Article |
Address correspondence to Angus I. Lamond, Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee. Dundee DD1 5EH, Scotland, UK. Tel.: 44-1382-345473. Fax: 44-1382-345695. email: a.i.lamond{at}dundee.ac.uk
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Abstract |
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Key Words: nucleolus; nucleus; mitosis; fluorescent protein; 4D imaging
G. Miller's present address is Fred Hutchinson Cancer Research Center, MS A1-162, 1100 Fairview Ave. N., Seattle, WA 9810.
C. Lyon's present address is Cyclacel, James Lindsay Place, Dundee Technopole, Dundee DD1 5JJ, UK.
Abbreviations used in this paper: B23, nucleophosmin/nucleolar phosphoprotein B23/numatrin; DFC, dense fibrillar component; FC, fibrillar centre; FIB, fibrillarin; FP, fluorescent protein; GC, granular component; IBB, importin-ß binding; LB1, lamin B1; LBR, lamin B receptor; NE, nuclear envelope; NOR, nucleolar organizing region; PNB, prenucleolar body; rDNA, ribosomal DNA; RL27, ribosomal protein L27; RPA39, RNA polymerase I subunit RPA39; RRN3, RNA polymerase I transcription factor RRN3; UBF, upstream binding transcription factor.
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Introduction |
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During mitosis, mammalian nucleoli disassemble and their components disperse. When cells exit mitosis nucleolar components reassemble around the respective NORs, which can later coalesce to form either one or multiple functional nucleoli. Partially processed rRNA transcripts, together with associated processing factors, form structures during mitosis termed "prenucleolar bodies" (PNBs; Jimenez-Garcia et al., 1994; Dundr et al., 2000; Savino et al., 2001). Components are subsequently transferred from the PNBs into the reforming nucleoli at NORs (Dundr et al., 2000; Savino et al., 2001). In contrast, the process of nucleolar disassembly when cells enter mitosis is not well characterized. Both the timing and mechanism of breakdown is unclear although it has been reported that a subset of nucleolar factors, including RNA polymerase I (Scheer and Rose, 1984; Gilbert et al., 1995) and the RNA Pol I upstream binding transcription factor (UBF) and transcription factor SL1 (Roussel et al., 1993; Jordan et al., 1996), remain associated with chromosomes at NORs, although no nascent rRNA transcripts are synthesized during mitosis (Weisenberger and Scheer, 1995).
Fluorescent protein (FP)-tagged nuclear factors provide markers in live cells for the location of specific nuclear bodies, such as nucleoli, or even subcompartments within nucleoli (Gerlich and Ellenberg, 2003a; Janicki and Spector, 2003). Proteins tagged with different spectral variants of either GFP, or RFP, can be coexpressed to detect two or more subnuclear structures in a single cell. The 3D localization of these markers can be recorded and quantitated over time, i.e., "4D" imaging, to provide detailed information about their dynamic movement and kinetic behavior (Gerlich et al., 2001; Dundr et al., 2002b; Gerlich and Ellenberg, 2003a). Here, we have applied quantitative 4D imaging using cell lines expressing different combinations of two or three FP-tagged markers to analyze the processes of nucleolar breakdown and reassembly during mitosis in live cells and to correlate this with parallel events affecting other nuclear structures.
