Endocytosis is a basic function of eukaryotic cells that leads to the internalization of fluid from the extracellular medium, nutrient uptake and the recycling of membrane components. In multicellular organisms, endocytosis has also been adapted by specialized cells for specific functions, including signaling down regulation, synaptic vesicle recycling, and antigen presentation. There are various routes by which endocytosis is accomplished, involving different cellular structures. The endocytic pathway utilized and the particular mechanisms used to regulate it depend on the ligand being internalized and the cell type examined. The multiplicity of options adds complexities that suggest that endocytosis should be studied using several different approaches and experimental systems. The coelomocytes of C. elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. We describe here a more detailed analysis of
cup-5 one of the mutants identified from this screen. We show that the
cup-5 loss of function mutation results in an enhanced rate of uptake of fluid-phase markers, decreased degradation of endocytosed protein, and accumulation of large vacuoles. Based on pulse chase experiments in coelomocytes marked with RME-8, a late endosomal marker, we identify these large vacuoles as late endosomes and/or lysosomes. Furthermore, CUP-5 itself localizes to compartments that are not labeled with EEA1, an early endosome marker, or RME-8, a late endosome marker. The predicted CUP-5 protein is 611 amino acids with six predicted membrane spanning domains. CUP-5 is homologous to mucolipin-1 in humans. Loss of human mucolipin-1 underlies Mucolipidosis Type IV (MLIV), a lysosomal storage disease that results in severe developmental neuropathology. MLIV cells display an enhanced rate of uptake and accumulate large vesicles, suggesting that a defect in endocytosis may underlie the disease. This similarity in phenotypes between human and worm cells suggests that the C. elegans
cup-5 mutant may be a useful model for studying conserved aspects of mucolipin-1 structure and function and for analyzing its function in endocytosis. Thanks to Iva Greenwald, in whose lab much of this work was done.