RESEARCH ARTICLE 1397 -specific targeting of -1 to , secretory vesicles, cytoplasm or mitochondria

Wei-Ping Li, Pingsheng Liu, Brian K. Pilcher and Richard G. W. Anderson* Department of Cell , University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039, USA *Author for correspondence (e-mail: [email protected])

Accepted 15 January 2001 Journal of Cell Science 114, 1397-1408 © The Company of Biologists Ltd

SUMMARY

In commonly used tissue culture cells, caveolin-1 is accumulate caveolin-1 in modified mitochondria. The embedded in caveolae membranes. It appears to reach this cytosolic and the secreted forms appear to be incorporated location after being cotranslationally inserted into ER into a soluble, complex. We conclude that caveolin-1 membranes, processed in the Golgi and shipped to the cell can be targeted to a variety of intracellular destinations, surface. We now report that caveolae are not the preferred which suggests a novel mechanism for the intracellular location for caveolin-1 in all cell types. cells traffic of this . and keratinocytes target caveolin-1 to the cytosol while in exocrine and endocrine cells it accumulates in the secretory pathway. We also found that airway epithelial cells Key words: Caveolin-1, Intracellular trafficking, Caveolae

INTRODUCTION will target the molecule to caveolae by determining with immunocytochemistry if caveolin-1 in different tissue cells is Caveolin-1 was originally identified as a novel tyrosine kinase always in this membrane domain. Unexpectedly, we found substrate in Rous sarcoma transformed cells (Glenney, 1989). examples of cells that preferentially target caveolin-1 to either Immunogold cytochemistry localized the protein to the the cytoplasm, mitochondria or elements of the secretory distinctive striated coat structure that decorates the inner pathway but had little caveolin-1 in invaginated caveolae. The membrane surface of fibroblast caveolae (Rothberg et al., 1992) behavior of caveolin-1 in these cells suggests the existence of and to elements of the Golgi apparatus (Kurzchalia et al., 1992). a novel pathway of intracellular and extracellular molecular Studies carried out concurrently implicated caveolin-1 in the trafficking that may be specialized for delivering to sorting of molecules during vesicular trafficking to the apical multiple cellular compartments. surface of polarized epithelial cells (Kurzchalia et al., 1992). Subsequently, several laboratories used caveolin-1 as an integral protein marker to prepare cell fractions enriched in caveolae MATERIALS AND METHODS (reviewed in Anderson, 1998). Several methods of purification yield vesicles that are approximately the size of caveolae (Smart Cyclophilin A pAb was purchased from ABR (Golden, CO, USA). et al., 1995; Westermann et al., 1999), sometimes with an ApoA 1 pAb was a gift from Dr Helen Hobbs (University of Texas apparent striated coat (Chang et al., 1994). These and other Southwestern Medical Center, USA). Purified human HDL was studies suggest that caveolin-1 plays a role in the traffic of prepared by standard methods. OptiPrep was purchased from caveolae and caveolae-related membranes within the cell Accurate Chemical & Scientific Corporation (Westbury, NY, USA). (Anderson, 1993; Anderson, 1998). Caveolin-2 and caveolin-3 Caveolin-1 pAb and mAb were either raised in our laboratory or obtained from Transduction Laboratories (Lexington, KY, USA). are two homologous that may have similar functions Caveolin-2 mAb and caveolin-3 pAb were obtained from to caveolin-1 (Smart et al., 1999). Transduction Laboratories (Lexington, KY, USA). FITC-goat anti- Studies on the function of caveolin-1 follow two main rabbit IgG was from Zymed Laboratories (San Francisco, CA, USA). themes. One idea is that caveolin-1 is a scaffolding protein Protein A gold was from Dr J. W. Slot (Utrecht University, The involved in organizing the activity of multiple signaling Netherlands). BSA, proline, leupeptin, soybean trypsin inhibitor, molecules in caveolae (Okamoto et al., 1998). The other is pepstatin A, Dulbecco’s modified Eagle’s Medium (DMEM), based on the - (Murata et al., 1995) and fatty acid- benzamidine, adenine-free base, human apo-transferrin, aprotinin, L- (Trigatti et al., 1999) binding properties of caveolin-1 and the ascorbic acid, bovine pancreatic insulin, ethylenediaminetetraacetic possibility that it mediates the intracellular transport of lipids acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), hydrocortisone, such as cholesterol (Smart et al., 1996). The exact function of ATP, secretin, dexamethasone, cholecystokinin, mitomycin C, sodium chloride, sodium deoxycholate, sodium fluoride, sodium vanadate and a protein is often reflected in how the molecule is used 3,3′,5-triiodo-L-thyronine sodium salt were from Sigma (St Louis, by various tissue cells. If, for example, caveolin-1 were a MO, USA). A23187 was from Biomol (Plymouth Meeting, PA, USA). scaffolding protein, then the molecule should be in caveolae- Bio-Rad Protein Assay was from Bio-Rad Laboratories (Hercules, related structures whenever it is expressed. In the current study, CA, USA). F-12K medium was from GibcoBRL (Grand Island, NY, we tested the hypothesis that all cells expressing caveolin-1 