Correspondence to: Frederick R. Maxfield, Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Ave., Room E-215, New York, NY 10021., frmaxfie{at}mail.med.cornell.edu (E-mail), (212) 746-6405 (phone), (212) 746-8875 (fax)
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Abstract |
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Furin and TGN38 are membrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N., W.G. Mallet, T.T. Soe, T.E. McGraw, and F.R. Maxfield. 1998. J. Cell Biol. 142:923936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independent pathways for endosomal transport of proteins to the TGN from the plasma membrane.
Key Words: endocytosis, endosomes, Golgi complex, protease, transport
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Introduction |
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UPON internalization from the plasma membrane, most solutes, ligands, lipids, and transmembrane proteins enter compartments known as sorting endosomes (-amidating monooxygenase (
Endocytic recycling of membrane proteins, such as transferrin receptors in CHO cells, does not require specific sorting motifs (
We described recently the transport of a chimeric transmembrane protein, Tac-TGN38, to the TGN of CHO-derived TRVb-1 cells via the endocytic recycling pathway (
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Materials and Methods |
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Cells and Constructs
Chimeric Tac-furin protein constructs TTF and TFF in the plasmid pCDM8.1 incorporating the cytomegalovirus promoter were obtained from Michael Marks (University of Pennsylvania, Philadelphia, PA) (
Antibodies and Fluorescent Reagents
Monoclonal antibodies (IgG1) against Tac were purified from ascites fluid prepared from the hybridoma cell line 2A3A1H (ATCC) using protein G affinity chromatography. Antibodies were conjugated to Cy3 (Cy3anti-Tac) (Amersham North America), Alexa 488 (A488anti-Tac) (Molecular Probes), or fluorescein isothiocyanate (FITCanti-Tac) (Molecular Probes) according to the manufacturers' instructions. For some experiments, antibodies were labeled with Na125I as described previously (-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythro-sphingosine] and fixable 70-kD dextrans conjugated to rhodamine were purchased from Molecular Probes. Polyclonal antibodies against the cytoplasmic domain of rat TGN38 were obtained from Keith Stanley (Heart Research Institute, Sydney, Australia) (
Fluorescence Staining Methods
For microscopy, cells were passaged onto poly-D-lysinetreated Number 1 coverslips affixed beneath holes cut into the bottoms of 35-mm Petri dishes. For incubations of live cells with antibodies or ligands, cells were treated as described previously (
Microscopy
Digital epifluorescence microscopy (see Figure 3, Figure 4 D, and 5, E and F) was performed as described previously (
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Kinetic Experiments
To determine the rate of Tac-furin externalization by accumulation of fluorescent antibodies, TRVb-1/Tac-furin cells cultured on coverslips were incubated with Cy3anti-Tac IgG (3 µg/ml) in McCoy's 5A + 0.1% BSA for 5, 10, 15, 20, 30, 40, 50, 60, 75, or 90 min. Unbound antibody was removed by washing, and cells were fixed. Cy3 fluorescence was imaged by epifluorescence microscopy. Fluorescence power per cell was determined as described below. Fluorescence power was relatively uniform among all cells for each time point.
