EXPERIMENTAL

CELL

RESEARCH

192,

433-444 (1991)

Association of Glycosphingolipids with Intermediate Filaments of Human Umbilical Vein Endothelial Cells BAIBAKURINS Departments

GILLARD,*,~JULIAN P. HEATH,? LISAT. THURMON,* ANDDONALDM. MARCUS*,$

of *Medicine, ?Cell Biology, and $Microbiology

and Immunology,

Our previous studies of glycosphingolipids (GSLs) of human umbilical vein endothelial cells (HUVECs) established that globoside and ganglioside G,, are the most abundant GSLs of HUVECs. Both compounds are located intracellularly, as well as on the cell surface. In this study, we demonstrate that the intracellular globoside and G,, antigens are associated with the vimentin intermediate filaments of the HUVEC cytoskeleton. Immunofluorescence staining of fixed, permeabilized HUVECs showed colocalization of globoside and G,, with vimentin but not with tubulin or actin. Both GSLs remained associated with intermediate filaments after perinuclear collapse of the filaments induced by colcemid. Indirect evidence that the globoside epitope is present on a GSL is the loss of staining by anti-globoside after methanol fixation and the absence of anti-globoside reactivity with HUVEC proteins on immunoblots. Colocalization of anti-globoside and anti-vimentin was also demonstrated in cryosections of endothelial cells, which indicates that the observed association was not an artifact induced by exposure of cells to detergent or organic solvent. Association of globoside with intermediate filaments was confirmed by immunoelectron microscopy, which demonstrated the presence of antigen along intermediate filaments, as well as on the cell surface and on lipid vesicles. Interferon-y decreased the ratio of surface to filamentous globoside staining, but had the opposite effect on GM3 distribution. Less abundant HUVEC GSLs, including Gb,, nLc, , IV2FucnLc,, and IV3NeuAcnLc,, were not detected along filaments. This is the first report of the association of GSLs with intermediate filaments. We suggest that intermediate filaments may play a role in the transport of GSLs. 6’ 1991

Academic

Press,

Inc.

INTRODUCTION Glycosphingolipids (GSLS)~ are amphipathic molecules that are composed of a hydrophobic ceramide

1TO whom correspondence and reprint requests should be addressedat Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. FAX: (713) 790-0681.

Baylor College

of Medicine, Houston, Texas 77030

moiety and a hydrophilic carbohydrate portion (Table 1). They are located primarily in the outer leaflet of the plasma membrane, and in polarized epithelial cells they are concentrated on the apical surface of the cell [ 1, 21. Glycolipids are also found intracellularly in the secretory granules of mast cells and neutrophils [3-51. Cell surface GSLs are very immunogenic and are useful markers of cellular differentiation and malignant transformation [6, 71. These compounds also modulate the activity of membrane-associated kinases and epidermal growth factor receptor [8-121. We have characterized the major GSLs of human umbilical vein endothelial cells (HUVEC) [13] and demonstrated by immunofluorescence that globoside, the most abundant neutral GSL of these cells, is expressed on the cell surface and intracellularly [14]. Treatment of HUVECs with interferon-y (IFN-7) changes the morphology of these cells from cobblestone to fibroblast-like and induces expression of class II major histocompatibility antigens [15]. We found that IFN-7 increases expression of G,, and decreases expression of globoside on the cell surface, with a concomitant increase in intracellular globoside [14]. We now report that intracellular globoside and G,, are associated with intermediate filaments. The association of a GSL with intermediate filaments has not been reported previously, and it suggests that intermediate filaments may play a role in the intracellular transport of GSLs.

METHODS Cells. Endothelial cells were isolated from human umbilical cord veins [ 161 and cultured in Medium 199 containing 20% (v/v) newborn calf serum (Hyclone, Logan, UT), 0.1 mg/ml penicillin, 100 fig/ml streptomycin, 200 rg/ml neomycin, and 2 mM L-glutamine (GIBCO, Grand Island, NY), as described previously [13]. The HUVEC medium was supplemented with 50 pg/ml endothelial cell growth supple-

’ Abbreviations used: GSL, glycosphingolipid, HUVEC, human umbilical vein endothelial cell; IgG, isotype G immunoglobulin; IgM, isotype M immunoglobulin. GSL structures are abbreviated according to the IUPAC-IUB recommendations [44] except that ganglia series gangliosides are abbreviated according to Svennerholm [45] and the suffix OseCer is omitted. 433

