Abstract
Collective motion of cell sheets plays a role not only in development and repair, but also in devastating diseases such as cancer. However, unlike single-cell motility, collective motion of cell sheets involves complex cell-cell communication during migration; therefore, its mechanism is largely unknown. To elucidate propagation of signaling transduced by cell-cell interaction, we designed a hydrogel substrate that can cause local mechanical stretching of cell sheets. Poly (N-isopropyl acrylamide) (PNIPAAm) hydrogel is a temperature-responsive polymer gel whose volume changes isotropically in response to temperature changes below 37 °C. We designed a combined hydrogel substrate consisting of collagen-immobilized PNIPAAm as the local stimulation side and polyacrylamide (PAAm) as the non-stimulation side to assess propagation of mechanical transduction. Mardin-Darby canine kidney (MDCK) cells adhered to the collagen-immobilized PNIPAAm gel increased it area and were flattened as the gel swelled with temperature decrease. E-cadherin in these cells became undetectable in some domains, and actin stress fibers were more clearly observed at the cell base. In contrast, E-cadherin in cells adhered to the collagen-immobilized PAAm side was equally stained as that in cells adhered to the collagen-immobilized PAAm side even after temperature decrease. ERK1/2 MAPK activation of cells on the non-stimulated substrate occurred after partial stretching of the cell sheet suggesting the propagation of signaling. These results indicate that a change in the balance of mechanical tension induced by partial stretching of cell sheets leads to activation and propagation of the cell signaling.
Highlights
Collective motion of cell sheets plays a crucial role in many fundamental biological processes ranging from dynamical processes to those involved in homeostatic regulation such as morphogenesis, regeneration, and wound repair or devastating diseases such as cancer [1]
To perform local stimulation of cell sheets, we designed a combined hydrogel substrate consisting of a PNIPAAm gel, which provided mechanical stimulation with temperature change, and a PAAm gel, which did not provide mechanical stimulation, as shown in PNIPAAm and PAAm gels were tightly bonded after the temperature was changed from 37 °C to 32 °C several times
Because an increase of area and perimeter were consisted to the swelling ratio of the gel, e.g., distances of micro-beads embedded in the gel, an observation of the cellular shape on PNIPAAm side (Figure 3A) was consequence of stretching cells according to the gel swelling after temperature decrease
Summary
Collective motion of cell sheets plays a crucial role in many fundamental biological processes ranging from dynamical processes to those involved in homeostatic regulation such as morphogenesis, regeneration, and wound repair or devastating diseases such as cancer [1]. Many reports suggest that motorgenic activity is propagated via cytoskeletal mechanical transduction; it is difficult to determine whether such motility signaling is actively induced by cell-cell transduction or is a consequence of a passive migratory signal from individual cells Assessment of this question requires a method to analyze propagation of signaling among cells after imposing local mechanical stimulation on cell sheets without cellular migration. We expected that use of the NIPAAm/PAAm combined culture substrate would allow analysis of local mechanical stimuli, independently of individual migratory signaling Using this culture substrate, we found that the cell sheet on the NIPAAm side of the combined gel was mechanically stretched and that area of the adhered cell was increased and height was flattened. Because the ERK activation in cells on PAAm side occurred within 15 min after the cells were stretched, it appeared that local mechanical stimulus was propagated by interaction via cell-cell communication and not by migration of individual cells
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