Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA
* Author for correspondence (e-mail: laing{at}id.wustl.edu)
Accepted 15 February 2005
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Summary |
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Key words: Connexin, Gap junction, ZO-1, Lipid rafts
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Introduction |
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ROS 17/2.8 is a rat osteoblastic cell line that expresses only Cx43 on the plasma membrane. We recently demonstrated that Cx45 and Cx43 associate with ZO-1 in Cx45-transfected ROS (ROS/Cx45) cells (Laing et al., 2001). ZO-1 is a member of the family of proteins called membrane-associated guanylate kinases, and it has been studied primarily in the context of tight junctions in epithelial cells and adherens junctions in non-epithelial cells. ZO-1 has at least five different domains that mediate protein-protein interactions, including three PDZ domains, an SH3 domain, and a catalytically inactive guanylate kinase (GUK) domain. These domains mediate interactions between ZO-1 and other proteins in the tight junction (ZO-2, ZO-3, occludin, JAM and claudin), or the adherens junction (
-catenin and afadin) (Gonzalez-Mariscal et al., 2000
; Yokoyama et al., 2001
). Recent studies indicated that ZO-1 specifically binds to actin and cross-links the tight junction protein occludin to the actin cytoskeleton in epithelial cells (Fanning et al., 1998
; Fanning et al., 2002
).
ZO-1 also interacts with several different gap junction proteins, including Cx43 (Toyofuku et al., 1998; Toyofuku et al., 2001
; Giepmans and Moolenar, 1998
), Cx45 (Laing et al., 2001
; Kausalya et al., 2001
), Cx31.9 (Nielsen et al., 2002
), Cx36 (Li et al., 2004a
), Cx46, and Cx50 (Nielsen et al., 2003
) and Cx47 (Li et al., 2004b
). A recent NMR-based study demonstrated that the last 20 amino acids in the carboxyl terminus of Cx43 interacts with the second PDZ domain within ZO-1 (Sorgen et al., 2004
).
Different laboratories have used different strategies to disrupt interaction between Cx43 and ZO-1. Studies in which cells were transfected with mutant connexin polypeptides that lack the carboxyl-terminal PDZ binding domain, or in which the carboxyl terminus is blocked with an added epitope, suggest gap junctions can form without an interaction with ZO-1 (Giepmans and Moolenar, 1998; Falk and Lauf, 2001
; Jordan et al., 1999
; Bukauskas et al., 2000
; Windoffer et al., 2000
; Hunter et al., 2003
). However, these gap junction plaques are abnormally large and a large percentage of the channels in these plaques are inactive, suggesting that ZO-1 may play a role in gap junction function (Bukauskas et al., 2000
; Hunter et al., 2003
). A recent study identified a frame-shift mutation in Cx43 that deletes the Cx43 ZO-1 binding site and is linked to ODDD (van Steensel et al., 2004
). In contrast, expression of a connexin-binding fragment derived from ZO-1 disrupts gap junction formation and diminishes gap junction permeability in neonatal rat ventricular myocytes and transfected HEK cells (Toyofuku et al., 1998
), through an unelucidated mechanism.
In the current study, we altered the function of ZO-1 in ROS cells by stably transfecting these cells with a connexin-binding amino-terminal fragment of ZO-1 or the full length ZO-1. We assessed the importance of the ZO-1 interaction by determining if there were any changes in connexin abundance, localization and gap junctional communication in these transfected cells.
