Journal of Cell Science 107, 3545-3555 (1994) 3545 Printed in Great Britain © The Company of Biologists Limited 1994

Biosynthesis of glycosphingolipids is reduced in the absence of a vimentin intermediate filament network

Baiba K. Gillard1,*, Lisa T. Thurmon1, Rhonda G. Harrell1, Yassemi Capetanaki2, Megumi Saito3, Robert K. Yu3 and Donald M. Marcus1,4 1Departments of Medicine and 2Cell Biology and Microbiology, Baylor College of Medicine, Houston, Texas, USA 3Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Richmond, Virginia, USA 4Department of Immunology, Baylor College of Medicine, Houston, Texas, USA *Author for correspondence

SUMMARY

Our previous observations on the immunocytochemical co- biosynthesis, mouse vimentin cDNA was transfected into localization of intermediate filaments and glycosphin- vim− cells. Transfected cells that expressed a mouse golipids led us to analyze the role of filaments in the biosyn- vimentin network demonstrated a twofold or greater thesis and intracellular transport of glycosphingolipids. increase in the rate of biosynthesis of neutral glycosphin- Cells with (vim+) and without (vim−) vimentin intermedi- golipids and gangliosides. There was no difference between ate filaments were cloned from the adrenal carcinoma cell vim+ and vim− cells in the synthesis of ceramide or sphin- line SW13. There was no difference between vim+ and vim− gomyelin, or in their content of phospholipids or choles- cells in the proportion of newly synthesized C6-NBD-glu- terol. The nature of the biochemical defect(s) underlying cosylceramide transported to the plasma membrane. The the diminished incorporation of radiolabeled sugars into vim+ cells synthesized glycosphingolipids, especially lacto- glycosphingolipids is unclear. Possibilities include alter- sylceramide and globotriosylceramide, and to a lesser ations in the ultrastructure of the Golgi and/or abnormal- − extent GM3 ganglioside, more rapidly than vim cells. The ities in a portion of the endocytic pathway. altered rate of biosynthesis did not result from differences in the levels of the glycosyltransferases that synthesize those compounds. To determine whether the presence of a Key words: glycosphingolipid biosynthesis, vimentin, intermediate vimentin network was responsible for the differences in filament function

INTRODUCTION clear whether the GSL associated with IF are in vesicles or are bound directly to the filaments. Several years ago we noted that the neutral glycosphingolipid These observations raised questions about the possible role (GSL) Gb4Cer* and ganglioside GM3 (Table 1) colocalized of IF in the transport or metabolism of GSL. To explore these with vimentin intermediate filaments (IF) of cultured human questions, we have studied the biosynthesis and transport of umbilical vein endothelial cells (Gillard et al., 1991). We GSL in cells that lack IF networks. The SW13 cell line was demonstrated subsequently that Gb4Cer co-localized with derived from a human carcinoma of the adrenal cortex other IF, desmin, keratin and GFAP, in a variety of cell types (Leibovitz et al., 1973). The cell line contains a mixture of cells − (Gillard et al., 1992). Our biochemical analysis of umbilical with (vim+) and without (vim ) vimentin IF, and stable cell vein cells indicated that most of the cell GSL were detectable lines can be cloned (Hedberg and Chen, 1986). In this study − in a cell fraction enriched in IF. Our inablility to detect GSL we report that vim cells exhibit a decrease in the biosynthesis other than Gb Cer and G in association with IF by immuno- of some GSL, and that this abnormality can be corrected by 4 M3 − fluorescence probably results from technical limitations such expression of a mouse vimentin network in vim cells. as the lower concentration of other GSL, the varying affinity of anti-GSL antibodies, and the poor reactivity of antibodies with GSL that contain short oligosaccharide chains. It is not MATERIALS AND METHODS Cells *Glycosphingolipid structures are abbreviated according to the IUPAC-IUB recommen- The SW13 cell line (ATCC CC1 105), derived from a human small dations (IUPAC-IUB Commission on Biochemical Nomenclature, 1977) except that ganglio series gangliosides are abbreviated according to Svennerholm (Svennerholm, cell carcinoma of the adrenal cortex (Leibovitz et al., 1973), was 1964) and the suffix Cer is used in place of OseCer. obtained from the American Type Culture Collection (Rockville,

