bioRxiv2025
The viscoelastic properties of the substrate are known to affect many aspects of cellular behavior from migration to differentiation. Cells are also known to sense deformations in the extracellular matrix (ECM). However, these mechanotransduction events have mostly been studied separately, and the emphasis has been on purely elastic materials although most tissues in the body are viscoelastic. We wanted to understand how the viscosity and elasticity of the substrate affect cells mechanosensation, i.e., how cells integrate passive (substrate viscoelasticity) and active cues (dynamic substrate deformation) into calcium transients. We used a light-controllable Disperse Red 1-glass (DR1-glass) coating together with a thin polyacrylamide (PAA) hydrogel with controllable viscoelasticity. This allowed us to generate local deformations in the cell-substrate interface on substrates with different viscoelastic properties. Inspecting the resulting calcium transients in a Madin Darby canine kidney type II (MDCK II) monolayer with the genetically encoded calcium indicator jRCaMP1b revealed that cells responded to deformation differently depending on the viscoelasticity of the substrate. On stiff elastic gels cells exhibited all in all the largest calcium responses as well as increased sensitivity to the magnitude of deformation, with larger deformations leading to stronger calcium signals, whereas on soft elastic and soft viscoelastic gels the magnitudes of deformation had less effect on the degree of calcium signals. Indeed, immunostainings showed that cells formed the strongest focal adhesions (FA) on stiff gels, albeit differences were surprisingly small. Instead, computational modeling revealed that forces generated at FAs were strongly dependent on the viscoelasticity of the substrate, with increased elasticity and decreased viscosity leading to larger forces. Moreover, viscoelasticity affected the dynamics of the force generation. On stiff elastic gels force increased in fast steps, whereas on soft gels the buildup was gradual and was further slowed down by increased viscosity. Surprisingly, experiments with PIEZO1 KO cells showed that the calcium responses to the substrate deformation did not require PIEZO1 channels. Instead, depleting the ER with thapsigargin (TG), depolymerizing the actin cytoskeleton with cytochalasin D (cyto D) and latrunculin A (Lat A), and inhibiting actomyosin II with y-27632, showed that the calcium was mainly released from the ER in an actin, but not actomyosin, dependent mechanism. The data therefore suggests that the forces subjected to FAs could be directly transmitted to the ER via an actin mediated tension that results in the opening of ER residing calcium release channels. Our results illustrate that the viscoelasticity of the cell niche controls cell behavior in two ways. Firstly, it affects how cells build adhesions to the ECM and thus the accumulation of mechanosensitive proteins. Secondly, it affects the dynamics of the deformations and forces that are sensed by FAs. In line with our previous findings, we show that the mechanically induced calcium responses are dependent on dynamics, with fast mechanical cues resulting in larger calcium responses. Moreover, our research suggests that cells may possess distinct response mechanisms that are selectively activated depending on the mechanical properties of their niche, and the type and dynamics of the mechanical stimulation.