Volume 58(1): 83–93, 2010Journal of Histochemistry & Cytochemistry

http://www.jhc.org

ARTICLE

Gangliosides Are Important for the Preservation of the Structureand Organization of RBL-2H3 Mast Cells

Adriana Maria Mariano Silveira e Souza, Edvaldo S. Trindade, Maria Célia Jamur,and Constance Oliver

Department of Cell and Molecular Biology and Pathogenic Bioagents, Faculdade de Medicina de Ribeirão Preto, Universidadede São Paulo, Ribeirão Preto, São Paulo, Brazil (AMMSS,MCJ,CO), and Department of Cell Biology, Universidade Federal doParaná, Curitiba, Paraná, Brazil (EST)

SUMMARY Gangliosides are known to be important in many biological processes. How-ever, details concerning the exact function of these glycosphingolipids in cell physiologyare poorly understood. In this study, the role of gangliosides present on the surface of ro-dent mast cells in maintaining cell structure was examined using RBL-2H3 mast cells and twomutant cell lines (E5 and D1) deficient in the gangliosides, GM1 and the a-galactosyl deriva-tives of the ganglioside GD1b. The two deficient cell lines were morphologically differentfrom each other as well as from the parental RBL-2H3 cells. Actin filaments in RBL-2H3and E5 cells were under the plasma membrane following the spindle shape of the cells,whereas in D1 cells, they were concentrated in large membrane ruffles. Microtubules inRBL-2H3 and E5 cells radiated from the centrosome and were organized into long, straightbundles. The bundles in D1 cells were thicker and organized circumferentially under theplasma membrane. The endoplasmic reticulum, the Golgi complex, and the secretory gran-ule matrix were also altered in the mutant cell lines. These results suggest that the mast cell–specific a-galactosyl derivatives of ganglioside GD1b and GM1 are important in maintainingnormal cell morphology. (J Histochem Cytochem 58:83–93, 2010)

KEY WORDS

mast cells

cytoskeleton

endoplasmic reticulum

Golgi complex

secretory granules

gangliosides

GLYCOSPHINGOLIPIDS, PARTICULARLY GANGLIOSIDES, are im-portant constituents of eukaryotic plasma membranes(Knoll et al. 2000; Crespo et al. 2004; Sonnino et al.2007). Gangliosides are distinguished from other glyco-sphingolipids because they contain at least one sialicacid linked to their oligosaccharide chain (Birklé et al.2003). Gangliosides are anchored in the lipid bilayerby a hydrophobic ceramide moiety and expose a hydro-philic oligosaccharide chain in the extracellular space(Sonnino et al. 2007). The biological importance ofgangliosides in cellular processes such as growth, differ-entiation, and adhesion is well recognized (Bremer et al.1986; Hakomori 2000; Allende and Proia 2002). Varia-tions in the type, number, and linkage of sugar residuesin the oligosaccharide chain give rise to the wide range

of naturally occurring glycosphingolipids. These formcell-type-specific patterns at the cell surface that changewith cell growth, differentiation, viral transformation,and oncogenesis (Hakomori 1981,1996; Yamashitaet al. 1999). At the cell surface, the gangliosides can in-teract with toxins, virus, and bacteria, as well as withmembrane-bound receptors and enzymes (Karlsson1989). They are also involved in modulation of enzymeproperties, cell signaling, and protein sorting (Palestiniet al. 2000; Lin and Shaw 2005; Jou et al. 2006). Finally,the glycosphingolipids form a protective layer on bio-logical membranes, protecting them from inappropri-ate degradation (Knoll et al. 2000).

Another unique feature of gangliosides is their enrich-ment in plasma membrane microdomains (lipid rafts),

Correspondence to: Constance Oliver, PhD, Department of Cell andMolecular Biology and Pathogenic Bioagents, Faculdade de Medicinade Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes,3900, Ribeirão Preto, SP, Brazil, Cep: 14049-900. E-mail: coliver@fmrp.usp.br

Received for publication August 10, 2009; accepted September 18,2009 [DOI: 10.1369/jhc.2009.954776].

© 2009 Silveira e Souza et al. This article is distributed under theterms of a License to Publish Agreement (http://www.jhc.org/misc/ltopub.shtml). JHC deposits all of its published articles into the U.S.National Institutes of Health (http://www.nih.gov/) and PubMedCentral (http://www.pubmedcentral.nih.gov/) repositories for publicrelease twelve months after publication.

