Abstract
Above a threshold electric field strength, 600 ns-duration pulsed electric field (nsPEF) exposure substantially porates and permeabilizes cellular plasma membranes in aqueous solution to many small ions. Repetitive exposures increase permeabilization to calcium ions (Ca2+) in a dosage-dependent manner. Such exposure conditions can create relatively long-lived pores that reseal after passive lateral diffusion of lipids should have closed the pores. One explanation for eventual pore resealing is active membrane repair, and an ubiquitous repair mechanism in mammalian cells is lysosome exocytosis. A previous study shows that intracellular lysosome movement halts upon a 16.2 kV/cm, 600-ns PEF exposure of a single train of 20 pulses at 5 Hz. In that study, lysosome stagnation qualitatively correlates with the presence of Ca2+ in the extracellular solution and with microtubule collapse. The present study tests the hypothesis that limitation of nsPEF-induced Ca2+ influx and colloid osmotic cell swelling permits unabated lysosome translocation in exposed cells. The results indicate that the efforts used herein to preclude Ca2+ influx and colloid osmotic swelling following nsPEF exposure did not prevent attenuation of lysosome translocation. Intracellular lysosome movement is inhibited by nsPEF exposure(s) in the presence of PEG 300-containing solution or by 20 pulses of nsPEF in the presence of extracellular calcium. The only cases with no significant decreases in lysosome movement are the sham and exposure to a single nsPEF in Ca2+-free solution.
Highlights
Exposure to nanosecond pulsed electric fields creates nanopores within lipid bilayer membranes of cells
One hypothesized mechanism of pore formation that is supported by molecular dynamics simulations is that penetration of interfacial water molecules into the hydrophobic core of the bilayer increases with localized electric field strength [1]
Given exposure to a 600-ns PEF at 16.2 kV/cm, the entire plasma membrane becomes thoroughly porated [4], and pore lifetimes are on the order of minutes, which extends beyond the timeframe for resealing via passive lateral diffusion of lipids [5]
Summary
Exposure to nanosecond pulsed electric fields (nsPEF) creates nanopores within lipid bilayer membranes of cells. Whether the pore is irreversible or reversible, and its lifetime, correlates with pulse duration, localized electric field magnitude, and exposure dosage. Living mammalian cells possess a host of active mechanisms for repairing plasma membrane damage, including lysosomal exocytosis as a restoration mechanism. Microtubule-associated motor proteins transport lysosomes to the damage site, where the localized [Ca2+] accelerates fusion of accumulated lysosomes with the plasma membrane. We qualitatively observed that nsPEF exposure in a Ca2+-containing solution can disrupt cytoskeletal structure and halt lysosomal movement. NsPEF-induced interruption of lysosomal translocation is quantified using a mean square displacement (MSD) analysis [7], confirming observations that decreases in the transport of lysosomes occur when microtubules are constrained or likely disrupted
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