Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus.

Abstract

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts . 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2°C . The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2°C ; however, when the cells were warmed to 37°C, internalization of some of the fluorescent lipid occurred . Most of the internalized C6-NBD-PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37°C was blocked by the presence of inhibitors of endocytosis .Incubation of cells containing C6-NBD-PC at 37°C resulted in a rapid degradation of the fluorescent lipid . This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium.We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface . Both fluorescent lipid and protein remained at the plasma membrane at 2°C and neither were internalized at 37°C in the presence of inhibitors of endocytosis . However, when incubated at 37°C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites.Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer . The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane .

Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.

“…Mutant Tf receptors, differing in internalization rates due to cytoplasmic tail mutations or deletions also recycled efficiently, and exited the cell at the same rate as wild type receptors (Jing et al, 1990;McGraw and Maxfield, 1990;McGraw et al, 1991). Furthermore, other Ct-NBD-derivatized lipids, glucosylceramide (Kok et al, 1989), galactosylceramide, and PC (Sleight and Abanto, 1989;Sleight and Pagano, 1984; and this report) also enter the endocytic route, colocalized with transferrin, and are recycled.…”

“…Sphingomyelin and phosphatidylcholine are asymmetrically located in the exoplasmic leaflet of the plasma membrane and have a very low rate of transbilayer flip-flop, at least at the plasma membrane (for review see Devaux, 1990, and exogenously introduced fluorescent analogs of phosphatidylcholine (Ct-NBD-PC) and sphingomyelin (Ct-NBD-SM) do not translocate to the cytoplasmic surface during the endocytic process (Koval and Pagano, 1989;Sleight and Pagano, 1984). Furthermore, the rate of internalization of Ct-NBD-SM measured in CHO cells (~1.5 % internalized per minute; [Koval and Pagano, 1989] and unpublished observations) is comparable to bulk membrane endocytic rates (*1.6% of plasma membrane area internalized per minute) determined by stereological analyses and rates of bulk fluid phase endocytosis in BHK cells .…”

Uptake and metabolism of fluorescent ceramide analogs by rat oligodendrocytes in culture

“…1B). The mechanism of incorporation was further investigated by incubating the cells under energy-depleted (5 mM axide/ 50 mM deoxyglucose [24]) or low-temperature (4°C; GO-90 min) conditions that block endocytosis. The internalization of the fluid-phase marker FTTC-dextran [25] in oligodendrocytes in culture was blocked effectively under these conditions .…”

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

“…For these studies, it was necessary to use the nonhydrolyzable dstereoisomer of NBD-PC, because the natural l-isomer is rapidly degraded by cells (Sleight and Pagano, 1984), pre- . DsRed-cav-1 colocalization with BODIPY-LacCer and fluorescent albumin.…”