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Vol. 19, Issue 9, 3652-3666, September 2008
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*Institut Curie, Centre de Recherche, Centre National de la Recherche Scientifique Unité Mixte de Recherche 144, @Centre National de la Recherche Scientifique Unité Mixte de Recherche 218, 75248 Paris Cedex 05, France; Centre National de la Recherche Scientifique, Centre de Génétique Moléculaire, Unité Propre de Recherche 2167, Gif-sur-Yvette, F-91198; ||Université Paris-Sud, F-91405 Orsay Cedex, France; and ¶Université Pierre et Marie Curie, F-75005, Paris, France
Submitted November 19, 2007;
Revised May 19, 2008;
Accepted June 11, 2008
Monitoring Editor: Susan Wente
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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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30 different proteins termed nucleoporins (Nups), most of which are organized into subcomplexes that associate with each other to build up the mature NPCs (for reviews, see Hetzer et al., 2005
; Schwartz, 2005; Lim and Fahrenkrog, 2006; Tran and Wente, 2006).
During cell division, the NE and NPCs are subjected to major rearrangements. However, the extent to which the NE and NPCs disassemble at mitotic entry varies among organisms (for reviews, see Margalit et al., 2005; Prunuske and Ullman, 2006). Unlike in most yeast and fungi, characterized by a "closed mitosis," NE disassembly is required in animal cells to allow spindle microtubule access to chromosomes. In vertebrates, cell division leads to complete NE breakdown at the prophase–prometaphase transition. During this "open mitosis," integral membrane proteins of the NE and the soluble subcomplexes of the NPCs redistribute throughout the endoplasmic reticulum and the mitotic cytoplasm (for reviews, see Hetzer et al., 2005; Margalit et al., 2005; Prunuske and Ullman, 2006). In Drosophila and Caenorhabditis elegans embryos, however, the NE only partially disassembles near spindle poles in early mitosis. NPCs disassemble during prometaphase (or even after metaphase in early C. elegans embryos), thus leaving behind a fenestrated or leaky nuclear envelope (Stafstrom and Staehelin, 1984; Lee et al., 2000; Kiseleva et al., 2001). In Drosophila embryos, the spindles of syncytial mitoses remain confined within a membranous structure partly derived from the NE and previously termed spindle envelope (SE) (Stafstrom and Staehelin, 1984; Harel et al., 1989, and references therein). Accordingly, the term "semi-closed" has been used to describe Drosophila mitosis. In all metazoans, NE and NPC reassembly at mitotic exit is initiated around the two sets of chromosomes in late anaphase, and it continues until late telophase/early G1, leading to the reformation of two daughter nuclei (for reviews, see Hetzer et al., 2005; Margalit et al., 2005; Prunuske and Ullman, 2006; Gorjanacz et al., 2007).
These various aspects of NE and NPC remodeling during mitosis need to be spatially and temporally coordinated with other cell cycle events. Key regulators that contribute to this spatio-temporal coordination are mitotic kinases and phosphatases as well as the small GTPase Ran and the transport receptors of the karyopherin/importin β family (for reviews, see Dasso, 2002; Harel and Forbes, 2004; Hetzer et al., 2005; Margalit et al., 2005; see also Onischenko et al., 2005). In addition, there is growing evidence that NPCs functionally participate in mitosis. Indeed, several kinetochore constituents, including the checkpoint proteins Mad1 and Mad2, are localized to NPCs in interphase; conversely, a growing number of nucleoporins have been found on the mitotic spindle or at kinetochores during mitosis (for reviews, see Stukenberg and Macara, 2003; Hetzer et al., 2005; see also Babu et al., 2003, Liu et al., 2003; Arnaoutov et al., 2005; Galy et al., 2006; Fernandez and Piano, 2006; Rasala et al., 2006).
Among them, the vertebrate Nup107-160 subcomplex, which is composed in vertebrates of nine subunits (Belgareh et al., 2001; Vasu et al., 2001; Loiodice et al., 2004), contributes to various aspects of cell cycle progression. Besides playing crucial role at a very early stage of NPC reassembly (Harel et al., 2003; Walther et al., 2003a), a fraction of human Nup107-160 subcomplex and of C. elegans Nup107p localizes at kinetochores during mitosis (Belgareh et al., 2001, Harel et al., 2003; Loiodice et al., 2004; Galy et al., 2006) and was recently demonstrated to contribute to proper kinetochore functions in human cells (Zuccolo et al., 2007). In addition, this complex was reported to localize to spindle poles and proximal spindle fibers in prometaphase mammalian cells and throughout reconstituted spindles in Xenopus egg extracts (Orjalo et al., 2006).
