Abstract
BackgroundNanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Flow cytometric and fluorescence microscopy evidence has associated phospholipid rearrangement directly with nanoelectropulse exposure and supports the hypothesis that the potential that develops across the lipid bilayer during an electric pulse drives phosphatidylserine (PS) externalization.ResultsIn this work we extend observations of cells exposed to electric pulses with 30 ns and 7 ns durations to still narrower pulse widths, and we find that even 3 ns pulses are sufficient to produce responses similar to those reported previously. We show here that in contrast to unipolar pulses, which perturb membrane phospholipid order, tracked with FM1-43 fluorescence, only at the anode side of the cell, bipolar pulses redistribute phospholipids at both the anode and cathode poles, consistent with migration of the anionic PS head group in the transmembrane field. In addition, we demonstrate that, as predicted by the membrane charging hypothesis, a train of shorter pulses requires higher fields to produce phospholipid scrambling comparable to that produced by a time-equivalent train of longer pulses (for a given applied field, 30, 4 ns pulses produce a weaker response than 4, 30 ns pulses). Finally, we show that influx of YO-PRO-1, a fluorescent dye used to detect early apoptosis and activation of the purinergic P2X7 receptor channels, is observed after exposure of Jurkat T lymphoblasts to sufficiently large numbers of pulses, suggesting that membrane poration occurs even with nanosecond pulses when the electric field is high enough. Propidium iodide entry, a traditional indicator of electroporation, occurs with even higher pulse counts.ConclusionMegavolt-per-meter electric pulses as short as 3 ns alter the structure of the plasma membrane and permeabilize the cell to small molecules. The dose responses of cells to unipolar and bipolar pulses ranging from 3 ns to 30 ns duration support the hypothesis that a field-driven charging of the membrane dielectric causes the formation of pores on a nanosecond time scale, and that the anionic phospholipid PS migrates electrophoretically along the wall of these pores to the external face of the membrane.
Highlights
Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis
Pulse polarity and membrane perturbation patterns are consistent with electric field-driven PS translocation We have previously reported that PS externalization after exposure to unipolar, 30 ns, 2.5 MV/m pulses is confined to the anode-facing pole of the cell [14]
We report the influx of YO-PRO-1, a fluorescent dye that binds to nucleic acids, and which is a sensitive indicator of early apoptosis [42] and of activation of P2X7 receptor channels [43], after exposure of Jurkat cells to a sufficiently large number (30) of 4 ns pulses at fields above 6 MV/m at high pulse repetition rates (1 kHz), under conditions where detectable levels of propidium iodide continue to be excluded from the cell interior
Summary
Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Nanosecond, megavolt-per-meter pulsed electric fields nondestructively perturb the intracellular environment, causing calcium bursts [1,2], eosinophil sparklers [3], vacuole permeabilization [4], and the appearance of apoptotic indicators such as release of cytochrome c into the cytoplasm [5] and caspase activation [6,7] In addition to these effects in the cell interior, nanoelectropulse exposure induces phosphatidylserine (PS) externalization – translocation of PS from the cytoplasmic face of the plasma membrane to the cell exterior – a normal event in platelet activation and blood coagulation [8], a diagnostic feature of apoptotic cells which serves as a physiological semaphore for their phagocytic removal [9], and a means of intramembrane signal transduction in lymphocytes [10]. By employing the fluorescent dye FM1-43 as an indicator of relative changes in the phospholipid composition of the external leaflet of the plasma membrane [13], we have achieved real-time microscopic visualization of pulse-induced phospholipid scrambling, which occurs within milliseconds of pulse delivery, is pulse count- and field-dependent, and is confined always to the anode-directed pole of the cell [14]
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