Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts.
We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.
Koval
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Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210-3301.
Pagano
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D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002451:Cell Compartmentation / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002502:Centrioles / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects; D008985:Monensin / Q000494:pharmacology; D015739:Nocodazole; D013109:Sphingomyelins / Q000378:metabolism*
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D013109:Sphingomyelins; D008985:Monensin; D015739:Nocodazole