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Results |
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We next used the HeLaYFP-RPA39 cells to monitor the localization of FP-RPA39 throughout an entire mitosis, using both confocal and deconvolution fluorescence microscopy. Time-lapse microscopy showed that RPA39 remained concentrated in chromatin-associated foci for most of mitosis, consistent with previous data (Scheer and Rose, 1984; Gilbert et al., 1995). However, detailed time-lapse analysis of single live cells consistently showed a window during metaphase, lasting 30 min, when RPA39 is no longer detected in chromatin foci (Fig. 2, Metaphase, arrowheads; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200405013/DC1). Loss of RNA polymerase I subunits from chromatin, specifically during metaphase, was also observed by immunolocalization, using monoclonal antibodies specific for the RPA20 subunit (see Online supplemental material). Although RPA20 has been reported as a core subunit of RNA polymerases I, II, and III, during interphase the immunofluorescence signal of RPA20 detected using this antibody predominantly colocalizes in bright foci within nucleoli that contain RRN3 (Jones et al., 2000; see Online supplemental material). An independent study has also found that RNA polymerase I subunits RPA194, RPA39, and RPA16, but not RPA43, are transiently lost from chromatin-associated foci during metaphase (Dundr, N., and T. Misteli, personal communication). We suggest that RNA polymerase I either transiently leaves chromosomes during metaphase, or else that multiple subunits dissociate transiently from the polymerase ternary complex. However, immunolabeling with antibodies specific for the RNA polymerase I UBF, which binds to the rRNA gene repeats, showed that UBF associated with chromatin foci throughout mitosis (Fig. 2, UBF). Thus, although UBF colocalizes in foci with RPA39 during prophase, prometaphase, anaphase, and telophase, it remains in similar foci throughout metaphase when the RNA polymerase I subunits are no longer detected (Fig. 2). The loss of RNA polymerase I subunits from chromatin foci is therefore not a detection problem at this stage of metaphase. In summary, the data indicate that the previous view that RNA polymerase I remains associated with chromatin throughout the entire period of mitosis may need to be revised.
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To quantitate the pathway of nucleolar disassembly, we measured the levels of FP-fluorescence in defined cellular structures during mitotic progression (Fig. 3 D; see Online supplemental material). Data obtained from 5 to 25 separate experiments for each fluorescent marker show that the rate of loss of RPA39 from nucleoli is slower than either RL27 or B23, though comparable to FIB (Fig. 3 D). However, the loss of RPA39 is initiated earlier than any of the other markers tested. A comparison of the times at which the levels of fluorescence for each marker dropped to 50% of their initial values in nucleoli indicates that loss of RPA39 precedes the other markers by 4 min (Fig. 3, D and E). The loss of RNA polymerase I subunits is therefore the earliest event we have detected at the onset of nucleolar disassembly.
Comparison of nucleolar and nuclear envelope (NE) disassembly
We next compared the relative timing of nucleolar and NE disassembly using stable cell lines expressing either YFP-Lamin B receptor (HeLaYFP-LBR), or YFP-lamin B1 (HeLaYFP-LB1). These are the first and last components, respectively, to disassemble from NE during mitosis (Beaudouin et al., 2002). We performed 4D analyses using both these cell lines with DsRed-B23 expressed transiently (Fig. 4). Nucleoli containing B23 are still detected when lamin B receptor (LBR) starts to dissociate from NE (Fig. 4 A). This is most clear on the xz projection (Fig. 4 A, bottom). Loss of B23 from nucleoli occurs 1.5 min after the decrease in LBR signal from NE (Fig. 5 D). In contrast, analysis of HeLaYFP-LB1 cells shows that the loss of B23 from nucleoli occurs
2 min before dissociation of lamin B1 (LB1) from NE (Fig. 4 B; Fig. 5 D). Therefore, nucleolar disassembly occurs predominantly within the window during which NE components dissociate.
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Reassembly of NORs
We next analyzed nuclear and nucleolar reassembly after mitosis in cells stably expressing YFP-FIB, and CFP-H2B and transiently expressing HcRed-IBB (Fig. 6). FIB is associated with chromatin in the daughter nuclei at an early stage, before the re-import of IBB (Fig. 6 A). This chromatin association is at least 5 min before the detection of any foci corresponding to the reformation of nucleoli (Fig. 6 A and Fig. 7 A). RPA39 also associates early with daughter nuclei, before the nuclear accumulation of IBB (Fig. 6 B). However, RPA39 is immediately detected in chromatin-associated foci, which only accumulate FIB 5 min later (Fig. 6 C, foci, arrowheads). Interestingly, although FIB is already present in the nuclei, it only accumulates at the RPA39 foci when IBB accumulates in the daughter nuclei (Fig. 6 B and C). At the same time we observe a clear increase in the number of foci containing both FIB and RPA39 (Fig. 6 B, 05:2612:59). This suggests that reestablishment of nuclear protein import is important for stepwise reformation of nucleoli.
