Surface morphology live-cell imaging reveals how macropinocytosis inhibitors affect membrane dynamics

Macropinocytosis is a unique endocytic pathway that involves dynamic membrane ruffling and is important in various cellular functions beyond nutrient uptake. Due to its impact, macropinocytosis has been the subject of various studies and macropinocytosis inhibitors have been widely used. However, inhibitors exhibit offtarget effects and could be less specific for macropinocytosis, thereby affecting various cellular mechanisms with effects on nanoscale membrane dynamics that remain elusive. In this study, we revealed how five different inhibitors affect membrane dynamics before and after treatment using scanning ion conductance microscopic imaging on living cells. Our results indicated that cytochalasins and sodium azide exhibited similar slowing effects on horizontal ruffle movements, even though their inhibitory mechanisms are different. In contrast, ruffle defor-mation and elongation were less strongly inhibited compared to the movements along cell membrane direction. These results suggested the involvement of mechanisms beyond actin polymerization. The ruffles disappeared and the cell surface smoothened upon the 5-(N-ethyl-N-isopropyl)-Amiloride treatment. In the presence of wortmannin, we observed active ruffles but no cup-like structures. These findings and scanning ion conductance microscopy live-cell imaging would potentially contribute to a better understanding of inhibitor effects.

Automated summary

In this study, we used scanning ion conductance microscopy to investigate the effects of five different inhibitors on membrane dynamics in living cells. Our results showed that cytochalasins and sodium azide both slowed down horizontal ruffle movements, although their inhibitory mechanisms are different. We also found that ruffle deformation and elongation were less affected compared to movements along the cell membrane direction, suggesting the involvement of mechanisms beyond actin polymerization. The disappearance of ruffles and smoothing of the cell surface were observed after treatment with 5-(N-ethyl-N-isopropyl)-Amiloride, while active ruffles without cup-like structures were observed in the presence of wortmannin. These findings contribute to a better understanding of the effects of inhibitors.