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Identification of caveolae and caveolin in C6 glioma cells
Int. J. Devl Neuroscience, Vol. 17, No. 7, pp. 705±714, 1999 # 1999 ISDN. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0736-5748/99 $20.00 + 0.00
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IDENTIFICATION OF CAVEOLAE AND CAVEOLIN IN C6 GLIOMA CELLS W.I. SILVA,$* H.M. MALDONADO,% M.P. LISANTI,} J. DEVELLIS,} G. CHOMPREÂ,$ N. MAYOL,% M. ORTIZ,$ G. VELAÂZQUEZ,6 A. MALDONADO6 and J. MONTALVO$ $Department of Physiology and Biophysics, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico; %Department of Pharmacology, Universidad Central del Caribe, School of Medicine, Bayamon, Puerto Rico; }Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA; kMental Retardation Research Center, University of California, Los Angeles, CA, USA; Department of Biology, University of Puerto Rico, Cayey, Puerto Rico (Received 4 December 1998; received in revised form 26 April 1999; accepted 3 May 1999) AbstractÐCaveolae (CAV) constitute a novel subcellular transport vesicle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling events. One of the principal components of CAV are caveolin protein isoforms. Here, we have undertaken the immunochemical identi®cation of CAV and the known caveolin isoforms (1a, 1b, 2 and 3) in cultured rat C6 glioma cells. Immunoblot analysis revealed that particulate fractions from rat C6 glioma cells express caveolin-1 and caveolin-2. The relative detergent-insolubility of these caveolin isoforms was also determined by Western blot analysis. Indirect immuno¯uorescence analysis with caveolin-1 and -2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized by intensely ¯uorescent puncta throughout the cytoplasm and diuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin-1 and -2 immunoreactivity seen in Western blots and immunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin-enriched membrane complexes using two dierent methods. Light-density, Triton X-100-insoluble caveolin-1- and caveolin2-enriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5±40% discontinuous sucrose-density gradients. Similar fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. These results further support the localization of CAV and caveolins in glial cells. In addition, they demonstrate that cultured C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in glial cells and the brain. # 1999 ISDN. Published by Elsevier Science Ltd. All rights reserved. Key words: C6 glial cells, Caveolae, Caveolin, Plasmalemmal vesicles.
INTRODUCTION Caveolae (CAV) are ¯ask-shaped, non-clathrin-coated invaginations of the plasma membrane. CAV were ®rst identi®ed as an endocytic compartment in endothelial cells, where they appear to move molecules across the cell by transcytosis.21 More recently, they have been found to be sites where small molecules are concentrated and internalized by a process called potocytosis.1±3 A growing body of biochemical and morphological evidence indicates that a variety of molecules known to function directly or indirectly in signal transduction are enriched in caveolae.13,15,17,19 Based on these observations, a ``caveolae signaling hypothesis'' has been forwarded,13±15,19 which states that caveolar localization of signaling molecules could provide a compartmental basis for a subset of signaling events. Sequestration of signaling molecules *To whom correspondence should be addressed. Tel.: +1-787-758-2525/1608; Fax: +1-787-753-0120; E-mail: [email protected] Abbreviations: CAV, caveolae, Tris±HCl, tris(hydroxymethyl)aminomethane hydrochloride, Mes, 4-morpholineethanesulfonic acid, MBS, Mes-buered saline, Hepes, N-[2-hydroxyethyl]piperazine-N-[2-ethane sulfonic acid]10, DTT, 1,4dithiothreithol, PBS, phosphate-buered saline, BSA, bovine serum albumin, EDTA, ethylenediaminetetraacetic acid, mAb, monoclonal antibody, pAb, polyclonal antibody, MeOH, methanol. 705
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within caveolae could explain the extensive ``cross-talk'' that is observed between dierent signaling pathways or modules.1±3,13±15,19 The multifaceted structural and functional properties of CAV can be attributed largely to one of its principal protein components, caveolin. Caveolin is a 21-kDa membrane protein, originally described as a primary v-src tyrosine kinase substrate, and referred to as VIP21.7,12,22 It is now known that caveolin 1 is the ®rst member of a multi-gene family. At least two caveolin-1 isoforms (alpha and beta, 24 and 21 kDa, respectively) are known, and their expression seems to be dependent on the availability of two distinct translational initiation sites.16,25 Caveolin appears to function as a multivalent scaolding protein that interacts with dierent classes of signal transducers.19 These caveolin-associated proteins include lipidanchored molecules such as heterotrimeric G-protein subunits, H-Ras, eNOS, Src-family tyrosine kinases and signaling receptors.19 More recently, additional caveolin variants have been identi®ed and cloned, these have been referred to as caveolin-2 and caveolin-3.24,28,30 The expression of caveolin-3 is muscle-speci®c and may play a role in the pathogenesis of muscular dystrophy.29,30 Although caveolae are present in most cells, they are most abundant in terminallydierentiated cell types: endothelia, adipocytes, muscle cells and type I pneumocytes. In contrast, caveolin and caveolae are reduced or absent in ®broblasts transformed by certain activated oncogenes, such as v-Abl or H-Ras (G12V).11 Since caveolin's original characterization, the identi®cation of caveolin and CAV in the brain had been elusive.1,4,8,9,13,15,20,26 Yet, analysis of detergent-resistant membrane complexes from 1-day-old chick brains revealed the presence of a caveolin-enriched membrane subpopulation.9 These caveolin-enriched membrane complexes were particularly prevalent in the chick cerebellum.9 Even more recently, caveolin-1 and caveolin-2 were found to be expressed in dierentiated PC12 cells and in dorsal root ganglion neurons.6 Still, thorough identi®cation of caveolae and caveolins in brain tissue requires analysis of glial cells and glial cell model systems in addition to neurons and neuronal cellular model systems. In this regard Cameron et al.5 were able to demonstrate the presence of CAV, caveolin-1a and apparently novel caveolin-1 molecular variants in primary cultures of type 1 astrocytes from rat brain. Indirect immuno¯uorescence analysis with a caveolin-1 antibody also demonstrated the labeling of process-bearing astrocytes and oligodendrocytes.5 Thus, glial cells express a caveolin-caveolae compartment similar to the one expressed in peripheral tissues and cells. The study in primary culture of glial cells5 evaluated the expression of the caveolin-1 isoform. Furthermore, Ikezu et al.,10 detected expression of the dierent caveolin isoforms in primary cultures of rat astrocytes. A more thorough analysis of expression of the dierent caveolin isoforms in brain tissue would bene®t from studies on established cultured brain cell lines. A valuable cultured cell line model system would represent a most valuable experimental system for the analysis of the role and functions of CAV and caveolins in brain tissue, particularly glial cells. Consequently in this study we evaluated the expression of the dierent caveolin variants in cultured rat C6 glioma cells by a combination of experimental approaches. Here we corroborate the expression of CAV and caveolin-1 in glial cells, by documenting their expression in cultured rat C6 glioma cells. In addition using immunoblot analysis and indirect immuno¯uorescence analysis we demonstrate the expression of caveolin-2 in cultured rat C6 glioma cells. In this way we establish the validity of cultured C6 glial cells as a model system for the assessment of the emerging physiological relevance of this novel subcellular transport and cell signaling compartment. EXPERIMENTAL PROCEDURES Materials Cell culture reagents were obtained from Sigma, Co. (St. Louis, MO): glutamine, penicillin, streptomycin, fungizone, EDTA (ethylenediaminetetraacetic acid), Ca2+, Mg2+-free Hank's F10 balanced salt solution and fetal bovine serum (FBS). Immunoblotting and cellular fractionation reagents were also from Sigma, Co. (St. Louis, MO): Hepes buer, DTT, MgCl2, phenylmethyl-sulfonyl-¯uoride (PMSF), leupeptin, antipain, bestatin, chymostatin, pestatin A,