USA). Fetal bovine serum (FBS) and fetal calf serum (FCS) were 1398 JOURNAL OF CELL SCIENCE 114 (7) from HyClone (Logan, UT, USA). C57Bl/6J mice were from Jackson fibroblast-populated collagen gels (2×104 cells/ml, rat tail type I Laboratory (Bar Harbor, MI, USA). Sprague Dawley rats were from collagen) and placed into Transwell™ polycarbonate cell culture Harlan Sprague Dawley (Indianapolis, IN, USA). Bovine type I inserts (4.0 µm pore size). Following keratinocyte attachment (approx. collagen (Vitrogen-100) was purchased from Celltrix Laboratories 4 hours), the cloning cylinders were removed and cultures were (Palo Alto, CA, USA) and rat tail type I collagen was obtained from immersed in FAD medium containing 50 µg/ml L-ascorbic acid. Upstate Biotechnology (Lake Placid, NY, USA). DMEM/F-12 Following a 6 day culture period to allow the keratinocytes to reach medium, recombinant human epidermal growth factor, and confluence, the cultured skin equivalents were raised to the air-liquid penicillin/streptomycin were obtained from Life Technologies, Inc. interface to stimulate keratinocyte differentiation and cornification. (Rockville, MD, USA). Recombinant Cholera toxin B subunit was FAD medium with additives was replaced every 2 days until cultured obtained from Calbiochem-Novabiochem corporation (Cambridge, skin equivalents were harvested. MA, USA). Borosilicate cloning cylinders (8 mm inner diameter) were purchased form BellCo Glass, Inc. (Vineland, NJ, USA). Isolation of caveolae Transwell™ polycarbonate cell culture inserts (4.0 µm pore size) were Caveolae were isolated by the method of Smart et al. (Smart et al., obtained from Corning Costar Corp. (Cambridge, MA, USA). Hybond 1995). Briefly, confluent normal human fibroblasts were collected in N+ blotting membrane, Rediprime II random priming label and ECL hypotonic buffer and dounced 20 times on ice. Plasma membrane (PM) immunoblotting systems were purchased from Amersham Pharmacia was isolated on a 30% Percoll gradient from the post-nuclear Biotech, Inc. (Piscataway, NJ, USA). [γ32P]-dCTP was obtained from supernatant (PNS) and then sonicated. The sonicated sample was NEN Life Science Products, Inc. (Boston, MA, USA). Immobilon-P mixed with OptiPrep (final OptiPrep concentration, 23%) in a TH641 polyvinylidene difluoride (PVDF) membrane was purchased from tube. A linear 20%-10% OptiPrep gradient was overlaid on the sample Millipore Corp. (Bedford, MA, USA). and centrifuged at 52,000 g for 90 minutes at 4°C. The bottom 1 ml Buffer A: 20 mM Tricine, pH 7.8, 250 mM sucrose, 1 mM EDTA. was designated non-caveolae membranes (NCM). The top 5 ml were Buffer B: 20 mM Tris-HCl, pH 7.6, 130 mM NaCl, 0.2% Tween- collected and mixed with 4 ml of 50% OptiPrep in a second TH641 20. tube. 2 ml of 5% OptiPrep were overlaid and the sample centrifuged Buffer C: 100 mM sodium phosphate, pH 7.4, 0.15 M NaCl, 4 mM at 52,000 g for 90 minutes at 4°C. The gradient was fractionated in KCl, 2 mM MgCl2, and 0.02% (wt/vol) sodium azide. 0.7 ml fractions. Fraction 3 was designated caveolae membranes (CM). Buffer D: Buffer C containing 1% BSA, 0.05% Tween 20, 0.05% Triton X-100. Immunoblotting Buffer E: Buffer C containing 1% BSA, 0.01% Tween 20, 0.01% Equal amounts of the indicated mouse tissue (40 mg wet mass) was Triton X-100. sonicated in 200 µl 2× SDS-sample buffer (100 mM Tris-HCL, pH Buffer F: 10 mM Tris-HCl pH 7.5, 250 mM sucrose, 0.1 mM 6.8, 20% glycerol, 4% SDS, 4% 2-mercaptoethanol). The 50 µl EDTA. sample was diluted with 950 µl of water before adding 100 µl of 72% TCA to precipitate the protein. The precipitate was redissolved in Cell culture 1000 µl 2× SDS-sample buffer. Equal fractions (30 µl, approx. 300 µg Normal human fibroblasts (Goldstein et al., 1983) grown to wet mass tissue) were heated at 95°C for 5 minutes in SDS-sample confluence in 150 mm plates were cultured in DMEM supplemented buffer (Laemmli, 1970) before being separated by electrophoresis at with penicillin and 10% FBS. 20 hours before each experiment, the 25 mA per gel. The proteins were transferred to PVDF membranes. medium was changed to MEM plus 200 µg/ml of BSA. The pituitary After blocking with buffer B containing 5% nonfat dry milk, the cell line GH3 was cultured (100 mm plates) in F-12K medium membranes were incubated with the first antibody followed by the supplemented with 2.5% FBS and 15% horse serum (HS) for 4 days second antibody conjugated with HRP in buffer B containing 1% before each experiment. nonfat dry milk. Bound antibody was detected using an ECL detection system. To isolate protein from keratinocyte cultures, cell layers were Keratinocyte cell culture washed with PBS and treated for 10 minutes at 4°C with cell lysis Human primary keratinocytes were harvested from healthy adult skin buffer (1.0% NP-40, 20 mM Tris, pH 7.5, 2.0 mM sodium vanadate, obtained as surgical discard tissue. Subcutaneous fat and deep dermis 1.0 mM NaF, 100 mM NaCl, 5.0 µg/ml sodium deoxycholate, 2.0 mM were removed, and the remaining tissue was incubated in 0.25% EDTA, 2.0 mM EGTA, and 25 µg/ml each of leupeptin, aprotinin and trypsin in PBS at 25°C. After 16 hours, the epidermis was separated pepstatin). Cell lysates were then dounce-homogenized and the lysate from the dermis with forceps, and