Cellular regulation of extension and retraction of pseudopod-like blebs produced by nanosecond pulsed electric field (nsPEF).

Recently we described a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells exposed to nanosecond pulsed electric field (nsPEF). In Ca(2+)-free buffer such exposure initiates formation of pseudopod-like blebs (PLBs), protrusive cylindrical cell extensions that are distinct from apoptotic and necrotic blebs. PLBs nucleate predominantly on anode-facing cell pole and extend toward anode during nsPEF exposure. Bleb extension depends on actin polymerization and availability of actin monomers. Inhibition of intracellular Ca(2+), cell contractility, and RhoA produced no effect on PLB initiation. Meanwhile, inhibition of WASP by wiskostatin causes dose-dependent suppression of PLB growth. Soon after the end of nsPEF exposure PLBs lose directionality of growth and then retract. Microtubule toxins nocodazole and paclitaxel did not show immediate effect on PLBs; however, nocodazole increased mobility of intracellular components during PLB extension and retraction. Retraction of PLBs is produced by myosin activation and the corresponding increase in PLB cortex contractility. Inhibition of myosin by blebbistatin reduces retraction while inhibition of RhoA-ROCK pathway by Y-27632 completely prevents retraction. Contraction of PLBs can produce cell translocation resembling active cell movement. Overall, the formation, properties, and life cycle of PLBs share common features with protrusions associated with ameboid cell migration. PLB life cycle may be controlled through activation of WASP by its upstream effectors such as Cdc42 and PIP2, and main ROCK activator-RhoA. Parallels between pseudopod-like blebbing and motility blebbing may provide new insights into their underlying mechanisms.

Electric field exposure triggers and guides formation of pseudopod-like blebs in U937 monocytes.

We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to "regular," round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10-20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb formation. In contrast to round-shaped blebs, PLBs retracted within several minutes after exposure. Cells treated with 1 nM of the actin polymerization blocker cytochalasin D were unable to form PLBs and instead produced stationary, spherical blebs with no elongation or retraction capacity. Live cell fluorescent actin tagging showed that during elongation actin promptly entered the PLB interior, forming bleb cortex and scaffold, which was not seen in stationary blebs. Overall, PLB formation was governed by both passive (physicochemical) effects of membrane permeabilization and active cytoskeleton assembly in the living cell. To a certain extent, PLB mimics the membrane extension in the process of cell migration and can be employed as a nonchemical model for studies of cytomechanics, membrane-cytoskeleton interaction and cell motility.

Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells.

BACKGROUND: Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-µs EP. METHODS: Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry. RESULTS: 10-ns EP caused apoptotic or necrotic death within 2-20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S=alphaD((-K)), where coefficients K and alpha determined the slope and the "shoulder" of the survival curve. K was similar in all groups, whereas alpha was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only. CONCLUSIONS: 1.8- and 9-µs EP cause cell death efficiently and indiscriminately (LD50 1-3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD50 50-80 J/g for Jurkat and 400-500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores ("nanopores"), triggering different cell death mechanisms. GENERAL SIGNIFICANCE: Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.

Gadolinium blocks membrane permeabilization induced by nanosecond electric pulses and reduces cell death.

It has been widely accepted that nanosecond electric pulses (nsEP) are distinguished from micro- and millisecond duration pulses by their ability to cause intracellular effects and cell death with reduced effects on the cell plasma membrane. However, we found that nsEP-induced cell death is most likely mediated by the plasma membrane disruption. We showed that nsEP can cause long-lasting (minutes) increase in plasma membrane electrical conductance and disrupt electrolyte balance, followed by water uptake, cell swelling and blebbing. These effects of plasma membrane permeabilization could be blocked by Gd(3+) in a dose-dependent manner, with a threshold at sub-micromolar concentrations. Consequently, Gd(3+) protected cells from nsEP-induced cell death, thereby pointing to plasma membrane permeabilization as a likely primary mechanism of lethal cell damage.