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Sigmacote, Triton X-100, sucrose, bovine serum albumin (BSA), Mes buer and NaCl. Electrophoresis reagents were from Bio-Rad (Melville, NY): SDS, Tris±HCl, bmercaptoethanol, glycine, methanol, nitrocellulose membranes, Tween 20, and Coomasie Blue. The rabbit anti-caveolin-1 (anity-puri®ed pAb; directed against caveolin-1 residues 1±97; catalog #C13630), anti-caveolin-2 (mAb, directed against the extreme C-terminus of caveolin-2; clone 65, catalog #57820) and anti-caveolin 3 (anity-puri®ed polyclonal antibodies pAb; catalog #38330) antibodies were purchased from Transduction Laboratories (Lexington, KY). Cell cultures Rat C6 glioma cells (Cell Systems, Seattle, WA) were routinely propagated as con¯uent monolayers grown in tissue culture ¯asks (T75). Late passage rat C6 glioma cells were grown in Ham's F10 media in 25 mM Hepes, 2.5% sodium bicarbonate supplemented with 1% glutamine, 1% antibiotic/antimycotic and 10% FBS. Medium removal was done every other day. Cell passage was done weekly. For immuno¯uorescence analysis cells were seeded on coverslips of dual-chamber slides at a density of approximately 1105 cells/ml and incubated at 378C, under an atmosphere of 5% CO2. Subcon¯uent monolayer cells were used for experiments at 1 or 2 days after passage. Cellular fractionation All cellular fractionation procedures were done at 48C. For the preparation of cytosolic and particulate fractions, the medium was removed from the con¯uent monolayers of C6 glioma and the cells washed with phosphate buer saline (PBS). Three ml of lysis buer (20 mM Hepes pH 7.0, 2 mM DTT, 10 mM MgCl2, 0.1 mg/ml BSA) plus a cocktail of protease inhibitors (100 mg/ ml each of leupeptin, antipain, bestatin, chymostatin and pepstatin A) was added to each ¯ask. The cells were scraped from the ¯ask and transferred to a 15 ml centrifuge tube pre-treated with Sigmacote (Sigma, St. Louis). The cells were vortexed during 10 s and sonicated for 30 s twice with 30 s given to settle. The homogenate was centrifuged at 5000 rpm for 3 min at 48C in a clinical centrifuge, and the resulting supernatant spun at 37,500 rpm (100,000 g) for 30 min at 48C in a 80Ti rotor (Beckman Instruments, Palo Alto, CA). This step yielded the corresponding cytosolic and particulate fractions. To generate the caveolin detergent-soluble and -insoluble fractions, C6 glioma cells, particulate fractions were processed as previously described.13,23 Brie¯y, 1% Triton X-100 was added to the lysis buer described above, samples incubated on ice for 5 min, followed by centrifugation at 12,000 g for 20 min. Soluble proteins in the supernatant were separated from the insoluble material in the pellet and solubilized as described for the other cell fractions prior to SDS-PAGE. Total homogenate fractions from rat skeletal muscle and adipocytes were obtained using a modi®cation of the above procedures. Isolation of caveolin-enriched membrane fractions was done using both a Triton X-100-based method,13,23 and a detergent-free method.29 In the ®rst method, light-density, Triton X-100insoluble complexes were obtained using the following procedures. Brie¯y, C6 glioma cells were rinsed twice with Mes buered saline (25 mM Mes, pH 6.5, 0.15 M NaCl) (MBS). Then MBS containing 2% Triton X-100 and 1 mM PMSF was added and the cells scraped. The scraped cells were homogenized with 10±12 strokes in a Dounce homogenizer. A discontinous sucrose density gradient was prepared by adjusting the homogenate to 40% sucrose by addition of 80% sucrose in MBS, and overlaying with 4 ml of 30% sucrose in MBS and 4 ml 5% sucrose in MBS. The sample was centrifuged at 39,000 rpm for 22 h in a SW 41 rotor (Beckman Instruments, Palo Alto, CA). Twelve fractions of 1 ml each were collected from the top of the gradient after the centrifugation, diluted threefold with 25 mM Mes buer, pH 6.5, (MB) and centrifuged at 14,000g for 30 min in a microcentrifuge. The resulting pellets were resuspended in MB and stored at ÿ808C, or alternatively directly resuspended in SDS-PAGE sample buer. Caveolin-enriched complexes were obtained in the absence of detergent as described by Song et al.28 Brie¯y, rat C6 glioma were rinsed twice with MBS. Then MBS containing 500 mM sodium carbonate (Na2CO3) and 1 mM PMSF was added and the cells scraped. The scraped cells were homogenized with 10±12 strokes in a Dounce homogenizer. A discontinous sucrose density gradient was prepared by adjusting the homogenate to 45% sucrose by addition of 90%
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sucrose in MBS, and overlaying with 4 ml of 35% sucrose in MBS containing 250 mM Na2CO3 and 4 ml 5% sucrose in MBS containing 250 mM Na2CO3. The sample was centrifuged at 39,000 rpm for 22 h in a SW 41 rotor (Beckman Instruments, Palo Alto, CA). Twelve 1-ml fractions were collected from the top of the gradient, diluted threefold with MB and centrifuged at 14,000g for 30 min in a microcentrifuge. The resulting pellets were resuspended in MB and stored at ÿ808C, or alternatively directly resuspended in SDS-PAGE sample buer.
SDS-PAGE and immunoblotting SDS-PAGE and immunoblotting analysis of the pellets and cytosolic fractions from above was done as previously described.13,24,27 In brief, equal total protein amounts (cytosolic and particulate fractions) or volumes (gradient fractions), plus the molecular weight standards were loaded in a 1 mm thick 12% SDS polyacrylamide gel and electrophoresed for 45 min at a constant 200 V in a Mini Protean II (Bio-Rad, Melville, NY). After electrophoresis the gel was equilibrated in transfer buer (25 mM Tris, 190 mM Glycine, 20% MeOH, 0.1% SDS) for 30 min, and then transferred overnight to a PVDF membrane using a Mini Trans-Blot apparatus (Bio-Rad, Melville, NY) at 48C and a constant 25 V. The PVDF membranes were stained with India ink to verify transfer eciency. The PVDF membranes were incubated in blocking solution (buer A: 3% nonfat dry milk in 10 mM Tris pH 7.5, 100 mM NaCl, 0.5% Tween 20) at room temperature for 1 h. Subsequently membranes were incubated for 1 h (at room temperature) or overnight (at 48C) with a primary polyclonal antibody against caveolin-1 and -3, and monoclonal antibody against caveolin-2 (1:20,000 dilution). Primary antibodies were prepared in blocking solution. After incubation the antibody solution was discarded and the membrane washed with blocking solution for 30 min using agitation and changing the buer every 10 min. Washing buer was discarded and the membrane incubated with the secondary antibody (enzyme conjugate anti-rabbit IgG horseradish peroxidase or antimouse IgG1 horse radish peroxidase conjugate) diluted 1:20,000 in blocking solution for 1 h. After incubation with the secondary antibody, the washing step was repeated, and an additional wash with Tris/NaCl performed for 15 min. The washing solution was removed and the membrane subsequently processed as described by the manufacturer of the enhanced chemiluminescence assay (Pierce, Rockford, IL).
Immuno¯uorescence All steps were performed at room temperature and as previously described.30 In brief, media was decanted and the cells rinsed three times (10 min) in phosphate-buered saline (PBS). Fixation was done for 45 min with PBS containing 3% paraformaldehyde, 10 mM sodium mperiodate (NaIO4) and 70 mM L-lysine. The slides were rinsed for 10 min (three times) and treated with 100 mM NH4Cl in PBS for 10 min to quench free aldehyde groups. Cells were then rinsed three times with PBS and permeabilization done for 10 min in PBS containing 0.1% Triton-100. After permeabilization cells were rinsed in blocking solution (2% BSA in PBS) for 30 min. The slides were incubated for 1 h at room temperature in primary antibody (1:100 for anti-caveolin-1 or 1:10 anti-caveolin-2) diluted into blocking solution. Cells were rinsed three times with PBS and incubated during 1 h with secondary antibody: anti-rabbit IgG conjugated with FITC (1:100) for caveolin-1, or anti-mouse IgG1 conjugated with FITC (1:10) for caveolin2 (Sigma, St. Louis, MO). Cells were rinsed three times with PBS, the walls of the slides were removed and 10 ml of mounting media (Dako Co., Carpinteria, CA) added. Coverslips were sealed with clear nail enamel and samples dried for 3 min prior to microscopic analysis.
Other procedures Protein concentration of subcellular fractions was determined using the Coomasie Blue method (Pierce, Rockford, IL).
