Status of caveolin-1 in various membrane domains of the bovine lens

Experimental Eye Research 85 (2007) 473e481 www.elsevier.com/locate/yexer

Status of caveolin-1 in various membrane domains of the bovine lens Richard J. Cenedella a,*, Patricia S. Sexton a, Lawrence Brako c, Woo-Kuen Lo c, Robert F. Jacob b a

Department of Biochemistry, A.T. Still University of Health Sciences, Kirksville, MO 63501, USA b Elucida Research, Beverly, MA 01915-0091, USA c Department of Anatomy and Neurosciences, Moorehouse School of Medicine, Atlanta, GA 30310, USA Received 10 January 2007; accepted in revised form 24 May 2007 Available online 28 June 2007

Abstract Recent studies of the distribution and relative concentration of caveolin-1 in fractions of bovine lens epithelial and fiber cells have led to the novel concept that caveolin-1 may largely exist as a peripheral membrane protein in some cells. Caveolin-1 is typically viewed as a scaffolding protein for caveolae in plasma membrane. In this study, membrane from cultured bovine lens epithelial cells and bovine lens fiber cells were divided into urea soluble and insoluble fractions. Cytosolic lipid vesicles were also recovered from the lens epithelial cells. Lipid-raft domains were recovered from fiber cells following treatment with detergents and examined for caveolin and lipid content. Aliquots of all fractions were Western blotted for caveolin-1. Fluorescence microscopy and double immunofluorescence labeling were used to examine the distribution of caveolin-1 in cultured epithelial cells. Electron micrographs revealed an abundance of caveolae in plasma membrane of cultured lens epithelial cells. About 60% of the caveolin-1 in the epithelial-crude membrane was soluble in urea, a characteristic of peripheral membrane proteins. About 30% of the total was urea-insoluble membrane protein that likely supports the structure of caveolae. The remaining caveolin was part of cytosolic lipid vesicles. By contrast, most caveolin in the bovine lens fiber cell membrane was identified as intrinsic protein, being present at relatively low concentrations in caveolae-free lipid raft domains enriched in cholesterol and sphingomyelin. We estimate that these domains occupied 25e30% of the fiber cell membrane surface. Thus, the status of caveolin-1 in lens epithelial cells appears markedly different from that in fiber cells. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: caveolin-1; caveolae; plasma membrane; peripheral protein; lens epithelial cell membranes; lens fiber cell membranes; lipid-like rafts; lipid vesicles

1. Introduction Through its structural role in caveolae, caveolin-1 influences the activity of many signaling molecules (Li et al., 1995; Mineo et al., 1996; Lui et al., 1997, 2002). It also participates in the movement of cholesterol from the endoplasmic reticulum to the plasma membrane (Smart et al., 1996; Uittenbogaard and Smart, 2000) and the movement of oxidized membrane cholesterol to the golgi system (Smart et al., 1994). The cycling of caveolin between cellular compartments appears to be mediated in part by microtubules for movement of caveolin from plasma * Corresponding author. Tel.: þ1 660 626 2347; fax: þ1 660 626 2981. E-mail address: [email protected] (R.J. Cenedella). 0014-4835/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2007.05.011

membrane to internal membranes (Conrad et al., 1995) and by lipid vesicles for movement from the endoplasmic reticulum to plasma membrane (Pol et al., 2001; Robenek et al., 2004). The complexity of caveolin transport in cells may be partially due to the highly variable subcellular distribution of caveolin1 from one cell type to another. Depending on the cell type, caveolin-1 can be found principally in plasma membrane, intracellular membranes, or in the cytosol destined for secretion. For example, in cultured fibroblasts, endothelial cells and epithelial cells, caveolin-1 is found concentrated in caveolae of the plasma membrane (Li et al., 2001). In skeletal muscle under fasting conditions intracellular membranes accounted for most of the available caveolin (Munoz et al., 1996). In cultured pituitary somatotropes,