Determination of the externalization rate by accumulation of radiolabeled antibodies, measurement of the level of surface expression of Tac-furin, and determination of the endocytic rate constant of Tac-furin (
To determine the exit rate of antibody-labeled Tac-furin from the cell, TRVb-1/Tac-furin cells were incubated with FITCanti-Tac for 60 min, followed by a 30-min chase in McCoy's/BSA. After this procedure, anti-Tac is mostly detected in the TGN (see Figure 3 D). Cells were further incubated for 5, 10, 15, 20, 30, 40, 50, 60, 75, or 90 min in the presence of antifluorescein antibodies (10 µg/ml) in the chase medium and fixed. We have shown previously that antifluorescein antibodies are not internalized by fluid-phase pinocytosis sufficiently to cause significant intracellular quenching (
Nocodazole and Wortmannin Studies
Experiments examining the effects of nocodazole and wortmannin on protein transport were performed in parallel. For all steps, BSA was omitted from incubation media to prevent adsorption and deactivation of reagents, and all cells received 0.1% (vol/vol) DMSO to control for effects of the solvent. Nocodazole-treated cells were pretreated with nocodazole (33 µM) for 30 min at 4°C, then 30 min at 37°C. To maintain identical conditions of temperature and DMSO exposure, wortmannin-treated cells were incubated for 30 min at 4°C in the presence of 0.1% DMSO, then pretreated with wortmannin (100 nM) for 30 min at 37°C. Untreated cells were incubated for 30 min at 4°C and 30 min at 37°C in the presence of 0.1% DMSO. After pretreatments, cells were incubated with ligands for 15 min followed by a 45-min chase in the continuous presence of nocodazole, wortmannin, or DMSO alone, respectively.
Image Processing and Quantification
Processing of digitized images was performed using the MetaMorph image processing software package (Universal Imaging). All images were corrected for background fluorescence and crossover between channels. To quantify fluorescence power per cell (see Figure 3a and Figure c, and Figure 4 D), the background fluorescence value was subtracted from images, and the remaining fluorescence power in the field was summed and divided by the number of cells in the field; typically, 10 fields of ~20 cells per field were analyzed for each data point in a single experiment. For quantitative microscopic analyses and 125I-antibody experiments, data points were fit using the SigmaPlot software program (SPSS Inc.).
To quantify the colocalization of internalized fluorescent anti-Tac and LDL over a time course, cells were imaged using the MRC600 confocal microscope. Using routines available in the MetaMorph software package, images from the green and red channels were thresholded to detect labeled objects above background fluorescence, then labeled endosomes were selected on the basis of size (between 10 and 50 square pixels). Double-labeled endosomes were identified by performing a logical AND operation with the endosomes detected in the green and red channels. Intensities of singly and doubly labeled endosomes were transferred to Microsoft Excel for statistical analyses.
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Results |
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Internalized Tac-Furin Is Delivered to the TGN
Tac-furin (TTF) was shown by Bonifacino and co-workers to cycle between the plasma membrane and the TGN, maintaining a steady-state enrichment in the TGN (
When TRVb-1/TTF cells were incubated with fluorescently labeled anti-Tac antibodies, the antibodies were internalized and transported over time to the TGN, which was identified using anti-TGN38 antibodies (Figure 2), using antibodies against furin, or using the fluorescent lipid analogue NBD-C6-ceramide (data not shown). Tac-furin was detected in the TGN after ~30 min of internalization (Figure 2E and Figure F), becoming further enriched there after 60 min (Figure 2G and Figure H). These results demonstrate the transport of Tac-furin to the TGN in our system and confirm previous reports in other cell types. Internalized anti-Tac Fab fragments were transported identically to intact IgG (data not shown), demonstrating that our findings are not an artifact of protein aggregation by divalent IgG. The delivery of internalized anti-Tac to the TGN in TRVb-1/TTF cells allowed us to describe the kinetics of Tac-furin trafficking by incubating cells with anti-Tac under various conditions.
Kinetic Parameters of Tac-Furin Transport
To determine the rate of exit of Tac-furin from TRVb-1/TTF cells, the cells were incubated at 37°C with Cy3anti-Tac, and the cells were imaged by epifluorescence microscopy. The accumulation of the Cy3anti-Tac was then quantified in terms of fluorescence power per cell versus incubation time (Figure 3 A, solid line). Under these conditions, the rate of accumulation of anti-Tac is equal to the rate of appearance of unlabeled intracellular Tac-furin at the plasma membrane (
Alternatively, cells in a 24-well plate were incubated at 37°C for various times with 125Ianti-Tac antibodies, and the cell-associated radioactivity was measured for each time point. We found that the cells accumulated anti-Tac with a half-time of ~36 min (k = 0.019 min-1) (Figure 3 A, dashed line). The difference in the exit rate constants obtained from the two different methods is small compared with other parameters of Tac-furin trafficking (see below), and may be due to the differences in the experimental procedures. From the specific activity of the 125Ianti-Tac, the asymptote of the accumulation curve, and the number of cells in each well, we calculate that ~2 x 105 copies of Tac-furin are expressed per cell. Tac-furin and endogenous furin localize predominantly to the TGN in these cells (Figure 1 and data not shown), so it is unlikely that sorting or retention mechanisms are saturated at this expression level.
