Article |
Address correspondence to Mitsuo Ikebe, Dept. of Physiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Tel.: (508) 856-1954. Fax: (508) 856-4600. email: mitsuo.ikebe{at}umassmed.edu
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Abstract |
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Key Words: migration; siRNA; nonmuscle; localization; Rho-kinase
Abbreviations used in this paper: DIC, differential interference contrast; MBS, myosin binding subunit; MLC20, myosin regulatory light chain; MLCK, myosin light chain kinase; MLCP, myosin phosphatase; Rho-kinase, Rho-associated kinase; RNAi, RNA interference; siRNA, small interfering RNA; ZIP, zipper-interacting protein.
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Introduction |
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Although MLCK is thought to be responsible for myosin II phosphorylation, recent studies have suggested that other protein kinases might also contribute to phosphorylation of myosin II. Amano et al. (1996) showed that Rho-associated kinase (Rho-kinase) phosphorylates MLC20 at Ser19. Recently it was reported that zipper-interacting protein (ZIP) kinase (Murata-Hori et al., 1999) and integrin-linked kinase (Deng et al., 2001) phosphorylate MLC20 at Ser19 and Thr18. Interestingly, ZIP kinase phosphorylate Ser19 and Thr18 of MLC20 with similar potency in contrast to MLCK (Niiro and Ikebe, 2001). These findings have raised a hypothesis that myosin II can be phosphorylated by various protein kinases in cells by diverse stimulations. On the other hand, it has been realized that the phosphorylation level of myosin II is also controlled by regulating myosin phosphatase (MLCP). Kimura et al. (1996) showed that the myosin binding subunit (MBS) of MLCP is phosphorylated by Rho-kinase and the phosphorylation down-regulates MLCP activity. The phosphorylation site of MBS responsible for the down-regulation of MLCP is Thr641 (rat sequence; Feng et al., 1999) and it was found subsequently that MBS can be phosphorylated at Thr641 by various kinases including ZIP kinase like kinase (MacDonald et al., 2001) and integrin-linked kinase (Kiss et al., 2002; Muranyi et al., 2002) suggesting that MLCP activity is regulated via multiple signaling pathways.
Phosphorylation of myosin II has been thought to be critical for the various actin-based contractile events in nonmuscle cells (Tan et al., 1992; Komatsu et al., 2000). Because the assembly and the motor activity of myosin II is regulated by MLC20 phosphorylation, the localization of phosphorylated MLC20 would reflect the distribution of activated phosphorylated myosin II in motile cells. A critical question is whether the localization of myosin II phosphorylated at only Ser19 and at both Ser19 + Thr18 of MLC20 are different from each other and whether this is related to the function of myosin at particular cellular compartment, because the diphosphorylation of MLC20 significantly facilitates the formation of stable myosin filaments (Ikebe et al., 1988; Kamisoyama et al., 1994).
Here, we identified that ZIP kinase is responsible for myosin phosphorylation in motile fibroblasts. The present paper also showed that there is a significant amount of diphosphorylated myosin in motile cells whose localization is different from the monophosphorylated one and plays an important role in the maintenance of cell morphology and migration. ZIP kinase phosphorylating Thr18 and Ser19 of MLC20 with the same potency is primarily responsible for this event.
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Results |
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The specificity of pTS Ab was examined by immunoblot analysis. MLC20 was phosphorylated by MLCK, PKC, and ZIP kinase. The unphosphorylated, mono-, and diphosphorylated MLC20s were separated on an urea/glycerol gel (Fig. 1 A, top), followed by immunoblotting with pTS Ab (Fig. 1 A, middle). The pTS Ab only recognized the diphosphorylated MLC20 by MLCK and ZIP kinase, but did not recognized unphosphorylated, monophosphorylated by MLCK and ZIP kinase, or monophosphorylated (Thr9) MLC20 by PKC. It was shown previously that ZIP kinase phosphorylates Ser19 and Thr18 of MLC20 with same rate constant thus yielding the same amount of Ser19 phosphorylated MLC20 and Thr18 phosphorylated MLC20 (Niiro and Ikebe, 2001). Therefore, the results shown in Fig. 1 indicate that pTS Ab specifically recognizes diphosphorylated MLC20. Fig. 1 B shows the immunoblotting of whole cell lysates of REF-2A fibroblast and NRK epithelial cells. The result indicates that the antibodies specifically recognize MLC20 but not other proteins. pTS Ab and pSer19 Ab recognizing the Ser19 phosphorylated MLC20 were used as probes to determine the distribution of di- and monophosphorylated myosin II at MLC20 in motile fibroblasts.