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RESULTS Immunoblot analysis of the known caveolin isoforms (1a, 1b, 2 and 3) in cultured rat C6 glioma cells In the present study we have undertaken the immunochemical identi®cation of CAV and the known caveolin isoforms (1a, 1b, 2 and 3) in cultured rat C6 glioma cells. As a ®rst approach a series of subcellular fractions were prepared from C6 glioma cells and evaluated by immunoblot analysis for the expression of the dierent caveolin isoforms. Fig. 1 displays the results obtained in C6 glial cells and a series of controls. Fig. 1 (top panel) demonstrates the expression of caveolin-1 alpha in the total homogenate (lane 1) and particulate (lane 2) fractions of C6 glial cells. No caveolin-1 immunoreactivity was detected in the C6 glial cell's cytosolic fraction. The following samples were all positive for caveolin-1 immunoreactivity: the human endothelial cell fraction (lane 6), rat skeletal muscle total homogenate fraction (lane 7) and a rat adipocytes total homogenate fraction (lane 8). The middle panel of Fig. 1 shows that C6 glial cells also express the caveolin-2 isoform in the total homogenate (lane 1) and particulate (lane 2) fractions. Also yielding positive immunoreactivity were the human endothelial total homogenate fraction (lane 6) and the rat adipocyte total homogenate fraction (lane 8). Rat skeletal muscle did not display caveolin 2 immunoreactivity. In contrast, only rat skeletal muscle yielded caveolin-3 immunoreactivity (bottom panel of Fig. 1). It must be noted that for both caveolin-1 and -2 a signi®cantly higher immunoreactivity was obtained in the Triton-X-100 detergent soluble fraction (lane 5) than in the detergent-insoluble fractions of C6 glial cells (lane 4).
Fig. 1. Immunoblot analysis of the expression of caveolin-1 (top panel), caveolin-2 (middle panel), and caveolin-3 (bottom panel) in fractions from rat C6 glioma cells. Equal amounts of sample proteins (10 mg protein) were separated by SDS-PAGE and processed for immunoblot analysis using the dierent caveolin isoforms antibodies (see Methods). In all panels: lane 1, total homogenate from C6 glial cells; lane 2, particulate fraction from C6 glial cells; lane 3, cytosolic fraction from rat C6 glial cells; lane 4, detergent-insoluble fraction from rat C6 glioma cells; lane 5, detergent-soluble fraction from rat C6 glioma cells; lane 6, a human endothelial total homogenate fraction supplied by Transduction Laboratories; lane 7, rat skeletal muscle total homogenate fraction; and, lane 8 is a rat adipocyte total homogenate fraction. Molecular mass 10ÿ3 for each of the caveolin isoforms is indicated at the left side of all panels.
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Indirect immuno¯uorescence analysis of C6 glioma cells using caveolin-1 and -2 antibodies The expression of caveolin has been shown to correlate with the presence of morphologically identi®able CAV.11,15,18 In order to demonstrate that the caveolin immunoreactive species seen in Western blots can be ascribed to CAV or CAV-like membrane domains and vesicles we performed indirect immuno¯uorescence analysis of rat C6 glioma cells using caveolin-1, and -2 antibodies. Immunocytochemical staining of C6 glioma cells with a caveolin-1 antibody [Fig. 2(a)] was characterized by intensely ¯uorescent puncta throughout the cytoplasm and diuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and
Fig. 2. Indirect immuno¯uorescence localization of caveolin-1 (a) and caveolin-2 (b) in cultured rat C6 glioma cells. (a) the caveolin-1 staining pattern is characterized by caveolin's characteristic dotted distribution throughout the cytoplasm and presence of micropatches particularly at leading cell edges (open arrows). Perinuclear staining (closed arrows) was also observed, consistent and suggestive of caveolin's presence in the Golgi region of C6 glioma cells. (b) a similar staining pattern was observed with the caveolin-2 antibody, with the presence of cytoplasmic dots and micropatches (open arrows), plus perinuclear staining (closed arrows). N, nucleus. Scale bar: 10 Fm.
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suggestive of caveolin's localization in C6 glioma cells in the trans Golgi region. In addition, the labeling with a caveolin-2 antibody [Fig. 2(b)] was characterized by a similar dotted staining throughout the cytoplasm, diuse micropatches at the level of the plasma membrane and perinuclear staining. Isolation of caveolin-enriched membrane complexes using a nonionic detergent-based method and a detergent-free method To further demonstrate that the caveolin-CAV membrane compartments detected in cultured C6 glioma cells by immunoblot analysis and indirect immuno¯uorescence display physical characteristics to those of CAV from other cell types, we performed the isolation of caveolinenriched membrane complexes using a nonionic detergent-based method and a detergent-free method. In the ®rst method C6 glioma cells were solubilized in Triton X-100 and detergent lysates were fractionated by equilibrium centrifugation on 5±40% discontinuous sucrose density gradients. Consistent with the known properties of CAV, light-density, Triton-X-100-insoluble membrane fractions enriched in both caveolin-1 and caveolin-2, were obtained as demonstrated by the peak of caveolin immunoreactivity in the light-density fractions 4±5 or the 5±30% sucrose interphase [Fig. 3(A) upper immunoblot, and Fig. 3(B) upper immunoblot]. The pattern of distribution of the caveolin-enriched fractions isolated from cultured rat C6 glioma cells in the presence of Triton X-100 was similar to that demonstrated in human ®broblasts, MDCK cells and primary cultures of rat type 1 astrocytes.5,11,24,25 In addition our study shows that rat C6 glioma cell caveolin-enriched membrane fractions isolated in the absence of a nonionic detergent display a similar buoyant density to those of CAV isolated from other cell types. As seen in Fig. 3, light-density, caveolin-1- and -2-enriched fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. Again the primary caveolin-1 and -2 immunoreactivity was obtained in fractions 4±5 or the 5± 35% sucrose interphase [Fig. 3(A) lower immunoblot, and Fig. 3(B) lower immunoblot].
Fig. 3. Isolation of caveolin-1- (A) and caveolin-2- (B) -enriched membrane fractions from rat C6 glioma cells using a Triton X-100-based method (upper immunoblot of both A and B) and a detergentfree, sodium carbonate-based method (lower immunoblot of both A and B). Equal volumes of fractions collected across the gradients were resolved by SDS-PAGE and immunoblotted using the indicated anti-caveolin antibodies. Using both methods with either caveolin-1 or caveolin-2, we obtained the characteristic light-density, caveolin-enriched membrane fractions, fractions 4±6. Fraction number is indicated horizontally.
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DISCUSSION In resemblance to other cell types, at least two caveolin isoforms are expressed in C6 glial cells. This scenario resembles the case for dierentiated PC12 cells and dorsal root ganglion cells that also express these two isoforms.6 As seen in the immunoblots of Fig. 1, human endothelial cells and rat adipocytes also express both of these isoforms. In contrast, rat skeletal muscle expresses the caveolin-1 and -3 isoforms. Thus the muscle-speci®c expression of caveolin 3, is again evident in our study. This contrasts the results of Ikezu et al.10 on primary cultures of rat astrocytes, where caveolin-3 was found. This may be related to the fact that the authors had to immunoprecipitate to detect caveolin-3. Alternatively, the dierence may relate to the dierentiation states of the cells used. Furthermore, although caveolin-1 and -2 are expressed in dierentiated PC12 and dorsal root ganglion neurons, the existence of neuron-speci®c caveolins can not be ruled out.6 Similarly, the existence of glial-speci®c caveolins remains a possibility, and is supported by the study of Cameron et al.5 that also demonstrated the existence of potentially novel caveolin 1 isoforms or variants in primary cultures of rat type I astrocytes and oligodendrocytes. The caveolin variants detected in the previously cited study could be related to the caveolin-2 isoform as we document in our study. Ultimately, these caveolin isoforms could correlate to the expression of caveolae subpopulations involved in diverse aspects of subcellular vesicular tracking and targeting, and cell signaling in brain glial cells. The observations that caveolin-1 in NIH 3T3 ®broblasts is down-regulated in response to cell transformation, and up-regulated during dierentiation of adipocytes and PC12 cells may help explain the prior failure to detect the expression of caveolins in undierentiated PC12 cells and N2a neuroblastoma cells.8,24,25 Our ®ndings in rat C6 glioma cells are interesting since this represents a transformed cell line, which expresses both caveolin-1 and -2. The ability to detect these caveolins in C6 glial cells could be related to the fact that C6 glioma cells express oligodendrocyte- or astrocyte-like properties. The expression of these dierentiated phenotypes is dependent on hormonal stimulation and passage number, although not determined in this study. In our cell cultures the relative expression of the caveolins could be intimately linked to the preponderance of one of these two dierentiated phenotypes. Consequently, the expression of the dierent caveolin isoforms in dierentiated oligodendrocytes and/or astrocytes remains an open question. The immuno¯uorescence staining patterns obtained with both the caveolin-1 and -2 antibodies in C6 glial cells is consistent with the patterns reported for most cell types where caveolae and caveolins have been identi®ed, i.e. adipocytes, NIH 3T3 ®broblasts. The pattern observed using the caveolin-1 and -2 antibodies in some aspects resembled the patterns observed in primary cultures of type 1 astrocytes and oligodendrocytes from rat brain.5 Though the extent of perinuclear and centrosomal region staining was more marked in the study of Cameron et al.5 The dierences could be related to dierences in methodology, speci®cally the need to quench free aldehyde groups, and/or the dierentiation state of our C6 glial cells. In addition similar to the results in primary cultures of type 1 astrocytes and oligodendrocytes from rat brain, when cell processes were evident, linear arrays of the cytoplasmic dotted distributions or puncta were observed extending from the nucleus to the cell periphery of C6 glial cells (unpublished observations). The ®ndings of the present study further support the localization of CAV and caveolin in glial cells. They also demonstrate the expression of two of the three known caveolin isoforms in C6 glial cells, constituting the ®rst demonstration of the expression of caveolin-2 in a C6 glial cell model system. The relationship of the detected caveolins in C6 glial cells to CAV is further supported by the indirect immuno¯uorescence analysis and the isolation of CAV-like membrane microdomains by two dierent methods. In addition, this study establishes the validity of cultured C6 glial cells as a model system for the assessment of the emerging physiological relevance of this novel subcellular transport and cell signaling compartment in the brain, particularly glial cells. In view of these ®ndings, the existence of CAV-caveolin in glial cells strongly suggest that glial cells possess subcellular transport routes that shall prove to be as fascinating, intriguing, challenging and revealing as the axonal transport and synaptic vesicle recycling pathways of neurons.