To determine the steady-state surface expression of Tac-furin, TRVb-1/TTF cells were incubated with 125Ianti-Tac antibody at 0°C to prevent internalization, and the bound counts were compared with the asymptote of the 37°C 125Ianti-Tac accumulation curve. We estimate that 5% of Tac-furin is at the plasma membrane at steady state (data not shown).
We also measured the internalization rate constant of Tac-furin in TRVb-1/TTF cells. As determined from the ratios of internal to surface antibody over a brief time course at 37°C, the protein is internalized with a rate constant of 0.36 min-1, which is consistent with the presence of rapid internalization signals in the furin cytoplasmic domain (Figure 3 B). At steady state, the relative rates of internalization and externalization determine the relative amounts of protein in internal compartments and at the plasma membrane. The ratio of the measured rates of endocytosis (0.36 min-1) and externalization (0.019 min-1) of 125Ianti-Tac is about 19, which agrees well with the estimated internal-to-surface ratio of Tac-furin (also about 19). This indicates that our kinetic data accurately describe the rates of trafficking of Tac-furin.
To demonstrate that antibody labeling did not perturb the kinetics of Tac-furin trafficking, we measured the externalization rate by another method. TRVb-1/TTF cells were incubated for 60 min with FITCanti-Tac, followed by a 30-min chase. At this time, the FITCanti-Tac predominantly labeled the TGN (Figure 3 D). Subsequently, antifluorescein was applied to the medium, and the cells were incubated over a long time course to allow externalization of Tac-furin. Over this time period, the pericentriolar fluorescence signal decreased, indicating that the antibody was externalized from the TGN to the plasma membrane (Figure 3 E). Cells were then imaged by epifluorescence microscopy, and the fluorescein fluorescence power per cell was quantified for each time point (Figure 3 C). The fluorescence power declined in a monoexponential fashion (k = 0.026 min-1), with a half-time of about 26 min indicating the rate of exit of FITCanti-Taclabeled Tac-furin from the cells. This rate is similar to the rate of externalization of unlabeled Tac-furin from cells (Figure 3 A), confirming that antibody labeling has not altered the kinetics of Tac-furin transport. Since the majority of the FITCanti-Tac was externalized from the TGN under this procedure, the measured rate constant mainly reflects the rate of transport of Tac-furin from the TGN to the plasma membrane. The exact pathway and the rate of exit of Tac-furin from the TGN per se are not directly shown by these studies.
The agreement of this rate constant with that for whole-cell antibody accumulation is consistent with the existence of a single major exit route for Tac-furin (see below). Also, the fluorescein quenching procedure measures only the kinetics of externalization of the cycling Tac-furin pool. Our results suggest either that the cycling and biosynthetic pools are externalized at the same rate or that the contribution of the biosynthetic pool to these kinetics is small. Finally, the transport of FITCanti-Tac back to the plasma membrane demonstrates that the antibody remains associated with Tac-furin throughout its trafficking itinerary. Otherwise, dissociated FITCanti-Tac would accumulate in lysosomes, which was not observed. The low level of residual fluorescence power observed in these experiments after a prolonged chase is probably due to autofluorescence and the incomplete quenching of fluorescein by antifluorescein antibodies, as little TGN or endosomal anti-Tac staining could be detected at the longest time points. The rapid internalization of Tac-furin at the plasma membrane and the relatively slow movement from the TGN to the plasma membrane account for the steady-state localization of the chimera.