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along with sphingomyelin, cholesterol, and a number offunctionally related proteins (Pike 2003, Ng et al. 2005;Sonnino et al. 2007). There is growing evidence sug-gesting that lipid rafts play an important role in theregulation of membrane–cytoskeleton interactions andcytoskeleton-dependent cellular processes, particularlyin the control of cellular biomechanics (Levitan andGooch 2007). Lipid rafts are critical for actin organiza-tion, membrane trafficking, and cell polarity, as well ascytokinesis (Ng et al. 2005). They are implicated in thefunctioning of diverse signaling pathways, such as thosemediated by growth factors, morphogens, integrins(Simons and Toomre 2000), and antigen receptors onimmune cells (Field et al. 1995; Xavier et al. 1998;Cheng et al. 2001; Dráber and Dráberová 2001;Holowka and Baird 2001; Miceli et al. 2001). Thesemembrane microdomains also act as portals of entryfor bacterial toxins such as cholera toxin, the B sub-unit of which binds to the lipid raft–enriched GM1

ganglioside (Wolf et al. 2008). Indeed, this propertyof cholera toxin B subunit has been widely exploitedto visualize lipid rafts on a variety of cell types (Guptaand DeFranco 2003; Mitchell et al. 2009).

The a-galactosyl derivatives of the ganglioside GD1b

are unique gangliosides present on the surface of ro-dent mast cells and are specifically recognized by mono-clonal antibody (MAb) AA4 (Guo et al. 1989). Thesegangliosides derived from GD1b have been identifiedas components of lipid rafts in the plasma membraneof RBL-2H3 cells (Sheets et al. 1999; Silveira e Souzaet al. 2008). However, the functional role of thesegangliosides, which are close to FcyRI (Basciano et al.1986), in the lipid rafts is still not clear. It is known thatwhen the a-galactosyl derivatives of ganglioside GD1b

are bound by MAb AA4, histamine release is inhibitedin a time- and concentration-dependent manner andthat morphological and biochemical changes similarto those seen with activation of FcyRI are produced(Basciano et al. 1986; Oliver et al. 1992; Swaim et al.1994; Stephan et al. 1997). Using two cell lines deficientin the a-galactosyl derivatives of ganglioside GD1b, aswell as the parent cell line, RBL-2H3, Silveira e Souzaet al. (2008) demonstrated the importance of thesegangliosides for the correct assembly of lipid rafts andconsequently for FcyRI-mediated degranulation in ro-dent mast cells.

There is accumulating evidence showing that cel-lular function is highly affected by gangliosides; how-ever, few studies have evaluated the influence of theselipids on cell structure. Using the mutant cell linesB6A4A2III-E5 and B6A4C1III-D1, which are deficientin a-galactosyl derivatives of ganglioside GD1b (Silveirae Souza et al. 2008), as well as the parent cell line, RBL-2H3, the present study evaluated the role of these gan-gliosides and GM1 in maintaining cell structure.

Materials and Methods

Cell Lines

Mutant RBL-2H3 cells designated B6A4A2III-E5 (E5)and B6A4C1III-D1 (D1) were generated by exposureto ethyl methane sulfonate, followed by subcloningand identification of sublines deficient in IgE-mediateddegranulation as well as in binding of the monoclonalantibody AA4 (Stracke et al. 1987) that is specific fora-galactosyl derivatives of ganglioside GD1b (Guo et al.1989). The wild-type RBL-2H3 cells (a rat mast cellline) and the mutant cell lines were grown as mono-layer, as previously described (Barsumian et al. 1981).

Antibodies

The following primary antibodies were used: mouseMAb anti-b-tubulin (MAB3408, Clone KMX-1,0.75 mg/ml; Millipore, Billerica, MA) (Birkett et al.1985); goat anti-GRP 78 [sc-1050, raised against a pep-tide mapping at the N terminus of the 78-kDa glucose-regulated protein (GRP 78) of human origin, 15 mg/ml,Santa Cruz Biotechnology, Inc.; Santa Cruz, CA](Hatayama et al. 1992); rabbit anti-g-tubulin (T3320,raised against a peptide corresponding to the C ter-minus of Xenopus g-tubulin, aa 437–451, 1:15,000;Sigma-Aldrich, St. Louis, MO) (Oakley and Oakley1989); and mouse MAb anti-GM130 (610823, Clone35, 10 mg/ml; Transduction Laboratories, Lexington,KY) (Nakamura et al. 1997). The following secondaryantibodies were used for immunofluorescence: goat anti-mouse IgG F(ab)′2-Alexa 488 or 594, and donkey anti-goat IgG F(ab)′2-Alexa 488 and goat anti-rabbit IgGF(ab)′2-Alexa 594 (1/300 in PBS; Molecular Probes,Eugene, OR).

