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Sphingolipid Metabolism in Cultured Fibroblasts: Microscopic And Proc. Nati Acad. Sci. USA Vol. 80, pp. 2608-2612, May 1983 Cell Biology Sphingolipid metabolism in cultured fibroblasts: Microscopic and biochemical studies employing a fluorescent ceramide analogue (Golgi apparatus/sphingomyelin/cerebrosides/liposomes/fluorescence) NAOMI G. LPSKY AND RICHARD E. PAGANO Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, Maryland 21210 Communicated by Harden M. McConnell, December 30, 1982 ABSTRACT A fluorescent analogue of ceramide, N-[7-(4-ni- HI trobenzo-2-oxa-1,3-diazole)]-e-aminocaproyl sphingosine (C6-NBD- ceramide), was used to investigate sphingolipid metabolism in HO-C-H Chinesehamster fibroblasts. C6-NBD-ceramide was incorporated | /~~~~0 into small unilamellar dioleoyl phosphatidylcholine vesicles and N N incubated with cells in monolayer culture at 20C, resulting in rapid 0 /9 and preferential transfer of the labeled ceramide from vesicles to HI ° cells. The cells were then washed and subsequently incubated at H-C-N- C-(CH2)5-N NO2 37°C for various intervals. The metabolism of C6-NBD-ceramide was monitored by lipid extraction and analysis, and the intracel- lular distribution of the labeled molecule was followed by fluo- H rescence microscopy. Initially, fluorescence was detected almost HO-C-C =C-(CH2)12- CH3 exclusively in mitochondria, with over 90% of the extractable lipid I fluorescence due to C6-NBD-ceramide. After 30 min at 370C, in- H H tense fluorescence. appeared in the Golgi apparatus. This organ- elle was identified by colocalization of NBD fluorescence with a FIG. 1. Structure of C6-NBD-ceramide. Golgi-apparatus-specific stain. At later times the plasma mem- brane became visibly labeled as well, at which point 90% of the orescent of the cell-associated fluorescence was recovered as NBD-labeled sphin- tracer allows direct microscopic observation gomyelin and NBD-labeled cerebroside. These metabolites were translocation and sequestering of the labeled precursor and its identified by enzymatic and biochemical analysis and by thin-layer metabolites as it occurs in living cells. Thus, artifacts and delays chromatography of the fluorescent lipid extracts. The finding that due to tissue fixation or subcellular fractionation are avoided. C6-NBD-ceramide is used by these cells in standard pathways of Here we report that C6-NBD-ceramide is metabolized by sphingolipid biosynthesis suggests that this fluorescent precursor cultured fibroblasts to fluorescently labeled sphingomyelin and will be a valuable tool for correlating the metabolism of sphin- cerebroside. During this metabolism, the intracellular distri- golipids with their intracellular distribution and translocation. In bution of fluorescence, involving the mitochondria, Golgi ap- addition, during its metabolism by Chinese hamster fibroblasts, paratus, and plasma membrane, also changes in a striking man- this compound acts as a vital stain for the Golgi apparatus. ner. The sphingolipids are important membrane lipids which have METHODS been implicated in a number of cellular properties, including Materials. Chemicals were purchased as indicated: Clostri- blood group specificity, receptor action, nerve conduction, dium perfringens phospholipase C, human placenta sphingo- membrane stability, oncogenic transformation, and aging (for myelinase, taurodeoxycholate, sphingosine-l-phosphocholine, reviews see refs. 1-4). Although all of the biosynthetic path- psychosine, and bovine brain sphingolipids (Sigma); Eagle's ways for the sphingolipids are not yet completely understood minimal essential medium (GIBCO); lectins (Vector Labora- (5-7), it is well known that defects in their metabolism or break- tories, Burlingame, CA); and rhodamine 3B (Eastman). down can have serious consequences, such as Tay-Sachs or Lipids and Lipid Vesicles. N-Rhodamine B Farber diseases (8, 9). All the sphingolipids have in common sulfonyl dioleoyl the ceramide backbone, which is either.glycosylated to yield phosphatidylethanolamine (N-Rh-PtdEtn) was synthesized as cerebrosides and gangliosides or coupled to phosphocholine to described (10). C6-NBD-ceramide, C6-NBD-galactosylceram- give sphingomyelin. The sites of synthesis of these molecules ide, and NBD-sphingomyelin were synthesized (9), using sphin- inside the cell remain to be determined, as do the molecular Abbreviations: NBD, 4-nitrobenzo-2-oxa-1,3-diazole; NBD-sphingo- mechanisms underlying translocation and insertion of the dif- myelin, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminoacyl sphingosine-1- ferent sphingolipids into different subcellular membranes. phosphocholine; NBD-cerebroside, N-[7-(4-nitrobenzo-2-oxa-1,3-dia- In order to examine the correspondence between sphingo- zole)]aminoacyl-sphingosine monoglycoside; C6-NBD