IntroductionThe functional role of vimentin, a class III intermediatefilament (IF) component of the cytoskeleton, represents afascinating enigma in biology. Cytoplasmic vimentin networkis widely developed in most growing cells in culture, whateverthe cell-type specific IF they express in vivo (Franke et al.,1979). In vivo, vimentin is expressed differentially inembryonic and postnatal life: in mesenchymal and endodermiccells in embryos, but only in mesenchyma-derived cells inadults (Bachmann et al., 1983; Holthöfer et al., 1984). Theparadigm of vimentin-null mice, which develop and reproducewithout an obvious phenotype (Colucci-Guyon et al., 1994),confirms that vimentin is not required for the survival ofindividual cells and apparently argues against a major role ofthis IF, at least under normal physiological conditions.However, various cell culture models, including those derivedfrom vimentin-null mice, suggest that vimentin could play arole in: (1) organization of other cytoplasmic structures, suchas microtubules and microfilaments, and maintenance ofcellular and nuclear shape; (2) cellular migration and stabilityin response to mechanical stress; (3) membrane traffic (Faigleet al., 2000) and granule secretion (Pryzwansky and Merricks,1998); (4) accumulation of lipid droplet in adipocytes,metabolism of lipoprotein-derived cholesterol and recycling ofsphingosine and glycosphingolipids; (5) cell cycle (vimentin is

considered as an immediate-early gene); and (6) interactionwith several non-structural proteins, such as protein kinase C,stress response proteins (Evans, 1998) or ubiquitin-relatedproteins (Wu et al., 1999). The fact that these findings have notalways been confirmed indicates that the functional role ofvimentin is still a matter of controversy. Indeed, it has beenshown that the absence of vimentin network in cells fromvimentin-null mice (Colucci-Guyon et al., 1994; Holwell et al.,1997) as well as disruption of this network by injection of anti-vimentin antibodies (Klymkowsky et al., 1989) or IF mimeticpeptides (Goldman et al., 1999) could either modify or not bothcell morphology and cytoskeleton organization. Moreover, ithas been suggested that vimentin participates in lipidtrafficking (Klymkowsky, 1995) although, until now, no IF-associated proteins with motor-like properties have beenidentified.

In normal adult kidney, vimentin is expressed in glomeruli,vessels and interstitial cells, but not in tubular cells, in contrastto its expression in developing kidney (Bachmann et al., 1983;Holthöfer et al., 1984). However, vimentin is expressed inproximal tubular cells during the recovery phase that followsischemia or nephrotoxic tubular necrosis (Gossrau et al., 1989;Gröne et al., 1987; Nouwen et al., 1994; Wallin et al., 1992;Witzgall et al., 1994), in renal cell carcinoma (Dierick et al.,1991; Holthöfer et al., 1983; Waldherr and Schwechheimer,

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It has been reported that vimentin, a cytoskeleton filamentthat is expressed only in mesenchymal cells after birth, isre-expressed in epithelial cells in vivo under pathologicalconditions and in vitro in primary culture. Whethervimentin re-expression is only a marker of cellulardedifferentiation or is instrumental in the maintenance ofcell structure and/or function is a matter of debate. Toaddress this issue, we used renal proximal tubular cells inprimary culture from vimentin-null mice ( Vim–/–) and fromwild-type littermates (Vim+/+). The absence of vimentindid not affect cell morphology, proliferation and activityof hydrolases, but dramatically decreased Na-glucosecotransport activity. This phenotype was associated with aspecific reduction of SGLT1 protein in the detergent-resistant membrane microdomains (DRM). In Vim+/+ cells,disruption of these microdomains by methyl-β-cyclodextrin

decreased SGLT1 protein abundance in DRM, a changethat was paralleled by a decrease of Na-glucose transportactivity. Importantly, we showed that vimentin is located toDRM, but it disappeared after methyl-β-cyclodextrintreatment. In Vim–/– cells, supplementation of cholesterolwith cholesterol-methyl-β-cyclodextrin complexescompletely restored Na-glucose transport activity.Interestingly, neither cholesterol content nor cholesterolmetabolism changed in Vim–/– cells. Our results areconsistent with the view that re-expression of vimentin inepithelial cells could be instrumental to maintain thephysical state of rafts and, thus, the function of DRM-associated proteins.

Key words: Vimentin, Rafts, SGLT1

Summary

Vimentin affects localization and activity of sodium-glucose cotransporter SGLT1 in membrane raftsIsabelle Runembert 1, Guillaume Queffeulou 1, Pierre Federici 2, François Vrtovsnik 1, Emma Colucci-Guyon 3,Charles Babinet 3, Pascale Briand 2, Germain Trugnan 4, Gérard Friedlander 1 and Fabiola Terzi 1,*1INSERM U426 and Department Physiology, Faculté de Médecine Xavier Bichat, IFR 02, Université Paris 7, Paris, France2INSERM U380, Institut Cochin de Génétique Moléculaire, Paris, France3URA 1960 CNRS, Institut Pasteur, Paris, France4INSERM U538, Faculté de Médecine Saint-Antoine, Paris, France*Author for correspondence (e-mail: terzi@cochin.inserm.fr)

Accepted 4 November 2001Journal of Cell Science 115, 713-724 (2002) © The Company of Biologists Ltd

Research Article

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1985) and in proximal tubular cells in culture (Franke etal., 1979; Hatzinger et al., 1988). In all these conditions,vimentin expression is considered as a marker of cellulardedifferentiation, but the question still remained whethervimentin is directly involved in cell recovery. In a previousstudy, we showed that the absence of vimentin did notaffect cell proliferation, cell differentiation and structuralorganization of injured tubules in post-ischemic kidneys fromvimentin-null mice, suggesting that vimentin is not mandatoryto restoration of a differentiated morphological phenotype ofproximal cells (Terzi et al., 1997). The purpose of the presentstudy was to investigate whether vimentin plays a role in themaintenance of cell function. One of the principal functions ofproximal tubular cells is to ensure vectorial transport of solutes.It has been shown that disruption of cytoskeleton impaired bothion and water channels and sodium-dependent transportactivities (Steel and Hediger, 1998). In kidney, ischemia andreperfusion induce a disorganization of microtubule andmicrofilament networks in proximal tubules, a phenotypeassociated with alterations of cell polarity and protein sorting(Molitoris et al., 1989). These morphological changes result ina decrease of vectorial transport activities. Interestingly, ratstreated with colchicine or nocodazole exhibited the samemorphological and functional modifications as those observedin post-ischemic kidneys (Hansch et al., 1993). We thereforehypothesized that the development of vimentin network ininjured or cultured epithelial cells could be instrumental in themaintenance of transport capabilities. To test this hypothesis,we investigated the consequences of vimentin inactivation oncell function by growing proximal tubular cells in primaryculture obtained from mice bearing a null mutation of thevimentin gene.

