Cell Motility and the Cytoskeleton 21:255-271 (1992)

Association of Glycosphingolipids With Intermediate Filaments of Mesenchymal, Epithelial, Glial, and Muscle Cells Baiba Kurins Gillard, Lisa T. Thurmon, and Donald M. Marcus Departments of Medicine (B.K.G., L. T.T., D.M.M.) and Microbiology and Immunology (0.M.M.), Baylor College of Medicine, Houston, Texas We reported recently that two glycosphingolipids (GSLs), globoside (Gb,) and ganglioside G, , colocalized with vimentin intermediate filaments of human umbilical vein endothelial cells. To determine whether this association is unique to endothelial cells or to vimentin, we analyzed a variety of cell types. Doublelabel immunofluorescent staining of fixed, permeabilized cells, with and without colcemid treatment, was performed with antibodies against glycolipids and intermediate filaments. Globoside colocalized with vimentin in human and mouse fibroblasts, with desmin in smooth muscle cells, with keratin in keratinocytes and hepatoma cells, and with glial fibrillary acidic protein (GFAP) in glial cells. Globoside colocalization was detected only with vimentin in MDCK and HeLa cells, which contain separate vimentin and keratin networks. G, ganglioside also colocalized with vimentin in human fibroblasts. Association of other GSLs with intermediate filaments was not detected by immunofluorescence, but all cell GSLs were detected in cytoskeletal fractions of metabolically labelled endothelial cells. These observations indicate that globoside colocalizes with vimentin, desmin, keratin and GFAP, with a preference for vimentin in cells that contain both vimentin and keratin networks. The nature of the association is not yet known. Globoside and G, may be present in vesicles associated with intermediate filaments (IF), or bound directly to IF or IF associated proteins. The prevalence of this association suggests that colocalization of globoside with the intermediate filament network has functional significance. We are investigating the possibility that intermediate filaments participate in the intracellular transport and sorting of glycosphingolipids.

Key words: cytoskeleton, globoside, vimentin, desmin, keratin, glial fibrillary acidic protein

INTRODUCTION

Glycosphingolipids (GSLs)* are amphipathic molecules composed of a hydrophobic ceramide moiety and a hydrophilic carbohydrate portion (Table I). They are located in cell membranes and lipoproteins. In cells they are located primarily in the outer leaflet of the plasma membrane, but they are also found intracellularly in the secretory granules of mast cells and neutrophils [Katz and Austen, 1986; Symington et al., 1987; Symington, 19891. In polarized epithelial cells they are concentrated in the apical surface of the plasma membrane [Rodriquez-Boulan and Nelson, 1989; Nichols et al., 19871. 0 1992 Wiley-Liss, Inc.

Received September 27, 1991; accepted November 13, 1991 Address reprint requests to Baiba Kurins Gillard, Ph.D., Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Abbreviations used: GFAP, glial fibrilary acidic protein; GSL, glycosphingolipid; IF, intermediate filament; IgG, isotype G immunoglobulin; IgM, isotype M immunoglobulin. GSL structures are abbreviated according to the IUPAC-IUB recommendations [IUPAC-IUB Commission on Biochemical Nomenclature, 19771 except that ganglio series gangliosides are abbreviated according to Svennerholm [Svennerholm, 19641 and the suffix OseCer is omitted.

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Gillard et al. TABLE I. Glycosphingolipid Structures and Antibodies ,OH CH3

\ / \ / \ / \ / \ / \ / \ / CH3 Structure (R-Cer)”

GalNAc~l-3Galal-4Gal~l-4GlcP 1-Cer NeuAca2-3Galp 1-4GlcP 1-Cer GalP 1-3GalNAcP 1-4[NeuAca2-3]Gal@1-4GlcP 1-Cer GalNacP 1-4[NeuAca2-8NeuAca2-3]Galp 1-4GlcP I-Cer

Name

Antibodyb

Globoside, Gb, GM, GM,, G,,

9G7 M2590 Cholera toxin 126.4

“Gal = D-galactose; Glc = D-glucose; GalNAc = N-acetyl-D-galactosamine; GlcNAc = N-acetyl-D-glucosamine; NeuAc = N-acetylneuraminic acid; Cer = ceramide (n-acylsphingosine). The ceramide is microheterogeneous in the composition of the sphingosine base and the fatty acid [Wiegandt, 19851. bAntibodies bind the indicated carbohydrate determinants on both glycolipids and glycoproteins. G,, was detected by labelling with cholera toxin rather than an antibody.

Cell surface GSLs are very immunogenic and are useful markers of cellular differentiation and malignant transformation [Hakomori, 1981; Hakomori, 1989; Hakomori and Kannagi, 19831. These compounds also modulate the activity of membrane-associated kinases and epidermal growth factor receptor [Hanai et al., 1987; Hanai et al., 1988a,b; Hannun and Bell, 1989; Kreutter et al., 1987; Igarashi et al., 19891. Recently the carbohydrate moieties of glycolipids have been identified as the receptors for two adhesion molecules, LECAM-2 and LECAM-3, that mediate adhesive reactions of neutrophils, platelets and T lymphocytes to endothelial cells [Brandley et al., 1990; Phillips et al., 1990; Larsen et al., 19901. Rates of transport of GSLs from the Golgi apparatus to the plasma membrane have been determined [Miller-Prodraza and Fishman, 1984; van Meer, 1989; van’t Hof and van Meer, 1990; Schwarzmann and Sandhoff, 19901, but the mechanism of transport and sorting of GSLs to the cell surface and to intracellular organelles is not known. We recently observed that the neutral GSL globoside and GM, ganglioside colocalized with vimentin intermediate filaments (IF) of human umbilical vein endothelial cells [Gillard et al., 19911. Nagai and coworkers had previously reported association of galactosylceramide with microtubules [Sakakibara et al., 19811. Our study was the first report of an association of GSLs with intermediate filaments. We also noted that interferon-y alters the distribution of GSLs between the plasma membrane and intracellular organelles [Gillard et al., 1990, 19911. To determine the generality of this association and its modulation by interferon-y, we have analyzed a variety of cell types. We report that globoside colocalizes with intermediate filaments in many cell types, but interferon-y modulates subcellular GSL distribution only in endothelial cells.

