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Vol. 19, Issue 8, 3544-3553, August 2008
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*Department of Biology, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan; Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe 651-2492, Japan; and Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan
Submitted April 23, 2008;
Revised May 29, 2008;
Accepted June 4, 2008
Monitoring Editor: Fred Chang
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Yamamoto et al., 1997). Sporulation consists of two overlapping processes, meiosis and spore morphogenesis. The most important event of spore morphogenesis is the assembly of double-layered intracellular membranes, termed forespore membranes (FSMs), which develop into the spore plasma membrane (Yoo et al., 1973). During meiosis II, FSMs are assembled by the fusion of membrane vesicles. At this stage, the spindle pole body (SPB), which plays a crucial role in spindle microtubule formation, undergoes a morphological transformation into a multilayered structure as revealed by electron microscopy (Hirata and Tanaka, 1982; Tanaka and Hirata, 1982). As analyzed by fluorescence microscopy using an anti-Sad1 (an SPB component) antibody, compact dots of SPB are transformed into crescent shapes coincident with the initiation of FSM formation (Hagan and Yanagida, 1995; Ikemoto et al., 2000). These morphological alterations of the SPB are called "SPB modification" (Nakase et al., 2008). After SPB modification, membrane vesicles are then recruited to the vicinity of the modified SPBs, and subsequently fuse there to generate forespore membranes (Hirata and Tanaka, 1982; Tanaka and Hirata, 1982). As the nucleus divides in meiosis II, the FSM expands, and eventually it encapsulates a haploid nucleus generated by two rounds of meiosis, producing the membrane-bounded precursor of the spore, the prespore. In the space between the inner and outer membranes of the FSM, spore wall materials are deposited to form layers of spore walls (Yoo et al., 1973; Hirata and Tanaka, 1982). Finally, the inner layer of the FSM becomes the spore plasma membrane, whereas the outer layer of the membrane autolyzes. Mature spores are then liberated from an ascus upon autolysis of the ascus walls (Tanaka and Hirata, 1982). The basic process of sporulation in the budding yeast Saccharomyces cerevisiae (Byers, 1981; Kupiec et al., 1997; Neiman, 2005), is very similar to that of S. pombe as described above.
To address the molecular mechanism of sporulation, we have identified and analyzed a number of sporulation-specific genes (Bresch et al., 1968; Ikemoto et al., 2000; Asakawa et al., 2001; Nakamura et al., 2001, 2002, 2005; Nakase et al., 2001, 2004, 2008; Nakamura-Kubo et al., 2003; Yoshida et al., 2005; Ye et al., 2007). Spo2, Spo13, and Spo15 proteins localize to the SPB. The morphological transformation of the SPB is not detected in either spo2, spo13, or spo15 mutants, suggesting that these proteins are involved in the initiation of FSM formation (Ikemoto et al., 2000; Nakase et al., 2008). spo3+ is a sporulation-specific gene encoding an FSM-integrated transmembrane protein. Anucleated spores are often observed in the spo3 mutant, because FSM formation is impaired (Hirata and Shimoda, 1992; Nakamura et al., 2001). In addition to these genes, the general secretion apparatus is also required for vesicle transport during sporulation. The S. pombe Sec12 homologue, Spo14/Stl1, is necessary for proper construction of the FSM (d'Enfert et al., 1992; Nakamura-Kubo et al., 2003). Sec12 is responsible for vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus in S. cerevisiae (Nakano et al., 1988). Spo20 is structurally and functionally related to the major S. cerevisiae phosphatidylinositol/phosphatidylcholine-transfer protein Sec14, which is required for vesicle formation from the Golgi apparatus (Bankaitis et al., 1990; Nakase et al., 2001). Spo20 regulates formation of the FSM, in addition to its known roles in post-Golgi vesicle trafficking (Nakase et al., 2001). Additional sec genes are also involved in FSM formation (Nakamura et al., 2001, 2005).
The spatiotemporal coordination of meiotic nuclear divisions and FSM formation is essential for accurate distribution of the genome into four haploid spores. Cytological observations by electron and fluorescence microscopy described above have been obtained with fixed specimens. To gain a better understanding of sporulation in S. pombe, appreciation for the dynamic aspect of FSM formation is essential. Here, we made time-lapse observations of living cells, which provides information and insights that cannot be obtained from fixed specimens. We report the behavior of FSMs in individual living cells of wild-type and sporulation-deficient mutant strains. Based on these observations, we propose a model for FSM assembly in S. pombe.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Gutz et al., 1974; Moreno et al., 1990). S. pombe cells were grown at 30°C and sporulated at 28°C.
