Epithelial sphingolipid sorting is insensitive to reorganization of the Golgi by nocodazole, but is abolished by monensin in MDCK cells and by brefeldin A in Caco-2 cells

Abstract

In epithelial MDCK and Caco-2 cells, short-chain analogs of glucosylceramide and sphingomyelin are delivered from the Golgi to the cell surface with different apical/basolateral polarities, which results in an apical enrichment of the glycolipid glucosylceramide over the phospholipid sphingomyelin. Here, we have interfered with the integrity of the Golgi complex in various ways and tested the effects on lipid transport and sorting. Nocodazole, which depolymerizes microtubules, dispersed the Golgi over the cytoplasm of MDCK cells and reduced transport of newly synthesized C6-NBD-(N6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminocaproyl)-gluco-sylceramide and C6-NBD-sphingomyelin to the apical surface by 40%. The lipids were not mistargeted to the basolateral surface and upon removal of nocodazole, apical transport recovered. Nocodazole did not affect the apical enrichment of glucosylceramide over sphingomyelin. The ionophore monensin led to swelling of the Golgi of MDCK cells and inhibited lipid transport to the cell surface by 30-50%. Whereas sphingomyelin transport to both surface domains was equally affected, monensin mainly inhibited apical transport of glucosylceramide. At 10-20 μM of monensin, the two lipids displayed the same polarity of delivery: sorting between the two lipids was abolished. Brefeldin A at 1 μg/ml, which resulted in disruption of the Golgi in HepG2 cells and completely inhibited protein secretion, had no inhibitory effect on transport of the C6-NBD-lipids to the surface. The same was observed in Caco-2 cells. However, brefeldin A selectively shifted transport of sphingomyelin towards the apical direction which abolished the apical enrichment of glucosylceramide over sphingomyelin. Caco-2 cells were used because in MDCK cells brefeldin A did not change Golgi structure nor lipid transport and sorting. In summary, modification of the Golgi by monensin and brefeldin A, but not nocodazole, interfered with the sorting event by which glucosylceramide is enriched over sphingomyelin in the transport pathway from the Golgi to the apical surface.