Correspondence to John G. Collard: j.collard{at}nki.nl
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Irene H.L. Hamelers's present address is Dept. of Biochemistry of Membranes, Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, Netherlands.
Cristina Olivo's present address is Dept. of Hematology, University Medical Center Utrecht, 3508 GA Utrecht, Netherlands.
Abbreviations used in this paper: Col IV, collagen IV; ERK, extracellular-signal regulated kinase; FN, fibronectin; GEF, guanine nucleotide exchange factor; LN, laminin; NPAG, p-nitrophenyl N-acetyl-ß-D-glucosaminide; PAK, p21-activated kinase; PLL, poly-L-lysine; Tiam1, T-lymphoma invasion and metastasis 1; Tiam1/, Tiam1-deficient; VN, vitronectin; WT, wild-type.
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Introduction |
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Small GTPases of the Rho family are involved in downstream signaling of a large number of growth factor receptors and adhesion molecules, including integrins. Integrin-induced activation of Rho proteins regulates many different processes, ranging from cell survival, adhesion, and spreading to the secretion of matrix proteins and their assembly into a basement membrane. The linkage of the ECM receptors to the actin cytoskeleton is crucial for cell matrix assembly (Bishop and Hall, 2000). Through Rho proteinmediated cytoskeletal remodeling and contraction, cells are able to remodel the ECM (Chiquet et al., 1996; Wierzbicka-Patynowski and Schwarzbauer, 2003). In fibroblasts, Rho and Rho kinase activation are necessary for the proper organization of a fibronectin (FN) matrix (Danen et al., 2002), whereas Rac has been implicated in LN1 assembly and in apical pole orientation in epithelial cells (O'Brien et al., 2001; DeHart et al., 2003).
Previously, we identified the Rac activator Tiam1 (T-lymphoma invasion and metastasis 1), which plays an important role in Rac-mediated E-cadherinbased cellcell adhesions (Michiels et al., 1995; Hordijk et al., 1997; Sander et al., 1998; Malliri and Collard, 2003). We have investigated whether Tiam1 is involved in Rac-mediated cell-matrix signaling. We found that both wild-type (WT) and Tiam1-deficient (Tiam1/) keratinocytes adhere to and spread on various exogenous ECM components. However, Tiam1/ keratiocytes are unable to spread properly on an inert glass substrate, on which these cells have to deposit their own LN5 substrate. Our studies identify Tiam1 as a key molecule in 3ß1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for spreading and migration of keratinocytes.
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Results |
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F-actin stress fibers terminate at focal adhesion sites, at which integrins connect cells to the ECM. To visualize the distribution of the focal adhesions, cells were stained with a paxillin antibody. Consistent with the F-actin distribution, WT keratinocytes showed many small adhesion complexes and only few focal adhesions at the ends of actin cables (Fig. 1 C). In contrast, Tiam1/ keratinocytes displayed fewer, but larger, focal adhesions at the end of stress fibers.
Because Rac activity is involved in the regulation of cell spreading, we analyzed the effect of Tiam1 deficiency on the level of Rac activity in the keratinocytes grown on a Col IV matrix. Consistent with the phenotypic differences on a Col IV substrate, RacGTP levels were reproducibly lower (3050%) in Tiam1/ than in WT keratinocytes (Fig. 1 D).
From these studies we concluded that Tiam1 deficiency leads to reduced basal Rac activity and reduced cell spreading of keratinocytes when seeded on a Col IV substrate. This reduction in cell spreading is accompanied by the appearance of a large number of thick focal adhesions and actin stress fibers and a decrease in lamellipodial extensions.