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however, cellular caveolin was contained mostly in the cytosol, where it is collected prior to secretion (Li et al., 2001). Caveolin distribution can also depend on cell treatment. Changes in laminar flow induce translocation of caveolin-1 from the plasma membrane to intracellular sites (Sun et al., 2003), heat shock treatment (43  C) of NIH3T3 cells causes an internalization of caveolin from the plasma membrane to the perinucleus (Kang et al., 2000) and growth of NIH3T3 cells to confluence promotes a shift of caveolin-1 from endogenous sites to areas of cell- to- cell contact in the plasma membrane (Volonte et al., 1999). As seen in the current study and before (Sexton et al., 2004), the caveolin-1 of confluent cultured bovine lens epithelial cells (BLEC) is also found mainly in the plasma membrane and within the membrane it appears to concentrate in cholesterol enriched domains (Rujoi et al., 2004). The goal of this study was to describe the quantitative distribution of caveolin between various subcellular domains of lens epithelial and fiber cells and the structure of fiber cell lipid rafts. In addition, a serendipitous observation that most membraneassociated caveolin may be present as peripheral protein became a topic for investigation, since caveolin-1 is typically viewed as an integral membrane protein. An average of over 60% of the caveolin-1 in the water insoluble fraction of cultured BLEC was soluble in 7 M urea, a characteristic of membrane peripheral protein. Extraction of membrane with concentrated solutions of urea is an established means of removing peripheral membrane proteins (Huber et al., 1996; Lim and Neubig, 2001; May et al., 2005). About 30% of the water insoluble fraction of BLEC was urea insoluble (intrinsic) protein and an average of 7.5% of the total cellular caveolin was recovered from cytosolic lipid vesicles. By contrast, caveolin-1 was found almost exclusively as an intrinsic protein in bovine lens fiber cell membrane, being present in caveolae-free lipid raft domains. Lens membrane lipid rafts have been investigated largely because of their relationship to connexins and gap junctions. Connexins are important to the structure of gap junctions (Goodenough, 1992) and are targeted to lipid rafts through interaction with caveolin-1 (Schubert et al., 2002). Lipid-rafts have been examined in both cultured lens epithelial cells (Lin et al., 2003; Lin and Takemoto, 2005; Perdue and Yan, 2006) and fiber cells of intact lenses (Lin et al., 2004; Zampighi et al., 2005). Interpreting the results of these studies is complicated because the structures of lipid rafts of lens epithelial and fiber cells are likely different, the fiber cell plasma membrane contains few caveolae (Lo et al., 2004), and the rat lenses used in some studies (Lin and Takemoto, 2005; Perdue and Yan, 2006) contain little to no caveolin (Lo et al., 2004 and unpublished observations).

were generally used at confluence in the 3rd or 4th passage. Cells layers from 4 to 12 dishes were scraped into 0.6 ml per dish of Buffer A (5 mM Tris, pH 8, 5 mM b-mercaptoethanol, 1 mM EDTA, 2 mM EGTA, 1 mM TLCK), plus the protease inhibitor cocktail ‘‘Complete’’, one tablet per 50 ml (Roche Diagnostics, Mannheim, Germany). Cells were Dounce homogenized on ice (50e100 strokes) and centrifuged at 100,000  g  1 h at 4  C (SW 28 Beckman rotor). The density of the recovered water soluble supernatant was adjusted to 1.2 g/ml with solid KBr and centrifuged at 100,000  g  16 h. The upper 20% and lower 80% of total volume were collected and dialyzed against large volumes of 5 mM Tris (pH 7.4). The upper phase contained lipid vesicles and the lower phase contained the bulk of water soluble proteins. The water insoluble pellet was washed with several ml of Buffer A (expelled through a 25 gauge needle and syringe) and recentrifuged (100,000  g  1 h). The pellet was then extracted with 1e2 ml of 7 M urea in Buffer A and centrifuged (100,000  g  1 h, SW60 Beckman rotor, at 4  C). The recovered urea soluble fraction (USF) was dialyzed against 5 mM Tris and the urea insoluble fraction (UIF) was washed with Buffer A, centrifuged and suspended in 200 ml of Buffer A. The four fractions (WSF d > 1.2 g/ml, WSF d < 1.2 g/ml, USF and UIF) were assayed for protein (Lees and Paxman, 1972) and 50 mg samples of protein taken for Western blotting for caveolin-1 as done before (Sexton et al., 2004; Cenedella et al., 2006). Proteins in sample buffer were generally boiled for 5 min. The polyclonal antibody used was BD Biosciences 610059 at 1/400 dilution. Caveolin-1 was detected by chemiluminescence and relative caveolin concentrations estimated by densiometric scanning of exposed X-ray film. Total relative content of caveolin-1 in each fraction was calculated accounting for the percent of the total protein represented by the 50 mg applied to the gel (see Table 1).