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), yielding pREP1(CFP). pREP1(CFP-atb2) was constructed as follows. Two oligonucleotides were used to amplify the atb2+ gene by PCR using 5'-CCCGTCGAC(SalI)AATGAGAGAGATCATTTCCAT-3' and 5'-CCCGAGCTC(SacI)GGAAGAGAAATTAGTTTCAAA-3' as primers. The PCR product was digested with SalI and SacI, and then they were ligated into the corresponding site of pREP1 (Maundrell, 1993), yielding pREP1(CFP-atb2).
Live Analysis
Time-lapse microscopy was performed in growth chambers kept at 28°C with inverted fluorescence microscopes (IX-71; Olympus, Tokyo, Japan) equipped with a 60x/1.25 numerical aperture (NA- or 100x/1.35 NA oil immersion objective (Plan-Apo; Olympus), a cooled charge-coupled device (CCD) camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu, Japan), and a filter wheel (Mac5000; Ludl Electronic Products, Hawthorne, NY) populated with Chroma filters (Chroma Technology, Rockingham, VT). The CCD camera, a filter wheel, and image acquisition were controlled by AQUACOSMOS software (Hamamatsu Photonics). Digital images were processed with Adobe Photoshop version 7.0 (Adobe Systems, San Jose, CA).
To observe green fluorescent protein (GFP)-tagged Psy1 and chromatin, living cells scraped from an agar plate were suspended in 1 µg/ml Hoechst 33342, a DNA-specific fluorescence dye, and incubated for 5 min. Cells were collected by centrifugation, suspended in SSL-N medium, and placed onto a thin film of 2% agarose gel. The film was sandwiched between a pair of coverslips and placed on the stage. Live analysis was performed using a Chroma 86013 filter set (Chroma Technology). To observe CFP-Atb2 and YFP-Psy1 simultaneously, samples were prepared as described above without staining by Hoechst 33342. In this case, a Chroma 86006 filter set (Chroma Technology) was used. Both GFP- and YFP-tagged Psy1 are functional, because these fusion genes complemented the lethality of psy1
(Nakamura et al., 2001; data not shown).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our observations suggest that FSM formation is highly coordinated with meiosis II. To examine spatiotemporal relationships between FSM formation and meiosis II, we observed both FSMs and meiosis II in more detail. For this purpose, we tagged the psy1+ gene with the YFP gene and the atb2+ gene, encoding an 
-tubulin, with the CFP gene and performed two-color imaging. A previous report revealed that, in meiotic cells expressing low levels of GFP-Atb2, meiosis proceeded normally and viable spores were formed (Ding et al., 1998). We confirmed that CFP-Atb2 also behaved like GFP-Atb2 in meiosis (data not shown). The normal spindle dynamics in mitosis consists of three distinct periods: phase I, phase II, and phase III (Nabeshima et al., 1998). In phase I, the spindle elongates to an average length of 2.5 µm. Phase II is characterized by a period of constant spindle length. Phase III is the period at which the spindle elongates rapidly to an average length of 10 µm. Phase I corresponds to a prophase-like stage. Most of phase II is prometaphase and metaphase, whereas sister chromatid separation (anaphase A) occurs at the end of this phase. Phase III begins immediately after or simultaneously with the onset of anaphase B. These three phases have a strong resemblance to the principal events occurring in mitosis in higher eukaryotes (Nabeshima et al., 1998). Essentially the same phases are observed in meiosis (Yamamoto and Hiraoka, unpublished data). Therefore, simultaneous observation of YFP-Psy1 and CFP-Atb2 allows examination of the temporal relationship between FSM formation and meiosis II. The contour length of FSM was measured to monitor its expansion (Figure 1B). As shown in Figure 1A and in Supplemental Movie 2, shortly after meiosis II started (when spindle microtubules were formed), the YFP-Psy1 signal showed up as four dots at both ends of the meiotic spindles (6 min), confirming that FSM formation initiates at SPB. Both electron microscopy and fluorescence microscopy by using fixed specimens reveal that FSM formation initiates during meiosis II. However, the stage of meiosis II at which the assembly of FSM initiates remains unclear. We next examined the appearance of YFP-Psy1 dots in greater detail. Because the FSM is initially assembled on the SPB by the fusion of membrane vesicles, we defined the initiation of FSM formation as the appearance of GFP- or YFP-Psy1 dots at the end of the spindle microtubules. As shown in Figure 2, soon after the spindle microtubules seemed during meiosis II, two dots of YFP-Psy1 were observed at both ends of each spindle microtubule. Table 2 shows the timing of appearance of the YFP-Psy1 dot at the ends of the spindle microtubules. The duration from the onset of meiosis II (appearance of spindle microtubules) to the appearance of YFP-Psy1 dots at the ends of the microtubule spindles (Table 2, dot) was apparently shorter than phase I, indicating that FSM formation initiates during phase I. Because phase I corresponds to prophase II, FSM formation initiates at prophase II. The YFP-Psy1 signal then expanded at a constant rate. After meiosis II ended (the spindle microtubules collapsed at 34 min), the FSM continued to expand for 
20 min until closing of the FSM to form a prespore (Figure 1A). Finally, the sacs became spherical. The duration from closure of FSM to becoming spherical was 18 min (Table 2, to sphere).