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Interestingly, there is a close connection between the timing of FIB accumulation in the DFC and the time at which a functional NE is reestablished. Similarly, during nucleolar disassembly, loss of FIB from the DFC closely correlates with the time at which the functional integrity of NE is compromised (compare Fig. 5 with Fig. 6). This raises the interesting possibility that one or more factors normally excluded from the nucleus by an intact NE can contribute to signaling the dissociation of FIB from the nucleolus.
Reassembly of functional nucleoli
We define the reformation of functional nucleoli as requiring the presence of all the major interphase markers for the FC, DFC and GC subcompartments. To monitor this in live cells we analyzed cell lines stably expressing both CFP-B23 and YFP-FIB and transiently expressing DsRed-RL27 (Fig. 7). Together with the data from Fig. 6 showing the stepwise assembly of FC and DFC, these data showed for both the B23 and RL27 markers that the GC forms later than either the FC or DFC, consistent with previous immunofluorescence data (Dundr et al., 2000; Savino et al., 2001). Quantitative analysis showed that the GC forms 18 min after the DFC and
27 min after the FC. The appearance of the GC after mitosis correlates closely with the time at which BrUTP-labeled RNA appears in the GC in interphase pulse-chase experiments (unpublished data). Next, we addressed quantitatively the concentration kinetics of nucleolar factors in cells exiting mitosis (Fig. 7 B). These experiments indicate that FIB accumulates before both GC markers B23 and RL27, which reassemble with similar initial kinetics. In summary, these data support the view that the reassembly of nucleoli is normally coupled to the activation of ribosome subunit synthesis. The relationship between different nucleolar subcompartment markers in terms of their timing in nucleolar reassembly is summarized in Fig. 7 C.
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Discussion |
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The advantages of live cell imaging have recently been applied to study several dynamic nuclear processes (Clute and Pines, 1999; Gerlich et al., 2001; Beaudouin et al., 2002; Gerlich and Ellenberg, 2003a; Prasanth et al., 2003). A feature of the live cell approach used here is that we can conduct quantitative studies on nucleolar dynamics during mitosis at the single cell level. Moreover, we have directly correlated quantitatively the temporal changes in each of the separate nucleolar, NE and chromatin components within the nucleus. This has allowed us to detect small differences in the timing of events that would not be apparent by conventional immunofluorescence approaches, where temporal information is not available. Similarly, biochemical methods sample the mean properties of cell populations, rather than the behavior or sequence of events in individual cells. For example, we could reproducibly detect a timing difference as small as 4 min in the loss of RPA39 from the FC, before loss of DFC or GC markers during nucleolar breakdown. Quantitative analyses of these time-lapse data obtained from multiple cells in each experiment also provided information regarding the kinetic behavior of nuclear proteins across the cell population and showed the degree of variation between nuclei.
