SEIX 17-20 octobre 2005 - Atelier Calcium

of both the transcription and alternative splicing of nAChR subunits in Cha-1 neurons. The
expression of the D 2, D 6, D 7, D 1 and D 2 subunits is also detected in embryonic and
larval Cha-1 RNA by microarray analysis. The D 2 and D 2 subunits are the most
abundantly expressed subunits at both developmental stages. By combining calcium imaging
and knock-down of individual nAChR subunits by RNA interference (RNAi), we have shown
that a reverse genetic approach can be used to demonstrate roles for the D 2 and D 6 nAChR
subunits in cultured Cha-1 neurons.
THE INTERACTOME AND GENOME-WIDE RNA INTERFERENCE: PROSPECTS
FOR IDENTIFYING NOVEL CALCIUM SIGNALLING/MODULATING
COMPONENTS
The ease with which RNAi can be applied to S2 cells makes them particularly suitable for
genome-wide RNAi screens. Genes affecting calcium signalling can be detected in a number
of ways. The most obvious is to look for genes whose knockdown results in an abolition of
store-operated entry. This has been done in one study [7] by applying dsRNA for each gene
to S2 cells and subsequently using FLIPR to measure the calcium transients in response to
thapsigargin-evoked store depletion. The detection of only one gene product (STIM1) using
this approach implies either that only one gene is necessary for store-operated calcium entry
or, more likely, that the screen is not sufficiently sensitive to detect other essential genes, or
that there is a high degree of redundancy in the calcium signalling pathway. The latter can be
addressed using combinatorial RNAi screens, whose scale should perhaps be limited by
restricting the screen to a set selected on the basis of the presence of calcium-binding motifs
or other sequence elements, such as PZD domains, that suggest they might interact with
calcium-binding proteins. Future screens might use a different end-point for the assay, such
as thapsigargin-evoked apoptosis. Thus, the knockdown of genes that regulate calcium
signalling might result in changes in the probability of apoptosis resulting from challenges to
the calcium homeostasis machinery.
In addition to genome-wide screens, more direct approaches combining RNAi and
bioinformatics are possible. Alternatives to genome-wide screens include direct tests of
proteins that are recorded in the Drosophila interactome database as being interactors with
calcium-regulating genes. We have identified a number of genes on the basis of text-mining
approaches, the roles of each of which in calcium signalling can be tested using RNAi.
CONCLUSIONS
1) Drosophila cell lines and cell cultures lend themselves well to RNAi and calcium imaging
studies designed to uncover new calcium signalling components.
2) Genome – wide RNAi screens are feasible and when combined with use of known mutants
will afford functional screens for functionally linked molecules in signalling pathways.
3) Such studies will in future serve as a substrate for systems biology.
REFERENCES
1. Adams M.D. et al (2000) Science 287, 2185-2189.
2. Towers and Sattelle (2002) Bioessays 24, 1066-1073.
3. Clemens et al. (2000) PNAS 97, 6499-6503).
4. Millar et al (1995) 1843-1850.
5. Raymond et al (2004) Cell Calcium 35, 131-139.
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