MATERIALS AND METHODS Cells

Human endothelial cells from umbilical cord veins were obtained as described [Gillard et al., 19871. Human fibroblasts from foreskin were provided by Drs. Yi Ning and Olivia M. Pereira-Smith. Smooth muscle cells from dog saphenous vein were provided by Dr. Charles Seidel. Glial and neuronal cell preparations from 15-day fetal rat dorsal root ganglia and spinal cord and new-born rat hippocampus were obtained with the assistance of Eun-hye Joe and Dr. Kim Angelides. HaCaT 11-3 keratinocytes were from Dr. Dennis Roop [Ryle et al., 19891. Established cell lines were obtained from colleagues or purchased from the ATCC. Antibodies

Sources of anti-carbohydrate antibodies are cited in our previous publications [Gillard et al., 1990, 19911. In particular, anti-globoside monoclonal antibody 9G7 was kindly provided by Drs. Marjolein Bos, A.E.G. von dem Borne and P.A.T. Tetteroo [von dem Borne et al., 19861, anti-G,, monoclonal antibody M2590 by Drs. Y. Hirabayashi and T. Itoh [Hirabayashi et al., 19851, antiGalCer monoclonal antibody by Dr. Barbara Ranscht [Ranscht et al., 1982; Bansal et al., 19891 and anti-GD, monoclonal antibodies 126.4 and 14.18 by Drs. David Cheresh and Ralph Reisfeld [Cheresh et al., 1984; Hersey et al., 19891. FITC-Cholera toxin B subunit (List Biochemical Laboratories, Campbell, CA) was used at 2.4 pg/ml for detection of GM, ganglioside [Gasner et al., 1983; Holmgren, 1981; Holmgren et al., 19731. Antibodies to cytoskeletal proteins were from commercial sources: tubulin, rat IgG clone YL1/2 (Serotec, distributed by Bioproducts for Science, Indianapolis, IN); vimentin, mouse IgG clone V9 and goat polyclonal IgG to

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mouse vimentin, both from ICN, Costa Mesa, CA; desmin, rabbit polyclonal IgG to chicken gizzard desmin (Sigma, St. Louis, MO) and human IgG clone 9 (ICN); GFAP, mouse IgG clone GA-5 (ICN and Biogenex, San Ramone, CA); neurofilament, mouse IgG, clone NR4, (Boehringer Mannheim, Indianapolis, IN) and mouse IgG, clone 2F11 (Biogenex); and actin, mouse IgG clone C4 (ICN). Actin filaments were also detected with Texas Red-phalloidan (Sigma). Fluorescent labelled second antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA and Cappel Research Products, West Chester, PA) were demonstrated in control experiments to be specific for the appropriate first antibody. Antibodies were titered and used at appropriate concentrations to avoid bleedthrough between FITC and Texas Red or rhodamine channels. FIuorescence Microscopy Cells grown on glass coverslips were processed for single and double label fluorescence microscopy as described [Gillard et al., 19911. In brief, surface antigens were detected by staining live cells at 4°C in the presence of NaN,, or cells fixed in 4% formaldehyde. Fixed cells were double-stained with an antibody to an intracellular antigen to confirm that cells were not permeabilized by fixation alone. To detect intracellular antigens, cells were fixed in 4% formaldehyde and permeabilized with either 0.005% digitonin (Sigma) or 0.1% Triton X-100 (Sigma). All cell types were tested by single labelling to determine patterns of staining with antibodies to GSLs, IF and tubulin, and by double labelling for comparison of GSL colocalization to that for IF and tubulin. For some experiments, cells were fixed in methanol and extracted with ch1oroform:methanol to remove glycolipid antigens [Umesaki, 1984; Suzuki and Yamakawa, 19811.

Colcemid Treatment

Colcemid treatment of cells was performed with the colchicine derivative, demecolcine (trademark name colcemid, Sigma) as described [Gillard et al., 19911. Cells were incubated with 0.25 pg/ml colcemid for 2 hours at 37°C prior to fixation and staining. Disruption of microtubules and retraction of intermediate filaments was assessed by immunofluorescence microscopy, as above. Purification of GSLs Associated With the Cytoskeleton

Human endothelial cell GSLs were metabolically radiolabelled with 14C-labeled galactose and glu(New Nuc1ear7 Boston, MA), 0’5 pCi/ml media, as Previously described [Ladisch et al.9 1983; Gillard et al., 19901. Labelled cells were harvested

‘Osamine

Fig. 1. Immunofluorescence micrographs of surface and intracellular globoside antigen in HeLa cells. A: Cells stained for surface antigen with anti-globoside 9G7. Cells fixed with formaldehyde and pemeabilized with digitonin (B) or Triton X-100 (C) and then stained for surface and intracellular antigen with 9G7. Scale bar = 20 pm.

Fig. 2. Immunofluorescent double labeling of permeabilized human fibroblasts by antibodies against globoside (A,C,E) and tubulin (B) or vimentin (D,F). The cells in E and F were treated with colcemid prior to fixation. Scale bar = 20 km.

by EDTA and scraping, and vimentin-enriched cytoskeleton fractions were prepared by two methods. In the first method vimentin is enriched in a high-salt insoluble pellet [Franke et al., 19811. Cells were lysed with 1% Triton

X-100 (Fraction B l , soluble lysate) and the pellet was washed in high salt with 0.5% Triton X-100 (Fraction B2, cytoskeleton wash). The residual pellet, Fraction B3, was solubilized in 8M urea for protein analysis and

Glycolipid Association With Intermediate Filaments

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Fig. 3. Irnmunofluorescent double labelling of human fibroblasts with antibodies to G,, ganglioside (A,C) and vimentin (B,D). Cells in C and D were treated with colcemid. Arrows point to a retracted filament bundle. Scale bar = 20 brn.