Here, we have examined the in vivo dynamics of the Nup107 nucleoporin during mitosis in Drosophila, directly comparing it with the dynamics of other fluorescently tagged proteins of the nuclear envelope and with the checkpoint protein Mad2. We studied both the rapid mitosis of late syncytial embryos (cycles 10 to 13), in which synchronous nuclear divisions take place in a common cytoplasm close to the embryo cortex (Foe and Alberts, 1983; Stafstrom and Staehelin, 1984) and also the more typical mitosis of larval-stage neuroblasts or cellularized embryos, about which less is known concerning NPC and nuclear envelope dynamics. We have refined the temporal resolution of the order of events at the molecular level that take place in this organism during nuclear disassembly and reassembly. This study revealed both similarities and unique differences with other metazoan systems.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The nup107E8 allele lacks 976-base pairs of the nup107 open reading frame (ORF), including the starting ATG, as confirmed by polymerase chain reaction (PCR) and sequencing (Figure 1A).
For transgenic mRFP-Nup107 and GFP-Nup107 flies, a 7.7-kb fragment of the bacterial artificial chromosome (BAC) clone BACR19N18 (base pairs 4,540–12,268), containing the nup107/CG6743 gene (Fbgn0027868) was first subcloned into pBluescriptSK–. Next, a 4.2-kb XhoI–NsiI fragment, including 1.5 kb upstream and 0.8 kb downstream of nup107 ORF was introduced into the P transformation vector CasPer (Pirrotta, 1988). PCR-amplified monomeric red fluorescent protein 1 (mRFP) cDNA (Campbell et al., 2002) was inserted in-frame at a unique AgeI site located 18 base pairs upstream of the starting ATG of nup107. For the GFP-Nup107 transgene, the green fluorescent protein (GFP) DNA fragment (from the pEGFPC2 vector; Clontech, Mountain View, CA) was inserted in-frame as AgeI-XmaII (12 base pairs upstream of the starting ATG of nup107) in the above-described nup107-CasPer construct. The integrity of the transgenes was checked by sequencing. The mRFP-Nup107 and GFP-Nup107 transgenes were introduced into the germline of yw67 flies by standard methods (Ashburner et al., 2005). Two independent mRFP-nup107 insertion lines were tested for rescue of the lethality of the null nup107E8 allele, of which the mRFP-nup1077.1 rescued the nup107E8 mutation in a single copy. The stock w–; nup107E8; mRFP-nup1077.1/TM2 where the transgene is the only source of Nup107 (referred to as the rescued line in the text) was used for studying Nup107 dynamics.
Live Embryo Imaging
For live imaging, embryos were hand-dechorionated on double-sided adhesive tape and aligned on a coverslip covered with embryo glue in a Ludin chamber (Johansen and Johansen, 2004). Imaging was performed at 24–26°C by using an inverted confocal microscope equipped with an LSM5 META laser confocal imaging system, using a 40x oil/1.3 numerical aperture (N.A.) objective (Carl Zeiss MicroImaging, Jena, Germany). A 488-nm argon laser line with a 505- to 530-nm emission filter for GFP, and a 543-nm HeNe laser line with a 560- to 615-nm emission filter for rhodamine were used. The z-stacks (usually 2–4 planes, 0.5–0.8 µm apart) were acquired at zoom 6 or 10 every 10–30 s. Photobleaching was achieved after 1 or 2 prebleach image acquisitions, with 30 iterations of 60–85% 488-nm laser intensity within regions of interest of varying areas (as indicated on the figures). After acquisition, images were analyzed with MetaMorph software (Molecular Devices, Sunnyvale, CA) and converted to 8-bit images before being imported to Adobe Photoshop software 7.0 (Adobe Systems, Mountain View, CA). A median filter (MetaMorph) was applied to most images. Quantitative analyses were performed using MetaMorph software as described in the corresponding supplemental figure legends, and graphs were generated using Excel (Microsoft, Redmond, WA).