We show that the absence of vimentin did not affect cellproliferation and differentiation, but decreased selectively Na-glucose transport activity. This phenotype is associated with areduction of SGLT1 cotransporter in specialized membranemicrodomains, the rafts. We propose that, in injured or culturedepithelial cells, vimentin participates in preserving propertransport functions possibly through the maintenance ofmembrane physical state.

Materials and MethodsVimentin null miceTargeted inactivation of vimentin gene in mice have been reportedpreviously (Colucci-Guyon et al., 1994). Wild-type (Vim+/+) andhomozygous (Vim–/–) mice were obtained by intercrosses betweenVim+/– mice.

Proximal tubular cell (PTC) culturePrimary cultures of renal PTC were prepared as described previously(Menaa et al., 1995), using kidneys from 3-4 week-old Vim+/+ andVim–/– mice originating from the same litter. Culture mediumconsisted of 50:50 DMEM/Ham’s F-12 (Gibco BRL, France) with25 mM Hepes, 21.5 mM HCO–3, 1 mM sodium pyruvate, 10 ml/literof a 100× non-essential amino-acid mixture, 4 mM glutamine, 50IU/ml penicillin, 50 µg/ml streptomycin, 100 nM sodium selenite, 35µg/ml transferrin, 5 nM T3, 25 ng/ml PGE1, 0.5 µg/ml bovine insulin,100 nM dexamethasone and 500 nM retinoic acid. Hormones werepurchased from Sigma Chemical Co. (St Louis, MO). Fetal calfserum (1%; FCS, Gibco BRL) was added during the first 48 hoursof culture.

Experimental protocolAll experiments were performed at day 7 after seeding, except for thestudies of cell proliferation, which were performed at day 2, 4, 6, 8and 10 for cell number and DNA cell content and at day 2, 4 and 6for radiolabeled thymidine incorporation.

To modulate the plasma membrane cholesterol content, cells wereincubated with or without 10 mM of methyl-β-cyclodextrin (MCD,Sigma) or cholesterol-MCD inclusion complexes (corresponding to 3mg cholesterol per 10 ml medium) for two hours at 37°C withintermittent shaking.

Cell morphology and differentiationLight microscopy

Cells were examined every day under an inverted microscopeequipped with phase-contrast optics.

Immunocytochemistry and immunofluorescenceCells were fixed in ice-cold ethanol 95%-acetic acid 5% (v/v) forvimentin staining, in ice-cold methanol for ZO-1 and cytokeratinstaining and in 4% formaldehyde and ice-cold methanol for α-tubulinstaining. Cells were first incubated overnight at 4°C with the primaryantibody, and then for 1 hour at room temperature with the secondaryantibody. Peroxidase activity was detected using diaminobenzidinetetrahydrochloride (DAB, Dako, France).

For colocalization experiments, cells were fixed in 4%formaldehyde and permeabilized in ice-cold methanol. Cells were firstincubated overnight at 4°C with the two primary antibodies, and thenfor 1 hour at room temperature with the specific secondary FITC- orTRITC-conjugated antibodies.

For cross-linking experiments, cells were first incubated for 1 hourat 4°C and 10 minutes at 37°C with the primary antibodies. Then,cells were washed briefly with PBS-BSA 0.2% and incubated for 1hour at 4°C and 10 minutes at 37°C with the secondary specific FITC-and TRITC-conjugated antibodies. Cells were washed and re-incubated with the secondary antibodies for 20 minutes at 37°C andfinally fixed in 4% formaldehyde and ice-cold methanol.

The primary antibodies used were: (1) a monoclonal anti-vimentinantibody (Sigma), diluted 1/200 and a polyclonal anti-vimentinantibody (kindly provided by A. M. Hill, Institut Pasteur, Paris,France), diluted 1/50; (2) a rat monoclonal anti-cytokeratins K8-K18(kindly provided by A. Vandewalle, Faculté de Médecine XavierBichat, Paris, France), diluted 1/5; (3) a mouse monoclonal anti-α-tubulin antibody, diluted 1/2000; (4) a rat monoclonal anti-ZO-1antibody (Chemicon, Temecula, CA), diluted 1/50; (5) a polyclonalanti-SGLT1 antibody (Chemicon) diluted 1/50; (6) a monoclonal anti-5′-nucleotidase antibody (kindly provided by B. Kaissling, Universityof Zurich-Irchel, Switzerland), diluted 1/50. The secondary antibodiesused were: (1) a sheep anti-mouse horseradish peroxidase-linked Igantibody (Amersham, France), diluted 1/50 for both vimentin and α-tubulin staining; (2) an anti-rat IgG FITC-conjugated antibody(Sigma), diluted 1/100, for both cytokeratin and ZO-1 staining; (3) ananti-rabbit IgG FITC-conjugated antibody, an anti-mouse IgGTRITC-conjugated antibody, an anti-mouse IgG FITC-conjugatedantibody and an anti-rabbit IgG TRITC-conjugated antibody forcolocalization experiments, all diluted 1/80 and purchased by Sigma.

For actin staining, cells were fixed in 4% formaldehyde,permeabilized with 0.1% Triton X-100, and incubated with phalloidinconjugated to TRITC, diluted 1/1000, for 20 minutes at 37°C.

β-galactosidase stainingβ-galactosidase activity was determined on 0.5% glutaraldehyde fixedcells as previously described (Terzi et al., 1997).

Enzymatic activitiesEcto-5′-nucleotidase (EC 3.1.3.5) activity was determined using

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715Vimentin decreases SGLT1 activity in rafts

a method previously described (Siegfried et al., 1995). γ-Glutamyltranspeptidase (EC 2.3.2.2) activity was determined using anadaptation of the technique of Orlowski and Meister (Orlowski andMeister, 1965). Alkaline phosphatase (EC 3.1.3.1) activity wasrevealed on intact 4% paraformaldehyde-fixed cells by adding asolution of nitroblue tetrazolium (300 µg/ml, Sigma) and 5-bromo-4-chloro-3-indolylphosphate (150 µg/ml, Sigma) for 4-5 hours in thedark at room temperature. Negative controls were obtained by treatingcells with levamisole (1 mM, Sigma).

Cyclic AMP productioncAMP production was determined after stimulation of cells with 10–7

M hPTH (1-34; Sigma) or 10–6 M dDAVP (Sigma), as previouslydescribed (Silve et al., 1990).

