Cells penetrating into confinement undergo mesenchymal-to-amoeboid transition. The topographical features of the microenvironment expose cells to different hydraulic resistance levels. How cells respond to hydraulic resistance is unknown. We show that the cell phenotype shifts from amoeboid to mesenchymal upon increasing resistance. By combining automated morphological tracking and wavelet analysis along with fluorescence recovery after photobleaching (FRAP), we found an oscillatory phenotypic transition that cycles from blebbing to short, medium, and long actin network formation, and back to blebbing. Elevated hydraulic resistance promotes focal adhesion maturation and long actin filaments, thereby reducing the period required for amoeboid-to-mesenchymal transition. The period becomes independent of resistance upon blocking the mechanosensor TRPM7. Mathematical modeling links intracellular calcium oscillations with actomyosin turnover and force generation and recapitulates experimental data. We identify hydraulic resistance as a critical physical cue controlling cell phenotype and present an approach for connecting fluorescent signal fluctuations to morphological oscillations.

Cells migrating in vivo can encounter microenvironments with varying physical properties. One such physical variable is the viscosity of the fluid surrounding the cell. Increased fluid viscosity is expected to increase the hydraulic resistance experienced by the migrating cell and therefore decrease the cell speed.We demonstrate that contrary to this expected result, cells migrate faster in high viscosity media on 2D substrates. To reveal the molecular mechanism, we examined both actin dynamics and water dynamics driven by ion channel activity. Results show that cells increased in area in high viscosity and actomyosin dynamics remained similar, except that actin retrograde flow speed is reduced. Inhibiting ion channel fluxes in high viscosity media results in a large reduction in cell speed, suggesting that water flux contributes to the observed speed increase. Moreover, inhibiting actin-dependent vesicular trafficking that transports ion channels from the ER to the cell boundary changes ion channel spatial positioning and reduces cell speed in high viscosity media. Cells also displayed altered Ca 2+ -activity in high viscosity media, and when cytoplasmic Ca 2+ is sequestered, cell speed reduction and altered ion channel positioning were observed.Taken together, we find that the cell cytoplasmic actin-phase and water-phase are coupled during cell migration in high viscosity media. Directional water fluxes are mediated by ion channels whose position depend on actin-based vesicular trafficking. There are no significant changes in ion channel total content in high viscosity, in agreement with physical modeling that also predicts the observed cell speedup in high viscosity environment.

Cells migrating in vivo can encounter microenvironments with varying physical properties. One such physical variable is the viscosity of the fluid surrounding the cell. Increased fluid viscosity is expected to increase the hydraulic resistance experienced by the migrating cell and therefore decrease the cell speed. We demonstrate that contrary to this expected result, cells migrate faster in high viscosity media on 2D substrates. To reveal the molecular mechanism, we examined both actin dynamics and water dynamics driven by ion channel activity. Results show that cells increased in area in high viscosity and actomyosin dynamics remained similar, except that actin retrograde flow speed is reduced. Inhibiting ion channel fluxes in high viscosity media results in a large reduction in cell speed, suggesting that water flux contributes to the observed speed increase. Moreover, inhibiting actin-dependent vesicular trafficking that transports ion channels from the ER to the cell boundary changes ion channel spatial positioning and reduces cell speed in high viscosity media. Cells also displayed altered Ca2+-activity in high viscosity media, and when cytoplasmic Ca2+ is sequestered, cell speed reduction and altered ion channel positioning were observed. Taken together, we find that the cell cytoplasmic actin-phase and water-phase are coupled during cell migration in high viscosity media. Directional water fluxes are mediated by ion channels whose position depend on actin-based vesicular trafficking. There are no significant changes in ion channel total content in high viscosity, in agreement with physical modeling that also predicts the observed cell speedup in high viscosity environment.