Light Microscopy

For routine examination by differential interference con-trast (DIC), 1–5 3 104 cells were cultured overnight on13-mm round coverslips. The cells were rinsed twice inPBS and fixed for 20 min with 2% formaldehyde (EMSciences; Fort Washington, PA) in PBS at room tem-perature. Some samples were fixed with Carnoy’s solu-tion (3% chloroform, 1% acetic acid, and 6% ethanol)for 15 min at room temperature and stained with AlcianBlue (1% Alcian Blue in 120 mM hydrochloric acid,pH 1.0) for 15 min at room temperature; then thesamples were rinsed twice in 70% ethanol and once inmilli-Q water. The samples were counterstained withWeigert’s Fucsin-Resorcin (1% basic fucsin, 2% res-orcin, 90% ethanol, 240 mM hydrochloric acid, and30% FeCl3) for 15 min at room temperature, dehy-drated in a graded series of ethanol (50, 70, 80, 90,and 100%), cleared in xylol:ethanol, xylol, and mountedwith Permount (Fischer Scientific; Hanover Park, IL).To stain F-actin, 1–53 104 cells were cultured overnighton 13-mm round coverslips. Cells were fixed with 4%

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formaldehyde in PBS for 20 min at room tempera-ture, rinsed in PBS, briefly washed with 0.1 M glycinein PBS, permeablized with 0.3% Triton X-100 (Sigma-Aldrich) for 10 min, rinsed in PBS, and then incubatedfor 45 min with 2.6 U/ml Phalloidin-Alexa 488 (Molec-ular Probes). To visualize the gangliosides GM1 (Molec-ular Probes), 1–5 3 104 cells were cultured overnighton 13-mm round coverslips. The cells were rinsedin PBS, fixed for 20 min with 4% formaldehyde (EMSciences) in PBS, rinsed in PBS, briefly washed with0.1 M glycine in PBS and incubated with cholera toxinB conjugated to Alexa 488 (6 mg/ml) for 1 hr at roomtemperature. The cells were then rinsed in PBS, andcoverslips were mounted with Fluoromount-G (EMSciences). For immunostaining of the endoplasmic re-ticulum (ER; goat anti-GRP 78) and the Golgi complex(GM130), 1–5 3 104 cells were rinsed in PBS, fixed for20 min with 2% formaldehyde (EM Sciences) in PBS,rinsed in PBS, briefly washed with 0.1 M glycine inPBS, and permeabilized with acetone at 220C for4 min. To visualize the microtubules, the cells were fixedwith 4% formaldehyde, 50 mM taxol (Sigma-Aldrich),and 50 mM EGTA (Sigma-Aldrich) in PBS for 20 minat 37C, and permeabilized with 0.3% Triton X-100in PBS for 10 min at room temperature. The cells werethen processed as above. After fixation and perme-abilization, the cells were rinsed in PBS, briefly washedwith 0.1 M glycine in PBS, and blocked for 30 minat room temperature in PBS containing 1% BSA and5 mg/ml donkey IgG. The cells were then labeled withthe primary antibody diluted in PBS 1 1% BSA for1 hr at room temperature. After incubation, the cellswere rinsed thoroughly in PBS, and the samples wereincubated for 45 min at room temperature with thesecondary antibody diluted in PBS. All samples werethen rinsed in PBS, and coverslips were mounted withFluoromount-G (EM Sciences). For nuclear staining,after incubation with secondary antibodies, the cellswere incubated for 15 min at room temperature with4′,6-diamidino-2-phenylindole (DAPI) (MolecularProbes) at a concentration of 0.2 mg/ml in PBS. Controlsconsisted of omitting the primary antibody or substi-tuting normal mouse or rabbit IgG for the primaryantibody. All controls were negative.

The cells were examined with a Nikon microscope(Nikon E 800; Nikon Instruments, Inc., Melville, NY)equipped with a digital camera (DXM 1200; Nikon),or a LEICA TCS-NT scanning confocal microscope(Leica Microsystems; Heidelberg, Germany) or anOlympus BX50 (Olympus; Melville, NY) microscopeequipped with a Nikon DXM 1200 digital camera.

Flow Cytometry

The cells were harvested with trypsin-EDTA (InvitrogenLife Technologies; Carlsbad, CA) and washed by cen-trifugation in PBS (2000 rpm/5 min) and blocked for

30 min at room temperature in PBS containing 1%BSA and 5 mg/ml donkey IgG. Then the cells were in-cubated with cholera toxin B conjugated to Alexa 488(6 mg/ml) for 1 hr at room temperature. The cells werethen washed 5 times in PBS (2000 rpm/5 min). Afterrinsing, the cells were analyzed with a BD FACS Caliburflow cytometer, using the CellQuest program (Becton-Dickinson Labware; San Jose, CA).