the keratinocytes were scraped into cleared by centrifugation (14,000 g) at 4°C. Proteins were separated DMEM. The keratinocyte suspension was added to fresh DMEM by gel electrophoresis and immunoblotted using the same procedure supplemented with 5% FCS and 0.1% penicillin/streptomycin and a as for the mouse tissue. sample of keratinocyte suspension was then plated onto tissue culture dishes coated with 1 mg/ml type I collagen. Under these culture RNA isolation and northern hybridization conditions, keratinocytes proliferate, migrate, differentiate and cornify Total RNA was isolated by phenol-chloroform extraction (Pentland and Needleman, 1986). Cultures of dermal fibroblasts, (Chomczynski and Sacchi, 1987). RNA (10 µg) was denatured and obtained from surgical discard tissue, were outgrown from dermal resolved by electrophoresis through a 1% formaldehyde-agarose gel, explant cultures and maintained in DMEM supplemented with 10% transferred overnight to Hybond N+ (Amersham Pharmacia Biotech, FCS and 0.1% penicillin/streptomycin. Cells used for the cultured skin Inc., Piscataway, NJ, USA), and hybridized with a radiolabeled equivalents were passaged 5-10 times. Fibroblast feeder layers were caveolin-1 cDNA probe. The cDNA probe was labeled by random prepared using mouse 3T3 cells treated with mitomycin C (10 µg/ml) priming with [α32P]-dCTP. Following hybridization, the membranes for 2 hours at 37°C. Normal epidermal keratinocytes were plated onto were washed and visualized using the Typhoon 8600 phosphorimager feeder layers in FAD medium (DMEM:Hams F12, 3:1) supplemented system (Molecular Dynamics, Sunnyvale, CA, USA). with 0.1% penicillin/streptomycin, 5% FCS, 5 µg/ml insulin, 20 ng/ml recombinant human epidermal growth factor, 5 µg/ml transferrin, Immunofluorescence microscopy 10−10 M cholera toxin, 180 µM adenine, 0.4 µg/ml hydrocortisone and Adult C57Bl/6J mice or adult Sprague Dawley rats were fixed by 0.02 nM triiodothyronine. Cultured skin equivalents were generated perfusing euthanized animals via the left cardiac ventricle with 60 ml using a technique modified from Stark et al. (Stark et al., 1999). Briefly, of 3% (w/v) paraformaldehyde in buffer C containing 3 mM normal epidermal keratinocytes (1×106/cm2, passages 2-5) were trinitrophenol for 5 minutes. Tissues were removed and fixed further in seeded within 8 mm borosilicate cloning cylinders onto precontracted a mixture of 60% (v/v) methanol, 10% (v/v) glacial acetic acid, 30% Tissue distribution of caveolin 1399

(v/v) inhibisol (1,1,1-trichloroethane) for 24 hours. Lung was perfusion- RESULTS fixed via the right cardiac ventricle. Tissues were then embedded in paraffin. Paraffin sections (5 µm thick) were dewaxed in three changes We used immunoblotting to compare the caveolin-1 expression of xylene (10 minutes each), rehydrated into buffer C. The sections were level in various mouse tissues (Fig. 1A). Equal weights washed with 50 mM NH4Cl in buffer C for 30 minutes, rinsed in buffer (~300 µg) of each tissue was separated by gel electrophoresis C followed by buffer C containing 1% BSA and 1.5% normal goat and immunoblotted with caveolin-1 pAb (caveolin-1). On a serum for 60 minutes at room temperature. This was followed by an overnight incubation in the presence of either pAb (10 µg/ml) or mAb tissue weight basis, the lung had the highest expression level (30 µg/ml) caveolin-1 IgG, pAb (10 µg/ml) caveolin-3 IgG, non- of all the tissues, while the lowest amount of caveolin-1 was immune rabbit IgG (10 µg/ml) or non-immune mouse IgG (30 µg/ml). found in the liver. Samples of tissue rich in Primary antibodies were localized by incubating sections for 2 hours in (e.g., intestine and uterus) and adipocytes expressed high levels 15 µg/ml affinity-purified, goat anti-rabbit IgG or goat anti-mouse IgG of caveolin-1, consistent with the large numbers of invaginated both conjugated to FITC in buffer C containing 0.5% BSA. The sections caveolae present in these cells (Carpentier et al., 1977). were rinsed in buffer C containing 0.2% BSA, mounted on slides and Interestingly, however, we found high expression in tissues photographed with a Zeiss Photomicroscope III. such as adrenal, thymus and kidney, which are composed of Light microscopic immunogold labeling cells not usually thought to have large numbers of caveolae. Heart also expressed high levels of this protein and skeletal Normal human foreskin was fixed by immersion in 3% (w/v) paraformaldehyde/0.1% glutaraldehyde in buffer C. Following muscle appeared to contain as much caveolin-1 as the adrenal. fixation and a brief rinse with buffer C, the sample was embedded The tissue distribution of caveolin-2 in mice (Fig. 1A) was in paraffin and sectioned. For immunolabeling, deparaffinized and similar to that of caveolin-1, although the staining intensity of rehydrated tissue sections were rinsed with buffer D for 30 minutes the immunoblot for each tissue was generally lower. Caveolin- at room temperature followed by incubation with pAb (10 µg/ml) 3 was most prominent in skeletal and heart tissue, although caveolin-1 IgG, or non-immune rabbit IgG (10 µg/ml) in buffer D for small intestine and lung also contained significant amounts of 18 hours at 4°C. The sections were warmed to 25°C, and