Visualization of the Dynamic World of Individual Membrane Lesions in Live Electroporated Cells
2023 Frank Reidy Research Center for Bioelectrics Retreat
The phenomenon of cell electropermeabilization induced by applied electric fields (EF) has been known for several decades. However, due to the small size and dynamic response of individual membrane lesions to changes in EF, their visualization remains a challenging task. The lack of adequate tools for direct observation and tracking of these lesions hinders the investigation of electropermeabilization. To address this challenge, methodologies such as molecular dynamic simulations, artificial membrane and cellular models have been applied. However, several aspects of the phenomenon, including long-lasting membrane permeabilization, remain incompletely understood. In this study, we have developed a novel approach for detecting and monitoring individual electropores in live HEK293 cells. The method involves combining the whole-cell patch clamp technique on a cell attached to an indium tin oxide (ITO) surface and total internal reflection fluorescence (TIRF) imaging to visualize Ca2+ "puffs". Application of EF between a patch pipette and ITO surface induced membrane polarization to approximately -260 mV, followed by a sudden decrease in membrane resistance (indication of electroporation). The use of TIRF time-lapse imaging allowed for the identification and tracking of individual lesions at the membrane level in both time and space, while simultaneously recording the applied potential and corresponding current.
The applied EF created short-lived electropores (few milliseconds) as well as electropores that remained permeable to Ca2+ for over a minute. The respective changes in the transmembrane current corresponded to lesion conductance of 80-200 pS, suggesting that each puff represents an individual membrane pore. This study presents, for the first time, the visualization of individual electropores in live cells. The method provides a valuable tool for investigating the mechanisms of electropermeabilization and understanding the dynamics of individual electropores. Furthermore, it lays a foundation for future studies on electroporation using nanosecond EF pulses.
Silkunas, Mantas and Pakhomov, Andrei G., "Visualization of the Dynamic World of Individual Membrane Lesions in Live Electroporated Cells" (2023). 2023 Frank Reidy Research Center for Bioelectrics Retreat. 4.