Scanning Electron Microscopy (SEM)

Cells (3 3 104/coverslip) were plated on 13-mm roundcoverslips. Cells were rinsed in warm PBS (37C) andfixed in 2% glutaraldehyde (Ladd Research Industries;Burlington, VT) in warm PBS for 2 hr at room tempera-ture. Cells were postfixed in 1% OsO4 (EM Sciences)for 2 hr, rinsed in Milli-Q water, incubated with a satu-rated solution of thiocarbohydrazide (EM Sciences), fol-lowed by 1% OsO4. This step was repeated once. Thecells were dehydrated in a graded series of ethanol andcritically point-dried with liquid CO2 in a BAL-TEC-CPD 030 Critical-Point Dryer (BAL-TEC; Liechtenstein,AG), mounted on aluminum stubs with silver paint(EM Sciences), and coated with gold in a BAL-TECSCD 050 Sputter Coater (BAL-TEC). Samples wereexamined with a JEOL JSM-5200 scanning electronmicroscope (Jeol, Ltd.; Tokyo, Japan).

Transmission Electron Microscopy (TEM)

Cells were plated at 2 3 105 cells/well in 6-well tissueculture plates and cultured for 3 days before fixation.Media was changed 16–24 hr before fixation. Cellswere fixed in 2% glutaraldehyde (Ladd Research) plus2% formaldehyde (EM Sciences) in 0.1 M cacodylatebuffer, pH 7.4, containing 0.05% CaCl2 for 1 hr atroom temperature. Cells were postfixed in 1% OsO4

(EM Sciences) in 0.1 M cacodylate buffer, pH 7.4,for 2 hr, rinsed in milli-Q water, dehydrated in a gradedseries of ethanol. Cells were removed from the tissueculture plates with propylene oxide and embedded inEMBED 812 (EM Sciences). Thin sections were cut witha diamond knife, mounted on copper grids, and stainedfor 10 min each in Reynolds lead citrate (Reynolds1963) and 0.5% aqueous uranyl acetate and examinedwith a Philips EM 208 transmission electron micro-scope (Fei Company; Eindhoven, The Netherlands).

Identification and Quantificationof Glycosaminoglycans

Synthesis of [35S]-sulfated Glycosaminoglycans. Post-confluent RBL-2H3, E5, and D1 cells were grown in35-mm culture dishes in DMEM (Invitrogen) contain-ing 150 mCi/ml of [35S]-sulfate for 24 hr. The condi-tioned medium was removed, and the cells werewashed three times with 0.1 M PBS and then scrapedfrom the dishes using 3.5 M urea in 0.05 M Tris-HCl,

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pH 7.4. Protein-free glycosaminoglycan chains wereprepared from the cells and conditioned medium afterincubation with maxatase (0.1 mg/ml in 0.05 M Tris-HCl, pH 8.0, containing 0.15 M NaCl, 4 hr, 60C, finalvolume 100 ml) and identified and quantified usingagarose gel electrophoresis and enzymatic degradationwith glycosaminoglycan lyases (chondroitinases AC andABC, heparinase, and heparitinases I and II) as previouslydescribed (Nader et al. 1989; Trindade et al. 2008).

Results

Expression of GM1 Is Lower in D1 Cells

As previously reported (Silveira e Souza et al. 2008),RBL-2H3 cells highly express the derivatives of GD1b

ganglioside on their surface, whereas the E5 cells express35% and the D1 cells express ,1% of these gan-gliosides. To verify whether the expression of anotherganglioside was also affected in the E5 and D1 cells,RBL-2H3 cells and the mutant cell lines were incubatedwith cholera toxin B subunit, which recognizes GM1.

Similar to that observed for the ganglioside derivativesof GD1b, RBL-2H3 cells highly express the gangliosideGM1 on their surface (Figure 1A), whereas in the E5cell line, the level of expression of GM1 is only slightlyreduced when compared with RBL-2H3 cells (Fig-ure 1B). However, the D1 cells show only a minimumlevel of expression of these gangliosides (Figure 1C).By flow cytometry, the E5 and D1 cell lines express89.2% and ,1%, respectively, of GM1 relative to theamount present on RBL-2H3 cells (Figure 1D). There-fore, the level of expression of gangliosides in the E5mutants varies with the ganglioside type, whereas inthe D1 cell line, these two types of gangliosides arevirtually absent.