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As shown in Fig. 5 C, Rho-kinase inhibitor, Y27632 (maximal 100 µM), had no significant effect on myosin phosphorylation in the cell extracts. To examine whether Rho-kinase in the cell extracts is active, we used MBS as a substrate for Rho-kinase (Kimura et al., 1996). MBS was incubated with the extracts and followed by immunoblotting with pThr 641 Ab or pThr 799 Ab that recognizes the phosphorylated MBS at the two Rho-kinaseinduced phosphorylation sites, Thr641 or Ser799, respectively (Kawano et al., 1999). As shown in Fig. 5 D, the cell extracts phosphorylated MBS, and the phosphorylation was inhibited by Y27632. The results indicate that there is significant Rho-kinase activity in the cell extracts, but Rho-kinase does not significantly phosphorylate myosin.
In contrast, the MLCK inhibitor, ML-7, inhibited myosin II phosphorylation by the cell extracts (Fig. 5 C). Myosin phosphorylation in Ca2+ by the cell extracts was decreased by 50% with 30 µM ML-7 (Fig. 5 C), whereas the phosphorylation by isolated Rho-kinase was not significantly inhibited by ML-7 even with 100 µM (Fig. 5 E, middle). However, myosin phosphorylation activity in the cell extracts was not affected by the elimination of Ca2+ (Fig. 5 C, +EGTA) and this is contradictory to the fact that MLCK requires Ca2+/CaM for its activity. One possibility to account for this discrepancy is that MLCK might become the constitutively active form by proteolysis during the preparation steps of cell extracts (Ikebe et al., 1987b). To address this possibility, MLCK-specific peptide inhibitor, SM-1, which strongly inhibits both native and constitutively active MLCK (Ikebe et al., 1987b), was examined for the inhibition of the kinase activity in the cell extracts. As shown in Fig. 6, SM-1 peptide inhibitor did not inhibit the kinase activity in the cell extracts, whereas it significantly inhibited the 61-kD constitutively active form of MLCK. These results indicate that major myosin II kinase activity in the cell extract was neither Rho-kinase nor MLCK. These results together with the results of Figs. 46 suggest that ML-7 sensitive kinases other than Ca2+/CaM-dependent MLCK are responsible for myosin phosphorylation in fibroblast cells.
