Fusion of lipid vesicles with ascites tumor cells and their lipid-depleted variants. Studies with radioactive- and fluorescent-labeled vesicles.
Cultured ascites tumor cells and their lipid-depleted variants, which contained 35-40% less membrane phospholipid and cholesterol, were used for fusion experiments with unilamellar lipid vesicles which were between 300 and 600 nm in diameter. Vesicle-cell interaction was followed by tracer studies using vesicles double-labeled in the lipid moiety, by vesicle-encapsulated [3H] dextran, and by measurements of energy transfer between N-(10-[1-pyrene]decanoyl)sphingomyelin-labeled vesicles and alpha-parinaric acid-labeled cells in the presence of poly(ethylene glycol) (PEG) as fusogen. The reaction rates measured with the radiolabeled vesicles were found to follow patterns similar to those obtained with the resonance energy transfer assay. This latter method revealed a vesicle-cell membrane fusion reaction, which was substantiated by radiolabeling the internal cellular compartment after treatment of the cells with [3H]dextran-encapsulated vesicles as shown by electron microscopic autoradiography on semi-thin sections. Endocytosis as a reaction mechanism can be excluded, since no energy transfer was observed at 25 degrees C in the absence of PEG. Investigations of vesicle bilayer order and fluidity on vesicle-cell interaction revealed optimal reactivity, with intermediate fluidity corresponding to cholesterol/phospholipid ratios between 0.7 and 1.0 and fluorescence depolarization (P) values of 0.18 and 0.21. Lipid depletion decreased the reaction velocity between cells and vesicles by about 20%, exhibiting V values of 33.2 mumol/min, as compared to the control of 41.4 mumol/min determined for 10(7) cells. The affinity constants for vesicle lipid were affected only slightly with Km values of 0.195 mM (0.210 mM). The activation energies for the reaction were calculated to give values of EA = 22.44 kJ/mol for the control and of EA = 20.4 kJ/mol for the modified cells. These data indicate that the decrease in membrane lipid content apparently has no major influence on the extent of the interaction.
Seibicke
S
S
Institut für Zell- und Tumorbiologie, Deutsches Krebsforschungszentrum, Heidelberg, F.R.G.
Zimmermann
H P
HP
Haeffner
E W
EW
D000818:Animals; D001201:Ascites; D002462:Cell Membrane / Q000502:physiology*; D002784:Cholesterol / Q000502:physiology; D003911:Dextrans; D007700:Kinetics; D008081:Liposomes; D008561:Membrane Fusion; D008563:Membrane Lipids / Q000502:physiology*; D051379:Mice; D008854:Microscopy, Electron; D014407:Tumor Cells, Cultured / Q000502:physiology*
D003911:Dextrans; D008081:Liposomes; D008563:Membrane Lipids; D002784:Cholesterol