated more individually. Even at 3 hours after the confinement release, the N-cadherin staining was mainly detected in the cytoplasm, and only trace signal or no signal was observed at the cell-cell contact regions on the nanostructured surfaces, even AZ 960 abmole bioscience though N-cadherin was localized at the cell-cell contact regions before the induction of migration. When we focused on the individual cells each cell migrated back and forth, frequently changing its migration direction. The cells gradually acquired elongated shape and extended more protrusions to random directions. The quantification of migration rate and directional persistence for the cells originally located at the periphery of the circular clusters are shown in Figure 6O and 6P. The data show that the average migration rate was significantly greater in the cells migrating on the nanopatterns than in the cells migrating on the homogenous substrates, whereas the average directional persistence was lower on the nanopatterned substrates. These results drove us to further examine the intracellular.