Vimentin affects localization and activity of sodium-glucose cotransporter SGLT1 in membrane rafts

Isabelle Runembert1, Guillaume Queffeulou1, Pierre Federici2, François Vrtovsnik1, Emma Colucci-Guyon3, Charles Babinet3, Pascale Briand2, Germain Trugnan4, Gérard Friedlander1 and Fabiola Terzi1,*

1 INSERM U426 and Department Physiology, Faculté de Médecine Xavier Bichat, IFR 02, Université Paris 7, Paris, France
2 INSERM U380, Institut Cochin de Génétique Moléculaire, Paris, France
3 URA 1960 CNRS, Institut Pasteur, Paris, France
4 INSERM U538, Faculté de Médecine Saint-Antoine, Paris, France

* Author for correspondence (e-mail: terzi{at}cochin.inserm.fr )

Accepted 4 November 2001


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It has been reported that vimentin, a cytoskeleton filament that is expressed only in mesenchymal cells after birth, is re-expressed in epithelial cells in vivo under pathological conditions and in vitro in primary culture. Whether vimentin re-expression is only a marker of cellular dedifferentiation or is instrumental in the maintenance of cell structure and/or function is a matter of debate. To address this issue, we used renal proximal tubular cells in primary culture from vimentin-null mice (Vim-/-) and from wild-type littermates (Vim+/+). The absence of vimentin did not affect cell morphology, proliferation and activity of hydrolases, but dramatically decreased Na-glucose cotransport activity. This phenotype was associated with a specific reduction of SGLT1 protein in the detergent-resistant membrane microdomains (DRM). In Vim+/+ cells, disruption of these microdomains by methyl-ß-cyclodextrin decreased SGLT1 protein abundance in DRM, a change that was paralleled by a decrease of Na-glucose transport activity. Importantly, we showed that vimentin is located to DRM, but it disappeared after methyl-ß-cyclodextrin treatment. In Vim-/- cells, supplementation of cholesterol with cholesterol-methyl-ß-cyclodextrin complexes completely restored Na-glucose transport activity. Interestingly, neither cholesterol content nor cholesterol metabolism changed in Vim-/- cells. Our results are consistent with the view that re-expression of vimentin in epithelial cells could be instrumental to maintain the physical state of rafts and, thus, the function of DRM-associated proteins.

Key words: Vimentin, Rafts, SGLT1


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The functional role of vimentin, a class III intermediate filament (IF) component of the cytoskeleton, represents a fascinating enigma in biology. Cytoplasmic vimentin network is widely developed in most growing cells in culture, whatever the cell-type specific IF they express in vivo (Franke et al., 1979Go). In vivo, vimentin is expressed differentially in embryonic and postnatal life: in mesenchymal and endodermic cells in embryos, but only in mesenchyma-derived cells in adults (Bachmann et al., 1983Go; Holthöfer et al., 1984Go). The paradigm of vimentin-null mice, which develop and reproduce without an obvious phenotype (Colucci-Guyon et al., 1994Go), confirms that vimentin is not required for the survival of individual cells and apparently argues against a major role of this IF, at least under normal physiological conditions. However, various cell culture models, including those derived from vimentin-null mice, suggest that vimentin could play a role in: (1) organization of other cytoplasmic structures, such as microtubules and microfilaments, and maintenance of cellular and nuclear shape; (2) cellular migration and stability in response to mechanical stress; (3) membrane traffic (Faigle et al., 2000Go) and granule secretion (Pryzwansky and Merricks, 1998Go); (4) accumulation of lipid droplet in adipocytes, metabolism of lipoprotein-derived cholesterol and recycling of sphingosine and glycosphingolipids; (5) cell cycle (vimentin is considered as an immediate-early gene); and (6) interaction with several non-structural proteins, such as protein kinase C, stress response proteins (Evans, 1998Go) or ubiquitin-related proteins (Wu et al., 1999Go). The fact that these findings have not always been confirmed indicates that the functional role of vimentin is still a matter of controversy. Indeed, it has been shown that the absence of vimentin network in cells from vimentin-null mice (Colucci-Guyon et al., 1994Go; Holwell et al., 1997Go) as well as disruption of this network by injection of anti-vimentin antibodies (Klymkowsky et al., 1989Go) or IF mimetic peptides (Goldman et al., 1999Go) could either modify or not both cell morphology and cytoskeleton organization. Moreover, it has been suggested that vimentin participates in lipid trafficking (Klymkowsky, 1995Go) although, until now, no IF-associated proteins with motor-like properties have been identified.

In normal adult kidney, vimentin is expressed in glomeruli, vessels and interstitial cells, but not in tubular cells, in contrast to its expression in developing kidney (Bachmann et al., 1983Go; Holthöfer et al., 1984Go). However, vimentin is expressed in proximal tubular cells during the recovery phase that follows ischemia or nephrotoxic tubular necrosis (Gossrau et al., 1989Go; Gröne et al., 1987Go; Nouwen et al., 1994Go; Wallin et al., 1992Go; Witzgall et al., 1994Go), in renal cell carcinoma (Dierick et al., 1991Go; Holthöfer et al., 1983Go; Waldherr and Schwechheimer, 1985Go) and in proximal tubular cells in culture (Franke et al., 1979Go; Hatzinger et al., 1988Go). In all these conditions, vimentin expression is considered as a marker of cellular dedifferentiation, but the question still remained whether vimentin is directly involved in cell recovery. In a previous study, we showed that the absence of vimentin did not affect cell proliferation, cell differentiation and structural organization of injured tubules in post-ischemic kidneys from vimentin-null mice, suggesting that vimentin is not mandatory to restoration of a differentiated morphological phenotype of proximal cells (Terzi et al., 1997Go). The purpose of the present study was to investigate whether vimentin plays a role in the maintenance of cell function. One of the principal functions of proximal tubular cells is to ensure vectorial transport of solutes. It has been shown that disruption of cytoskeleton impaired both ion and water channels and sodium-dependent transport activities (Steel and Hediger, 1998Go). In kidney, ischemia and reperfusion induce a disorganization of microtubule and microfilament networks in proximal tubules, a phenotype associated with alterations of cell polarity and protein sorting (Molitoris et al., 1989Go). These morphological changes result in a decrease of vectorial transport activities. Interestingly, rats treated with colchicine or nocodazole exhibited the same morphological and functional modifications as those observed in post-ischemic kidneys (Hansch et al., 1993Go). We therefore hypothesized that the development of vimentin network in injured or cultured epithelial cells could be instrumental in the maintenance of transport capabilities. To test this hypothesis, we investigated the consequences of vimentin inactivation on cell function by growing proximal tubular cells in primary culture obtained from mice bearing a null mutation of the vimentin gene.

We show that the absence of vimentin did not affect cell proliferation and differentiation, but decreased selectively Na-glucose transport activity. This phenotype is associated with a reduction of SGLT1 cotransporter in specialized membrane microdomains, the rafts. We propose that, in injured or cultured epithelial cells, vimentin participates in preserving proper transport functions possibly through the maintenance of membrane physical state.


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Vimentin null mice
Targeted inactivation of vimentin gene in mice have been reported previously (Colucci-Guyon et al., 1994Go). Wild-type (Vim+/+) and homozygous (Vim-/-) mice were obtained by intercrosses between Vim+/- mice.

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

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

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

Cell morphology and differentiation
Light microscopy
Cells were examined every day under an inverted microscope equipped with phase-contrast optics.