Tiam1 is essential for keratinocyte adhesion to, and spreading on, a glass surface
The capacity of WT and Tiam1/ keratinocytes to adhere to a LN5-, FN-, vitronectin (VN)-, or Col IV-coated surface was not significantly different (Fig. 2, AC), although spreading of Tiam1/ cells was consistently found to be slightly reduced (Fig. 1 C and not depicted). If no exogenous matrix is available, adhesion and spreading of keratinocytes depends on the ability of these cells to secrete and deposit their own LN5 matrix. To investigate how the loss of Tiam1 affects keratinocyte adhesion under such conditions, Tiam1/ and WT cells were seeded on an inert glass surface. After 12 h, 75% of the WT cells had adhered to and spread on the glass. In contrast, only 510% of the Tiam1/ keratinocytes had adhered to the glass (Fig. 2, B and C). The few adherent cells were rounded and refractile, as if they were blocked in an early stage of spreading (Fig. 2 B). Similar results were found when cells were seeded on plastic (not depicted).
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We next investigated the pathways controlled by Rac that are responsible for the adhesion and spreading defect. We used effector loop mutants of GTPases previously shown to differentially bind and activate downstream effectors (Lamarche et al., 1996). The constitutively active L61Y40C mutant of Rac1 has lost its ability to interact with p21-activated kinase (PAK)1 and is unable to activate c-Jun NH2-terminal kinase activity, but it still induces F-actin polymerization and membrane ruffling. Conversely, the L61F37A mutant of Rac1 is unable to remodel the cytoskeleton, but interacts with p65PAK and activates c-Jun NH2-terminal kinase. As shown in Fig. 3, the expression of the L61Y40C mutant of Rac1, but not that of the L61F37A mutant, strongly increased the number of Tiam1/ cells adhering to and spreading on glass.
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Tiam1/ keratinocytes do not migrate in a scrape woundhealing assay because of their inability to deposit LN5
Next, we studied the migration of WT and Tiam1/ keratinocytes into a scrape wound, a process also dependent on the ability of keratinocytes to produce and secrete LN5. Confluent monolayers of WT and Tiam1/ keratinocytes, cultured on Col IVcoated surfaces in keratinocyte medium with defined growth factors but without ECM components, were scrape wounded and the migration of keratinocytes was investigated. As expected, WT keratinocytes migrated into the denuded area and closed the wound within 24 h (Fig. 4 A). In contrast, Tiam1/ keratinocytes did not migrate into the wound (Fig. 4, A and C), where the LN5 and Col IV coating was removed by scraping (Fig. 4 C). However, when ECM components were provided by the addition of chelated fetal calf serum to the medium after scraping, the Tiam1/ cells did migrate, albeit less efficiently than WT keratinocytes (Fig. 4 B). These data are consistent with our earlier conclusion that Tiam1/ keratinocytes are unable to produce and secrete sufficient amounts of LN5 substrate, resulting in their inability to spread and migrate onto an uncoated surface.
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We then studied the activation of Rac upon adhesion of keratinocytes to exogenous LN5 substrate. In WT cells, a small increase in active GTP-bound Rac could be measured 5 min after seeding, and this increase was much more pronounced after 30 min (Fig. 6 A). Rac activity remained elevated for at least 1 h and returned to basal levels within 3 h (Fig. 6 B). In contrast, in Tiam1/ keratinocytes we did not detect any Rac activation upon adhesion to LN5 after 5 and 30 min, or even after 3 h (Fig. 6, A and B), indicating that 3ß1-mediated Rac signaling is impaired in Tiam1/ cells.
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Although Tiam1/ keratinocytes spread less well on exogenous LN5 matrix than WT cells (Fig. 7 A), they are able to spread without any detectable change in Rac activity. Earlier studies have demonstrated that cross talk exists between Rac and Rho GTPases, and that changes in the balance between the activities of these proteins can influence cell morphology (van Leeuwen et al., 1997; Kodama et al., 1999; Sander et al., 1999). Therefore, we hypothesized that spreading of Tiam1/ keratinocytes might be regulated by a change in Rho activity, rather than Rac activity. In WT cells, a small decrease (1015%) in Rho activity could be detected 5, 30, and 60 min after seeding on LN5 (Fig. 7, B and C). However, in Tiam1/ cells a much larger decrease in Rho activity was found 5, 30, and 60 min after seeding (45, 55, and 60%, respectively). In both WT and Tiam1/ cells, the decrease in Rho activity was observed during at least 1 h and returned to basal levels within 3 h (Fig. 7, B and C). This suggests that spreading of Tiam1/ keratinocytes is caused by a substantial decrease in Rho activity, which leads to cytoskeletal relaxation. In this manner, in Tiam1/ keratinocytes Rac activity can be relatively increased, as compared with Rho activity, upon adhesion to exogenous LN5, thereby allowing cell spreading.
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Discussion |
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Keratinocytes can bind to LN5 via two integrins, 3ß1 and
6ß4, and several studies have addressed the mechanism by which keratinocytes adhere to LN5 in vitro. The
3ß1 integrin links the ECM to the actin cytoskeleton (Hodivala-Dilke et al., 1998) and thereby triggers adhesion and spreading of keratinocytes (Carter et al., 1991). The
6ß4 integrin mediates a long-term stable adhesion by inducing hemidesmosome assembly (Borradori and Sonnenberg, 1999). Keratinocytes isolated from
3-null epidermis (expressing
6ß4) retained the ability to attach to LN5, but did not spread properly (DiPersio et al., 1997), indicating that
3ß1 and
6ß4 have distinct but overlapping functions in keratinocytes. Both integrins can support initial cell attachment to LN5, but
3ß1 is required for subsequent cell spreading. Consistent with this conclusion are the findings that the spreading defect on LN5 in ß1-deficient cells cannot be rescued by expression of the
6ß4 integrin (Kikkawa et al., 2004). We found that Tiam1/ keratinocytes spread less well on several matrix components. On glass, where keratinocytes have to deposit their own LN5 matrix, initial adhesion occurred, but the subsequent spreading was impaired, suggesting that Tiam1 is required for
3ß1-mediated cell spreading. Indeed, no major differences in adhesion between WT and Tiam1/ cells were found on an exogenous LN1 substrate, for which keratinocytes use the
6ß4 integrin only. A role for Tiam1 in
3ß1 signaling is further supported by the observation that Tiam1/ keratinocytes display several defects that are also observed in
3-deficient keratinocytes. Experiments with WT and
3-null keratinocytes showed that both spread on FN. However, WT cells spread approximately two times better and displayed typical, well-extended lamellipodia, whereas
3-null keratinocytes exhibited fewer and smaller lamellipodia (Hodivala-Dilke et al., 1998). This is consistent with the phenotype of the Tiam1/ keratinocytes on various substrates including Col IV and LN5. In
3-null keratinocytes, cells display thick actin stress fibers and robust peripheral focal adhesions (DiPersio et al., 1997; Hodivala-Dilke et al., 1998), similar to those found in Tiam1/ keratinocytes.
Cell spreading is initiated by integrin-mediated cell-matrix interaction and requires the activation of multiple signaling pathways including Rac (for review see Schwartz and Shattil, 2000). More than 60 GEFs for Rho GTPases have been identified (Etienne-Manneville and Hall, 2002) but only a few have been implicated in integrin-mediated Rac signaling. Vav1, which is expressed in hematopoietic cells only, was shown to be essential for the spreading of T cells. The closely related GEF Vav2 is widely distributed and has been implicated in the spreading of fibroblasts (Marignani and Carpenter, 2001). An alternative pathway from ß1 integrinECM ligands to Rac activation has been proposed in human lung adenocarcinoma cells (Gu et al., 2001). This pathway depends on FAK/Cas/Crk-mediated activation of the RacGEF DOCK180 upon adhesion to LN10/11 via 3ß1. This might indicate that different cells are using different GEFs in
3ß1 signaling toward Rac (i.e., DOCK180 or Tiam1). Alternatively, tumor cells might have acquired additional means to activate Rac. In our study, we demonstrate that keratinocytes, which lack the RacGEF Tiam1, have a defect in
3ß1-mediated Rac activation and cell spreading, as a result of decreased LN5 production and secretion.
The in vitro data on adhesion, spreading, and migration of keratinocytes and the results in vivo in skins of WT and Tiam1/ mice resemble the data on the cells and skin of mice with a null mutation for the 3 integrin subunit (Itga3; DiPersio et al., 1997), although the phenotype of the Tiam1/ mice is less severe. The
3ß1-deficient mice die shortly after birth because of defects in kidney and lung organogenesis (Kreidberg et al., 1996). However, in contrast to
6ß4 knockout mice that show extensive skin blistering (Georges-Labouesse et al., 1996; van der Neut et al., 1996; DiPersio et al., 2000), the skin of
3 knockout mice develops normally. In these mice, regions were occasionally observed in which the LN matrix was deposited in a disorganized manner, which caused microblistering at sites of the body susceptible to high mechanical stress, such as the feet (DiPersio et al., 1997; Hodivala-Dilke et al., 1998). We did not find that targeted deletion of Tiam1 affects development of the epidermis and its adhesion to the basement membrane. Most likely, various mechanisms are active in vivo that may compensate for the loss of Tiam1. However, we found that Tiam1 expression is important for the reepithelialization of full-thickness excision wounds in the mouse skin. Specifically, Tiam1 deficiency leads to a significant delay in wound closure of the epidermis. This is consistent with the delay in wound closure of Tiam1/ versus WT keratinocytes in "scratch wound assays" in vitro. It has been well established that migration and proliferation of keratinocytes is crucial for reepithelialization of cutaneous wounds. Recently, a study in mice with an epidermis-specific knockout of ß1 integrins showed that cutaneous wounds failed to heal properly owing to a defect in the initiation of cell migration (Grose et al., 2002). Although ß1 integrins interact with several potential integrin ligands, the interaction between
3ß1 and LN5 is of particular importance in the impaired keratinocyte migration in K5ß1-null mice (Grose et al., 2002). Moreover, other studies have shown that expression of LN5 is required for the reepithelialization of cutaneous wounds (Ryan et al., 1999; Nguyen et al., 2000b) and that the
3ß1 integrin is an important player in the regulation of both basement membrane assembly and cutaneous wound repair (DiPersio et al., 1997; Hodivala-Dilke et al., 1998; Nguyen et al., 2000a,b; Choma et al., 2004). In light of the presented properties of Tiam1, with respect to LN5 secretion, adhesion, and impaired migration of epithelial cells in vitro, it is reasonable to assume that Tiam1 plays a similar role in epidermal wound closure in vivo, although we cannot exclude that Tiam1 may contribute to wound repair by other mechanisms as well.
In summary, our data indicate that Tiam1 is an essential GEF involved in the 3ß1-mediated activation of Rac upon adhesion of keratinocytes to LN5. The Tiam1-mediated Rac activation regulates the further production, secretion, and organized deposition of LN5, which is essential for the spreading and migration of keratinocytes.
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Materials and methods |
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To obtain immortalized cells, WT and Tiam1/ keratinocytes were transduced with supernatant containing pBabe puro SV40 LargeT (LT) antigen viruses. Expression levels of the SV40 LT antigen in WT and Tiam1/ cells were determined by Western blot analysis (Mertens et al., 2005).
Gene transfer into keratinocytes by retroviral transduction
SV40 LT antigen and the Rac mutants (a gift from L. van Aelst, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) were cloned into the LZRS-IRES-neo retroviral vector, whereas the Tiam1 coding sequence was cloned into the LZRS-IRES-blasticidin vector (Michiels et al., 2000). Retroviral constructs were transfected into Phoenix ecotropic packaging cells, and fresh viral supernatants were collected and used for infections, as previously described (Michiels et al., 2000).
Coating of dishes with ECM molecules
All ECM proteins except LN5 were coated to culturing dishes overnight at 4°C at the following concentrations: 10 µg/ml FN (isolated from human plasma); 10 µg/ml LN1 (Becton Dickinson); 10 µg/ml VN (Sigma-Aldrich); 20 µg/ml Col I (Vitrogen/Nutacon); and 25 µg/ml Col IV (Becton Dickinson). A LN5 matrix was obtained by culturing Rac-11P cells to confluency (Delwel et al., 1993), after which cells were detached with 10 mM EDTA in PBS containing a mix of protease inhibitors (Complete protease inhibitor cocktail tablets; Roche) at 4°C. Before use, the dishes were washed twice with PBS.
Cell culture
Keratinocytes were grown on a Col IV substrate and maintained in Epilife keratinocyte medium. For experiments, cells were used at a density of 7080% confluency. NIH 3T3 and Rac-11P cells were cultured in DME supplemented with 10% bovine calf serum. Cells were seeded 24 h before use to obtain a final density of 70% (NIH 3T3) and 100% (Rac-11P), respectively.
Immunoprecipitation and Western blotting
For immunoprecipitation, cells grown in 10-cm Ø dishes were lysed in 1 ml of buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 10% glycerol, 1% Nonidet P-40, and protease inhibitors. Extracts were clarified by centrifugation and precleared with GammaBind Sepharose beads (GE Healthcare). Precleared lysates were incubated with anti-Tiam1 antibody (C16; Santa Cruz Biotechnology, Inc.) and immune complexes were precipitated using GammaBind Sepharose. After overnight incubation, beads were washed three times and resuspended in SDSsample buffer.
For Western blotting, cell lysates or samples of precipitated proteins were boiled for 5 min and resolved by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories), blocked with BSA or skimmed milk, and probed using the indicated antibodies. Specific binding was detected using a secondary peroxidase-conjugated antibody (GE Healthcare) followed by chemiluminescence. Anti-Rac1 monoclonal antibody was obtained from Upstate Biotechnology. Activated FAK, ERK1/2, Src, and Stat3 were detected by antiphospho-FAK (Y397; Biosource), antiphospho-ERK1/2 (New England Biolabs, Inc.), antiphospho-Src (Y418; Biosource International), and antiphospho-Stat3 (Y705; Cell Signaling Technology), respectively. Polyclonal anti-Tiam1 (DH) has been previously described (Habets et al., 1994). AntiLN 2 (1109) was a gift from T. Sasaki (Max Planck Institute for Biochemistry, Martinsried, Germany). Antiß-actin was purchased from Sigma-Aldrich. Anti-Tiam1 (C16), anti-Rho (26C4), anti-myc, and antiSV40 LT were obtained from Santa Cruz Biotechnology, Inc.
Rac and Rho activity assays
GTPase activity was assayed essentially as previously described (Sander et al., 1999). In brief, after the adhesion of cells to a relevant surface, cells were washed and lysed with a 1% Nonidet P-40 buffer containing either 2 µg/ml PAK-CRIB peptide (Price et al., 2003) or GST-Rhotekin (Sander et al., 1999). Cell lysates were sheared though an insulin needle and cleared by centrifugation. Active complexes were precipitated with streptavidinagarose beads (Rac-PAK-CRIB; Sigma-Aldrich) or with glutathione beads (Rho-GST-Rhotekin; Sigma-Aldrich) and solubilized in SDSsample buffer. Rac and Rho were detected by Western blotting. Anti-Rac1 monoclonal antibody was purchased from Upstate Biotechnology and anti-Rho (26C4) was obtained from Santa Cruz Biotechnology, Inc.
Wound repair in vivo
Adult mice were anesthetized, shaved, and two full-thickness excision wounds (4 mm in diameter) were cut with small scissors on either side of the dorsal midline of each mouse. The four wounds per mouse were left uncovered. For histological and immunofluorescence analysis, the complete wounds including surrounding tissue (8 mm) of adjacent normal skin were excised at 3, 4, 5, and 6 d after injury. Cryo- and paraffin sections across the middle of the wounds were stained: the cryosections with LN
2 (1109) and DAPI, and the paraffin sections with hematoxylin and eosin. For each time point, wound diameters (distance between epithelial rims) were determined in three mice.
Phase-contrast microscopy and confocal microscopy
For phase-contrast microscopy, cells were seeded for 24 h onto either plastic coated with LN5 or Col IV or onto glass coverslips, viewed under a microscope (model Axiovert 25; Carl Zeiss MicroImaging, Inc.), and photographed. For immunofluorescence staining, keratinocytes were seeded on glass coverslips, which were either uncoated or coated with Col IV or LN5. After 16 h, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked with 2% BSA in PBS. Filamentous actin was labeled with 0.2 µM Alexa Fluor 568phalloidin (Invitrogen) for 30 min. The following primary antibodies were used: a mouse monoclonal against paxillin (BD Biosciences) and plectin (HD1; a gift from K. Owaribe, Nagoya University, Nagoya, Japan); rabbit antibody against mouse LN 2 (1109); and rat monoclonal antibody against integrin ß4 (346-11A; BD Biosciences). Primary antibodies were visualized with appropriate FITC- or Alexa Fluor 568labeled secondary antibodies (Zymed Laboratories). Images were collected by confocal microscopy (model TCS NT; Leica).
Adhesion assays
24-well microplates were coated overnight at 4°C with 25 µg/ml Col IV or LN5, which were secreted by Rac-11P cells (as described in Coating of dishes with ECM molecules). Plates were washed with PBS and saturated with 1% (wt/vol) BSA (Sigma-Aldrich), for 2 h at 37°C to block nonspecific adhesion. Keratinocytes were detached with EDTA and suspended in supplement-free keratinocyte medium. 6 x 104 cells/well were plated in triplicate and incubated for 612 h at 37°C. Nonadherent cells were removed by washing with PBS, and cell adhesion was estimated in a colorimetric assay, based on NPAG (p-nitrophenyl N-acetyl-ß-D-glucosaminide; Sigma-Aldrich) reaction. Absorbance was measured at 405 nm by using an ELISA reader (Bio-Rad Laboratories).
mRNA isolation and RT-PCR
Total cellular RNA was isolated from 70% confluent 10-cm Ø dishes using RNAzol B (Campro Scientific) and cDNA was synthesized by RT-PCR performed on 1 µg RNA using the Thermoscript RT-PCR system kit (Invitrogen). Specific transcripts were amplified with the following primers (Sigma Genosys): LN52 (forward, 5'-aaccagcaagtgagttacgg-3'; and reverse, 5'-ccattgtgacagggacatgg-3') and glyceraldehyde-3-phosphate dehydrogenase (forward, 5'-accacagtccatgccatcac-3'; and reverse, 5'-tccaccaccctgttgctgta-3') using the standard PCR protocol for the Platinum Taq PCRX DNA polymerase kit (Invitrogen). The PCR products were resolved by electrophoresis on 1.5% agarose gels and viewed after ethidium bromide staining.
Online supplemental material
Fig. S1 shows that both 3ß1 and
6ß4 are equally expressed in WT and Tiam1/ keratinocytes. Fig. S2 shows that on various other substrates such as Col IV, PLL, and FN, the degree of Rac activation in Tiam1/ keratinocytes was comparable to that in WT cells, suggesting that the activation of Rac as a result of adhesion to these substrates is regulated by other RacGEFs. In Fig. S3, analysis of intact skin revealed no differences in the deposition and processing of LN5, the expression of the ß1- and ß4-integrin subunits, and components of the basal lamina between WT and Tiam1/ mice. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200509172/DC1.
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Acknowledgments |
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This work was supported in part by a grant from the Dutch Cancer Society to A. Sonnenberg, by a fellowship from the European Community (Marie Curie) to C. Olivo, and by grants from the Dutch Cancer Society to J.G. Collard.
Submitted: 30 September 2005
Accepted: 31 October 2005
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