2. Experimental procedures

WSP 0.21 d < 1.2 USP 0.41 UIP 1.00

2.1. Fractionation of bovine lens epithelial cells Bovine eyes were obtained from a local abattoir and transported to the laboratory on ice. Primary cultures of bovine lens epithelial cells (BLEC) were established on 60 mm plastic dishes as done before (Hitchener and Cenedella, 1985). Cells

2.2. Immunoprecipitation and 2D electrophoresis of caveolin-1 multiforms Confluent layers of cultured BLEC were scraped into Buffer A (1 ml per dish, 6 dishes) supplemented with 1 mM PMSF, 1 mM Na2VO4, 5 mM NaF, plus 1 mg/ml of leupeptin Table 1 Calculation of caveolin-1 content of cultured BLEC protein fractions Fraction

a

Relative conc. Total protein (mg) Total relative % Total Cav-1 per 50 mg per fraction/50 mg content in whole content proteina fraction 533/50 (10.66)

2.24

5.3

2583/50 (51.66) 21.18 951/50 (19.02) 19.02

49.9 44.8

The relative concentration of caveolin-1 in 50 mg of protein from the low density water soluble protein (WSP d < 1.2) fraction, urea soluble protein (USP) fraction and the urea insoluble protein (UIP) fraction (from Fig. 1) multiplied by the number of 50 mg protein aliquots in the total fraction gives the total relative caveolin-1 content of each fraction.

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and aprotinin and expelled 20 times through a 25 gauge needle and syringe. The water soluble fraction recovered after ultracentrifugation (100,000  g  1 h at 4  C, Beckman SW 28 rotor) was lyophilized. The water insoluble proteins were extracted with 7 M urea and separated into urea soluble and insoluble fraction by centrifugation (100,000  g  1 h). The recovered USF was dialyzed and lyophilized, the UIF was washed with buffer A. The lyophilized WSF and USF and the pelleted UIF were dissolved in lysis buffer (Sexton et al., 2004), immunoprecipitated with rabbit anti-caveolin-1 polyclonal antibody, subjected to 2D-electrophoresis and the caveolin-1 multiforms detected by Western blotting as described before in detail (Cenedella et al., 2006). 2.3. Fractionation of bovine lenses Three decapsulated bovine lenses (about 5 g wet wt) were separated by dissection into superficial cortex, inner cortex and nucleus which accounted for an average 23%, 40% and 37% of total lens weight, respectively. The fractions were Dounce homogenized in 15e20 ml of Buffer A and separated into four fractions (WSF d > 1.2, WSF d < 1.2, USF and UIF) by differential centrifugation using the same protocol described above for fractionation of the cultured BLEC, except that proportionally larger volume of extracting and wash solutions were used. A total of 50 mg aliquots of protein from each fraction were subjected to SDS-PAGE and Western blotting. Based on the final total volume of each fraction, the protein content of each fraction, and the intensity of the caveolin-1 band seen by Western blot in the 50 mg protein aliquot, the relative content of caveolin-1 in the total WSF d < 1.2, USF and UIF was calculated. No caveolin-1 was detected in the WSF d > 1.2 g/ml. 2.4. Preparation of lipid raft domains from bovine lens fiber cell membrane