3546 B. K. Gillard and others

Maryland). Cells were grown in monolayer culture in DMEM with bovine brain gangliosides per sample) to minimize nonspecific loss of high glucose (Gibco, Grand Island, New York) supplemented with radiolabelled GSL during isolation. TLC plates containing radiola- 10% fetal bovine serum, 2 mM glutamine, 0.1 mg/ml penicillin and beled GSL were exposed to X-ray film for autoradiography, and 100 µg/ml streptomycin. Labeling and transport assays were done on relative amounts of GSL were determined from peak areas of densit- cell monolayers that were between 80 and 100% confluent. The SW13 ometric scans of autoradiograms obtained on a Visage 110 Image cell line contains a mixture of cells with and without vimentin IF analyzer. Autoradiograms of several different exposure times for each networks (Hedberg and Chen, 1986). Clones with high expression of TLC plate were scanned to ensure that peak areas were in the linear vimentin networks (vim+) or absence of vimentin (vim−) were range of the densitometer. Alternatively, radioactivity per band was selected by limiting dilution and screening by immunofluorscence quantitated directly from the TLC plate with a Betagen Betascope. microscopy. Vimentin content of cells was estimated by SDS-PAGE and western blotting, as described previously (Gillard et al., 1992). Transport assay We adapted the method originally developed by Pagano (1989) to Fluorescence microscopy study transport of GlcCer and sphingomyelin in fibroblasts (Lipsky Cells grown on glass coverslips were processed for single- and and Pagano, 1985) and epithelial cells (van Meer et al., 1987). To double-label fluorescence microscopy as described previously monitor biosynthesis and transport of all newly synthesized GSL, we (Gillard et al., 1993). In brief, cells were fixed in 4% EM grade included [14C]galactose and [14C]glucosamine in the labelling formaldehyde (Tousimis, Rockville, MD) and permeablized with mixture. Cells were incubated with a 1:1 complex of 5 µM C6NBD- 0.005% digitonin (Sigma, St Louis, MO) or 0.1% Triton X-100 Cer:defatted BSA and 1.5 µCi/ml each of the radiolabelled sugars. (Sigma), or were fixed and permeabilized with methanol. Cells were C6NBD-Cer was from Molecular Probes (Eugene, OR) and defatted then blocked with 1% bovine serum albumin (BSA), incubated with BSA was from Sigma (St Louis, MO). Labeled cell monolayers were first antibody for 45 minutes, washed, incubated with appropriate washed with cold DMEM, and back-exchanged with cold defatted second antibody, washed and mounted for observation. Human BSA twice for 30 minutes, to remove NBD-sphingolipids from the vimentin was detected with mouse IgG monoclonal antibody Clone outer leaflet of the plasma membrane. Lipids were purified from the V9 (ICN, Costa Mesa, CA). This monoclonal antibody binds to back-exchange media and the cells, and analyzed as described above. human but not to mouse vimentin. Mouse vimentin was detected with NBD-sphingolipid bands were detected on TLC plates by fluores- mouse IgM monoclonal antibody MAB 1635 (Chemicon, Temeula, cence and autoradiography. CA). This antibody binds both mouse and human vimentin. Anti- bodies to other cytoskeletal proteins and fluorescently-labelled second Assay of GSL synthetases antibodies were from commercial sources, as described previously Reaction mixtures for assay of UDP-Gal:GlcCer β1-4 galactosyl- (Gillard et al., 1992, 1993). Second antibodies were demonstrated in transferase (LacCer synthetase) contained 6.8 mM GlcCer, 0.5 mM control experiments to be specific for the appropriate first antibody. UDP-Gal (0.05 µCi), 0.5% Triton X-100 (w/v), 5 mM each MgCl2 Antibodies were titred and used at appropiate concentrations to avoid and MnCl2, 180-225 µg cell protein and 100 mM HEPES, pH 7.0, in bleedthrough between FITC and Texas Red channels. a final volume of 100 µl. Reactions were carried out at 37¡C for 2 hours. Reaction mixtures for assay of UDP-Gal:LacCer α1-4 galac- GSL purification and analysis tosyltransferase (Gb3Cer synthetase) consisted of 5 mM LacCer, 50 GSL isolation was performed as described previously (Gillard et al., µM UDP-Gal (0.1 µCi), 0.4% Triton X-100 (w/v), 10 mM MnCl2, 1992). In brief, GSL were extracted from cell pellets or lyophilized 50-250 µg cell protein and 100 mM Na cacodylate, pH 7.2, in a final media with chloroform:methanol:water, 50:50:10 (v/v/v). GSL were volume of 100 µl. Reactions were carried out at 37¡C for 1 hour. purified by DEAE-Sephadex A-25 ion exchange chromatography, Reactions were stopped by addition of 2-4 ml chloroform:methanol, base treatment, and desalting on C18-BondElut columns (Varian, 2:1 (v:v) and partitioned with 0.5 ml 0.9% NaCl. Lower phases were Harbor City, CA). Glycolipid composition of samples was analyzed evaporated and spotted on TLC plates. Plates were developed in chlo- by thin layer chromatography (TLC), on high performance Silica Gel roform:methanol:water, 65:35:4. Bands corresponding to the reaction 60 (E. M. Merck, Darmstadt, Germany). Plates were developed in products were scraped and counted in a scintillation counter. chloroform:methanol:water, 60:35:8 with 0.01% KCl for neutral GSL . and 50:40:10 with 0.025% CaCl2 2H2O for gangliosides. The GSL GlcCer, LacCer, Gb3Cer, and GM3 were tentatively identified on the Table 1. Structures of glycosphingolipids and related basis of their chromatographic mobility on TLC plates in comparison compounds to standard compounds of known structure. In addition, the presence of LacCer and Gb3Cer in SW13 cells was confirmed by immunoflu- Name Abbreviation Structure* orescent staining of the cell surface by specific monoclonal antibodies Neutral GSL (Gillard et al., 1990, 1993). Anti-GM3 gave very weak cell surface Glucosylceramide GlcCer Glcβ1-Cer staining of vim+ and vim− cells, and there are no monoclonal anti- Lactosylceramide LacCer Galβ1-4Glcβ1-Cer bodies available against GlcCer. Globo series Globotriosylceramide Gb3Cer Galα1-4Galβ1-4Glcβ1-Cer Metabolic radiolabelling Globotetraosylceramide Gb4Cer GalNAcβ1-3Galα1-4Galβ1- 4Glcβ1-Cer Metabolic radiolabelling of GSL with [14C]galactose and [14C]glu- Lacto series cosamine was performed as previously described (Gillard et al., Lactotriosylceramide Lc3Cer GlcNAcβ1-3Galβ1-4Glcβ1-Cer 1990), using 0.5-1.5 µCi/ml each of [14C]galactose and [14C]glu- Ganglio series β β β cosamine, both with a specific activity of 50 µCi/µmole (Dupont Gangliotriosylceramide Gg3Cer GalNAc 1-4Gal 1-4Glc 1-Cer NEN, Boston, MA). To determine GSL biosynthesis rates, cells were Ganglioside labelled for 15 to 120 minutes. To determine steady state GSL com- Sialolactosylceramide GM3 NeuAcα2-3Galβ1-4Glcβ1-Cer position, cells were labelled for 24 hours. To measure GSL turnover, Phospholipid + - cells were pulsed with radioactive sugars for 2 hours, and then chased Sphingomyelin SM (CH3)3N CH2CH2O3P - Cer for 2, 4, 8 or 24 hours. GSL were purified from the cells and the culture media after the two hour labelling period and at each chase *Cer, ceramide (N-acylsphingosine); Gal, D-galactose; Glc, D-glucose; time. Microscale isolation of radiolabeled GSL was facilitated by GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetyl-D-glucosamine; addition of cold carrier GSL (15 µg plasma neutral GSL and 15 µg NeuAc, N-acetyl-neuraminic acid. Vimentin affects glycolipid biosynthesis 3547

