Induction of endocytic vesicles by exogenous C6-ceramide

Ceramide is a newly discovered second messenger that has been shown to cause cell growth arrest and apoptosis, Here, we present evidence that exogenously added C-6-ceramide induces enlargement of late endosomes and lysosomes, 10 mu M C-6-ceramide caused the formation of numerous vesicles of varying sizes (2-10 mu m) in fibroblasts (3T3-L1 and 3T3-F442A), without toxic effects. Vesicle formation induced by C-6-ceramide was time- and dose-dependent, rapid, and reversible. Numerous small vesicles appeared within 8 h of treatment with 10 mu M C-6-ceramide, They enlarged with time, with large vesicles found in the perinuclear region and small ones observed at the cell periphery. Within 24 h of treatment, similar to 30% of the cells exhibited these vesicles. Removal of ceramide from the culture medium caused disappearance of the vesicles, which reappeared upon readdition of ceramide, Confocal immunofluorescence microscopic analysis using an anti-lysosome-associated membrane protein antibody identified the enlarged vesicles as late endosomes/lysosomes. The fluorescent C-6-NBD-ceramide, a vital stain for the Golgi apparatus, did not stain these vesicles. The effect on vesicle formation was influenced by ceramide structure; D-erythro-C-6-ceramide was the most active ceramide analogue tested. Short chain ceramide metabolites, such as sphingosine, sphingosine 1-phosphate, N-hexanoyl-sphingosylphosphorylcholine, N-acetylpsychosine, and C-2-ceramide G(M3), (G(M3), N-acetylneuraminosyl-alpha(2,3)-galactosyl-beta (1,4)-glucosylceramide), were inactive in causing vesicle formation when added exogenously, Together, these studies demonstrate that exogenous C-6-ceramide induces endocytic vesicle formation and causes enlarged late endosomes and lysosomes in mouse fibroblasts.