0014.4827191

$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE Glycosphingolipid

1

Structures

and Antibodies

Name Galactosylceramide Lactosylceramide Sialosyllactosylceramide (G& Globo series Globotriaosylceramide (CD77) Globotetraosylceramide (globoside) Forssman (Fors) Lacto series Lactoneotetraosylceramide (paragloboside) Blood group Hi 3Fucosyllactosamine (CD15) Sialosylparagloboside (SPG) Sialosylfucosyllactosamine a The indicated oligosaccharide b Antibodies bind the indicated

Structure” Gal Lc IPNeuAcLc

Anti-carbohydrate monoclonal antibodies were Antibodies. kindly provided by the following: SH-1, Dr. Byron Anderson [18]; 5B5, Dr. Moon Nahm [19]; 9G7 (also named CLB-ery-2), Drs. Marjolein Bos, A. E. G. von dem Borne, and P. A. T. Tetteroo [20]; lB2 [21]; BE2 [21]; and FH-6 [22], Dr. S. Hakomori; PM81 and PMNG, Dr. E. Ball 1‘231;M2590, Drs. Y. Hirabayashi and T. Itoh [24]. The properties of Fol serum, which contains a monoclonal IgM protein that binds sialosylparagloboside, were described previously [25]. The car bohydrate epitopes recognized by these antibodies are indicated in Table 1. Absorption of anti-globoside 9G7 prior to immunofluorescent staining was done by incubation of antibody with 300 pg/ml (Gb,),BSA (Chembiomed, Edmonton, Canada) for 60 min at 25OC. Antibodies to cytoskeletal proteins were purchased: anti-vimentin (mouse monoclonal IgG, ICN, Lisle, IL and mouse monoclonal IgM, ENZO, New York, NY); anti-tubulin (rat monoclonal IgG, Bioproducts, Indianapolis, IN). Actin was stained with rhodamine-conjugated phalloidin (Sigma, St. Louis, MO). The L243 cell line was a gift from Dr. Ron Levy. The second antibodies used were fluorescein isothiocyanate (FITC)-conjugated goat antibody to mouse IgM, tetramethylrhodamine isothiocyanate (Texas Red)-coupled goat antibody to mouse IgG, Texas Red-coupled goat antibody to rat IgG, or rhodamine-conjugated goat antibody to human IgM. Control experiments demonstrated that these second antibodies were specific for the appropriate first antibody. Second antibodies were purchased from Pandex (Mundelein, IL), Kirkegard and Perry (Gaithersburg, MD), and Southern Biotechnology Associates, Inc. (Birmingham, AL). Goat anti-mouse IgM conjugated to 10.nm gold particles was from E-Y Laboratories, Inc. (San Mateo, CA). Fluorescence microscopy. Cells were grown on glass coverslips coated with 10 pglml human fibronectin (New York Blood Center, New York, NY). To detect cell surface antigens, cells were fixed for 20 min in 1% paraformaldehyde (EM grade, Tousimis Research Corp., Rockville, MD) in PBS, pH 7.4, blocked with 1% BSA in PBS, incubated with first antibody for 45 min, washed, incubated with appro-

M2590

Gb, Gb, IV3GalNAcGb,

SH-1, 5B5 9G7

nLc, IV’FucnLc, 1113FucnLc, IV3NeuAcnLc, V13NeuAcV31113Fuc,nLc,

lB2 BE2 PM81, PMN6 Fol FH6

structures are linked /0-l to ceramide. carbohydrate determinants on both glycolipids

ment (Biomedical Technologies, Stoughton, MA) and 100 pg/ml porcine heparin (Sigma Chemical Co., St. Louis, MO) [17]. HUVECs were routinely passaged with 0.25% trypsin plus 0.09% EDTA (GIBCO) and seeded in flasks coated with 0.2% gelatin. After the first passage, cells were grown free of antibiotics. Cells were used for experiments between passages 2 and 8. Cells were identified as endothelial cells based on cobblestone morphology and positive staining with antibody against Factor VIII [16].

