Disassembly of actin structures by nanosecond pulsed electric field is a downstream effect of cell swelling

Abstract

Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance.

Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells

“…Using live-cell imaging we demonstrated that permeabilisation does not occur across the entire cell membrane, as YO-PRO-1 showed a polar entry and then diffused across the cell, in agreement with a previous study using 600 ns pulses31.…”

Direct plasma stimuli including electrostimulation and OH radical induce transient increase in intracellular Ca2+ and uptake of a middle‐size membrane‐impermeable molecule

“…Assuming that the electric field is not be distorted, the cell thickness should be sufficiently thin compared with the glass, and the dielectric constant of cell is almost equal to that of glass (ϵ r ≈ 4–5, borosilicate glass), the electric field along z‐axis (E z ) applied to the adherent cell should be calculated from V p and the thickness of dishes. Because the thickness of dishes used in this study was about 0.20–1.0 mm, the estimated intensity of the E z transiently applied to the adherent cells was 30–150 kV/cm at a maximum and could affect the cell membrane structure and cellular activities …”

Multiple nanosecond electric pulses increase the number but not the size of long-lived nanopores in the cell membrane

“…Cell culture methods, nsEP generation and delivery to cells, dosimetry, and image acquisition and processing were essentially the same as reported earlier [6,15,37,39] and will only be described in brief here. A novel method of calibrating the emission of DNA stains against their concentrations is introduced and discussed in section 3.2 under "Results".…”

“…The exposure procedures were similar to those described recently [6,15,39,40]. Nearly rectangular 60- or 600-ns pulses were generated in a custom-made transmission line circuit.…”