Abstract:
Epithelial monolayers serve fundamental physiological roles in development, homeostasis, and disease; yet how collective cell dynamics and substrate mechanics coordinate to drive three-dimensional morphogenesis remains poorly understood. Here, we investigate how substrate mechanics and pharmacological modulation of cell motility and contractility influence monolayer dynamics, dome formation kinetics, and cell adhesion in MDCKII epithelial cells.
Using epifluorescence and spinning disk confocal microscopy, we tracked monolayer dynamics and dome formation on glass, PDMS, and uniform or micropatterned protein coatings. Quantitative analysis of velocity fields revealed a transient motility peak 55-67 hours post-seeding. HGF increased peak velocities in a dose-dependent manner, while blebbistatin suppressed motility and contractility without altering adhesion strength. Blebbistatin-treated cells formed smaller domes that collapsed gradually rather than suddenly, with rapid dome turnover and shorter lifetimes. In contrast, PDMS—which reduces adhesion while increasing motility—produced larger domes with collapse dynamics similar to controls. Confocal imaging revealed that dome collapse involves transient loss of actin stress fibers in dome cells, which are then pulled outward by contractile neighbors before repolarizing and migrating back—a recovery abolished by blebbistatin. Centrifugation-based adhesion assays quantified substrate-specific detachment thresholds.[SV1.1][KK1.2]
These findings reveal that monolayer motility and dome kinetics are governed by interplay between actomyosin contractility, cell motility, and substrate adhesion, with implications for understanding epithelial morphogenesis, tissue mechanics, and biomaterial design.