Antibody6

and glycoproteins.

priate second antibody, washed, and mounted in PBS:glycerol 1:9, pH 9, containing 1 mg/ml p-phenylenediamine (Sigma) [26]. To detect intracellular antigens, cells were fixed in 4% paraformaldehyde and permeabilized for 10 min in 0.1% Triton X-100 (Sigma). For some experiments, cells were fixed in -70°C acetone or -20°C methanol. Permeabilized cells were blocked, incubated with antibodies, and mounted as above. For double labeling, we used a combination of FITC- and Texas Red-conjugated second antibodies. A Zeiss Axiophot with epi-illumination for fluorescence was used for fluorescence and phase microscopy. Zeiss filter combinations were used. For FITC, the exciter filter was 450-490, the beam splitter 510, and the barrier filter 515-565; for Texas Red, the filter combination was 530-585, 600, and 615, respectively, and for rhodamine, 510-560, 580, and 590. Cryosections. Endothelial cell monolayers in tissue culture flasks were fixed with paraformaldehyde, harvested by scraping, and embedded in agarose [27]. Small agarose blocks were infused with 2.3 M sucrose, mounted on specimen holders, and frozen and stored in liquid nitrogen [28]. Thin sections (0.25-0.5 pm) were cut with glass knives on an RMC ultramicrotome equipped with a cryounit. Cryosections were transferred to gelatin-coated glass slides and processed for immunofluorescent staining with antibodies as above. Colcemid treatment. The colchicine derivative, demecolcine (trademark name Colcemid, Sigma), was added to cell culture media at concentrations of 0 to 2.5 fig/ml, and cells were incubated at 37°C for the indicated times. Disruption of microtubules and retraction of intermediate filaments was assessed by immunofluorescence microscopy, as above. Electron microscopy. Cells were grown on fibronectin-coated Lux Thermanox coverslips (Miles Scientific, Naperville, IL). For analysis of normal morphology, cells were fixed in 2.5% EM grade glutaraldehyde in PBS for 1 h at 25”C, washed in PBS overnight at 4”C, postfixed with 1% buffered osmium tetroxide, dehydrated in ethanol, and embedded in Spurr’s media. Embedded cells were separated from the coverslips by plunging into liquid nitrogen. Selected areas were sectioned in the plane of the monolayer with a diamond knife and observed in a Philips EM 410 electron microscope. All EM chemicals were from Electron Microscopy Sciences (Ft. Washington, PA). For immunoelectron microscopy, cells were labeled with primary antibodies as for immunofluorescence followed by second antibody conjugated to 10.nm gold particles. Cells were then fixed in 2.5% glutaraldehyde and processed for EM as above. Quantitation of gold particle localization was done in 12 different Colcemid-treated cells on a total

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FIG. 1. Immunofluorescence micrographs of surface and intracellular globoside antigen in HUVECs. (A) Confluent endothelial cells were stained for cell surface antigen with anti-globoside 9G7. (B) Phase contrast photomicrograph of field in A. (C) Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 and stained for surface and intracellular antigen with anti-globoside 9G7. (D) Cells were fixed with methanol and stained with anti-globoside 9G7. Scale bar = 20 pm.

area of 2700 Km”. The area of three compartments, intermediate filament bundles, nucleus, and cytoplasm, was determined on electron micrographs at 13,500X final magnification using a superimposed square lattice sheet of 440 intersection points corresponding to an area of 228 km*. The total number of rold oarticles in each comoartment was counted and the density of labeling was expressed as number of gold particles/pm’. Interferon-y treatment. Cells were seeded onto coverslips coated with fibronectin. Confluent monolayers were treated with human recombinant interferon-y (Amgen, Thousand Oaks, CA) at 200 U/ml for 4 days, which induces optimal class II major histocompatibility antigen expression on HUVECs, as well as a change in cell morphology from a cobblestone to a fibroblast-like appearance 114, 151. Effcacy of treatment was confirmed by immunofluorescent staining with

antibody tigen.

