A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses.

Abstract

Abstract. Treatment of erythrocyte ghosts in random positions in a suspension with membrane fusion-inducing direct current electric field pulses causes the membranes to become fusogenic. Significant fusion yields are observed if the membranes are dielectrophoretically aligned into m e m b r a n e -m e m b r a n e contact with a weak alternating electric field as much as 5 min after the application of the pulses. This demonstrates that a long-lived membrane structural alteration is involved in this fusion mechanism. Other experiments indicate that the areas on the membrane which become fusogenic after treatment with the pulses may be very highly localized. The locations of these fusogenic areas coincide with where the transmembrane electric field strength was greatest during the pulse. The fusogenic membrane alteration, or components thereof, in these areas laterally diffuses very slowly or not at all, or, to be fusogenic, must be present at concentrations in the membrane above a certain threshold. The loss of soluble 0.9-3-nm-diameter fluorescent probes from resealed cytoplasmic compartments of randomly positioned erythrocyte ghosts occurs through electric field pulse-induced pores only during a pulse but not between pulses or after a train of pulses if the probe diameter is 1.2 nm or greater. For a given pulse treatment of membranes in random positions in suspensions, an increase in ionic strength of the medium results in (a) a decrease in loss during the pulse, (b) no difference in loss between pulses, and (c) an increase in fusion yield when membrane-membrane contact is established. The latter two results (b and c) are incompatible with a fusion mechanism that proposes a simple relationship between electric field-induced pores and fusion.

Electroporation in Biological Cell and Tissue: An Overview

“…The main difference between two processes is the critical voltage required for electrofusion, which is higher than for electroporation (Teissié and Rols 1993;Abidor et al 1994;Teissié and Ramos 1998), and the duration of fusogenic state, which is shorter than cell membrane resealing process. The resealing of the cell membrane after electroporation can take up to tens of minutes, while membrane fusion is only possible if the contact of permeabilized membranes is achieved within few minutes after pulse application (Teissié and Rols 1986;Sowers 1986;Ramos and Teissié 2000a). The contact needed for electrofusion can be obtained before or immediately after the electroporation pulse.…”

“…The contact needed for electrofusion can be obtained before or immediately after the electroporation pulse. When the cell contact is obtained before electroporation, most often dielectrophoresis is used (Zimmermann 1982), while the contact of cells after electroporation is obtained by centrifugation of fusogenic cells (Teissié and Rols 1986;Sowers 1986). …”

Electropermeabilization mediates a stable insertion of glycophorin A with Chinese hamster ovary cell membranes

“…Cell membrane electropermeabilization must occur in order for insertion to take place. Taking into account the observation that electropermeabilization for small molecules is a long-lived process (Neumann, 1989), as is the case for the associated property of electrofusion (Sowers, 1986(Sowers, , 1987Teissi6 and Rols, 1986), we investigated the lifetime of the electroinsertioncompetent state of the membrane by adding glycophorin to previously pulsed CHO cells. An increase in bound glycophorirdcell and of labelled cells was observed even when proteins were added several minutes after cell electropulsa- tion (Fig.…”

Membrane and actin reorganization in electropulse-induced cell fusion

“…In contrast, membrane fusion in erythrocyte ghosts can be delayed for several seconds or minutes after electric pulsing (Dimitrov and Sowers, 1990). This means, the electric pulses initiate a sequence of processes that, after termination of the electric field, continue to produce a long-lived fusogenic state that precedes mixing of the membranes (Sowers, 1986).…”

“…However, for energetic reasons cells may, by the tilting of lipids, directly pass from the stalk to a modified stalk and further, by expansion of a prepore, to a fusion pore (Kuzmin et al, 2001). Moreover, earlier studies on mammalian erythrocyte ghosts (Sowers, 1986) and CHO cells (Teissié and Ramos, 1998) have indicated that cells acquire a fusogenic state that can last for minutes before they are brought into contact, this means before hemifusion of apposed membranes has an opportunity to commence (Dimitrov and Sowers, 1990). In supplementary material Movie 5, the right pair of cells appears to lack contact for 18 seconds after electric pulsing before fusion commences.…”