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chromatography and phospholipids fractionated by 2D thin layer chromatography were quantitated by measurement of phospholipid phosphorus as done before (Cenedella and Belis, 1981). Membrane proteins were separated by SDS-PAGE, transferred to ProBlott membrane and immunoblotted with anti-caveolin-1 antibody as described above. Membranes were stripped and reprobed with polyclonal antibody against the main intrinsic protein (MIP26) at 1/5000 dilution (antibody supplied by Kevin Schey, Medical College of South Carolina, Charleston, SC). 2.5. Double-labeling lmmunofluorescence Confluent cultures of BLEC grown on glass slides were fixed in methanol, washed in PBS, and blocked in 4% calf serum plus 1% BSA. Cells were then exposed to a 1:500 dilution of anticaveolin-1 polyclonal antibody (BD Biosciences 2297) and to a 1:500 dilution of anti-protein disulfide isomerase monoclonal antibody (CalBiochem). Cells were then exposed to a 1:1000 dilution of goat anti-rabbit IgG Alexafluor 500 plus a 1:1000 dilution of anti-mouse IgG Alexafluor 555 (Molecular Probes). After washing in PBS and mounting the slides in ProLong mounting medium, the fluorescence was viewed and recorded as described before in detail (Sexton et al., 2004). 2.6. Immunofluorescence staining of lipid vesicles The water-soluble fraction of d < 1.2 g/ml recovered from 12 subconfluent dishes of BLEC was dialyzed, concentrated by lyophilization, thawed and spread as a thin coat on a glass slide, and air dried. The dried layer was fixed in methanol, washed in PBS, blocked as described above and exposed to a 1:500 dilution of anti-caveolin-1 polyclonal antibody. Layers were then exposed to goat anti-rabbit Alexafluor mounted ProLong mounting media, and examined for immunofluorescence. 2.7. Transmission electron microscopy

The total water insoluble fraction recovered from superficial cortex of 6 lenses was washed in TNE buffer (25 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.6) (Rujoi et al., 2004). The water insoluble pellet was extracted with 4 volumes of TNE buffer containing either1% (w/v) Triton X-100 alone or 1% Triton X-100 plus 35 mM octyl-b-D-glucopyranoside and 600 mM NaCl (Baron and Coburn, 2004). The membrane was kept on ice for 40 min and periodically Dounce homogenized. Two ml of the sample was mixed with 2 ml of 2.5 M sucrose in TNE and overlaid with a linear sucrose gradient (30%e6%). The gradient was centrifuged at 100,000  g for 22 h in a Beckman SW-28 rotor at 4  C. One ml fractions were collected, top-down, yielding 17 fractions. Each fraction was mixed with 1.5 ml of TNE buffer and centrifuged at 140,000  g  2 h in a Beckman SW-60 rotor. The resulting pellets were suspended in 100e200 ml of TNE buffer and aliquots taken for protein and lipid assays, immunoblotting, microscopic examination and X-ray diffraction analysis. Protein was assayed by a modified Lowry assay (Lees and Paxman, 1972). Cholesterol was measured by gas

BLEC cultured on glass slides were fixed in 2.5% (w/v) glutaraldehyde e 0.1 M cacodylate buffer, postfixed in aqueous OsO4 and examined for the presence of caveolae using a JEOL 1200EX electron microscope as recently described in detail (Lo et al., 2004). The high buoyancy-lipid raft fractions collected following centrifugation of the detergenttreated cortical membrane (see above) were diluted in TNE buffer and pelleted by centrifugation at 140,000  g  2 h in a Beckman SW-60 rotor. Pellets were fixed in the glutaraldehyde-cacodylate, sliced, and structure viewed by electron microscopy as described for intact lenses (Lo et al., 2004). 3. Results 3.1. Distribution of caveolin-1 in bovine lens epithelial cells The relative concentration of caveolin-1 in the various BLEC protein fractions was estimated from the immunoreactivity

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(evaluated by chemiluminescence) of caveolin-1 in 50 mg of protein separated by SDS-PAGE. Although the concentration of caveolin-1 was highest in the integral membrane protein fraction (urea insoluble protein, UIP) of BLEC (Fig. 1), the peripheral protein (urea soluble protein, USP) accounted for more of the total caveolin-1 content of these cells due to its greater mass. As illustrated by the sample calculation shown in Table 1, the USP of BLEC accounted for more than 70% of the total water insoluble protein (WIP) and about 50% of the total caveolin-1content. The average cellular content of caveolin-1 measured in four separate experiments was 62  7% (SEM) in USP and 30  6% in UIP. The remaining caveolin-1 of BLEC (about 8%) was recovered in the low density cytosolic fraction as a component of lipid vesicles. Examination of this fraction by fluorescence microscopy revealed abundant vesicles as large as 100 nm (data not shown). No detectable caveolin-1 was present in the truly water soluble fraction (i.e., the fraction recovered above density 1.21 g/ml). Two dimensional immunoblots of the three caveolin-1 containing fractions showed similar multiform patterns for caveolin-1 from USF and UIF (4 spots) as compared with the caveolin-1 found in the lipid vesicle fraction (2 spots) (Fig. 2). The multiforms all possess the same apparent molecular weight but differ in isoelectric point pH values. The multiforms reacted with neither caveolin specific anti-phosphotyrosine pY14 nor anti-phosphotyrosine antibody (Cenedella et al., 2006). We speculate that these multiforms represent variability in posttranslational modifications, including variable palmitoylation. Double immunofluorescence labeling was used to detect the distribution of caveolin-1 and anti-protein disulfide isomerase, a marker for endoplasmic reticulum, in confluent BLEC. The results revealed little or no colocalization of caveolin-1 with endoplasmic reticulum (Fig. 3). As seen earlier (Sexton et al., 2004), caveolin-1 was present mainly in cell borders indicating its presence in the plasma membrane.