1,243 to human

class II major

histocompatibility

an-

RESULTS

Globoside Is Present on the Cell Surface and Inside the Cell

Immunofluorescent staining of HUVECS with the anti-globoside monoclonal antibody 9G7 demonstratec that the globoside epitope is expressed both on the ccl: surface and intracellularly. On the cell surface the flue. rescence was a diffuse pattern of spots with some

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brighter patches. As seen in Fig. 1A and the corresponding phase photograph, Fig. lB, the intensity of surface expression varied from cell to cell, as reported previously for expression of other GSL surface epitopes [29] and for a glycophospholipid-anchored antigen [30]. Intracellular filamentous staining with anti-globoside was detectable in 50% of the cells. Typically the staining was bright around the nucleus with a discrete pattern of filaments at the cell periphery (Fig. 1C). The variation in staining was not accounted for by cell density: sparse and confluent cells gave similar variation in fluorescence intensity among cells (data not shown). Acetonefixed cells gave the same staining pattern as cells fixed in paraformaldehyde and permeabilized with Triton X-100. Although the globoside carbohydrate structure has been identified only on GSLs and not on glycoproteins, many carbohydrate epitopes are present on both glycolipids and glycoproteins. To test the nature of the 9G7 antigen, immunofluorescent staining of cells fixed in paraformaldehyde was compared to cells fixed in methanol, which extracts GSLs. The absence of staining after methanol fixation (Fig. 1D) suggests that the antigen is a glycolipid [31]. Anti-globoside binding to HUVEC proteins was negative by Western immunoblotting (not shown). In addition, binding of antibody was not detected after absorption of 9G7 with (Gb4),BSA, which confirms that globoside is the antigenie structure recognized by this antibody. Colocalization

of Globoside

with

Vimentin

To identify the filaments stained by 9G7 monoclonal antibody, we performed double-label immunofluorescence with anti-globoside and antibodies against the major proteins of the cell cytoskeleton. The filamentous staining pattern produced by 9G7 was found to coincide spatially with the vimentin intermediate filaments of HUVECs (Figs. 2A and 2B). However, 9G7 did not stain all of the vimentin filaments, and stained filaments in the perinuclear region more brightly than in the cell periphery. Anti-vimentin staining intensity was more uniform throughout the cell. Antibody to tubulin stained filaments in a pattern similar to 9G7 (Figs. 2C and 2D), but distinct differences were noted. As shown in Figs. 2C and 2D, some 9G7-stained filaments were not stained by anti-tubulin. In addition, while anti-tubulin staining was most intense and radiated from the microtubule organizing centers, the brightest 9G7-

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437

stained filaments were thick perinuclear structures. The pattern of actin filament networks seen in phalloidan-stained cells was very different from the pattern produced by 9G7 (Figs. 2E and 2F). The colocalization of globoside with vimentin filaments was further established by comparing staining patterns after Colcemid treatment. Colcemid treatment results in depolymerization of microtubules and collapse of vimentin filaments into thick perinuclear bundles and cytoplasmic strands [32]. Only short microtubule filaments attached to the microtubule organizing center remained after treatment of HUVECs with 0.1 pug/ml Colcemid for 2 h, while filaments stained by both 9G7 and anti-vimentin had retracted from the cell edges, and formed thick perinuclear bundles. The 9G7 staining continued to colocalize with vimentin filaments even after extended Colcemid treatment (0.25 pg/ml, 16 h, Figs. 3A and 3B) that totally disrupted microtubules (Fig. 3C), which indicates a tight association between globoside and vimentin filaments. Immunoelectron Colocalization

Microscopy Confirms Globoside with Intermediate Filaments

Association of globoside with intermediate filaments was also demonstrated by immunoelectron microscopy. Electron micrographs of cells processed to show the normal cytoplasmic organization of HUVECs demonstrated, as expected from the immunofluorescent staining patterns, that intermediate filaments and microtubules were often closely associated (not shown). Electron micrographs of Colcemid-treated cells contained thick perinuclear bundles of intermediate filaments (not shown). In cells labeled with anti-globoside 9G7 and anti-mouse IgM conjugated to lo-nm gold particles, the labeling was found along intermediate filaments, on the plasma membrane, and at the periphery of lipid granules. Figures 4A and 4B are high magnification electron micrographs showing the 9G7 labeling of intermediate filaments in Colcemid-treated cells. The degree of labeling varied from cell to cell, as was found in the fluorescent antibody experiments. In a statistical analysis of 12 Colcemid-treated cells, the density of label measured throughout the cytoplasm was 227 times greater on intermediate filaments than in the nucleus, where no antigen is expected, and 18 times greater on IF than in non-IF containing areas of the cytoplasm (Table 2).

