Calcium influx affects intracellular transport and membrane repair following nanosecond pulsed electric field exposure.
The cellular response to subtle membrane damage following exposure to nanosecond pulsed electric fields (nsPEF) is not well understood. Recent work has shown that when cells are exposed to nsPEF, ion permeable nanopores (<2  nm) are created in the plasma membrane in contrast to larger diameter pores (>2  nm) created by longer micro- and millisecond duration pulses. Nanoporation of the plasma membrane by nsPEF has been shown to cause a transient increase in intracellular calcium concentration within milliseconds after exposure. Our research objective is to determine the impact of nsPEF on calcium-dependent structural and repair systems in mammalian cells. Chinese hamster ovary (CHO-K1) cells were exposed in the presence and absence of calcium ions in the outside buffer to either 1 or 20, 600-ns duration electrical pulses at 16.2  kV/cm, and pore size was determined using propidium iodide and calcium green. Membrane organization was observed with morphological changes and increases in FM1-43 fluorescence. Migration of lysosomes, implicated in membrane repair, was followed using confocal microscopy of red fluorescent protein-tagged LAMP1. Microtubule structure was imaged using mEmerald-tubulin. We found that at high 600-ns PEF dosage, calcium-induced membrane restructuring and microtubule depolymerization coincide with interruption of membrane repair via lysosomal exocytosis.
Oak Ridge Institute for Science and Education, JBSA Fort Sam Houston, Texas 78234.
Gary Lee
GL
Thompson
University of Texas Health Science Center at San Antonio, Department of Radiological Sciences, San Antonio, Texas 78229.
Caleb C
CC
Roth
711th Human Performance Wing, JBSA Fort Sam Houston, Texas 78234.
Danielle R
DR
Dalzell
711th Human Performance Wing, JBSA Fort Sam Houston, Texas 78234.
Marjorie
M
Kuipers
711th Human Performance Wing, JBSA Fort Sam Houston, Texas 78234.
Bennett L
BL
Ibey
D000818:Animals; D001692:Biological Transport / Q000502:physiology* / Q000528:radiation effects*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism* / Q000528:radiation effects*; D006224:Cricetinae; D003412:Cricetulus; D004560:Electricity; D005456:Fluorescent Dyes; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism / Q000528:radiation effects; D036103:Nanotechnology; D016062:Porosity; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds
C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D002118:Calcium