Cellular segregation in cocultures is driven by differential adhesion and contractility on distinct timescales.

Cellular sorting and pattern formation are crucial for many biological processes such as development, tissue regeneration, and cancer progression. Prominent physical driving forces for cellular sorting are differential adhesion and contractility. Here, we studied the segregation of epithelial cocultures containing highly contractile, ZO1/2-depleted MDCKII cells (dKD) and their wild-type (WT) counterparts using multiple quantitative, high-throughput methods to monitor their dynamical and mechanical properties. We observe a time-dependent segregation process governed mainly by differential contractility on short (<5 h) and differential adhesion on long (>5 h) timescales. The overly contractile dKD cells exert strong lateral forces on their WT neighbors, thereby apically depleting their surface area. Concomitantly, the tight junction-depleted, contractile cells exhibit weaker cell-cell adhesion and lower traction force. Drug-induced contractility reduction and partial calcium depletion delay the initial segregation but cease to change the final demixed state, rendering differential adhesion the dominant segregation force at longer timescales. This well-controlled model system shows how cell sorting is accomplished through a complex interplay between differential adhesion and contractility and can be explained largely by generic physical driving forces.

Tight Junction ZO Proteins Maintain Tissue Fluidity, Ensuring Efficient Collective Cell Migration.

Tight junctions (TJs) are essential components of epithelial tissues connecting neighboring cells to provide protective barriers. While their general function to seal compartments is well understood, their role in collective cell migration is largely unexplored. Here, the importance of the TJ zonula occludens (ZO) proteins ZO1 and ZO2 for epithelial migration is investigated employing video microscopy in conjunction with velocimetry, segmentation, cell tracking, and atomic force microscopy/spectroscopy. The results indicate that ZO proteins are necessary for fast and coherent migration. In particular, ZO1 and 2 loss (dKD) induces actomyosin remodeling away from the central cortex towards the periphery of individual cells, resulting in altered viscoelastic properties. A tug-of-war emerges between two subpopulations of cells with distinct morphological and mechanical properties: 1) smaller and highly contractile cells with an outward bulging apical membrane, and 2) larger, flattened cells, which, due to tensile stress, display a higher proliferation rate. In response, the cell density increases, leading to crowding-induced jamming and more small cells over time. Co-cultures comprising wildtype and dKD cells migrate inefficiently due to phase separation based on differences in contractility rather than differential adhesion. This study shows that ZO proteins are necessary for efficient collective cell migration by maintaining tissue fluidity and controlling proliferation.