FIG. 2. Double labeling of HUVECs for globoside and the major proteins of the cell cytoskeleton. Endothelial cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and processed for immunofluorescence with the indicated antibodies. (A, C, E) The fluorescence pattern for anti-globoside 9G7. (B) Anti-vimentin fluorescence for the same field as A. (D) Anti-tubulin fluorescence for the same field as C. Arrows point to filaments stained by anti-globoside but not anti-tubulin; arrowheads point to filaments stained by both antibodies. (F) Phalloidan-stained actin fluorescence for the same field as E. The position of a thick filament stained by anti-globoside is indicated by “g”; the position of a cluster of actin filaments is indicated by “a”. Scale bar = 20 Wm.

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Globoside Colocalization with Vimentin in Endothelial Cell Cryosections

Is Demonstrated

Our results thus far demonstrate that in cells fixed and then permeabilized either with Triton X-100 or ace tone, globoside is associated with intermediate fila ments. To address the possibility that the observed asso ciation might be an artifact due to redistribution of membrane lipids by Triton X-100 or acetone, we pre pared thin cryosections of endothelial cells to avoid ex posure of cells to detergent or organic solvent. As shown in Fig. 5, immunofluorescence patterns of cryosections double labeled with anti-globoside and anti-vimentin clearly demonstrate globoside colocalization with intermediate filaments. We conclude that the observed association of globoside with intermediate filaments is not a detergent-induced artifact. The Ganglioside G,, Is Also Associated with HUVEC Intermediate Filaments The association of other HUVEC glycolipids [13] with intermediate filaments was tested by immunofluorescence staining with the anti-carbohydrate antibodies listed in Table 1. The ganglioside G,, is the major ganglioside of HUVECs. Immunofluorescent staining of monoclonal antibody M2590 HUVECs with anti-G,, [ 241 gave a punctate surface pattern as well as an intracellular filamentous pattern (Fig. 6A). In double-labeled cells the M2590 filamentous pattern colocalized with vimentin filaments more closely than with microtubules (not shown). The association with intermediate filaments was confirmed in Colcemid-treated cells, in which M2590 clearly stained the thick perinuclear intermediate filament bundles (Fig. 6B). Methanol fixation abolished M2590 staining (not shown). Other anti-carbohydrate antibodies did not give detectable filamentous staining. Sialosylparagloboside (IV3NeuAcnLc,) is the second most abundant ganglioside of HUVECs [ 131. Immunofluorescent staining with Fol serum, which binds the sialosyllactosamine epitope [ 251, gave a bright pattern of fine dots on the cell surface and larger dots concentrated in the perinuclear region (Fig. 7C), but no filamentous pattern. Methanol fixation resulted in loss of surface staining but not perinuclear staining (Fig. 7D), suggesting that the Fol epitope is expressed on GSLs on the cell surface and on glycoproteins inside the cell. Immunofluorescent staining with anti-carbohydrate antibodies to paragloboside (lB2), blood group Hl (BE2), and sialylfucosyllactosamine (FH6) gave patterns similar to Fol (Fig. 7C). No

FIG. 3. Colcemid treatment collapses filaments stained by antiglohoside and anti-vimentin. Cells were treated with 0.25 pg/ml Colcemid for 16 h, as described under Methods, and then double labeled with anti-globoside (A) and anti-vimentin (B), or with anti-tuhulin ((3. Scale bar = 20 pm.

FIG. 4. Immunogold localization of globoside in Colcemid-treated cells. (A) Label on loose networks of intermediate filaments. on dense bundles of intermediate filaments in the perinuclear region. Scale bar = 0.2.5 pm, final magnification = X82,600. 439

(B) Label

440

GILLARD

TABLE Localization

2

of Gold Particles in Colcemid-Treated Immunostained with Anti-Globoside”

HUVECs

Gold particles/pm’ mean k SD Intermediate Cytoplasm Nucleus

filaments

2.48 * 0.46 0.25 -+ 0.23 0.01 f 0.02

a Analysis of particle localization was done as described under Methods. Statistical analysis indicated that anti-globoside localization to intermediate filaments was signiticantly greater than to the cytoplasm or the nucleus (P < 0.001).