Reaction mixtures for assay of CMP-NeuAc:LacCer α2-3 sialyl- Berg, 1981) and surviving colonies were expanded and screened by transferase (GM3 synthetase) contained 400 µM LacCer, 40 nmol immunofluorescence assay for vimentin expression. Selected colonies [3H]CMP-sialic acid (0.2 µCi), 0.15% Triton X-100 (w/v), 10 mM were subcloned by limiting dilution. MnCl2, 80 µl of cell homogenate and 25 mM cacodylate buffer, pH 6.5, in a final volume of 100 µl. Controls were total assay mixtures Ceramide and sphingomyelin biosynthesis incubated with heat-denatured cell homogenates. Reactions were Confluent cell monolayers were metabolically labelled with carried out at 37¡C for 1 hour. Glycolipids were isolated by Sephadex [14C]palmitate, specific activity 56 mCi/mmol, 2 to 6 µCi/ml media for G-50 (Pharmacia, Piscataway, NJ) gel filtration according to a 15 to 120 minute time course, or for 45 minutes in triplicate at 37¡C. Nakamura and Sweeley (1987) and the radioactivity in the product [14C]Palmitate-labelled lipids were extracted from cells with chloro- was determined by liquid scintillation counting. form:methanol:water, 50:50:10 (v/v/v) followed by a second extraction with chloroform:methanol 2:1, base-treated to hydrolyze glycerolipids − Expression of mouse vimentin in SW13 vim cells and desalted on C18-BondElut columns. Lipid samples were analyzed The mouse vimentin expression construct was prepared by ligating a by TLC in three solvent systems. Ceramide was separated from palmitic full-length mouse vimentin cDNA (Capetanaki et al., 1990) into the acid and fatty acid methyl esters by development of plates in chloro- pEMSVscribe vector (Davis et al., 1987). In this construct, vimentin form:methanol:2.5 M NH4OH, 90:20:2. GlcCer, LacCer, Gb3Cer and expression is under the control of the constitutive MSV-LTR sphingomyelin were resolved in chloroform:methanol:0.25% − promoter. SW13 vim clones were co-transfected with this construct aq.CaCl2.2H2O, 65:25:4 (v/v/v). Gangliosides were resolved in chloro- and the selection plasmid pSV2neo (Mulligan and Berg, 1981) in a form:methanol:0.25% aq.CaCl2.2H2O, 50:50:10. 10:1 ratio, using the calcium phosphate precipitation procedure (Tsui et al., 1982; Wigler et al., 1977). Forty-eight hours after transforma- Lipid composition tion, cells were transfered to G418 selection medium (Mulligan and Total lipid composition of vim+ and vim− clones was determined by

