Mechanics regulates ATP-stimulated collective calcium response in fibroblast cells

Abstract

Cells constantly sense their chemical and mechanical environments. We study the effect of mechanics on the ATP-induced collective calcium response of fibroblast cells in experiments that mimic various tissue environments. We find that closely packed two-dimensional cell cultures on a soft polyacrylamide gel (Young's modulus E = 690 Pa) contain more cells exhibiting calcium oscillations than cultures on a rigid substrate (E = 36 000 Pa). Calcium responses of cells on soft substrates show a slower decay of calcium level relative to those on rigid substrates. Actin enhancement and disruption experiments for the cell cultures allow us to conclude that actin filaments determine the collective Ca(2+) oscillatory behaviour in the culture. Inhibition of gap junctions results in a decrease of the oscillation period and reduced correlation of calcium responses, which suggests additional complexity of signalling upon cell-cell contact. Moreover, the frequency of calcium oscillations is independent of the rigidity of the substrate but depends on ATP concentration. We compare our results with those from similar experiments on individual cells. Overall, our observations show that collective chemical signalling in cell cultures via calcium depends critically on the mechanical environment.

Calcium oscillations in wounded fibroblast monolayers are spatially regulated through substrate mechanics

“…When cultured on various substrates (E = 360 Pa -10 GPa), the wound-induced Ca 2+ oscillation period shows no general trend of dependence on the substrate Young's modulus (figure S6 in the supplementary material). The absence of a systematic dependence of oscillation period on substrate rigidity is consistent with results from our previous studies where ATP-induced Ca 2+ oscillations were investigated in a similar fibroblast monolayer system (49).…”

“…Our current and previous studies suggest that dynamic coupling of cells through gap junction is the dominant mode of Ca 2+ communication following wounding and ATP excitation in 3T3 cell monolayers (49,59). It is also known that gap junctions can close upon elevated intracellular Ca 2+ level (68).…”

“…We find that creation of an edge in a fibroblast monolayer can indeed trigger oscillations of the cytosolic calcium in the majority of the monolayer cells. Wound-induced oscillations are much more persistent than the previously reported oscillations following chemical stimulation by ATP (49). Measurable oscillations can last for > 2 hours after wounding, which is comparable to the time it takes to start cellular reorientation and migration.…”

Three‐Dimensional Osteosarcoma Models for Advancing Drug Discovery and Development

“…[36] In these models, the cells are frequently embedded into a matrix where they can acquire its native polarity and establish natural interactions with other cells and ECM components, overcoming the main drawbacks of 2D monolayer cultures. [37] Also, the spatial gradients and pharmacological kinetics due to constraints to the transport and distribution of soluble factors are accurately mimicked in 3D cultures, in contrast to the free and rapid diffusion in 2D cultures. [30] A simple example is the effect of substrate stiffness in cell responsiveness to external adenosine triphosphate (ATP) through calcium signaling pathway, dependent of cell-cell interaction via gap junctions.…”

“…[30] A simple example is the effect of substrate stiffness in cell responsiveness to external adenosine triphosphate (ATP) through calcium signaling pathway, dependent of cell-cell interaction via gap junctions. [37] Also, the spatial gradients and pharmacological kinetics due to constraints to the transport and distribution of soluble factors are accurately mimicked in 3D cultures, in contrast to the free and rapid diffusion in 2D cultures. [38] The increased precision of 3D cell cultures that may result in a greater predictive value for clinical outcomes has stimulated its application in drug discovery.…”