Microinjection of Embryos
For microinjections followed by imaging, 1- to 2-h embryo collections were hand-dechorionated, aligned in the center of an IWAKI plate coated with embryo glue, and covered with halocarbon oil (Voltalef 10S; Prolabo, Lutterworth, Leicestershire, United Kingdom) to avoid desiccation. To determine the embryo age, fluorescent and differential interference contrast images of the whole embryo were acquired before injection. Injections were performed with a manual microinjector (TransferMan NK2; Eppendorf, Hamburg, Germany) on a standard inverted microscope using a 10x objective. Colchicine (catalog no. 27620; Sigma-Aldrich, St. Louis, MO) was used at 1 mM in phosphate-buffered saline (PBS) or H20, and Alexa488-conjugated wheat germ agglutinin (WGA) (Invitrogen, Carlsbad, CA) at 0.1 mg/ml in PBS.
Larval Neuroblast Imaging
Larval neuroblasts were prepared and imaged as described previously (Buffin et al., 2005), using an Olympus IX-70 inverted microscope equipped with a focused xenon lamp and an OrcaER camera (Hammamatsu, Bridgewater, NJ), piloted by Cell-R hardware and software system (Olympus). Depending on the fly line, image acquisition times were 100–400 ms for GFP and 250–400 ms for red fluorescent protein (RFP). Images were usually collected at 15 s intervals (with the exception of the Lamin C/Nup107 movie, 30 s).
Antibodies, Western Blot Analysis, and Immunostaining
The anti-dmNup107 polyclonal rabbit serum was generated against the full-length Drosophila protein (cDNA LD18761 cloned in pBluescript and obtained from Invitrogen) fused to glutathione-S-transferase. For Western blot analysis, 5 µl of dechorionated embryos, two third-instar larvae, and two adult flies were homogenized in 1x SDS-Laemmli buffer. Samples were boiled for 5 min and centrifuged for 15 min at 14,000 rpm. Proteins were then separated on 8% SDS-polyacrylamide gel electrophoresis and transferred on nitrocellulose membrane. The membrane was probed with the anti-Drosophila Nup107 polyclonal antibody and mouse anti-tubulin 
antibody (mouse DM1A; Sigma-Aldrich).
Drosophila Mad1 (CG2072) is listed in Flybase as TXBP181-like. The full-length Mad1 cDNA GM14169 (cloned in vector pOT2) was obtained from the Drosophila Genomics Resource Center (Indiana University, Bloomington, IN). A BglII–XhoI restriction fragment of this plasmid encoding the last 334 amino acids of Mad1 was cloned into the BamI and SalI sites of pET28b expression vector. The protein was expressed and His-tag purified and used to inoculate mice (Kernov Antibody Services, St. Etienne en Cogles, France). A polyclonal serum (mouse no. 1) was used in this study.
Fixation and immunostaining of 1- to 3-h-old embryos were performed essentially as described previously (Johansen and Johansen, 2004). Incubations were performed overnight at 4°C for primary antibodies and for 2–3 h at room temperature for secondary antibodies. Primary antibodies and dilutions used were rabbit anti-dmNup153 (1/500) and rat anti-Mtor (1/750) (Mendjan et al., 2006), rabbit anti-dmNup107 (1/1000), and mouse anti-Mad1 (1/50). After a 5-min incubation with 4,6-diamidino-2-phenylindole (DAPI), embryos were mounted with Mowiol and imaged using either the above-described Zeiss confocal system or (for Figure 9) a CSU10 spinning head (Yokogawa Electric, Tokyo, Japan) coupled with four solids diodes (405, 491, 561, and 635 nm) adapted on a inverted DMIRB microscope (Leica, Wetzlar, Germany). For confocal spinning imaging, selection of fluorophores was realized using an acousto-optic tunable filter on excitation and a filter wheel on emission (Roper-Sutter-Errol). The whole setup was driven with MetaMorph 7 software (Molecular Devices). Acquisitions were performed using an oil immersion objective100x PL APO HCX, N.A. 1.4 mounted on a piezo-electric motor (Physik Instrument, Irvine, CA) and a CoolSnap HQ camera (Photometrics). Image stacks were acquired with a binning of 2, with a plane spacing of 0.5 µm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 kb of the 5' coding region of the nup107 gene, which is also recessive lethal (Figure 1, A and B). Further characterization of this phenotype will be reported elsewhere.