Tac-Furin Is Delivered to the TGN via Late Endosomes
We next determined the route by which Tac-furin is transported to the TGN. To evaluate if Tac-furin, like Tac-TGN38, transits through the endocytic recycling pathway, TRVb-1/TTF cells were incubated for 5 min with fluorescently-labeled anti-Tac and transferrin, then fixed immediately or chased in the continuous presence of transferrin to label the recycling pathway (Figure 4, AC). As reported previously (
We also found that endocytosed Tac-furin does not recycle rapidly to the plasma membrane. TRVb-1/TTF cells were incubated briefly with anti-Tac conjugated to fluorescein (FITCanti-Tac), then chased over a short time course in the absence or presence of antifluorescein antibodies in the medium. Cells were imaged by epifluorescence microscopy, and the fluorescence power per cell was determined for each chase time point (Figure 4 D). If Tac-furin is rapidly recycled, then the internalized FITCanti-Tac should reappear at the plasma membrane, where its fluorescence would be quenched by the antifluorescein antibodies. Instead, we observed no loss of cell-associated fluorescence over the time of the chase. This finding again contrasts with the rapid recycling of the bulk of internalized Tac-TGN38, which was demonstrated using the same approach (
The most common fate of an endocytosed protein that is not recycled is accumulation in endosomes that over time have matured and are segregated from the recycling pathway. This class of endosomes is defined as late endosomes (
The absence of Tac-furin from CI-MPRenriched endosomes may be due to transport into a novel pathway distinct from the classical degradative pathway, or may be explained by the exit of Tac-furin from endosomes before they have become significantly enriched in CI-MPR. The existence of multiple classes of matured endosomes with distinct properties has been reported in other systems (
To confirm that endocytosed Tac-furin enters late endosomes, TRVb-1/TTF cells were pulsed for 5 min with fluorescently labeled anti-Tac and LDL (Figure 6). After a 5-min chase, the two probes were detected in peripheral spots, colocalizing significantly although incompletely (Figure 6 A). The frequency of double-labeled endosomes remained high through about 1520 min of chase (Figure 6 B, yellow spots). At these chase times, the majority of LDL is in late endosomes (
The extent of colocalization of anti-Tac and LDL was quantified by two different computational methods. Endosomes labeled with LDL were selected, and the proportion that was also labeled with anti-Tac was quantified versus chase time. Also, the ratio of anti-Tac to LDL fluorescence was measured for each double-labeled endosome, and the distribution of these ratios was determined as a function of chase time. These analyses were performed on images similar to those in Figure 6, using two independent data sets. We found that the proportion of LDL-labeled endosomes containing anti-Tac declined sharply between 20 and 40 min of chase, coincident with the appearance of anti-Tac in the TGN (Figure 7 A). In contrast, the population of endosomes labeled with both probes exhibited no change in fluorescence power ratios during this same interval (Figure 7 B), although the size of that population decreased over time. The abrupt loss of double-labeled endosomes and the relative invariance of fluorescence power ratios suggest that anti-Tac may be sorted away from LDL by a highly concerted process, rather than by a more gradual or iterative mechanism; in this way, a double-labeled endosome would suddenly become singly labeled. We failed to detect an accumulation of endosomes labeled with anti-Tac and not with LDL, so it seems plausible that Tac-furin follows a relatively direct route from endosomes to the TGN. This event apparently precedes or coincides with the delivery of CI-MPR to late endosomes from the TGN, such that the two molecules mostly do not overlap.
