Electrofusion of Mammalian Cells and Giant Unilamellar Vesicles

Teissié, Justin; Rols, Marie Pierre; Blangero, C.

doi:10.1007/978-1-4899-2528-2_13

“…The behaviour of lipid vesicles was rather similar to that observed with cells (Chernomordik et al, 1987), i.e. permeabilization was reversible, a fusogenic state was induced (TeissiC et al, 1989;Biisch et al, 1982;Hub et al, 1982;Melikyan and Chernomordik, 1989) and plasmid transfer was observed (Chernomordik et al, 1990). Permeabilization was shown to be very transient in pure lipid systems (Benz and Zimmermann, 1981).…”

supporting

confidence: 64%

“…The methods were adapted from those previously described (TeissiC et al, 1989). Electropulsation was operated by use of a CNRS cell electropulsator (Jouan, France).…”

Section: Chemicalsmentioning

confidence: 99%

Insertion of Glycophorin A, A Transmembraneous Protein, in Lipid Bilayers can be Mediated by Electropermeabilization

Raffy

¹

,

Teissié

²

1995

European Journal of Biochemistry

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Transmembraneous back-insertion of a solubilized membrane protein, glycophorin A, has been obtained in 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (Pam,GroPCho) cell-size-like liposomes by submitting the lipidprotein mixture to calibrated electric field pulses. Field conditions which are prone to trigger glycophorin insertion are similar to those which mediate lipid layer electropermeabilization. The efflux of calcein, trapped in the liposomes during their preparation, was observed only when field strength is higher than 1.3 kV/cm. Electroinsertion was detected only above the same critical field intensity. Calcein efflux as well as glycophorin insertion were increased by increasing field intensity, pulse duration andor number of pulses. Experimental evidence of protein insertion was provided by physico-chemical as well as biochemical methods. Direct observation of the pulsed vesicles under the microscope revealed the insertion by means of immunofluorescence and fluorescence. Electroinsertion of fluorescent glycophorin A revealed that the inner bilayers were also labeled. The gel-to-liquid phase transition temperature of Pam,GroPCho decreased after insertion but its cooperativity was not affected. A narrow 3'P-NMR peak was observed after electroinsertion showing that the polar headgroups of phospholipids had been altered. Analysis of trypsin-digested peptides revealed that the two trans-orientations of the protein across the external lipid layer were present after electroinsertion. Localized perturbation of the polar headgroup region of phospholipids, which supports the transient permeabilization of lipid layers, allows spontaneous transinsertion of glycophorin across the lipid bilayers.

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“…Application of electric pulses to the vesicle. The methods were adapted from those previously described (TeissiC et al, 1989). Electropulsation was operated by use of a CNRS cell electropulsator (Jouan, France).…”

Section: Chemicalsmentioning

confidence: 99%

Insertion of Glycophorin A, A Transmembraneous Protein, in Lipid Bilayers can be Mediated by Electropermeabilization

Raffy¹,

Teissié²

1995

View full text Add to dashboard Cite

Transmembraneous back-insertion of a solubilized membrane protein, glycophorin A, has been obtained in 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (Pam,GroPCho) cell-size-like liposomes by submitting the lipidprotein mixture to calibrated electric field pulses. Field conditions which are prone to trigger glycophorin insertion are similar to those which mediate lipid layer electropermeabilization. The efflux of calcein, trapped in the liposomes during their preparation, was observed only when field strength is higher than 1.3 kV/cm. Electroinsertion was detected only above the same critical field intensity. Calcein efflux as well as glycophorin insertion were increased by increasing field intensity, pulse duration andor number of pulses. Experimental evidence of protein insertion was provided by physico-chemical as well as biochemical methods. Direct observation of the pulsed vesicles under the microscope revealed the insertion by means of immunofluorescence and fluorescence. Electroinsertion of fluorescent glycophorin A revealed that the inner bilayers were also labeled. The gel-to-liquid phase transition temperature of Pam,GroPCho decreased after insertion but its cooperativity was not affected. A narrow 3'P-NMR peak was observed after electroinsertion showing that the polar headgroups of phospholipids had been altered. Analysis of trypsin-digested peptides revealed that the two trans-orientations of the protein across the external lipid layer were present after electroinsertion. Localized perturbation of the polar headgroup region of phospholipids, which supports the transient permeabilization of lipid layers, allows spontaneous transinsertion of glycophorin across the lipid bilayers.

show abstract

“…However, the existence of the opposite effect, that is, the modification of protein skeleton under conditions of electroporation, was also reported in literature on the effects on the cytoskeleton of animal cells after electric treatment reported by Teissie et al [36]. However, the existence of the opposite effect, that is, the modification of protein skeleton under conditions of electroporation, was also reported in literature on the effects on the cytoskeleton of animal cells after electric treatment reported by Teissie et al [36].…”

Section: The Specificity Of Electroporation For Biomembranesmentioning

confidence: 65%