Lack of Gangliosides Affects Cell Structure

Because gangliosides are known to play a role in thestructure of the plasma membrane, the possibility thatthe decreased expression of GM1 and the a-galactosylderivatives of gangliosides GD1b (Silveira e Souza et al.2008) may produce morphological alterations in the

Figure 1 Expression of GM1 ganglio-sides. GM1 gangliosides were labeledwith the B subunit of cholera toxin con-jugated to Alexa 488. All of the RBL-2H3cells express GM1 gangliosides on theirsurface (A). E5 cells have a lower expres-sion of GM1 gangliosides (B), and D1 cellsvirtually do not express these gan-gliosides on their surface (C). Nuclei aremarked in blue with 4′,6-diamidino-2-phenylindole (DAPI). (D) Flow cytometryof RBL-2H3, E5, and D1 cells labeled forGM1 gangliosides. E5 and D1 cell linesexpress 89.2% and ,1% of GM1 ganglio-sides present on RBL-2H3 cells. Medianfluorescence intensity and percentagefluorescence intensity are shown (n53).

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mutant cell lines was investigated. DIC microscopy(Figure 2) shows that the cell lines RBL-2H3 and E5are primarily spindle-shaped, whereas the D1 cellsare round, somewhat smaller, and grow as small colo-nies, indicating that the lack of gangliosides affects thestructural characteristics of these cells. These morpho-logical differences were confirmed by scanning electronmicroscopy (Figure 2). Similar to that seen by DIC, byscanning electron microscopy, RBL-2H3 cells are elon-gated and spindle-shaped, and their surface is coveredwith small, short microvilli. E5 cells have morphologysimilar to that of RBL-2H3 cells. The majority of D1cells are round and somewhat smaller, and their surfaceis dramatically altered, possessing a few large rufflesinstead of fine microvilli (Figure 2, arrows).

Because the D1 cells have a distinct morphology, in-cluding extensive membrane ruffles, it was of interestto compare the distribution of the cytoskeleton in thethree cell lines. In RBL-2H3 and E5 cells (Figure 3), theactin filaments are under the plasma membrane fol-lowing the spindle shape of the cells and in associationwith microvilli. In D1 cells, the actin filaments are con-centrated in large membrane ruffles (Figure 3, arrows),and extend into the cytoplasmic projections markingthe entire cell. The distribution of the microtubules isalso altered in the D1 cells (Figure 3). Microtubulesin RBL-2H3 and E5 cells radiate from the centrosomeand are organized into long, straight bundles that givea fusiform shape to the cells. In D1 cells, the microtu-bules are also associated with a centrosome, but thebundles in D1 cells appear thicker and are organizedcircumferentially in a basket shape and are largely re-stricted to the cell body.

Because organization of the cytoskeleton was alteredin the mutant cells, especially in D1 cells, it was of in-terest to examine the structure of two organelles, theGolgi apparatus and the rough ER, which are depen-dent on the cytoskeleton for their organization. Boththe ER and the Golgi complex (Figure 4) are alteredin the mutant cell lines. The ER in RBL-2H3 cells has

a filamentous distribution throughout the cytoplasm.In contrast, in the E5 and D1 cells, the ER is more ve-sicular, especially in the perinuclear region. By TEM,the ER in the RBL-2H3 cells appears filamentous withlong, thin cisternae. The E5 cells have numerous shortcisternae, whereas in the D1 cells, the cisternae appearslightly dilated. The Golgi complex in all three cell lines,RBL-2H3, E5, and D1, is located in a perinuclear po-sition (Figure 4). By TEM, the RBL-2H3 cells have awell-organized Golgi complex, with their cisternaeshowing the typical arrangement of flattened saccules.In the E5 cells, although the Golgi complex is well-developed, the cisternae are not as well-organized asin the RBL-2H3 cells and appear slightly dilated. TheGolgi complex of the D1 cells is smaller, and the cis-ternae are shorter and disorganized. Therefore, the factthat these organelles are altered in the mutant cell linescould affect ganglioside synthesis and/or transport.

Because both the ER and the Golgi apparatus arealtered in the mutant cells, the matrix of the secretorygranules, produced by these organelles, was also ex-amined. The cells were stained with Alcian Blue, whichlabels the glycosaminoglycans, to visualize the granulesand to view their distribution. In RBL-2H3 cells, thegranules are present throughout the cytoplasm (Fig-ure 5, arrows). In E5 cells, the granules appear to besmaller (Figure 5, arrow), and the D1 cells containfewer but larger granules than do the RBL-2H3 cells(Figure 5, black arrows). Clear vacuoles (Figure 5,green arrow) are also present in the cytoplasm of theD1 cells. The difference in the cytoplasmic granuleswas even more apparent when the cells were examinedby TEM. The granules in the RBL-2H3 cells containelectron-dense material (Figure 5, arrows), most likelygranule matrix. In contrast, the cytoplasmic granules inthe E5 (Figure 5, arrows) and D1 (Figure 5, arrows)cells are largely electron-lucid and the granule matrixappears different.