processed this isoform. We were unable to detect any caveolin-3 in uterus, for streptavidin/biotin immunogold-silver labeling (Roth et al., 1992). even though this tissue is rich in smooth muscle cells. Immunoelectron microscopy We explored further the expression of caveolin-1 in skeletal and heart tissue (Fig. 1B,C) because other laboratories have Euthanized mice were perfused with 60 ml of 3% (w/v) paraformaldehyde-0.1% glutaraldehyde in buffer C for 5 minutes. reported that caveolin-1 is not expressed in muscle (Parton et Animal tissues were removed, cut into small pieces and fixed for al., 1997). The caveolin-1 pAb did not react with caveolin-3 an additional 30 minutes in the same fixative. Some samples were because the caveolin-3 pAb and the caveolin-1 pAb recognized embedded in Lowicryl K4M at low temperature as previously described protein bands with distinctly different molecular masses that (Roth, 1989). Ultrathin K4M sections were incubated for 17 hours in corresponded to the size of the respective proteins (compare left the presence of 20 µg/ml of either pAb anti-caveolin-1 or non-immune and right panels, Fig. 1B). Antibodies that recognize only α- rabbit IgG in buffer E. The IgG was localized by incubating each set of caveolin-1 (mAb 2) did not react with skeletal muscle caveolin- sections for 1 hour in protein A-gold (10 nm diameter) diluted 1:65 in 1 (Fig. 1C) while those that recognized the α and β isoforms buffer E. Electron micrographs were taken with a JEOL 1200 electron (pAb 1-3, mAb 1) demonstrated prominent staining, suggesting microscope operating at 80 kV. To localize caveolin-1 in isolated liver that only β-caveolin-1 is expressed in skeletal muscle cells. mitochondria by immunogold labeling, mitochondria were fixed, immersed in 2.3 M sucrose containing 15% polyvinylpyrrolidone Tissue cells that target caveolin-1 to caveolae (10 kDa) and processed for immunogold localization in ultrathin cryosections using pAb Caveolin-1 IgG (Tokuyasu, 1980). Cultured fibroblasts, endothelial cells and polarized epithelial cells are commonly used to study caveolin-1 targeting to CsCl fractionation invaginated caveolae. Previous studies suggest that blood Cytosol of human fibroblasts was fractionated on CsCl gradients by vessel endothelium (Esser et al., 1998; Feng et al., 1999) and standard methods (Kongshaug et al., 1989). A step gradient was gizzard smooth muscle (Chang et al., 1994) are tissue cells prepared in a Ti60 rotor tube consisting of 2 ml 1.35 g/ml CsCl at the where caveolin-1 is localized in invaginated caveolae in situ. bottom followed by 9 ml 1.21 g/ml CsCl and 7 ml 1.063 g/ml CsCl. We also found that the caveolin-1 in mouse endothelial and The gradient was overlaid with 7 ml cytosol of human fibroblasts and alveolar type I cells was concentrated in typical, flask-shaped centrifuged at 214,000 g for 23 hours before being separated into 14 or 24 fractions. From each fraction, 1 ml was precipitated by TCA caveolae (data not shown). We conclude that in situ a variety precipitation and the proteins separated by gel electrophoresis. of tissue cells have flask-shaped caveolae rich in caveolin-1 and that the antibodies used in the current study recognize caveolin- Isolation of mitochondria 1 when it is in this location. Mitochondria were isolated using the method of Weinbach (Weinbach, 1961). 1 g rat liver was homogenized with a dounce Tissue cells that target caveolin-1 to the secretory homogenizer (30 strokes) in 10 ml buffer F. The homogenate was pathway centrifuged at 610 g for 10 minutes. The supernatant was collected Previously we reported that most of the caveolin-1 in and centrifuged at 8600 g for 10 minutes. The mitochondria (pellet) pancreatic acinar cells is located in the lumen of secretory was washed with buffer F three times and processed either for vesicles (Liu et al., 1999). In these cells, caveolin-1 is immunoblotting, immunoprecipitation or immunogold labeling of ultrathin frozen sections. cosecreted with amylase through a regulated pathway. The secreted caveolin-1 is in a lipid complex that has the Other methods properties of a high-density (HDL) particle. We Immunoprecipitation of caveolin-1 from mitochondria fractions was examined other exocrine secretory cells to determine if they carried out as previously described (Liu et al., 1996). also target caveolin-1 to the secretory pathway (Fig. 2). 1400 JOURNAL OF CELL SCIENCE 114 (7)

Fig. 1. Tissue distribution of . Tissue (40 mg wet mass) was sonicated in 200 µl 2× SDS-sample buffer to solubilize proteins and inhibit proteinase activity. Proteins were precipitated with TCA, separated by SDS-PAGE and processed for immunoblotting (approx. 300 µg/lane). (A) Gels were immunoblotted with the indicated antibody or stained for protein. (B) Samples of heart, small intestine or skeletal muscle were either immunoblotted with caveolin-3 pAb alone (left side) or with a mixture of α,β-caveolin-1 pAb and caveolin-3 pAb. (C) Samples of heart, small intestine and skeletal muscle were processed for immunoblotting with five different antibodies against caveolin-1. All of the pAb plus mAb 1 react with both caveolin-1 isoforms while mAb 2 is specific for α-caveolin-1. The positions of marker proteins (kDa) are shown.