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AcknowledgementsÐThis work was partially supported by NIH grants RR03035 and GM 8239 to WI Silva, and GM50695 to H Maldonado. It was also supported in part by a Howard Hughes Medical Institute grant through the Undergraduate Biological Sciences Education Program to University of Puerto Rico, Cayey.
REFERENCES 1. Anderson, R. G., Caveolae: where incoming and outgoing messengers meet. Proc. Natl. Acad. Sci. USA, 1993, 90, 10909±10913. 2. Anderson, R. G., Plasmalemmal caveolae and GPI-anchored membrane proteins. Curr. Opin. Cell. Biol., 1993, 5, 647±652. 3. Anderson, R. G., Multiple endocytic pathway. J. Invest. Dermatol., 1992, 5, 7S±9S. 4. Bouillot, C., Prochiantz, A., Rougon, G. and Allinquant, B., Axonal amyloid precursor protein expressed by neurons in vitro is present in a membrane fraction with caveolae-like properties. J. Biol. Chem., 1996, 271, 7640±7644. 5. Cameron, P. L., Run, J. W., Bollag, R., Rasmussen, H. and Cameron, R. S., Identi®cation of caveolin and caveolin-related proteins in the brain. J. Neurosci., 1997, 17, 9520±9535. 6. Galbiati, F., VolonteÂ, D., Gil, O., Zanazzi, G., Salzer, J., Sargiacomo, M., Scherer, P., Engelman, J., Schlegen, A., Parenti, M., Okamoto, T. and Lisanti, M., Expression of Caveolin-1 and -2 in dierentiating PC12 cells and dorsal root ganglion neurons: caveolin-2 is up-regulated in response to cell injury. Proc. Natl. Acad. Sci. USA, 1998, 95, 10257±10262. 7. Glenney, J. R. Jr. and Soppet, D., Sequence and expression of caveolin, a protein component of caveolae plasma membrane domains phosphorylated on tyrosine in Rous sarcoma virus-transformed ®broblast. Proc. Natl. Acad. Sci. USA, 1992, 89, 10517±10521. 8. Gorodinsky, A. and Harris, D. A., Glycolipid-anchored proteins in neuroblastoma cells from detergent-resistant complexes without caveolin. J. Cell. Biol., 1995, 129, 619±627. 9. Henke, R. C., Hancox, K. A. and Jerey, P. L., Characterization of two distinct populations of detergent resistant membrane complexes isolated from chick brain tissues. J. Neurosci. Res., 1996, 45, 617±630. 10. Ikezu, T., Ueda, H., Trapp, B. D., Nishiyama, K., Sha, J. F., Volonte, D., Galbiati, F., Byrd, A. L., Bassell, G., Serizawa, H., Lane, W. S., Lisanti, M. P. and Okamoto, T., Anity-puri®cation and characterization of caveolins from the brain: dierential expression of caveolin-1, -2 and B3 in brain endothelial and astroglial cell types. Brain Res., 1998, 804(2), 177±192. 11. Koleske, A. J., Baltimore, D. and Lisanti, M. P., Reduction of caveolin and caveolae in oncogenically transformed cells. Proc. Natl. Acad. Sci. USA, 1995, 92, 1381±1385. 12. Kurzchalia, T. V., Dupree, P. and Monier, S., VIP21-caveolin, a protein of the trans-Golgi network and caveolae. FEBS Lett., 1994, 346, 88±91. 13. Lisanti, M. P., Scherer, P. E., Tang, Z., KuÈbler, Koleske, A. J. and Sargicomo, M., Caveolae and human disease: functional roles in transcytosis, potocytosis, signalling and cell polarity. Dev. Biol., 1995, 6, 47±58. 14. Lisanti, M. P., Scherer, P. E., Tang, Z. and Sargicomo, M., Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends. Cell. Biol., 1994, 4, 231±235. 15. Lisanti, M. P., Scherer, P. E., Vidugiriene, J., Tang, Z., Hermanowski-Vosatka, A., Tu, Y. H., Cook, R. F. and Sargiacomo, M., Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J. Cell. Biol., 1994, 126, 111±126. 16. Lisanti, M. P., Tang, Z. L. and Sargiacomo, M., Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae. J. Cell. Biol., 1993, 123, 595±604. 17. Lisanti, M. P., Tang, Z., Scherer, P. E., Kubler, E., Koleske, A. J. and Sargiacomo, M., Caveolae, transmembrane signalling and cellular transformation. Mol. Membr. Biol., 1995, 12, 121±124. 18. Lisanti, M. P., Tang, Z., Scherer, P. E. and Sargiacomo, M., Caveolae puri®cation and glycosylphosphatidylinositollinked protein sorting in polarized epithelia. Methods. Enzymol., 1995, 250, 655±668. 19. Okamoto, T., Schlegel, A., Scherer, P. E. and Lisanti, M. P., Caveolins, a family of scaolding proteins for organization `preassembled signaling complexes' at the plasma membrane. J. Biol. Chem., 1998, 273, 5419±5422. 20. Olive, S., Dubois, C., Schachner, M. and Rougon, G., The F3 neuronal glycosylphosphatidylinositol-linked molecule is localized to glycolipid-enriched membrane subdomains and interacts with L1 and fyn kinase in cerebellum. J. Neurochem., 1995, 65, 2307±2317. 21. Palade, G. E., Fine structure of blood capillaries. J. Appl. Physics., 1953, 24, 1424. 22. Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y. S., Glenney, J. R. and Anderson, R. G., Caveolin, a protein component of caveolae membrane coats. Cell, 1992, 68, 673±682. 23. Sargiacomo, M., Sudol, M., Tang, Z. and Lisanti, M. P., Signal transducing molecules and glycosyl-phosphatidylinositolo-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J. Cell. Biol., 1993, 122, 789±807. 24. Scherer, P. E., Okamoto, T., Chun, M., Nishimoto, I., Lodish, H. F. and Lisanti, M. P., Identi®cation, sequence and expression of caveolin-2 de®nes a caveolin gene family. Proc. Natl. Acad. Sci. USA, 1996, 93, 131±135. 25. Scherer, P. E., Tang, Z., Chun, M., Sargiacomo, M., Lodish, U. F. and Lisanti, M. P., Caveolin isoforms dier in their N-terminal protein sequence and subcellular distribution. Identi®cation and epitope mapping of an isoformspeci®c monoclonal antibody probe. J. Biol. Chem., 1995, 270, 16395±16401. 26. Shyng, S. L., Heuser, J. E. and Harris, D. A., A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J. Cell. Biol., 1994, 125, 1239±1250. 27. Silva, W. I., Detection of G protein in bovine brain clathrin coated vesicles with common alpha and beta subunits antibodies. Jpn. J. Pharm., 1991, 56, 97±100. 28. Song, K. S., Scherer, P. E., Tang, Z., Okamoto, T., Li, S., Chafel, M., Chu, C., Kohtz, D. S. and Lisanti, M. P., Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and cofractionates with dystrophin and dystrophin-associated glycoproteins. J. Biol. Chem., 1996, 271, 15160±15165.