GSL purification. Since GSLs could be present in the residual pellet due to non-specific sticking, we also used a second method in which the vimentin-enriched fraction is obtained in a soluble form [Traub et al., 19851. Cells were lysed with 0.5% Triton X-100 (Fraction C1, soluble lysate). Vimentin was solubilized from the pellet by a low salt wash (Fraction C2, vimentin-enriched fraction). The residual pellet (Fraction C3) was solubilized in 8M urea for analysis. GSLs were extracted and purified from each fraction as described [Gillard et al., 19901. Protein composition of each fraction was analyzed by SDSPAGE on 7.5% gels. Total protein composition was assessed by silver staining, and cytoskeleton proteins were identified by Western blotting. Glycolipid composition of each fraction was analyzed by TLC on high perfor-

mance Silica Gel 60 (E. Merck, Darmstedt, Germany) and autoradiography, as described [Gillard et al., 19901. Interferon-y Treatment

The effect of interferon-? on the cellular distribution of GSLs was assessed in human fibroblasts, HeLa and HepG2 cells. Cell monolayers were treated with human recombinant interferon-? (Amgen, Thousand Oaks, CA) at 200 U/ml for 4 days. Efficacy of treatment was confirmed by immunofluorescent staining with monoclonal antibodies W632 and L243 to human class I and class I1 major histocompatibility antigens, respectively [Gillard et al., 19901. The effect on GSL cellular distribution was assessed by immunofluorescence microscopy and by flow cytometry.

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Fig. 4. Anti-globoside immunofluorescent staining of mouse fibroblasts. A,B: 3T3 cells. B shows the same field as A, double-labelled with anti-tubulin. Dividing cells are marked with an asterisk. Note that globoside remains associated with intermediate filaments during cell division. C: L929 cells. D: 10T1/2 cells. The filamentous anti-globoside pattern in these cells colocalized with anti-vimentin (not shown). Scale bar = 20 )*m.

Flow Cytometry

RESULTS

Cell monolayers were harvested non-enzymatically by treatment with 5 mM EDTA and scraping. For analysis of surface antigen expression, washed cells were blocked with phosphate buffered saline containing 1% w/v bovine serum albumin and 0.1 % NaN, for 30 min on ice, incubated with first and second antibodies, fixed in 1% paraformaldehyde and analyzed with a Coulter Epics V flow cytometer, as described [Gillard et al., 19901. For analysis of intracellular antigens, washed cells were fixed in 0.25% paraformaldehyde, permeabilized with 0.005% digitonin and blocked with 1% bovine serum albumin prior to incubation with antibodies [Anderson et al., 19891.

The anti-globoside monoclonal antibody 9G7 produces abundant cell surface immunofluorescent staining of many cells. A typical pattern is presented in Figure 1A. Surface staining of HeLa cells produced a fine punctate pattern with localized areas of higher antigen density. HeLa cells permeabilized with either digitonin or Triton X- 100 showed intracellular filamentous and vesicular staining, in addition to the punctate surface staining (Fig. lB,C). Cells fixed in acetone showed a similar filamentous pattern, but cells fixed in methanol showed loss of staining with anti-globoside 9G7 (data not shown), consistent with the glycolipid nature of the antigen [Umesaki, 1984; Suzuki and Yamakawa, 19811. To

Glycolipid Association With Intermediate Filaments

Fig. 5 . Immunofluorescent staining of primary cultured embryonic rat glial cells. Glial cells were obtained from neuronal cell preparations from newborn rat hippocampus (A,B) or 15-day-old fetal rat dorsal root ganglia (C-F). Cells were double labelled with anti-globoside in the left panels and anti-GFAP in the right panels. Scale bar = 20 p,m.

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Fig. 6.

Glycolipid Association With Intermediate Filaments

test the association of globoside and other GSLs with various types of intermediate filaments, we performed single- and double-label immunofluorescent staining of fixed, permeabilized cells with antibodies to GSLs and IF. Cells were analyzed with an without colcemid treatment. Colcemid treatment depolymerizes microtubules and causes condensation of IF around the nucleus. This allowed clear differentiation between colocalization of GSLs with IF or microtubules, resolution of the separate vimentin and keratin networks in some cell lines, and facilitated identification of the coalesced IF bundles and associated GSLs. Human Fibroblasts

Immunofluorescent staining by anti-globoside produced very bright punctate staining of the plasma membrane and intracellular vesicles of human fibroblasts (Fig. 2C). In contrast to human endothelial cells [Gillard, et al., 19911 and HeLa cells, a filamentous pattern was detected in only a small subset of cells (Fig. 2A). After colcemid treatment, however, thick filament bundles around the nucleus were prominent (Fig. 2E). The filamentous anti-globoside staining colocalized with staining by anti-vimentin antibodies (Fig. 2E,F) but not with anti-tubulin (Fig. 2A,B). The reactive antigen is probably a GSL because the immunofluorescent staining was abolished by fixation with methanol, and no glycoproteins reacting with anti-globoside were detected by Western blotting (data not shown). Antibody against G,, ganglioside also produced immunofluorescent staining that colocalized with vimentin in human fibroblasts (Fig. 3), weaker than that with anti-globoside. Thus in human fibroblasts as in human endothelial cells [Gillard et al., 19911 both globoside and G, colocalized with vimentin. Mouse Fibroblasts To test for colocalization of GSLs with vimentin in other species, mouse fibroblast lines were analyzed. In contrast to human fibroblasts, in which filamentous staining by anti-globoside was seen in only a small portion of the cells, filamentous staining was seen in essentially all cells in three different mouse fibroblast lines

Fig. 6. Immunofluorescent staining of muscle cells. A,B: Primary cultures of dog saphenous vein smooth muscle cells double labelled with anti-globoside (A) and anti-desmin (B). C,D: BC3Hl smooth muscle cells labelled with anti-globoside. The filaments in C colocalize with anti-desmin (not shown). &I: C2C12 skeletal muscle myoblasts labelled with anti-globoside (E-G) and anti-desmin (HJ). H and I are the same field as G. H was photographed at the same focus as G, I was focused on the desmin filaments just above the nucleus. Scale bars = 20 p,m for A,B and C-I.