Caveolin-1 was found in secretory vesicles of mouse serous endocrine cells with the morphology of corticotropes and secreting (Fig. 2A), but not mucous secreting (Fig. 2B), cells mammatropes (Fig. 3B,C, respectively) did not contain of the salivary gland. In addition, caveolin-1 was targeted to caveolin-1. We confirmed that pituitary cells are able to secret secretory vesicles of chief cells (Fig. 2C) and the apical caveolin-1 (Fig. 3D) by culturing the pituitary cell line GH3 cytoplasm of mammary epithelial cells (compare Fig. D overnight and immunoblotting either the medium (lanes 7-12) with E). or the cells (lane 1-6) with caveolin-1 pAb. The culture HDL purified from plasma does not contain detectable medium was positive for caveolin-1, indicating that the caveolin-1 (Liu et al., 1999), which may mean that secretion endogenous caveolin-1 synthesized by these cells was secreted of caveolin-1 is restricted to exocrine cells. Indeed, we found into the medium. Unlike pancreatic acinar cells (Liu et al., very little caveolin-1 protein in hepatocytes, the major source 1999), caveolin-1 secretion was not regulated by any of the of in the body (Fig. 1). A survey of various agents we tested (Fig. 3D, lanes 8-12) and little caveolin-1 was endocrine tissues, however, revealed that anterior pituitary cells detected in the cells (lanes 1-6). We examined the subcellular with the morphologic characteristics of somatotropes had distribution of caveolin-1 in these cells by immunoblotting caveolin-1 concentrated in secretory vesicles (Fig. 3A). either the postnuclear supernatant (lane 1), cytosol (lane 2), By contrast, the secretory vesicles of neighboring pituitary plasma membrane (lane 3), non-caveolae membrane (lane 4) Tissue distribution of caveolin 1401

Fig. 2. Cells target caveolin-1 to secretory vesicles. Immunogold (A-C) and immunofluorescence (D,E) localization of caveolin-1 in mouse (A-C) and rat (D,E) exocrine cells. (A-C) Mouse salivary gland (A,B) and stomach (C) were fixed and embedded in Lowikryl K4M. Ultrathin sections were processed for immunogold localization of caveolin-1 (10 nm gold) in secretory vesicles of serous secreting cells (A), mucous secreting cells (B) and Chief cells (C). (D,E) Lactating rat mammary gland was embedded in paraffin, sectioned and processed for indirect immunofluorescence using either caveolin-1 pAb (D) or non-immune pAb (E). Bars, 0.4 µm (A-C); 5 µm (D,E). Asterisks in B indicate secretory droplets.

Fig. 3. Caveolin-1 is secreted by pituitary somatotropes. (A-C) Samples of mouse pituitary were embedded in Lowikryl K4M and processed for immunogold labeling with caveolin-1 pAb. Somatotropes (A), which can be distinguished by the large size of their secretory granules, expressed caveolin-1 and targeted it to secretory vesicles. The secretory vesicles in neighboring corticotropes (B) and mammatropes (C) did not contain caveolin-1. (D) The pituitary cell line GH3 was cultured for 18 hours in medium containing the indicated factors. Cells were separated from medium by centrifugation, the protein in both the medium and the cells TCA precipitated and the precipitates processed for immunoblotting with caveolin-1 pAb. Approximately twice as much cell precipitate was loaded on each lane than medium precipitate. (E) GH3 cells were cultured in F-12K medium plus 2.5% FBS and 15% HS. After 4 days in culture, the cells were collected and washed with F-12K medium. Cells were fractionated into the indicated fractions, the protein TCA precipitated and processed for immunoblotting using a caveolin-1 pAb. Each lane was loaded with 10 µg of protein. Bar, 0.2 µm. or caveolae membrane (lane 5) with caveolin-1 pAb (Fig. 3E). Tissue cells that target caveolin-1 to the cytoplasm The only place caveolin-1 was detected was in the cytosol A portion of the caveolin-1 expressed in cultured fibroblasts fraction (lane 2). An EM examination of these cells showed appears to be soluble in the cytoplasm of the cell that they contained few secretory granules (data not shown). (Uittenbogaard et al., 1998). We searched for tissue cells that These results suggest that most of the newly synthesized might selectively target caveolin-1 to the cytosol (Fig. 4). caveolin-1 in GH3 cells is shunted into an unregulated Initially, we focused on skeletal muscle because caveolin-3 secretory pathway. appears to substitute for caveolin-1 in invaginated caveolae 1402 JOURNAL OF CELL SCIENCE 114 (7)

Fig. 4. Identification of tissue cells that target caveolin-1 to the cytoplasm. Mouse skeletal muscle (A-D) and human skin (E-H) were embedded either in paraffin (A,B,E) or Lowikryl K4M (C,D,F-H). For immunofluorescence, paraffin sections were stained with either caveolin-1 pAb (A) or caveolin-3 pAb (B). Ultrathin sections of skeletal muscle (C,D) and skin (F-H) were processed for immunogold (10 nm gold) localization using either a caveolin-1 pAb that recognizes both isoforms (C,F,G), a caveolin-1 mAb that is specific for the α isoform (D), or a non-immune IgG (H). Paraffin sections of human foreskin (E) were processed for light microscopic immunogold labeling using caveolin-1 pAb. Arrowheads in C mark the Z-line of the sarcomere. Bar, 5 µm (A,B,E), 0.4 µm (C,D,F-H).