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To further evaluate the role of ZIP kinase in myosin II phosphorylation in mammalian cultured cells, we diminished ZIP kinase expression by using RNA interference (RNAi) technique. NIH3T3 cells were transfected with small interfering RNA (siRNA) oligoduplex corresponding to the coding region of the ZIP kinase mRNA. The siRNA-transfected cells were harvested and subjected to Western blot analysis using the specific antibodies as probes. Immunoblots showed that two molecular mass proteins recognized by ZIP kinase Ab (58 and 34 kD, respectively) were diminished in the cells transfected with siRNA (Fig. 8 A, left). It was reported that there are two ZIP kinase variants having different molecular weights (Kawai et al., 1998; Kogel et al., 1998; MacDonald et al., 2001), and it is expected that these two molecular weight bands are corresponding to the longer and shorter forms of ZIP kinase variants. Consistently, the myosin II kinase activity in cytosol fraction that is predominantly due to ZIP kinase activity was significantly decreased. Myosin II kinase activity in cytoplasmic fractions from cells transfected with either mock or ZIP kinase siRNA were measured. The activity was significantly diminished compared with that of mock-transfected cells (Fig. 8 D; 67% decrease). The extent of the decrease in activity was comparable to the decrease in the expression level of ZIP kinase (65% decrease). The siRNA specific to ZIP kinase attenuated neither Rho-kinase nor MLCK (Fig. 8 A, right). Consistently, MBS phosphorylation at Thr799, a Rho-kinasespecific site, was unaffected by the ZIP kinasespecific siRNA (Fig. 8 A, right). We also found that the siRNA treatment did not affect MBS phosphorylation at Thr641, another Rho kinase site. It was reported that ZIP kinase-like kinase can phosphorylate MBS at Thr641 in vitro (MacDonald et al., 2001). As shown in Fig. 8 A, both mono- and diphosphorylated MLC20 were markedly decreased in ZIP kinase siRNA-transfected cells compared with that of mock-transfected cells. These results suggest that the decrease in myosin phosphorylation via the suppression of ZIP kinase causes failure of direct myosin phosphorylation by ZIP kinase but not down-regulation of MLCP through the MBS phosphorylation. Fig. 8 B shows immunostaining of the siRNA-transfected cells with the phosphorylation site-specific antibodies. Consistent with the Western blot analysis, the intensity of immunofluorescence signals of pTS Ab was significantly reduced in the ZIP kinase siRNA-transfected cells (Fig. 8 B, e) compared with the control cells (Fig. 8 B, a). The decrease in MLC20 phosphorylation in the siRNA-transfected cells was observed at the central stress fibers (Fig. 8 B, e), similar to that observed with ML-7treated cells (Fig. 4 A). It was also recognized that the filamentous structure of myosin II was low in the cells transfected with ZIP kinase siRNA (Fig. 8 C, c). This is consistent with the lower myosin II phosphorylation in siRNA-transfected cells because it is known that myosin II phosphorylation induces the filament formation of myosin (Ikebe and Hartshorne, 1985b; Sellers et al., 1985). To warrant that the effect of ZIP kinase siRNA on the decrease in MLC20 phosphorylation is due to the elimination of ZIP kinase, we examined whether human ZIP kinase that is resistant to siRNA can rescue the decrease in MLC20 phosphorylation of the cells treated with ZIP kinase siRNA. The cells treated by mouse ZIP kinase siRNA were transfected by human ZIP kinase and MLC20 phosphorylation level was monitored. The cells expressing human GFP-ZIP kinase showed strong diphosphorylation signals at the stress fiber (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200309056/DC1). The focal adhesion was also reduced in the siRNA-transfected cells (Fig. 8 B, g; decreased by 45.3 ± 9.6%). Consistent with this observation, a large number of nonadhesive cells were observed when siRNA-treated cells were cultured on noncoated or poly-D-lysinecoated glass bottom culture dishes (unpublished data). These results suggest that ZIP kinase plays a critical role in the change in MLC20 phosphorylation and the regulation of cytoskeletal structure in mammalian cultured cells.