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29. Song, S. K., Li Shengwen, Okamoto, T., Quilliam, L. A., Sargiacomo, M. and Lisanti, M. P., Co-puri®cation and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free puri®cation of caveolae microdomains. J. Biol. Chem., 1996, 271, 9690±9697. 30. Tang, Z., Scherer, P. E., Okamoto, T., Song, K., Chu, C., Kohtz, D. S., Nishimoto, I., Lodish, H. F., and Lisanti, M. P., Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle. J. Biol. Chem., 1996, 271, 22558±22561
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PII: S0736-5748(99)00040-4
IDENTIFICATION OF CAVEOLAE AND CAVEOLIN IN C6 GLIOMA CELLS W.I. SILVA,$* H.M. MALDONADO,% M.P. LISANTI,} J. DEVELLIS,} G. CHOMPREÂ,$ N. MAYOL,% M. ORTIZ,$ G. VELAÂZQUEZ,6 A. MALDONADO6 and J. MONTALVO$ $Department of Physiology and Biophysics, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico; %Department of Pharmacology, Universidad Central del Caribe, School of Medicine, Bayamon, Puerto Rico; }Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA; kMental Retardation Research Center, University of California, Los Angeles, CA, USA; Department of Biology, University of Puerto Rico, Cayey, Puerto Rico (Received 4 December 1998; received in revised form 26 April 1999; accepted 3 May 1999) AbstractÐCaveolae (CAV) constitute a novel subcellular transport vesicle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling events. One of the principal components of CAV are caveolin protein isoforms. Here, we have undertaken the immunochemical identi®cation of CAV and the known caveolin isoforms (1a, 1b, 2 and 3) in cultured rat C6 glioma cells. Immunoblot analysis revealed that particulate fractions from rat C6 glioma cells express caveolin-1 and caveolin-2. The relative detergent-insolubility of these caveolin isoforms was also determined by Western blot analysis. Indirect immuno¯uorescence analysis with caveolin-1 and -2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized by intensely ¯uorescent puncta throughout the cytoplasm and diuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin-1 and -2 immunoreactivity seen in Western blots and immunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin-enriched membrane complexes using two dierent methods. Light-density, Triton X-100-insoluble caveolin-1- and caveolin2-enriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5±40% discontinuous sucrose-density gradients. Similar fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. These results further support the localization of CAV and caveolins in glial cells. In addition, they demonstrate that cultured C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in glial cells and the brain. # 1999 ISDN. Published by Elsevier Science Ltd. All rights reserved. Key words: C6 glial cells, Caveolae, Caveolin, Plasmalemmal vesicles.
INTRODUCTION Caveolae (CAV) are ¯ask-shaped, non-clathrin-coated invaginations of the plasma membrane. CAV were ®rst identi®ed as an endocytic compartment in endothelial cells, where they appear to move molecules across the cell by transcytosis.21 More recently, they have been found to be sites where small molecules are concentrated and internalized by a process called potocytosis.1±3 A growing body of biochemical and morphological evidence indicates that a variety of molecules known to function directly or indirectly in signal transduction are enriched in caveolae.13,15,17,19 Based on these observations, a ``caveolae signaling hypothesis'' has been forwarded,13±15,19 which states that caveolar localization of signaling molecules could provide a compartmental basis for a subset of signaling events. Sequestration of signaling molecules *To whom correspondence should be addressed. Tel.: +1-787-758-2525/1608; Fax: +1-787-753-0120; E-mail: [email protected] Abbreviations: CAV, caveolae, Tris±HCl, tris(hydroxymethyl)aminomethane hydrochloride, Mes, 4-morpholineethanesulfonic acid, MBS, Mes-buered saline, Hepes, N-[2-hydroxyethyl]piperazine-N-[2-ethane sulfonic acid]10, DTT, 1,4dithiothreithol, PBS, phosphate-buered saline, BSA, bovine serum albumin, EDTA, ethylenediaminetetraacetic acid, mAb, monoclonal antibody, pAb, polyclonal antibody, MeOH, methanol. 705
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within caveolae could explain the extensive ``cross-talk'' that is observed between dierent signaling pathways or modules.1±3,13±15,19 The multifaceted structural and functional properties of CAV can be attributed largely to one of its principal protein components, caveolin. Caveolin is a 21-kDa membrane protein, originally described as a primary v-src tyrosine kinase substrate, and referred to as VIP21.7,12,22 It is now known that caveolin 1 is the ®rst member of a multi-gene family. At least two caveolin-1 isoforms (alpha and beta, 24 and 21 kDa, respectively) are known, and their expression seems to be dependent on the availability of two distinct translational initiation sites.16,25 Caveolin appears to function as a multivalent scaolding protein that interacts with dierent classes of signal transducers.19 These caveolin-associated proteins include lipidanchored molecules such as heterotrimeric G-protein subunits, H-Ras, eNOS, Src-family tyrosine kinases and signaling receptors.19 More recently, additional caveolin variants have been identi®ed and cloned, these have been referred to as caveolin-2 and caveolin-3.24,28,30 The expression of caveolin-3 is muscle-speci®c and may play a role in the pathogenesis of muscular dystrophy.29,30 Although caveolae are present in most cells, they are most abundant in terminallydierentiated cell types: endothelia, adipocytes, muscle cells and type I pneumocytes. In contrast, caveolin and caveolae are reduced or absent in ®broblasts transformed by certain activated oncogenes, such as v-Abl or H-Ras (G12V).11 Since caveolin's original characterization, the identi®cation of caveolin and CAV in the brain had been elusive.1,4,8,9,13,15,20,26 Yet, analysis of detergent-resistant membrane complexes from 1-day-old chick brains revealed the presence of a caveolin-enriched membrane subpopulation.9 These caveolin-enriched membrane complexes were particularly prevalent in the chick cerebellum.9 Even more recently, caveolin-1 and caveolin-2 were found to be expressed in dierentiated PC12 cells and in dorsal root ganglion neurons.6 Still, thorough identi®cation of caveolae and caveolins in brain tissue requires analysis of glial cells and glial cell model systems in addition to neurons and neuronal cellular model systems. In this regard Cameron et al.5 were able to demonstrate the presence of CAV, caveolin-1a and apparently novel caveolin-1 molecular variants in primary cultures of type 1 astrocytes from rat brain. Indirect immuno¯uorescence analysis with a caveolin-1 antibody also demonstrated the labeling of process-bearing astrocytes and oligodendrocytes.5 Thus, glial cells express a caveolin-caveolae compartment similar to the one expressed in peripheral tissues and cells. The study in primary culture of glial cells5 evaluated the expression of the caveolin-1 isoform. Furthermore, Ikezu et al.,10 detected expression of the dierent caveolin isoforms in primary cultures of rat astrocytes. A more thorough analysis of expression of the dierent caveolin isoforms in brain tissue would bene®t from studies on established cultured brain cell lines. A valuable cultured cell line model system would represent a most valuable experimental system for the analysis of the role and functions of CAV and caveolins in brain tissue, particularly glial cells. Consequently in this study we evaluated the expression of the dierent caveolin variants in cultured rat C6 glioma cells by a combination of experimental approaches. Here we corroborate the expression of CAV and caveolin-1 in glial cells, by documenting their expression in cultured rat C6 glioma cells. In addition using immunoblot analysis and indirect immuno¯uorescence analysis we demonstrate the expression of caveolin-2 in cultured rat C6 glioma cells. In this way we establish the validity of cultured C6 glial cells as a model system for the assessment of the emerging physiological relevance of this novel subcellular transport and cell signaling compartment. EXPERIMENTAL PROCEDURES Materials Cell culture reagents were obtained from Sigma, Co. (St. Louis, MO): glutamine, penicillin, streptomycin, fungizone, EDTA (ethylenediaminetetraacetic acid), Ca2+, Mg2+-free Hank's F10 balanced salt solution and fetal bovine serum (FBS). Immunoblotting and cellular fractionation reagents were also from Sigma, Co. (St. Louis, MO): Hepes buer, DTT, MgCl2, phenylmethyl-sulfonyl-¯uoride (PMSF), leupeptin, antipain, bestatin, chymostatin, pestatin A,
