(41c) Substratum Stiffness Regulates Erk Signaling Dynamics through Receptor-Level Control

The Ras/Erk cascade is classically stimulated by extracellular ligands to regulate processes such as cell growth, proliferation, and migration. During normal development and cancer progression, cells make fate decisions in response to the dynamics of Erk signaling, which depend on how stimuli from the microenvironment are interpreted by cells. While the mechanical properties of the microenvironment are also known to affect Erk signaling, their effects on the dynamics of Erk signaling are poorly understood. Here, we characterize how the stiffness of the underlying substratum affects the dynamics of Erk signaling in human mammary epithelial cells. We find that soft microenvironments attenuate Erk dynamics, both at steady-state and after stimulation with ligands. Optogenetically manipulating Ras and epidermal growth factor (EGF) receptor results in comparably sustained Erk signaling in cells on both soft and stiff substrata, revealing that long-term intracellular signaling is unaffected by substratum stiffness. Instead, we find that cells on soft microenvironments activate and internalize EGFR to lesser extents compared to cells on stiff microenvironments. Treatment with a fluorescently tagged EGF revealed less EGF localization at the membranes of cells on soft substrata, suggesting that cells on soft microenvironments activate EGFR less efficiently due to impaired receptor-ligand interactions. Finally, overexpression of EGFR in mammary epithelial cells is sufficient to increase EGF membrane binding, EGF internalization, and Erk signaling on soft microenvironments. These findings underscore how multiple microenvironmental signals, both biochemical and mechanical, may be jointly processed through a highly conserved pathway that regulates tissue development, homeostasis, and cancer progression.