Recent studies on the dynamics of nucleoli during mitosis have focused mainly on the process of postmitotic nucleolar reformation (Dundr et al., 2000; Savino et al., 2001). Two previous studies have included the use of live cell imaging to analyze events during the formation of nucleoli (Dundr et al., 2000; Savino et al., 2001). Both studies used a combination of immunofluorescence and cell lines expressing a single GFP-tagged marker to analyze the temporal order of formation of PNBs and nucleoli. In particular, both the composition of PNBs, which accumulate partially processed rRNA precursors and associated components, and how these components are subsequently transferred into NORs for nucleolar assembly have been studied (Jimenez-Garcia et al., 1994; Dundr et al., 2000; Savino et al., 2001). Here, we have focused on quantitating the temporal pathway of nucleologenesis as well as studying the spatial organization of reforming NORs. In each of the live cells analyzed, multiple FP-tagged markers used for different nucleolar subcompartments allows us to distinguish reforming NORs and nucleoli from PNBs. For example, PNBs do not contain RNA polymerase I, which reassociates with the NOR early during nucleologenesis. Thus, we could perform a correlative as well as quantitative analysis of components from all three nucleolar subcompartments in parallel. More importantly, we have extended these analyses by comparing the processes of nucleolar breakdown as well as reassembly in the same cells. A novel conclusion from this work is that RNA polymerase I subunits RPA39 and RPA20 transiently leave the NORs during metaphase, whereas UBF remains associated with NORs throughout mitosis. Our findings are supported by the data from a recent independent study showing that other RNA polymerase I subunits, specifically, RPA194, RPA39, and RPA16, but not RPA43, also leave the NORs during metaphase (Dundr, M., and T. Misteli, personal communication). These results differ from the current view that RNA polymerase I, as well as UBF, remains associated with NORs throughout mitosis, based on immunofluorescence data using fixed cells, where it is difficult to detect a transient absence of RNA polymerase I. Our data in contrast indicate that multiple RNA polymerase I subunits either leave the chromosomes transiently, or else decrease in concentration below our detection limit, for a brief period at metaphase. We note that this can explain the previous observation based on high resolution in situ hybridization studies that metaphase chromosomes do not contain nascent rRNA (Weisenberger and Scheer, 1995). Our data are also consistent with the findings from run-on assays that rRNA genes in metaphase chromosomes, whereas still in the same open configuration as interphase chromosomes, are less transcriptionally active (Conconi et al., 1989). Our present data therefore indicate that the mitotic behavior of RNA polymerase I may be more similar to RNA polymerase II than was previously apparent.
We observe distinct kinetic behavior of individual proteins during nucleolar disassembly (Fig. 3 D and Fig. 5 C). For example, although dissociation of B23 and RL27 from nucleoli initiates later than RPA39 or FIB, their dissociation rate is higher. Both GC components dissociate from nucleoli at similar rates, with comparable kinetics to cytoplasmic dispersal of IBB upon NE breakdown, which is most likely a diffusion-limited event. Although the molecular mechanism of nucleolar disassembly remains poorly understood, the present data raise the possibility that distinct processes could operate sequentially and/or independently during the disassembly of FC, DFC, and GC subcompartments. We observe that RPA39 leaves the nucleolus before breakdown of nuclear lamina components, whereas the DFC and GC markers are lost during the period of nuclear lamina breakdown. It is known from previous studies that lamina disassembly is triggered via cyclin B-CDK1 mediated phosphorylation of multiple components, including LB1 (Pines and Rieder, 2001; Burke and Ellenberg, 2002). Coincidently, the same cyclin complex is involved in repression of mitotic ribosomal transcription and nucleolar reformation. For example, phosphorylation of UBF and transcription factor SL1 by CDK1 causes shut-off of RNA polymerase I transcription (Heix et al., 1998; Klein and Grummt, 1999). The CDK1 inhibitor roscovitine also causes reactivation of RNA polymerase I transcription during mitosis but not the recruitment of rRNA processing factors to the rRNA gene repeats (Sirri et al., 2000, 2002). Our data indicate that the loss of RNA polymerase I and hence transcription of rRNA genes is likely to be the initial event during mitotic disassembly of nucleoli. However, loss of rRNA gene transcription alone may not be sufficient to cause subsequent disassembly of the entire nucleolus. For example, although inhibition of ribosomal transcription during interphase causes RNA polymerase I subunits to leave nucleoli in vivo (unpublished data), the inhibition of ribosomal transcription by Actinomycin D in isolated nucleoli does not cause nucleoli to disintegrate in vitro (unpublished data). Therefore, we propose that the mitotic disassembly of the DFC and GC subcompartments is a result of an active mechanism rather than an indirect effect of the loss of transcriptional activity. This is consistent with a recent study that germ cell proteins FRGY2a and FRGY2b can reversibly disassemble somatic nucleoli in Xenopus egg cytoplasm independent of rRNA transcription (Gonda et al., 2003), suggesting that transcription activity and nucleolar integrity may not be obligatorily coupled. It will thus be interesting in future to test whether molecular mechanisms such as phosphorylation by the cyclin BCDK1 complex may play a role in either RPA39 dissociation from FCs or in other steps in the nucleolar breakdown pathway.