Because sulfated glycosaminoglycans are a majorcomponent of the mast cell granule matrix, the content

Figure 2 D1 cells are morphologicallydistinct from RBL-2H3 and E5 cell lines.By differential interference contrast (DIC),the RBL-2H3 and E5 cells are spindle-shaped. D1 cells are round and grow closeto each other as small colonies. By scan-ning electron microscopy (SEM), RBL-2H3cells are spindle-shaped and their surfaceis covered with short microvilli. E5 cellshave morphology similar to that of RBL-2H3 cells. The majority of D1 cells areround, and their surface is dramaticallyaltered possessing only a few large ruf-fles (arrows).

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of sulfated glycosaminoglycans from the three cell lineswas analyzed by agarose gel electrophoresis. All threecell lines have chondroitin sulfate and heparin. Analy-sis of metabolically [35S]-sulfate–labeled lysates fromthe E5 and D1 cells (Figure 6) showed that they con-tained, respectively, 19% and 56% of the amount ofchondroitin sulfate seen in the RBL-2H3 cells. However,the E5, and especially the D1 cells (206% and 549%,respectively), contain much more heparin than do theRBL-2H3 cells, suggesting that the synthesis of glycos-aminoglycans and the reduction in the amount of gan-gliosides in the mutant cell lines may be related.

DiscussionThe importance of the a-galactosyl derivatives of theganglioside GD1b for mast cell degranulation via FcyRIand for the correct assembly of lipid rafts was dem-onstrated by Silveira e Souza et al. (2008). Ganglio-sides are components of all animal cell membranes,and the plasma membrane is intimately related to cellmorphology. In this study, the influence of ganglio-sides on cell structure and organization was examinedusing the ganglioside-deficient cell lines E5 and D1

(Silveira e Souza et al. 2008) and the parent cell line,RBL-2H3.

The morphological analysis by DIC and SEM showedthat the mutant cell line D1 has a cellular morphologydistinct from that of the mutant E5 and RBL-2H3 celllines, suggesting that the gangliosides are importantin the maintenance of normal cell morphology. This rolefor gangliosides was also observed by Yates et al. (1989)and Ikami et al. (2000). The morphological changes ob-served in D1 cells could be related to the lipid composi-tion of these cells. In this cell line, there is a large decreasein glycosphingolipids that may affect many physico-chemical properties of the plasma membrane (Katoet al. 2003) as well as the anchorage of the cytoskele-ton to the plasma membrane (Palestini et al. 2000).

Meivar-Levy et al. (1997) demonstrated in 3T3 fi-broblasts that long-term incubation with mycotoxinfumonisin B1 (FB1), a specific inhibitor of sphingolipidsynthesis, resulted in a decrease in synthesis of gan-glioside GM3 and caused profound changes in a num-ber of processes associated with the actin cytoskeleton,including cell spreading, microvilli formation, cyto-kinesis, formation of long processes, and disruption ofDNA synthesis and cell proliferation. Remarkably, all

Figure 3 Cytoskeleton is altered in the D1 cells. Actin filaments in RBL-2H3 and E5 cells lie under the plasma membrane following the spindleshape of the cells and in association with microvilli. In D1 cells, actin filaments are concentrated in large membrane ruffles (arrows) andextend into the cytoplasmic projections. F-actin was labeled with phaloidin-Alexa 488. In RBL-2H3 cells, microtubules labeled with anti-b-tubulin are organized into bundles that followed the fusiform shape of the cells, whereas in E5 cells, they appear less organized, and arecompletely disorganized in D1 cells. In D1 cells, microtubules appear as thick bundles that are restricted to the cell body and are not present inthe cytoplasmic extensions. Centrosomes (arrows) were labeled with anti-g-tubulin. Nuclei are marked in blue with DAPI.

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Figure 4 Lack of gangliosides also affects the endoplasmic reticulum (ER) and the Golgi complex. ER was labeled with anti-GRP-78, and alter-ations were assessed in the three cell lines. ER in RBL-2H3 cells is filamentous in form (arrow). In the E5 and D1 cell lines, ER appears vesiculated(arrows). By transmission electron microscopy (TEM), ER in the RBL-2H3 cells is filamentous with long, thin cisternae. E5 cells have numerousshort cisternae, and in D1 cells, the cisternae seem slightly dilated. Golgi complex in the RBL-2H3, E5, and D1 cell lines has perinuclear localization(arrows). The size of the Golgi complex is reduced in D1 cells. Cis-Golgi cisternae were labeled with anti-GM130, and nuclei were stained withDAPI. By TEM, the RBL-2H3 cells have a well-organized Golgi complex, with their cisternae showing the typical arrangement of flattenedsaccules (arrows). In E5 cells, the Golgi cisternae (arrow) are not as well-organized and appear slightly dilated. Golgi complex of the D1 cells issmaller and the cisternae are disorganized (arrows).