Fig. 1. Immunofluorescent staining of SW13 clones with monoclonal antibody V9 against human vimentin. Clones 2CB5 (A) and 2AG10 (B) are vim+, and 1FE3 (C) and 1HF5 (D) are vim−. Arrows in C and D, small aggregates of vimentin. Bar, 20 µm. 3548 B. K. Gillard and others

the method of Macala et al. (1983). Cholesterol and cholesterol ester were also assayed using the Zlatkis reagent (Zlatkis et al., 1953).

RESULTS Cloning of SW13 cells The SW13 cell line is known to contain a mixture of cells with (vim+) and without (vim−) vimentin IF networks (Fig. 1). In the SW13 cell line that we obtained from the ATCC, 96% of cells did not contain vimentin networks, 3% of cells contained complete networks and 1% contained partial networks. Vim+ (Fig. 1A and B) and vim− (Fig. 1C and D) cells were obtained by limiting dilution. The phenotype of the vim− clones is quite stable. The percentage of vim+ cells that contained networks varied from 40-100% in different cloned lines, and the per- centage of vim+ cells remained quite stable in some lines, but tended to decrease over a period of months in other lines when they were maintained in culture. The percentage of cells that expressed vimentin filaments was assayed by indirect immuno- fluorescence prior to each experiment, and the range for the four subclones studied was 2AG10, 95-100%; 2CB5, 72-98%; 1FE3, 0-10%; and 1HF5, 0-1%. Small aggregates of vimentin − Fig. 2. Transport of newly synthesized C6-NBD-GlcCer to the were detected in the periphery of vim cells by immunofluo- plasma membrane in vim+ and vim− SW13 clones. Cells were rescence (arrows, Fig. 1C,D), and very small quantities of − − labelled for the indicated time periods with [14C]galactose, vimentin could be detected in vim clone 1FE3 but none in vim [14C]glucosamine and C6-NBD-Cer. Newly synthesized C6-NBD- clone 1HF5 by western blotting of cell lysates (data not shown). GlcCer was removed from the plasma membrane by back exchange. (A and C) Experiment 1 with clones 2CB5 (vim+) and 1FE3 (vim−). Transport of glycolipids (B and D) Experiment 2, with clones 2AG10 (vim+) and 1HF5 − In the initial experiments, cells were incubated for 2 hours with (vim ). (A and B) Rate of synthesis of C6-NBD-GlcCer. [14C]galactose, [14C]glucosamine and C6-NBD-ceramide, and (C and D) Percentage of newly synthesized C6-NBD-GlcCer in the newly synthesized GSL that were transported to the plasma back exchange fraction. membrane were analyzed by the back exchange technique.

Fig. 3. Utilization of C6-NBD-Cer for GSL biosynthesis in vim+ 2CB5 SW13 cells. The pattern was the same in 2AG10, 1FE3 and 1HF5 cells (data not shown). Cells were labelled for 4 hours with [14C]galactose, [14C]glucosamine and C6-NBD-Cer and then treated by back exchange as described in Materials and Methods. Purified neutral sphingolipids (A) and gangliosides (B) were analyzed by TLC. In each panel, the autoradiogram is on the left and the corresponding fluorescence image is on the right. Cell lanes: cell GSL after back exchange. BkEx lanes: GSL in back exchange media. Mobility of standard GSL is indicated along each panel. Vimentin affects glycolipid biosynthesis 3549

