ARTICLE |
Correspondence to: Roland Govers, Garvan Inst. of Medical Research, 384 Victoria St., Darlinghurst NSW 2010, Australia. E-mail: r.govers@garvan.org.au
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Summary |
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Caveolin-1 is a member of a subset of intracellular proteins that regulate endothelial nitric oxide synthase (eNOS) activity. In caveolae, caveolin-1 inhibits eNOS activity via a direct interaction with the enzyme. Previous work has indicated that both eNOS and caveolin-1 are also localized at the perinuclear Golgi complex. Whether caveolin-1 is involved in eNOS regulation in this cell compartment is unknown. Here we studied the localization of eNOS and caveolin-1 in the perinuclear region of primary bovine aortic endothelial cells. By immunofluorescence microscopy we show that both eNOS and caveolin-1 co-localize with Golgi markers. On treatment of the cells with the microtubule-depolymerizing drug nocodazole, the Golgi complex is scattered and caveolin-1 is found in vesicles at the periphery of the cell, while eNOS is localized at large structures near the nucleus. The nocodazole-induced redistribution of eNOS is similar to that of cis-, medial-, and trans-Golgi markers, while the caveolin-1 redistribution resembles that of sec22, a marker for the intermediate compartment. The localization of eNOS and caveolin-1 at distinct perinuclear compartments that behave differently in the presence of nocodazole indicates that eNOS activity is not regulated by caveolin-1 in the Golgi complex.
(J Histochem Cytochem 50:779788, 2002)
Key Words: eNOS, caveolin-1, endothelium, immunolocalization
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Introduction |
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THE ENZYME endothelial nitric oxide synthase (eNOS) generates nitric oxide (NO), which is essential for vascular function (-subunits (
At present it is not clear whether eNOS that is localized at the Golgi complex is active or might be activated by specific stimuli. If Golgi-resident eNOS can be activated, it may also be associated with caveolin-1. Caveolin-1 is reported to be present at the Golgi complex and is believed to recycle between this organelle and the caveolae at the plasma membrane (
In this study we used the microtubule-depolymerizing agent nocodazole to determine whether eNOS and caveolin-1 are localized at the same subcompartment of the Golgi. By depolymerizing the microtubules, nocodazole induces fragmentation of the Golgi complex (
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Materials and Methods |
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Materials
BAECs were purchased from Clonetics (San Diego, CA). Normal goat serum and secondary fluorescent antibodies were obtained from Jackson Immunochemicals (West Grove, PA). Monoclonal anti-eNOS antibody (immunofluorescence) was purchased from BioMol (Plymouth Meeting, PA) and monoclonal anti-eNOS (immuno-EM), anti-caveolin-1 (#C37120), and anti-GM130 from Transduction Laboratories (San Diego, CA). Polyclonal anti-caveolin-1 (#C13630), anti-myc, and anti-mannosidase II antibodies were obtained from Transduction Laboratories (San Diego, CA), Upstate Biotechnologies (Lake Placid, NY), and K.W. Moremen (University of Georgia; Athens, GA), respectively. Polyclonal anti-GOS-28 antibody was raised in rabbits using the His6-tagged cytoplasmic portion of GOS-28 (His6-GOS-28-TM) as immunogen (
-2,6-sialyltransferase cDNA in SMH4 vector was kindly provided by Sean Munro (MRC, Cambridge, UK). Nocodazole was obtained from Sigma (St Louis, MO).
Cell Culture
Primary BAECs were cultured according to the supplier's instructions. BAECs were cultured in EGM BulletKit medium (Clonetics), split 1:6 on reaching confluence, and used between passages 3 to 7. For immunofluorescence, cells were grown on glass coverslips in 24-well dishes. The different passage number of the cells used for the experiments did not affect the localization of any of the studied proteins. All experiments were performed at 37C. Because of a better morphology, cells were used at a subconfluent stage. Cell confluence did not affect the Golgi localization of any of the proteins described in this study.
For transfection of BAECs with the eukaryotic expression vector pcDNA3.1 (Invitrogen; Carlsbad, CA) containing myc--2,6-sialyltransferase cDNA (cloned in HindIII/XbaI sites), cells were incubated with DNALipofectamine complexes according to the supplier's instructions (Life Technologies; Rockville, MD).
Immunofluorescence Microscopy
BAECs were immunostained by indirect fluorescent labeling. Cells were incubated with 20 µM nocodazole for the time periods indicated. To study the reversibility of the nocodazole-induced Golgi scattering, cell cultures were incubated with nocodazole for 60 min, washed once with preconditioned medium without nocodazole, and incubated with medium for the indicated periods of time.
After nocodazole incubation, cells were fixed with 3% paraformaldehyde, permeabilized, blocked with normal goat serum, incubated with primary antibodies for 60 min, washed three times with PBS, incubated with secondary antibodies (goat anti-rabbitFITC and goat anti-mouseTexas Red) for 30 min, washed three times with PBS, and embedded in Mowiol. Confocal laser scanning microscopy was performed using a Leica TCS 4D system. FITC and Texas Red label were scanned sequentially and overlaid using Adobe Photoshop software. Control cells that were labeled with the primary or secondary antibody only showed no fluorescence (not shown).