kDa

WSP d<1.2

WIP

USP

UIP

0.21

0.79

0.41

1.00

Cav. Std

30

20.1 Relative Conc.

Fig. 1. Distribution of caveolin-1 in cellular fractions of cultured bovine lens epithelial cells (BLEC). Cells were homogenized and crude membrane recovered after ultracentrifugation. Lipid vesicles were recovered from the water soluble supernatant following adjusting density to 1.2 g/ml and ultracentrifugation. Water insoluble protein (WIP) was divided into 7 M urea soluble and insoluble protein fractions. Aliquots of protein (50 mg/lane) were Western blotted with BD polyclonal antibody for caveolin-1 and detected by chemiluminescence. The urea insoluble protein (UIP) gave the greatest reaction and, therefore, served as the reference sample. WSP d < 1.2 ¼ water soluble fraction of density less than 1.2, WIP ¼ total water insoluble protein, USP ¼ urea soluble protein. The caveolin-1 standard is from human endothelial cells (BD Transduction Labs).

IEF PAGE 22 kDa

WSF

22 kDa

USF

22 kDa

UIF

multiform ~pH

4.5

4

3

2

1 5.7

Fig. 2. Two-dimensional electrophoresis of caveolin-1 ‘‘multiforms’’ from the WSF (d < 1.2 g/ml), USF, and UIF of confluent BLEC. Multiforms of 22 kDa caveolin-1 display multiple isoelectric pH values. These multiple forms are suggested to reflect variable posttranslational modification of caveolin-1 (Cenedella et al., 2006). Caveolin-1 immunoprecipitated from the water soluble fraction (WSF), urea soluble fraction (USF) and urea insoluble fraction (UIF) was subjected to 2D-electrophoresis, transferred to ProBlott membrane and the multiforms detected by chemiluminescence following exposure to the BD caveolin-1 antibody. Reactive proteins detected at 22 kDa for each of the three protein fractions (WSF, USF, UIF) are shown for the three-2D separations.

This observation is consistent with the presence of abundant caveolae in the plasma membrane of the BLEC (Fig. 4). 3.2. Concentration and distribution of caveolin-1 in bovine lens regions After removal of the capsule with its adhering epithelial cell layer, the bovine lens body was divided into superficial cortex (23% of lens weight), inner cortex (40% of lens weight) and nucleus (37% lens weight). Each lens region was then divided into water soluble, urea soluble and urea insoluble fractions. The urea insoluble fraction is the fiber cell plasma membrane with its intrinsic proteins (Russell, 1981). No caveolin was detected with the water soluble fraction of density <1.21 g/ml (would contain lipid vesicles) in any lens region (Fig. 5). Although the immunoreactivity of caveolin-1 in 50 mg of urea soluble proteins from the superficial cortex was weak relative to that in 50 mg of urea insoluble protein (Fig. 5), after accounting for the total mass of the urea soluble proteins, the urea soluble fractions represented about 30% of the total caveolin-1 in this lens region. In contrast to the bovine lens epithelial cells where most of the caveolin was extracted with urea from the total water insoluble protein, all of the caveolin-1 of the lens inner cortex and nucleus remained in the urea insoluble fraction and therefore represented intrinsic membrane protein (Fig. 5). But, the relative concentration of caveolin-1 in the fiber cell membrane appeared much lower than in the lens epithelial membranes. Caveolin-1 in 50 mg of urea insoluble protein from the lens epithelial cells gave 5e6