(Parton et al., 1997). Immunofluorescence staining of mouse The skin keratinocyte is another tissue cell that targets skeletal muscle with a pAb that recognizes both α and β- caveolin-1 to the cytoplasm. Light microscopic, immunogold caveolin-1 showed a cytoplasmic distribution of the protein staining of human skin (Fig. 4E) showed that keratinocytes with a marked striated pattern (Fig. 4A). The caveolin-3 pAb, stained positively with caveolin-1 pAb. EM immunogold by contrast, exclusively stained the cell surface (Fig. 4B). labeling of cells in the stratum spinosum (Fig. 4F) with Immunogold localization of β-caveolin-1 showed gold caveolin-1 pAb showed that gold particles were primarily particles scattered in the cytoplasm of the cell with associated with intermediate filaments that appeared to accumulations along the Z-line (arrowheads, Fig. 4C). No emanate from desmosomes. The cytoplasm of fully immunogold staining was seen with an mAb that only differentiated, cornified keratinocytes was densely labeled with recognizes α-caveolin-1 (Fig. 4D). Thus, the striated pattern caveolin-1 pAb gold particles (Fig. 4G). No gold labeling of seen by immunofluorescence appears to be due to the the cornified keratinocyte was seen with a non-immune IgG accumulation of β-caveolin-1 at Z lines. (Fig. 4H). The same result was obtained with five separate Tissue distribution of caveolin 1403

Fig. 5. Caveolin-1 expression in skin (A) and differentiating keratinocytes (B-E). (A) Samples (40 mg wet mass) of the indicated tissue were sonicated in 200 µl 2× SDS sample buffer to solubilize proteins and inhibit proteinase activity. Equal volume samples of each tissue were precipitated with TCA, separated by SDS-PAGE and processed for immunoblotting (approx. 300 µg/lane). (B) Normal human skin was processed for keratinocyte isolation as described. During the preparation of the cells, a portion was taken for RNA isolation (0 hours). The remaining keratinocytes were plated onto type I collagen- coated dishes in high calcium- containing DMEM. Total RNA was isolated from keratinocytes on collagen at the indicated times, 10 µg/lane was resolved on a formaldehyde-agarose gel, and probed with a radiolabeled caveolin-1 cDNA probe. (C) Cell lysates were prepared at the indicated times as described and 3 µg/lane of cell protein was separated by SDS-PAGE. Resolved proteins were transferred to PVDF membrane and immunoblotted with either caveolin-1, involucrin, or fillagrin pAbs. (D-E) Cultured skin equivalents were prepared as described. After 3 weeks to allow for keratinocyte differentiation and cornification, the samples were harvested and fixed in 10% neutral buffered formalin. Sections (5 µm) of paraffin-embedded tissue were processed for immunofluorescence using either caveolin-1 (D) or non-immune pAb (E). The arrows mark the boundaries of the epidermal equivalent and the asterisks indicate the collagen matrix, dermal equivalent. Bar, 5 µm. caveolin-1 antibodies that are directed against epitopes found determine if caveolin-1 expression is regulated during in both α and β caveolin-1 (data not shown). Immunoblots keratinocyte differentiation (Fig. 5B-E). Undifferentiated cells indicated that mouse skin has as much caveolin-1 on a tissue did not contain any detectable caveolin-1 mRNA (Fig. 5B, mass basis as fat (Fig. 5A). 0 hours). Caveolin-1 mRNA was detected after 4 hours of Undifferentiated human keratinocytes growing on a induction, and continued to increase for the next 44 hours. collagen matrix in culture can be induced to differentiate Likewise, we saw a progressive increase in the amount of (Pentland and Needleman, 1986). We used this system to caveolin-1 protein during differentiation, which matched the 1404 JOURNAL OF CELL SCIENCE 114 (7) increase of the two differentiation marker proteins, involucrin smooth muscle and alveolar cells, but a routine and fillagrin (Fig. 5C). Compared to day 1 cells, we estimated immunofluorescence survey indicated that mouse terminal from gel scans that there was an 8.3-fold increase in the amount airway epithelial cells expressed high levels of caveolin-1 as of caveolin-1 expression in day 4 cells. Immunofluorescence well (data not shown). We used immunogold labeling of staining of day 5 skin equivalent tissue showed abundant Lowikryl K4M embedded material to obtain high-resolution caveolin-1 staining of keratinocytes (between arrows, Fig. 5D) images of the caveolin-1 pAb labeling pattern in these cells with little staining of fibroblasts in the underlying collagen (Fig. 7). The gold was concentrated in numerous large, matrix (asterisk, Fig. 5D). No staining was observed in this membrane-bound structures that had the appearance of cultured skin model when we used a non-immune pAb (Fig. 5E). secretory vesicles (Fig. 7A). No labeling was evident at the cell The dramatic upregulation of caveolin-1 expression surface or in other compartments of these cells. The same result during keratinocyte differentiation confirms our immunohistochemistry findings that caveolin-1 is strongly expressed in suprabasal epidermal cells. In fibroblasts, soluble caveolin-1 appears to be in a complex with three heat shock proteins plus cholesterol (Uittenbogaard et al., 1998). Since caveolin-1 secreted by pancreatic acinar cells behaves like an HDL particle (Liu et al., 1999), we reasoned that the cytoplasmic caveolin-1 might also be in a lipoprotein complex (Fig. 6). We used samples of cytoplasm from normal human fibroblasts for this analysis. We found that 1-2% of the total caveolin-1 in these cells was soluble. The cytosol was loaded on the top of a CsCl gradient and centrifuged for 23 hours. 