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The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei
Correspondence to Jennifer Lippincott-Schwartz: jlippin{at}helix.nih.gov
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Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.
Abbreviations used in this paper: BFA, brefeldin A; FLIP, fluorescence loss in photobleaching; GalT, galactosyltransferase; ROI, region of interest.
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Introduction |
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In most animal cell types, the organization of the ER and Golgi apparatus is intimately tied to the localization of the nucleus and astral microtubules derived from centrosomes. The ER extends off the nuclear envelope along microtubules as a tubule network (Terasaki et al., 1986; Waterman-Storer and Salmon, 1998), whereas the Golgi apparatus localizes adjacently to centrosomes or to ER export sites (Barr and Egerer, 2005). Interestingly, this arrangement of organelles is absent in the preblastoderm Drosophila embryo. There, maternally derived ER and Golgi membranes are localized distinctly at the embryo periphery (Ripoche et al., 1994; Bobinnec et al., 2003), whereas nuclei and centrosomes are found deep within the embryo interior (Freeman et al., 1986; Raff and Glover, 1989). A result of this arrangement is that when nuclei and centrosomes arrive at the embryo periphery, ER and Golgi membranes must somehow become segregated among the thousands of syncytial nuclei.
In this study, we address how the secretory membrane system partitions among nuclei in preparation for cellularization in the Drosophila syncytial blastoderm. Toward this end, we have used GFP-tagged ER, Golgi, and plasma membrane markers in a variety of biophysical-based experiments to examine membrane continuity and organellar dynamics in living Drosophila embryos. We report that distinct nuclear-associated secretory units of ER and Golgi emerge across the embryo in the absence of plasma membrane boundaries during the syncytial blastoderm stage. The secretory units are shown to mediate localized protein delivery to the plasma membrane and to require centrosome-derived astral microtubules for their maintenance. We discuss how this organization helps to equivalently partition ER and Golgi into daughter cells at cellularization, and we propose potential roles for it in the establishment and maintenance of localized gene and protein expression patterns within the early embryo.
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Results |
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To investigate whether microtubules in the cortex of the preblastoderm embryo contributed to the observed organization of the ER, we examined the distribution of tubulin in GFP-tubulin–expressing embryos (see Materials and methods). Pronounced GFP-tubulin fluorescence was seen 
10–20 µm beneath the plasma membrane (Fig. 1 C) and included filamentous, tubulin-rich structures surrounding spherical particles that excluded fluorescence (Fig. 1 D, arrows), presumably representing yolk particles with associated microtubules as reported in other systems (Jaffe and Terasaki, 1994; Mehlmann et al., 1995; Terasaki et al., 1996). These observations led us to conclude that short microtubules were indeed present in the embryo cortex before nuclear migration.
We then examined whether these short microtubules played a role in ER organization in the preblastoderm embryo. Nocodazole (a microtubule-disrupting agent) was microinjected into preblastoderm embryos expressing Lys-GFP-KDEL, and changes in the distribution of Lys-GFP-KDEL were examined. Notably, in the region of the embryo in which nocodazole was injected, both the spherical cluster and tubule network patterns of the ER observed before nocodazole injection (Fig. 1 E, left) disappeared and were replaced by long, parallel strands of ER (Fig. 1 E, right). Injection of DMSO alone did not result in any obvious morphological change of ER clusters (unpublished data). These results indicated that cortical microtubules are necessary for the ER to become organized into spherical clusters and tubule networks during the preblastoderm stage.
ER membranes are interconnected before nuclear migration
We next asked whether ER membranes in the preblastoderm embryo existed as a single, interconnected system. To test this, we performed FRAP experiments in embryos expressing Lys-GFP-KDEL. A small region of ER at the embryo cortex was photobleached using a high intensity laser beam, abolishing GFP fluorescence in this area. Low power imaging was then used to assess fluorescence recovery into the photobleached region. As predicted for a continuous ER system, a rapid and complete recovery of Lys-GFP-KDEL fluorescence into the photobleached area was observed with no gross structural changes in ER membranes (Fig. 2 A).
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ER reorganization after nuclear migration and its dependence on astral microtubules
We next examined whether the morphology and continuity of the ER membrane changes upon nuclear migration to the embryo cortex, which occurs during interphase of nuclear cycle 10 (Foe and Alberts, 1983). In time-lapse sequences, no change in the total level of Lys-GFP-KDEL fluorescence occurred before or immediately after nuclear arrival (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200601156/DC1), strongly suggesting that nuclei do not bring a significant amount of their own ER during this period. Nevertheless, the organization of already existing ER was significantly affected by the migrating nuclei (Fig. 3 A and Video 2). In particular, the spiral clusters of the ER membrane appeared to unwind as nuclei arrived at the cortex. Interestingly, the membrane from these clusters, together with other tubule ER elements, then reassembled around individual nuclei.
