IMAGING BIOLUMINESCENT INDICATORS SHOWS CA2+ AND ATP PERMEABILITY THRESHOLDS IN LIVE CELLS ATTACKED BY COMPLEMENT

A series of permeability thresholds to Ca2+ metabolites and macromolec ules, occurring at different times when cells are attacked by compleme nt, has been established by imaging HeLa cells transiently expressing a recombinant cytosolic fusion protein of firefly luciferase and aequo rin (luciferase-aequorin) to measure changes in ATP and cytosolic free Ca2+. Nuclear fluorescence of propidium was used as a measure of perm eability to small molecules, and luciferase activity imaged to assess lysis, The rise in cytosolic free Ca2+ observed after C9 attack preced ed by at least 60 s both the increase in propidium fluorescence, measu red in single cells, and the decrease in ATP monitored by luciferase l ight emission. These effects were dependent on the concentration of C9 . At concentrations of C9 up to 4 mu g/ml no loss of luciferase-aequor in protein was detected at the end of the experiment. Thus the membran e integrity of the cells remained intact, even though the cells were p ermeable to propidium. These results confirmed our earlier observation s that propidium permeability in cells attacked by complement was not a reliable measure of cell death. They also show that it is vital to t ake account of cellular heterogeneity if the mechanisms by which cells respond to membrane pore former attack are to be correctly interprete d.