G-protein signal transduction is one of the most widely used mechanisms for cells to communicate with their environment. In a single cell several different G-protein signalling cascades are present, and recent evidence suggests extensive crosstalk between these individual cascades. How this is organised, while maintaining specificity in signalling is thus far poorly understood. To address this problem we study chemosensory behaviours in C. elegans. The animal is capable of detecting at least 60 odorants and to discriminate between many of them using only two pairs of cells, AWA and AWC. Interestingly, in these cells a specific subset of 6 G subunits is expressed (Jansen ea. 1999). To study the functions of the individual G subunits we use behavioural assays including an odorant discrimination assay (Bargmann ea 1993). Our preliminary data shows that the G subunit ODR-3 is sufficient to discriminate between at least some odorants. Many downstream effectors of G-proteins are known and are being tested for their function in these pathways. However, many of these genes are expressed ubiquitously and some have severe locomotory defects. This renders behavioural assays useless to study the effects of these proteins in signalling. Therefore we are developing imaging tools to visualise responses of individual cells to olfactory or gustatory stimuli. We will use two constructs, the Yellow Cameleon (YC) (Kerr ea. 2000) and the Voltage Sensitive Fluorescent Protein (VSFP) (Sakai ea. 2001). Both constructs utilise the Fluorescence Resonance Energy Transfer (FRET) principle to measure either the calcium fluxes in the cell or changes in the membrane potential, respectively. We will drive expression using cell-specific promotors, which allows us to elucidate the signal processing on a cellular level. To study effects in olfaction we will express the constructs in AWA using the promoter of the
odr-10 gene and in AWC with the
gpa-13 promoter, for gustation we will use ASE (
flp-5 promoter) and ASI (
gpa-4 promoter). Together with the data from behavioural assays, this will provide insight in the G-protein signalling pathways in olfaction in C. elegans.