Immunocytochemistry and immunofluorescence
Cells were fixed in ice-cold ethanol 95%-acetic acid 5% (v/v) for vimentin staining, in ice-cold methanol for ZO-1 and cytokeratin staining and in 4% formaldehyde and ice-cold methanol for {alpha}-tubulin staining. Cells were first incubated overnight at 4°C with the primary antibody, and then for 1 hour at room temperature with the secondary antibody. Peroxidase activity was detected using diaminobenzidine tetrahydrochloride (DAB, Dako, France).

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

For cross-linking experiments, cells were first incubated for 1 hour at 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 1 hour at 4°C and 10 minutes at 37°C with the secondary specific FITC-and TRITC-conjugated antibodies. Cells were washed and reincubated with the secondary antibodies for 20 minutes at 37°C and finally fixed in 4% formaldehyde and ice-cold methanol.

The primary antibodies used were: (1) a monoclonal anti-vimentin antibody (Sigma), diluted 1/200 and a polyclonal anti-vimentin antibody (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 Xavier Bichat, Paris, France), diluted 1/5; (3) a mouse monoclonal anti-{alpha}-tubulin antibody, diluted 1/2000; (4) a rat monoclonal anti-ZO-1 antibody (Chemicon, Temecula, CA), diluted 1/50; (5) a polyclonal anti-SGLT1 antibody (Chemicon) diluted 1/50; (6) a monoclonal anti-5'-nucleotidase antibody (kindly provided by B. Kaissling, University of Zurich-Irchel, Switzerland), diluted 1/50. The secondary antibodies used were: (1) a sheep anti-mouse horseradish peroxidase-linked Ig antibody (Amersham, France), diluted 1/50 for both vimentin and {alpha}-tubulin staining; (2) an anti-rat IgG FITC-conjugated antibody (Sigma), diluted 1/100, for both cytokeratin and ZO-1 staining; (3) an anti-rabbit IgG FITC-conjugated antibody, an anti-mouse IgG TRITC-conjugated antibody, an anti-mouse IgG FITC-conjugated antibody and an anti-rabbit IgG TRITC-conjugated antibody for colocalization 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 phalloidin conjugated to TRITC, diluted 1/1000, for 20 minutes at 37°C.

ß-galactosidase staining
ß-galactosidase activity was determined on 0.5% glutaraldehyde fixed cells as previously described (Terzi et al., 1997Go).

Enzymatic activities
Ecto-5'-nucleotidase (EC 3.1.3.5) activity was determined using a method previously described (Siegfried et al., 1995Go). {gamma}-Glutamyltranspeptidase (EC 2.3.2.2) activity was determined using an adaptation of the technique of Orlowski and Meister (Orlowski and Meister, 1965Go). Alkaline phosphatase (EC 3.1.3.1) activity was revealed on intact 4% paraformaldehyde-fixed cells by adding a solution of nitroblue tetrazolium (300 µg/ml, Sigma) and 5-bromo-4-chloro-3-indolylphosphate (150 µg/ml, Sigma) for 4-5 hours in the dark at room temperature. Negative controls were obtained by treating cells with levamisole (1 mM, Sigma).

Cyclic AMP production
cAMP production was determined after stimulation of cells with 10-7 M hPTH (1-34; Sigma) or 10-6 M dDAVP (Sigma), as previously described (Silve et al., 1990Go).

Cell proliferation
Cells were incubated with [3H]thymidine (1 µCi/ml; Amersham) for 7 hours, and subjected to 5% trichloroacetic acid for 45 minutes at 4°C. The precipitate was dissolved in 0.2 N sodium hydroxide and the incorporation of radioactivity was measured by scintillation counting. DNA content was measured as described by Labarca and Paigen (Labarca and Paigen, 1980Go). Cell number was counted in a Malassez chamber after cell trypsinization.

Uptake studies
Uptakes of methyl-{alpha}-D-glucopyranoside (MGP) and alanine were assayed in the presence and in the absence of Na as previously described (Vrtovsnik et al., 1992Go).

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). The uptake was stopped by washing the cells with a solution containing 137 mM NaCl, 5 mM Hepes and 3 mM BaCl2. Cells were then solubilized in 0.5% Triton X-100 and radioactivity was counted by liquid scintillation.

Northern blot analysis
Total RNA was extracted from cells using the RNAzol kit (Bioprobe, Montreuil-sous-Bois, France). RNA (20 µg per lane) was separated on a 1.2% agarose-formaldehyde gel and transferred onto a nylon membrane (Zetabind, CUNO, Inc., Meridien, CT). Prehybridation, hybridization and washing were carried out according to the manufacturer's recommendations. RNA was quantified by densitometric computer analysis in a series 400 PhosphoImager (Molecular Dynamics, Inc., Sunnyvale, CA). cDNA probes were labeled by random priming (Boehringer-Mannheim, France) using [{alpha}-32P]dCTP. The following probes were used: rat SGLT1, rat SGLT2 and murine GAPDH (kindly provided by E. Solito, Imperial College School of Medicine, London, UK). To generate the SGLT1 and SGLT2 probes, the 1816-1994 bp fragment of rat SGLT1 cDNA and the 1553-1787 bp fragment of rat SGLT2 cDNA were amplified by reverse transcriptase and polymerase chain reaction, respectively.

Cellular membrane isolation
Brush border membranes (BBM)
Cells were scraped and homogenized with a Dounce homogenizer in a buffer consisting of 300 mM mannitol, 5 mM EGTA, 0.5 mM PMSF and 12 mM Tris-HCl pH 7.4. BBM were then prepared by the MgCl2 precipitation and differential centrifugation procedures, as described (Biber et al., 1981Go). The final pellet was resuspended in a buffer containing 300 mM mannitol and 16 mM Hepes, 100 mM Tris, pH 7.5. The enrichment in BBM content of the preparation was assessed by measuring alkaline phosphatase activity in the homogenate and in the final membrane preparations. A tenfold enrichment factor was routinely obtained in both Vim+/+ and Vim-/- membrane preparations.

Cell surface biotinylation
Specific cell surface biotinylation was performed as previously described (Gottardi et al., 1995Go). Then, BBM were prepared as detailed above and biotinylated proteins were recovered, separated and immunoblotted as described (Gottardi et al., 1995Go).

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

Immunoprecipitation
An 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) and incubated overnight at 4°C with a rabbit anti-caveolin polyclonal antibody (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% ß-mercaptoethanol, heated to 95°C, centrifuged, and the supernatant was subjected to SDS-PAGE.

Western blot analysis
Immunoblotting
Proteins were separated on 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Biorad, France). The membrane was first incubated overnight at 4°C with the primary antibody, then, for 2 hours at room temperature with the peroxidase-conjugated secondary antibody. Immunoreactive proteins were detected by enhanced chemiluminescence (ECL kit, Amersham). The films were scanned using a Scan-Jet 3c/ADF (Hewlett Packard) and the signals were quantified using the NIH image software.

Antibodies
The primary antibodies used were: (1) a rabbit polyclonal anti-SGLT1 antibody (Chemicon), diluted 1/2000; (2) a rabbit polyclonal anti-5'-nucleotidase antibody (a gift from B. Kaissling), diluted 1/5000; (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 antibodies used were: (1) a sheep anti-mouse horseradish peroxidase-linked Ig antibody (Amersham), diluted 1/4000 for vimentin; and (2) a donkey anti-rabbit horseradish peroxidase-linked Ig antibody (Amersham), diluted 1/2000 for SGLT1 and 1/8000 for both 5'-nucleotidase and caveolin.