Cell proliferationCells were incubated with [3H]thymidine (1 µCi/ml; Amersham) for7 hours, and subjected to 5% trichloroacetic acid for 45 minutes at4°C. The precipitate was dissolved in 0.2 N sodium hydroxide and theincorporation of radioactivity was measured by scintillation counting.DNA content was measured as described by Labarca and Paigen(Labarca and Paigen, 1980). Cell number was counted in a Malassezchamber after cell trypsinization.

Uptake studiesUptakes of methyl-α-D-glucopyranoside (MGP) and alanine wereassayed in the presence and in the absence of Na as previouslydescribed (Vrtovsnik et al., 1992).

Activity of Na,K-ATPase was determined by measuring ouabaine-sensitive rubidium uptake. Cells were incubated in the presence of[86Rb]Cl (1 µCi/ml; Amersham) with or without ouabain (1 mM). Theuptake was stopped by washing the cells with a solution containing137 mM NaCl, 5 mM Hepes and 3 mM BaCl2. Cells were thensolubilized in 0.5% Triton X-100 and radioactivity was counted byliquid scintillation.

Northern blot analysisTotal RNA was extracted from cells using the RNAzol kit (Bioprobe,Montreuil-sous-Bois, France). RNA (20 µg per lane) was separatedon a 1.2% agarose-formaldehyde gel and transferred onto a nylonmembrane (Zetabind, CUNO, Inc., Meridien, CT). Prehybridation,hybridization and washing were carried out according to themanufacturer’s recommendations. RNA was quantified bydensitometric computer analysis in a series 400 PhosphoImager(Molecular Dynamics, Inc., Sunnyvale, CA). cDNA probes werelabeled by random priming (Boehringer-Mannheim, France) using[α-32P]dCTP. The following probes were used: rat SGLT1, ratSGLT2 and murine GAPDH (kindly provided by E. Solito, ImperialCollege School of Medicine, London, UK). To generate the SGLT1and SGLT2 probes, the 1816-1994 bp fragment of rat SGLT1cDNA and the 1553-1787 bp fragment of rat SGLT2 cDNA wereamplified by reverse transcriptase and polymerase chain reaction,respectively.

Cellular membrane isolation Brush border membranes (BBM)

Cells were scraped and homogenized with a Dounce homogenizer ina buffer consisting of 300 mM mannitol, 5 mM EGTA, 0.5 mM PMSFand 12 mM Tris-HCl pH 7.4. BBM were then prepared by the MgCl2precipitation and differential centrifugation procedures, as described(Biber et al., 1981). The final pellet was resuspended in a buffercontaining 300 mM mannitol and 16 mM Hepes, 100 mM Tris, pH

7.5. The enrichment in BBM content of the preparation was assessedby measuring alkaline phosphatase activity in the homogenate and inthe final membrane preparations. A tenfold enrichment factor wasroutinely obtained in both Vim+/+ and Vim–/– membrane preparations.

Cell surface biotinylationSpecific cell surface biotinylation was performed as previouslydescribed (Gottardi et al., 1995). Then, BBM were prepared asdetailed above and biotinylated proteins were recovered, separatedand immunoblotted as described (Gottardi et al., 1995).

Detergent-resistant membranes (DRM)Cells were scraped in a buffer (150 mM NaCl, 10 mM Hepes, 1 mMEDTA, pH 7.4) and broken by passage through a 25-gauge needle.The suspension was centrifuged and the pellet was resuspended in aTNE buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.4)containing 1% Triton X-100. DRM were isolated by flotation onsucrose gradient after centrifugation (250,000 g, SW41 Beckmanrotor) for 18-20 hours at 4°C. Then, fractions 4-7 corresponding toDRM were pooled, washed with TNE buffer, centrifuged for 1 hourat 4°C and the pellet was resuspended in the TNE buffer. To confirmthat vimentin is located to DRM, the pooled 4-7 DRM fractions wereresuspended in TNE buffer containing 1% Triton X-100 and a secondsucrose gradient was performed, as described above. Moreover, toanalyze the distribution of SGLT1 and vimentin proteins, the 12fractions of sucrose gradient were individually collected andprocessed separately as done for the pooled fractions 4-7.

ImmunoprecipitationAn aliquot of DRM fractions (50 µg proteins) was diluted in IP buffer(50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM NaF, 1 mM EDTA,1 mM EGTA, 1 mM NaVO4, 1 mM PMSF, 1% Triton X-100) andincubated overnight at 4°C with a rabbit anti-caveolin polyclonalantibody (Transduction Laboratories, France) or with a control non-immune rabbit serum. Then, protein A-sepharose beads (Amersham)were added for 2 hours at 4°C. The beads were washed with IP buffer,resuspended in sample buffer with 2% β-mercaptoethanol, heated to95°C, centrifuged, and the supernatant was subjected to SDS-PAGE.

Western blot analysisImmunoblotting

Proteins were separated on 10% SDS-polyacrylamide gel andtransferred onto a nitrocellulose membrane (Biorad, France). Themembrane was first incubated overnight at 4°C with the primaryantibody, then, for 2 hours at room temperature with the peroxidase-conjugated secondary antibody. Immunoreactive proteins weredetected by enhanced chemiluminescence (ECL kit, Amersham). Thefilms were scanned using a Scan-Jet 3c/ADF (Hewlett Packard) andthe signals were quantified using the NIH image software.

AntibodiesThe primary antibodies used were: (1) a rabbit polyclonal anti-SGLT1 antibody (Chemicon), diluted 1/2000; (2) a rabbit polyclonalanti-5′-nucleotidase antibody (a gift from B. Kaissling), diluted1/5000; (3) a mouse monoclonal anti-vimentin antibody (Sigma),diluted 1/1000; and (4) a rabbit polyclonal anti-caveolin antibody(Transduction Laboratory), diluted 1/5000. The secondary antibodiesused were: (1) a sheep anti-mouse horseradish peroxidase-linked Igantibody (Amersham), diluted 1/4000 for vimentin; and (2) a donkeyanti-rabbit horseradish peroxidase-linked Ig antibody (Amersham),diluted 1/2000 for SGLT1 and 1/8000 for both 5′-nucleotidase andcaveolin.

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Cholesterol analysisLipids were extracted from total cell extracts or DRMs according tothe procedure of Bligh and Dyer (Bligh and Dyer, 1959) and separatedby TLC on silicagel plates. Then, the spot of cholesterol was scraped,extracted with chloroform/methanol (2:1, v:v) and cholesterol contentwas assayed by gas-chromatography, as previously described (Lirbatet al., 1997).

Cholesterol biosynthesis was measured after 48 hour cellincubation with [14C]mevalonate (1 µCi/ml). [14C]cholesterol wasextracted from total cells as described (Bligh and Dyer, 1959) andanalyzed by thin layer chromatography.

