Tight junction ZO proteins maintain tissue fluidity, ensuring efficient collective cell migration

Abstract

Tight junctions are pivotal components of epithelial tissues connecting neighboring cells to provide protective barriers. However, explicit knowledge of their role during other crucial biological processes, such as collective cell migration, remains sparse. Here, the importance of the tight junction 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 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 cell populations with distinct morphological and mechanical properties: 1) smaller and highly contractile cells with an outwards bulged 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. These smaller cells are particularly immobile and therefore drive jamming. Knockout of only ZO1 induces a similar but less pronounced behavior. This study shows that ZO proteins are necessary for efficient collective cell migration by maintaining tissue fluidity and controlling proliferation.

Cellular segregation in co-cultures driven by differential adhesion and contractility on distinct time scales

“…In accordance with previous work(23), we observed a strong actomyosin upregulation at the apical-lateral cell periphery of dKD cells (Figure 2A). Particularly, activated (phosphorylated) myosin accumulated at the apical cell-cell junctions.…”

“…First, we acquired AFM indentation maps (Figure 3A) and examined the apparent local stiffness, which is reflected in the slope of the force-distance curve. Here, we observed a similar picture as in pure dKD monolayers, 23 dKD cells were softer at the central cortex and extremely stiff at the perijunctional actomyosin ring (vide supra). In contrast, neighboring WT cells showed only slightly pronounced cell boundaries but an increased stiffness at the center in comparison with dKD neighbors.…”

“…ZO1/2 knockdown was effected by Beutel et al using Crispr/Cas. (55) WT-GFP cells were created as described in Skamrahl et al(23) Clones of MDCKII cells expressing GFP-myosin-2-A were generated by transfecting cells with pTRA-GFP-NMCH II-A plasmid (Addgene plasmid # 10844). Clones expressing the GFP tag in a stable manner were selected via Neomycin resistance (G418).…”

“…Recently, it has been shown that in tight junction-depleted epithelial cells (ZO1 and 2 knockdown; abbreviated as dKD) two distinct cell populations emerge: some cells contract and by that adopt a roundish shape; pulling on their neighbors eventually results in laterally elongated cells that coexist with the contracted cells. (23, 24) In initial experiments, the stretched cell population was successfully replaced by less contractile wildtype (WT) cells, substantially increasing the mismatch in mechanical properties. In this co-culture, overly contractile dKD cells inhibited layer fluidity and migration by means of jamming.…”

Reciprocal regulation between cell mechanics and ZO-1 guides tight junction assembly and epithelial morphogenesis

“…Our data thus indicate that ZO-1 depletion leads to cell-wide changes in cell mechanics that are modulated by the overall actin organisation of the cell. This is supported by the recent observation that ZO proteins regulate tissue fluidity in migrating cells (Skamrahl et al, 2021). It is thus likely that the biological importance of ZO-1 and its impact on morphogenetic processes are dependent on other factors that regulate organisation of the actin cytoskeleton, such as the cell type or ECM properties.…”