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) under the control of the nup107 promoter (see Materials and Methods). The mRFP-nup107 transgene rescued in a single copy the lethal phenotype of nup107E8 flies (Figure 1, B and C, and Supplemental Figure 1), proving both that nup107 is an essential gene and that the transgene is fully functional. As anticipated, mRFP-Nup107 localized to the nuclear envelope in all Drosophila tissues and cell types examined (Figures 2 and 6; our unpublished data). Except when indicated, all of the studies described here used a rescued line in which all of Nup107 is derived from the mRFP-nup107 transgene.
To examine the dynamics of Nup107 during mitosis, we first followed by time-lapse scanning confocal microscopy (TLSCM) the last three syncytial mitotic cycles (cycles 10–13) of early Drosophila embryos expressing mRFP-Nup107 together with the GFP-tagged histone H2A variant His2AvD (Clarkson and Saint, 1999). Representative panels from one cleavage cycle (cycle 12) are shown in Figure 2A (Supplemental Movie 1). After entry into mitosis, the NE staining of mRFP-Nup107 persisted until prometaphase. At that stage, as the SE developed (Stafstrom and Staehelin, 1984, and references therein), the mRFP-Nup107 signal decreased at the rim and accumulated in the region delimited by the SE. Photobleaching analyses performed at this stage of the cell cycle by using the GFP-Nup107 line revealed that only a minor fraction of Nup107 remains stably associated with the NE in early prometaphase, whereas no NE-associated signal could be detected from late prometaphase on (Figure 2B). In metaphase, mRFP-Nup107 remained concentrated in the spindle region, but it was excluded from the area occupied by the chromosomes. This homogeneous, diffuse mRFP-Nup107 signal in the spindle region was independent of microtubules (see below, Figure 10A). During late anaphase, the signal faded within the spindle area, and Nup107 then began to accumulate on the two sets of separating chromatids. Finally, a rim-like staining reappeared at the periphery of the decondensing chromatin during telophase. This behavior was confirmed using polyclonal antibodies directed against dmNup107 in fixed wild-type syncytial embryos (Supplemental Figure 2).
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–5).
Live imaging of embryos expressing mRFP-Nup107 and GFP-Lamin C (from a protein trap transgenic line described in Schulze et al., 2005) (Figure 3A and Supplemental Movie 2) revealed that the GFP-Lamin C signal at the NE declined more progressively in prometaphase than mRFP-Nup107 (see quantifications in Figure 4A). GFP-Lamin C persisted at the SE into metaphase, where it surrounded the diffuse mRFP-Nup107 signal. In anaphase, however, GFP-Lamin C disappeared from the SE whereas Nup107 remained enriched within the spindle region (Figure 3A). Finally, GFP-Lamin C accumulated at the NE in telophase only once a full Nup107 rim signal was detected (a stage at which mRFP-Nup107 reached its half maximal intensity at the NE) and its recruitment was completed more than a minute later than Nup107 (see quantifications in Figure 5A). The dynamics of GFP-Lamin C during mitosis, in particular its persistence in the spindle envelope until late metaphase, are consistent with earlier observations of lamin behavior based on microinjection of embryos with fluorescently labeled anti-lamin Fab fragments (Paddy et al., 1996).
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). In many species, the lectin WGA recognizes several Nups containing phenylalanine-glycine (FG)-repeated motifs, but in Drosophila it was demonstrated previously to reflect the localization of one FG-nucleoporin, Nup58 (Onischenko et al., 2004). For simplicity, we refer to the signal detected by WGA as "WGA-Nup." Microinjection of Alexa488-labeled WGA into mRFP-Nup107 embryos (Figure 3B and Supplemental Movie 3) revealed that the gradual release of mRFP-Nup107 from the NE and its concomitant appearance in the nucleoplasm in prometaphase occurred before the decline of the WGA-Nup at the NE (Figure 4B). The WGA-Nup persisted at the NE into late prometaphase, longer than Nup107. As reported previously (Onischenko et al., 2005), and in marked contrast to Nup107, the WGA-Nup became evenly distributed in the cytoplasm and showed no enrichment in the spindle area during metaphase and anaphase. In telophase, the first reappearance of both markers at the NE was nearly coincident, although mRFP-Nup107 reached its half-maximal intensity at the NE slightly earlier than the WGA-Nup (Figure 5B).