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Materials and Methods |
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In vitro cell free peptide binding assay
The peptide-binding assay was performed as previously published (Laing et al., 2001). Briefly, radioactively labeled ZO-17-444 fusion protein was generated by in vitro transcription/translation using TNT T7 Quick coupled in vitro translation/translation system according to the manufacturer's instructions (Promega Corp., Madison WI, USA). Carboxyl-terminal peptides from Cx43 and Cx45 were synthesized previously (Laing et al., 2001
). Peptides corresponding to last 10 amino acids in the carboxyl terminus of Cx46 (amino acid residues 408-416) with an added amino-terminal cysteine residue (CRARPGDLAI) and the last ten amino acids in Cx32 (amino acid residues 274-283) (AEKSDRCAEC) were synthesized by Invitrogen, and conjugated to a Sulfolink or an Amino Link resin (Pierce Biotechnology Inc., Rockford IL, USA). These affinity resins were incubated with equal aliquots of the in vitro translated [35S]methionine-labeled ZO-17-444 fusion protein, and washed extensively with RIPA buffer (1% Triton X-100 and 0.6% SDS in PBS). The bound material was eluted by boiling for 5 minutes in 15 µl of sample buffer (2% SDS, 10% glycerol, 5% ß-mercaptoethanol, 0.1 M Tris-HCl pH 6.8) and analyzed by SDS-PAGE and fluorography.
Cell culture and transfection
ROS 17/2.8 is an osteosarcoma cell line that expresses Cx43 at the cell surface and forms functional Cx43 gap junctional channels (Steinberg et al., 1994). ROS cells were cultured in minimum Eagle's medium containing 10% heat inactivated bovine calf serum containing 2 mM glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, 5 units/ml penicillin and 5 µg/ml streptomycin. ROS cells were transfected using Lipofectamine (Invitrogen, Carlsbad, CA, USA) with the plasmid encoding the ZO-17-444 fusion protein or the ZO-1myc protein and stable clones were selected in G418 and isolated by limiting dilution. Stably transfected ROS cells were selected and cultured in the same culture medium, which contains 400 µg/ml G418. These transfectants are called ROS/ZO-1dn cells and ROS/ZO-1myc throughout this manuscript.
Immunoprecipitation
Co-immunoprecipitation studies were performed as published previously (Laing et al., 2001). Two 100-mm dishes of transfected ROS cells were washed, solubilized in immunoprecipitation (IP) buffer containing 1% Triton X-100, 0.5% CHAPS, 0.1% SDS and a 0.1% Sigma protease inhibitor cocktail in PBS, and lysates were precipitated at 4°C for 2 hours with our anti-Cx43 antibody. The antigen-antibody complexes were collected with protein A Sepharose and extensively washed with lysis buffer. Precipitated proteins were eluted by boiling in sample buffer, separated by SDS-PAGE, transferred to Immobilon-P membranes, and ZO-1 was identified by immunoblotting with 1:1000 dilution of monoclonal antibody directed against ZO-1 or the myc epitope. These blots were then probed with anti-mouse affinity purified peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) and developed with the SuperSignal West Pico chemiluminescence system (Pierce Biotechnology Inc.).
ZO-17-444 fusion protein-associated proteins were isolated from transfected ROS cells by affinity chromatography. Transfected ROS cells were lysed in lysis buffer [50 mM Tris pH 8, 5 mM MgCl2, 1% Triton X-100, 60 mM n-octyl D-glucoside and 1% Sigma protease inhibitor cocktail (v/v)]. Lysates were clarified by centrifugation and fractionated with 50 µl of a cobalt chelate affinity resin (Pierce Biotechnology Inc.), a resin that binds to 6-His modified proteins. The resins were then washed 4 times with 1 ml of lysis buffer, boiled for 5 minutes in 15 µl sample buffer and the eluted material was analyzed by immunoblotting with Cx43 antiserum. These blots were then probed with anti-rabbit affinity purified peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) and developed with the SuperSignal West Pico chemiluminescence system (Pierce Biotechnology Inc.).
Lucifer Yellow dye transfer
Gap junction permeability was assessed by microinjecting Lucifer Yellow into single cells in adherent monolayers and counting the number of neighboring cells that received dye in 3 minutes using previously published techniques (Laing et al., 1994; Koval et al., 1995
). Data was analyzed with the Student's t-test.
