Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, Phone: 44 141 330 6228 FAX: 44 141 330 5994 E-mail: prosay@udcf.gla.ac.uk
Mobilisation and turn-over of intracellular calcium are integral aspects of cell regulation during responses to several classes of extracellular stimuli. Our laboratory is developing a powerful method for measuring cytosolic calcium changes in Drosophila involving a transgene that directs expression of the calcium-sensitive luminescent protein, aequorin. Compared with fluorescent calcium reporters, changes can be monitored for a long period without damage to the cell, autofluorescence is eliminated, and aequorin is not sequestered into organellar compartments. Cell-type specificity of expression is obtained by use of the binary GAL4/UASG system, allowing us to capitalise on the large number of P[GAL4] enhancer trap lines that we and others have generated.
The Malpighian tubule provides a unique phenotype for the genetic dissection of cell signalling pathways. Tubule function can be assessed by fluid secretion assays and electrophysiology, and tubules are known to be stimulated by elevation of cAMP, cGMP (via nitric oxide) or by agents which are thought to manipulate intracellular calcium (Dow, J.A.T., Maddrell, S.H.P., Gortz, A., Skaer, N.V., Brogan, S. Kaiser, K. (1994) J. exp. Biol. 197:421-428). However, it is difficult to measure these changes directly, because tubules do not label effectively with calcium sensitive fluorescent dyes, and the cells are too small to be impaled with ion-selective electrodes. Additionally, by screening over 700 P[GAL4] lines for patterns of expression specific to tubule sub-regions, we have recently shown that the tubules are complex structures, with at least 5 genetically-defined sub-regions and 5 distinct cell types in a tissue which totals just 145 cells. Accordingly, we have developed a UASG-aequorin construct which allows intracellular calcium peaks to be detected in genetically defined compartments of tubules, or indeed of any Drosophila tissue. The results show that thapsigargin-induced calcium release from intracellular stores and ionophore-induced calcium entry are both easily detectable in living tubules. Additionally, diuretic peptides appear to elicit a rise in intracellular calcium in only certain cell types. Our current working hypothesis allocates different aspects of tubule ion transport to different identified cell types.
The Malpighian tubule also provides a context within which we can develop the technology for more exacting applications. In particular, we are extending application of the aequorin system to the intrinsic (Kenyon) cells of the mushroom bodies, higher brain centres implicated in learning and memory (Yang, M-Y., Armstrong, J.D., Vilinsky, I., Strausfeld, N.J. Kaiser, K. (1995) Neuron 15:45-54). Several different classes of Kenyon cell can be identified on the basis of GAL4 expression and functional characteristics. However, little is known about the transmitter systems that operate within them. The aequorin system, particularly in conjunction with signal transduction mutations, may be a powerful way to address this issue.
