Real-time measurements of chemically-induced membrane fusion in cell monolayers, using a resonance energy transfer method.
Fusion of mouse melanoma cells grown in monolayers has been directly monitored by fluorescence resonance energy transfer between fluorescein and rhodamine probes attached to octadecanoic acid. Various poly(ethylene glycol)s (PEG), either alone or in combination with amphipathic molecules, have been used as fusogens. Fusion starts at a maximum rate as soon as PEG is removed from the medium and reaches a plateau after 20-30 min. Both the initial rate and extent of fusion have been recorded for each experiment. The extent of fusion shows in general a positive correlation with the initial rate, although PEGs with different molar masses appear to induce fusion at different rates, but to a similar extent. A good correlation has been found between the extent of fusion, as measured by fluorescence, and the 'fusion index' computed from cell and nucleus counting; a calibration curve is provided for the interconversion of both parameters. Optimum fusion values are obtained with 50% (w/v) PEG 1500. The effect of pre-treatments with surfactants (Triton X-100, sodium dodecylsulphate) on PEG-induced fusion has also been tested. Sodium dodecylsulphate, but not Triton, enhances considerably both the rate and extent of cell fusion. The in situ generation of the amphipathic molecule diacylglycerol, through the catalytic activity of a phospholipase C, also enhances significantly the fusion parameters. These results are in good agreement with previous studies based on syncytia counting.
Partearroyo
M A
MA
Department of Biochemistry, University of the Basque Country, Bilbao, Spain.
Cabezón
E
E
Nieva
J L
JL
Alonso
A
A
Goñi
F M
FM
D000818:Animals; D002459:Cell Fusion / Q000187:drug effects*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D008561:Membrane Fusion / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D011092:Polyethylene Glycols / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000494:pharmacology
D005456:Fluorescent Dyes; D011092:Polyethylene Glycols; D010738:Type C Phospholipases