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A model for how ER membranes reorganize as nuclei and their associated centrosomes migrate to the embryo cortex based on our results is depicted in Fig. 3 E. In this scheme, the existing tubulin at the cortex becomes incorporated into centrosome-derived astral microtubules as nuclei and their associated centrosomes migrate to the cortex. Growth of the astral microtubules recruits and retains ER membrane to areas specifically surrounding nuclei.
The ER membrane surrounding an individual nuclei in the syncytial blastoderm behaves as a distinct, isolated unit
Time-lapse images of a Lys-GFP-KDEL–expressing embryo during the syncytial blastoderm stage revealed the ER to be tightly organized around individual nuclei during interphase and mitosis (Fig. 4 A and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200601156/DC1). This is in accordance with earlier observations of ER dynamics during the syncytial mitoses (Bobinnec et al., 2003). Upon photobleaching a small area of ER adjacent to one nuclei, rapid recovery into this area occurred as a result of fluorescent molecules outside the bleached box redistributing into the bleached area (Fig. 4, B and C). The fluorescent redistribution appeared to occur primarily from nonbleached ER surrounding the one nuclei and not from ER surrounding other nuclei. This raised the possibility that ER membranes that surround individual nuclei at this developmental stage exist as segregated units that do not exchange components.
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To determine whether this property of ER depended on astral microtubules, we performed a parallel FLIP experiment in Lys-GFP-KDEL–expressing syncytial blastoderm embryos microinjected with nocodazole. Upon repetitive photobleaching of a small region of ER, fluorescence in areas extending 20 µm away from the FLIP ROI center now showed a significant drop in fluorescence (40%; Fig. 4, F and G) compared with the slight drop observed in untreated cells (i.e., 5%; Fig. 4, B and C). Hence, microtubules were necessary for the ER to behave as isolated units surrounding individual nuclei.
It is known that the plasma membrane in the syncytial blastoderm partly invaginates around each nuclei (Karr and Alberts, 1986; Miller et al., 1989). To address whether this could explain the restricted exchange of ER proteins between ER associated with different nuclei, we examined the depth of such plasma membrane invaginations using the plasma membrane marker Spider-GFP, a casein kinase I encoded by the gene gilgamesh that associates with the plasma membrane and secretory vesicles destined for the plasma membrane (see Materials and methods; Babu et al., 2002). In syncytial blastoderm embryos expressing Spider-GFP, plasma membrane invaginations were seen extending down 5 µm from the embryo surface (Fig. 5 A, Spider-GFP). In contrast, ER membranes observed in Lys-GFP-KDEL–expressing embryos extended far deeper (15 µm; Fig. 5 A, Lys-GFP-KDEL). Therefore, if ER compartmentalization was dependent on plasma membrane invaginations, no compartmentalization should exist at depths >5 µm below the embryo surface.
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We also performed FLIP experiments using Lys-GFP-KDEL in preblastoderm embryos whose ER had not yet become compartmentalized. The rate of fluorescence loss from regions lateral to the bleach area was significantly faster than that observed in the syncytial blastoderm embryo (Fig. 5, F and G), with a 60% drop in fluorescence (vs. 10% in the blastoderm) from a region of comparable distance from the bleached box within the same time period. Together, these data suggested that compartmentalization of ER in the embryo (1) only occurs after nuclei have migrated to the cortex, (2) does not rely on plasma membrane invaginations, and (3) requires microtubules.
Organization of the Golgi apparatus in preblastoderm and syncytial blastoderm embryos
Given our observation that the ER reorganizes into compartmentalized, nuclear-associated units in the syncytial blastoderm, we asked whether other organelles that exchange components with the ER also exhibit such compartmentalization. The Golgi apparatus receives all soluble and membrane-bound cargo exported out of the ER and sorts these components either back to the ER or to the plasma membrane or endosomes (Lippincott-Schwartz et al., 2000). To gain insight into Golgi distribution and whether any organizational changes of the Golgi occur in the developing Drosophila embryo, we imaged transgenic embryos expressing galactosyltransferase (GalT)-GFP (GalT-GFP), a resident Golgi enzyme. The same transgenic lines have been used to look at Golgi structure in Drosophila ovaries (Snapp et al., 2004).