immunofluorescent staining was produced by antibodies SH-1 and 5B5 to Gb,, one of the major neutral GSLs in HUVECs, or by antibodies PM81 and PMN6 to the minor HUVEC GSL, fucosyllactosamine. Interferon-y and G,

Effects a Redistribution

of Globoside

We previously demonstrated by cell surface and metabolic radiolabeling that interferon-y activation of HUVECs results in decreased surface expression of globoside and increased surface expression of G,, [ 141. Immunofluorescent staining of interferon-y-treated cells by antibodies 9G7 and M2590 confirms this observation. The punctate surface staining by 9G7 is decreased, and filamentous staining is enhanced by interferon-y activation (Figs. 7A-7D). In contrast, surface expression of the M2590 antigen is significantly increased by interferon-y activation (Figs. 7E and 7F). Thus the relative distribution of these two GSL antigens between the cell surface and the vimentin cytoskeleton is differentially modulated by interferon-y. DISCUSSION

The monoclonal antibody 9G7 reacts strongly with the carbohydrate chain of globoside, very weakly with Gb,, and not with Forssman or other GSLs. We believe that the intermediate filament-associated antigen that binds 9G7 is a GSL because immunofluorescent staining is greatly decreased by fixation with methanol, which solubilizes GSLs but not glycoproteins [31]. In addition, the globoside carbohydrate sequence is not known to occur on any purified glycoproteins. There is one report that rabbit anti-globoside antibodies bound to a crude protein extract of human erythrocytes, but there is no clear demonstration that the determinant was present on a purified glycoprotein [33]. We have found that globoside and other glycolipids can be extracted from an insoluble cytoskeletal fraction of these cells, and no glycoprotein reactive with anti-globoside can be demonstrated in this fraction or in the whole cell lysate by immunoblotting (B. K. Gillard and L. T. Thurmon, unpublished observations).

ET AL.

Our initial observation of globoside association with the cell cytoskeleton was done with cells that had been fixed and permeabilized with acetone [14], a method known to preserve glycolipid antigens [3,4]. For most of the work presented in this paper, we used paraformaldehyde fixation followed by permeabilization with Triton X-100 because this method gives better preservation of cell morphology. These two methods of fixing and permeabilizing HUVECs gave the same filamentous staining pattern with anti-globoside. More recently, we have found that permeabilization with 0.005% digitonin gives the same pattern of filamentous staining with anti-globoside as does permeabilization with Triton X-100 (B. K. Gillard and L. T. Thurmon, unpublished observations). However, all of these methods expose cells to detergent or organic solvent, which could cause redistribution of membrane glycolipids. To address this we also analyzed cryosections of endothelial cells by double immunofluorescence. Our results (Fig. 5) demonstrate colocalization of filaments stained by anti-globoside and anti-vimentin in endothelial cell cryosections, indicating that the observed association is not a detergent or solvent-induced artifact. We also tested for association of other HUVEC glycolipids with intermediate filaments by immunofluorescent staining. Antibodies against G,, produced a staining pattern similar to that of anti-globoside (Figs. 6A and 6B) but antibodies against another HUVEC glycolipid, Gb3, did not produce surface or intracellular staining. This glycolipid is not as abundant as globoside, and it is known that cell membrane glycolipids with short carbohydrate chains may not react well with antibodies. Antibodies against other HUVEC glycolipids, such as sialosylparagloboside and blood group Hi, produced strong surface staining but no detectable filamentous staining (Fig. 6C). Work in progress with more sensitive radiolabeling methods will determine if all cell GSLs, or only a subset, are associated with intermediate filaments. Although the intracellular distribution of intermediate filaments and microtubules is similar, our conclusion that 9G7 is associated with intermediate filaments is based on several lines of evidence. First, the immunofluorescent staining pattern produced by 9G7 parallels that produced by antibodies against vimentin more closely than the anti-tubulin pattern. Second, treatment with Colcemid depolymerized microtubules and collapsed intermediate filaments into thick perinuclear bundles which were stained by 9G7 and anti-vimentin (Fig. 3). Third, decoration of intermediate filaments by 9G7 was demonstrated by electron microscopy (Fig. 4). Taken together, these observations indicate that the intracellular filamentous fluorescent pattern produced by 9G7 coincides with intermediate filaments, but does not rule out some association of glycolipids with microtubules. Nagai and collaborators reported previously that antibodies against galactosylceramide produced a mi-

