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.

Simultaneous Quantification of the Interplay Between Molecular Turnover and Cell Mechanics by AFM-FRAP.

Quantifying the adaptive mechanical behavior of living cells is essential for the understanding of their inner working and function. Yet, despite the establishment of quantitative methodologies correlating independent measurements of cell mechanics and its underlying molecular kinetics, explicit evidence and knowledge of the sensitivity of the feedback mechanisms of cells controlling their adaptive mechanics behavior remains elusive. Here, a combination of atomic force microscopy and fluorescence recovery after photobleaching is introduced offering simultaneous quantification and direct correlation of molecule kinetics and mechanics in living cells. Systematic application of this optomechanical atomic force microscopy-fluorescence recovery after photobleaching platform reveals changes in the actin turnover and filament lengths of ventral actin stress fibers in response to constant mechanical force at the apical actin cortex with a dynamic range from 0.1 to 10 nN, highlighting a direct relationship of active mechanosensation and adaptation of the cellular actin cytoskeleton. Simultaneous quantification of the relationship between molecule kinetics and cell mechanics may thus open-up unprecedented insights into adaptive mechanobiological mechanisms of cells.

Epithelial cells sacrifice excess area to preserve fluidity in response to external mechanical stress.

Viscoelastic properties of epithelial cells subject to shape changes were monitored by indentation-retraction/relaxation experiments. MDCK II cells cultured on extensible polydimethylsiloxane substrates were laterally stretched and, in response, displayed increased cortex contractility and loss of excess surface area. Thereby, the cells preserve their fluidity but inevitably become stiffer. We found similar behavior in demixed cell monolayers of ZO-1/2 double knock down (dKD) cells, cells exposed to different temperatures and after removal of cholesterol from the plasma membrane. Conversely, the mechanical response of single cells adhered onto differently sized patches displays no visible rheological change. Sacrificing excess surface area allows the cells to respond to mechanical challenges without losing their ability to flow. They gain a new degree of freedom that permits resolving the interdependence of fluidity ß on stiffness [Formula: see text]. We also propose a model that permits to tell apart contributions from excess membrane area and excess cell surface area.

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.

Mechanical and morphological response of confluent epithelial cell layers to reinforcement and dissolution of the F-actin cytoskeleton.

The F-actin cytoskeleton and its connection to the plasma membrane provide structure and shape of epithelial cells. In this study we focus on the impact of the F-actin cytoskeleton on the morphology and mechanical behaviour of confluent epithelial cells. F-actin depolymerisation was fostered by Latrunculin A, while depolymerisation was allayed by Jasplakinolide. The impact of drug treatment on cellular mechanics was measured using atomic force microscopy based active microrheology and force-indentation curves, while morphology was monitored by AFM imaging, electric cell-substrate impedance sensing (ECIS) experiments and fluorescence microscopy. A softening and fluidisation of the cells upon dissolution of F-actin was observed, accompanied by reduction of cell-substrate and cell-cell contacts and an altered topography. The strengthening of actin filaments upon Jasplakinolide treatment was mirrored in several mechanical properties. The largest impact was on the cellular viscosity. The cells were, however, capable of restoring their initial phenotypes, e.g., amount of actin, intercellular and cell-substrate interactions.

Cytoskeletal Control of Antigen-Dependent T Cell Activation.

Cytoskeletal actin dynamics is essential for T cell activation. Here, we show evidence that the binding kinetics of the antigen engaging the T cell receptor influences the nanoscale actin organization and mechanics of the immune synapse. Using an engineered T cell system expressing a specific T cell receptor and stimulated by a range of antigens, we found that the peak force experienced by the T cell receptor during activation was independent of the unbinding kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the synapse revealed a striking dependence on the antigen unbinding kinetics. These findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalize the force experienced by the T cell receptor in an antigen-specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus be a mechanism that allows T cells to adjust the lengthscale and timescale of T cell receptor signaling.