We also investigated the localization of another FG-repeat nucleoporin, Nup153, in fixed mRFP–Nup107-expressing embryos by using specific anti-Nup153 antibodies (Mendjan et al., 2006). This analysis revealed that unlike WGA, Nup153 localized together with Nup107 within the spindle area during metaphase (Supplemental Figure 3). In this respect, the behavior of Nup153 is similar to that reported for the RL1 antibody (Snow et al., 1987) that recognizes FG nucleoporins in a variety of species and labels four nucleoporins (including an 150-kDa protein) in Drosophila (Onischenko et al., 2004). In addition, Nup153 seemed to be released from the NE before Nup107 in prophase, and it gave rise to a weaker signal than Nup107 on the reforming NE during telophase. This suggests that Nup153 and Nup107, although they both remain concentrated in the spindle area in metaphase, have slightly different behaviors during mitotic entry and exit.
Importin β, a key component of the nuclear import machinery, also plays essential roles in the NE, NPCs, and spindle assembly in vertebrates (for reviews, see Harel and Forbes, 2004). Additionally, it has been shown to interact with several nucleoporins, including Nup107 and Nup153 in Xenopus mitotic extracts (Walther et al., 2003b). In Drosophila, importin β is encoded by the ketel gene (Lippai et al., 2000). Here, we used an exon trap allele of ketel called ketelGFP in which a GFP exon is inserted between amino acids 18 and 19 of ketel within its Ran binding domain (Morin et al., 2001, Villanyi et al., 2008). Previous studies performed in vertebrates demonstrated that deletion of the first 10 or 32 residues of importin β impairs its interaction with RanGTP and leads to its increased residency at NPCs, probably at the import termination sites (i.e., the nuclear side of NPCs) (Kutay et al., 1997, and references therein). Consistent with this, fluorescence recovery after photobleaching analyses (Supplemental Figure 4) revealed that fluorescent recovery of ketelGFP at the NE and in the nucleus seemed to be slower compared with full-length GFP-human importin β expressed in HeLa cells (Rabut et al., 2004). Despite the reduced function of the ketelGFP allele compared with wild-type Ketel in the fly (Villanyi et al., 2008), its expression in one copy does not impair mitotic progression in the syncytial embryos (Figure 3C and Supplemental Movie 4). Live imaging of embryos expressing ketelGFP and mRFP-Nup107 further revealed their similar mitotic dynamics (Figure 3C and Supplemental Movie 4). Both proteins persisted within the spindle area throughout mitosis, although ketelGFP showed an increased persistence in the spindle area in anaphase, which may reflect the previously reported interaction between ketel and microtubules (Tirian et al., 2003) (Figure 3C; frame 4:20 min). In addition, ketelGFP was released from the NE slightly earlier than Nup107 (20–30 s) at mitotic entry and reached its half-maximal intensity at the NE slightly later than mRFP-Nup107 in telophase (Figures 4C and 5C and Supplemental Figure 5). The dynamics of ketelGFP differed from the dynamics described previously for wild-type Ketel (Trieselmann and Wilde, 2002) and rather reproduced the behavior of Nup153 (compare Supplemental Figure 3 and Figure 3C). Because importin β has been reported to have higher affinity for Nup153 compared with other vertebrate FG-repeat nucleoporins (Ben-Efraim and Gerace, 2001), the increased residency of the ketelGFP fusion at NPCs, and potentially its dynamics in mitosis, may thus reflect its binding to Nup153. Accordingly, although the ketelGFP allele does not seem to be an appropriate tool to study the dynamics of wild-type importin β in flies, it nevertheless provides a valuable marker to follow some features of NPCs dynamics in Drosophila.
NPC and NE Dynamics in Dividing Larval Neuroblasts
The rapid (10–12 min) cell cycles of the syncytial embryo are very unusual. To examine the behavior of Nup107 in a more conventional Drosophila mitosis, we recorded divisions in third-instar larval neuroblasts, another well characterized Drosophila mitotic cell type whose asymmetric division generates a large daughter neuroblast and a small ganglion mother cell (Knoblich, 2001; Chia and Yang, 2002).
Time-lapse analysis of living neuroblasts expressing mRFP-Nup107 and ketelGFP confirmed an overall behavior for both proteins similar to that seen in syncytial embryos (Figure 6A and Supplemental Movies 5 and 6). In particular, we observed that 1) in prophase ketelGFP is released from the NE before Nup107 (Figure 6A, top, frames 3:30 and 4:00 min); 2) both proteins are enriched within the spindle area in prometaphase (albeit to a lesser extent than in syncytial embryos); 3) ketelGFP persists in the spindle area in late metaphase and anaphase, whereas Nup107 becomes gradually fainter and may even be slightly excluded from the spindle area (Figure 6A, bottom, frames 6:30–11:45 min); and 4) both proteins, although always excluded from chromatin before this stage, are simultaneously recruited to decondensing chromatin in early telophase (Figure 6A, bottom, frames 12:00–13:00 min).