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Fl .G. 5. Immunofluorescent double labeling of HUVEC cryosectic ms for globoside and vimentin. Cryosections were prepared and stai ned as daescribed under Methods. (A, C, E) The fluorescence pattern for anti-globoside 9G7. (B, D, F) Anti-vimentin fluorescence for the serme field s as A, C, E. (G) A lower power view of a section through a group cif cells stained with anti-globoside. Scale bar = 10 pm for A-F, 16 pm for c:.

trot ubule-like

staining pattern with several epithelial cell lines [34]. Colchicine treatment of these cells resuli ;ed in diffuse staining throughout the cell cytoplasm rat1-rer than staining of perinuclear filament bundles as

might be expected for an intermediate filament-as sociated antigen. Thus, in some epithelial cell lines gal actosylceramide appears to be associated with micro kubules, and in HUVECs globoside and G,, are associa ted

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FIG. 6. Binding of anti-carbohydrate antibodies to HUVECs. (A) Immunofluorescent pattern of anti-G,, M2590. (B) Anti-G,:, pattern 01 colcemid-treated cells. (C) Immunofiuorescent pattern of anti-sialosylparagloboside Fol. (D) Fol pattern in cells fixed with methanol. Cells in A, B, and C were fixed with paraformaldehyde and permeabilized with Triton X-100. Scale bar = 20 Wm.

with intermediate filaments. We are in the process of analyzing other cells with other types of intermediate filament networks to determine if the observed association of glycolipids with IF in HUVECs is a general phenomenon. To date we have observed the association of globoside with vimentin, desmin, and keratin networks in a variety of mesenchymal, smooth muscle, and epithelial cell lines (B. K. Gillard and L. T. Thurmon, unpublished observations). The vimentin network undergoes reversible rearrangement during mitosis, and this is accompanied by increased phosphorylation of vimentin [35, 361. Our immunofluorescence data on dividing 3T3 fibroblasts show that globoside remains associated with vimentin filaments throughout mitosis. The nature of the association between globoside and

intermediate filaments and its functional implications remains to be determined. Other classes of lipids have also been found in close association with intermediate filaments. Traub [37-391 has reported the binding of phospholipids to hydrophobic regions of the N-terminal head piece of intermediate filaments and binding of cholesterol to the rod domain. Franke et al. [40] have reported that lipid globules of adipocytes are surrounded by a cage of intermediate filaments. Globoside and other glycolipids could be associated with intermediate filaments as a component of vesicles, or they could be bound directly to a hydrophobic region of intermediate filaments or associated proteins. If the glycolipids are in the form of vesicles, intermediate filaments could be involved in their transport. The transport of

FIG. 7. Surface expression of intermediate filament-associated GSLs is modulated by interferon-y. HUVECs were treated with interferon-y as described under Methods, fixed with paraformaldehyde, permeahilized with Triton X-100, and processed for immunofluorescent staining with the indicated antibodies. (A-D) Two separate experiments showing anti-glob&de staining in control cells (A, C) and the respective IFN-y-treated cells (B, D). (E-F) M2590 staining of control cells (E) and IFN-y-treated cells (F). Scale har = 20 pm. 443

444

GILLARD

vesicles by microtubules has been studied extensively [41, 421, but there is no evidence to support a similar transport function for intermediate filaments. There is, however, increasing evidence for intermediate filamentassociated proteins [35], which could constitute a transport system distinct from that of microtubules. In addition, microtubules and intermediate filaments are cross-linked in neuronal cells [43], and might be connected in other tissues as well. Experiments to examine the transport of glycolipids by intermediate filaments are currently in progress. This work was supported by research grants from the National Institutes of Health (HL 38788 and AI 17712) and the National Science Foundation (DCB 8820262). The Cell Biology Electron Microscopy Laboratory is supported by NIH Reproductive Biology Center Grant HD 07495. The authors thank Dr. Lhszlh G. Kiimtives and Donna Turner for assistance with electron microscopy, cryosectioning, and preparation of’ the illustrations, and Charlene Shackelf’ord f’or preparation of’ the manuscript.

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