In contrast with the precise temporal regulation in the order of events during both disassembly and reformation of nucleoli during mitosis, higher order aspects of nucleologenesis appear to be less strictly controlled. A statistical evaluation of the number of FC foci and functional nucleoli that appear after mitosis suggested that daughter nuclei are more similar to each other than to unrelated nuclei exiting mitosis elsewhere, consistent with the expected conservation in global chromosome positioning (Gerlich and Ellenberg, 2003b; Walter et al., 2003). Nonetheless, we observe statistically significant differences between the two daughter nuclei. At least from the point at which RNA polymerase I subunits reassociate with NORs, it appears that local effects, which can differ between daughter nuclei, influence the overall pathway of nucleologenesis. Effects acting at the level of the local chromosome environment, such as variations in chromosome orientation and decondensation (Thomson et al., 2004), variable activation of rRNA genes within the repeat clusters and variation in protein concentration kinetics (Dundr et al., 2002a), may all influence both the association of RNA polymerase I with NORs and the probability of NOR fusion and hence the number of active nucleoli formed. It is interesting to compare this view with a recent FISH analysis on the process of induced RNA polymerase II gene activation at the single cell level (Levsky et al., 2002). Both this FISH analysis and our work suggest that the pattern and level of gene activation varies at the single cell level, which had not been apparent from previous biochemical studies from cell populations. Although it is increasingly appreciated that nuclear structure, including the relative 3D distribution of chromosomes (Parada and Misteli, 2002; Bickmore and Chubb, 2003), can influence gene expression by RNA polymerase II, we infer from this work that nuclear structure may also have an important effect on events connected with RNA polymerase I transcription.
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Materials and methods |
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Immunostaining
To prepare the fixation buffer, 2x PHEM buffer (18.14 g Pipes, 6.5 g Hepes, 0.99 g MgSO4, 3.8 g EGTA in 500 ml H2O, pH 7.0) were added to freshly prepared 37% PFA in 1:5 ratio. The fixation buffer was then warmed to 37°C and added directly to the cells slowly after pouring the media out of the dish. The cells were fixed for 10 min before being washed thrice with 1x PBS very gently. The cells were then permeabilized, stained and mounted as previously published (Leung and Lamond, 2002). Primary antibodies used were anti-UBF (1:5; Santa Cruz Biotechnology, Inc.) and anti-RPA20 (1:1; B6-2; Jones et al., 2000).
Immunoprecipitations and in vitro transcription assays
50 µg of HeLaYFP-RPA39 or HeLa nuclear extract were precleared for 30 min with 5 µl of protein GSepharose beads and used in immunoprecipitations with 4 µg of anti-GFP antibodies bound to 7.5 µl of protein GSepharose beads in 0.25 M KCl/TM10 (50 mM TrisHCl, pH 7.9, 12.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM sodium metabisulfite and 1 mM DTT) buffer. The beads were incubated with nuclear extract for 1 h, with shaking, at 4°C. After immunoprecipitation the beads were washed in TM10/0.25 M KCl buffer, then equilibrated in TM10/0.05 M KCl buffer and used in in vitro transcription assays. In vitro transcription reactions were performed as described previously (Miller et al., 2001) at a final salt concentration of 5070 mM KCl. Supercoiled prHu3 plasmid DNA, which contains the human rRNA gene promoter from 515 to +1548, were used as templates in the transcription reaction. The resulting transcripts were analyzed in an S1 nuclease protection assay after annealing the RNA to a 5'-endlabeled oligonucleotide, which was identical to the region between 20 and +40 of the promoter template strand.