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Discussion |
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Protein kinases responsible for the phosphorylation of myosin II in migrating cells
A critical question to understand the regulation of myosin II phosphorylation in migrating cells is the identity of the protein kinases phosphorylating MLC20. MLCK has been thought to be a predominant kinase responsible for myosin II phosphorylation in mammalian nonmuscle cells. This is partly because the possibility of other kinases to be physiologically important myosin II kinase has been overlooked. Recently, it was shown that ZIP kinase can phosphorylate MLC20 at Ser19 and Thr18. A critical finding is that ZIP kinase unlike MLCK phosphorylates Ser19 and Thr18 with same rate constant yielding diphosphorylated MLC20 effectively. Successful production of the antibody recognizing diphosphorylated MLC20 enables us to study the nature of critical myosin II kinase in nonmuscle cells. Our results indicate that the protein kinases responsible for the phosphorylation of myosin II are the kinases that phosphorylate MLC20 at Ser19 and Thr18 with similar rate constant because (a) a significant level of the diphosphorylated MLC20 was present in the cells; (b) the kinases in the cell extract produced diphosphorylated MLC20 at the time when a significant portion of unphosphorylated MLC20 remained unlike MLCK; and (c) SM-1 as well as wortmannin, MLCK inhibitors, had no detectable effect on myosin II kinase activity in the cell extract. It has been shown in vitro that Rho-kinase can phosphorylate myosin II at Ser19 (Amano et al., 1996). The incubation of the cells with Rho-kinase inhibitor, Y27632, a specific inhibitor of Rho-kinase, significantly inhibited MLC20 phosphorylation in cells. However, Y27632 did not inhibit myosin phosphorylation activity in the cell extracts. We think that the inhibition of MLC20 phosphorylation in cells by Y27632 is not due to the inhibition of myosin II kinase but due to the activation of MLCP. Supporting this view, we found that MLCP activity obtained from the cells treated with Y27632 was 1.8 times higher than that obtained from the untreated cells (unpublished data). It has been reported that the inhibition of MLCP by phosphatase inhibitor or microinjection of MBS Ab into mammalian cultured cells increases MLC20 phosphorylation (Chartier et al., 1991; Totsukawa et al., 2000). Together with our present paper, we think that Rho-kinase mainly contributes to myosin phosphorylation through the regulation of MLCP but not direct myosin II phosphorylation in vivo.
Interestingly, ML-7 diminished the myosin II kinase activity in the cell extracts. Consistently, ML-7 also attenuated the MLC20 phosphorylation in cells. However, the kinase activity in the cell extracts was neither inhibited by EGTA nor SM1 peptide, suggesting that ML-7 inhibit the kinases other than MLCK in the cell extract. Supporting this idea, ML-7 inhibited the purified ZIP kinase with similar concentration dependence against the inhibition of the kinases in the cell extracts. Recently, it was reported that ZIP kinase like kinase purified in smooth muscle is inhibited by ML-9 that is similar to ML-7 and the present result is consistent with this observation (Borman et al., 2002). The present result indicates that ML-7 is not specific to MLCK but also inhibits ZIP kinase, therefore, earlier results using ML-7 as a MLCKspecific inhibitor may need to be reevaluated.
To further ensure the importance of ZIP kinase for myosin phosphorylation in mammalian cultured cells, we have used several approaches. First, identity of the major myosin II kinase in the cell extracts as ZIP kinase was demonstrated by the immunodepletion experiment. The depletion of ZIP kinase by the specific antibodies markedly reduced the myosin II kinase activity in the cell extracts, indicating that ZIP kinase is the major kinase responsible for myosin phosphorylation in the cell extracts. Consistently, the immunoprecipitation of the extracts using the ZIP kinase Ab recovered the myosin II kinase activity. Second, the microinjection of ZIP kinase into serum-starved NIH3T3 cells induced myosin phosphorylation (unpublished data). Supporting the idea that ZIP kinase participates in myosin phosphorylation, the overexpression of ZIP kinase in HeLa cells induced myosin phosphorylation (Murata-Hori et al., 2001). It was also reported that ZIP kinase increases myosin phosphorylation of smooth muscle strips and induces contraction (Niiro and Ikebe, 2001). These previous results support the idea that ZIP kinase can increase myosin II phosphorylation and activate the contractile activity of actomyosin.
Further evidence that ZIP kinase is critical for myosin phosphorylation in mammalian cells was obtained using a recently developed siRNA technique (Fire et al., 1998). The depletion of endogenous ZIP kinase in NIH3T3 fibroblasts by the specific siRNA decreased mono- and diphosphorylation of MLC20 without changing myosin expression level. Furthermore, immunocytochemical analysis revealed that diphosphorylated myosin filaments at the central region were remarkably diminished by transfection of the ZIP kinase siRNA. Interestingly, myosin phosphorylation at the cortical region was not completely abolished in the ZIP kinasedepleted cells, suggesting that other kinases may be involved in myosin phosphorylation at this region. Recently, it was shown that MLCK contributes to myosin phosphorylation at the cortical region but not in the center (Totsukawa et al., 2000, 2004), therefore, myosin phosphorylation at the cell cortical region may be in part mediated by MLCK.
