Developmental cell fusion is an important process in multicellular organisms.
eff-1 is a gene encoding
type-1 membrane proteins essential for all epithelial cell fusion events in C. elegans (1). Alternative splicing results in at least four different isoforms of EFF-1. EFF-1A and EFF-1B predicted structures show five putative domains: a signal peptide, a phospholipase A2 site, a fusion peptide, a transmembrane and a cytoplasmic tail, while EFF-1C and EFF-1D are predicted to be secreted proteins containing only the first two domains. Understanding how and where EFF-1 proteins work will help us to better understand the puzzle of the fusion mechanism. Until recently only two
eff-1 alleles were known. The more severe allele
eff-1(
hy21ts) causes total hypodermal fusion arrest and a series of different abnormal phenotypes such as Unc, Dpy, Pvl, Egl and Ste. RNAi experiments and analysis of
eff-1(
hy21ts)/Df show more severe Eff-1 phenotypes, implying for a correlation between EFF-1 dosage and fusion activity. These experiments also show that
hy21ts is not a null allele. To understand the structure and function of
eff-1 we looked for new alleles using a non-complementation screen. Young adult
ajm-1::gfp males were mutagenized with EMS, mated with
eff-1(
hy21ts)
unc-104(
e120)II hermaphrodites and the Fl hermaphrodite progeny were screened at 15 degrees C for non-Unc animals exhibiting Eff-1 phenotypes. Suspected mutants that showed fusion defects and failed to complement
eff-1(
hy21) were sequenced using
eff-1 specific primers. Out of 18,000 Fl hermaphrodites screened, two independent animals were identified as non-complementing
eff-1 alleles, both with the same point mutation (
hy32 and
hy33). The
hy32 and
hy33 mutation is a C135Y change at the phospholipase A2 site next to the putative aspartic acid active site. This mutation results in Eff-1 phenotypes, more severe than the original
hy21ts allele. The
hy40 and
hy41 alleles of
eff-1 were recently isolated in a two-step clonal screen by T. Gattegno. These worms are very Dpy and have a small brood size.
hy41 is a change of a conserved G316E.
hy40 is a stop codon after aa 224, resulting in a short secreted protein containing the phospholipase A2 site. Another short EFF-1 protein probably exists in the
np29 allele, which was recently isolated in a Mos mutagenesis screen by N. Pujol and characterized by J. Novelli (2). We will show a phenotypic comparison between
eff-1 alleles as well as the
hy21ts/Df strain. We will also show analysis of worms expressing a translational EFF-1A::GFP construct and discuss the implications of these results. 1. Mohler et al, Dev Cell 2002. 2. We thank N. Pujol, J. Novelli and J. Hodgkin for kindly provided
np29.