To examine the (intra)cellular fate of a glycolipid, normally residing at the cell surface, a fluorescent analog of glucosylceramide, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsphingosine (C6-NBD-glucosylceramide), was inserted into the plasma membrane of baby hamster kidney cells at low temperature. Upon warming the cells to 370C, part of the glycolipid analog was internalized. A comparison with receptor-mediated uptake of transferrin revealed that after 2 min of warming, both C6-NBD-glucosylceramide and the transferrin-transferrin receptor complex are localized in the same intracellular compartment (early endosomes). We conclude that C6-NBD-glucosylceramide is internalized along the pathway of receptor-mediated endocytosis. When, after internalization of part of the membrane-inserted glycolipid analog, the residual pool of plasma membrane C6-NBD-lipid was removed by "back exchange" with a lipid acceptor, C6-NBD-glucosylceramide molecules can be shown to return intact to the plasma membrane. This demonstrates that glycolipids, analogous to a variety of protein receptors, are able to recycle to the plasma membrane after internalization.
Primary cultures of rat oligodendrocytes were incubated with a fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoylceramide+ ++ (C6-NBD-ceramide). This compound is known to stain the Golgi complex specifically. Within 30 min of incubation at 37 degrees C most of the C6-NBD-ceramide was incorporated into the perinuclear Golgi system, as revealed by conventional and confocal laser fluorescence microscopy. Interestingly, C6-NBD-ceramide was found to accumulate also in smaller, oval-shaped structures in many of the processes, at distances up to 30 microns from the nucleus. This implies the possibility that these structures are Golgi (-derived) complexes. Indeed, after incubation of oligodendrocytes with C6-NBD-ceramide and rhodamine-labeled transferrin both fluorescent labels colocalized in the Golgi system of the cell body as well as in the structures in the processes. Additional support for the Golgi character of these structures was obtained by transmission electron microscopy. Particularly in oligodendrocytes cocultured with neurons, many Golgi structures were present all over the processes. The results lead us to conclude that, in the oligodendrocyte, the Golgi complex does not only reside in the perikaryon, but also in the processes. One can speculate that a polarized biosynthetic activity, involving the presence of the Golgi near the site of myelin synthesis, may be advantageous to the oligodendrocyte for assembly and/or repair of the myelin membrane at the distal end of the processes.
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