Matrix stiffness and confinement influence YAP localization in clustered epithelial cells

Epithelial cell clusters reside in complex extracellular matrices (ECMs) of varying mechanical properties including stiffness, topography, dimensionality, and confinement. Through mechanotransduction, cells sense and translate the mechanical cues presented by the surrounding ECM into biochemical signals, which control fundamental aspects of cell behavior including differentiation, proliferation, and motility. While it is well established that nuclear translocation of Yes-associated-protein (YAP) in single cells serves as a key sensor of ECM stiffness, it remains unknown whether grouped epithelial cells exhibit a similar nuclear YAP localization response on stiff substrates. Moreover, the regulation of YAP activity in clustered cells in confined microenvironments has remained unexplored. In this study, we cultured epithelial cell monolayers on flat polyacrylamide (PA) gels of varying stiffness and measured nuclear and cytoplasmic localization of YAP. We found that nuclear YAP localization in grouped cells increased on stiffer gels. However, this stiffness-dependent nuclear localization of YAP was not as effective in densely packed monolayers. To understand how ECM stiffness and confinement independently influence YAP activity, we fabricated a PA-microchannels platform and cultured epithelial cell clusters in channels of tunable width and stiffness. Our measurements demonstrate that the likelihood of nuclear YAP localization increases in cell clusters confined within narrower channels. In wide channels, cells neighboring the channel walls exhibit less roundedness and more nuclear YAP compared to those in the interior of the channels. Taken together, these findings reveal that the mechanosensitive nuclear localization of YAP in clustered cells depends not only on ECM stiffness, but also on the cell density and the degree of matrix confinement.