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of the morphological effects of FB1 could be reversedby the addition of low concentrations of GM3.

The primary intracellular determinant of cell mor-phology is the cytoskeleton. Pfeiffer et al. (1985) andSahara et al. (1990) have previously shown that po-lymerized actin in RBL-2H3 cells is mainly associatedwith membrane ruffles after stimulation similar to that

seen in unstimulated D1 cells. However, unlike acti-vated RBL-2H3 cells, the D1 cells are not spread oversubstrate. The actin filaments are important for de-granulation and serve to transport the granules for shortdistances at the cellular periphery (Kuznetsov et al.1992). In addition, in resting cells, subcortical actinacts as a physical barrier, preventing the secretion ofgranules. Frigeri and Apgar (1999) demonstrated thatRBL-2H3 cells exposed to latrunculin, an inhibitor ofactin polymerization, showed a decrease in F-actin andan increase in degranulation after cell activation.

A growing amount of evidence indicates that lipidrafts are essential for membrane–cytoskeleton coupling(Levitan and Gooch 2007; Pike 2009). The factors thatgovern the formation of lipid rafts continue to be elu-cidated, but lipid raft formation often requires actinfilaments. The connection between lipid raft proteinsand actin filaments can affect the lateral distributionand mobility of these membrane proteins (Rodgers andGlaser 1993; Lenne et al. 2006). The presence of actinand other cytoskeletal proteins in lipid raft fractionshas been known since the earliest description of thesestructures (Jordan and Rodgers 2003; Badizadeganet al. 2004), and proteomic analyses have documentedthe presence of numerous cytoskeletal proteins in thesefractions (Nebl et al. 2002). Furthermore, cholesteroldepletion, which is often regarded as a functional test

Figure 6 Secretory granule matrix is also altered in the mutant celllines. The amount of chondroitin sulfate is reduced in D1 and E5 cellsin comparison to RBL-2H3 cells, but there is more heparin in the mu-tant cells (E5 and D1).

Figure 5 Secretory granules are altered in the mutant cell lines. In RBL-2H3 cells, the granules are distributed through the cytoplasm(arrows). In E5 cells, the granules are smaller (arrow) and stain less intensely. D1 cells have a few large granules (black arrows), and theircytoplasm appears to be filled with clear vacuoles (green arrow). Alcian Blue staining counterstained with Weigert’s Fucsin-Resorcin. By TEM,RBL-2H3 cells contain electron-dense granules (arrows). However, in the E5 and D1 cells, the granules are electron-lucid (arrows). N, nucleus.

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of dependence on lipid rafts, is associated with loss ofphosphatidylinositol 4,5-biphosphate from the plasmamembrane and a global reorganization of the actincytoskeleton (Kwik et al. 2003), suggesting a possiblerole for actin in the organization and/or function of lipidrafts. However, the extent to which the actin cytoskele-ton participates in establishing membrane rafts is notyet established. Importantly, the actin cytoskeleton isa dynamic structure that changes in response to extracel-lular signals, and it may therefore represent one mecha-nism for governing the establishment and distributionof membrane rafts in the plasma membrane (Chichiliand Rodgers 2007). In a recent review, Chichili andRodgers (2009) show that there is evidence of struc-turing of rafts by a synergistic interaction between thecortical actin cytoskeleton and lipid rafts, and that manyof the structural and functional properties of rafts re-quire an intact actin cytoskeleton. The data of Fujitaet al. (2009) indicate that the GM1 and GM3 clustersin the cell membrane are regulated in different waysand that segregation of the two gangliosides dependson an intact actin cytoskeleton.