Cholesterol analysis
Lipids were extracted from total cell extracts or DRMs according to the procedure of Bligh and Dyer (Bligh and Dyer, 1959Go) and separated by TLC on silicagel plates. Then, the spot of cholesterol was scraped, extracted with chloroform/methanol (2:1, v:v) and cholesterol content was assayed by gas-chromatography, as previously described (Lirbat et al., 1997Go).

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

Expression of data and statistical analysis
Results were expressed as means±s.e.m. of four to six separate experiments performed in triplicate. Differences between the experimental groups were evaluated using one-way analysis of variance, which was followed, when significant, by Student's t-test.


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All experiments were performed on renal proximal tubular cells in primary culture obtained from animals by intercrosses between Vim+/- mice.

Effect of vimentin inactivation on cell morphology, differentiation and proliferation
It has been reported that vimentin is expressed in many epithelial and non-epithelial cell types in culture, including renal proximal tubular cells (Franke et al., 1979Go; Hatzinger et al., 1988Go). Immunocytochemical analysis confirmed that, under our experimental conditions, a typical vimentin network developed after seeding and was maintained thereafter in wild-type cells. In Vim-/- cells, vimentin could not be detected, but X-gal staining revealed a ß-galactosidase activity in nuclei of these cells throughout the culture time (data not shown).

The first step of this study was to evaluate whether the absence of vimentin modified proximal tubular cell morphology, differentiation and/or proliferation. Both Vim+/+ and Vim-/- cells grew as a single monolayer, formed domes soon after they reached confluence at day 6-7, and developed epithelial cell tight junctions, as judged by ZO-1 immunostaining (data not shown). Cytokeratin, microtubular and actin filaments formed well-organized networks from nucleus to plasma membrane in both Vim+/+ and Vim-/- cells (data not shown).

Differentiation of Vim+/+ and Vim-/- cells was evaluated by measuring brush border membrane enzyme activities, as well as 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 {gamma}-glutamyltranspeptidase activities, high hPTH response and low dDAVP response. Moreover, more than 90% of cultured cells were positive for alkaline phosphatase (data not shown). No difference appeared for these parameters between the two cell types (Table 1).


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Table 1. Effect of vimentin gene inactivation on proximal tubular cell differentiation

 

The ability of proximal tubular cells to proliferate was then investigated by measuring [3H]thymidine incorporation, DNA content and cell number under basal conditions and after stimulation with 2.5% FCS. The pattern of cell proliferation was similar in Vim+/+ and Vim-/-cells, regardless the experimental conditions and the method used (data not shown).

Effect of vimentin inactivation on membrane transports
Finally, we evaluated whether the absence of vimentin affected transport activities. Na-glucose cotransport activity, assessed by Na-dependent uptake of MGP, a non-metabolized analogue of D-glucose, was significantly reduced in Vim-/- cells compared with Vim+/+ cells (Table 2). By contrast, absence of vimentin did not affect Na-dependent neutral amino acid transport: indeed, alanine uptake was comparable in Vim+/+ and Vim-/- cells (Table 2). Similarly, ouabain-sensitive rubidium uptake, which reflects the activity of Na,K-ATPase, was similar in Vim+/+ and Vim-/- cells (Table 2).


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Table 2. Effect of vimentin gene inactivation on proximal tubular cell transport activity

 

To better characterize the changes of Na-glucose cotransport activity in cells lacking vimentin, we determined the kinetic parameters of Na-dependent MGP uptake in Vim+/+ and Vim-/- cells. Analysis by the Eadie-Hofstee plot showed that the absence of vimentin affected the Vmax but not the Km value of the MGP transport system (Fig. 1). Indeed, the Vmax value was 50.4±1.8 and 24.3±1.9 nmol/mg protein/10 minutes in Vim+/+ and Vim-/- cells, respectively, (P<0.001), whereas the Km value was 453±59 and 441±123 µM in Vim+/+ and Vim-/- cells, respectively (P=NS).



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

 

Effect of vimentin inactivation on Na-glucose cotransport mRNA and protein levels
The second part of this study aimed to elucidate the molecular and cellular mechanisms whereby the absence of vimentin affected the Na-glucose cotransport activity in proximal tubular cells. Since it has been reported that vimentin could be involved in transcription and trafficking of mRNAs (Skalli and Goldman, 1991Go; Traub and Shoeman, 1994Go), we analyzed the expression 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 both SGLT1 and SGLT2 mRNAs, with SGLT2 being more abundantly expressed (Fig. 2A). At variance with whole kidneys, proximal tubular cells in primary culture from wild-type mice expressed exclusively the SGLT1 transcript, whereas that of SGLT2 was undetectable by northern blot. The same pattern of expression was observed in cells lacking vimentin (Fig. 2A).



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Fig. 2. SGLT1 and SGLT2 mRNA and SGLT1 protein expression of proximal tubular cells in primary culture from vimentin-null mice (Vim-/-) and from wild-type littermates (Vim+/+). (A) Northern blot analysis. Total RNA was extracted from Vim+/+ and Vim-/- cultured cells and from whole kidney (K) of Vim+/+ mice using RNAzol kit. cDNA probes, labeled by a random priming method, were: the rat SGLT1, the rat SGLT2 and the mouse GAPDH. Blots are representative samples from six animals and six separate cultures. (B) Western blot analysis of brush border membranes (BBM). BBM were prepared by MgCl2 precipitation and differential centrifugation procedures. Proteins were immunoblotted with a rabbit polyclonal anti-SGLT1 antibody and a rabbit polyclonal anti-5'-nucleotidase antibody. Blots are representative samples from five separate cultures. (C) Western blot analysis of biotinylated proteins extracted from BBM. For specific cell surface biotinylation experiments, cells were incubated twice consecutively with NHS-ss-biotin, BBM were prepared and the biotinylated antigens were recovered with streptavidin agarose beads. Then, proteins were immunoblotted with a rabbit polyclonal anti-SGLT1 antibody. Blots are representative samples from two separate cultures. Statistical analysis: no difference was observed between Vim+/+ and Vim-/- cells for any of the parameters.

 

We next evaluated by western blot the abundance of SGLT1 protein in brush border membranes (BBM) prepared from Vim+/+ and Vim-/- cultured cells. In wild-type cells, anti-SGLT1 antibody reacted with a specific band of about 75 kDa (Fig. 2B), which was almost abolished by preabsorbing antibodies with excess of recombinant SGLT1 (data not shown). BBM from Vim-/- cells expressed the same amounts of SGLT1 and of 5'-nucleotidase, a BBM protein used as a control, as those from Vim+/+ cells (Fig. 2B). To exclude the possibility that submembranous SGLT1 proteins could adhere non-specifically during BBM preparation, confluent monolayers were used for cell surface biotinylation experiments. As shown in Fig. 2C, similar amounts of SGLT1 were detected in plasma biotinylated membranes from Vim+/+ and Vim-/- cells.