Approximately 80-85% of the newly synthesized C6-NBD- GlcCer, and a GSL that contained C6-NBD and had the chro- matographic mobility of Gb4Cer, were transported to the plasma membrane in both vim+ and vim− cells (data not shown). These data were confirmed and extended by analysis of transport over a period of 30 minutes to 4 hours (Fig. 2). The upper panels in Fig. 2 show the rate of synthesis of C6-NBD-GlcCer in the vim+ clones 2AG10 and 2CB5, and the vim− clones 1FE3 and 1HF5. The lower panels show the proportion of newly synthesized C6-NBD- GlcCer that has reached the plasma membrane. Although the absolute amount of C6-NBD-GlcCer synthesized is lower in the vim− cell lines 1FE3 and 1HF5, the proportion of newly synthe- sized GSL transported to the plasma membrane over a four hour time period is the same in vim+ and vim− cells. Transport of C6- NBD-GlcCer was proportional to the amount synthesized, even when the synthesis rate was 6 to 10 times higher, as in the 2AG10 vim+ cells. Since C6-NBD-ceramide rapidly accumulates in the distal Golgi (Pagano et al., 1989), this assay probably does not provide information about earlier steps in ceramide or GSL transport. There were two surprising aspects of these data. First, utilization of C6-NBD-Cer for biosynthesis of GSLs differed from that of native ceramide. Only two neutral GSL containing C6- NBD, GlcCer and a compound with the chromatographic mobility + − of Gb4Cer, were synthesized in any quantity by vim and vim cells (Fig. 3A, fluorescent bands), in comparison to the much more Fig. 4. Biosynthesis of neutral GSL and gangliosides by SW13 complex pattern seen when the GSL were synthesized from clones. Cells were incubated with [14C]galactose and normal ceramide (Fig. 3A, autoradiogram bands without corre- [14C]glucosamine for the time periods indicated. The purified GSL sponding fluorescent bands). Four ganglioside bands containing were analyzed by TLC and incorporation of radioactive sugars into C6-NBD were detected (Fig. 3B, fluorescent bands), compared to TLC bands was quantitated by autoradiography. (A and B) Neutral eight ganglioside bands containing native ceramide (autoradi- − GSL bands with mobility of standard GlcCer, LacCer, Gb3Cer, and ogram). Second, the vim cells synthesized a much smaller Gb4Cer. (C and D) Total gangliosides (Total), GM3, the sum of four amount of small neutral GSL than vim+ cells. In view of these bands with mobility between GD1a and GD1b (a+b+c+d) and the sum data, we analyzed the rates of GSL biosynthesis in SW13 cells. of 2 bands with mobility between GT1b and GQ1b (e+f). B

A

Fig. 5. Incorporation of C [14C]galactose and [14C]glucosamine into GSL over a 2 hour time period. Purified lipid extracts were analyzed by TLC. (A) Autoradiogram of TLC. (B and C) Densitometric scans of autoradiogram. Mobility of standard GSL are indicated to the right of the TLC panel and above the peaks in the densitometric scans. Bands below GM3 contain both complex neutral GSL and gangliosides. 3550 B. K. Gillard and others