Expression of data and statistical analysisResults were expressed as means±s.e.m. of four to six separateexperiments performed in triplicate. Differences between theexperimental groups were evaluated using one-way analysis ofvariance, which was followed, when significant, by Student’s t-test.

Results All experiments were performed on renal proximal tubularcells in primary culture obtained from animals by intercrossesbetween Vim+/– mice.

Effect of vimentin inactivation on cell morphology,differentiation and proliferationIt has been reported that vimentin is expressed in manyepithelial and non-epithelial cell types in culture, includingrenal proximal tubular cells (Franke et al., 1979; Hatzingeret al., 1988). Immunocytochemical analysis confirmed that,under our experimental conditions, a typical vimentin networkdeveloped after seeding and was maintained thereafter in wild-type cells. In Vim–/– cells, vimentin could not be detected, butX-gal staining revealed a β-galactosidase activity in nuclei ofthese cells throughout the culture time (data not shown).

The first step of this study was to evaluate whether theabsence of vimentin modified proximal tubular cellmorphology, differentiation and/or proliferation. Both Vim+/+

and Vim–/– cells grew as a single monolayer, formed domessoon after they reached confluence at day 6-7, and developedepithelial cell tight junctions, as judged by ZO-1immunostaining (data not shown). Cytokeratin, microtubularand actin filaments formed well-organized networks fromnucleus to plasma membrane in both Vim+/+ and Vim–/– cells(data not shown).

Differentiation of Vim+/+ and Vim–/– cells was evaluated bymeasuring brush border membrane enzyme activities, as wellas the response to hormones. As shown in Table 1, both Vim+/+

and Vim–/– cells exhibited a typical proximal phenotype with

high ecto-5′-nucleotidase and γ-glutamyltranspeptidaseactivities, high hPTH response and low dDAVP response.Moreover, more than 90% of cultured cells were positive foralkaline phosphatase (data not shown). No difference appearedfor these parameters between the two cell types (Table 1).

The ability of proximal tubular cells to proliferate was theninvestigated by measuring [3H]thymidine incorporation, DNAcontent and cell number under basal conditions and afterstimulation with 2.5% FCS. The pattern of cell proliferationwas similar in Vim+/+ and Vim–/–cells, regardless theexperimental conditions and the method used (data not shown).

Effect of vimentin inactivation on membrane transportsFinally, we evaluated whether the absence of vimentin affectedtransport activities. Na-glucose cotransport activity, assessedby Na-dependent uptake of MGP, a non-metabolized analogueof D-glucose, was significantly reduced in Vim–/– cellscompared with Vim+/+ cells (Table 2). By contrast, absence ofvimentin did not affect Na-dependent neutral amino acidtransport: indeed, alanine uptake was comparable in Vim+/+ andVim–/– cells (Table 2). Similarly, ouabain-sensitive rubidiumuptake, which reflects the activity of Na,K-ATPase, was similarin Vim+/+ and Vim–/– cells (Table 2).

To better characterize the changes of Na-glucose cotransportactivity in cells lacking vimentin, we determined the kineticparameters of Na-dependent MGP uptake in Vim+/+ and Vim–/–

cells. Analysis by the Eadie-Hofstee plot showed that theabsence of vimentin affected the Vmax but not the Km value ofthe MGP transport system (Fig. 1). Indeed, the Vmax value was50.4±1.8 and 24.3±1.9 nmol/mg protein/10 minutes in Vim+/+

and Vim–/– cells, respectively, (P<0.001), whereas the Km valuewas 453±59 and 441±123 µM in Vim+/+ and Vim–/– cells,respectively (P=NS).

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Table 1. Effect of vimentin gene inactivation on proximaltubular cell differentiation

Enzymatic activity Intracellular cAMP content(nmol/mg protein/minutes) (pmol/mg protein)

5′-Nu γ-GT Basal PTH dDAVP

Vim+/+ 15.5±0.74 306±15 15±3 547±101 165±53Vim–/– 15.0±2.08 298±17 20±3 436±85 138±71

5′-Nu, 5′-nucleotidase; γ-GT, γ-glutamyltranspeptidase; PTH, parathyroidhormone (added at 10–7 M); dDAVP, arginine vasopressin (added at 10–6 M).Experiments were performed in proximal tubular cells in primary culturefrom vimentin-null mice (Vim–/–) and from wild-type littermates (Vim+/+).Data are means±s.e.m. of five separate cultures; experiments were performedin triplicate. No significant difference was observed between Vim–/–andVim+/+ cells for all the parameters.

Table 2. Effect of vimentin gene inactivation on proximal tubular cell transport activityNa-dependent MGP uptake Na-dependent alanine uptake Ouabain-sensitive rubidium uptake

(nmol/mg protein/10 minutes) (nmol/mg protein/10 minutes) (nmol/mg protein/10 minutes)

Vim+/+ 29±3.5 2.6±0.32 146±40Vim–/– 17±1.1* 2.7±0.31 185±11

Na-dependent [14C]-MGP (1 mM, 10 minutes), L-[3H]-alanine (0.1 mM, 10 minutes) and ouabain-sensitive [86Rb]-Cl uptake (K: 140 mM, 10 minutes) wasmeasured as described in Materials and Methods. Experiments were performed in proximal tubular cells in primary culture from vimentin-null mice (Vim–/–) andfrom wild-type littermates (Vim+/+). Data are means±s.e.m. of five separate cultures; experiments were performed in triplicate. MGP, methyl-α-D-glucopyranoside.

*ANOVA: Vim–/–vs Vim+/+ cells, P<0.002.

717Vimentin decreases SGLT1 activity in rafts

Effect of vimentin inactivation on Na-glucose cotransportmRNA and protein levelsThe second part of this study aimed to elucidate the molecularand cellular mechanisms whereby the absence of vimentinaffected the Na-glucose cotransport activity in proximaltubular cells. Since it has been reported that vimentin could beinvolved in transcription and trafficking of mRNAs (Skalli andGoldman, 1991; Traub and Shoeman, 1994), we analyzed theexpression of SGLT1 and SGLT2, the two proximal Na-glucose cotransporters, in whole kidneys and in cultured cells.As expected, whole kidneys from control mice expressed bothSGLT1 and SGLT2 mRNAs, with SGLT2 being moreabundantly expressed (Fig. 2A). At variance with wholekidneys, proximal tubular cells in primary culture from wild-type mice expressed exclusively the SGLT1 transcript, whereasthat of SGLT2 was undetectable by northern blot. The samepattern of expression was observed in cells lacking vimentin(Fig. 2A).

