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.

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.

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.