Permeabilization of the nuclear envelope following nanosecond pulsed electric field exposure.

Permeabilization of cell membranes occurs upon exposure to a threshold absorbed dose (AD) of nanosecond pulsed electric fields (nsPEF). The ultimate, physiological bioeffect of this exposure depends on the type of cultured cell and environment, indicating that cell-specific pathways and structures are stimulated. Here we investigate 10 and 600 ns duration PEF effects on Chinese hamster ovary (CHO) cell nuclei, where our hypothesis is that pulse disruption of the nuclear envelope membrane leads to observed cell death and decreased viability 24 h post-exposure. To observe short-term responses to nsPEF exposure, CHO cells have been stably transfected with two fluorescently-labeled proteins known to be sequestered for cellular chromosomal function within the nucleus - histone-2b (H2B) and proliferating cell nuclear antigen (PCNA). H2B remains associated with chromatin after nsPEF exposure, whereas PCNA leaks out of nuclei permeabilized by a threshold AD of 10 and 600 ns PEF. A downturn in 24 h viability, measured by MTT assay, is observed at the number of pulses required to induce permeabilization of the nucleus.

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.

Disruption of the actin cortex contributes to susceptibility of mammalian cells to nanosecond pulsed electric fields.

Nanosecond pulsed electric fields (nsPEFs) perturb membranes of cultured mammalian cells in a dose-dependent manner with different types of cells exhibiting characteristic survivability. Adherent cells appear more robust than non-adherent cells during whole-cell exposure. We hypothesize that cellular elasticity based upon the actin cytoskeleton is a contributing parameter, and the alteration of a cell's actin cortex will significantly affect viability upon nsPEF exposure. Chinese hamster ovary (CHO) cells that are (a) untreated, (b) treated with latrunculin A to inhibit actin polymerization, or (c) exposed to nsPEFs have been probed using atomic force microscopy (AFM) force-indentations. Exposure to 50 or 100 pulses of 10 ns duration and 150 kV/cm in a single dosage approximately lowers average CHO cell elastic modulus by half, whereas latrunculin lowers it more than 75%. Latrunculin pre-treatment disrupts the actin cortex enough that it negates cumulative damage by equally fractionated (i.e., two rounds of 50 pulses each, separated by 10 min) dosages of nsPEFs as seen in untreated and dimethyl sulfoxide (DMSO)-treated cells with propidium uptake, phosphatidylserine externalization, and 24 h viability according to MTT and CellTiter Glo assays. These results suggest a correlation among cell stiffness, cytoskeletal integrity, and susceptibility to recurrent exposures to nsPEFs, which emphasizes a mechanobiological underpinning of nsPEF bioeffects.

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

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.

Activation of intracellular phosphoinositide signaling after a single 600 nanosecond electric pulse.

Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways--phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2 kV/cm, 600 ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions.

Nanosecond pulsed electric field thresholds for nanopore formation in neural cells.

The persistent influx of ions through nanopores created upon cellular exposure to nanosecond pulse electric fields (nsPEF) could be used to modulate neuronal function. One ion, calcium (Ca(2+)), is important to action potential firing and regulates many ion channels. However, uncontrolled hyper-excitability of neurons leads to Ca(2+) overload and neurodegeneration. Thus, to prevent unintended consequences of nsPEF-induced neural stimulation, knowledge of optimum exposure parameters is required. We determined the relationship between nsPEF exposure parameters (pulse width and amplitude) and nanopore formation in two cell types: rodent neuroblastoma (NG108) and mouse primary hippocampal neurons (PHN). We identified thresholds for nanoporation using Annexin V and FM1-43, to detect changes in membrane asymmetry, and through Ca(2+) influx using Calcium Green. The ED50 for a single 600 ns pulse, necessary to cause uptake of extracellular Ca(2+), was 1.76 kV/cm for NG108 and 0.84 kV/cm for PHN. At 16.2 kV/cm, the ED50 for pulse width was 95 ns for both cell lines. Cadmium, a nonspecific Ca(2+) channel blocker, failed to prevent Ca(2+) uptake suggesting that observed influx is likely due to nanoporation. These data demonstrate that moderate amplitude single nsPEF exposures result in rapid Ca(2+) influx that may be capable of controllably modulating neurological function.

Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload.

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.

Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone.

Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian initiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites.