Article |
Address correspondence to Yoshiaki Ishigatsubo, The First Dept. of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan. Tel.: (81) 45-787-2630. Fax: (81) 45-786-3444. email: ishigats{at}med.yokohama-cu.ac.jp
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Abstract |
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Key Words: affixin; ILK; -actinin; zyxin; Mena
Abbreviations used in this paper: ABD, actin-binding domain; CH, calponin homology; DIC, differential interference contrast; FA, focal adhesion; FN, fibronectin; ILK, integrin-linked kinase; siRNA, small interference RNA; SF, stress fiber.
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Introduction |
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Integrin-linked kinase (ILK) is a ubiquitously expressed serine/threonine protein kinase capable of interacting with the cytoplasmic domains of integrin ß1 and ß3 (Hannigan et al., 1996). Several reports have demonstrated that ILK is involved in the integrin-dependent cell adhesion, spreading, and cell shape change in cultured cells (Hannigan et al., 1996; Huang and Wu, 1999; Zhang et al., 2002b). Recently, we have shown that ILK binds to a novel focal adhesion (FA) protein, named affixin (ß-parvin), that consists of two tandem calponin homology (CH) domains and belongs to a novel family of FA proteins, together with other homologous proteins such as actopaxin/CH-ILKBP/-parvin (
-parvin) and
-parvin (Olski et al., 2001; Tu et al., 2001; Yamaji et al., 2001; Nikolopoulos and Turner, 2002). In CHO-K1 cells replated on fibronectin (FN), affixin and ILK are concentrated on the cell surface in blebs and then recruited into nascent substrate adhesion sites in advance of other FA components. In well-spread cells, affixin is then distributed at FA and leading edge with ILK as well as along stress fibers (SFs; Yamaji et al., 2001). Consistent with these subcellular localizations, affixin, as well as
-parvin, were suggested to mediate integrinILK signaling for actin organization and thus have roles in cell spreading and adhesion. For example, the overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage. The coexpression of ILK enhances this effect. Thus, we suggested that affixin is involved in integrinILK signaling required for the development of nascent cellsubstrate adhesion structures to mature FAs (Yamaji et al., 2001). In platelets, we noted that ILK stably forms a complex with affixin, and thrombin stimulation induces their association with integrin ß3, which is followed by ILK activation and their subsequent incorporation into the Triton X-100insoluble membranecytoskeletal fraction (Yamaji et al., 2002). On the basis of these results, we suggested that ILK and affixin play critical roles in cell spreading, particularly for the initial formation of FA and integrincytoskeletal linkage. Interestingly,
-parvin was reported to form a complex with not only ILK, but also with PINCH and paxillin, and the analysis of the binding-defective point mutant revealed that a correct complex formation is essential for their FA localization (Nikolopoulos and Turner, 2002; Zhang et al., 2002a). Although these results indicated that affixin and its family are closely correlated with the initial formation of FA via integrinILK signaling, the downstream target and the precise roles of affixin in the initial maturation process of FA are unknown.
In the present work, we demonstrated that affixin interacts with -actinin through its second CH domain. Their association depends on kinase activity of ILK in vitro and the FN-induced integrin stimulation in vivo. The overexpression of the specific
-actininbinding site of affixin fused with GFP (GFP-affixin249272) resulted in the unique inhibition of affixin
-actinin interaction and the perturbation of Mena localization, which in turn resulted in the blockade of cell spreading. Furthermore, we designed small interference RNA (siRNA) that specifically down-regulated affixin expression, and noted that affixin knockdown cells formed multiple blebs, which was followed by the inhibition of FA formation and cell spreading as observed in GFP-affixin249272overexpressing cells. These results suggest that affixin plays a critical role in integrincytoskeletal linkage during maturation of nascent FA and SF extensions.
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Results |
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Endogenous affixin binds to -actinin in a substrate adhesiondependent manner
Next, we examined whether affixin endogenously interacts with -actinin in mammalian cells. Recently, we have reported that in resting platelets, affixin is recruited into the integrin complex together with ILK in response to thrombin stimulation. The tripartite complex then gradually translocates to the Triton X-100insoluble fraction, biochemically corresponding to the actin-based membranecytoskeletal fraction (Yamaji et al., 2002). On the basis of these observations, we speculated that the interaction of affixin with
-actinin may be dependent on integrin stimulation. In Fig. 2 A, CHO-K1 cells were cultured on FN-coated dishes or remained in suspension on nonadhesive plastic dishes for 3 h, and the cell lysates were subjected to immunoprecipitation analysis using the anti-affixin antibody. As shown in Fig. 2 A, a moderate amount of
-actinin was coimmunoprecipitated with affixin from the cells cultured on FN dishes, but not from those cultured on plastic dishes. In contrast, paxillin, which was reported to interact with
-parvin, as well as actin and vinculin, failed to coimmunoprecipitate with affixin under either condition. These results indicate that affixin interacts with
-actinin, but not with paxillin, in an adhesion-dependent manner in mammalian fibroblasts.
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Affixin binds to -actinin in vitro in an ILK kinase activitydependent manner
To confirm the direct interaction between affixin and -actinin, we performed an in vitro pull-down assay using bacterially purified GST-affixin, or GST-CH2 (affixin213364; previously called RP2). For this purpose, the GST fusion proteins were incubated with purified
-actinin, and the bound proteins were subjected to immunoblotting with the anti-
-actinin antibody. As shown in Fig. 3 A, the results reproducibly did not exhibit any interaction between purified affixin and
-actinin. However, the result that the endogenous interaction between affixin and
-actinin was only observed when cells were plated on FN-coated dishes (Fig. 2 A) made us hypothesize further that some post-translational modification may be required for the interaction. In this respect, it should be noted that ILK has been shown to be acutely activated in response to integrin signaling (Delcommenne et al., 1998), and that the CH2 domain of affixin can be effectively phosphorylated by ILK in vitro (Yamaji et al., 2001). Together with the present results that the
-actininbinding site of affixin is very close to the ILK-binding site, it is plausible that the affixin
-actinin interaction is induced by the phosphorylation of the CH2 domain of affixin by ILK. The results in Fig. 3 (B and C) strongly support this notion: when preincubated with immunoprecipitated ILK under a condition in which GST-affixin was effectively phosphorylated by ILK (Yamaji et al., 2001), GST-affixin as well as GST-CH2, but not GST alone, became competent to interact with
-actinin. Importantly, similar effects were not observed when a kinase-dead mutant of ILK, ILK(K220M), was used for preincubation instead of ILK (Fig. 3 C; Yamaji et al., 2001). Considering that ILK (K220M) can associate with affixin (Yamaji et al., 2001), these results indicated that the interaction of affixin with
-actinin is dependent on ILK kinase activity, but not on its ability to associate with ILK. To further confirm direct interaction between affixin and
-actinin, we purified GST-affixin by SDS-PAGE after preincubation with in vitrotranslated ILK in the presence or absence of ATP, and examined its binding to purified
-actinin by blot overlay assay. Fig. 3 D shows again that GST-affixin specifically interacts with
-actinin, only when it was preincubated with ILK in the presence of 10 µM ATP in phosphorylation buffer. These results indicate that affixin directly associates with
-actinin only when its CH2 domain is phosphorylated by ILK.
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To obtain further insight into the roles of affixin-actinin interaction in cell spreading, cells overexpressing GFP-affixin249272 were observed by time-lapse video microscopy (Fig. 7 A; Videos 14, available at http://www.jcb.org/cgi/content/full/jcb.200308141/DC1). 24 h after transfection, cells overexpressing GFP-affixin249272 with a round shape were noted to actively protrude many blebs peripherally. However, these blebs did not further develop into lamellipodia as observed in normal cells, but were retracted into the cell body within 30 min (Fig. 7 A, d; indicated by arrows). It should be noted that these round cells still attached on the slides and actively moved in a very limited area, indicating that this round morphology did not reflect the process of apoptosis. These effects of GFP-affixin249272 suggest that affixin
-actinin interaction does not contribute to the initial formation of the cell protrusions in itself, but rather to their stabilization and further development into lamellipodia, which establish firm and broad interaction with the substrate.
Loss of affixin expression results in the blockade of FA formation, lamellipodial development, and cell spreading
To further confirm the essential role of affixin in cell spreading, we next used the siRNA to monitor the effects of knockdown of affixin expression. We synthesized and introduced three affixin siRNAs or an irrelevant control RNA into human fibroblasts (HT1080 and IMR-90) or HeLa cells. As shown in Fig. 8, the two affixin-targeted siRNAs #1 and #3 specifically suppressed the expression of affixin in HT1080, IMR-90 (Fig. 8 A), and HeLa cells (unpublished data), whereas the expression of other affixin-interacted or cytoskeletal proteins such as ILK, -actinin, vinculin, and actin were not affected. The differential interference contrast (DIC) images of these affixin-deficient cells transfected with affixin siRNA #3 revealed a marked increase in the number of multiple blebbing cells compared with control cells (Figs. 8, B and C). Time-lapse observations by DIC microscopy revealed that these blebs were repeatedly protruding from and retracting into the cell bodies, but could not develop into mature lamellipodia, which was quite similar to those of CHO-K1 cells overexpressing GFP-affixin249272 (Fig. 8 C; Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200308141/DC1). We also confirmed by TUNEL staining that affixin RNA interference does not induce apoptosis within 48 h after siRNA transfection (unpublished data). In the immunofluorescence microscopy, well-developed thick SFs and FAs were not observed in these affixin-deficient cells, but actin condensation in the cell periphery (Fig. 9 E) and diffuse staining of
-actinin (Fig. 9 K) and vinculin (Fig. 9 Q) in cytosol were detected instead. These results indicate that affixin plays a critical role in the process of FA formation, lamellipodial development, and cell spreading.
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Discussion |
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In this work, we demonstrated that an ILK-binding protein, affixin, directly interacts with -actinin through its second CH domain; thereby providing a novel molecular basis of
-actinin targeting to FAs. Previously, we demonstrated that during cell spreading observed after replating, ILK and affixin are both recruited into cell surface blebs formed at a very early stage before FAK and vinculin. Later, affixin localizes as small dots in the lamellipodia from which short actin bundles emanate. These features of affixin localization during the initial establishment of integrin-based adhesion are well consistent with the dynamics of
-actinin stated in the previous paragraph, suggesting a possibility that both proteins cooperate to transmit initial integrin signal to F-actin organization. In the present work, we confirmed that affixin and
-actinin are mainly colocalized at the tip of lamellipodia, where nascent cellsubstrate interactions intensively occur. Furthermore, we also revealed biochemically that in vivo interaction between endogenous affixin and
-actinin was dependent on integrin-mediated substrate adhesion, and peaked at an initial phase of cell spreading. The functional importance of the affixin
-actinin interaction in the initial integrin signaling was then demonstrated by the observation that introduction of the minimum
-actininbinding sequence of affixin, affixin249272, which disrupts endogenous interaction of affixin with
-actinin, severely interfered with cell spreading. The resultant round cells lacked FAs, well-developed lamellipodia, and SFs, but manifested peripheral small blebs in which F-actin often aggregates. Observation by time-lapse video microscopy revealed that these cells showed jerky movement and actively protruding small blebs, but failed to spread by developing lamellipodia from these blebs. These results are essentially reproduced in siRNA experiments in which affixin expression was specifically knocked down. Together, these data strongly support the notion that the affixin
-actinin interaction plays an essential role in the recruitment of
-actinin to nascent FAs, where
-actinin is considered to trigger robust actin polymerization by interacting with a zyxinMena complex and thus induces the stabilization of the nascent substrate adhesion, lamellipodial development, and formation of SFs (Drees et al., 1999; Fradelizi et al., 2001).
Kinase activity of ILK is essential for affixin-actinin interaction
ILK has been demonstrated to be activated by integrin signaling evoked by the interaction with the substrate (Delcommenne et al., 1998). Our analysis of thrombin-activated platelets revealed that its kinase activity is acutely enhanced within 90 s after the stimulation, which is followed by the incorporation of the integrinILKaffixin complex into 1% Triton X-100insoluble, membrane-skeletal structures (Yamaji et al., 2002). Because the CH2 domain of affixin is efficiently phosphorylated by ILK in vitro, and because the binding regions of ILK and -actinin on affixin are located close to each other (Yamaji et al., 2001), it is plausible that the interaction between
-actinin and affixin is triggered by phosphorylation of affixin by ILK in response to substrate adhesion. Indeed, we demonstrated here that the direct interaction between the CH2 domain of affixin and
-actinin was only observed when the CH2 domain was preincubated with ILK, but not with the kinase-dead mutant of ILK, in vitro. Although further analyses including the determination of ILK phosphorylation sites on affixin are required to completely verify our hypothesis, the present results are well consistent with the concept that ILK activation by integrin signaling and the subsequent phosphorylation of affixin is critical to the initial establishment of cellsubstrate adhesion. It should be also noted that the time course of the increase in affixin
-actinin interaction after cell replating (Fig. 2 B) well correlates with that of ILK activation induced by cellsubstrate adhesion (Delcommenne et al., 1998).
Previously, we demonstrated that the overexpression of affixin258364 corresponding to the entire CH2 domain in well-spread CHO-K1 cells does not severely interfere with cell adhesion unless ILK, but not the kinase-defective point mutant of ILK (K220M), is coexpressed or the cells are subjected to replating. On the other hand, we observed here that the overexpression of the smaller affixin fragment, affixin249272, corresponding to the first portion of the CH2 domain and lacking ILK-binding activity, exerts significant deleterious effects on cellsubstrate adhesion even without ILK coexpression or replating. This may indicate that phosphorylation of the CH2 domain by ILK induces a conformational change of the CH2 domain of affixin, which enables affixin to interact with -actinin to evoke the subsequent maturation of the FA complex.
Functional divergence between affixin and actopaxin/CH-ILKBP/-parvin
In the course of the present work, we also noted that affixin does not interact with paxillin. This is in sharp contrast with -parvin, which has been demonstrated to interact with paxillin through the paxillin-binding sequence domain located in the first half of the CH2 domain. Considering that
-parvin does not interact with
-actinin (Nikolopoulos and Turner, 2000, 2002), these results provide very interesting functional divergence between the two closely related members of the parvin family, both of which bind to ILK. It was reported that
-parvin directly interacts with F-actin, but affixin does not (Yamaji et al., 2001). This functional divergence may explain the difference in the effect of the CH2 domain overexpression on cell spreading: in contrast with affixin, the morphological effect of the overexpression of the
-parvin CH2 domain was revealed to be rather weak (Nikolopoulos and Turner, 2000; Tu et al., 2001). Furthermore, the
-parvin mutant, F271D, which exhibits impairment in binding to paxillin, has not been reported to cause any morphological changes without the inhibition of the correct FA localization of this mutant (Tu et al., 2001; Nikolopoulos and Turner, 2002). Finally,
-parvin siRNA have revealed no obvious effect on their morphology and FA formation (Fukuda et al., 2003). These results suggest that affixin has some distinct roles from
-parvin in cell spreading despite their close similarity in their amino acid sequence.
Role of affixin during early FA formation
Recently, Rosenberger et al. (2003) have reported that affixin interacts with PIX, a PAK-interacting protein that has GEF activity for Rac1 and possibly for Cdc42. In a previous report, we demonstrated that, in contrast to the CH2 domain, overexpression of the CH1 region of affixin promotes cell spreading of CHO-K1 cells, and speculated that this region may be the site that interacts with the downstream target of ILKaffixin signaling (Yamaji et al., 2001). Consistent with this prediction, we confirmed that the CH1 domain is the site that interacts with
PIX, and the overexpression of CH1 enhances Rac and Cdc42 activities via
PIX (Mishima et al., 2004). These results suggest that affixin also participates in the activation of Rac and Cdc42 by associating with
PIX through its CH1 domain. This activity of affixin should result in enhanced actin polymerization through the activation of various downstream effectors of Rac1/Cdc42, including Mena/VASP and WASP-Arp2/3. In addition, PIX was suggested to be responsible for the recruitment of PAK1 to integrin-based focal contacts, which is activated by Rac1/Cdc42 (Manser et al., 1998) and causes filopodia formation and membrane ruffles via the LIM kinaseADF/cofilin pathway (Edwards et al., 1999; Zebda et al., 2000). Therefore, these results suggest that affixin is not only a downstream mediator of integrinILK signaling, but is also a scaffold protein on which all these key players of actin polymerization converge in concert with
-actinin and PIX (Fig. 10). This protein complex formed around the initial integrin-based substrate adhesion site may synergistically evoke acute actin polymerization, which results in the rapid stabilization of the nascent cellsubstrate interaction, lamellipodia development, and SF formation. Dramatic effects of affixin knockdown on cell spreading are consistent with this hypothesis on the central role of ILKaffixin signaling in initial integrin signaling. Then, the fact that the introduction of the minimum
-actininbinding site caused essentially similar effects on affixin siRNA suggests that the interaction between affixin and
-actinin is one of the critical components of this nascent integrin signaling mediated by the ILKaffixin system.
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Materials and methods |
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Cell culture
CHO-K1, Cos-7, IMR-90, and HT1080 cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in Ham's F12 medium (CHO-K1), or DME (Cos-7 and IMR-90) or MEM (HT1080), containing 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. cDNA transfection was performed by either electroporation for the immunoprecipitation assay or lipofection using a FuGENETM 6 transfection reagent (Roche) for immunofluorescence analysis.
Immunoprecipitation assay
Cells cultured in 10-cm dishes were suspended in 200 µl lysis buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 µg/ml leupeptin, 1 mM PMSF, 2 mM sodium fluoride, and 1.5% Triton X-100. In experiments to analyze cell spreading on FN, cells were trypsinized, washed three times in PBS, plated onto FN-coated 10-cm dishes, and collected at appropriate times using another lysis buffer containing 20 mM Hepes, pH 7.5, 50 mM NaCl, 10 µg/ml leupeptin, 1 mM PMSF, 2 mM sodium fluoride, 100 µM CaCl2, and 0.75% Triton X-100. After a 30-min incubation on ice, the lysates were clarified by centrifugation at 15,000 rpm for 30 min (clearance of cell extracts with high speed centrifugation was always performed to exclude large cytoskeletal structures), and then incubated with 10 µl protein GSepharose (Amersham Biosciences) conjugated with 2 µg anti-affixin pAb, anti-flag mAb, or an equal amount of control normal IgG, for 1 h at 4°C. After washing with each lysis buffer, the immunocomplex was solubilized by adding SDS sample buffer to the resin and subjected to standard Western blot analysis.
Pull-down and blot overlay assay
Immunoprecipitates obtained using protein GSepharose conjugated with anti-flag mAb from Cos-7 lysates overexpressing flag-ILK or flag-ILK-KD (K220M) were mixed with purified recombinant GST-affixin, CH2, or GST control (15 µg) in a phosphorylation buffer (50 mM Hepes, pH 7.0, 10 mM MnCl2, 10 mM MgCl2, and 2 mM sodium fluoride) with 10 µM ATP as described previously (Yamaji et al., 2001). After incubation for 60 min at 30°C, glutathione-Sepharose 4B was added to the mixture and was incubated for 30 min at 4°C. After washing with PBS containing 0.05% Tween 20, resins (containing both protein GSepharose and glutathione-Sepharose 4B) were resuspended in binding buffer (50 mM Hepes, pH 7.0, 20 mM NaCl, and 0.5% Triton X-100) containing 20 µg/ml purified chicken -actinin (Sigma-Aldrich) and were incubated for 3 h at 4°C. After incubation, resins were washed again with binding buffer three times, and were resolved by 10% SDS-PAGE, blotted on PVDF membranes, and treated with the anti-
-actinin monoclonal or anti-ILK polyclonal antibody.
Blot overlay was performed as follows: after preincubation with in vitrotranslated ILK (Promega) in the phosphorylation buffer, GST-affixin was purified by SDS-PAGE and transferred to a PVDF membrane. After blocking, the membrane was overlaid with 10 µg/ml purified -actinin (Sigma-Aldrich) in overlay buffer (20 mM Hepes, pH 7.5, 140 mM NaCl, 1 mM MgCl2, 100 µM CaCl2, 2 mM sodium fluoride, 0.1% BSA, 0.3% NP-40, and 4 mM DTT) for 3 h at RT. Bound
-actinin was revealed by standard Western blot analysis using anti-
-actinin antibody.
Immunofluorescence microscopy
CHO-K1 cells or those transfected with expression plasmids were cultured on FN-coated coverslips for 24 h, and after washing with PBS, were fixed with 2% formaldehyde in PBS for 15 min and then permeabilized with 0.1% Triton X-100 in PBS for 15 min at RT. In anti-affixin staining experiments, cells were fixed with 100% methanol. After blocking, the cells were treated with appropriate primary antibodies for 45 min at 37°C, washed with PBS containing 0.05% Tween 20, and incubated with secondary antibodies (Cy3-conjugated goat antirabbit [Amersham Biosciences] and Alexa 488®conjugated [Molecular Probes, Inc.] or Cy5-conjugated [Amersham Biosciences] goat antimouse Ig antibodies) at 37°C for 45 min.
siRNA
21-base sequences of the human affixin gene, targeting #1; 5'-AAGCUGAAUGUGGCUGAGGUG-3', #2; 5'-AAGCAGUACAUGACCUGCUGC-3', and #3; 5'-AAGCUGAAUUUGGAGGUGACG-3' (sense sequences), were designed on the basis of a method described previously (Elbashir et al., 2002) and blasted to assess specificity. The target siRNA duplexes and a control nonsilencing siRNA (16-base overlap with that of Thermotoga maritimia) were synthesized and purchased from QIAGEN. HT1080, IMR-90, and HeLa cells were transfected with each siRNA duplex using TransMessengerTM transfection reagent (QIAGEN). For immunofluorescence microscopy, HT1080 cells were cultured on FN-coated coverslips for 48 h, fixed with 0.5% PFA in PBS for 15 min, and then similarly treated with other immunostains.
Online supplemental material
CHO-K1 cells cultured on FN-coated plates were transfected with either the GFP vector or GFP-affixin249272. For the replating experiment, cells were trypsinized 24 h after transfection, washed three times in PBS, and plated onto FN-coated plates. HT1080 cells transfected with either control or affixin-targeted siRNA duplex were cultured on glass-bottom dishes for 36 h before time-lapse observations. Immunofluorescence and DIC images were collected using and inverted microscope (model DM IRB; Leica) with the 10x objective every 30 min or the 40x every 10 min, as indicated. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200308141/DC1.
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Acknowledgments |
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This work was partly supported by grants from the Yokohama City University Center of Excellence Program of the Ministry of Education, Sports, Science and Technology of Japan (to Y. Ishigatsubo), the Kanagawa Nanbyou foundation (to S. Yamaji), the Yokohama Foundation for Advancement of Medical Science (to W. Mishima), and the Research Grant (14B-4) for Nervous and Mental Disorders from The Ministry of Health, Labor and Welfare, Japan (to A. Suzuki).
Submitted: 26 August 2003
Accepted: 9 April 2004
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