SGLT1 is located to detergent-resistant membranes in Vim+/+ cells
Recent studies suggested that sphingolipid- and cholesterol-enriched microdomains, also named detergent-resistant membranes (DRM), within the plasma membrane of eukaryotic cells, are implicated in many important cellular processes, including segregation and oligomerization of proteins (Smart et al., 1999Go). Since Na-glucose cotransport activity was repeatedly shown to be exquisitely sensitive to changes in the physical state (fluidity/viscosity) of the plasma membranes (Friedlander et al., 1988Go; Vrtovsnik et al., 1992Go), we hypothesized that SGLT1 could localize to DRM. To test this hypothesis, cell membranes from wild-type cells were solubilized in Triton X-100, DRM were purified on a sucrose flotation gradient, and the 12 fractions obtained were analyzed by western blot. As shown in Fig. 3A, the anti-SGLT1 antibody revealed that most SGLT1 proteins (60%) were in fractions 4-7. This pattern of detection matched the one of caveolin, a marker of DRM. Moreover, immunoprecipitation experiments of Vim+/+ DRM using a rabbit polyclonal anti-caveolin antibody showed that SGLT1 co-precipitated with caveolin (data not shown), suggesting that the two proteins form a physical complex.



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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 of each 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 was used, followed by a secondary FITC-conjugated antibody. (C) SGLT1 (left panel) and 5'-nucleotidase (5'-Nu, middle panel) crosslinking in Vim+/+ cells. An overlay of SGLT1 and 5'-nucleotidase images is shown in the right panel. A rabbit polyclonal anti-SGLT1 antibody and a mouse 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.

 

An important criteria to establish the association of a new protein with rafts, is to demonstrate, using antibody crosslinking experiments, that the protein co-clusters with a well-known raft-associated protein in patches at the plasma membrane. Thus, we first investigated, by immunoflurescence and confocal microscopy, the subcellular localization of SGLT1 in Vim+/+ cells. Control samples were fixed and then incubated with the anti-SGLT1 antibody, while experimental samples were treated with the specific antibody before fixation, a procedure causing in vivo cross-linking of a protein on the cell surface. The results are shown in Fig. 3B. In control samples SGLT1 was distributed over the entire plasma membrane, whereas upon antibody crosslinking the protein was found in patch-like clusters on the surface of the cells. Then, we analyzed whether SGLT1 co-clustered in plasma membrane with 5'-nucleotidase, a well-known GPI raft-anchored protein. As presented in Fig. 3C, the overlay of SGLT1 and 5'-nucleotidase crosslinking images showed that there was a complete colocalization of SGLT1 and 5'-nucleotidase in the patches of plasma membrane.

Effect of vimentin inactivation on SGLT1 content in detergent-resistant membranes
In light of the above findings, we wondered whether the absence of vimentin reduces the abundance of SGLT1 in DRM of Vim-/- cells. Thus, we screened, via immunoblot, the Tritoninsoluble complexes (corresponding to fractions 4-7) from Vim+/+ and Vim-/- cells. The amount of SGLT1 in DRM significantly declined in cells lacking vimentin, compared with Vim+/+ cells (Fig. 3B). By contrast, the amounts of two other DRM-associated proteins, 5'-nucleotidase and caveolin, were similar in Vim-/- and Vim+/+ cells (Fig. 4).



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Fig. 4. Effect of vimentin gene inactivation on SGLT1 expression in detergent-resistant membranes (DRM) of proximal tubular cells in primary culture from vimentin-null mice (Vim-/-; hatched bars) and from wild-type littermates (Vim+/+; open bars). Cells were solubilized in Triton X-100, DRM were purified on a sucrose flotation gradient and fractions 4-7 were pooled and immunoblotted with a rabbit polyclonal 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. Data are means±s.e.m. ANOVA: Vim-/- vs Vim+/+ cells, **P<0.005.

 

Effect of DRM disruption on SGLT1 protein content and activity in Vim+/+ cells
To provide further evidence that SGLT1 localization to DRM could be mandatory to its function, we investigated whether disruption of these microdomains affects Na-glucose transport activity. The integrity of microdomains has been shown to depend on cholesterol membrane content. We therefore analyzed the effect of methyl-ß-cyclodextrin (MCD), an extracellular cholesterol acceptor which can extract cholesterol from membranes, on Vim+/+ and Vim-/- cells. Under our experimental conditions, about 45% of total cholesterol was removed by MCD from plasma membranes in Vim+/+ (Fig. 5A). As shown in Fig. 5B, treatment of Vim+/+ cells with MCD reduced glucose uptake to a value close to that observed in untreated Vim-/- cells. By contrast, MCD treatment did not affect significantly glucose uptake in Vim-/- cells, despite a similar cholesterol depletion of plasma membranes (Fig. 5). Interestingly, treatment of cells with MCD led to a significant decrease of SGLT1 protein, but not of 5'-nucleotidase and caveolin, in cholesterol-depleted Vim+/+ DRM, compared with non-depleted ones (Fig. 6A). By contrast, it affected neither the abundance of SGLT1, nor that of 5'-nucleotidase in total BBM from Vim+/+ MCD-treated cells (Fig. 6B).



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Fig. 5. Effect of cholesterol-affecting drugs on cholesterol content and sodium-glucose cotransport activity of proximal tubular cells in primary culture from vimentin-null mice (Vim-/-; hatched bars) and from wild-type littermates (Vim+/+; open bars). Cells were treated or not with methyl-ß-cyclodextrin (MCD, 10 mM at 37°C for 2 hours) or cholesterol-methyl-ß-cyclodextrin inclusion complexes (MCD-chol, 0.03% cholesterol at 37°C for 2 hours), then (A) total plasma membrane cholesterol content and (B) Na-dependent [14C]-methyl-{alpha}-D-glucopyranoside (MGP) uptake (1 mM, 10 minutes) were measured as described. Data are means±s.e.m. of four separate cultures; 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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Fig. 6. Effect of methyl-ß-cyclodextrin (MCD) on SGLT1 expression in detergent-resistant membranes (DRM) and brush border membranes (BBM) of proximal tubular cells in primary culture from wild-type mice. Cells were treated or not with MCD (10 mM at 37°C for 2 hours), then DRM (A) and BBM (B) were prepared and analyzed by western blotting using a rabbit polyclonal 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. Data are means±s.e.m. ANOVA: treated vs untreated cells: §§P<0.01.

 

Vimentin is located to detergent-resistant membranes in Vim+/+ cells
Finally, we evaluated by which mechanism vimentin could affect SGLT1 localization to DRM. In view of previous data concerning vimentin, two hypotheses could be raised: a role of vimentin in structural raft's organization or in raft's cholesterol content. We first investigated whether vimentin is located to DRM. As shown in Fig. 7A, we found that an amount of vimentin was resistant to Triton X-100 and located to DRM. Since previous work (Melkonian et al., 1999Go) has suggested that cytoskeleton elements could adhere non-specifically during DRM preparation, additional experiments were performed to exclude this possibility. We run a second gradient on the pooled 4-7 DRM fractions and showed, by western blotting, that vimentin and caveolin still co-sedimented (Fig. 7B). Moreover, we immunoprecipitated DRM from Vim+/+ cells using a rabbit polyclonal anti-caveolin antibody, and showed, by western blotting, that vimentin co-precipitated with caveolin (Fig. 7C), suggesting that vimentin and caveolin form a physical complex. Finally, we showed that MCD treatment, which engendered the same phenotype that vimentin gene inactivation, induced the disappearance of vimentin from DRM of MCD-treated Vim+/+ cells (Fig. 7D).



