Aequorin is a photoprotein originally characterised in the luminescent jellyfish Aequorea victoria. The jellyfish is capable of producing flashes of blue light, which in turn is transduced to green by the famous green fluorescent protein.
Critically, the intracellular signal used to elicit this flash in vivo is an increase in intracellular calcium. As the dynamic range of calcium signalling concentrations in jellyfish is very similar to those in plants and animals generally (50-500 nM), this means that aequorin is a useful calcium indicator.
Aequorin was first used by direct extraction from jellyfish and microinjection, but over the last decade, recombinant aequorin has become available, and it has also been used as a transgene, originally in plants.
Our group, in collaboration with that of Kim Kaiser, has characterised a UAS::aequorin transgene that allows us to measure intracellular calcium in any population of Drosophila cells that can be marked with GAL4 enhancer trap technology. We have applied this to the Drosophila Malpighian tubule, and have produced some provocative results.
The work is described in:
Rosay, P., Davies, S. A., Yu, Y., Sozen, M. A., Kaiser, K. and Dow, J. A. T. (1997). Cell-type specific calcium signalling in a Drosophila epithelium. Journal Of Cell Science 110, 1683-1692.
Technically, the transgene encodes apoaequorin. The functional photoprotein -aequorin- is generated by reconstitution with the cell-permeable cofactor coelenterazine. To acheive this, tubules are acutely dissected from progeny of a cross between a GAL4 cell-specific driver line and a line containing the UAS/apoaequorin construct. These are incubated with coelenterazine for typically 4 h, then transferred to a luminometer (basically a VERY EXPENSIVE light-tight, mirrored box with a photomultiplier tube). Agonists can be injected through a light-tight seal, while the light emission is measured throughout.
The actual amount of light given out at any instant is a function of both the calcium activity and the amount of reconstituted aequorin that remains unused. The photon emission data can be converted to "real" values by permeabilising the cells at the end of the experiment with triton, causing massive luminescence that can be measured. It's then possible to integrate backwards in time to every instant of the experiment, and so get quantitative values.