Table 2. Rates of biosynthesis of GSL by vim+ and vim− Table 4. GSL composition of SW13 vim+ and vim− cells SW13 cells Counts per band per 3×106 cells* 7 GSL cpm/per 10 cells/ min* vim− clones vim+ clones Experiment 1 Experiment 2 1FE3 1HF5 2CB5 2AG10 2CB5 1FE3 2AG10 1HF5 Total neutral GSL 7,420 10,590 19,070 66,190 + − + − Clone (vim ) (vim ) (vim ) (vim ) GlcCer 2,830 6,080 10,410 22,710 Total neutral GSL 150.5±6.2 60.0±1.8 189.1±15.7 66.3±5.5 LacCer 260 430 1,220 10,120 GlcCer 67.3±1.4 37.7±1.8 87.3±7.0 46.4±6.6 Gb3Cer 420 588 2,440 26,726 LacCer 21.2±2.1 6.6±0.7 22.0±2.5 1.9±0.7 Complex GSL 909 600 2,112 828 Gb3Cer 46.6±2.8 5.6±0.6 38.1±7.6 0.2±0.1 Total gangliosides 13,800 19,138 25,513 71,424 Complex GSL 11.2±1.0 9.2±1.1 1.3±1.4 14.7±2.1 Total gangliosides 71.1±3.5 65.7±2.0 211.3±29.4 16.2±1.3 *Cells were labelled with [14C]galactose and [14C]glucosamine for 24 hours. Quantitation of label incorporation per band was done on a Betagen *Cells were labelled over a time course of 15, 30, 45, 60 and 120 minutes Betascope analyzer by direct counting of the TLC plates for 23 hours. with [14C]galactose and [14C]glucosamine. Rates of label incoporation were Comparable results were obtained by densitiometry of autoradiograms. These calculated by a linear regression fit to the data points of cpm/band/107 cells vs values are representative of three separate experiments. time; values given are the slope ± s.e. cpm/band/107 cells were determined by densitometry of TLC autoradiograms. Data are representative of two to four experiments for each clone. LacCer synthetase activity was greater in vim− cells than in vim+ cells, in contrast to the lower rate of biosynthesis of − Biosynthesis of glycolipids LacCer in the vim cells. The content of small neutral GSL in vim− cells, as determined by metabolic radiolabeling for 24 The rate of incorporation of radiolabeled sugars into the small hours (Table 4), was also lower than that of vim+ cells. These neutral GSL LacCer and Gb Cer was slower in vim− cells than 3 differences in the GSL composition of vim+ and vim− cells vim+ cells over a time course from 15 minutes to 2 hours (Fig. were confirmed by immunofluorescent staining and flow cyto- 4). A smaller difference was seen in the biosynthesis of metric analysis. LacCer and Gb Cer were detected on the GlcCer, and ganglioside biosynthesis was decreased in 1HF5 3 surface of 22% and 27%, respectively, of vim+ cells, but only cells, but not in 1FE3. The data from a 2 hour time point are 9% and 6% of vim− cells. presented in Fig. 5. As seen in the autoradiogram on the left and the densitometric scan on the right, vim− cells contain very + − little LacCer and Gb3Cer in comparison to vim cells. The Expression of mouse vimentin in SW13 vim cells rates of GSL biosynthesis over a 2 hour time period, as deter- increases the biosynthesis and cell content of GSL mined by linear regression analysis of data obtained at 15, 30, In order to determine whether the decreased biosynthesis of 45, 60 and 120 minutes, are summarized in Table 2. Although neutral GSL is a direct consequence of the absence of there is some heterogeneity among vim+ and vim− clones, it vimentin, or a result of other phenotypic changes in vim− − is clear that vim cells synthesize Gb3Cer and LacCer at a cells, we transfected constructs expressing mouse vimentin much slower rate than vim+ cells, and that there is a smaller (mvim) into these cells. From the vim− line 1FE3 we estab- difference in the rate of synthesis of GlcCer. These differences lished cell line FV1, a stable transfectant that expressed were not the result of differential turnover of newly synthe- vimentin filaments in approximately 50% of the cells, and sized GSL, because pulse-chase experiments over a period of from 1HF5 we established two cell lines, HV1 and HV2, 24 hours revealed no differences in the rate of disappearance that expressed vimentin in over 90% of the cells (Fig. 6). of newly synthesized GSL between vim+ and vim− cells, and Subcloning of FV1 did not yield clones with a higher per- the culture medium contained only approximately 5% of the centage of vimentin-positive cells. Over a time course of 15 total lipid-extractable radioactivity (data not shown). There minutes to 2 hours, all three of the transfected clones was also no correlation between the activities of LacCer, demonstrated a marked increase in the biosynthesis of small Gb3Cer and GM3 synthetases (Table 3) and the rates of biosyn- and large neutral GSL and gangliosides. Data obtained with thesis of these compounds in vim+ and vim− cells. Indeed, the vim− clone 1HF5 and its two transfectants are presented in Fig. 7. The rates of GSL biosynthesis over the 2 hour time period, as determined by linear regression analysis of data − Table 3. GSL synthetase activity of SW13 vim+ and vim obtained at 20, 40, 60, 90 and 120 minutes, are summarized clones in Table 5. The increased biosynthesis of long chain neutral Synthetase activity (pmol/h/mg cell protein)* GSL and gangliosides observed in the mvim+ transfectants Clone % vim+† LacCer Gb Cer G was surprising because no appreciable differences were 3 M3 noted in these compounds between the original vim+ and 2AG10 99.5 33.9±4.2 (n=6) 3.88±0.29 (n=6) 21.7±4.6 (n=6) vim− cells. GSL content of mvim+ cells was also increased 2CB5 93.7 38.8±5.0 (n=8) 2.53±0.34 (n=8) nd − 1FE3 3.4 62.5±7.0 (n=8) 2.18±0.42 (n=6) 23.6±5.7 (n=6) compared to the parent vim cells (Table 6). These obser- 1HF5 0.0 59.5±14.6 (n=6) 1.33±0.34 (n=8) nd vations demonstrate that the presence of a vimentin network modulates the biosynthesis and cell content of GSL. *Values are mean ± s.d.; number of replicates are indicated in parentheses. †Percentage of cells which express vimentin networks was determined by immunofluorescence assay. Values are the average for two assays. Biosynthesis of ceramide and sphingomyelin nd, not done. In view of the data on the decreased biosynthesis of GSL in Vimentin affects glycolipid biosynthesis 3551 vim− cells, we analyzed the biosynthesis of ceramide, the significant difference between vim+ and vim− cells in the biosynthetic precursor of sphingolipids, and of sphin- incorporation of [14C]palmitate into ceramide or sphin- gomyelin, the most abundant sphingolipid. There was not a gomyelin (Table 7).

Fig. 6. Immunofluorescent staining of SW13 clones transfected with mouse vimentin cDNA (mvim+). Clones FV1 (A,B), HV1 (C,D) and HV2 (E,F) were double-labelled with antibody MAB 1635 to detect mouse vimentin (left panels) and antibody Clone V9 to detect human vimentin (right panels). Left and right panels show the same field. The asterisk in A and B indicates a cell with human vimentin. All other cells contain only mouse vimentin. Bars, 20 µm.