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The observed enrichment of ketelGFP and to some extent of mRFP-Nup107 within the spindle region of metaphase neuroblasts, together with the persistence of the GFP–Lamin C-labeled structure, suggested the existence of a spindle envelope in these cells. Although neuroblasts are well-studied cells, little is known concerning the dynamics of their NE in mitosis. In particular, it is unknown whether they undergo a semi-closed mitosis as reported for syncytial embryos (Stafstrom and Staehelin, 1984) and embryonic-derived cells in culture (Debec and Marcaillou, 1997), in which the mitotic spindles are enclosed in membranous structures that become permeable to 70-kDa molecules in prophase (Paddy et al., 1996). To further address this question, we examined dividing neuroblasts expressing GFP-protein disulfide isomerase (PDI), a component of the endoplasmic reticulum lumen (Bobinnec et al., 2003). This analysis revealed the presence of GFP–PDI-labeled membranous structures surrounding the mitotic spindle that persisted into anaphase (Supplemental Figure 6B).
In summary, our study has determined that the order of events governing the disassembly and reassembly of the NE and NPCs are quite similar in neuroblasts and syncytial embryos, despite differences in the mitotic kinetics of these two developmental stages. In particular, neuroblasts seem to undergo a semi-closed mitosis, similar to the mitosis reported previously for the embryonic tissues. The higher cytoplasmic signal of both ketelGFP and mRFP-Nup107 observed in neuroblasts (compared with syncytial embryos) could be due either to an increased permeability of the spindle envelope in differentiated tissues, or to the longer duration of their mitoses, allowing the progressive diffusion of large complexes.
The Association of Mad2 with NPCs at the End of Mitosis Is a Two-Step Process
Although best characterized with respect to its checkpoint function and kinetochore localization, Mad2 associates with NPCs during interphase in both human and yeasts (Campbell et al., 2001; Iouk et al., 2002). In Drosophila neuroblasts, Mad2 is primarily in the nucleoplasm, but a fraction of it is associated with the NE (Buffin et al., 2005). To further explore Mad2's association with the NE, we directly compared the behavior of GFP-Mad2 with that of mRFP-Nup107 during mitotic entry and exit.
Somewhat surprisingly, NE-associated Mad2 was only obvious in the later interphases (cycles 12 and 13) of syncytial embryos. Mad2 localization at the NPCs of interphase nuclei 10 and 11 was almost undetectable compared with the strong intranuclear fluorescence (Figure 7and Supplemental Movies 9–11). One explanation for the almost undetectable NE-associated Mad2 signal in interphases 10 and 11 was that the intense nucleoplasmic GFP-Mad2 signal was masking a weaker signal at the NE. To test this, we performed photobleaching of small regions (corresponding to areas of 1–16 µm2) on interphase syncytial embryos whose only source of Mad2 is GFP-Mad2 (Buffin et al., 2007) (Figure 7D). The intranuclear pool of Mad2 seemed to be freely diffusible, because even a small photobleached spot (marked as nucleus 1 of the various cycles in Figure 7D) resulted in a nearly uniform 30–50% drop of fluorescence throughout the nucleoplasm. More extensive photobleaching of Mad2 to background levels revealed that Mad2 is also present in the cytoplasm in these syncytial embryos (compare the fluorescence intensities outside vs. inside the nuclei marked 2–4). Most importantly, these photobleaching experiments revealed that a fraction of Mad2 did in fact associate with the NE of cycles 10 and 11. This indicates that Mad2 is present at the NE as well as in the nucleus in all syncytial embryonic interphases, but the NE pool is masked by the intensity of the intranuclear Mad2 signal in cycles 10 and 11.