Before and after nuclear migration, the Golgi appeared as several thousand punctate structures located at the periphery of the embryo (Fig. 6 A), as previously reported (Ripoche et al., 1994; Stanley et al., 1997). The dispersed Golgi puncta were localized to areas of the embryo enriched in ER and, in time-lapse videos, exhibited a jostling motion throughout successive nuclear cycles (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200601156/DC1). The appearance of these dispersed Golgi puncta resembled that of Golgi puncta found in plants (Nebenfuhr and Staehelin, 2001), sea urchin embryos (Terasaki, 2000), and mammalian cells after treatment with nocodazole (Cole et al., 1996; Storrie et al., 1998). In the case of plant and nocodazole-treated mammalian cells, the Golgi puncta were shown to be localized next to ER exit sites (Cole et al., 1996). An advantage of this type of Golgi distribution is that it allows the Golgi both to receive ER-derived secretory cargo and to recycle proteins back to the ER in the absence of long-range vesicular transport (Cole et al., 1996). Therefore, a similar function might be served by the localization of Golgi puncta near ER in the Drosophila embryo.
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Membrane exchange between the Golgi and ER is compartmentalized to areas surrounding individual nuclei
Previous studies have shown that Golgi enzymes undergo constitutive cycling through the ER (Miles et al., 2001; Ward et al., 2001), a property that allows Golgi structures to continually modify their size and distribution within cells (Altan-Bonnet et al., 2004). Given this characteristic, we investigated whether Golgi protein exchange between the ER and Golgi was restricted to ER and Golgi structures surrounding a particular nucleus in the syncytial blastoderm.
We began by examining the rate of GalT-GFP exchange between Golgi puncta in embryos at the preblastoderm stage, in which the ER exists as a single continuous system and nuclei are deep within the embryo. Repetitive photobleaching of Golgi puncta in a preblastoderm embryo expressing GalT-GFP revealed a 40% drop in fluorescence from all areas in a 30-µm radius of distance from the FLIP ROI center within 3–5 min of photobleaching (Fig. 7 A; red box is the photobleached area, and blue box is the area being monitored; B shows quantification). As an individual Golgi puncta did not move laterally across the embryo for >30 µm (not depicted) and the extent of the lateral loss of Golgi fluorescence was similar to that observed in FLIP experiments with the ER marker (Fig. 2, B and C), the data were consistent with Golgi enzymes cycling through an interconnected ER membrane network.
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To address whether the compartmentalized exchange of GalT-GFP between Golgi puncta extended to deeper areas within the embryo, we repetitively photobleached an area located below several nuclei (Fig. 7 E, red box). Significant fluorescence loss was observed in Golgi puncta directly above the bleached region (Fig. 7 E, green box) but not from Golgi puncta lateral to the bleached region (Fig. 7 E, blue box). Quantification revealed a 60% loss of fluorescence occurring in the region of Golgi puncta above the bleached area compared with a 5–10% drop in the region lateral to the bleached area (Fig. 7 F). Thus, both the ER and Golgi behave as compartmentalized units surrounding individual nuclei in the syncytial blastoderm.
Evidence for the localized delivery of secretory cargo to the plasma membrane from compartmentalized sources
Given the compartmentalized character of the ER and Golgi around individual syncytial nuclei, we wondered whether their secretory products were delivered to restricted areas of the plasma membrane near an individual nuclei. To test this possibility, we investigated whether blocking the secretory pathway in one area of the embryo resulted in the reduced delivery of secretory cargo to the plasma membrane only in that area or in other areas as well. To perturb the secretory pathway in these experiments, we used the drug brefeldin A (BFA), which blocks the transport of secretory cargo from the ER to the Golgi apparatus (Sciaky et al., 1997; Ward et al., 2001). To monitor secretory transport, we observed the invagination of the plasma membrane during cellularization, which is dependent on newly synthesized membrane moving through the secretory pathway (Sisson et al., 2000; Pelissier et al., 2003).
In embryos expressing Spider-GFP to label the plasma membrane, the injection of BFA resulted in a dramatic slowdown in membrane invagination during cellularization (Fig. 8 A). Notably, the slowing occurred only in the area of the embryo where BFA was injected, with sites far from the injection site invaginating their plasma membrane normally (i.e., up to 35 µm as found in wild-type embryos; unpublished data). Although the effect of BFA on plasma membrane invagination at cellularization has already been described (Sisson et al., 2000), the localized effect we observed is new. It suggested that delivery of material to the plasma membrane could not be compensated by surrounding secretory units and implied, therefore, that membrane insertion at the plasma membrane occurred in a localized manner in the embryo.