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(Fig. 4). Again, the anti-globoside staining colocalized with anti-vimentin but not with anti-tubulin (Fig. 4A,B). The pattern of anti-globoside staining varied with cell type. In 3T3 cells (Fig. 4A,B) and L929 cells (Fig. 4C) the pattern was primarily filamentous, with little cell surface punctate staining, whereas in 10T1/2 cells punctate staining was prominent (Fig. 4D). The association of globoside with intermediate filaments was maintained during cell division (Fig. 4A,B). Anti-G,, antibody produced cell surface punctate staining of L929 and 10T1/2 cells, but not 3T3 cells, but no filamentous staining was observed (data not shown). These results indicate that association of globoside with vimentin is not restricted to human cells. We next tested for association with other types of intermediate filaments. Glial and Neuronal Cells

Association of GSLs with glial fibrillary acid protein (GFAP) and neurofilaments was tested in primary rat neuronal cell preparations from fetal rat hippocampus, spinal cord and dorsal root ganglions, which contain a mixture of neuronal cells, glial cells and fibroblasts. Anti-globoside stained glial cells and fibroblasts, but not neuronal cells. Globoside colocalized with GFAP in type I (Fig. 5A,B) and I1 (Fig. 5C,D) astrocytes, and remained associated after colcemid treatment (not shown). The staining pattern produced by anti-GFAP was smooth and continuous, in contrast to the discontinuous pattern produced by anti-globoside. Most, but not all, glial cells showed intense filamentous staining with anti-globoside. In Fig. 5E, anti-globoside exhibited bright vesicular staining in both the GFAP+ and GFAP- cells. The GFAP filaments surrounded the vesicular structures in the GFAP+ cell (Fig. 5F). Globoside was not detected in primary rat neuronal cells. Anti-G,, and cholera toxin, which binds GM1, gave bright surface staining of neuronal processes (data not shown). Since light microscopy cannot resolve the plasma membrane of the neuronal process from the neurofilaments, we could not determine if these GSLs were also associated with neurofilaments. Globoside is present in PC12 cells [Ariga et al., 19881, which express neurofilaments after treatment with nerve growth factor. In these cells, globoside was expressed both on the plasma membrane and intracellularly ,but did not colocalize with neurofilaments (data not shown). Muscle Cells

Association of GSLs with desmin was tested in three types of muscle cells. In dog saphenous vein smooth muscle cells globoside colocalized with vimentin (not shown) and desmin (Fig. 6A,B), which probably form a single network [Klymkowsky et al., 19891, but

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Fig. 7. Immunofluorescent double labelling of Hacat 11-3 keratinocytes (A,B) and HepG2 hepatoma cells (C-F) with anti-globoside (A,C,E) and anti-keratin (B,D,F). Cells in panels E and F were treated with colcemid prior to labelling. Scale bar = 20 pm.

not with tubulin (not shown). In the smooth muscle cell line BC3H1, a filamentous pattern of globoside staining, which colocalized with desmin, was seen in a small subset of cells (Fig. 6C). In most BC3H1 cells, globoside

was localized at the plasma membrane and on intracellular vesicles that clustered in the region of the microtubule organizing center (Fig. 6D). Cell surface and vesicular globoside was also abundant in C2C12 skeletal

fig. 8.

lmmunofluorescent double labelling of MDCK cells with anti-globoside (left) and anti-vimentin (B,D) or anti-keratin (F,H). Cells in C, D, G and H were treated with colcemid. Scale bar = 20 pm.

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TABLE 11. Colocalization of Anti-Globoside With Intermediate Filaments ~~

Immunofluorescent pattern Cell type* (cell line) Hu endothelial Hu fibroblast Mu fibroblast Dog smooth muscle Mu smooth muscle (BC3Hl) Mu myoblast (C2C12) Hu hepatoma (HepG2) Hu keratinocyte (HacatII-3) Hu epithelial (HeLa) Dog epithelial (MDCK) Rat glial cell Rat neuronal cell Rat adrenal pheochromocytoma (PC12)

IF type Vimentin Vimentin Vimentin Desmin and vimentin Desmin and vimentin Desmin and vimentin Keratin

Colocalization with IF

Surface or vesicular

Yes Yes Yes Yes

Yes Yes Yes Yes

Yes

Yes

May colocalize with the nuclear IF, lamin Yes

Yes

Keratin

Yes

Yes

Keratin and vimentin Keratin and vimentin GFAP Neurofilament Peripherin, neurofilament, keratin, vimentin

Yes, with vimentin but not keratin Yes, with vimentin but not keratin Yes No Yes, but not with neurofilaments

Yes

Yes

Yes Yes No Yes

*Hu = human; Mu = mouse.

muscle cells. No filamentous staining was detected either in control (Fig. 6E) or colcemid treated C2C12 cells (not shown). Anti-globoside gave a fine punctate staining of the nuclear envelope in some C2C12 cells (Fig. 6E-G). This did not colocalize with cytoplasmic IF (Fig. 6G vs H,I). Nuclear envelope staining by anti-globoside was also seen in HeLa cells (data not shown). This suggests that globoside is present in the nuclear envelope or is associated with the nuclear IF, lamin, in these cells.

