A micropattern-based assay to study contact inhibition of locomotion and entosis of adherent human and canine cells in vitro.

We present a protocol for using micropatterns to study post-collision locomotion and entosis of human and canine cells in vitro. We describe steps for lentiviral transduction and the preparation of micropatterned slides consisting of narrow matrix-coated stripes separated by cytophobic spacers. We then detail cell seeding, chamber assembly, and live cell analysis. We provide steps for analysis by live cell imaging using fluorescence microscopy as well as fixing for subsequent analysis by confocal microscopy or correlative light and electron microscopy. For complete details on the use and execution of this protocol, please refer to Kummer et al. (2022)1 and Schwietzer et al. (2022).2.

Loss of contact inhibition of locomotion in the absence of JAM-A promotes entotic cell engulfment.

Entosis is a cell competition process during which tumor cells engulf other tumor cells. It is initiated by metabolic stress or by loss of matrix adhesion, and it provides the winning cell with resources derived from the internalized cell. Using micropatterns as substrates for single cell migration, we find that the depletion of the cell adhesion receptor JAM-A strongly increases the rate of entosis in matrix-adherent cells. The activity of JAM-A in suppressing entosis depends on phosphorylation at Tyr280, which is a binding site for C-terminal Src kinase, and which we have previously found to regulate tumor cell motility and contact inhibition of locomotion (CIL). Loss of JAM-A triggers entosis in matrix-adherent cells but not matrix-deprived cells. Our findings strongly suggest that the increased motility and the perturbed CIL response after the depletion of JAM-A promote entotic cell engulfment, and they link a dysregulation of CIL to entosis in breast cancer cells.

A JAM-A-tetraspanin-αvß5 integrin complex regulates contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvß5 integrin. JAM-A binds Csk and inhibits the activity of αvß5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell-cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell-matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvß5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.

JAM-A interacts with α3ß1 integrin and tetraspanins CD151 and CD9 to regulate collective cell migration of polarized epithelial cells.

Junctional adhesion molecule (JAM)-A is a cell adhesion receptor localized at epithelial cell-cell contacts with enrichment at the tight junctions. Its role during cell-cell contact formation and epithelial barrier formation has intensively been studied. In contrast, its role during collective cell migration is largely unexplored. Here, we show that JAM-A regulates collective cell migration of polarized epithelial cells. Depletion of JAM-A in MDCK cells enhances the motility of singly migrating cells but reduces cell motility of cells embedded in a collective by impairing the dynamics of cryptic lamellipodia formation. This activity of JAM-A is observed in cells grown on laminin and collagen-I but not on fibronectin or vitronectin. Accordingly, we find that JAM-A exists in a complex with the laminin- and collagen-I-binding α3ß1 integrin. We also find that JAM-A interacts with tetraspanins CD151 and CD9, which both interact with α3ß1 integrin and regulate α3ß1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with α3ß1 integrin and tetraspanins CD151 and CD9 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3ß1 integrin or tetraspanins CD151 and CD9 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A exists with α3ß1 integrin and tetraspanins CD151 and CD9 in a functional complex to regulate collective cell migration of polarized epithelial cells.

Tetraspanins: integrating cell surface receptors to functional microdomains in homeostasis and disease.

Tetraspanins comprise a family of proteins embedded in the membrane through four transmembrane domains. One of the most distinctive features of tetraspanins is their ability to interact with other proteins in the membrane using their extracellular, transmembrane and cytoplasmic domains, allowing them to incorporate several proteins into clusters called tetraspanin-enriched microdomains. The spatial proximity of signaling proteins and their regulators enables a rapid functional cross-talk between these proteins, which is required for a rapid translation of extracellular signals into intracellular signaling cascades. In this article, we highlight a few examples that illustrate how tetraspanin-mediated interactions between cell surface proteins allow their functional cross-talk to regulate intracellular signaling.