Immunoelectron Microscopy
BAECs were fixed by mixing the culture medium 1:1 with double-strength fixative (4% paraformaldehyde, 2% acrolein in 0.1 M sodium phosphate buffer, pH 7.4) at ambient temperature. After 4 min the mixture was replaced by single-strength fixative (2% paraformaldehyde, 1% acrolein in phosphate buffer) and fixation proceeded for 2.5 hr at room temperature. Then cells were scraped and processed for ultracryotomy and immuno-EM as described (60-nm-thick cryosections were thawed and immunolabeled for eNOS by incubating them with a monoclonal anti-eNOS antibody, rabbit anti-mouse IgG, and protein Agold successively. Sections were then stained with uranyl acetate and dried in methylcellulose. Control cryosections that were labeled with secondary antibody and protein Agold only showed no labeling (not shown).
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Results |
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Immunofluorescence observations revealed a typical perinuclear localization for eNOS (Fig 1A). By immuno-EM these eNOS-positive areas were recognized as the Golgi apparatus, in which eNOS appeared to be distributed in a rather diffuse fashion over all recognizable Golgi elements, including the cisternae and vesicular/tubular membranes in their surroundings (Fig 1C). Anti-caveolin-1 antibody gave identical perinuclear labeling patterns by immunofluorescence as eNOS (Fig 1B). However, immuno-EM was not successful for caveolin-1 localization, which is probably due to differences in the processing of the cells. Therefore, nocodazole in combination with immunofluorescence was used to study the localization of eNOS and caveolin-1 in detail and to see whether these proteins are actually present at the same subcompartment of the Golgi. BAECs were treated with nocodazole for various periods of time, after which the cells were fixed, labeled with monoclonal antibodies against eNOS and caveolin-1, and analyzed by confocal immunofluorescence microscopy (Fig 2). An antibody against the Golgi SNARE [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor] GOS-28 (
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The reversibility of the drug treatment was also studied. BAECs were incubated for 1 hr with nocodazole, washed, and incubated for various time periods in the absence of nocodazole (Fig 3). eNOS and GOS-28 returned to their original perinuclear Golgi-like distribution between 15 and 60 min after drug removal. Most of the caveolin-1 co-localized with GOS-28 (and therefore also with eNOS) within 10 minutes after the nocodazole was removed from the cells. This co-localization was complete after 15 min. This clearly demonstrates that on nocodazole removal, the caveolin-1-positive vesicles quickly redistribute from the cell periphery towards the large nocodazole-induced eNOS- and GOS-28-containing Golgi fragments before these structures regain their original perinuclear morphology.
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To define the Golgi subcompartments in which eNOS and caveolin-1 are localized, BAECs were incubated for 1 hr with nocodazole and labeled with monoclonal antibodies directed against eNOS, caveolin-1, and Golgi matrix protein GM130 (
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Because our data indicated that caveolin-1 was not present in Golgi stacks or TGN, BAECs were double labeled with antibodies against caveolin-1 and sec22 (Fig 4G). Sec22 is a marker for the intermediate compartment (IC). The sec22 antibody labeled the Golgi region as well as peripheral vesicular structures, most likely representing vesicular tubular clusters (VTCs) near the Golgi and peripheral VTCs adjacent to ER exit sites, respectively (
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Discussion |
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The data presented here demonstrate that eNOS and caveolin-1 are localized at distinct perinuclear cell compartments that behave differently when exposed to the Golgi-disrupting drug nocodazole. A 1-hr incubation of BAECs with nocodazole results in a shift in the cellular distribution of eNOS and caveolin-1 from a dense perinuclear Golgi-like structure into a vesicular staining pattern, with virtually no overlap between eNOS and caveolin-1. The caveolin-1-positive vesicles, in contrast to the structures containing eNOS, are highly dynamic. Their formation is initiated after 5 min of drug treatment and within 10 min after nocodazole removal most of these vesicles can no longer be distinguished from eNOS-containing structures. Unfortunately, it was impossible to confirm these data by immuno-EM because in these cells none of the caveolin-1 antibodies that were tested labeled caveolin-1 in or near the Golgi complex using immuno-EM techniques.
The Golgi markers GOS-28, GM130, mannosidase II, -2,6-sialyltransferase, and the IC marker sec22 were studied to determine the perinuclear (sub)compartments where eNOS and caveolin-1 are localized. The Golgi v-SNARE GOS-28 is localized predominantly at vesicles at the terminal rims of the Golgi stacks, where it is involved in intra-Golgi transport (
-2,6-sialyltransferase are enzymes involved in carbohydrate modification and are localized at the cis/medial-Golgi and trans-Golgi/TGN, respectively (
The Golgi localization of eNOS is dependent on its first 35 amino acid residues (
Previous studies have shown that in BAECs eNOS is activated by shear stress (
Our studies indicate that eNOS and its inhibitor caveolin-1 do not co-localize within the Golgi complex. Because Golgi-resident eNOS is probably not involved in NO production, this may imply the presence of other regulatory proteins in the Golgi complex that reduce eNOS activity. An important step in understanding the complex cellular regulation of eNOS will now be to reveal the mechanism by which eNOS activity is regulated in this cell compartment.
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Acknowledgments |
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Supported by grants to R. Govers from the Netherlands Organization for Scientific Research (NWO; 902-26-224) and from the Netherlands Heart Foundation (99.041).
We thank Dr Richard Scheller for the anti-sec22 antibody, Dr Sean Munro for the myc--2,6-sialyltransferase cDNA-containing SMH4 plasmid, and Dr Judith Klumperman for stimulating discussions.
Received for publication December 5, 2001; accepted January 9, 2002.
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