Immunoblotting
Cells were scraped in PBS containing 1% SDS. The samples were sonicated and the DNA was sheared by passing the lysate through a tuberculin syringe. Protein concentrations were determined with the BCA protein assay. Approximately 10 µg of protein from each of these cells were separated by SDS-PAGE and analyzed by immunoblotting with our antibody directed against Cx43 that we characterized previously (Laing et al., 1997). Aliquots of SDS extracts were also analyzed by immunoblotting with antibody directed against ZO-1 (Zymed Laboratories) and N-cadherin (Zymed Laboratories).
Biotinylation of cell surface proteins
Confluent monolayers of cells from two 100 mm plates were incubated in PBS containing 100 µg/ml EZ-link NHS-LC-Biotin (Pierce Biotechnology Inc.) at 4°C for 1 hour, and then the excess biotin was quenched by adding 5 ml 0.1 M glycine/PBS. The cells were washed 6 times in ice cold PBS, harvested by scraping and solubilized in IP buffer. Biotinylated proteins were isolated by fractionating these lysates with Neutravidin-agarose (Pierce Biotechnology Inc.), eluted by boiling for 5 minutes in 15 µl sample buffer and analyzed by SDS-PAGE and immunoblotting with our Cx43 antibodies. The blot was stripped with Restore western blot stripping buffer (Pierce Biotechnology Inc.) and reprobed with an anti-actin antibody (1:1000 dilution). Ten micrograms of ROS cell extracts were run as a positive control for Cx43 and actin expression.
Immunofluorescence microscopy
Transfected ROS cells were processed for immunofluorescence microscopy as described previously (Laing et al., 2001). The cells were fixed in a 1:1 solution of methanol and acetone for 2 minutes at room temperature, permeabilized with 1% Triton X-100, and incubated in monoclonal antibody against ZO-1, Cx43, N-cadherin, the myc or the Xpress tag epitope and the appropriate secondary antibodies. We also examined Cx43 expression in the transfected ROS cells with two different polyclonal antibodies directed against Cx43 that we have used previously (Laing et al., 1997
; Koval et al., 1995
) or a commercially available antibody directed against the carboxyl-terminal 20 amino acids of Cx43 (Sigma-Aldrich, St Louis, MO, USA). In co-localization experiments the cells were stained simultaneously with the Sigma Cx43 polyclonal antibody (1:1000 dilution) and the Zymed ZO-1 monoclonal antibody (1:1000 dilution) or N-cadherin monoclonal antibody and subsequently with Cy3-conjugated goat anti-mouse IgG antibodies and Alexa 488-conjugated goat anti-rabbit IgG antibodies.
Sucrose gradient fractionation of ROS cell lysates
Adherent cells from two 100-mm diameter plates were washed three times in cold PBS, scraped into 750 µl of MES-buffered saline (MBS, 25 mM MES pH 6.5, 150 mM NaCl) containing 1% Triton X-100, and disrupted in a tight-fitting Dounce homogenizer five times. The sample was mixed with an equal volume of 80% sucrose in MBS without Triton X-100, transferred to a 5 ml ultracentrifuge tube, and overlaid with 1.5 ml of 30% sucrose and 1.5 ml of 5% sucrose in MBS lacking Triton X-100. The samples were centrifuged for 18 hours at 200,000 g (44,000 rpm in a Sorval rotor, TH-660). Nine 500 µl fractions were collected and analyzed by SDS-PAGE and immunoblotting with antibodies directed against Cx43, ZO-1 or caveolin-1.
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Results |
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We then investigated whether the ZO-17-444 fusion protein associated with Cx43 in ROS/ZO-1dn cells. Cell lysates were passed over a cobalt chelate affinity resin and the bound and eluted material was analyzed by immunoblotting with a Cx43 monoclonal antibody. Cx43 was isolated on the affinity resin from the lysates derived from transfected ROS cells expressing larger quantities of the ZO-17-444 fusion protein (ROS/ZO-1dn clones E, J and K) (Fig. 3A).