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Fig. 3. Double immunofluorescence labeling of cultured BLEC for caveolin-1 and endoplasmic reticulum. Confluent cultures of bovine lens epithelial cells grown on glass slides were exposed to rabbit anti-caveolin-1 polyclonal antibody and mouse anti-protein disulfide-isomerase (PDI, a resident protein of endoplasmic reticulum), monoclonal antibody. Fluorescent secondary antibody detected caveolin-1 as green (B) and PDI as red (A). Merged fields are shown in panel C.

times more reactivity than the same mass of urea insoluble protein from fiber cells of the three lens regions (Fig. 6). 3.3. Bovine lens lipid rafts Because caveolin-1 is present in plasma membrane as part of lipid rafts (Lui et al., 2002; Rujoi et al., 2004; Stan, 2005; Hardin and Vallejo, 2006) and since most of the lens caveolin is an integral membrane protein of the lens fiber cell, we examined lens fiber cell membrane for the presence of caveolin-containing lipid rafts. When we applied a typical approach to isolation of lipid rafts from lens membrane (Rujoi et al., 2004), treatment with 1% Triton-X 100 at 4  C and recovery of the resulting high buoyancy fraction from a continuous sucrose gradient, only trace amounts of caveolin and cholesterol were recovered from the low density fractions (Fig. 7). Baron and Coburn similarly obtained poor recovery of lipid rafts from highly ordered trachea smooth muscle cells. However, when these cells were treated with 35 mM octyl-b-D-glucopyranoside (octylglucoside) and 600 mM NaCl in addition to the 1% Triton X-100 greatly increased amounts of rafts were recovered (Baron and Coburn, 2004). Essentially all of the caveolin-1 was recovered from the high buoyancy fractions when this method was applied to preparation of lipid-raft domains from the lens fiber cell membrane (Fig. 8). These fractions were also enriched in cholesterol and phospholipids and the phospholipids were almost

two fold enriched in sphingomyelin, from 18e19% to 34% (Fig. 9). The lipid-raft fractions (gradient fractions 3e8 combined) accounted for between 24 and 29% of the total cholesterol and phospholipids separated by the gradients. Aliquots of the high buoyancy fractions were centrifuged, washed and fixed for examination by electron microscopy. Unilamellar vesicles of between about 100 and 400 nm were seen that contained no apparent caveolae (data not shown). 4. Discussion The ocular lens consists of a monolayer of epithelial cells which cover the anterior surface. These cells are attached to a basement membrane which encapsulates the lens. Epithelial cells in a germinative zone of the epithelia undergo migration and differentiation into vastly elongated fiber cells which Lens Fraction SC

N

IC

Exp. A WSF USF UIF WSF USF UIF WSF USF UIF Cav. Std

22 kDa

SC Exp.B

WSF

USF

IC UIF

WSF USF UIF

N WSF USF UIF Cav. Std

22 kDa

Fig. 4. Identification of caveolae in cultured BLEC by transmission electron microscopy. Cells cultured on glass sides were fixed and examined for the presence of caveolae using a JEOL 1200EX electron microscope. Arrows locate caveolae. Bar ¼ 200 nm.

Fig. 5. Distribution of caveolin-1 between multiple fractions from various lens regions. Decapsulated bovine lenses were divided into superficial cortex (SC, 23% lens wt.), inner cortex (IC, 40% lens wt.) and nucleus (N, 37% lens wt.). 50 mg aliquots of protein from the recovered water soluble fraction, d < 1.2 g/ ml (WSF), urea soluble fraction (USF) and urea insoluble fraction (UIF) were separated by SDS-PAGE and immunoblotted with rabbit anti-caveolin-1 polyclonal antibody. Caveolin-1 was detected by chemiluminescence. The contribution of each fraction to the total relative content of caveolin-1 in the lens was determined from the relative concentration of caveolin-1 in the 50 mg of protein determined by densiometric scanning of exposed X-ray film and the total protein content of each fraction. The results from 2 separate experiments (A and B) are shown.