We next evaluated by western blot the abundance of SGLT1protein in brush border membranes (BBM) prepared fromVim+/+ and Vim–/– cultured cells. In wild-type cells, anti-SGLT1antibody reacted with a specific band of about 75 kDa (Fig.2B), which was almost abolished by preabsorbing antibodieswith excess of recombinant SGLT1 (data not shown). BBMfrom Vim–/– cells expressed the same amounts of SGLT1 andof 5′-nucleotidase, a BBM protein used as a control, as thosefrom Vim+/+ cells (Fig. 2B). To exclude the possibility that sub-membranous SGLT1 proteins could adhere non-specifically

during BBM preparation, confluent monolayers were used forcell surface biotinylation experiments. As shown in Fig. 2C,similar amounts of SGLT1 were detected in plasmabiotinylated membranes from Vim+/+ and Vim–/– cells.

SGLT1 is located to detergent-resistant membranes inVim+/+ cells Recent studies suggested that sphingolipid- and cholesterol-enriched microdomains, also named detergent-resistantmembranes (DRM), within the plasma membrane ofeukaryotic cells, are implicated in many important cellularprocesses, including segregation and oligomerization of

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Fig. 1.Sodium-glucose cotransport activity of proximal tubular cellsin primary culture from vimentin-null mice (Vim–/–; closed circles)and from wild-type littermates (Vim+/+; open circles). The uptake ofmethyl-α-D-glucopyranoside (MGP) was evaluated in the presenceof [14C]-MGP (0.5 µCi/ml) and appropriate concentrations of MGP.Na-dependent glucose uptake (insert) was calculated as thedifference between MGP uptakes measured in the presence ofsodium or glucamine. Eadie-Hofstee plot shows the Vmax and theKmof the MGP transport system of the two cell types. Data aremeans±s.e.m. of four separate cultures, experiments were performedin triplicate. Statistical analysis: ANOVA, Vim–/– vs Vim+/+ cells,P<0.005.

Fig. 2.SGLT1 and SGLT2 mRNA and SGLT1 protein expression ofproximal tubular cells in primary culture from vimentin-null mice(Vim–/–) and from wild-type littermates (Vim+/+). (A) Northern blotanalysis. Total RNA was extracted from Vim+/+ and Vim–/– culturedcells and from whole kidney (K) of Vim+/+ mice using RNAzol kit.cDNA probes, labeled by a random priming method, were: the ratSGLT1, the rat SGLT2 and the mouse GAPDH. Blots arerepresentative samples from six animals and six separate cultures.(B) Western blot analysis of brush border membranes (BBM). BBMwere prepared by MgCl2 precipitation and differential centrifugationprocedures. Proteins were immunoblotted with a rabbit polyclonalanti-SGLT1 antibody and a rabbit polyclonal anti-5′-nucleotidaseantibody. Blots are representative samples from five separatecultures. (C) Western blot analysis of biotinylated proteins extractedfrom BBM. For specific cell surface biotinylation experiments, cellswere incubated twice consecutively with NHS-ss-biotin, BBM wereprepared and the biotinylated antigens were recovered withstreptavidin agarose beads. Then, proteins were immunoblotted witha rabbit polyclonal anti-SGLT1 antibody. Blots are representativesamples from two separate cultures. Statistical analysis: no differencewas observed between Vim+/+ and Vim–/– cells for any of theparameters.

718

proteins (Smart et al., 1999). Since Na-glucose cotransportactivity was repeatedly shown to be exquisitely sensitive tochanges in the physical state (fluidity/viscosity) of the plasmamembranes (Friedlander et al., 1988; Vrtovsnik et al., 1992),we hypothesized that SGLT1 could localize to DRM. To testthis hypothesis, cell membranes from wild-type cells weresolubilized in Triton X-100, DRM were purified on a sucroseflotation gradient, and the 12 fractions obtained were analyzedby western blot. As shown in Fig. 3A, the anti-SGLT1 antibodyrevealed that most SGLT1 proteins (60%) were in fractions 4-7. This pattern of detection matched the one of caveolin, amarker of DRM. Moreover, immunoprecipitation experimentsof Vim+/+ DRM using a rabbit polyclonal anti-caveolinantibody showed that SGLT1 co-precipitated with caveolin(data not shown), suggesting that the two proteins form aphysical complex.

An important criteria to establish the association of a newprotein with rafts, is to demonstrate, using antibodycrosslinking experiments, that the protein co-clusters with awell-known raft-associated protein in patches at the plasmamembrane. Thus, we first investigated, by immunoflurescenceand confocal microscopy, the subcellular localization ofSGLT1 in Vim+/+ cells. Control samples were fixed and thenincubated with the anti-SGLT1 antibody, while experimentalsamples were treated with the specific antibody before fixation,a procedure causing in vivo cross-linking of a protein on thecell surface. The results are shown in Fig. 3B. In controlsamples SGLT1 was distributed over the entire plasmamembrane, whereas upon antibody crosslinking the proteinwas found in patch-like clusters on the surface of the cells.Then, we analyzed whether SGLT1 co-clustered in plasmamembrane with 5′-nucleotidase, a well-known GPI raft-

Journal of Cell Science 115 (4)

Fig. 3. SGLT1 protein expression in detergent-resistant membranes (DRM) of proximal tubular cells in primary culture from wild-type animals.(A) Cell membranes from wild-type cells were solubilized in Triton X-100, DRM were purified on a sucrose flotation gradient and an aliquot ofeach 1 ml gradient fraction (lanes 1-8=5-30% sucrose; lanes 9-12=40% sucrose) was analyzed by western blotting. A rabbit polyclonal anti-SGLT1 antibody and a rabbit polyclonal anti-caveolin antibody were used. (B) SGLT1 protein localization in a Vim+/+ cell after 4%formaldehyde and ice-cold methanol fixation (left panel) or antibody crosslinking (right panel). A rabbit polyclonal anti-SGLT1 antibody wasused, followed by a secondary FITC-conjugated antibody. (C) SGLT1 (left panel) and 5′-nucleotidase (5′-Nu, middle panel) crosslinking inVim+/+ cells. An overlay of SGLT1 and 5′-nucleotidase images is shown in the right panel. A rabbit polyclonal anti-SGLT1 antibody and amouse monoclonal anti-5′-nucleotidase antibody were used, followed by the specific secondary FITC- and TRITC-conjugated antibodies.Finally, cells were fixed in 4% formaldehyde and ice-cold methanol.

719Vimentin decreases SGLT1 activity in rafts

anchored protein. As presented in Fig. 3C, the overlay ofSGLT1 and 5′-nucleotidase crosslinking images showed thatthere was a complete colocalization of SGLT1 and 5′-nucleotidase in the patches of plasma membrane.