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) and time-lapse observation of living cells (Figures 1 and 2) suggest that the four FSMs expand synchronously. To confirm this, the contour length of each of the FSMs was measured. As shown in Figure 3(wild type), four FSMs expanded in a synchronous manner.
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). However, we have no direct evidence that Spo15 is involved in the initiation of FSM formation. Therefore, we examined the behavior of FSM by time-lapse observation in spo15 mutant cells. Strain, TN242, which expresses GFP-Psy1, was cultured in sporulation medium, stained with Hoechst 33342, and then GFP-Psy1 was traced by time-lapse fluorescence microscopy. Like wild-type cells, GFP-Psy1 was dispersed in the cytoplasm of the spo15 zygote before meiosis II (Supplemental Figure 2, 0–4 min). However, no FSM-like structure was observed when meiosis II progressed (20–44 min; Supplemental Movie 3). Next, we observed the behavior of FSMs and microtubules simultaneously in the spo15 null mutant. In wild-type cells, YFP-Psy1 dots showed up at the ends of the spindle microtubules during prophase II (Figure 2). When meiosis II initiated, YFP-Psy1 dots were not detected at the ends of spindle microtubules. The FSM-like structures were not formed even after meiosis II ended (Figure 4). Given its localization to SPB, these results demonstrate that Spo15 plays an essential role in initiation of FSM formation.
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). Electron microscopy revealed that membrane vesicles accumulated in the cytoplasm of immature spo3 asci, suggesting that Spo3 is essential for the assembly of the FSM. In most wild-type cells, the FSM encapsulates each haploid nucleus. About 70% of the spo3 zygotes form four aggregates of GFP-Psy1 near nuclei (class I). These aberrant structures may represent remnants of collapsed membranes. The rest (30%) of the spo3 zygotes contains four, albeit remarkably small, nucleated prespores (class II) (Nakamura et al., 2001). These phenotypes have also been observed in electron microscopic studies showing aberrant prespores called spore-like bodies in spo3 mutants (Hirata and Shimoda, 1992). To clarify these defects in FSM formation in more detail, we made time-lapse observations of FSM formation in spo3 mutant cells. Figure 5, A and B, shows an example of GFP-Psy1 behavior in class I and class II phenotypes, respectively. In class I mutant cells (Figure 5A and Supplemental Movie 4), the FSMs seemed to initiate and to expand normally at an early stage of meiosis II (–18 min). Interestingly, at late meiosis II, expansion of the FSMs was severely impaired (22–32 min). After the meiosis II completed, the FSMs closed without engulfing the nuclei. Finally, four small sacs formed near the nuclei (34–50 min). In class II phenotype cells, the FSMs behaved essentially as the class I phenotype cells. However, despite the apparent defect in FSM expansion, the FSM somehow engulfed the nucleus, resulting in formation of prespores that were remarkably small (Figure 5B and Supplemental Movie 5). These data suggest that the defect in FSM formation between class I and class II cells is identical, but that the difference in phenotype is due to the degree of FSM expansion; the defect in class I being more severe than in class II.