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Membrane Source std. kDa

BLEC

SC

IC

N

97 66 45 30

20 Relative Conc.

1.00

0.15

0.15

0.20

Fig. 6. Relative concentration of urea insoluble caveolin-1 (intrinsic protein) in membrane of bovine lens epithelial cells and in fiber cell membrane from bovine lens superficial cortex (SC), inner cortex (IC) and nucleus (N). Each lane contained 50 mg of membrane protein. Caveolin-1 was detected by chemiluminescence following exposure to rabbit anti-caveolin polyclonal antibody (BD Biosciences, 1/400). Relative concentration of caveolin-1 was estimated from densiometric scanning of exposed X-ray film. The caveolin signal from the BLEC was set at 100% of scale.

eventually contain plasma membrane as the only subcellular organelle. This process continues throughout life with one layer of fiber cells overlaying another. The outer part of the lens (about 60% of fresh weight) is the cortex and the inner and older part consisting of tightly assembled fiber cells is the nucleus. Synthesis of proteins and lipids is confined to about the outer 10% of the lens radius (Cenedella, 1993) due to loss of organelles (Bassnett, 1997). The principal observation of the present study is that 50% or more of the total caveolin-1 present in bovine lens epithelial cells is soluble in concentrated urea but little to no caveolin of bovine fiber cells is urea soluble. This indicates that most of the membrane associated caveolin of the lens epithelial cell is present as extrinsic protein and essentially all of the caveolin-1 of lens fiber cells is intrinsic protein. Extraction of total membrane with 7 M urea is an established method for removing non-integral proteins from plasma membrane (May et al., 2005). For example, extraction of CHO cell membranes with 7 M urea caused a 56% reduction of total membrane protein (May et al., 2005). Since a domain of caveolin-1 (residues 102e122 out of 178) has been shown to integrate into the cytoplasmic leaflet of plasma membrane (Spisni et al., 2005; Williams and Lisanti, 2004), one would not predict that this caveolin can be extracted with concentrated urea solutions. The water insoluble fraction of cultured bovine lens epithelial cells would contain total cellular membrane, including that of the endoplasmic reticulum. However, the caveolin present in

lens epithelial cells appears to be largely plasma membrane associated since double immunofluorescence labeling showed caveolin to be mainly located at the cell surface and failed to demonstrate co-localization of caveolin and endoplasmic reticulum (Fig. 3). What might explain the urea solubility of most of the water insoluble caveolin of BLEC? Although classified as an integral membrane protein, perhaps caveolin is partially extractable because it is shallowly inserted into membrane through only 21 amino acid residues. The principal argument against this possibility is that 7 M urea did not similarly extract caveolin from the fiber cell plasma membranes of the inner cortex or nucleus of bovine lenses where it is an integral protein. Integral proteins are typically resistant to urea extraction. For example, urea failed to extract integral lipid-anchored G-proteins from Sf9 insect cell membrane (Lim and Neubig, 2001), transient receptor potential protein from fly photoreceptor membrane (Huber et al., 1996) or rhodopsin from retinal rod outer-disk membrane of bovine retina (Willardson et al., 1993). Caveolin monomers readily associate into heptameric oligomers through interactions between their a-helices and with each monomer inserted into the membrane (Fernandez et al., 2002). Perhaps the urea soluble caveolin reflects binding of peripheral caveolin to integral caveolin oligomers solely through a-helix-a-helix interactions without membrane insertion. Why should two populations of caveolin-1 behave differently since we assume that the urea soluble and urea insoluble caveolin possess identical structure? The similarity in the isoelectric profiles of 2D separated urea soluble and insoluble caveolin-1 of BLEC further indicates that even posttranslational modifications of the two populations of caveolin-1 are very similar, if not identical (Fig. 2). The binding of alphacrystallin to lens fiber cell membrane in aging and cataract formation may provide some insight into caveolin-1 binding. In aging and cataracts there is extensive binding of alpha crystallin, one of the lens’ major water-soluble proteins, to the plasma membrane (Chandrasekher and Cenedella, 1997; Cobb and Petrash, 2002). Alpha crystallin is envisioned to bind as an aggregate of monomers, although only a fraction of the monomers is in direct contact with the membrane (Chandrasekher and Cenedella, 1997). In contrast to the epithelial cells, urea soluble caveolin was found only in fiber cells of the lens outer cortex where it accounted for about 30% of the total caveolin-1. The remaining caveolin in this and deeper lens regions was urea insoluble. The disappearance of urea soluble caveolin could reflect either loss by degradation or salvage for lipid-raft formation. Because the concentration of caveolin-1 was many fold greater in epithelial cell than fiber cell membrane, it seems likely that the caveolin present in the fiber cells largely reflects caveolin imported into the lens with differentiated epithelial cells rather than caveolin generated by de novo synthesis in the cortex. Regardless of the source, caveolin-1 becomes integrated into lipid raft domains of the fiber cells since it is confined to high buoyancy membrane fractions that are also enriched in cholesterol and sphingomyelin, signature lipids of liquid-ordered membrane domains (Baron and Coburn, 2004). Since the high buoyancy

