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Imaging bioluminescent indicators shows Ca 2+ and ATP permeability thresholds in live cells attacked by complement
Author(s) -
Graciela B. Sala-Newby,
KM Taylor,
Michael Norman Badminton,
Christopher M. Rembold,
Anthony K. Campbell
Publication year - 1998
Publication title -
immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.297
H-Index - 133
eISSN - 1365-2567
pISSN - 0019-2805
DOI - 10.1046/j.1365-2567.1998.00004.x
Subject(s) - bioluminescence , complement (music) , biophysics , permeability (electromagnetism) , chemistry , microbiology and biotechnology , biology , biochemistry , membrane , gene , complementation , phenotype
A series of permeability thresholds to Ca 2+ metabolites and macromolecules, occurring at different times when cells are attacked by complement, has been established by imaging HeLa cells transiently expressing a recombinant cytosolic fusion protein of firefly luciferase and aequorin (luciferase–aequorin) to measure changes in ATP and cytosolic free Ca 2+ . Nuclear fluorescence of propidium was used as a measure of permeability to small molecules, and luciferase activity imaged to assess lysis. The rise in cytosolic free Ca 2+ observed after C9 attack preceded by at least 60 s both the increase in propidium fluorescence, measured in single cells, and the decrease in ATP monitored by luciferase light emission. These effects were dependent on the concentration of C9. At concentrations of C9 up to 4 μg/ml no loss of luciferase–aequorin protein was detected at the end of the experiment. Thus the membrane integrity of the cells remained intact, even though the cells were permeable to propidium. These results confirmed our earlier observations that propidium permeability in cells attacked by complement was not a reliable measure of cell death. They also show that it is vital to take account of cellular heterogeneity if the mechanisms by which cells respond to membrane pore former attack are to be correctly interpreted.