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Expression of the ZO-17-444 fusion protein reduces gap junctional communication in ROS/ZO-1dn transfectants
Gap junctional communication was assessed in ROS and ROS/ZO-1dn transfectants by microinjecting single cells with the dye Lucifer Yellow and counting the number of cells that received dye within 3 minutes. Dye transfer was reduced in three independent ROS/ZO-1dn clones: Lucifer Yellow spread to an average of 20.6 cells per microinjected ROS cell, and to 2.2-3.4 cells per microinjected ROS/ZO-1dn transfectants (Fig. 4). Student's t-test analysis of this data showed that Lucifer Yellow transfer was reduced in all of the transfected cell lines (P<0.00).
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Expression of the ZO-17-444 fusion protein alters plasma membrane localization of Cx43
We next asked whether the reduction in gap junctional communication in ROS/ZO-1dn cells was accompanied by a reduction in the total amount of Cx43 expressed by the transfectants. SDS lysates derived from cells were analyzed by immunoblotting with our polyclonal Cx43 antiserum. These blots were then stripped and reprobed with a monoclonal antibody directed against GAPDH. Immunoblotting of SDS-derived lysates of these cells showed that there was less Cx43 in clone E but little difference in the amount of Cx43 in the lysates derived from the untransfected ROS cells and the ROS/ZO-1dn clones J and K (Fig. 5A).
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We then examined the localization of Cx43 by immunofluorescence microscopy. In these experiments transfected ROS cells were simultaneously stained with a Cx43 polyclonal antibody and a ZO-1 monoclonal antibody. As can be seen in Fig. 6, there was punctate appositional membrane and perinuclear staining for Cx43 in the untransfected ROS cells. There was very little membrane staining for Cx43 in the ROS/ZO-1dn cells, where the Cx43 staining was found in a perinuclear region. In contrast, there was little change in the appositional membrane staining for ZO-1 in the transfected ROS cells. In addition, these micrographs showed that most of the membrane-associated Cx43 co-localized with ZO-1 in the ROS cells. Immunoblotting experiments showed that there was more ZO-1 in SDS lysates derived from ROS/ZO-1dn (clone E) than in ROS cells but there was less ZO-1 in lysates derived from ROS/ZO-1dn (clone J and K; Fig. 6B). A loss of gap junctional staining was also seen in ROS/ZO-1dn clones E and K (data not shown). Immunofluorescence microscopy with three different Cx43 antibodies, the Chemicon monoclonal antibody and two different polyclonal Cx43 antibodies generated in our laboratory, also showed a loss of punctate appositional membrane staining for Cx43 in ROS/ZO-1dn cells (data not shown).
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N-cadherin localization is not altered in ROS/ZO-1dn cells
As adherens junctions may regulate gap junction formation, we determined whether the adherens junctions were altered in the ROS/ZO-1dn cells. In these experiments cells were stained simultaneously with the Sigma Cx43 polyclonal antibody and the Zymed N-cadherin monoclonal antibody. These experiments showed that there was plentiful junctional N-cadherin staining in both the ROS and ROS/ZO-1dn cells (clone E) (Fig. 8A). As we had shown before, there was a loss of most of the junctional staining for Cx43 in the ROS/ZO-1dn cells. Immunoblotting showed that there were similar amounts of N-cadherin in ROS cells and in the different ROS/ZO-1dn cells, confirming that expression of the ZO-17-444 fusion protein did not substantially alter the expression of N-cadherin in these cells (Fig. 8B). Similar staining patterns were produced with the N-cadherin antibodies in the other ROS/ZO-1dn clones (data not shown).