3552 B. K. Gillard and others

exhibit decreased incorporation of radiolabelled galactose and glucosamine into GSL compared to either vim+ cells cloned from the SW13 cell line (Tables 2 and 4; Figs 4 and 5), or to cells that express a mouse vimentin network (Tables 5 and 6; Fig. 7). The decreased incorporation was not uniform, it was most marked for LacCer and Gb3Cer. Since there was no dif- ference between vim+ cells and vim− cells in the rate of turnover of newly synthesized GSL, as measured by pulse- chase experiments, or in shedding into the medium (data not shown), the vim− cells exhibit a defect in the biosynthesis of GSL. A possible explanation for the decreased incorporation of radiolabeled sugars is that the sugar nucleotide pool of vim− cells is larger than that of vim+ cells, which would result in a lower specific activity of the sugar incorporated into vim− cells. This explanation is improbable in view of the selective decrease in GSL biosynthesis. The most reasonable interpreta- tion of these data is that vim− cells have a defect in biosyn- thesis of GSL, and that this defect is corrected by expression of a vimentin network. It is interesting that the defect in biosyn- thesis is not secondary to decreased levels of glycosyltrans- ferases (Table 3), which is the basis for changes in biosynthe- sis of GSL during development and in cells that have undergone malignant transformation (Hakomori, 1989). What are the possible explanations of this abnormality? To Fig. 7. Autoradiograms of TLC plates. Incorporation of place the discussion in perspective, we will briefly review [14C]galactose and [14C]glucosamine into neutral GSL (A) and current information about the biosynthesis and transport of gangliosides (B) of the vim− clone 1HF5 (lanes −) and its vim+ GSL and sphingomyelin. Ceramide, the precursor of GSL and transfectants HV1 and HV2 (lanes +). The time of labelling ranged sphingomyelin, is synthesized from serine and palmitate on the from 20 to 120 minutes. cytoplasmic face of the endoplasmic reticulum (Merrill and Wang, 1992; Mandon et al., 1992) (Fig. 8). GlcCer is synthe- sized on the cytoplasmic surface of pre-Golgi and Golgi Lipid composition (Trinchera et al., 1991; Futerman and Pagano, 1991; Jeckel et Analysis of lipid composition revealed no difference between al., 1992), and there is conflicing evidence as to the topology vim+ and vim− cells in their content of phospholipids, sphin- of LacCer biosynthesis (Trinchera et al., 1991; Lanert et al., gomyelin, or cholesterol (data not shown). 1994). Data from immunolocalization of glycosyl transferases and treatment of cells with either brefeldin A or monensin (van Echten et al., 1990; Young et al., 1990; van Meer, 1993; DISCUSSION Young, 1993) indicates that extension of GSL chains and addition of terminal sugar residues takes place within Golgi Cell lines that lack a vimentin network, 1FE3 and 1HF5, cisternae as GSL proceed from the cis- to trans-Golgi, appar-

Table 5. Effect of expression of mouse vimentin in SW13 vim− cells on the rate of biosynthesis of GSL GSL cpm/ per 107 cells/ min* vim− mvim+ Ratio vim− mvim+ mvim+ Ratio FV1 HV1 HV2 Clone 1FE3 FV1 1FE3 1HF5 HV1 HV2 1HF5 1HF5 % vim+†,‡ 4 50 1 86 95 Total neutral GSL 51.5±5.9 87.7±14.7 1.7 36.4±3.8 105.1±11 129.5±2.1 2.9 3.6 GlcCer 40.9±8.0 55.7±11.2 1.4 28.4±2.9 93.1±10.1 110.1±2.9 3.3 3.9 LacCer 4.1±0.6 3.4±0.9 0.8 0.4±0.1 9.5±1.3 13.5±0.8 2 3.8 33.8 Gb3Cer 0.9±0.1 2.2±0.1 2.4 0.2±0.0 1.4±0.3 8.8±0.8 7.0 44.0 Complex GSL 6.9±1.7 28.8± + 6.2 4.2 1.8±1.6 7.9±4 4.5±3.1 4.4 2.5 Total gangliosides 53.0±7.7 89.0 ±10.8 1.7 27.6±4.0 63.1±4.9 81.9±8.4 2.3 3.0

*Cells were labeled with [14C]galactose and [14C]glucosamine for 20, 40, 60, 90 and 120 minutes. Rates of label incoporation were calculated by a linear regression fit to the data points of cpm/band/107 cells vs time; values given are the slope ± s.e. Cpm/band/107 cells were determined by densitometry of TLC autoradiograms. †Clones expressing mouse vimentin networks (mvim+) were obtained by transfection of vim− clones with mouse vimentin cDNA, as described in Materials and Methods. Clone FV1 was derived from 1FE3, clones HV1 and HV2 were derived from 1HF5. ‡Percentage of cells expressing vimentin networks was determined by immunofluorescence assay. Values are the sum of mouse and human vimentin network expression. Vimentin affects glycolipid biosynthesis 3553

Table 6. Effect of expression of mouse vimentin in SW13 vim− cells on GSL composition Counts per band per 106 cells* vim− mvim+ Ratio vim− mvim+ Ratio FV1 HV1 HV2 Clone 1FE3 FV1 1FE3 1HF5 HV1 HV2 1HF5 1HF5 % vim+†,‡ 4 50 1 86 95 Total neutral GSL 7,361 13,389 1.8 4,357 4,706 8,51 1.1 2.0 GlcCer 2,836 6,701 2.4 1,745 2,864 4,105 1.6 2.4 LacCer 325 590 1.8 154 24 479 1.6 3.1 Gb3Cer 336 1,698 5.1 267 387 1,024 1.4 3.8 Complex GSL 3,410 4,376 1.3 1,697 1,158 2,136 0.7 1.3 Total gangliosides 9,529 23,539 2.5 8,493 12,550 18,511 1.5 2.2