Effect of vimentin inactivation on SGLT1 content indetergent-resistant membranes In light of the above findings, we wondered whether theabsence of vimentin reduces the abundance of SGLT1 in DRMof Vim–/– cells. Thus, we screened, via immunoblot, the Triton-insoluble complexes (corresponding to fractions 4-7) fromVim+/+ and Vim–/– cells. The amount of SGLT1 in DRMsignificantly declined in cells lacking vimentin, compared withVim+/+ cells (Fig. 3B). By contrast, the amounts of two otherDRM-associated proteins, 5′-nucleotidase and caveolin, weresimilar in Vim–/– and Vim+/+ cells (Fig. 4).

Effect of DRM disruption on SGLT1 protein content andactivity in Vim+/+ cellsTo provide further evidence that SGLT1 localization to DRMcould be mandatory to its function, we investigated whetherdisruption of these microdomains affects Na-glucosetransport activity. The integrity of microdomains has beenshown to depend on cholesterol membrane content. Wetherefore analyzed the effect of methyl-β-cyclodextrin(MCD), an extracellular cholesterol acceptor which canextract cholesterol from membranes, on Vim+/+ and Vim−/−

cells. Under our experimental conditions, about 45% of total

cholesterol was removed by MCD from plasma membranesin Vim+/+ (Fig. 5A). As shown in Fig. 5B, treatment of Vim+/+

cells with MCD reduced glucose uptake to a value close tothat observed in untreated Vim–/– cells. By contrast, MCDtreatment did not affect significantly glucose uptake in Vim–/–

cells, despite a similar cholesterol depletion of plasmamembranes (Fig. 5). Interestingly, treatment of cells withMCD led to a significant decrease of SGLT1 protein, but notof 5′-nucleotidase and caveolin, in cholesterol-depletedVim+/+ DRM, compared with non-depleted ones (Fig. 6A).By contrast, it affected neither the abundance of SGLT1, northat of 5′-nucleotidase in total BBM from Vim+/+ MCD-treated cells (Fig. 6B).

Vimentin is located to detergent-resistant membranes inVim+/+ cells Finally, we evaluated by which mechanism vimentin couldaffect SGLT1 localization to DRM. In view of previous dataconcerning vimentin, two hypotheses could be raised: a role ofvimentin in structural raft’s organization or in raft’s cholesterolcontent. We first investigated whether vimentin is located toDRM. As shown in Fig. 7A, we found that an amount ofvimentin was resistant to Triton X-100 and located to DRM.Since previous work (Melkonian et al., 1999) has suggested

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Fig. 4.Effect of vimentin gene inactivation on SGLT1 expression indetergent-resistant membranes (DRM) of proximal tubular cells inprimary culture from vimentin-null mice (Vim–/–; hatched bars) andfrom wild-type littermates (Vim+/+; open bars). Cells were solubilizedin Triton X-100, DRM were purified on a sucrose flotation gradientand fractions 4-7 were pooled and immunoblotted with a rabbitpolyclonal anti-SGLT1 antibody, a rabbit polyclonal anti-5′-nucleotidase antibody and a rabbit polyclonal anti-caveolin antibody.Blots are representative samples from three separate cultures. Dataare means±s.e.m. ANOVA: Vim–/–vs Vim+/+ cells, **P<0.005.

Fig. 5.Effect of cholesterol-affecting drugs on cholesterol contentand sodium-glucose cotransport activity of proximal tubular cells inprimary culture from vimentin-null mice (Vim–/–; hatched bars) andfrom wild-type littermates (Vim+/+; open bars). Cells were treated ornot with methyl-β-cyclodextrin (MCD, 10 mM at 37°C for 2 hours)or cholesterol-methyl-β-cyclodextrin inclusion complexes (MCD-chol, 0.03% cholesterol at 37°C for 2 hours), then (A) total plasmamembrane cholesterol content and (B) Na-dependent [14C]-methyl-α-D-glucopyranoside (MGP) uptake (1 mM, 10 minutes) weremeasured as described. Data are means±s.e.m. of four separatecultures; experiments were performed in triplicate. ANOVA: Vim–/–

vs Vim+/+ cells, *P<0.05; treated vs untreated cells: §P<0.05,§§P<0.01, §§§P<0.005.

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that cytoskeleton elements could adhere non-specificallyduring DRM preparation, additional experiments wereperformed to exclude this possibility. We run a second gradienton the pooled 4-7 DRM fractions and showed, by westernblotting, that vimentin and caveolin still co-sedimented (Fig.7B). Moreover, we immunoprecipitated DRM from Vim+/+

cells using a rabbit polyclonal anti-caveolin antibody, andshowed, by western blotting, that vimentin co-precipitated with

caveolin (Fig. 7C), suggesting that vimentin and caveolin forma physical complex. Finally, we showed that MCD treatment,which engendered the same phenotype that vimentin geneinactivation, induced the disappearance of vimentin from DRMof MCD-treated Vim+/+ cells (Fig. 7D).

To provide further evidence in favor of a physical interactionbetween vimentin and rafts, we investigated, byimmunofluorescence and confocal microscopy, whethervimentin colocalized to plasma membrane with a well-knownraft-associated protein, such as 5′-nucleotidase. As expected,confocal microscopy showed that 5′-nucleotidase wasdistributed along the entire plasma membrane (Fig. 8B). Usinga polyclonal anti-vimentin antibody, we observed that vimentinintermediate filaments were anchored in plasma membrane too,resulting in a thin staining of the cell border (Fig. 8A). Asshown in Fig. 8C, overlay of vimentin and 5′-nucleotidaseimages showed that there was a significant colocalization ofthe two proteins at the plasma membrane. Similarly, the doublestaining of SGLT1 (Fig. 8E) and vimentin (Fig. 8D) showedthat the two proteins colocalized to the plasma membrane (Fig.8F).

Second, we analyzed the consequences of vimentin geneinactivation on cholesterol level and metabolism. Neither totalcellular, nor DRM cholesterol content was affected by theabsence of vimentin. Indeed, total cell cholesterol was9.4±2.25 and 8.2±1.71 nmol/20 µg protein, whereas DRMcholesterol was 102±19 and 99±29 nmol/20 µg protein inVim+/+ and Vim–/– cells, respectively. Moreover, cholesterolmetabolism, as judged by [14C]mevalonate incorporation, wassimilar in Vim+/+ and Vim–/– cells (51405±8803 vs 55514±8061cpm/mg protein).

Although cholesterol content was not decreased in Vim–/–

cells compared with Vim+/+ cells, we wondered whetherloading Vim–/– cells with an excess of cholesterol might restorethe Na-glucose cotransport activity through a stiffening ofplasma membrane. Once DRM were enriched with cholesterol(Fig. 5A), the Na-glucose transport activity increased in Vim–/–

cells and was not different from that observed in Vim+/+ cells(Fig. 5B). By contrast, in wild-type cells, glucose uptake wasunaffected by this treatment.