ZIP kinase is important for NIH3T3 cell polarity and migration
We found that the disruption of ZIP kinase causes the change in cell morphology and migratory behavior of NIH3T3 fibroblasts. It is widely believed that the reorganization of actomyosin is an essential process for progress of cell migration and that myosin phosphorylation is involved in this process. In the present paper, we found that the interference of ZIP kinase inhibits cell migration activity. Interestingly, attenuation of ZIP kinase induced elongated cell morphology. We think that the decrease in myosin phosphorylation via the depletion of ZIP kinases causes failure of stable myosin filament formation in stress fiber structure and thus changing cytoskeletal structure and cell morphology. This might allow cells to become elongated and lose motility.
In summary, based upon the present paper, we propose that ZIP kinase promotes dynamic rearrangement of myosin structure through the myosin phosphorylation in motile fibroblast cells and contributes to the cell motile processes involving in spreading and migration.
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Materials and methods |
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Antibodies
A phosphopeptide KKRPQRAphosphoTSNVFAMC was coupled to keyhole limpet hemocyanin at COOH-terminal cysteine residue. A pTS Ab was affinity purified using the phosphopeptide and then absorbed with unphosphopeptide. A pSer19 Ab, ZIP kinase Ab, and phosphorylation-specific Ab against MBS at Thr 641 or Ser799 were described previously (Komatsu et al., 2000; Niiro and Ikebe, 2001; Takizawa et al., 2002). A rabbit Ab against heavy chain of myosin IIB, MLC20, and MLCK were provided by R. Adelstein (National Institutes of Health, Bethesda, MD), J. Stull (University of Texas Southwestern Medical Center, Dallas, TX), and P. de Lanerolle (University of Illinois, Chicago, IL), respectively. Anti-MLC20, MBS, ROK, ß-actin, and paxillin Abs were purchased from Sigma-Aldrich, Covance Research Products Inc., and Transduction Laboratories, respectively.
Cell culture, microinjection, and transfection
REF-2A cells (a gift from F. Matsumura, Rutgers University, Piscataway, NJ) and NIH3T3 fibroblast cells were maintained in DME containing 10% newborn calf serum. NRK cells (NRK52E; a gift from Y.-L. Wang, University of Massachusetts, Worcester, MA) and COS 7 cells were cultured in F12 medium (Sigma-Aldrich) containing 10% FBS (GIBCO BRL), 2 mM L-glutamine or DME containing 10% FBS, respectively. Microinjection was performed using a micromanipulator (Transjector 5246; Eppendorf). 0.1mg/ml of ZIP kinase was coinjected with FITC-dextran.
For RNAi, the selected sequences were submitted to a BLAST search to ensure that only ZIP kinase gene was targeted. The targeting sequence of mouse ZIP kinase (AB007143), AAGACAGATGTGGTGCTGATC, corresponding to the coding region 256276 of ZIP kinase was used for siRNA and synthesized by Dharmacon Research. Double strand siRNA was prepared according to the manufacturer's protocol (Dharmacon), and transfected using Lipofectamine 2000 (Invitrogen). As a negative control (nonspecific siRNA), human ZIP kinase (AB022341) siRNA (AAGACGGACGTGGTCCTCATC) was used. siRNA-transfected cells were cultured on the fibronectin (10 µg/ml)-coated glass coverslips.
Preparation of cell extracts
REF-2A cells were washed and then lysed in buffer I (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 0.1 mM EGTA, 5 mM DTT, 5% glycerol, 0.2 mM N-p-tosyl-L-lysine chloromethl ketone, 0.2 mM N-tosyl-L-phenylal-anine chloromethl ketone, 2 mM PMSF, and 0.05% NP-40). After added 0.4 M NaCl, cell lysates were sonicated and centrifuged at 10,000 g for 15 min. Protein concentration was determined by the method of Bradford (1976) by using BSA as a standard. For NIH3T3 cells, nuclear and cytosol fractions were prepared from cells treated with siRNA using Nuclear/Cytosol Fractionation Kit (BioVision, Inc.).
