Document Type

Article

Publication Date

2026

Publication Title

The Journal of Membrane Biology

Volume

259

Pages

19

DOI

10.1007/s00232-026-00384-5

Abstract

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.

Rights

© The Authors 2026.

This article is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original authors and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

Data Availability

Article states: "Data will be made available on request."

Original Publication Citation

Silkunas, M., Enos, A., Semenov, I., Silkuniene, G., Zivla, E., Pakhomova, O. N., & Pakhomov, A. G. (2026). Peculiarities of phosphatidylserine externalization by nano- and microsecond electric pulses. Journal of Membrane Biology, 259, Article 19. https://doi.org/10.1007/s00232-026-00384-5

ORCID

0000-0002-4568-9265 (Silkunas), 0009-0009-7372-1136 (Enos), 0000-0002-0302-1355 (Semenov), 0000-0001-8436-9718 (Silkuniene), 0009-0006-8465-6817 (Zivla), 0000-0003-4950-4130 (Pakhomova)

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