The
eff-1 gene is required for fusion of epithelial (1), as well as of myoepithelial cells. It was recently shown that
eff-1 is also sufficient to form syncytia in different tissues in C. elegans (2). This gene is predicted to encode at least four isoforms; two of them are Type I proteins: EFF-1A (74.4kDa) and EFF-1B (67.3kDa) and two are secreted forms: EFF-1C (15kDa) and EFF-1D (27kDa). It is still not clear which isoform is required for normal cell fusion. In order to biochemicaly characterize EFF-1 and to analyze EFF-1 isoforms distribution at different developmental stages, it was necessary to obtain a large arsenal of antibodies designed against a spectrum of epitopes in the protein. We have raised rabbit polyclonal antibodies against six peptides representing different domains of the predicted proteins, as well as full length proteins of the different isoforms. We first chose the antibodies that present high titer by an ELISA assay.Our first goal was to identify the EFF-1 isoforms, which are overexpressed following heat shock in worms carrying an hsp: EFF-1 extrachromosomal array (2), in comparison with non heat shocked worms and N2. Heat shock promoter fused to a heterologous gene served as a control for heat shock proteins. An affinity purified antibody raised against a predicted Phospholipase A2 active site in
eff-1 sequence detected 67kDa and 27kDa bands in the over expressed
eff-1 extracts, whereas a different antibody, developed against another peptide in the same region of the protein, detected 97kDa, 50kDa and 40kDa bands. In order to confirm the identification of these proteins and to learn about possible post translational modifications we will show immunoprecipitation and subcellular fractionation of worm lysates using antibodies against different domains of the protein. Currently we are testing several mutants of
eff-1. The western blot results suggest that there are at least two EFF-1 variant forms but it is still unclear whether these fragments are degradation products of EFF-1 or actively expressed proteins, resulting from alternative splicing or post translational modifications. Work is in progress to stain animals in order to learn the pattern of protein expression. Preliminary results show immunostaining of the tested antibodies in the intestine, the excretory canal and the seam cells in N2 worms. The cellular localization of EFF-1 together with the characterization of expression pattern of the isoforms will allow us to better understand the mechanism of
eff-1 dependent cell fusion. (1) Dev Cell, 2002, 2, 355-362. (2) Curr Biol, 2004, 14, 1587-1591.