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cells first occurred at the both sides of the spindle microtubule at phase I (Figure 6A, Table 2, and Supplemental Movie 6). The FSMs expanded at a rate similar to that observed in wild-type cells until phase II ended (Figure 6B, Table 3, and Supplemental Movie 6). Interestingly, the expansion of the FSMs was severely inhibited at about the time of onset of phase III (Table 3). After completion of meiosis II, the FSMs closed. These results indicate that FSM expansion during late and postmeiosis II is impaired in the spo3 mutant. The duration of phase I, II, and III in spo3 cells was similar to that in wild-type cells (Table 2), supporting our previous observation that meiosis II proceeds normally in spo3 cells (Nakamura et al., 2001).
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). Therefore, we examined FSM formation in the spo14-B221 mutant. As shown in Supplemental Figure 3 and Supplemental Movie 7, GFP-Psy1 dots occurred at opposite sides of nuclei as in wild-type cells (0–4 min), but expansion occurred to be inhibited at early meiosis (0–18 min). The GFP-Psy1 signal in spo14 mutant cells had a smaller curvature than in wild-type and spo3 mutant cells. Anaphase II progressed without further expansion of FSMs (20–24 min), resulting in formation of four tiny aggregates adjacent to the nuclei (44–54 min). To examine FSM formation in a spo14 mutant in more detail, the FSM and microtubules were visualized by YFP-Psy1 and CFP-Atb2, respectively. FSM formation initiated at phase I as in wild-type cells (Table 2, 2–4 min), but the expansion of the FSMs was severely impaired during phase II (8–16 min). Indeed, the length of the FSMs at the end of phase I in spo14 mutant cells was not different from in wild-type cells (Table 3). At late and postmeiosis, the FSMs expanded very slowly (Figure 7, A and B, Supplemental Movie 8, and Table 3). This is in marked contrast to the spo3 mutant, where FSM formation seems to proceed normally until phase III. These data suggest that FSM formation is impaired from initiation to closure in spo14 mutants.
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). However, our live observations in the present study show that the form of the FSMs in the class I phenotype seems to be similar to that observed during meiosis (Supplemental Figure 3, 28–42 min). Eventually, the FSMs closed to form small aggregates, corresponding to the class II phenotype. Thus, we conclude that the FSM-like structure in the class I phenotype is not a terminal phenotype, but rather an intermediate form of the FSM. Thus, the class II phenotype is the terminal phenotype uniformly observed in spo14 mutants. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initiation of FSM Formation
Because FSM formation takes place coincident with meiosis II, these two events must be highly coordinated. We observed FSM and microtubules simultaneously and analyzed the spatiotemporal relationship between FSM formation and meiosis II. The precise stage at which FSM formation begins was not determined, although previous studies have shown that initiation of FSM formation occurs during meiosis II. Our time-lapse analysis revealed a pair of YFP-Psy1 dots sandwiching a nucleus, which corresponds to the initiation of FSM formation, at the SPBs seemed during phase I of meiosis II. Given that phase I corresponds to prophase, these data indicate that FSM formation begins at prophase II.
Expansion of the FSM
The FSM expands continuously during meiosis. The FSM continued to expand even after meiosis II ended. In spo3 cells, the FSM expanded normally until the onset of anaphase IIB, after which the expansion is severely impaired (Figure 6 and Table 3). In contrast, a spo14 mutation, which causes a defect in ER-to-Golgi membrane vesicle transport, compromised FSM formation throughout meiosis II. From these results, we suggest that FSM expansion is composed of at least two steps: "early meiotic FSM expansion" and "late and postmeiotic FSM expansion." Early meiotic FSM expansion takes place coincident with the initiation of meiosis II. In this step, the FSM expands by fusion with vesicles that are likely derived from the ER through the Golgi. In contrast, late and postmeiotic FSM expansion requires Spo3 function in addition to the general secretory machinery. The form of the FSM at anaphase II may be considerably different in spo3 and spo14 mutants. The FSM seems to be more rigid in the spo14 mutant than in wild type or in the spo3 mutant, possibly due to differences in composition.
The coordination of cell cycle events during cell proliferation is attained in part through surveillance systems called "checkpoint controls." When there are defects in critical cell cycle events, checkpoint mechanisms arrest or delay progression of the cell cycle to prevent aneuploidy and cell lethality (Hartwell and Weinert, 1989). In addition, S. pombe has a cytokinesis or contractile ring checkpoint, which monitors formation and integrity of the medial actomyosin ring, and thereby delays subsequent nuclear division (Le Goff et al., 1999; Liu et al., 2000). We examined whether progression of meiosis II is delayed when FSM formation is inhibited. Inhibition of FSM formation by either spo3, spo14, or spo15 mutations was found not to affect the progression of meiosis II (Table 2). Conversely, some mutants, such as rec mutants, which undergo abnormal meiotic nuclear divisions are able to form spores, though they are apparently misshapen (Ponticelli and Smith, 1989). Thus, unlike mitosis, no checkpoint-like control coordinating meiosis and FSM formation seems to exist.