It is possible that the abnormal distribution of actinfilaments observed in D1 cells is a result of the disorga-nized lipid rafts in these cells (Silveira e Souza et al.2008), and that the disorganization of both lipid raftsand actin filaments leads to impaired degranulation inthe mutant mast cell lines. A physical association be-tween an outer-membrane lipid and the actin cytoskel-eton requires the presence of other structural signalingor regulatory proteins or lipids, or both (Badizadeganet al. 2004). The results obtained previously (Silveira eSouza et al. 2008) and in this study suggest an associ-ation between the gangliosides in the outer leaflet of theplasma membrane and actin filaments on the cytoplas-mic surface of the plasma membrane. Therefore, lipidrafts might be involved in regulating cytoskeleton or-ganization, thus regulating cytoskeleton-dependent cel-lular processes. Holowka et al. (2000) demonstratedthat functionally important interactions between thehigh-affinity Fcy receptor in activated mast cells andlipid rafts are dependent on the actin cytoskeleton. Asimilar role for the actin cytoskeleton in the biologyof lipid rafts has been suggested in other systems, suchT-cell signaling (Badizadegan et al. 2004). Disruptionof lipid rafts by cholesterol depletion in T-cells inhibitsthe inducible tyrosine phosphorylation of the T-cell re-ceptor (TCR) and prevents its association with actin(Moran and Miceli 1998). The idea that actin plays acentral role in T-cell signaling via lipid rafts is furthersupported by the observation that activation of TCR in-duces reorganization of actin in a cholesterol-dependentmanner (Valensin et al. 2002). Mitchell et al. (2009)found that clustering GM1 lipid rafts in T-cells canregulate b1 integrin function and activate adhesion-strengthening processes that are accompanied by a re-

modeling of the contact area with the adhesive plane,the induction of high-affinity integrin states, and therearrangement of integrin-cytoskeleton networks.

Microtubules are one of the major determinants of cellshape and polarity (Ilschner and Brandt 1996; Condeand Cáceres 2009). The ganglioside-deficient D1 cellsare round, and the arrangement of their microtubulesis completely disorganized. Several studies have showna relationship between microtubules and gangliosides.Microtubules are essential for ganglioside-mediatedneurite elongation (Spero and Roisen 1984; Spero et al.1986), and exogenously added GM1 induced typicalramified morphological changes in microglia in a dose-dependent manner (Park et al. 2008).

The abnormal distribution of microtubules in D1 cellsmay be one of the causes of the disorganized arrange-ment of the Golgi complex observed by TEM in thiscell line. Both the structure and positioning of the Golgicomplex are highly dependent on the microtubule cy-toskeleton. Rivero et al. (2009) observed that whenthe microtubules are disorganized, the Golgi complexfragments, giving rise to discrete Golgi elements, whichare dispersed throughout the cell, exactly as observedin the D1 mutant cell line. This suggests that microtu-bules are required to link stacks into a single organelleand to ensure its pericentrosomal location (Thybergand Moskalewski 1999). Additionally, the specific gly-cosyltransferases that synthesize the oligosaccharidemoiety of the gangliosides (Gillard et al. 1994; Crespoet al. 2004) are located in this organelle. Therefore, dis-ruption of the Golgi complex could also interfere withganglioside synthesis.

The disorganization of the ER, Golgi apparatus, andmicrotubules may explain the alterations in the secre-tory granule matrix seen in D1 cells. The ER, and espe-cially the Golgi complex, are involved in the biosynthesisof heparan sulfate/heparin and chondroitin sulfate(Silbert and Sugumaran 2002; Gorsi and Stringer2007). The distribution of glycosyltransferases alongthe Golgi cisternae dictates the overall structure ofglycosylated compounds (de Graffenried and Bertozzi2004). A disruption in the orderly passage of glycosy-lated molecules through the Golgi complex could leadto the production of alternate products, such as heparininstead of chondroitin sulfate.

The results of this study demonstrate that decreasedganglioside expression in the mutant cells, E5 and D1,affects the normal cell structure of the RBL-2H3 mastcells. Various cell processes, such as signal transduction,vesicular transport, endocytosis, and cell adhesion,could be compromised by the decrease in gangliosidesthat leads to lipid raft disorganization.

Acknowledgments

We thank Coordenação de Aperfeiçoamento de Pessoal deNível Superior, Fundação de Apoio ao Ensino, Pesquisa e

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Assistência, and Fundação de Amparo à Pesquisa do Estadode São Paulo (individual grant 01/10752-2 to CO) for finan-cial support.

We thank Reuben P. Siraganian, MD, PhD, National In-stitutes of Health, Bethesda, MD, who generously providedthe cell lines; Márcia Sirlene Graeff for assistance with theconfocal microscopy; Maria Dolores S. Ferreira, Tereza P.Maglia, and José Augusto Moulin for assistance with thescanning and transmission electron microscopy; and AndersonRoberto de Souza for technical assistance; all from the De-partment of Cell and Molecular Biology and PathogenicBioagents, FMRP-USP, Ribeirão Preto, SP. We also thankPatrícia Vianna Bonini Palma, Fabiana Rossetto, and CamilaCristina Branquinho de Oliveira Menezes, Hemocentro–Faculdade de Medicina de Ribeirão Preto-Universidade deSão Paulo, Ribeirão Preto, Brazil, for assistance with theflow cytometry.

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