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Fig. 7. Vimentin protein expression in detergent-resistant membranes (DRM) of proximal tubular cells in primary culture from wild-type animals. Cell membranes were solubilized in Triton X-100, then DRM were purified on a sucrose flotation gradient. (A) An aliquot of each 1 ml gradient fraction (lanes 1-8=5-30% sucrose; lanes 9-12=40% sucrose) was collected and analyzed by western blotting using a mouse monoclonal anti-vimentin antibody. (B) Fractions 4-7 of DRM were pooled, washed, resuspended in TNE buffer containing 1% Triton X-100 and a second sucrose gradient was performed as described above, followed by western blot analysis. A mouse monoclonal antivimentin antibody and a rabbit polyclonal anti-caveolin antibody were used. (C) The pooled 4-7 DRM fractions were immunoprecipitated with a rabbit polyclonal anti-caveolin antibody (lines 1 and 2) or non-immune rabbit serum (line 3). The immunoprecipitates were analyzed by western blotting using either a rabbit polyclonal anti-caveolin antibody (left and right) or a mouse monoclonal anti-vimentin antibody (middle). (D) Effect of methyl-ß-cyclodextrin (MCD) on DRM vimentin expression. Cells were treated or not with MCD (10 mM at 37°C for 2 hours), then DRM were prepared as described above, followed by western blotting using a mouse monoclonal anti-vimentin antibody. All blots are representative samples from three separate cultures.

 

To provide further evidence in favor of a physical interaction between vimentin and rafts, we investigated, by immunofluorescence and confocal microscopy, whether vimentin colocalized to plasma membrane with a well-known raft-associated protein, such as 5'-nucleotidase. As expected, confocal microscopy showed that 5'-nucleotidase was distributed along the entire plasma membrane (Fig. 8B). Using a polyclonal anti-vimentin antibody, we observed that vimentin intermediate filaments were anchored in plasma membrane too, resulting in a thin staining of the cell border (Fig. 8A). As shown in Fig. 8C, overlay of vimentin and 5'-nucleotidase images showed that there was a significant colocalization of the two proteins at the plasma membrane. Similarly, the double staining of SGLT1 (Fig. 8E) and vimentin (Fig. 8D) showed that the two proteins colocalized to the plasma membrane (Fig. 8F).



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Fig. 8. Vimentin protein localization in proximal tubular cells in primary culture from wild-type animals. (A-C) Cells were fixed in 4% formaldehyde and ice-cold methanol, then incubated with a rabbit polyclonal anti-vimentin antibody (A) and a mouse monoclonal anti-5'-nucleotidase antibody (B), followed by the specific secondary TRITC-and FITC-conjugated antibodies. The overlay is shown in panel C. (D-F) Cells were fixed in 4% formaldehyde and ice-cold methanol, then incubated with a mouse monoclonal antivimentin anti-vimentin antibody (D) and a rabbit polyclonal anti-SGLT1 antibody (E), followed by the specific secondary TRITC- and FITC-conjugated antibodies. The overlay is shown in F.

 

Second, we analyzed the consequences of vimentin gene inactivation on cholesterol level and metabolism. Neither total cellular, nor DRM cholesterol content was affected by the absence of vimentin. Indeed, total cell cholesterol was 9.4±2.25 and 8.2±1.71 nmol/20 µg protein, whereas DRM cholesterol was 102±19 and 99±29 nmol/20 µg protein in Vim+/+ and Vim-/- cells, respectively. Moreover, cholesterol metabolism, as judged by [14C]mevalonate incorporation, was similar in Vim+/+ and Vim-/- cells (51405±8803 vs 55514±8061 cpm/mg protein).

Although cholesterol content was not decreased in Vim-/- cells compared with Vim+/+ cells, we wondered whether loading Vim-/- cells with an excess of cholesterol might restore the Na-glucose cotransport activity through a stiffening of plasma 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 was unaffected by this treatment.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we provide evidence that the absence of vimentin affected the activity of sodium-glucose cotransport in cultured renal proximal tubular cells. We show that this effect was associated with a parallel decrease of this transporter in detergent-resistant domains of brush border membranes. We finally demonstrated that vimentin localized to the rafts, too. These results suggest that vimentin plays a key role in the function of SGLT1, possibly by maintaining the membrane structure of rafts.

The effect of vimentin on glucose transport activity is specific
Our findings showed that the absence of vimentin has a specific impact on sodium-glucose transporter. Indeed, cultured cells originating from Vim-/- animals did not differ from those cultured from wild-type littermates for any other parameter. Particularly, proliferation or differentiation, as judged from the activity of brush border enzymes and of apical or basolateral transporters (sodium-alanine cotransport, Na,K-ATPase), were similar in Vim+/+ and Vim-/- cells. These results are in agreement with those reported previously in vivo showing that, after renal ischemia-induced tubular necrosis, regeneration and differentiation were not impaired in mice lacking vimentin (Terzi et al., 1997Go). It is noteworthy that, in both in vitro and in vivo models, the absence of vimentin did not result in overexpression or massive rearrangement of other intermediate filaments such as cytokeratins (Colucci-Guyon et al., 1994Go; Holwell et al., 1997Go).

SGLT1, but not SGLT2, is expressed in cultured proximal tubular cells
In the cultured renal proximal tubular cells used in the present study, sodium-glucose cotransport activity was ensured exclusively by SGLT1, as showed by the kinetic parameters of MGP uptake with an apparent Km value close to 450 µM, and the detection of SGLT1 mRNA but not of SGLT2 transcript. This pattern contrasts with the abundance of SGLT2 mRNA in mouse whole kidney and raises the possibility that, under our culture conditions, SGLT2 is downregulated. Alternatively, these results could suggest that cells originated predominantly from the S3 segment of the proximal tubule, in which SGLT1 is predominantly expressed. However, the activity of brush border enzymes in the cultured cells argues against this possibility. Nevertheless, it is important to point out that the exclusive expression of SGLT1 mRNA was similar in Vim-/- and Vim+/+ cultured cells, showing that vimentin does not affect transcription of SGLT1, as suggested for other proteins by previous work (Skalli and Goldman, 1991Go; Traub and Shoeman, 1994Go).

SGLT1 is located to DRM
Our study provides unequivocal evidence that SGLT1 is located to detergent-resistant membrane domains or rafts. This is, to our knowledge, the first report of the presence of a sodium-dependent cotransport system in this cholesterol-and sphingomyelin-enriched domain of the apical plasma membrane. It is noteworthy that fractions enriched in SGLT1 are also enriched in caveolin, a protein well known to localize to 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 plasma membrane. This is an important criterion to establish the association of a new protein with rafts. The observation that SGLT1 is localized in rafts suggests that optimal activity of this cotransporter would be achieved when its lipidic microenvironment has a low fluidity. Several data are in agreement with this hypothesis. First, in the present work, cholesterol depletion by methyl-ß-cyclodextrin decreased sodium-glucose cotransport activity in Vim+/+ cells. Second, previous reports (Friedlander and Amiel, 1989Go) have shown that fluidification of plasma membranes of renal epithelial cells by aromatic alcohols dramatically decreased sodium-glucose cotransport activity. Finally, exposure of proximal cells to sphingomyelinase, a treatment that resulted in depletion of plasma membranes in both sphingomyelin and cholesterol, gave similar results (Vrtovsnik et al., 1992Go). Interestingly, repletion of sphingomyelinase-treated cells with cholesterol-enriched liposomes restored sodium-glucose cotransport activity (Vrtovsnik et al., 1992Go). This observation corroborates the present studies showing that enrichment of DRM by cholesterol-cyclodextrin complexes restored glucose transport in Vim-/- cells. Taken together, these results demonstrate that membrane-bound cholesterol is an important modulator of glucose transport.