fatty acid, N-[7- lipid metabolism and the intracellular distribution of these lip- (4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproic acid; C6-NBD-cer- ids, we have initiated a study using N-[7-(4-nitrobenzo-2-oxa- amide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl sphin- gosine; C6-NBD-galactosylceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-dia- 1,3-diazole)]-6-aminocaproyl sphingosine (C6-NBD-ceramide), zole)]-6-aminocaproyl sphingosine galactoside; N-Rh-PtdEtn, N-rho- a fluorescent derivative of ceramide (Fig. 1). The use of a flu- damine B sulfonyl dioleoyl phosphatidylethanolamine; Oe2-PtdCho, dioleoyl phosphatidylcholine; HMEM, 18 mM Hepes-buffered Eagle's The publication costs ofthis article were defrayed in part by page charge minimal essential medium, pH 7.4, with 0.62 mM phosphate; HMEMB, payment. This article must therefore be hereby marked "advertise- HMEM with choline, ethanolamine, serine, and myo-inositol at0.5 mM ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. each. 2608 Downloaded by guest on September 27, 2021 Cell Biology: Lipsky and Pagano Proc. Natl. Acad. Sci. USA 80 (1983) 2609 gosine, psychosine, or sphingosyl phosphocholine, respec- ments, filter combinations were chosen that eliminated cross- tively. N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproic acid over between rhodamine and NBD fluorescence channels. (C6-NBD fatty acid) and dioleoyl phosphatidylcholine (Ole2- Thin-Layer Chromatography. Lipid extracts were routinely PtdCho) were purchased from Avanti Biochemicals. Concen- applied to silica gel 60 thin-layer plates (Merck) or, where in- trations of lipid stock solutions were determined by phosphorus dicated, to silica gel G/1% sodium borate plates (Analtech, measurement (11), or, for C6-NBD-ceramide, by reference to Newark, DE). For two-dimensional chromatography, solvent 1 known concentrations of fluorescent standards (see below). was CHC13/CH3OH/H2O, 65:25:4 (vol/vol), and solvent 2 was Small unilamellar vesicles were formed by ethanol injection tetrahydrofuran/methylal/CH30H/H20, 10:6:4: 1 (vol/vol) (20). (12): 12 mol % C6-NBD-ceramide and 88 mol % Ole2-PtdCho One-dimensional plates were routinely developed with solvent (unless otherwise indicated) were mixed, dried under argon, 1. Anothersolventsystem was solvent3, CHC13/CH3OH/H2O/ and then, under reduced pressure, made to 2.8 mM in ethanol NH40H, 72:48:9:2 (vol/vol) (6). Fluorescent lipids were de- and injected while vortex mixing into 13 vol of 10 mM Hepes- tected on plates by UV illumination. The spots were scraped buffered calcium- and magnesium-free Puck's saline. This and the lipids were extracted as described for cell extracts. preparation was dialyzed at 20C overnight against the same Identification of Fluorescent Lipid Metabolites. All pro- buffered saline, then diluted to a final concentration of 22-44 cedures were first tested on appropriate bovine brain sphin- AtM with 18 mM Hepes-buffered Eagle's minimal essential me- golipid standards to ensure their efficacy. Acid hydrolysis was dium, pH 7.4, with 0.62 mM phosphate and choline, ethanol- performed as described (21). Lability to sphingomyelinase was amine, serine, and myo-inositol at 0.5 mM each (HMEMB) for assayed (22, 23) with modifications as follows: 0.25-3.0 nmol of incubations. fluorescent compound were desiccated in a screw-capped test Cell Culture, Vesicle-Cell Incubations, and Lipid Extrac- tube, then 200 A1 of taurodeoxycholate (62 ,ug/ml in 0.1 M po- tion. Monolayer cultures of Chinese hamster V79 fibroblasts tassium acetate, pH 5.0) was added and the solution was briefly (13) were grown to confluency in Eagle's minimal essential me- sonicated. Twenty units of enzyme or an equal volume of buff- dium supplemented with 12% horse serum in a water-saturated er was added, and the mixture was incubated at room tem- atmosphere of 5% CO2 in air. The plates were washed two times perature for 2 hr. Lipids were extracted with 1.2 ml of CHC13/ with Hepes-buffered Eagle's minimal essential medium, pH CH30H, 2:1 (vol/vol). Under these conditions, no contami- 7.4, with 0.62 mM phosphate (HMEM). Monolayers were in- nating phospholipase C activity on natural or fluorescent phos- cubated with vesicles at 20C for 90 min, then washed three times phatidylcholine was detected. Phospholipase C hydrolysis of with HMEM. For time course experiments, the plates were lipid substrates (24) and cerebroside ring oxidation, reduction, subsequently incubated at 370C in HMEMB. Cells were re- and removal (25) were performed. moved by trypsin treatment (14) for counting in a hemacytome- ter or were scraped for extraction, in medium, with a rubber RESULTS policeman, and lipids were extracted (15). Total NBD (excita- Cellular Uptake of C6-NBD-Ceramide from Vesicles. When
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