The Transmembrane Domain of Furin Does Not Encode Essential Endosomal Sorting Information
In addition to cytoplasmic domain sorting signals, the transmembrane domains of some membrane proteins have been shown to perform a sorting function. In particular, the transmembrane domain of TGN38 has been shown to play a role in the localization of that protein to the TGN (
Transport Pathways of Tac-Furin and Tac-TGN38 to the TGN Are Differentially Sensitive to Nocodazole and Wortmannin
The transport of Tac-furin to the TGN via late endosomes is distinct from the pathway that is followed by Tac-TGN38. The point at which the trafficking of the two proteins diverges is the sorting endosome: Tac-furin is retained as the sorting endosome matures into a late endosome, whereas Tac-TGN38 is transported to the ERC. To show directly the divergent routes taken by these two chimeras, we performed a series of pulse-chase experiments under conditions that are known to selectively alter different properties of the endosomal system.
First, we took advantage of the effects of the microtubule-disrupting compound, nocodazole. Nocodazole has been shown to inhibit endosome maturation (
In TRVb-1/TTF cells treated with DMSO alone, internalized antibody colocalized well with NBD-C6-ceramide (Figure 8 A) and had very little overlap with LDL, which is expected to label mainly late endosomes after this time course (Figure 8 B). However, nocodazole treatment resulted in a substantial redistribution of anti-Tac into LDL-containing structures, and overlap with NBD-C6-ceramide was greatly diminished (Figure 8C and Figure D). Note the dispersion of the NBD-C6-ceramide staining, which is a consequence of the fragmentation of the TGN caused by microtubule disassembly (
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A second reagent having relevant effects on vesicular transport is the phosphatidylinositol 3-OH kinase (PI3 kinase) inhibitor wortmannin. Wortmannin exerts numerous effects on endosomal trafficking, including the inhibition of early endosome fusion (
After 45-min chase, anti-Tac mostly labeled the TGN in mock-treated cells, as observed previously (data not shown). The trafficking of Tac-furin was severely inhibited by wortmannin treatment. Under these conditions, internalized Tac-furin was delivered to the TGN only very inefficiently, instead remaining colocalized with LDL (Figure 9, AD). Note the nebulous appearance of the anti-Tac and LDL labeling in Figure 9A, Figure C, and Figure D; this pattern is presumably due to the effects of wortmannin on the morphology of endosomes. The appearance of Tac-TGN38 in the TGN also was reduced upon treatment with the drug, presumably due to slower transit of the chimera through the endocytic recycling pathway. However, significant colocalization of Cy3anti-Tac and NBD-C6-ceramide was apparent at the end of the 45-min chase (Figure 9E and Figure F), increasing with a more prolonged chase (data not shown). Before delivery to the TGN, Tac-TGN38 remained colocalized with transferrin, and at no point was overlap with internalized LDL observed (data not shown). Given the reported activities of wortmannin and our own observations, the most straightforward conclusion is that Tac-furin trafficking is blocked from late endosomes to the TGN. This may represent a direct dependence of this transport event on PI3-kinase activity, or may be an indirect consequence of inhibiting the delivery of transport factors to late endosomes. Irrespective of the precise point of action of wortmannin, the absence of accumulation of Tac-TGN38 in LDL-containing endosomes under these conditions underscores the different pathways that these chimeras take in the endosomal system.
