Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes

Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central seco nd messenger in cellular signaling for apoptosis (programmed cell death), d ifferentiation, and senescence. We used microinjection and either Hoffman m odulation contrast or fluorescence microscopy of giant liposomes composed o f 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphin gomyelin (C16: 0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (mol ar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, i n addition to rapid domain formation (capping), vectorial budding of vesicl es, i.e., endocytosis and shedding, can be induced by the asymmetrical sphi ngomyelinase-catalyzed generation of ceramide in either the outer or the in ner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin lipos omes. These results are readily explained by I) the lateral phase separatio n of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented be nding rigidity of the ceramide-containing domains, leading to membrane inva gination and vesiculation of the bilayer.