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Sigmacote, Triton X-100, sucrose, bovine serum albumin (BSA), Mes buer and NaCl. Electrophoresis reagents were from Bio-Rad (Melville, NY): SDS, Tris±HCl, bmercaptoethanol, glycine, methanol, nitrocellulose membranes, Tween 20, and Coomasie Blue. The rabbit anti-caveolin-1 (anity-puri®ed pAb; directed against caveolin-1 residues 1±97; catalog #C13630), anti-caveolin-2 (mAb, directed against the extreme C-terminus of caveolin-2; clone 65, catalog #57820) and anti-caveolin 3 (anity-puri®ed polyclonal antibodies pAb; catalog #38330) antibodies were purchased from Transduction Laboratories (Lexington, KY). Cell cultures Rat C6 glioma cells (Cell Systems, Seattle, WA) were routinely propagated as con¯uent monolayers grown in tissue culture ¯asks (T75). Late passage rat C6 glioma cells were grown in Ham's F10 media in 25 mM Hepes, 2.5% sodium bicarbonate supplemented with 1% glutamine, 1% antibiotic/antimycotic and 10% FBS. Medium removal was done every other day. Cell passage was done weekly. For immuno¯uorescence analysis cells were seeded on coverslips of dual-chamber slides at a density of approximately 1105 cells/ml and incubated at 378C, under an atmosphere of 5% CO2. Subcon¯uent monolayer cells were used for experiments at 1 or 2 days after passage. Cellular fractionation All cellular fractionation procedures were done at 48C. For the preparation of cytosolic and particulate fractions, the medium was removed from the con¯uent monolayers of C6 glioma and the cells washed with phosphate buer saline (PBS). Three ml of lysis buer (20 mM Hepes pH 7.0, 2 mM DTT, 10 mM MgCl2, 0.1 mg/ml BSA) plus a cocktail of protease inhibitors (100 mg/ ml each of leupeptin, antipain, bestatin, chymostatin and pepstatin A) was added to each ¯ask. The cells were scraped from the ¯ask and transferred to a 15 ml centrifuge tube pre-treated with Sigmacote (Sigma, St. Louis). The cells were vortexed during 10 s and sonicated for 30 s twice with 30 s given to settle. The homogenate was centrifuged at 5000 rpm for 3 min at 48C in a clinical centrifuge, and the resulting supernatant spun at 37,500 rpm (100,000 g) for 30 min at 48C in a 80Ti rotor (Beckman Instruments, Palo Alto, CA). This step yielded the corresponding cytosolic and particulate fractions. To generate the caveolin detergent-soluble and -insoluble fractions, C6 glioma cells, particulate fractions were processed as previously described.13,23 Brie¯y, 1% Triton X-100 was added to the lysis buer described above, samples incubated on ice for 5 min, followed by centrifugation at 12,000 g for 20 min. Soluble proteins in the supernatant were separated from the insoluble material in the pellet and solubilized as described for the other cell fractions prior to SDS-PAGE. Total homogenate fractions from rat skeletal muscle and adipocytes were obtained using a modi®cation of the above procedures. Isolation of caveolin-enriched membrane fractions was done using both a Triton X-100-based method,13,23 and a detergent-free method.29 In the ®rst method, light-density, Triton X-100insoluble complexes were obtained using the following procedures. Brie¯y, C6 glioma cells were rinsed twice with Mes buered saline (25 mM Mes, pH 6.5, 0.15 M NaCl) (MBS). Then MBS containing 2% Triton X-100 and 1 mM PMSF was added and the cells scraped. The scraped cells were homogenized with 10±12 strokes in a Dounce homogenizer. A discontinous sucrose density gradient was prepared by adjusting the homogenate to 40% sucrose by addition of 80% sucrose in MBS, and overlaying with 4 ml of 30% sucrose in MBS and 4 ml 5% sucrose in MBS. The sample was centrifuged at 39,000 rpm for 22 h in a SW 41 rotor (Beckman Instruments, Palo Alto, CA). Twelve fractions of 1 ml each were collected from the top of the gradient after the centrifugation, diluted threefold with 25 mM Mes buer, pH 6.5, (MB) and centrifuged at 14,000g for 30 min in a microcentrifuge. The resulting pellets were resuspended in MB and stored at ÿ808C, or alternatively directly resuspended in SDS-PAGE sample buer. Caveolin-enriched complexes were obtained in the absence of detergent as described by Song et al.28 Brie¯y, rat C6 glioma were rinsed twice with MBS. Then MBS containing 500 mM sodium carbonate (Na2CO3) and 1 mM PMSF was added and the cells scraped. The scraped cells were homogenized with 10±12 strokes in a Dounce homogenizer. A discontinous sucrose density gradient was prepared by adjusting the homogenate to 45% sucrose by addition of 90%
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sucrose in MBS, and overlaying with 4 ml of 35% sucrose in MBS containing 250 mM Na2CO3 and 4 ml 5% sucrose in MBS containing 250 mM Na2CO3. The sample was centrifuged at 39,000 rpm for 22 h in a SW 41 rotor (Beckman Instruments, Palo Alto, CA). Twelve 1-ml fractions were collected from the top of the gradient, diluted threefold with MB and centrifuged at 14,000g for 30 min in a microcentrifuge. The resulting pellets were resuspended in MB and stored at ÿ808C, or alternatively directly resuspended in SDS-PAGE sample buer.
SDS-PAGE and immunoblotting SDS-PAGE and immunoblotting analysis of the pellets and cytosolic fractions from above was done as previously described.13,24,27 In brief, equal total protein amounts (cytosolic and particulate fractions) or volumes (gradient fractions), plus the molecular weight standards were loaded in a 1 mm thick 12% SDS polyacrylamide gel and electrophoresed for 45 min at a constant 200 V in a Mini Protean II (Bio-Rad, Melville, NY). After electrophoresis the gel was equilibrated in transfer buer (25 mM Tris, 190 mM Glycine, 20% MeOH, 0.1% SDS) for 30 min, and then transferred overnight to a PVDF membrane using a Mini Trans-Blot apparatus (Bio-Rad, Melville, NY) at 48C and a constant 25 V. The PVDF membranes were stained with India ink to verify transfer eciency. The PVDF membranes were incubated in blocking solution (buer A: 3% nonfat dry milk in 10 mM Tris pH 7.5, 100 mM NaCl, 0.5% Tween 20) at room temperature for 1 h. Subsequently membranes were incubated for 1 h (at room temperature) or overnight (at 48C) with a primary polyclonal antibody against caveolin-1 and -3, and monoclonal antibody against caveolin-2 (1:20,000 dilution). Primary antibodies were prepared in blocking solution. After incubation the antibody solution was discarded and the membrane washed with blocking solution for 30 min using agitation and changing the buer every 10 min. Washing buer was discarded and the membrane incubated with the secondary antibody (enzyme conjugate anti-rabbit IgG horseradish peroxidase or antimouse IgG1 horse radish peroxidase conjugate) diluted 1:20,000 in blocking solution for 1 h. After incubation with the secondary antibody, the washing step was repeated, and an additional wash with Tris/NaCl performed for 15 min. The washing solution was removed and the membrane subsequently processed as described by the manufacturer of the enhanced chemiluminescence assay (Pierce, Rockford, IL).
Immuno¯uorescence All steps were performed at room temperature and as previously described.30 In brief, media was decanted and the cells rinsed three times (10 min) in phosphate-buered saline (PBS). Fixation was done for 45 min with PBS containing 3% paraformaldehyde, 10 mM sodium mperiodate (NaIO4) and 70 mM L-lysine. The slides were rinsed for 10 min (three times) and treated with 100 mM NH4Cl in PBS for 10 min to quench free aldehyde groups. Cells were then rinsed three times with PBS and permeabilization done for 10 min in PBS containing 0.1% Triton-100. After permeabilization cells were rinsed in blocking solution (2% BSA in PBS) for 30 min. The slides were incubated for 1 h at room temperature in primary antibody (1:100 for anti-caveolin-1 or 1:10 anti-caveolin-2) diluted into blocking solution. Cells were rinsed three times with PBS and incubated during 1 h with secondary antibody: anti-rabbit IgG conjugated with FITC (1:100) for caveolin-1, or anti-mouse IgG1 conjugated with FITC (1:10) for caveolin2 (Sigma, St. Louis, MO). Cells were rinsed three times with PBS, the walls of the slides were removed and 10 ml of mounting media (Dako Co., Carpinteria, CA) added. Coverslips were sealed with clear nail enamel and samples dried for 3 min prior to microscopic analysis.
Other procedures Protein concentration of subcellular fractions was determined using the Coomasie Blue method (Pierce, Rockford, IL).
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RESULTS Immunoblot analysis of the known caveolin isoforms (1a, 1b, 2 and 3) in cultured rat C6 glioma cells In the present study we have undertaken the immunochemical identi®cation of CAV and the known caveolin isoforms (1a, 1b, 2 and 3) in cultured rat C6 glioma cells. As a ®rst approach a series of subcellular fractions were prepared from C6 glioma cells and evaluated by immunoblot analysis for the expression of the dierent caveolin isoforms. Fig. 1 displays the results obtained in C6 glial cells and a series of controls. Fig. 1 (top panel) demonstrates the expression of caveolin-1 alpha in the total homogenate (lane 1) and particulate (lane 2) fractions of C6 glial cells. No caveolin-1 immunoreactivity was detected in the C6 glial cell's cytosolic fraction. The following samples were all positive for caveolin-1 immunoreactivity: the human endothelial cell fraction (lane 6), rat skeletal muscle total homogenate fraction (lane 7) and a rat adipocytes total homogenate fraction (lane 8). The middle panel of Fig. 1 shows that C6 glial cells also express the caveolin-2 isoform in the total homogenate (lane 1) and particulate (lane 2) fractions. Also yielding positive immunoreactivity were the human endothelial total homogenate fraction (lane 6) and the rat adipocyte total homogenate fraction (lane 8). Rat skeletal muscle did not display caveolin 2 immunoreactivity. In contrast, only rat skeletal muscle yielded caveolin-3 immunoreactivity (bottom panel of Fig. 1). It must be noted that for both caveolin-1 and -2 a signi®cantly higher immunoreactivity was obtained in the Triton-X-100 detergent soluble fraction (lane 5) than in the detergent-insoluble fractions of C6 glial cells (lane 4).