Glycosphingolipid Composition of Cytoskeleton Fractions

As summarized in Table 11, globoside colocalized with intermediate filaments in a variety of cell types, but G, ganglioside associated with IF was detected only in human endothelial cells and fibroblasts. Antibodies to other GSLs (GalCer, LcCer, Gb,, nLc,, Fuc21VnLc,, Fuc3111nLc,, Gg,, GalGb,, GalNAc31VGb4, G,, , NeuAc31VnLc,, GD3,G,,, GT3)localized to the cell surface and to various intracellular organelles, but did not Epidermal and Epithelial Cells give detectable staining of IF (data not shown). A more Anti-globoside staining colocalized with keratin sensitive biochemical method was used to determine if filaments in untreated and colcemid-treated HaCaT 11-3 other GSLs might be associated with intermediate filakeratinocytes (Fig. 7A,B) and HepG2 hepatoma cells ments. Cellular GSLs of human endothelial cells were (Fig. 7C-F). Vimentin filaments were not detected in metabolically radiolabelled with 14C-galactoseand 14Cthese cells. MDCK and HeLa cells contain both vimentin glucosamine, vimentin-enriched cytoskeleton fractions and keratin in separate IF networks [Klymkowsky et al., were prepared by two procedures, as outlined in Meth19891. Although globoside colocalized with keratin in ods, and the GSL composition of these fractions was keratinocyte and HepG2 cells, in MDCK (Fig. 8) and determined by thin layer chromatography. Protein comHeLa cells (not shown) colocalization was seen only position of the cytoskeleton fractions is shown in Figure with the vimentin network. Differences between the pat- 9. Vimentin was enriched in fractions B3 and C2 (Fig. tern of staining produced by anti-globoside and anti-ker- 9B), which also contained actin (Fig. 90). Tubulin was atin antibodies are seen most clearly in the cells treated detected only in fractions B1 and C1 (Fig. 9B), which with colcemid (Fig. 8G,H). Thus globoside has a pref- are essentially total cell lysates. Three to eight percent of erence for vimentin over keratin filaments in cells which the total cell GSLs were recovered in the vimentin-encontain both types of networks. riched fractions. TLC autoradiogram patterns of the neu-

Glycolipid Association With Intermediate Filaments

A. Protein Silver Stain

267

A. Neutral Glycosphingolipids - 200 - 97

- 68 - 42 - 24

GlcCerC LcCer [

c

Gb3 Gb4 nLc4~ Hl H2H3Origin-

B. Anti-Vimentin

A

- 84

B1

82

B3 c1

c2

c3

C2

G

6. Gangliosides

- 47 A B1 B1B2 8 3 c 1 c2 c 3 GM3C SPG c

C. Anti-Tubulin

- 84 - 47 Origin-

A B1 B l B 2 8 3 c 1 c2 c 3

A D. Anti-Actin

- 84 - 47 A B1 B1 82 8 3 C1 C2C3

act

Fig. 9. Protein composition of cytoskeleton fractions of human umbilical vein endothelial cells metabolically radiolabeled with I4C-galactose and 14C-glucosamine. Cytoskeleton fractions were obtained by two procedures, designated Method B and Method C, as outlined in the Methods section. Lanes A: Total cell protein. Lanes Bl,B2,B3: Detergent soluble lysate, high salt wash, and vimentin-enriched pellet obtained by Method B. Lanes Cl,C2,C3: Detergent soluble lysate, vimentin-enriched low salt wash, and residual pellet obtained by Method C. A. Silver Stain. E D Western blots with antibodies against vimentin, tubulin, and actin, respectively. The amount loaded per lane for fraction A corresponds to 1.2 X lo4 cells, for fraction B 1, 2.4 or 4.8 x lo4 cells and for the other fractions, 4.8 x lo" cells. Lane act contains 0.6 pg smooth muscle actin (Sigma).

Bi

B2

B3 Ci

Fig. 10. Autoradiograph of neutral glycosphingolipids (A) and gangliosides (B) extracted from cytoskeleton fractions of human umbilical vein endothelial cells metabolically labeled with 14C-galactose and 14C-glucosamine. Fractions were obtained as described in the legend to Figure 9. The glycolipids were separated by high-performance thin layer chromatography and compounds were visualized by autoradiography. Each lane contains 2000 cpm purified GSL, except ganglioside lane C2 contains 680 cpm. The solvent system for A was chloroform: methanol:O.l% aq KCI :: 60:35:8 and for B, ch1oroform:methanol: 0.25% aq CaCI,.H,O :: 50:40:10. Mobility of standard GSLs is indicated on the left.

tral GSLs and the gangliosides (Fig. 10) indicated that all cell GSLs were present in the vimentin-enriched fractions. Ganglioside patterns (Fig. 10B) indicated that the vimentin-associated GSLs were enriched in shorter-chain ceramide moieties which migrate more slowly than the long-chain analogues.