DiscussionIn the present study, we provide evidence that the absence ofvimentin affected the activity of sodium-glucose cotransport incultured renal proximal tubular cells. We show that this effectwas associated with a parallel decrease of this transporter indetergent-resistant domains of brush border membranes. Wefinally demonstrated that vimentin localized to the rafts, too.These results suggest that vimentin plays a key role in thefunction of SGLT1, possibly by maintaining the membranestructure of rafts.

The effect of vimentin on glucose transport activity isspecificOur findings showed that the absence of vimentin has a specificimpact on sodium-glucose transporter. Indeed, cultured cellsoriginating from Vim–/– animals did not differ from thosecultured from wild-type littermates for any other parameter.Particularly, proliferation or differentiation, as judged from the

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Fig. 6.Effect of methyl-β-cyclodextrin (MCD) on SGLT1 expressionin detergent-resistant membranes (DRM) and brush bordermembranes (BBM) of proximal tubular cells in primary culture fromwild-type mice. Cells were treated or not with MCD (10 mM at 37°Cfor 2 hours), then DRM (A) and BBM (B) were prepared andanalyzed by western blotting using a rabbit polyclonal anti-SGLT1antibody, a rabbit polyclonal anti-5′-nucleotidase antibody and arabbit polyclonal anti-caveolin antibody. Blots are representativesamples from three separate cultures. Data are means±s.e.m.ANOVA: treated vs untreated cells: §§P<0.01.

721Vimentin decreases SGLT1 activity in rafts

activity of brush border enzymes and of apical or basolateraltransporters (sodium-alanine cotransport, Na,K-ATPase),were similar inVim+/+ and Vim–/– cells. These results are inagreement with those reported previously in vivo showing that,after renal ischemia-induced tubular necrosis, regeneration anddifferentiation were not impaired in mice lacking vimentin(Terzi et al., 1997). It is noteworthy that, in both in vitro andin vivo models, the absence of vimentin did not result inoverexpression or massive rearrangement of other intermediatefilaments such as cytokeratins (Colucci-Guyon et al., 1994;Holwell et al., 1997).

SGLT1, but not SGLT2, is expressed in culturedproximal tubular cellsIn the cultured renal proximal tubular cells used in the presentstudy, sodium-glucose cotransport activity was ensuredexclusively by SGLT1, as showed by the kinetic parameters ofMGP uptake with an apparent Km value close to 450 µM, andthe detection of SGLT1 mRNA but not of SGLT2 transcript.This pattern contrasts with the abundance of SGLT2 mRNA inmouse whole kidney and raises the possibility that, under ourculture conditions, SGLT2 is downregulated. Alternatively,these results could suggest that cells originated predominantlyfrom the S3 segment of the proximal tubule, in which SGLT1is predominantly expressed. However, the activity of brushborder enzymes in the cultured cells argues against thispossibility. Nevertheless, it is important to point out that theexclusive expression of SGLT1 mRNA was similar in Vim–/–

and Vim+/+ cultured cells, showing that vimentin does not affecttranscription of SGLT1, as suggested for other proteins

by previous work (Skalli and Goldman, 1991; Traub andShoeman, 1994).

SGLT1 is located to DRMOur study provides unequivocal evidence that SGLT1 islocated to detergent-resistant membrane domains or rafts. Thisis, to our knowledge, the first report of the presence of asodium-dependent cotransport system in this cholesterol-and sphingomyelin-enriched domain of the apical plasmamembrane. It is noteworthy that fractions enriched in SGLT1are also enriched in caveolin, a protein well known to localizeto rafts. Moreover, using antibody crosslinking experiments,we have shown that SGLT1 co-clustered with 5′-nucleotidase,a GPI anchored raft protein, in patches at the plasmamembrane. This is an important criterion to establish theassociation of a new protein with rafts. The observation thatSGLT1 is localized in rafts suggests that optimal activity ofthis cotransporter would be achieved when its lipidicmicroenvironment has a low fluidity. Several data are inagreement with this hypothesis. First, in the present work,cholesterol depletion by methyl-β-cyclodextrin decreasedsodium-glucose cotransport activity in Vim+/+ cells. Second,previous reports (Friedlander and Amiel, 1989) have shownthat fluidification of plasma membranes of renal epithelial cellsby aromatic alcohols dramatically decreased sodium-glucosecotransport activity. Finally, exposure of proximal cells tosphingomyelinase, a treatment that resulted in depletion ofplasma membranes in both sphingomyelin and cholesterol,gave similar results (Vrtovsnik et al., 1992). Interestingly,repletion of sphingomyelinase-treated cells with cholesterol-

Fig. 7. Vimentin proteinexpression in detergent-resistantmembranes (DRM) of proximaltubular cells in primary culturefrom wild-type animals. Cellmembranes were solubilized inTriton X-100, then DRM werepurified on a sucrose flotationgradient. (A) An aliquot of each 1ml gradient fraction (lanes 1-8=5-30% sucrose; lanes 9-12=40%sucrose) was collected andanalyzed by western blotting usinga mouse monoclonal anti-vimentinantibody. (B) Fractions 4-7 ofDRM were pooled, washed,resuspended in TNE buffercontaining 1% Triton X-100 and asecond sucrose gradient wasperformed as described above,followed by western blot analysis.A mouse monoclonal anti-vimentin antibody and a rabbitpolyclonal anti-caveolin antibodywere used. (C) The pooled 4-7DRM fractions wereimmunoprecipitated with a rabbit polyclonal anti-caveolin antibody (lines 1 and 2) or non-immune rabbit serum (line 3). Theimmunoprecipitates were analyzed by western blotting using either a rabbit polyclonal anti-caveolin antibody (left and right) or a mousemonoclonal anti-vimentin antibody (middle). (D) Effect of methyl-β-cyclodextrin (MCD) on DRM vimentin expression. Cells were treated ornot with MCD (10 mM at 37°C for 2 hours), then DRM were prepared as described above, followed by western blotting using a mousemonoclonal anti-vimentin antibody. All blots are representative samples from three separate cultures.

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enriched liposomes restored sodium-glucose cotransportactivity (Vrtovsnik et al., 1992). This observation corroboratesthe present studies showing that enrichment of DRM bycholesterol-cyclodextrin complexes restored glucose transportin Vim–/– cells. Taken together, these results demonstrate thatmembrane-bound cholesterol is an important modulator ofglucose transport.