The mechanisms by which localization to DRM allows SGLT1 to function remain to be elucidated. Several reports (Giudicelli et al., 1998Go; Stevens et al., 1990Go; Takahashi et al., 1985Go; Turner and Kempner, 1982Go) have suggested that functional SGLT1 is an oligomeric protein, resulting by homodimerization of two identical subunits, or heterodimerization between a SGLT1 monomer and RS1, a regulatory protein with a molecular weight very close to that of SGLT1 (Veyhl et al., 1993Go). Since DRM were reported to act as concentration platforms at the cell surface allowing proteins to oligomerize (Abrami et al., 1998Go) or to form supramolecular signalling complexes (Field et al., 1997Go; Green et al., 1999Go), it can be proposed that this property extends to sodium-glucose cotransporter and that dimers, whatever their composition, are located preferentially in rafts. The experimental condition used to prepare DRM together with the ability of the transporter to dissociate under chemical and physical forces because of non-covalent interactions between units accounts for the observation that only one 75 kDa band was apparent in DRM preparations.

Interaction of vimentin with DRM components
Previous reports (Deckert et al., 1996Go; Lisanti et al., 1994Go; Mallard et al., 1998Go; Moran and Miceli, 1998Go; Oliferenko et al., 1999Go) have shown that cytoskeleton elements participate to the constitution of rafts. Microtubules were reported to play a role in the trafficking of lipids and proteins of rafts to the apical domain of the plasma membrane. Actin was found to be associated with rafts and to play an important role in clustering of raft proteins (Fujimoto et al., 1995Go; Oliferenko et al., 1999Go). Our study provides the first evidence that intermediate filaments are associated with DRM. Binding of vimentin to plasma membrane has been previously shown and confirmed by immufluorescence analysis in the present study, where we showed a colocalization at the cell border of vimentin with two well-known membrane-associated proteins, the 5'-nucleotidase and SGLT1. Vimentin network can interact with membrane-adhesion proteins, such as spectrin, ankyrin, plectin, SNAP23 or PLIC and/or directly with the lipid bilayer (Faigle et al., 2000Go; Georgatos and Marchesi, 1985Go; Mangeat and Burridge, 1984Go; Perides et al., 1986Go; Seifert et al., 1992Go; Wu et al., 1999Go). It has been suggested that cytoskeleton elements, such as actin, adhere non-specifically during DRM preparations (Melkonian et al., 1999Go). However, data from the present work argue against this idea. In fact, we showed that: (1) in pull-down experiments, vimentin co-immunoprecipitated with caveolin; (2) vimentin and caveolin still co-sedimented when DRM fractions underwent a double sucrose gradient; and (3) vimentin disappeared from DRM after treatment of cells with methyl-ß-cyclodextrin, a procedure known to disorganize these domains. Taken together these results strongly suggest that vimentin is closely associated, or located to DRM and forms a physical complex with caveolin.

With regard to the mechanism that underlies the effect of vimentin on rafts, at least two possibilities can be put forward. First, vimentin that has been shown to modify glycosphingolipid (Gillard et al., 1998Go) and cholesterol (Sarria et al., 1992Go) metabolism could directly affect the lipidic composition of DRM, which might influence protein localization to rafts (chemical hypothesis). Alternatively, as previously suggested for actin (Oliferenko et al., 1999Go), vimentin could maintain the structure of rafts, limiting the extent of the lateral mobility of raft-associated proteins (physical hypothesis). The observation that total cell and DRM cholesterol contents, as well as cholesterol metabolism were similar in Vim+/+ and Vim-/- cells argues strongly against the first hypothesis. By contrast, the fact that increased membrane cholesterol content to supra-physiological value restored the sodium-glucose transport activity in Vim-/- cells is in favor of the second idea. Indeed, this treatment, which increases the viscosity of cell plasma membranes (Le Grimellec et al., 1992Go), could lead to a physical state of the Vim-/- membranes that is normally achieved in wild-type cells in the presence of vimentin.

Possible existence of different types of DRM
Recent studies suggest that the plasma membrane may contain different kinds of microdomains, differing by their composition in both lipids and proteins. Ostermeyer et al. showed the existence of glycosphingolipid-depleted DRM (Ostermeyer et al., 1999Go), whereas Iwabuchi et al. reported that of caveolin-depleted microdomains (Iwabuchi et al., 1998Go). Moreover, several reports have shown that cholesterol depletion does not alter the entire protein composition of rafts (Abrami et al., 1998Go; Furuchi and Anderson, 1998Go; Sheets et al., 1999Go), and that the changes induced by cholesterol-modulating drugs can differ from one cell type to another (Ilangumaran and Hoessli, 1998Go). The present work provides further evidence in favor of this idea and suggests that vimentin is associated with a specific subset of membrane proteins. Indeed, the absence of vimentin affects exclusively SGLT1 expression in DRM, but not expression of caveolin and 5'-nucleotidase, two other well-known DRM-associated proteins. Similarly, the activity of sodium-glucose transport was reduced in cells lacking vimentin, whereas that of 5'-nucleotidase was unaffected (data not shown). Whether vimentin acts to cluster particular types of DRM in plasma membranes is an attractive idea.

In conclusion, our study provides the first evidence that: (1) vimentin is located to DRM and this localization is essential to maintain SGLT1 association with a subset of DRM; (2) the absence of vimentin reduces the activity of sodium-glucose cotransport activity, through a decrease of SGLT1 localization to rafts of BBM. Since vimentin is re-expressed in renal proximal tubular cells, in vivo, in pathologies characterized by an impairment of sodium-glucose transport activity and of apical membrane lipidic polarity, such as ischemic or toxic renal injury (Molitoris et al., 1989Go), we speculate that vimentin is a key element in the restoration of glucose transport under pathological conditions. The relevance of this hypothesis deserves further investigation.


    Acknowledgments
 
We are deeply grateful to K. Koumanov for cholesterol determination. We thank D. Paulin and A. Vandewalle for anti-cytokeratin antibodies, B. Kaissling for anti-5'-nucleotidase antibodies, A. M. Hill for anti-vimentin antibodies and E. Solito for GAPDH probe. This work was supported in part by grants from INSERM, Université René Descartes, Laboratoires Physiologiques, Association de la Recherche contre le Cancer (9896) and CEGETEL Company.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Abrami, L., Fivaz, M., Glauser, P. E., Parton, R. G. and van der Goot, F. G. (1998). A pore-forming toxin interacts with a GPI-anchored protein and causes vacuolation of the endoplasmic reticulum. J. Cell Biol. 140,525 -540.[Abstract/Free Full Text]

Bachmann, S., Kriz, W., Kuhn, C. and Franke, W. W. (1983). Differentiation of cell types in the mammalian kidney in immunofluorescence microscopy using antibodies to intermediate filament proteins and desmoplakins. Histochemistry 77,365 -394.[Medline]

Biber, J., Stieger, B., Haase, W. and Murer, H. (1981). A high yield preparation for rat kidney brush border membranes. Different behaviour of lysosomal markers. Biochim. Biophys. Acta. 647,169 -176.[Medline]

Bligh, E. and Dyer, W. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37,911 -917.