Fig. 1. Immunoblot analysis of the expression of caveolin-1 (top panel), caveolin-2 (middle panel), and caveolin-3 (bottom panel) in fractions from rat C6 glioma cells. Equal amounts of sample proteins (10 mg protein) were separated by SDS-PAGE and processed for immunoblot analysis using the dierent caveolin isoforms antibodies (see Methods). In all panels: lane 1, total homogenate from C6 glial cells; lane 2, particulate fraction from C6 glial cells; lane 3, cytosolic fraction from rat C6 glial cells; lane 4, detergent-insoluble fraction from rat C6 glioma cells; lane 5, detergent-soluble fraction from rat C6 glioma cells; lane 6, a human endothelial total homogenate fraction supplied by Transduction Laboratories; lane 7, rat skeletal muscle total homogenate fraction; and, lane 8 is a rat adipocyte total homogenate fraction. Molecular mass 10ÿ3 for each of the caveolin isoforms is indicated at the left side of all panels.
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Indirect immuno¯uorescence analysis of C6 glioma cells using caveolin-1 and -2 antibodies The expression of caveolin has been shown to correlate with the presence of morphologically identi®able CAV.11,15,18 In order to demonstrate that the caveolin immunoreactive species seen in Western blots can be ascribed to CAV or CAV-like membrane domains and vesicles we performed indirect immuno¯uorescence analysis of rat C6 glioma cells using caveolin-1, and -2 antibodies. Immunocytochemical staining of C6 glioma cells with a caveolin-1 antibody [Fig. 2(a)] was characterized by intensely ¯uorescent puncta throughout the cytoplasm and diuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and
Fig. 2. Indirect immuno¯uorescence localization of caveolin-1 (a) and caveolin-2 (b) in cultured rat C6 glioma cells. (a) the caveolin-1 staining pattern is characterized by caveolin's characteristic dotted distribution throughout the cytoplasm and presence of micropatches particularly at leading cell edges (open arrows). Perinuclear staining (closed arrows) was also observed, consistent and suggestive of caveolin's presence in the Golgi region of C6 glioma cells. (b) a similar staining pattern was observed with the caveolin-2 antibody, with the presence of cytoplasmic dots and micropatches (open arrows), plus perinuclear staining (closed arrows). N, nucleus. Scale bar: 10 Fm.
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suggestive of caveolin's localization in C6 glioma cells in the trans Golgi region. In addition, the labeling with a caveolin-2 antibody [Fig. 2(b)] was characterized by a similar dotted staining throughout the cytoplasm, diuse micropatches at the level of the plasma membrane and perinuclear staining. Isolation of caveolin-enriched membrane complexes using a nonionic detergent-based method and a detergent-free method To further demonstrate that the caveolin-CAV membrane compartments detected in cultured C6 glioma cells by immunoblot analysis and indirect immuno¯uorescence display physical characteristics to those of CAV from other cell types, we performed the isolation of caveolinenriched membrane complexes using a nonionic detergent-based method and a detergent-free method. In the ®rst method C6 glioma cells were solubilized in Triton X-100 and detergent lysates were fractionated by equilibrium centrifugation on 5±40% discontinuous sucrose density gradients. Consistent with the known properties of CAV, light-density, Triton-X-100-insoluble membrane fractions enriched in both caveolin-1 and caveolin-2, were obtained as demonstrated by the peak of caveolin immunoreactivity in the light-density fractions 4±5 or the 5±30% sucrose interphase [Fig. 3(A) upper immunoblot, and Fig. 3(B) upper immunoblot]. The pattern of distribution of the caveolin-enriched fractions isolated from cultured rat C6 glioma cells in the presence of Triton X-100 was similar to that demonstrated in human ®broblasts, MDCK cells and primary cultures of rat type 1 astrocytes.5,11,24,25 In addition our study shows that rat C6 glioma cell caveolin-enriched membrane fractions isolated in the absence of a nonionic detergent display a similar buoyant density to those of CAV isolated from other cell types. As seen in Fig. 3, light-density, caveolin-1- and -2-enriched fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. Again the primary caveolin-1 and -2 immunoreactivity was obtained in fractions 4±5 or the 5± 35% sucrose interphase [Fig. 3(A) lower immunoblot, and Fig. 3(B) lower immunoblot].
Fig. 3. Isolation of caveolin-1- (A) and caveolin-2- (B) -enriched membrane fractions from rat C6 glioma cells using a Triton X-100-based method (upper immunoblot of both A and B) and a detergentfree, sodium carbonate-based method (lower immunoblot of both A and B). Equal volumes of fractions collected across the gradients were resolved by SDS-PAGE and immunoblotted using the indicated anti-caveolin antibodies. Using both methods with either caveolin-1 or caveolin-2, we obtained the characteristic light-density, caveolin-enriched membrane fractions, fractions 4±6. Fraction number is indicated horizontally.
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DISCUSSION In resemblance to other cell types, at least two caveolin isoforms are expressed in C6 glial cells. This scenario resembles the case for dierentiated PC12 cells and dorsal root ganglion cells that also express these two isoforms.6 As seen in the immunoblots of Fig. 1, human endothelial cells and rat adipocytes also express both of these isoforms. In contrast, rat skeletal muscle expresses the caveolin-1 and -3 isoforms. Thus the muscle-speci®c expression of caveolin 3, is again evident in our study. This contrasts the results of Ikezu et al.10 on primary cultures of rat astrocytes, where caveolin-3 was found. This may be related to the fact that the authors had to immunoprecipitate to detect caveolin-3. Alternatively, the dierence may relate to the dierentiation states of the cells used. Furthermore, although caveolin-1 and -2 are expressed in dierentiated PC12 and dorsal root ganglion neurons, the existence of neuron-speci®c caveolins can not be ruled out.6 Similarly, the existence of glial-speci®c caveolins remains a possibility, and is supported by the study of Cameron et al.5 that also demonstrated the existence of potentially novel caveolin 1 isoforms or variants in primary cultures of rat type I astrocytes and oligodendrocytes. The caveolin variants detected in the previously cited study could be related to the caveolin-2 isoform as we document in our study. Ultimately, these caveolin isoforms could correlate to the expression of caveolae subpopulations involved in diverse aspects of subcellular vesicular tracking and targeting, and cell signaling in brain glial cells. The observations that caveolin-1 in NIH 3T3 ®broblasts is down-regulated in response to cell transformation, and up-regulated during dierentiation of adipocytes and PC12 cells may help explain the prior failure to detect the expression of caveolins in undierentiated PC12 cells and N2a neuroblastoma cells.8,24,25 Our ®ndings in rat C6 glioma cells are interesting since this represents a transformed cell line, which expresses both caveolin-1 and -2. The ability to detect these caveolins in C6 glial cells could be related to the fact that C6 glioma cells express oligodendrocyte- or astrocyte-like properties. The expression of these dierentiated phenotypes is dependent on hormonal stimulation and passage number, although not determined in this study. In our cell cultures the relative expression of the caveolins could be intimately linked to the preponderance of one of these two dierentiated phenotypes. Consequently, the expression of the dierent caveolin isoforms in dierentiated oligodendrocytes and/or astrocytes remains an open question. The immuno¯uorescence staining patterns obtained with both the caveolin-1 and -2 antibodies in C6 glial cells is consistent with the patterns reported for most cell types where caveolae and caveolins have been identi®ed, i.e. adipocytes, NIH 3T3 ®broblasts. The pattern observed using the caveolin-1 and -2 antibodies in some aspects resembled the patterns observed in primary cultures of type 1 astrocytes and oligodendrocytes from rat brain.5 Though the extent of perinuclear and centrosomal region staining was more marked in the study of Cameron et al.5 The dierences could be related to dierences in methodology, speci®cally the need to quench free aldehyde groups, and/or the dierentiation state of our C6 glial cells. In addition similar to the results in primary cultures of type 1 astrocytes and oligodendrocytes from rat brain, when cell processes were evident, linear arrays of the cytoplasmic dotted distributions or puncta were observed extending from the nucleus to the cell periphery of C6 glial cells (unpublished observations). The ®ndings of the present study further support the localization of CAV and caveolin in glial cells. They also demonstrate the expression of two of the three known caveolin isoforms in C6 glial cells, constituting the ®rst demonstration of the expression of caveolin-2 in a C6 glial cell model system. The relationship of the detected caveolins in C6 glial cells to CAV is further supported by the indirect immuno¯uorescence analysis and the isolation of CAV-like membrane microdomains by two dierent methods. In addition, this study establishes the validity of cultured C6 glial cells as a model system for the assessment of the emerging physiological relevance of this novel subcellular transport and cell signaling compartment in the brain, particularly glial cells. In view of these ®ndings, the existence of CAV-caveolin in glial cells strongly suggest that glial cells possess subcellular transport routes that shall prove to be as fascinating, intriguing, challenging and revealing as the axonal transport and synaptic vesicle recycling pathways of neurons.