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Altogether, these findings suggested that the membrane carriers responsible for plasma membrane delivery of secretory products were derived from ER/Golgi membranes directly adjacent to the areas of insertion. Thus, all elements of the secretory pathway (ER, Golgi, and transport carriers) appeared to be compartmentalized around individual nuclei in the embryo cortex.
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Discussion |
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The organization and continuity of ER membranes has been extensively studied during oocyte maturation and early embryogenesis of other organisms, including starfish (Jaffe and Terasaki, 1994; Terasaki et al., 1996), sea urchin (Terasaki, 2000), mouse (Mehlmann et al., 1995), and frog (Terasaki et al., 2001). In these studies, ER membranes labeled by injecting either the ER lipophilic dye DiI or mRNA encoding for an ER lumenal protein marker (ssGFP-KDEL) were shown to exist as an interconnected network of membrane sheets and tubules in the cortex of the fertilized egg, as is the case in the fertilized Drosophila preblastoderm embryo. In addition, prominent ER clusters directly beneath the plasma membrane were described in the cortex of the mouse and frog embryos (Mehlmann et al., 1995; Terasaki et al., 2001). These were postulated to have a role in the generation of transient calcium waves or in the propagation of calcium oscillations. The ER clusters that we observed in the Drosophila preblastoderm embryo may have a similar role, which the recent study on the existence of distinct calcium microdomains during syncytial divisions (Parry et al., 2005) would support.
Because the ER exists as a continuous system in the Drosophila preblastoderm, the embryo must have a mechanism for partitioning this organelle among nuclei so that at cellularization, the newly formed cells have equivalent amounts of ER membrane. Our data suggest that this occurs by a microtubule-driven process that causes the ER to be divided up among the nuclei, resulting in each interphase nucleus becoming surrounded by a single ER membrane system that is separate from adjacent ones. This was demonstrated in FLIP experiments in which resident proteins of the ER and Golgi were seen to be rapidly circulating only within ER and Golgi membrane that associated with a particular nuclei during the four rounds of nuclear division at the periphery. Microtubule depolymerization by the microinjection of nocodazole resulted in the loss of ER and Golgi compartmentalization around a given nucleus, indicating that an intact microtubule network is essential to keep ER and Golgi structures close to individual nuclei in the syncytium. The significance of microtubules in the biogenesis and maintenance of the ER network has been unequivocally demonstrated (Terasaki et al., 1986; Waterman-Storer and Salmon, 1998), so our data reinforce the role of intact microtubules in organizing ER membranes around individual nuclei. Based on these data, we propose a model for the organization and distribution of distinct and separate ER and Golgi membranes with individual nuclei, as illustrated in Fig. 9.
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Another function for the compartmentalization of ER and Golgi in the embryo could be to help establish and maintain localized gene and protein expression patterns (Blankenship and Wieschaus, 2001; Houchmandzadeh et al., 2002). Although zygotic transcription and protein synthesis increases dramatically during cellularization, transcription and protein synthesis has been reported to start as early as nuclear cycles 8–10 (Lamb and Laird, 1976; Edgar and Schubiger, 1986; Pritchard and Schubiger, 1996), resulting in highly localized expression patterns well before cellularization.
One simple and logical way that compartmentalization of the ER may affect gene expression patterning could be by the segregation of maternal mRNAs and proteins. In ascidians, some maternally loaded mRNAs have been shown to associate with the rough cortical ER and relocalize with the ER as it moves (Sardet et al., 2003; Prodon et al., 2005). Likewise, some maternal mRNAs in Drosophila have been shown to be anchored on ER membranes (Herpers and Rabouille, 2004). Indeed, recent work has shown that gurken mRNA anchors to specific transitional ER–Golgi units in the Drosophila oocyte (Herpers and Rabouille, 2004), allowing specific sorting and secretion to take place. If maternally loaded mRNAs anchored on the ER become compartmentalized around individual nuclei upon nuclear migration, as our results would suggest, then these transcripts are likely to be locally expressed. Thus, an already existing polarity in the distribution of maternal material would not only be preserved but would also be further maintained during cortical divisions. To this end, ER and Golgi compartmentalization might provide a mechanism for spatially and temporally restricting maternally derived material during the early stages of Drosophila embryogenesis.