The mechanisms by which localization to DRM allowsSGLT1 to function remain to be elucidated. Several reports(Giudicelli et al., 1998; Stevens et al., 1990; Takahashi et al.,1985; Turner and Kempner, 1982) have suggested thatfunctional SGLT1 is an oligomeric protein, resultingby homodimerization of two identical subunits, orheterodimerization between a SGLT1 monomer and RS1, aregulatory protein with a molecular weight very close to thatof SGLT1 (Veyhl et al., 1993). Since DRM were reported toact as concentration platforms at the cell surface allowingproteins to oligomerize (Abrami et al., 1998) or to formsupramolecular signalling complexes (Field et al., 1997; Greenet al., 1999), it can be proposed that this property extends tosodium-glucose cotransporter and that dimers, whatevertheir composition, are located preferentially in rafts. Theexperimental condition used to prepare DRM together with theability of the transporter to dissociate under chemical andphysical forces because of non-covalent interactions betweenunits accounts for the observation that only one 75 kDa bandwas apparent in DRM preparations.

Interaction of vimentin with DRM componentsPrevious reports (Deckert et al., 1996; Lisanti et al., 1994;Mallard et al., 1998; Moran and Miceli, 1998; Oliferenko etal., 1999) have shown that cytoskeleton elements participate tothe constitution of rafts. Microtubules were reported to play arole in the trafficking of lipids and proteins of rafts to the apicaldomain of the plasma membrane. Actin was found to beassociated with rafts and to play an important role in clusteringof raft proteins (Fujimoto et al., 1995; Oliferenko et al., 1999).

Our study provides the first evidence that intermediatefilaments are associated with DRM. Binding of vimentin toplasma membrane has been previously shown and confirmedby immufluorescence analysis in the present study, where weshowed a colocalization at the cell border of vimentin with twowell-known membrane-associated proteins, the 5′-nucleotidaseand SGLT1. Vimentin network can interact with membrane-adhesion proteins, such as spectrin, ankyrin, plectin, SNAP23or PLIC and/or directly with the lipid bilayer (Faigle et al.,2000; Georgatos and Marchesi, 1985; Mangeat and Burridge,1984; Perides et al., 1986; Seifert et al., 1992; Wu et al., 1999).It has been suggested that cytoskeleton elements, such as actin,adhere non-specifically during DRM preparations (Melkonianet al., 1999). However, data from the present work argueagainst this idea. In fact, we showed that: (1) in pull-downexperiments, vimentin co-immunoprecipitated with caveolin;(2) vimentin and caveolin still co-sedimented when DRMfractions underwent a double sucrose gradient; and (3)vimentin disappeared from DRM after treatment of cells withmethyl-β-cyclodextrin, a procedure known to disorganize thesedomains. Taken together these results strongly suggest thatvimentin is closely associated, or located to DRM and forms aphysical complex with caveolin.

With regard to the mechanism that underlies the effectof vimentin on rafts, at least two possibilities can be putforward. First, vimentin that has been shown to modifyglycosphingolipid (Gillard et al., 1998) and cholesterol (Sarriaet al., 1992) metabolism could directly affect the lipidiccomposition of DRM, which might influence proteinlocalization to rafts (chemical hypothesis). Alternatively, aspreviously suggested for actin (Oliferenko et al., 1999),vimentin could maintain the structure of rafts, limiting theextent of the lateral mobility of raft-associated proteins(physical hypothesis). The observation that total cell and DRMcholesterol contents, as well as cholesterol metabolism weresimilar in Vim+/+ and Vim–/– cells argues strongly against thefirst hypothesis. By contrast, the fact that increased membranecholesterol content to supra-physiological value restored the

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Fig. 8. Vimentin protein localization inproximal tubular cells in primary culture fromwild-type animals. (A-C) Cells were fixed in4% formaldehyde and ice-cold methanol, thenincubated with a rabbit polyclonal anti-vimentin antibody (A) and a mousemonoclonal anti-5′-nucleotidase antibody (B),followed by the specific secondary TRITC-and FITC-conjugated antibodies. The overlayis shown in panel C. (D-F) Cells were fixed in4% formaldehyde and ice-cold methanol, thenincubated with a mouse monoclonal anti-vimentin antibody (D) and a rabbit polyclonalanti-SGLT1 antibody (E), followed by thespecific secondary TRITC- and FITC-conjugated antibodies. The overlay is shownin F.

723Vimentin decreases SGLT1 activity in rafts

sodium-glucose transport activity in Vim–/– cells is in favor ofthe second idea. Indeed, this treatment, which increases theviscosity of cell plasma membranes (Le Grimellec et al., 1992),could lead to a physical state of the Vim–/– membranes that isnormally achieved in wild-type cells in the presence ofvimentin.

Possible existence of different types of DRMRecent studies suggest that the plasma membrane may containdifferent kinds of microdomains, differing by theircomposition in both lipids and proteins. Ostermeyer et al.showed the existence of glycosphingolipid-depleted DRM(Ostermeyer et al., 1999), whereas Iwabuchi et al. reportedthat of caveolin-depleted microdomains (Iwabuchi et al.,1998). Moreover, several reports have shown that cholesteroldepletion does not alter the entire protein composition of rafts(Abrami et al., 1998; Furuchi and Anderson, 1998; Sheets etal., 1999), and that the changes induced by cholesterol-modulating drugs can differ from one cell type to another(Ilangumaran and Hoessli, 1998). The present work providesfurther evidence in favor of this idea and suggests thatvimentin is associated with a specific subset of membraneproteins. Indeed, the absence of vimentin affects exclusivelySGLT1 expression in DRM, but not expression of caveolin and5′-nucleotidase, two other well-known DRM-associatedproteins. Similarly, the activity of sodium-glucose transportwas reduced in cells lacking vimentin, whereas that of 5′-nucleotidase was unaffected (data not shown). Whethervimentin acts to cluster particular types of DRM in plasmamembranes is an attractive idea.

In conclusion, our study provides the first evidence that: (1)vimentin is located to DRM and this localization is essentialto maintain SGLT1 association with a subset of DRM; (2) theabsence of vimentin reduces the activity of sodium-glucosecotransport activity, through a decrease of SGLT1 localizationto rafts of BBM. Since vimentin is re-expressed in renalproximal tubular cells, in vivo, in pathologies characterized byan impairment of sodium-glucose transport activity and ofapical membrane lipidic polarity, such as ischemic or toxicrenal injury (Molitoris et al., 1989), we speculate that vimentinis a key element in the restoration of glucose transport underpathological conditions. The relevance of this hypothesisdeserves further investigation.

We are deeply grateful to K. Koumanov for cholesteroldetermination. We thank D. Paulin and A. Vandewalle for anti-cytokeratin antibodies, B. Kaissling for anti-5′-nucleotidaseantibodies, A. M. Hill for anti-vimentin antibodies and E. Solito forGAPDH probe. This work was supported in part by grants fromINSERM, Université René Descartes, Laboratoires Physiologiques,Association de la Recherche contre le Cancer (9896) and CEGETELCompany.

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