Calcium is a ubiquitous intracellular signal responsible for controlling a diverse array of processes. The specificity of Ca2+ signals is determined, in part, by their spatio temporal pattern, which is achieved using different storage compartments and specialised binding and gating molecules. The inositol 1, 4, 5-trisphosphate (IP3) receptor (IP3R) plays a key role in regulating the flux of Ca2+ from the endoplasmic reticulum (ER) into the cytoplasm. It follows that the localisation of IP3Rs may be critical in determining the specificity of Ca2+ signals. Our aim is to explore the relationship between IP3R subcellular localisation and the function of this pathway in the biology of a whole animal. To this end we are establishing a system in C. elegans in which IP3Rs are tagged with GFP. The large size and complex pleiotropic phenotypes associated with the IP3R make this a challenging goal. C. elegans IP3Rs are encoded by a single gene, itr-1
. Previously the expression pattern of itr-1
has been identified in transgenic animals expressing truncated forms of ITR-1 fused to GFP (1, 2, 3) and by using anti-ITR-1 antibodies (2). To establish a system that is closer to the <sym06
> situation we have adopted two approaches. Firstly we recreated the full-length itr-1
gDNA fused to GFP in an extrachromosomal array. This was achieved using PCR fusion and in vivo homologous recombination of two DNA fragments. The resulting localisation pattern of ITR-1::GFP was validated by its ability to rescue itr-1
) mutant phenotypes. More recently we used a <sym06
> approach using biolistic transformation to GFP tag endogenous itr-1
by homologous recombination. The results of these experiments will be presented. 1. Dal Santo, P., Logan, M. A., Chisholm, A. D. & Jorgenson, E. M. (1999) Cell 98, 757-767.2. Baylis, H. A., Furuichi, T., Yoshikawa, F., Mikoshiba, K. & Sattelle, D. B. (1999) J. Mol. Biol. 294, 467-476.3. Gower, N. J. D., Temple, G. R., Schein, J. E., Marra, M., Walker, D. S. & Baylis, H. A. (2001) J. Mol. Biol. 306, 145-157.