Immunoprecipitation and immunodepletion
The cell extracts were incubated with either nonspecific rabbit IgGs or anti-ZIP kinase Ab at 4°C for 3 h and then protein A-Support (Bio-Rad Laboratories) was added. The immunocomplex was centrifuged, washed three times with wash buffer (0.1 M KCl and Tris-HCl, pH 8.8), and two times with buffer B and used for myosin phosphorylation assay.
Biochemical procedures
Urea/glycerol PAGE (Perrie and Perry, 1970) and SDS-PAGE (Laemmli, 1970) were performed as described previously. MLC20 was phosphorylated by MLCK and PKC (Ikebe and Hartshorne, 1985a; Ikebe et al., 1987a). Immunoblotting was done as described previously using nitrocellulose membranes (Yano et al., 1993; Komatsu et al., 2000). In vitro phosphorylation was performed using buffer containing 30 mM NaCl, 5 mM MgCl2, 1 µM microcystin-LR, 0.2 mM ATP, and 30 mM Tris-HCl, pH 7.5, and 0.2 mM CaCl2 for buffer A and 5 mM EGTA for buffer B. Myosin (0.4 mg/ml) or MBS was phosphorylated in the presence of kinase inhibitors (Y27632, ML-7 in buffer A or SM-1 peptide in buffer B) by 0.2mg/ml of cell extracts or exogenous kinases (1 µg/ml Rho-kinase, 1 µg/ml CaM, and 1 µg/ml MLCK in buffer A, Ca2+/CaM-independent 1 µg/ml MLCK or 1 µg/ml GST-ZIP kinase in buffer B) in the presence of kinase inhibitors. The reaction was done for 15 min at 30°C, and then phosphorylated MLC20 or MBS was detected by Western blotting analysis.
Immunofluorescence staining and image processing
Immunocytochemistry was performed as described previously (Komatsu et al., 2000). Cells were stained with Texas redconjugated phalloidin (Molecular Probes) for F-actin. For double staining with pSer19 Ab and pTS Ab, the cells were first stained with pTS Ab for 6 h at 4°C and then followed by pSer19 Ab overnight. DIC and fluorescence images were viewed using a DM IRB laser scanning confocal microscope (Leica) controlled by TCS SP II systems (Leica). All images were taken with same laser out put to directly compare the fluorescence signal intensities. Images were processed using Adobe® Photoshop® 5.5 software.
Migration assays
Cell migration was studied using transwell migration chambers (6.5-mm diam; 8-µm pore size; COSTAR Corp.) coated on both sides of the membrane with 10 µg/ml fibronectin in PBS for 16 h at 4°C. Mock- or siRNA-transfected NIH3T3 cells were cultured for 24 h in DME supplemented with 0.1% newborn calf serum and then detached by trypsinization. Assays were performed by the addition of the cells (5 x 104 cells/well) to the upper compartment of the transwell chamber and allowed to migrate to the membrane in the bottom chambers containing medium supplemented with 20 ng/ml PDGF for 3 h. Migrated cells attached to the bottom surface of the membrane were fixed in methanol, stained with Giemsa, and counted.
Online supplemental material
Fig. S1 shows that immunodepletion of ZIP kinase had no effect on the expression level of both ROK and MLCK (A) and that ZIP kinase was responsible for myosin II phosphorylation in the cell extracts from different cell types (B). Fig. S2 shows that the decrease in myosin II phosphorylation by mouse ZIP kinase siRNA was rescued by expression of human ZIP kinase. Fig. S3 shows the wound healing assay indicating that the migration of ZIP kinase siRNA-transfected cells is slower than that of mock-transfected cells. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200309056/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL60831, HL61426, and AR41653.
Submitted: 9 September 2003
Accepted: 16 March 2004
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References |
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