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expression of carbohydrate blood group A and H antigens in cultured cells [Clausen and Hakomori, 1989; Yamamoto et a]., 19901. Our previous studies demonstrated that treatment The selective nature of the association of globoside of human endothelial cells with interferon-? results in an with IF was demonstrated in epithelial cells and muscle enrichment of gangliosides in the plasma membrane and cells. Globoside was associated with keratin IF in keraa higher proportion of neutral GSLs in the cytoplasm, tinocytes and HepG2 cells, but it was associated with compared to untreated cells [Gillard et al., 19901. Interonly the vimentin network, and not keratin, of MDCK feron-? did not alter the distribution of GSLs in fibroand HeLa cells. Although globoside was demonstrated blasts, HeLa or HepG2 cells (data not shown). These both on the surface and intracellularly in C2C12 cells, it cells all responded to interferon-? by increasing their was not detected in association with cytoplasmic IF. surface expression of class I major histocompatibility These observations provide further evidence that the coantigens, and fibroblasts and HeLa cells were induced to localization of anti-globoside with IF is not an artifact of express class I1 major histocompatibility antigens. the permeabilization procedure. In our previous study [Gillard et al., 19911, association of globoside with intermediate filaments was demonstrated in cryosections of DISCUSSION endothelial cells which had not been exposed to deterThese data demonstrate that the colocalization of gent. In addition to globoside, anti-G,, produced filaGSLs with IF is not unique to human umbilical vein endothelial cells, and that GSLs can associate with mentous staining in some cells. However, this antibody, desmin, keratin, and GFAP, in addition to vimentin. and other antibodies against the carbohydrate portions of Although GSLs are seen to be clearly associated with IFs GSLs, produced only cell surface staining of many cells. after depolymerization of microtubles with colcemid, it Globoside and GM3 are the most abundant neutral glymay be that in intact cells GSLs are also loosely associ- colipid and ganglioside, respectively, of many non-neuated with microtubles. The association of GSLs with IF ronal cells. The failure of antibodies other than antipersisted during mitosis as well as in cells treated with globoside and anti-GM, to produce intracellular colcemid. The staining produced by antibodies against IF filamentous staining raised the question of whether there and GSLs differed in some aspects. In contrast to the was a selective association of globoside and G,, with uniform immunofluorescence produced by antibodies IF, whether it was a reflection of the lower concentration against IF, the anti-GSL staining had a discrete, punctate of other GSLs, or of variations in affinity among antiappearance. This appearance is compatible with the pres- GSL antibodies. Analysis of vimentin-enriched fractions ence of GSLs in vesicles associated with IF, or with the of cells demonstrated essentially the full complement of binding of GSLs by hydrophobic regions of IF or IF- cell GSLs. Although the presence of GSLs in these fracassociated proteins. Immunofluorescent staining of mi- tions could result from the nonspecific adherence of crotubule-associated protein MAP- l produces a similar GSLs to insoluble vimentin after lysis of the cell, these punctate pattern along microtubules [Ockleford, 1990, observations are compatible with the concept that all cell p. 1091. Immunofluorescence staining of the nuclear en- GSLs can associate with IF, but may not be readily develope was produced by anti-globoside in a subset of monstrable by immunofluorescence. HeLa and C2C12 cells. This fluorescent pattern is simIn human endothelial cells, the proportion of gloilar to that produced by anti-lamin antibodies [Ockleford, boside and G,, in the plasma membrane and intracellu1990, p. 311 and suggests that GSLs may also associate larly was altered by treatment with interferon-? [Gillard with the nuclear IF. et al., 1990, 19911. In this study, we found that GSL The percentage of cells that exhibit an association subcellular distribution in three other human cell lines of GSL with IF varied widely. Whereas a majority of was not altered by treatment with interferon-?. Intermouse fibroblasts and glial cells exhibited filamentous feron-? treatment induces a large number of responses in staining by anti-globoside, only a small subset of human endothelial cells, including a striking alteration in cell fibroblasts and BC3H1 cells showed filamentous stain- morphology and altered expression of cell adhesion moling. This variation does not appear to be related to the ecules [Pober et al., 1984; Stolpen et al., 19861. The abundance of globoside in these cells, because human alteration in localization of GSLs in endothelial cells fibroblasts and BC3H1 cells exhibited abundant cell sur- may be a secondary consequence of major changes in the face staining by anti-globoside. The explanation for this structure and physiological state of these cells, rather variation is not clear. It may be related to the cell cycle, than a direct consequence of interferon-? action. or to variations between cells in the activity of glycosylThe enrichment of GSLs in the apical plasma memtransferases, which accounts for the wide variation in brane of polarized epithelial cells [Simons and van Meer,

Effect of Interferon-y on Cellular Localization of GSLs

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1988; van Meer, 1989; Rodriquez-Boulan and Nelson, antibodies 01, 04, and R-mAb used in the analysis of oligodendrocyte development. J. Neurosci. Res. 24548-557. 1989; Nichols et al., 19871 and in the secretory granules of neutrophils and mast cells [Katz and Austen, 1986; Brandley, B.K., Swiedler, S.J., and Robbins, P.W. (1990): Carbohydrate ligands of the LEC cell adhesion molecules. Cell 63: Symington et al., 1987; Symington, 19891 indicates that 861-863. there can be selective transport of GSLs from the Golgi Chan, K.-F. J. (1989): Ganglioside-modulated protein phosphorylation in muscle. J. Biol. Chem. 264:18632-18637. to different organelles. Our observations of different patterns of GSL localization provide additional evidence for Cheresh, D.A., Harper, J.R., Schulz, G., and Reisfeld, R.A. (1984): Localization of the gangliosides GD, and GD, in adhesion the sorting for GSLs. For example, globoside was found plaques and on the surface of human melanoma cells. Proc. primarily intracellularly , colocalized with IF, in 3T3 and Natl. Acad. Sci. U.S.A. 815767-5771, L929 cells, both intracellularly and on the surface of 10T Clausen, H., and Hakomori, S.-I. (1989): ABH and related histoblood group antigens; Immunochemical differences in carrier 112 cells, and on the surface, intracellular vesicles, and isotypes and their distribution. Vox Sang. 56:l-20. nuclear envelope, but not cytoplasmic IF of C2C12 cells. Franke, W.W., Schiller, D.L., Moll, R., Winter, S . , Schmid, E., and Previous work has demonstrated that vesicular transport Englebrecht, I. (1981): Diversity of cytokeratins. Differentiaof glycoproteins from the Golgi to the plasma membrane tion specific expression of cytokeratin poly-peptides in epitheand the endoplasmic reticulum occurs along microtubles lial cells and tissues. J. Mol. Biol. 153:933-959. and is mediated by microtubule-associated motor pro- Gamer, A.L., Kirschner, D.A., and Willinger, M. (1983): Ganglioside localization on myelinated nerve fibres by cholera toxin teins [Matteoni and Kreis, 1987; Vale and Hotani, 1988; binding. J . Neurocytol. 12:921-938. Klausner, 1989; Lippincott-Schwartz et al., 19901. To Gillard, B.K., Jones, M.A., and Marcus, D.M. (1987): Glycosphindate, there is no evidence for a role for intermediate golipids of human umbilical vein endothelial cells and smooth filaments in intracellular transport. The association of muscle cells. Arch. Biochem. Biophys. 256:435-445. GSLs with intermediate filaments in a wide variety of Gillard, B.K., Jones, M.A., Turner, A.A., Lewis, D.E., and Marcus, D.M. (1990): Interferon-y alters expression of endothelial cellcell types suggests that the association plays a role in surface glycosphingolipids. Arch. Biochem. Biophys. 279: some general cellular function. We suggest that interme29. diate filaments may play a role in the intracellular trans- Gillard,122-1 B.K., Heath, J.P., Thurmon, L.T., and Marcus, D.M. port of GSLs. Other possible functions include GSL reg(199 1): Association of glycosphingolipids with intermediate ulation of IF assembly, in analogy to the reported filaments of human umbilical vein endothelial cells. Exp. Cell Res. 192:433-444. phospholipid modulation of IF polymer formation [Perides et al., 1987, 19861, and GSL modulation of Hakomori, S . (1981): Glycosphingolipids in cellular interaction, differentiation, and oncogenesis. Annu. Rev. Biochem. 50:733IF-associated kinase activity [Hanai et al., 1988a; Han764. nun and Bell, 1989; Kreutter et al., 1987; Chan, 1989; Hakomori, S . (1989): Aberrant glycosylation in tumors and tumorIgarashi et al., 19891. associated carbohydrate antigens. Adv. Cancer Res. 52:257ACKNOWLEDGMENTS