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Over-expression of ZO-1 enhances junctional plaques and gap junctional communication
To further examine the role that ZO-1 plays in the formation of Cx43 gap junctions, ROS cells were stably transfected with a plasmid containing the cDNA for the ZO-1 protein, with an added carboxyl-terminal myc epitope tag. Immunofluorescence analysis of the transfected ROS cells (ROS/ZO-1myc) with the myc monoclonal antibody showed that this protein was expressed at appositional membranes (Fig. 9A). Immunofluorescence analysis with the Chemicon monoclonal Cx43 antibody showed that there was decidedly more staining, both at the appositional membrane and in a perinuclear region, in the ROS/ZO-1myc cells than was seen in untransfected ROS cells. A similar increase in Cx43 appositional membrane staining was seen with the other Cx43 antibodies used in Fig. 6 (data not shown). There was also more appositional membrane staining for ZO-1 in the ROS/ZO-1 myc cells.
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Dye transfer was used to assess gap junctional communication in ZO-1myc-transfected ROS cells (Fig. 9C). In ROS cells, dye transferred to 19.2±4.5 cells per injected cell, while dye was transferred to 30.8±10.3 cells per injected cell in ROS/ZO-1myc cells. Student's t-test analysis of this data showed that expression of ZO-1myc significantly increased the permeability of the gap junctions in ROS cells (P<0.001).
We then assessed the abundance of Cx43 in the ROS/ZO-1myc cells by immunoblotting. Immunoblots of SDS extracts derived from the ROS/ZO-1myc cells showed that similar amounts of Cx43 could be found in the ZO-1myc-transfected cells, untransfected ROS cells and ROS/ZO-1dn cells (clone K) (Fig. 10A).
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We then examined how much Cx43 was found at the cell surface of ROS/ZO-1myc by subjecting these cells to cell surface biotinylation. The biotinylated material was isolated by neutravidin-agarose and subject to immunoblotting analysis with Cx43 antibody. The resulting immunoblot, Fig. 10C, shows that there was more Cx43 at the plasma membrane of the ROS/ZO-1myc cells than of the ROS or ROS/ZO-1dn cells (clone K).
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Discussion |
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The data presented in this study suggest that ZO-1 may regulate the formation of gap junction plaques. This conclusion is supported by the observations that there was a marked reduction in linear punctate staining, characteristic of gap junction plaques, in ROS/ZO-1dn transfectants; however, in ROS/ZO-1myc cells punctate appositional membrane staining was increased. This change in gap junction formation may be due to changes in the Cx43 ZO-1 interaction in the transfected cells.
The data presented in this study indicate that disruption of the Cx43 ZO-1 interaction does not substantially alter the amount of biotinylatable Cx43 found in the ROS/ZO-1dn cells, while increased expression of ZO-1 increases the amount of Cx43 at the plasma membrane. This finding suggests that transfer of Cx43 to the plasma membrane does not absolutely require an interaction with ZO-1, but is affected by changes in the abundance of ZO-1. It has been postulated that biotinylatable Cx43 is protein that is found at the plasma membrane but not incorporated into gap junctional plaques (Cooper and Lampe, 2002; Musil and Goodenough, 1991
), but the data presented here suggest that our biotinylation protocol cannot distinguish between connexins in plaques and connexins not in plaques at the cell surface.
Our finding that Cx43 is not associated with lipid rafts differs from recent studies that suggested that Cx43 is associated with lipid rafts in NIH3T3 cells (Schubert et al., 2002), lens epithelial cells (Lin et al., 2003
) and Sertolli cells (Mograbi et al., 2003
). However, our results are similar to those obtained with bronchial smooth muscle cells, where Cx43 was not associated with lipid rafts (Darby et al., 2000
). These studies suggest that different cell lines may package connexins into different plasma membrane domains. It is not entirely clear what might govern this difference in connexin packaging. One possibility is that gap junctions in cells that express more ZO-1 are associated with the actin cytoskeleton, and gap junctions in cells that express less ZO-1 are associated with lipid rafts. This hypothesis is supported by our results showing that expression of the ZO-17-444 fusion protein both disrupts the interaction between Cx43 and ZO-1 and increases the abundance of Cx43 in lipid rafts in transfected ROS cells. This leads to the possibility that the ZO-1-dependent association with the cytoskeleton may be an important step in the life cycle of gap junctions, possibly leading to the endocytosis of these gap junctions. Other explanations that may account for the lack of the lipid raft-associated gap junctions in the ROS cells could be differences in the lipid composition in the various cell lines or differences in our experimental protocol.