14 fractions were collected and immunoblotted with a caveolin-1 pAb (Fig. 6A, Caveolin-1). Most of the caveolin-1 was in fractions 8-12 and clearly separated from the bulk protein at the bottom of the gradient (Fig. 6B). A sample of plasma HDL was loaded on a companion gradient, fractionated and immunoblotted with an ApoA1 pAb (Fig. 6A, ApoA1). The HDL sample migrated on the gradient exactly in the same position as soluble cytoplasmic caveolin-1. One of the heat shock proteins associated with cytoplasmic caveolin-1 is cyclophilin A, which is also a prominent protein in caveolae (Uittenbogaard et al., 1998). We ran a second sample of the cytosol, this time taking 24 fractions, and immunoblotted each fraction with either caveolin-1 (Fig. 6C, Cav-1) or cyclophilin A (Fig. 6C, Cyclo-A) pAb. Remarkably, all the cyclophilin A floated in the light fractions rich in caveolin-1 (fractions 9-22). Most of the cytosolic protein, by contrast, was in the bottom six fractions of the gradient (Fig. 6D). We conclude that cytosolic Fig. 6. Soluble caveolin-1 in the cytoplasm cofractionates with plasma HDL. Normal caveolin-1, like secreted caveolin-1, behaves human fibroblasts were cultured to confluence in 150 mm plates and incubated in MEM as if it is associated with a lipoprotein particle for 20 hours. The cells were incubated in distilled water containing protease inhibitors. that has the buoyant density of HDL. The released material was centrifuged to remove cell debris and loaded onto a CsCl density gradient. Following centrifugation, either 14 (A,B) or 24 (C,D) fractions were Tissue cells that target caveolin-1 to collected and a 100 µl sample from each fraction was used for protein analysis. The mitochondria protein in a 1 ml sample of each fraction was then precipitated with TCA. The precipitates were solubilized in 60 µl of sample buffer and one half was separated by Tissue immunoblots (Fig. 1) indicated that the SDS-PAGE and immunoblotted using the indicated antibody. A sample of purified tissue with the highest caveolin-1 expression human HDL (17 µg) was processed on a companion CsCl gradient in the same level in mice was the lung. Much of the experiment and the fractions immunoblotted with an apoA 1 pAb. The protein in each caveolin-1 may be synthesized by endothelial, fraction was measured using standard techniques. Tissue distribution of caveolin 1405

Fig. 7. Targeting of caveolin-1 to mitochondria. (A-D) Samples of mouse lung were fixed and embedded in either Lowikryl K4M (A-C) or Epon-812 (D). Lowikryl K4M ultrathin sections were processed for immunogold (10 nm gold) localization of caveolin-1. Epon ultrathin sections were examined directly. Arrowheads indicate mitochondria cristae. Bars, 0.3 µm. (E) Mitochondria were isolated from liver homogenates and samples (25 µg/lane) of the initial homogenate (lane 1) and the mitochondria fraction (lane 2) were processed for immunoblot analysis of caveolin-1 (Cav-1) or cytochrome C (CytC). (F) Caveolin-1 was immunoprecipitated from fractions of mitochondria using pAb caveolin-1. Samples of the supernatant fraction (lane 1) and the pellet (lane 2) were resolved by gel electrophoresis and immunoblotted with an mAb caveolin-1. (G,H) Immunogold labeling of frozen thin sections of liver mitochondria fractions with either caveolin-1 pAb (G) or non- immune IgG (H). Bar, 0.2 µm. was obtained with several different caveolin-1 mAbs and pAbs mitochondria with attenuated cristae were easily identified in (data not shown). The location of these cells in the airway sections of Epon-embedded Clara cells (Fig. 7D, arrowheads). suggested they might be Clara cells. Previous comparative Examination of the Lowikryl K4M-embedded Clara cells at morphology studies have shown that mouse Clara cells contain high magnification showed that a double membrane was around numerous large mitochondria with attenuated cristae (Smith the caveolin-1 positive structures and that an occasional crista et al., 1979; Widdicombe and Pack, 1982). Indeed, large could be seen protruding into the matrix (arrowheads, Fig. 7C) 1406 JOURNAL OF CELL SCIENCE 114 (7) that was similar to the cristae seen in the Epon sections able to faithfully target the molecule to the secretory pathway (arrowheads, Fig. 7D). The same Clara cell also typically (Liu et al., 1999). We conclude that the molecule being contained secretory granules (Fig. 7B) that had few gold detected in these various compartments is authentic caveolin- particles. These results suggest that in Clara cells the majority 1 and that it has not been grossly altered by post-translational of the caveolin-1 is targeted to modified mitochondria, not to modification. secretory granules. We have identified four locations in the cell where caveolin- Immunogold particles were found associated with 1 can reside; caveolae, cytoplasm, mitochondria and elements mitochondria in many cell types labeled with caveolin-1 pAb of the secretory pathway. In some cells, caveolin-1 is in but the number of gold particles was very low (data not shown). multiple locations (Kurzchalia et al., 1992), or moves between We confirmed that mitochondria contain caveolin-1 by compartments in response to specific stimuli (Smart et al., isolating liver mitochondria and processing them for either 1994). The caveolin-1 that is not in caveolae or Golgi apparatus immunoblotting (Fig. 7E), immunoprecipitation (Fig. 7F) or membranes behaves as if it were soluble (Liu et al., 1999; EM immunogold detection of caveolin-1 (Fig. 7G,H). Uittenbogaard et al., 1998), which would require that the Caveolin-1 was detected in immunoblots of mitochondria hydrophobic portions of the molecule be sequestered away fractions (Fig. 7E, Cav-1), although the amount was low. The from the aqueous environment. We propose that the caveolin-1 pAb also stained a band of approx. 