This work was supported by National Institutes of Health Research Grants HL 38788 and A1 17712, and American Cancer Society Grant BE-63 173. The authors thank Dr. Kim Angelides for helpful discussions and critical reading of the manuscript, Drs. Julian Heath and Bruce Hollifield for assistance with microscopy, Donna Turner for assistance with micrograph prints and Charlene Shackelford for preparation of the manuscript. REFERENCES Anderson, P., Blue, M.-L., O’Brien, C., and Schlossman, S.F. (1989): Monoclonal antibodies reactive with the T cell receptor f chain: production and characterization using a new method. J . Immunol. 143:1899-1904. Ariga, T., Macala, L.J., Saito, M., Margolis, R.K., Greene, L.A., Margolis, R.U., and Yu, R.K. (1988): Lipid composition of PC 12 pheochromocytoma cells: characterization of globoside as a major neutral glycolipid. Biochemistry 2752-58. Bansal, R., Wanington, A.E., Card, A.L., Ranscht, B., and Pfeiffer, S.E. (1989): Multiple and novel specificities of monoclonal

331. Hakomori, S . , and Kannagi, R. (1983): Glycosphingolipids as tumorassociated and differentiation markers. J.N.C.I. 71:231-251. Hanai, N., Nores, G., Torres-Mendez, C., and Hakomori, S . (1987): Modified ganglioside as a possible modulator of transmembrane signaling mechanism through growth factor receptors: a preliminary note. Biochem. Biophys. Res. Commun. 147:127134. Hanai, N., Dohi, T., Nores, G.A., and Hakomori, S. (1988a): A novel ganglioside, De-N-acetyl-GM, (I13NeuNH,LacCer), acting as a strong promoter for epidermal factor receptor kinase and as a stimulator for cell growth. J. Biol. Chem. 263:62966301. Hanai, N., Nores, G.A., MacLeod, C., Torres-Mendez, C., and Hakomori, S . (1988b): Ganglioside-mediated modulation of cell growth. Specific effects of G, in tyrosine phosphorylation of the epidermal growth factor receptor. J. Biol Chem. 263: 10915-10921. Hannun, Y . A . , and Bell, R.M. (1989): Functions of sphingolipids and sphingolipid breakdown products in cellular regulation. Science 243500-507. Hersey, P., Schibeci, S . , and Cheresh, D. (1989): Augmentation of lymphocyte responses by monoclonal antibodies to the gangliosides GD3 and GD2: The role of protein kinase C, cyclic nucleotides, and intracellular calcium. Cell. Immunol. 119: 263 -278. Hirabayashi, Y.,Hamaoka, A , , Matsumoto, M., Matsubara, T.,

270

Gillard et al.

Tagawa, M., Wakabayashi, S., and Taniguchi, M. (1985): Syngeneic monoclonal antibody against melanoma antigen with interspecies cross-reactivity recognizes GM3, a prominent ganglioside of B16 melanoma. J. Biol. Chem. 260:1332813333. Holmgren, J. (1981): Actions of cholera toxin and the prevention and treatment of cholera. Nature 292:413. Holmgren, J., Lonnroth, I., and Svennerholm, L. (1973): Tissue receptor for cholera exterotoxin: postulated structure from studies with GM1 ganglioside and related glycolipids. Infect. Immun. 8:208. Igarashi, Y., Hakomori, S . , Toyokuni, T., Dean, B., Fujita, S . , Sugimoto, M., Ogawa, T., El-Ghendy, K., and Racker, E. (1989): Effect of chemically well-defined sphingosine and its N-methyl derivatives on protein kinase C and src kinase activities. Biochemistry 28:6796-6800. IUPAC-IUB Commission on Biochemical Nomenclature (1977): The nomenclature of lipids. Lipids 12:455-463. Katz, H.R., and Austen, K.F. (1986): Plasma membrane and intracellular expression of globotetraosylceramide (globoside) in mouse bone marrow-derived mast cells. J. Immunol. 136: 3819-3824. Klausner, R.D. (1989): Sorting and traffic in the central vacuolar system. Cell 57:703-706. Klymkowsky, M.W., Bachant, J.B., and Domingo, A. (1989): Functions of intermediate filaments. Cell Motility Cytoskeleton 14: 309-331. Kreutter, D., Kim, J.Y.H., Goldenring, J.R., Rasmussen, H., Ukomadu, C., DeLorenzo, R.J., and Yu, R.K. (1987): Regulation of protein kinase C activity by gangliosides. J. Biol. Chem. 262: 1633-1 637. Ladisch, S . , Gillard, B.K., Wong, C., and Ulsh, L. (1983): Shedding and immunoregulatory activity of YAC-1 lymphoma cell gangliosides. Cancer Res. 43:3808-3813. Larsen, E., Palabrica, T., Sajer, S . , Gilbert, G.E., Wagner, D.D., Furie, B.C., and Furie, B. (1990): PADGEM-Dependent adhesion of platelets to monocytes and neutrophils is mediated by a lineage-specific carbohydrate, LNF III(CD15). Cell 63:467474. Lippincott-Schwartz, J., Donaldson, J.G., Schweizer, A,, Berger, E.G., Hauri, H-P., Yuan, L.C., and Klausner, R.D. (1990): Microtubule-dependent retrograde transport of proteins into the ER in the presence of Brefeldin A suggests an ER recycling pathway. Cell 60:821-836. Matteoni, R., and Kreis, T.E. (1987): Translocation and clustering of endosomes and lysosomes depends on microtubles. J. Cell Biol. 105:1253-1265. Miller-Prodraza, H., and Fishman, P.H. (1984): Effect of drugs and temperature on biosynthesis and transport of glycosphingolipids in cultured neurotumor cells. Biochim. Biophys. Acta 804: 44-51. Nichols, G.E., Shiraishi, T., Allietta, M., Tillack, T.W., and Young, W.W., JR. (1987): Polarity of the Forssman glycolipid in MDCK epithelial cells. Biochim. Biophys. Acta 930:154-166. Ockleford, C.D. (1990): An Atlas of Antigens. New York: Stockton Press. Perides, G., Scherbarth, A., and Traub, P. (1986): Influence of phospholipids on the formation and stability of vimentin-type intermediate filaments. Eur. J. Cell Biol. 42:268-280. Perides, G., Harter, C., and Traub, P. (1987): Electrostatic and hydrophobic interactions of the intermediate filament protein vimentin and its amino terminus with lipid bilayers. J. Biol. Chem. 262:13742-13749. Phillips, M.L., Nudelman, E., Gaeta, F.C.A., Perez, M., Singhal,

