M.G. Malabarba1, E. Gerbino1, A. Croce1, H. Kloess1, A. E. Salcini2, G. Cassata1 and P. P. di Fiore1. Epsin belongs to a class of endocytic adaptors proteins whose function is not yet completely elucidated. C. elegans has proved to be a good model system to study endocytosis and it has the great advantage of studying the role of a protein in the contest of a whole organism. EPN-1 is the only C. elegans homologue of the mammalian family of Epsin and it retains the same modular organization. From the N-terminal, it displays the ENTH domain, two UIM domains, several AP2 binding motives (DPW) as well as two clathrin binding consensus, and at the C-terminal portion four EH binding motives (NPF). As initial approach to study the function of Epsin in the nematode, we downregulated the expression of the gene by RNAi. Interfered worms displayed retardation in growth, uncoordinated movements and egg laying defects. Endogenous EPN-1 protein is ubiquitously expressed with higher level of expression in the pharynx, vulva epithelial cells, and some unidentified neurons, judged both by the generation of transgenic worms, expressing
epn-1p::EPN-1::GFP fused protein and by immunofluorescence using specific antisera. To better analyze EPN-1 function, we generated a deletion mutant by EMS mutagenesis:
epn-1(
ng5).
epn-1(
ng5) display a lethal phenotype occurring in L1/L2 stage. The larvae accumulate vacuoles and have a general sick aspect until they die. The phenotype is recessive. The study of the phenotype associated to loss of
epn-1 gene function was approached by two main lines of experiments: the investigation of a possible cell specific requirement of the protein and a structurefunction analysis of the different EPN-1 domains. At present, we established that 1)
unc-119p::EPN-1::GFP, which drives the expression of the protein specifically in neurons, is the only array able to rescue larval lethality (besides arrays containing the endogenous promoter), while, when EPN-1 is expressed only in the pharynx or in the intestine the lethality persist; 2) the ENTH-UIM domains are the minimal region of the EPN-1 protein capable to restore viability. Rescued animals, although viable, show peculiar phenotypes that may contribute to identify the function(s) of EPN-1 in C. elegans. Preliminary results indicating a role for EPN-1 at the neuromuscular junctions and in neurotransmission will be presented and discussed. In conclusion, we determined that
epn-1 is an essential gene in C. elegans, as in yeast and D. melanogaster, moreover, we generated a valuable set of reagents, which will allow us to study its function in the context of the different molecular and genetic pathways in which the protein acts.