60 kDa in these hydrophobic regions of soluble caveolin-1 are embedded in a fractions. Most likely this band corresponds to oligomeric lipid particle surrounded by a shell that has the caveolin-1 that is sometimes seen in immunoblots because buoyant density of an HDL particle. immunoprecipitation of caveolin-1 from the mitochondria The presence of soluble caveolin-1 in a lipid complex fraction with a pAb caveolin-1 followed by immunoblotting implies that it has an important function in intracellular lipid with a mAb caveolin-1 (Fig. 7F, compare lane 1 with 2) showed transport. Previous work has shown that caveolin-1 can move both α and β caveolin-1 (Cav-1) and the 60 kDa band. between caveolae and the ER as a soluble protein in a complex Immunogold labeling of these fractions (Fig. 7G) detected with several heat shock proteins and cholesterol (Smart et al., caveolin-1 pAb reactivity in the matrix of the mitochondria, 1994; Uittenbogaard et al., 1998). Our results indicate that this just as was observed for Clara cell mitochondria (Fig. 7A). No mobile complex is a lipid particle. Thus, the soluble caveolin- staining was seen with a non-immune IgG (Fig. 7H). The same 1 that travels to ER, mitochondria and various cytosolic result was obtained with two different polyclonal antibodies locations may be embedded in a lipid particle. These particles directed against opposite ends of the molecule. Thus, low may also be able to utilize novel machinery to move across levels of caveolin-1 appear to be present in the mitochondria membrane barriers into the interior of the ER and mitochondria of many cell types but in Clara cells large amounts of the compartments. The lipoprotein form of caveolin-1 should protein are targeted to modified mitochondria that presumably be capable of delivering lipids to these intracellular are carrying out special functions for this cell. compartments, just as plasma lipoproteins carry lipids between tissues. The targeting of the particles to specific compartments may depend on the binding of caveolin-1, or associated DISCUSSION proteins, to receptors at these locations. Caveolin-rich lipid particles may belong to a family of The distribution of caveolin-1 in the various tissues we have intracellular lipid particles that have essential functions in the examined suggests an unprecedented behavior for a cellular delivery and storage of intracellular lipids. All cells contain protein. The 178 amino acid long sequence of caveolin-1 lipid particles. For example, yeast cells contain a uniform-sized predicts that it is an integral (Kurzchalia et population of lipid particles coated with specific sets of al., 1992) with both the COOH- and NH2- termini in the proteins that are essential for cell viability (Athenstaedt et al., cytoplasm. As expected of an integral membrane protein, the 1999; Leber et al., 1994). Most metazoan cells are populated caveolin-1 in caveolae is resistant to salt extraction (Rothberg with numerous large lipid droplets that function in lipid et al., 1992). Yet we found that in several different cellular storage. Many of these droplets are coated with proteins of compartments caveolin-1 is a soluble protein. For caveolin-1 unknown function such as vimentin, perilipin and ADRP to be both a soluble and a membrane protein during its lifetime, (Londos et al., 1999). Recently, MAP kinase and mechanisms must exist for converting it from one form to the phospholipase A2, two molecules involved in signal other and for moving it to specific locations in the cell. transduction, were localized to these coats (Yu et al., 1998). We suggest that caveolin-1 is a cholesterol binding Soluble caveolin-1 behaves like an apolipoprotein apolipoprotein for a smaller size of lipid particle (Murata et al., The various antibodies we used in this study recognize in tissue 1995) that shuttles cholesterol between intracellular blots a single species of protein with the same apparent compartments, including lipid storage droplets. Interestingly, a molecular mass as fibroblast caveolin-1 (Fig. 1). This suggests mutant form of caveolin-3 has been identified that associates that the immunogold and immunofluorescence staining is not with a cholesterol-rich, non-endocytic compartment that due to a cross-reacting epitope on another protein or that the resembles a lipid droplet (Roy et al., 1999). caveolin-1 in these cells has undergone a post-translational modification. There appear to be just two mRNAs for caveolin- Origin of soluble caveolin-1 1, one for the alpha and the other for the beta isoform (Ko et In vitro studies clearly indicate that caveolin-1 is al., 1998), which is consistent with the known genomic cotranslationally inserted into ER membranes (Dupree et al., structure of caveolin-1 (Engelman et al., 1998). Pancreatic 1993). Specific regions of the protein have been identified that acinar cells in culture transfected with a caveolin-1 cDNA are control its passage through ER and Golgi compartments on its Tissue distribution of caveolin 1407 way to the cell surface, all the while remaining a membrane assistance. This work was supported by grants from the National protein (Machleidt et al., 2000). Therefore, caveolae are the Institutes of Health, HL 20948, GM 52016, AR02153 and the Perot most likely site where caveolin-1 leaves the membrane and Family Foundation. enters the cytoplasm. 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