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AcknowledgementsÐThis work was partially supported by NIH grants RR03035 and GM 8239 to WI Silva, and GM50695 to H Maldonado. It was also supported in part by a Howard Hughes Medical Institute grant through the Undergraduate Biological Sciences Education Program to University of Puerto Rico, Cayey.
REFERENCES 1. Anderson, R. G., Caveolae: where incoming and outgoing messengers meet. Proc. Natl. Acad. Sci. USA, 1993, 90, 10909±10913. 2. Anderson, R. G., Plasmalemmal caveolae and GPI-anchored membrane proteins. Curr. Opin. Cell. Biol., 1993, 5, 647±652. 3. Anderson, R. G., Multiple endocytic pathway. J. Invest. Dermatol., 1992, 5, 7S±9S. 4. Bouillot, C., Prochiantz, A., Rougon, G. and Allinquant, B., Axonal amyloid precursor protein expressed by neurons in vitro is present in a membrane fraction with caveolae-like properties. J. Biol. Chem., 1996, 271, 7640±7644. 5. Cameron, P. L., Run, J. W., Bollag, R., Rasmussen, H. and Cameron, R. S., Identi®cation of caveolin and caveolin-related proteins in the brain. J. Neurosci., 1997, 17, 9520±9535. 6. Galbiati, F., VolonteÂ, D., Gil, O., Zanazzi, G., Salzer, J., Sargiacomo, M., Scherer, P., Engelman, J., Schlegen, A., Parenti, M., Okamoto, T. and Lisanti, M., Expression of Caveolin-1 and -2 in dierentiating PC12 cells and dorsal root ganglion neurons: caveolin-2 is up-regulated in response to cell injury. Proc. Natl. Acad. Sci. USA, 1998, 95, 10257±10262. 7. Glenney, J. R. Jr. and Soppet, D., Sequence and expression of caveolin, a protein component of caveolae plasma membrane domains phosphorylated on tyrosine in Rous sarcoma virus-transformed ®broblast. Proc. Natl. Acad. Sci. USA, 1992, 89, 10517±10521. 8. Gorodinsky, A. and Harris, D. A., Glycolipid-anchored proteins in neuroblastoma cells from detergent-resistant complexes without caveolin. J. Cell. Biol., 1995, 129, 619±627. 9. Henke, R. C., Hancox, K. A. and Jerey, P. L., Characterization of two distinct populations of detergent resistant membrane complexes isolated from chick brain tissues. J. Neurosci. Res., 1996, 45, 617±630. 10. Ikezu, T., Ueda, H., Trapp, B. D., Nishiyama, K., Sha, J. F., Volonte, D., Galbiati, F., Byrd, A. L., Bassell, G., Serizawa, H., Lane, W. S., Lisanti, M. P. and Okamoto, T., Anity-puri®cation and characterization of caveolins from the brain: dierential expression of caveolin-1, -2 and B3 in brain endothelial and astroglial cell types. Brain Res., 1998, 804(2), 177±192. 11. Koleske, A. J., Baltimore, D. and Lisanti, M. P., Reduction of caveolin and caveolae in oncogenically transformed cells. Proc. Natl. Acad. Sci. USA, 1995, 92, 1381±1385. 12. Kurzchalia, T. V., Dupree, P. and Monier, S., VIP21-caveolin, a protein of the trans-Golgi network and caveolae. FEBS Lett., 1994, 346, 88±91. 13. Lisanti, M. P., Scherer, P. E., Tang, Z., KuÈbler, Koleske, A. J. and Sargicomo, M., Caveolae and human disease: functional roles in transcytosis, potocytosis, signalling and cell polarity. Dev. Biol., 1995, 6, 47±58. 14. Lisanti, M. P., Scherer, P. E., Tang, Z. and Sargicomo, M., Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends. Cell. Biol., 1994, 4, 231±235. 15. Lisanti, M. P., Scherer, P. E., Vidugiriene, J., Tang, Z., Hermanowski-Vosatka, A., Tu, Y. H., Cook, R. F. and Sargiacomo, M., Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J. Cell. Biol., 1994, 126, 111±126. 16. Lisanti, M. P., Tang, Z. L. and Sargiacomo, M., Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae. J. Cell. Biol., 1993, 123, 595±604. 17. Lisanti, M. P., Tang, Z., Scherer, P. E., Kubler, E., Koleske, A. J. and Sargiacomo, M., Caveolae, transmembrane signalling and cellular transformation. Mol. Membr. Biol., 1995, 12, 121±124. 18. Lisanti, M. P., Tang, Z., Scherer, P. E. and Sargiacomo, M., Caveolae puri®cation and glycosylphosphatidylinositollinked protein sorting in polarized epithelia. Methods. Enzymol., 1995, 250, 655±668. 19. Okamoto, T., Schlegel, A., Scherer, P. E. and Lisanti, M. P., Caveolins, a family of scaolding proteins for organization `preassembled signaling complexes' at the plasma membrane. J. Biol. Chem., 1998, 273, 5419±5422. 20. Olive, S., Dubois, C., Schachner, M. and Rougon, G., The F3 neuronal glycosylphosphatidylinositol-linked molecule is localized to glycolipid-enriched membrane subdomains and interacts with L1 and fyn kinase in cerebellum. J. Neurochem., 1995, 65, 2307±2317. 21. Palade, G. E., Fine structure of blood capillaries. J. Appl. Physics., 1953, 24, 1424. 22. Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y. S., Glenney, J. R. and Anderson, R. G., Caveolin, a protein component of caveolae membrane coats. Cell, 1992, 68, 673±682. 23. Sargiacomo, M., Sudol, M., Tang, Z. and Lisanti, M. P., Signal transducing molecules and glycosyl-phosphatidylinositolo-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J. Cell. Biol., 1993, 122, 789±807. 24. Scherer, P. E., Okamoto, T., Chun, M., Nishimoto, I., Lodish, H. F. and Lisanti, M. P., Identi®cation, sequence and expression of caveolin-2 de®nes a caveolin gene family. Proc. Natl. Acad. Sci. USA, 1996, 93, 131±135. 25. Scherer, P. E., Tang, Z., Chun, M., Sargiacomo, M., Lodish, U. F. and Lisanti, M. P., Caveolin isoforms dier in their N-terminal protein sequence and subcellular distribution. Identi®cation and epitope mapping of an isoformspeci®c monoclonal antibody probe. J. Biol. Chem., 1995, 270, 16395±16401. 26. Shyng, S. L., Heuser, J. E. and Harris, D. A., A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J. Cell. Biol., 1994, 125, 1239±1250. 27. Silva, W. I., Detection of G protein in bovine brain clathrin coated vesicles with common alpha and beta subunits antibodies. Jpn. J. Pharm., 1991, 56, 97±100. 28. Song, K. S., Scherer, P. E., Tang, Z., Okamoto, T., Li, S., Chafel, M., Chu, C., Kohtz, D. S. and Lisanti, M. P., Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and cofractionates with dystrophin and dystrophin-associated glycoproteins. J. Biol. Chem., 1996, 271, 15160±15165.
714
W.I. Silva et al.
29. Song, S. K., Li Shengwen, Okamoto, T., Quilliam, L. A., Sargiacomo, M. and Lisanti, M. P., Co-puri®cation and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free puri®cation of caveolae microdomains. J. Biol. Chem., 1996, 271, 9690±9697. 30. Tang, Z., Scherer, P. E., Okamoto, T., Song, K., Chu, C., Kohtz, D. S., Nishimoto, I., Lodish, H. F., and Lisanti, M. P., Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle. J. Biol. Chem., 1996, 271, 22558±22561