While our data strongly suggest that the Cx43/ZO-1 interaction plays a role in the formation of Cx43 gap junction plaques, in a number of studies altered or epitope-tagged connexins that should not bind ZO-1 have been shown to make functional gap junctions (Windoffer et al., 2000; Giepmans and Moolenar, 1998
; Jordan et al., 1999
). However, this does not eliminate the possibility that ZO-1 plays a role in gap junction formation, as recent studies suggested that Cx43-GFP fusion proteins do not interact with ZO-1 and that the aberrantly large gap junction plaques made by the Cx43-GFP constructs are mostly non-functional (Hunter et al., 2003
; Bukauskas et al., 2000
). Still, it is not clear why connexins that cannot interact with ZO-1 can form gap junctions, while blocking the interaction between Cx43 and ZO-1 with a fusion protein inhibits gap junction formation. One possible explanation for this difference is that the interaction between Cx43 and the ZO-17-444 fusion protein inhibits the formation of gap junction plaques, while gap junctions can form passively in the absence of an interaction with ZO-1.
However, we cannot formally exclude the possibility that expression of the ZO-17-444 fusion protein alters gap junction formation and function indirectly, by disrupting the interaction between ZO-1 and another protein that subsequently affects Cx43. In epithelial cells, expression of an amino-terminal fragment of ZO-1 induces a epithelial-mesenchymal transition (Ryeom et al., 2000; Reichert et al., 2000
). Similarly, expression of the amino terminus of ZO-3, which is closely related to ZO-1, disrupts junctional assembly and produces global changes in the actin cytoskeleton in transfected MDCK cells (Wittchen et al., 2000
; Wittchen et al., 2003
). In each of these studies, expression of these fusion proteins alters the expression of E-cadherin in the transfected epithelial cells. As cadherins play a role in gap junction formation (Meyer et al., 1992
), this may be a route through which Cx43 gap junction formation is altered in these cells. However, the distribution and abundance of N-cadherin was not substantially altered in ROS/ZO-1dn cells (Fig. 8). This observation, along with our previously published data that ZO-1 is the predominant connexin-binding partner in ROS cells strongly suggest that the critical interaction in gap junction formation is the association of Cx43 with ZO-1.
Thus our studies suggest the following model for the role of ZO-1 in the formation of gap junction plaques. Gap junction hemichannels are delivered to the plasma membrane and reside transiently in a lipid raft domain where they are not involved in intercellular communication. If sufficient ZO-1 is present, Cx43 hexamers will rapidly be integrated into actin bound gap junctional plaques and little Cx43 resides in lipid rafts in the steady state. If, however, all of the ZO-1 does not interact with the actin cytoskeleton, the connexin/ZO-1 complex then accumulates in a lipid raft compartment. Because ZO-1 is an actin binding protein, interactions with the actin cytoskeleton may be involved in this recruitment of connexins to gap junctions.
Furthermore, these studies highlight ZO-1 as a protein that may have an important regulatory effect on gap junctional communication. In non-epithelial cells such as ROS, ZO-1 may nucleate gap junction formation at the site of adherens junctions (Fujimoto et al., 1997). In epithelial cells, where ZO-1 is primarily associated with tight junction (Stevenson et al., 1986
), Cx43 plaques may be formed in the vicinity of tight junctions. Studies in thyroid gland epithelial cells, which express Cx43 and Cx32, have shown that the ZO-1-binding connexin Cx43 is intimately associated with tight junctions, but that Cx32, which we show here does not associate with ZO-1, forms gap junctions at the lateral membranes of these cells (Guerrier et al., 1995
). It is thus probably that the regulation of connexin trafficking and gap junctional communication by ZO-1 is different in epithelial and non-epithelial tissues.
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Acknowledgments |
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