A.K., Hakomori, S . , and Paulson, J.C. (1990): ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Le". Science 250: 1130-1 132. Pober, J.S., Gimbrone, M.A., Jr., Collins, T., Cotran, R.S., Ault, K.A., Fiers, W., Krensky, A.M., Clayberger, C., Reiss, C.S., and Burakoff, S.J. (1984): Interactions of T lymphocytes with human vascular endothelial cells: Role of endothelial cells surface antigens. Immunobiology 168:483-494. Ranscht, B., Clapshaw, P.A., Price, J., Noble, M.,and Seifert, W. (1982): Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside. Proc. Natl. Acad. Sci. U.S.A. 79:2709-2713. Rodriquez-Boulan, E., and Nelson, W.J. (1989): Morphogenesis of the polarized epithelial cell phenotype. Science 245:718725. Ryle, C.M., Breitkreutz, D., Stark, H.J., Leigh, I.M., Steinert, P.M., Roop, D., and Fusenig, N.E. (1989): Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HACAT and in ras-transfected tumorigenic clones. Differentiation 40:42-54. Sakakibara, K., Momoi, T., Uchida, T., and Nagai, Y. (1981): Evidence for association of glycosphingolipid with a colchicinesensitive microtubule-like cytoskeletal structure of cultured cells. Nature 293:76-79. Schwarzmann, G., and Sandhoff, K. (1990): Metabolism and intracellular transport of glycosphingolipids. Biochemistry 29: 10865-10871. Simons, K., and van Meer, G. (1988): Lipid sorting in epithelial cells. Biochemistry 27:6197-6202. Stolpen, A.H., Guinan, E.C., Fiers, W., and Pober, J.S. (1986): Recombinant tumor necrosis factor and immune interferon act singly and in combination to reorganize human vascular endothelial cell monolayers. Am. J. Pathol. 123:16-24. Suzuki, A., and Yamakawa, T. (1981): The different distribution of asialo GM, and Forssman antigen in the small intestine of mouse demonstrated by immunofluorescence staining. J. Biochem. (Tokyo) 90:1541-1544. Svennerholm, L. (1964): The gangliosides. J. Lipid Res. 5:145-155. Symington, F.W. (1989): CDwl7: A neutrophil glycolipid antigen regulated by activation. J. Immunol. 142:2784-2790. Symington, F.W, Murray, W.A., Bearman, S.I., and Hakomori, S . (1987): Intracellular localization of lactosylceramide, the major human neutrophil glycosphingolipid. J. Biol. Chem. 262: 11356-1 1363. Traub, P., Perides, G., Scherbarth, A., and Traub, U. (1985): Tenacious binding of lipids to vimentin during its isolation and purification from Ehrlich ascites tumor cells. FEBS Lett. 193: 217-221. Umesaki, Y. (1984): Immunohistochemical and biochemical demonstraton of the change in glycolipid composition of the intestinal epithelial cell surface in mice in relation to epithelial cell differentiation and bacterial association. J. Histochem. Cytochem. 32:299-304. Vale, R.D., and Hotani, H. (1988): Formation of membrane networks in vitro by kinesin-driven microtubule movement. J. Cell Biol. 107:2233-2241. van Meer, G. (1989): Biosynthetic lipid traffic in animal eukaryotes. Annu. Rev. Cell Biol. 5:247-275. van? Hof, W., and van Meer, G. (1990): Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane. J. Cell Biol. 111:977-986. von dem Borne, A.E., Bos, M.J., Joustra-Maas, N., Tromp, J.F., vant Veer, M.B., van Wijngaarden du Bois, R., and Tetteroo,

Glycolipid Association With Intermediate Filaments P.A. (1986): A murine monoclonal IgM antibody specific for blood group P antigen (globoside). Br. J. Haematol. 63:35-46. Wiegandt, H. (1985): Glycolipids. The Netherlands: Elsevier Science.

271

Yamamoto, F., Clausen, H . , White, T., Marken, J., and Hakomori, S . 4 . (1990): Molecular genetic basis of the histo-blood group ABO system. Nature 345:229-233.