Cell-substrate interactions play a key role in the regulation of epithelial cell mechanics. Through a series of studies, we demonstrate how substrate interactions impact both the response to an oncogene and the cellular contractility and organization of a monolayer. We first examine the effects of oncogenic Ras in cells in adherent and suspended states. To accomplish this, we utilized atomic force microscopy and a microfluidic optical stretcher. We found that adherent cells stiffen and suspended cells soften with the expression of constitutively active Ras. The effect on adherent cells was reversed when contractility was inhibited with the ROCK inhibitor Y-27632, resulting in softer cells. These findings suggest that increased ROCK activity as a result of Ras has opposite effects on suspended and adhered cells. In a subsequent study, we examined the effects of a substrate on contracting and relaxing monolayers. We created a new methodology for measuring the mechanodynamics of epithelial monolayers by culturing cells at an air-liquid interface. These model monolayers were grown in the absence of any supporting structures in hanging drops. We found that the direction of strain in the unsupported monolayers was not correlated to nuclear alignment as observed when the monolayers were grown on soft deformable gels. It was also observed that both gel and glass substrates led to the promotion of long-range cell nuclei alignment not seen in the unsupported monolayers. To further characterize the morphology and mechanics of monolayers clusters observed in our experiments, we created a new computational model based on the vertex model. The energy function used in this model takes into account cell-cell and cell-substrate adhesion as well as anisotropic cellular mechanical properties. The results of these simulations suggest that the promotion of long-range alignment on solid substrates were due to cells having anisotropic elastic moduli with global alignment. They also suggest that the alignment observed in monolayers grown on air water interfaces is due to cells having low substrate adhesion and isotropic moduli. Our findings establish the importance of studying epithelial cell mechanics in different states of attachment.

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