Colucci-Guyon, E., Portier, M. M., Dunia, I., Paulin, D., Pournin, S. and Babinet, C. (1994). Mice lacking vimentin develop and reproduce without an obvious phenotype. Cell 79,679 -694.[Medline]

Deckert, M., Ticchioni, M. and Bernard, A. (1996). Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases. J. Cell Biol. 133,791 -799.[Abstract]

Dierick, A. M., Praet, M., Verbeeck, P., Robyns, C. and Oosterlinck, W. (1991). Vimentin expression of renal cell carcinoma in relation of DNA content and histological grading: a combined light microscopy, immunocytochemical, and cytophotometrical analysis. Histopathology 18,315 -322.[Medline]

Evans, R. M. (1998). Vimentin: the conundrum of the intermediate filament gene family. BioEssays 20, 79-86.[Medline]

Faigle, W., Colucci-Guyon, E., Louvard, D., Amigorena, S. and Galli, T. (2000). Vimentin filaments in fibroblasts are a reservoir for SNAP23, a component of the membrane fusion machinery. Mol. Biol. Cell 11,3485 -3494.[Abstract/Free Full Text]

Field, A. K., Holowka, D. and Baird, B. (1997). Compartmentalized activation of the high affinity immunoglobulin {epsilon} receptor within membrane domains. J. Biol. Chem. 272,4276 -4280.[Abstract/Free Full Text]

Franke, W. W., Schmid, E., Winter, S., Osborn, M. and Weber, K. (1979). Widespread occurence of intermediate-sized filaments of vimentin-type in cultured cells from diverse vertebrates. Exp. Cell Res. 123,25 -46.[Medline]

Friedlander, G. and Amiel, C. (1989). Protein kinase C activation has dissimilar effects on sodium-coupled uptakes in renal proximal tubular cells in primary culture. J. Biol. Chem. 264,3935 -3941.[Abstract/Free Full Text]

Friedlander, G., Shahedi, M., Le Grimellec, C. and Amiel, C. (1988). Increase in membrane fluidity and opening of tight junctions have similar effects on sodium-coupled uptakes in renal epithelial cells. J. Biol. Chem. 263,11183 -11188.[Abstract/Free Full Text]

Fujimoto, T., Myawaki, A. and Mikoshiba, K. (1995). Inositol 1,4,5-triphosphate receptor-like protein in plasmalemmal caveola is linked to actin filaments. J. Cell Sci. 108,7 -15.[Abstract/Free Full Text]

Furuchi, T. and Anderson, R. G. W. (1998). Cholesterol depletion of caveola causes hyperactivation of extracellular signal-related kinase (ERK). J. Biol. Chem. 273,21099 -21104.[Abstract/Free Full Text]

Georgatos, S. D. and Marchesi, V. T. (1985). The binding of vimentin to human erythrocyte membranes: a model system for the study of intermediate filament-membrane interactions. J. Cell Biol. 100,1955 -1961.[Abstract]

Gillard, B. K., Clement, R., Colucci-Guyon, E., Babinet, C., Schwarzmann, G., Taki, T., Kasama, T. and Marcus, D. M. (1998). Decreased synthesis of glycosphingolipids in cells lacking vimentin intermediate filaments. Exp. Cell Res. 242,561 -572.[Medline]

Giudicelli, J., Bertrand, M. F., Bilski, S., Tran, T. T. and Poiree, J. C. (1998). Effect of cross-linkers on the structure and function of pig-renal sodium-glucose cotransporters after papain treatment. Biochem. J. 330,733 -736.[Medline]

Goldman, R. D., Chou, Y. H., Prahlad, V. and Yoon, M. (1999). Intermediate filaments: dynamic processes regulating their assembly, motility, and interactions with other cytoskeletal systems. FASEB J. 13,261 -265.[Free Full Text]

Gossrau, R., Günther, T. and Graf, R. (1989). Enhancement of gentamicininduced nephrotoxicity by Mg deficiency in non-pregnant rats. Histochemistry 90,489 -496.[Medline]

Gottardi, C. J., Dunbar, L. A. and Caplan, M. J. (1995). Biotinylation and assessment of membrane polarity: caveats and methodological concerns. Am. J. Physiol. 268,F285 -F295.[Abstract/Free Full Text]

Green, J. M., Zhelesnyak, A., Chung, J., Lindberg, F. P., Sarfati, M., Frazier, W. A. and Brown, E. J. (1999). Role of cholesterol in formation and function of signiling complex involving {alpha}vß3, integrin-associated protein (CD47), and heterotrimeric G proteins. J. Cell Biol. 146,673 -682.[Abstract/Free Full Text]

Gröne, H. J., Weber, K., Gröne, E., Helmchen, U. and Osborn, M. (1987). Coexpression of keratin and vimentin in damaged and regenerating tubular epithelia of the kidney. Am. J. Pathol. 129,1 -8.[Abstract]

Hansch, E., Forgo, J., Murer, H. and Biber, J. (1993). Role of microtubules in the adaptative response to low phosphate of Na/Pi cotransport in opossum kidney cells. Pflüg Arch. Eur. J. Phys. 442,516 -522.

Hatzinger, P. B., Chen, Q., Dong, L. and Stevens, J. L. (1988). Alterations in intermediate filament proteins in rat kidney proximal tubule epithelial cells. Biochem. Biophys. Res. Commun. 157,1316 -1322.[Medline]

Holthöfer, H., Miettinen, A., Lehto, V. P., Lehtonen, E. and Virtanen, I. (1984). Expression of vimentin and cytokeratin types of intermediate filament proteins in developing and adult human kidneys. Lab. Invest. 50,552 -559.[Medline]

Holthöfer, H., Miettinen, A., Paasivuo, R., Lehto, V. P., Linder, E., Alfthan, O. and Virtanen, I. (1983). Cellular origin and differentiation of renal carcinomas. Lab. Invest. 49,317 -326.[Medline]

Holwell, T. A., Schweitzer, S. C. and Evans, R. M. (1997). Tetracycline regulated expression of vimentin in fibroblasts derived from vimentin null mice. J. Cell. Sci. 110,1947 -1956.[Abstract/Free Full Text]

Ilangumaran, S. and Hoessli, D. C. (1998). Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane. Biochem. J. 335,433 -440.[Medline]

Iwabuchi, K., Handa, K. and Hakomori, S. (1998). Separation of `glycosphingolipid signaling domain' from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling. J. Biol. Chem. 273,33766 -33773.[Abstract/Free Full Text]

Klymkowsky, M. W. (1995). Intermediate filaments: new proteins, some answers, more questions. Curr. Opin. Cell Biol. 7,46 -54.[Medline]

Klymkowsky, M. W., Bachant, J. B., and Domingo, A. (1989). Functions of intermediate filaments. Cell Motil. Cytoskeleton 14,309 -331.[Medline]

Labarca, C. and Paigen, K. (1980). A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102,344 -352.[Medline]

Le Grimellec, C., Friedlander, G., El Yandouzi, E. H., Zlatkine, P. and Giocondi, M.-C. (1992). Membrane fluidity and transport properties in epithelia. Kidney Int. 42,825 -836.[Medline]

Lirbat, B., Wolf, C., Chevy, F., Citadelle, D., Bereziat, G. and Roux, C. (1997). Normal and inhibited cholesterol synthesis in the cultured rat embryo. J. Lipid Res. 38, 22-34.[Abstract]

Lisanti, M. P., Scherer, P. E., Vidugiriene, J., Tang, Z. L., Hermanowski-Vosatka, A., Tu, Y. H., Cook, R. F. and Sargiacomo, M. (1994). Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J. Cell Biol. 126,111 -126.[Abstract]

Mallard, F., Antony, C., Tenza, D., Salamero, J., Goud, B. and Johannes, L. (1998). Direct pathway from early/recycling endosomes to the golgi apparatus revealed through the study of Shiga toxin B-fragment transport. J. Cell Biol. 143,973 -990.[Abstract/Free Full Text]

Mangeat, P. H. and Burridge, K. (1984). Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific antibodies: relation to cytoskeletal structures. J. Cell Biol. 98,1363 -1377.[Abstract]

Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G. and Brown, D. A. (1999). Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J. Biol. Chem. 274,3910 -3917.[Abstract/Free Full Text]

Menaa, C., Vrtovsnik, F., Friedlander, G., Corvol, M. and Garabedian, M. (1995). Insulin-like growth factor I, a unique calcium-dependent stimulator of 1,25-dihydroxyvitamin D3 production. Studies in cultured mouse kidney cells. J. Biol. Chem. 270,25461 -25467.[Abstract/Free Full Text]

Molitoris, B. A., Falk, A. S. and Dahl, R. H. (1989). Ischemia-induced loss of epithelial polarity. J. Clin. Invest. 84,1334 -1339.[Medline]

Moran, M. and Miceli, M. C. (1998). Engagement of GPI-linked CD48 contributes to TCR signals and cytoskeletal reorganization: a role for lipid rafts in T cell activation. Immunity 9, 787-796.[Medline]

Nouwen, E. J., Verstrepen, W. A., Buyssens, N., Zhu, M. Q. and De Broe, M. E. (1994). Hyperplasia, hypertrophy, and phenotypic alterations in the distal nephron after acute proximal tubular injury in the rat. Lab. Invest. 70,479 -493.[Medline]

Oliferenko, S., Paiha, K., Harder, T., Gerke, V., Schwarzler, C., Schwarz, H., Beug, Gunthert, U. and Huber, L. A. (1999). Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton. J. Cell Biol. 146,843 -854.[Abstract/Free Full Text]

Orlowski, M. and Meister, A. (1965). Isolation of {gamma}-glutamyl transpeptidase from hog dog. J. Biol. Chem. 240,338 -347.[Free Full Text]

Ostermeyer, A. G., Beckrich, B. T., Ivarson, K. A., Grove, K. E. and Brown, D. A. (1999). Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells. methyl-ß-cyclodextrin does not affect cell surface transport of a GPI-anchored protein. J. Biol. Chem. 274,34459 -34466.[Abstract/Free Full Text]

Perides, G., Scherbarth, A., Kuhn, S. and Traub, P. (1986). An electron microscopic study of the interaction in vitro of vimentin intermediate filaments with vesicles prepared from Ehrlich ascites tumor cell lipids. Eur. J. Cell Biol. 41,313 -325.[Medline]

Pryzwansky, K. B. and Merricks, E. P. (1998). Chemotactic peptide-induced changes of intermediate filament organization in neutrophils during granule secretion: role of cyclic guanosine monophosphate. Mol. Biol. Cell 9,2933 -2947.[Abstract/Free Full Text]

Sarria, A. J., Panini, S. R. and Evans, R. M. (1992). A functional role for vimentin intermediate filaments in the metabolism of lipoprotein-derived cholesterol in human SW-13 cells. J. Biol. Chem. 267,19455 -19463.[Abstract/Free Full Text]

Seifert, G. J., Lawson, D. and Wiche, G. (1992). Immunolocalization of the intermediate filaments-associated protein plectin at focal contacts and actine stress fibers. Eur. J. Cell Biol. 59,138 -147.[Medline]

Sheets, E. D., Holowka, D. and Baird, B. (1999). Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of Fc{epsilon}RI and their association with dertergent-resistant membranes. J. Cell Biol. 145,877 -887.[Abstract/Free Full Text]

Siegfried, G., Vrtovsnik, F., Prie, D., Amiel, C. and Friedlander, G. (1995). Parathyroid hormone stimulates ecto-5'-nucleotidase activity in renal epithelial cells: role of protein kinase-C. Endocrinology 136,1267 -1275.[Abstract]

Silve, C., Suarez, F., el Hessni, A., Loiseau, A., Graulet, A. M. and Gueris, J. (1990). The resistance to parathyroid hormone of fibroblasts from some patients with type Ib pseudohypoparathyroidism is reversible with dexamethasone. J. Clin. Endocrinol. Metab. 71,631 -638.[Abstract]

Skalli, O. and Goldman, R. D. (1991). Recent insights into assembly, dynamics, and function of intermediate filament networks. Cell Motil. Cytoskeleton 19, 67-79.[Medline]

Smart, E. J., Graf, G. A., McNiven, M. A., Sessa, W. C., Engelman, J. A., Scherer, P. E., Okamoto, T. and Lisanti, M. P. (1999). Caveolins, liquid-ordered domains, and signal transduction. Mol. Cell Biol. 19,7289 -7304.[Free Full Text]

Steel, A. and Hediger, M. A. (1998). The molecular physiology of sodium-and proton-coupled solute transporters. News Physiol. Sci. 13,123 -131.[Abstract/Free Full Text]

Stevens, B. R., Fernandez, A., Hirayama, B. and Wright, E. M. (1990). Intestinal brush border membrane Na/glucose cotranspoter functions in situ as a homotetramer. Proc. Natl. Acad. Sci. USA 87,1456 -1460.[Abstract]

Takahashi, M., Malathi, P., Preiser, H. and Jung, C. Y. (1985). Radiation inactivation studies on the rabbit kidney sodium-dependent glucose transporter. J. Biol. Chem. 260,10551 -10556.[Abstract/Free Full Text]

Terzi, F., Maunoury, R., Colucci-Guyon, E., Babinet, C., Federici, P., Briand, P. and Friedlander, G. (1997). Normal tubular regeneration and differentiation of the post-ischemic kidney in mice lacking vimentin. Am. J. Pathol. 150,1361 -1371.[Abstract]

Traub, P. and Shoeman, R. L. (1994). Intermediate filament and related proteins: potential activators of nucleosomes during transcription, initiation and elongation. Bioessays 16,349 -355.[Medline]

Turner, R. J. and Kempner, E. S. (1982). Radiation inactivation studies of the renal brush-border membrane phlorizin-binding protein. J. Biol. Chem. 257,10794 -10797.[Abstract]

Veyhl, M., Spangenberg, J., Puschel, B., Poppe, R., Dekel, C., Fritzsch, G., Haase, W. and Koepsell, H. (1993). Cloning of a membrane-associated protein which modifies activity and properties of the Na(+)-D-glucose cotransporter. J. Biol. Chem. 268,25041 -25053.[Abstract/Free Full Text]

Vrtovsnik, F., El Yandouzi, E. H., Le Grimellec, C. and Friedlander, G. (1992). Sphingomyelin and cholesterol modulate sodium coupled uptakes in proximal tubular cells. Kidney Int. 41,983 -991.[Medline]

Waldherr, R. and Schwechheimer, K. (1985). Co-expression of cytokeratin and vimentin intermediate-sized filaments in renal cell carcinomas. Virchows Arch. (Pathol. Anat.) 408, 15-27.

Wallin, A., Zhang, G., Jones, T. W., Jaken, S. and Stevens, J. L. (1992). Mechanism of nephrogenic repair response: studies on proliferation and vimentin expression after 35S-1,2-di-chlorovinyl-L-cysteine nephrotoxicity in vivo and in cultured proximal tubule epithelial cells. Lab. Invest. 66,474 -484.[Medline]

Witzgall, R., Brown, D., Schwarz, C. and Bonventre, J. V. (1994). Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells. J. Clin. Invest. 93,2175 -2188.[Medline]

Wu, A. L., Wang, J., Zheleznyak, A. and Brown, E. J. (1999). Ubiquitinrelated proteins regulate interaction of vimentin intermediate filaments with the plasma membrane. Mol. Cell 4,619 -625.[Medline]


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