Peculiarities of Phosphatidylserine Externalization by Nano- and Microsecond Electric Pulses

Phosphatidylserine (PS) externalization is a hallmark of apoptosis but can also be triggered within seconds by pulsed electric fields (PEFs). Whether PS reaches the outer leaflet by lateral migration through electropores or indirectly through Ca²⁺-dependent signaling and scramblase activation remains debated. We used total internal reflection fluorescence (TIRF) microscopy with Annexin V-Alexa Fluor 568 and/or bovine lactadherin-FITC to resolve PS externalization in HEK293 cells placed on an indium tin oxide (ITO) transparent electrode. PEF exposures produced discrete PS-positive puncta ("freckles") within ~ 10 s after a 20-µs pulse (1.2-2.5 kV/cm) or with a ~ 1-min delay after a 400-ns pulse (9.3-13.7 kV/cm). Their number gradually increased, reaching up to 80-120 freckles per 100 µm² of cell membrane area within 5 min. Freckles were round, brightest in the center, and well described by a Gaussian intensity profile, with a consistent full width at half maximum (FWHM) of ~ 0.35 µm despite up to ~ 50-fold differences in peak intensity. Freckle formation was inhibited in Ca²⁺-free medium but could be partially restored by applying 20-µs PEFs at higher strengths. Many freckles moved laterally and axially in an apparently stochastic manner, whereas others remained spatially confined. The uniform size and shape of freckles and their mobility patterns suggested that they are PS-positive vesicles rather than PS membrane patches. Further analyses identified freckles as extracellular vesicles of ~ 200-nm estimated diameter whose formation is triggered by applying PEFs at intensities above the electroporation threshold.