Closure of the FSM
Live analysis also showed that FSM closure takes place 20 min after meiosis II ends. In spo3 and spo14 mutants, FSM closure was also observed, suggesting that the degree of FSM expansion does not directly control its closure. By electron microscopy, the leading edge of FSM is observed as an electron dense structure, which is apparently distinct from the FSM (Hirata, unpublished data). Several proteins have been identified as leading edge components both in S. cerevisiae and S. pombe (Knop and Strasser, 2000; Moreno-Borchart et al., 2001; Nickas and Neiman, 2002; Okuzaki et al., 2003; Itadani et al., 2006). In S. pombe, Meu14 is one of the leading edge components. Meu14 possesses a coiled-coil domain and localizes to the leading edge of the FSM during meiosis II. A meu14 mutant consequently produces abnormal spores at a high frequency, suggesting that Meu14 is responsible for spore morphogenesis, especially determination of FSM closure. The ring structure of Meu14 forms in both spo3 or spo15 cells, supporting the notion that closure of the FSM occurs normally in these mutants (Okuzaki et al., 2003). Therefore, leading edge proteins such as Meu14, but not proteins related to FSM expansion (e.g., Spo3 and Spo14) might control FSM closure.
The expansion and closure of four FSMs in a zygote was found to occur in a synchronous manner. The mechanism responsible for this synchrony is not clear. Our live analysis revealed that the four FSMs expanded synchronously in both spo3 and spo14 mutants (Figure 3). Moreover, our preliminary data indicate that uncontrolled FSM expansion and closure occurred in the meu14 mutant (Ito, Shimoda, and Nakamura, unpublished data). The leading edge of the FSM may control the synchrony of FSM expansion as well as closure itself.
Spore Morphogenesis
Live analysis also revealed that the prespore became spherical in shape after FSM closure. A previous study by electron microscopy showed that spore wall materials are deposited between the inner and outer membranes of the prespore, after which the spore wall was formed (Yoo et al., 1973). Prespores might become spherical concomitantly with formation of the spore wall. Alternatively, at this stage, actin patches are dispersed at the periphery of the prespores (Petersen et al., 1998; Itadani et al., 2006; Ohtaka et al., 2007). Actin relocalization may be required for spherical shaping of spores. Simultaneous observation of actin and FSM will allow us to address this question.
Possible Model for the FSM Formation
Based on the present work and on previous electron and fluorescence microscopic observations using fixed specimens, we propose a new model for the morphogenesis of FSM relative to meiosis (Figure 8). 1) Initiation of FSM formation. During prophase I, outer plaques form on the cytoplasmic side of the SPB, where membrane vesicles accumulate and begin to fuse with one another. Although the molecular mechanism that triggers FSM formation is still unknown, Spo15 is involved in this step. 2) Early meiotic FSM expansion. Membrane vesicles derived from the ER through the Golgi apparatus fuse to the FSM. The general secretory machinery including Spo14, Psy1, and Sec9, a synaptosomal-associated protein of 25 kDa orthologue, are responsible for the membrane fusion. 3) Late and postmeiotic FSM expansion. At this stage, Spo3 is responsible for membrane fusion. 4) FSM closure. About 20 min after completion of meiosis II, the FSM closes. Leading edge components such as Meu14 are involved in this process. Based on the observation that the FSM can close in FSM formation-defective mutants (e.g., spo3 and spo14), FSM closure can occur independently of expansion. 5) Spherical shaping of prespores. After FSM closure, the resulting prespores becomes spherical as spore wall material accumulates in the space between the inner and outer membranes of the prespores leading to spore wall formation.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Radiation Biology Center, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. 
Address correspondence to: Taro Nakamura (taronaka{at}sci.osaka-cu.ac.jp)
Abbreviations used: CFP, cyan fluorescent protein; ER, endoplasmic reticulum; FSM, forespore membrane; GFP, green fluorescent protein; SPB, spindle pole body; YFP, yellow fluorescent protein.
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