*Quantitation of label incorporation per band was done by direct counting of TLC plates with the Betascope Betagen dot blot analyzer for 14 hours. Cells were labelled for 24 hours with [14C]galactose and [14C]glucosamine. †,‡Same as for Table 5. ently by vesicular transport. Recent data indicate the existence Sandhoff, 1990; Trinchera et al., 1990; Pagano et al., 1989; of two sites of GlcCer synthetase activity, one extending from Hoekstra and Kok, 1992). the smooth ER into the cis-Golgi (Futerman and Pagano, 1991) Our data indicate that ceramide is synthesized normally in and one that colocalizes with trans-Golgi markers (Jeckel et vim− cells, and that it is transported efficiently to the site of al., 1992), so the simple concept of vectorial GSL biosynthe- sphingomyelin synthesis in the cis- and medial-Golgi. It is sis is clearly an oversimplification. GSL are thought to proceed possible that there is a defect in the transport of ceramide to from the trans-Golgi to other intracellular organelles and the the site of GSL biosynthesis, which results in slower synthesis plasma membrane by vesicular transport (van Meer, 1989; of GlcCer and LacCer. LacCer is the common precursor for Wattenberg, 1990; van Meer and Burger, 1992), and they form the biosynthesis of all more complex GSL (Fig. 8), and limited detergent-insoluble ‘rafts’ that include GPI-linked proteins quantities of this compound might lead to competition among (Brown and Rose, 1992). In polarized epithelial cells, such as glycosyltransferases for substrate. (Young, 1993; Young et al., MDCK, there is preferential localization of GSL in the apical 1994). This could explain the low levels of LacCer and more membrane (Nichols et al., 1987; Rodriquez-Boulan and normal levels of most larger GSL, as well as the increased Nelson, 1989; van Meer, 1989), but there is no information biosynthesis of complex neutral GSL seen in cells that express concerning the mechanism of this preferential sorting or a mouse vimentin network (Table 5 and Fig. 7). The relatively retention. There are also recent data that GlcCer can be trans- normal levels of GlcCer might result from direct transport of ported from the Golgi to the plasma membrane in brefeldin A- a substantial amount of this compound to the plasma treated cells, presumably by a non-vesicular transport membrane, which would make it unavailable as a substrate for mechanism (Warnock et al., 1994). GSL re-cycle from the glycosyltransferases (Young, 1993; Warnock et al., 1994). plasma membrane to early endosomes, from which they may Another possibility is that vimentin plays a role in organizing re-cycle back to the plasma membrane unaltered, they may be the ultrastructure of the pre-Golgi (intermediate compartment) transported to the Golgi, or proceed to late endosomes and and Golgi, and that the defect in biosynthesis results from dis- lysosomes, from which either ceramide or partially hydrolyzed organization of arrays of biosynthetic enzymes. GSL may move to the Golgi and be used as substrates for gly- Another hypothesis, which is not mutually exclusive with cosyltransferases (Fishman et al., 1983; Schwarzmann and

Table 7. Biosynthesis of ceramide and sphingomyelin in SW13 mvim+ and vim− clones Ratio of counts per band per 106 cells* Clones Ceramide Sphingomyelin GlcCer ratio ratio ratio mvim+/vim− n mean ± s.d. mean ± s.d. mean ± s.d. FV1/1FE3 3 1.26±0.04† 1.23±0.23 1.37±0.08† HV1/1HF5 4 1.40±0.29 1.29±0.50 1.52±0.36 HV2/1HF5 4 1.14±0.09 0.99±0.29 1.11±0.14

*SW13 clones were labelled with [14C]palmitic acid as described in Materials and Methods. Radioactivity in bands on TLC plates was quantitated on the Betagen Betascope analyzer. Values are mean ± s.d. for three to four independent experiments, and are expressed as the ratio of palmitate incorporation in mvim+ cells over vim− cells. †Paired 2-tail t-test P<0.05. Other ratios are not significantly different from 1.0. Fig. 8. Scheme of sphingolipid biosynthesis. 3554 B. K. Gillard and others those above, is that there is an abnormality in recycling GSL work was supported by American Cancer Society Grant BE-88 and from endosomes or lysosomes to the Golgi. As noted above, National Institutes of Health Research Grants AI 17712 and NS11853. radiolabelled sugars can be incorporated into recycling GSL, or added to ceramide produced from recycling GSL. Evans REFERENCES and collaborators (Sarrai et al., 1992; Evans, 1994) have analyzed cholesterol metabolism in SW13 cells. 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