In summary, our data suggest that in the absence of plasma membrane boundaries surrounding nuclei but with the requirement of an intact microtubule network, the embryo is able to differentiate the secretory endomembrane system (i.e., ER and Golgi) into segregated nuclear-associated units. In a volume occupied by thousands of nuclei, this capacity for apportioning ER and Golgi among nuclei is likely to be vital for cellularization and for the establishment and maintenance of localized gene and protein expression patterns. The fact that many other organelles are organized by microtubules (i.e., endosomes, lysosomes, and mitochondria) further suggests that it is possible to have the functional equivalents of cells despite the complete absence of plasma membrane boundaries within a syncytium.
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Materials and methods |
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Other Drosophila stocks
Spider-GFP flies were a gift from A. Debec (Universite Pierre et Marie Curie, Observatoire Oceanologique, Villefranche-sur-mer, France). They were generated using a "protein trap" methodology (Morin et al., 2001) and can be obtained from the European Drosophila Stock Center in Szeged (http://expbio.bio.u-szeged.hu/fly/). GFP-tubulin flies were a gift from A. Spradling (Carnegie Institution, Baltimore, MD).
Imaging of live embryos by laser confocal microscopy
Embryos were collected on apple juice–agar plates, dechorionated in 2% bleach, and placed flat or upright on a Lab-Tech chambered coverglass (Nunc). Chambers were then filled with Drosophila Ringer's solution (Tübingen and Düsseldorf). Confocal microscope images of live embryos were captured on an inverted microscope (510 Meta or ConfoCor-2; Carl Zeiss MicroImaging, Inc.) using the 488-nm line of an Ar laser with a 505–530 emission filter for GFP and a 543-nm HeNe laser line with a 560–615 emission filter for rhodamine. To image yolk autofluorescence, a two-photon laser (Chameleon; Coherent) at 820 nm was used with a 435–485 infrared emission filter. Images were captured with a C-Apochromat 1.2 NA 40x water immersion objective (Carl Zeiss MicroImaging, Inc.). Images were analyzed with Image software (W. Rasband, National Institutes of Health [NIH], Bethesda, MD) and Image Examiner software (Carl Zeiss MicroImaging, Inc.) and prepared by Adobe Photoshop 7.0.
Imaging of live embryos by confocal microscopy after microinjection
Embryos were collected for 30 min, aged on collection plates for 50 min, and dechorionated in 2% bleach. Dechorionated embryos were microinjected as previously described (Smith and DeLotto, 1994). Rhodamine-tubulin was obtained from Cytoskeleton, Inc. BFA (Sigma-Aldrich) was microinjected at 5 mg/ml in DMSO. For nocodazole injections, embryos were dechorionated, microinjected with nocodazole (Sigma-Aldrich) at 10 mg/ml in DMSO, mounted in chambers, and placed on ice for 10 min to depolymerize microtubules. Embryos were then imaged at 25°C.
Photobleaching and analysis
FRAP and FLIP were performed by photobleaching a small ROI and monitoring fluorescence recovery or loss over time as described previously (Lippincott-Schwartz and Patterson, 2003; Snapp et al., 2003). To create the fluorescence recovery or loss curves, the background-corrected fluorescence intensities were transformed into a 0–1 scale and were plotted using Microsoft Excel X.
Online supplemental material
Video 1 is a 3D rendering of ER membranes at the surface of a Lys-GFP-KDEL–expressing embryo. Video 2 shows nuclear migration during nuclear cycle 10 interphase in a Lys-GFP-KDEL–expressing embryo as viewed from the embryo surface. Video 3 shows divisions of ER membranes during cycles 10–13 in a Lys-GFP-KDEL–expressing embryo as viewed from the embryo surface. Video 4 shows the dynamics of Golgi puncta during cycles 11–13 at a cross section of a GalT-GFP–expressing embryo. Video 5 shows the movement of Golgi structures around individual syncytial interphase nuclei in a GalT-GFP–expressing embryo as viewed from the embryo surface. Video 6 shows the same as Video 5 at a cross section of the embryo. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200601156/DC1.
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Acknowledgments |
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This work was supported, in part, by a grant from the NIH Office of the Director Undergraduate Scholarship Program as well as by the Intramural Research Program of the NIH in the National Institute of Child Health and Human Development.
Submitted: 27 January 2006
Accepted: 20 March 2006
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