Induction of endocytic vesicles by exogenous C(6)-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 microM C(6)-ceramide caused the formation of numerous vesicles of varying sizes (2-10 micrometers) 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 microM 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, approximately 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.
Glycobiology Program, Center for Cancer and Transplantation Biology, Children's Research Institute, Washington, D.C. 20010, USA. RLi@cnmc.org
R
R
Li
E J
EJ
Blanchette-Mackie
S
S
Ladisch
D016475:3T3 Cells; D000818:Animals; D002518:Ceramides / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000187:drug effects / Q000648:ultrastructure; D008247:Lysosomes / Q000187:drug effects / Q000648:ultrastructure; D051379:Mice; D018613:Microscopy, Confocal; D013997:Time Factors
D002518:Ceramides; C101954:N-caproylsphingosine