Extracellular Ca2+ drives cytoskeletal remodeling and cell swelling following nanosecond pulsed electric field exposure

Cell swelling and cytoskeletal disruption are known to be secondary effects of cell membrane permeabilization induced by nanosecond pulsed electric fields (nsPEFs). In this study, we used healthy and cancer urothelial cells to investigate the role of Ca 2+ influx on cytoskeleton remodeling and morphological changes of cells following exposure. A train of 200 nsPEFs (300 ns pulse duration, 10 Hz), delivered via contact electrodes, effectively permeabilized the cell membrane in an isosmotic physiological solution. Subsequent shrinkage of the actin cortex and a reduction in actin fluorescence were observed only in the presence of extracellular Ca 2+ . In its absence, no significant changes in the phalloidin-stained actin cortex were detected. Time-lapse imaging using scanning ion conductance microscopy (SICM) revealed that a significantly greater and more immediate increase in projected cell area and cell volume occurred after nsPEFs exposure in a solution containing Ca 2+ compared to a solution without Ca 2+ . These findings demonstrate that Ca 2+ is a key driver of actin cytoskeleton disintegration and morphological changes following membrane permeabilization with nsPEFs. • Ca 2+ influx drives actin cytoskeleton disintegration after nsPEFs. • Actin cortex shrinkage occurs only with extracellular Ca 2+ present. • SICM imaging shows strong Ca 2+ -dependent swelling dynamics post-nsPEFs. • Ca 2+ depletion minimizes cell swelling and structural remodeling. • Ca 2+ -dependent responses are consistent across healthy and cancer urothelial cells.