BrUTP incorporation
Coverslips seeded with HeLa cells were rinsed with hypotonic KH buffer (30 mM KCl, 10 mM Hepes, pH 7.4) briefly and incubated with 50 µl KH buffer containing 10 mM BrUTP (Sigma-Aldrich) for 5 min in a 5% CO2 incubator at 37°C. The cells on coverslips were "chased" with DME containing 20% FCS and 200 µg/ml (final concentration) G418 for a defined time in the incubator to chase the transcripts before fixation. Before methanol fixation for 20 min at 20°C, the coverslips were rinsed with PBS. The cells were then permeabilized with acetone for 30 s and air-dried for 10 min, followed by rehydration with PBS for 5 min and immunostaining using the anti-BrdU (1:5) antibody.
Mitotic studies of living cells
At each time point a 3D image stack spanning the entire nuclear volume was recorded for each of the three wavelengths. Low light imaging conditions were chosen to ensure that cells were able to progress through the entire mitosis. For these experiments a series of "double stable" cell lines were established that express combinations of two FP-tagged markers (Table SII). The third marker in each experiment was expressed by transient transfection (Table SI). Cells were seeded in a Labtek II chambered coverglasses on the previous night before imaging and an imaging medium containing 20% FCS and 0.5 mg/ml L-ascorbic acid replaced the growth medium for 34 h before imaging. 4D imaging was performed on a customized confocal laser scanning fluorescence microscope (model LSM510; Carl Zeiss MicroImaging, Inc.) kept at 37°C and equipped with a z-scanning stage (HRZ 200) for fast 4D acquisition using a Plan Apochromat 63x DIC oil immersion objective. Triple-color imaging of CFP, YFP and DsRed/HcRed was achieved by alternating the 413 nm Kr, 514 nm Ar, and 543 nm HeNe laser for selective excitation. Bidirectional scanning and detection were performed as published previously (Gerlich et al., 2001).
Quantitation and statistical analysis
For object identification, a reference channel was chosen that represented the structure of interest. For example, FP-H2B and FP-RPA39 are used to define the nuclear volume and reforming NORs, respectively. The images in this channel were processed by an anisotropic diffusion filtering and subsequent thresholding (Gerlich et al., 2001) to obtain a binary mask representing areas of interest. Mean fluorescence intensities were then measured in all channels of the original unfiltered images within these areas. Automated analysis of 4D data was achieved by implementation of a computer macro, which was executed in the image processing toolbox Heurisko 4.05 (Aeon). Importantly, a single threshold was selected for the analysis of entire 4D images. Although the specific threshold chosen by the user affects absolute values, the kinetics of protein concentrations measured during mitotic progression were largely unaffected (Gerlich et al., 2001). The data collected were then normalized and analyzed using GraphPad Prism 4.0 software.
Online supplemental material
Fig. S1 includes (A) representative Western blots showing expression levels of FP-fusion proteins in cell lines used, (B) FP-RPA39 localization at EM level, the localization of (C) pulsed labeled rRNA transcripts and (D) RPA20 in HeLaYFP-RPA39 cells. Distributions of quantitative measurements in Figs. 3, 57 are shown in Fig. S2. The statistical analysis for the differential behavior of daughter cells after mitosis is shown in Fig. S3. Fig. S4 shows that the loss of functionality of the NE precedes its structural disintegration. The plasmid and stable cell lines used in this work are tabulated in Table S1. The localization of RPA39, FIB and B23 are shown in Videos 1 and 2. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200405013/DC1.
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Acknowledgments |
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A.K.L. Leung was supported by a Croucher Foundation Scholarship, an EMBL Advanced Light Microscopy Fellowship and a Company of Biologists Ltd. travelling Fellowship. D. Gerlich is supported by an EMBO long-term fellowship. G. Miller was a Wellcome Trust Prize Student. A.I. Lamond is a Wellcome Trust Principal Research Fellow. J. Zomerdijk is a Wellcome Trust Senior Research Fellow. The Human Frontier Science Program is acknowledged for a research grant entitled "Functional organization of the cell nucleus investigated through proteomics and molecular dynamics".
Submitted: 4 May 2004
Accepted: 23 July 2004
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