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Fig. 7. Extraction of bovine lens cortex membrane with Triton X-100 yielded low amounts of lipid raft-like fractions. A. Sucrose gradient of bovine lens cortex water insoluble protein (crude membrane) was treated with 1% (w/v) Triton X-100. One ml fractions were collected from the top down. B. Cholesterol was present in only the pellet and in the higher density, low buoyancy, fractions. C. Distribution of caveolin-1 and MIP 26 between the membrane fractions. One-tenth of each fraction was mixed with an equal volume of 2 sample buffer, proteins separated on 13% SDS-PAGE gels and transferred to ProBlott membrane. Samples were first immunoblotted with caveolin-1 antibody, the membranes stripped and reprobed with MIP 26 antibody.

fractions contained about 25e30% of the total membrane cholesterol and sphingomyelin and almost all of the caveolin-1, the caveolin-rich lipid rafts appear confined to a subdomain which occupies perhaps 25e30% of the fiber cell membranes’ total

surface area. We recognize that detergent solubilization of membranes may lead to lipid reorganization that could result in overestimating the abundance of lipid-ordered domains (Edidin, 2003). However, membrane fractions isolated by

Fig. 8. Large amounts of high buoyancy, low density, lipid raft-like fractions were recovered from crude membrane of bovine lens cortex. A. Sucrose gradient of membrane treated with buffer containing 1% Triton X-100, 35 mM octylglucoside and 600 mM NaCl. B. Distribution of cholesterol, phospholipid and total protein between the 17 fractions. C. Distribution of caveolin-1 and MIP 26 among the membrane fractions recovered from the sucrose gradient. Gels were prepared as described in Fig. 1.

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Fig. 9. Sphingomyelin was enriched in the low density, high buoyancy membrane fractions. Phospholipids extracted from aliquots of the total crude membrane and from each of four high buoyancy fractions (4, 5, 6 þ 7, and 8) recovered from a gradient after detergent treatment were individually separated by 2D thin layer chromatography and the recovered sphingomyelin (SPH), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) quantitated by measuring phospholipid phosphorus. Open bars show the distribution of phospholipids in the total crude membrane. The distribution of phospholipids in the total membrane from a second experiment was 18% SPH, 48% PC, 14% PS and 20% PE. Filled bars show the average (SEM) composition in the four low density fractions.

detergent-free methods can be similar to those using detergentbased methods (Smart et al., 1995; Macdonald and Pike, 2005). Although lipid rafts of lens fiber cells are likely quite different than those associated with the epithelial membrane because caveolae are lost upon epithelial cell differentiation, the fiber cell rafts are nevertheless highly organized. Small angle X-ray analysis of lipid-rafts prepared from bovine lens cortical membrane revealed three periodicities of 33, 60 and ˚ , with the largest periodicity suggested to be a caveolin85 A containing, annular lipid domain (Jacob, unpublished observations). Whether these caveolin containing domains have functional significance to the lens [for example, in connexin trafficking or support of fiber junctions, gap junction-like structures which communicate fiber cells (Kistler et al., 1995)] or simply reflect the need to warehouse an obsolescent molecule is unknown. Acknowledgments Kendrick Labs (Madison, WI) and Jon Johansen are thanked for conducting the two-dimensional electrophoresis and Kevin Schey (Medical College of South-Carolina, Charleston, SC) is thanked for the antibody against MIP 26. This work was supported in part by the National Institutes of Health (EY02568). References Baron, C.B., Coburn, R.F., 2004. Smooth muscle raft-like membranes. J. Lipid Res. 45, 41e53.

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