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Discussion |
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Over the past several years, numerous reports have described the transport of certain membrane-associated proteins between the endosomal system and the secretory pathway, specifically the TGN. However, the exact route by which proteins are delivered to the TGN usually has not been specified. This is significant since the transport pathway determines where protein sorting must take place and also may suggest the mechanism of sorting. We demonstrated recently the postendocytic transport of a chimeric transmembrane protein, Tac-TGN38, to the TGN via the endocytic recycling pathway (
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The steady-state localization of a protein is determined by the slowest step in its trafficking. Irrespective of the transport itineraries and sorting mechanisms involved, both Tac-furin and Tac-TGN38 are localized to the TGN predominantly through their relatively slow rates of exit from that compartment. This is likely reflected in the rates of transport of the proteins from internal sites to the plasma membrane, which are slower than all other kinetic steps measured for each chimera. In the case of Tac-furin, the kinetics of exit from the cell appears to be independent of the transmembrane domain. However, this may not be the case for Tac-TGN38 (
The two distinct endosomal pathways, which diverge at the level of the sorting endosome, indicate at least two sorting steps that are involved in transport to the TGN. Tac-TGN38 traverses the recycling pathway that is followed by the bulk of internalized membrane proteins in CHO cells in the absence of specific targeting. From some point along this pathway, Tac-TGN38 is then diverted for delivery to the TGN. This process must require a specific property of Tac-TGN38, such as an amino acid motif or selective partitioning into membrane domains due to the protein's physical characteristics. The cytoplasmic domain sequence, SDYQRL, appears to fulfill at least part of this sorting function (
The itinerary followed by Tac-furin is now more completely described. This protein evades the endocytic recycling pathway, neither appearing in the ERC nor rapidly returning to the plasma membrane. Rather, Tac-furin transits from sorting endosomes to late endosomes, which must require high-fidelity, active sorting of Tac-furin at the sorting endosome. From late endosomes, Tac-furin is transported to the TGN, apparently before the accumulation of CI-MPR in late endosomes. This late endosome to TGN step may also require active sorting, or this may be the pathway taken by most membrane proteins that have accessed late endosomes, in the absence of a positive sorting signal. There is evidence that internalized membrane proteins such as the transferrin and LDL receptors may be transported from endosomes to the TGN at a low constitutive rate (
The different endosomal pathways followed by Tac-furin and Tac-TGN38 must require different sorting mechanisms, as supported by studies using nocodazole and wortmannin. The inhibition of Tac-furin transport by nocodazole is readily explained, since the chimera is delivered via late endosomes, and entry of endocytosed proteins into late endosomes is blocked by nocodazole. The absence of an effect on Tac-TGN38 transport is consistent with the properties of the endocytic recycling pathway, although it would not necessarily be predicted that a route linking the recycling pathway and the TGN would also be independent of microtubules. Our data also suggest that transport from late endosomes to the TGN depends on PI3 kinase activity since wortmannin apparently causes internalized Tac-furin to accumulate in late endosomes rather than in the TGN. This may reflect a general requirement for PI3 kinase in late endosome to TGN trafficking, or alternatively a specific role of PI3 kinase in the pathway that transports furin. Analyses of the effects of wortmannin treatment on endosome-to-TGN trafficking in other systems have yielded conflicting results (
An extensive series of studies by Thomas and co-workers has revealed possible roles for a number of proteins, including phosphofurin acidic cluster sorting protein 1 (PACS-1) (
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Acknowledgements |
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The authors thank Juan Bonifacino and Michael Marks for the Tac chimera plasmids, and Keith Stanley, George Banting, Yukio Ikehara, and Peter Lobel for antibodies used in these studies. We thank members of the Maxfield lab and Timothy McGraw for advice and suggestions, and members of the McGraw lab for extensive technical assistance. We are grateful to Anne Müsch, James Arden, and Sharron Lin (Weill Medical College of Cornell University, New York, NY) for critical reading of the manuscript. We also thank Dr. Xinwa Chang (Department of Medicine, Weill Medical College) for assistance with statistical analyses.
This work was supported by National Institutes of Health grant DK27083. W.G. Mallet was supported by a postdoctoral fellowship from the Pharmaceutical Research and Manufacturers of America Foundation.
Submitted: March 29, 1999; Revised: May 28, 1999; Accepted: June 18, 1999.
1.used in this paper: CI-MPR, receptor for insulin-like growth factor II and mannose 6-phosphate containing ligands; ERC, endocytic recycling compartment; LDL, low density lipoprotein; PACS-1, phosphofurin acidic cluster sorting protein 1; PI3 kinase, phosphatidylinositol 3-OH kinase
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References |
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