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Both the early dissociation of GFP-Mad2 and the late recruitment of Mad2 to the newly formed NE were also seen in the more conventional mitoses of larval neuroblasts (Figure 8). As already observed for Nup107 and ketelGFP (Figure 6A), GFP-Mad2 was, however, more broadly diffused throughout the cytoplasm during prometaphase and metaphase in neuroblasts compared with syncytial embryos (compare Figure 8A with Figure 7, A–C). As in syncytial embryos, no nuclear accumulation of Mad2 could be detected in neuroblasts during telophase (arrowheads in Figure 8). GFP-Mad2 began to stream into the nucleus only 3 min later (open arrows) and NE-associated Mad2 again became evident after six to seven additional minutes, in early G1 (arrows). Thus, both in embryos and neuroblasts, Mad2 seems to be first imported into the nucleoplasm of the reforming nucleus, and only subsequently does a fraction of this checkpoint protein associate with the NE.
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; Campbell et al., 2001; Luo et al., 2002) is also localized on the nuclear side of the NPCs in vertebrates and yeasts (Campbell et al., 2001). In budding and fission yeasts, the NE localization of Mad2 relies on Mad1 (Iouk et al., 2002; Ikui et al., 2002) and in Saccharomyces cerevisiae, the basket nucleoporins Mlp1/2 were further demonstrated to be major binding sites for Mad1 at NPCs (Scott et al., 2005). This prompted us to analyze the behavior of Drosophila Mad1 (CG2072, listed in Flybase as TXBP181-like) and Mtor (Megator, the Drosophila orthologue of S. cerevisiae Mlp1/2 and vertebrate Tpr; Qi et al., 2004) in fixed mRFP-Nup107 Drosophila embryos. As observed for GFP-Mad2, and unlike mRFP-Nup107 or Mtor, Mad1 was mainly nuclear in early embryos, and its NE localization only became apparent in late preblastoderm or cellularized embryos (Figure 9A, compare top and bottom panels, and Figure 9B). We therefore further investigated its behavior during the asynchronous mitotic divisions on cellularized embryos, in which Mad1 is best detected at the NE (Figure 9B). In prometaphase, a fraction of Nup107 was still detectable at the NE, whereas Mad1 and Mtor were localized within the spindle area (Figure 9B, bottom, brackets). In metaphase, Mtor displayed its typical localization at the spindle matrix (Qi et al., 2004), and a fraction of Mad1 also persisted within the spindle area, whereas mRFP-Nup107 had a more diffuse localization throughout the cell (Figure 9B, top, brackets). On NE reassembly, Mad1 was first excluded from the reforming nuclei, even from those already ringed with an apparently continuous mRFP-Nup107 signal (arrowheads in Figure 9B). In a subsequent stage, nuclear accumulation of Mad1 was observed, whereas Mtor was still exclusively localized in the cytoplasm (open arrows). Still later, some Mad1 was detected at the NE, and this was always coincident with NE localization of Mtor (arrows in Figure 9B).
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Nup107 Is Undetectable at Drosophila Kinetochores
Another feature of metazoan mitosis is the relocalization of several NPC constituents to mitotic structures. In both human cells and C. elegans embryos, a fraction of the Nup107-160 NPC-subcomplex localizes to kinetochores during mitosis (Belgareh et al., 2001; Galy et al., 2006). Surprisingly, none of the mRFP-Nup107 movies acquired in fly embryos or neuroblasts revealed any kinetochore association (Figures 2–3 and 6
–8). To further explore this issue, we compared the mitotic behavior of mRFP-Nup107 with that of GFP-tagged Mad2, which localizes to unattached kinetochores in mitosis (Buffin et al., 2005). Analysis of live embryos expressing both fusion proteins did not reveal any colocalization between mRFP-Nup107 and GFP-Mad2 at kinetochores (Figure 10A; also see Figure 7 and Supplemental Movies 9–11). Because mammalian Nup107 is enriched at kinetochores lacking microtubules (Orjalo et al., 2006; Zuccolo et al., 2007), we next examined embryos after injection of the microtubule-depolymerizing drug colchicine (Figure 10A). Under these conditions, which cause metaphase arrest and a notable accumulation of Mad2 at kinetochores, mRFP-Nup107 still showed no kinetochore association, and rather seemed to be excluded from the Mad2-labeled kinetochore area (Figure 10A). It is, however, noteworthy that the mRFP-Nup107 signal persisted within the area defined by the spindle envelope, indicating that this localization is independent of microtubules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NPC Disassembly and Reassembly in Drosophila Mitosis
During prophase and prometaphase, Nup107 and the other NPC-associated proteins examined were progressively released from the NE, most likely reflecting the loose synchrony of NPC disassembly (Stafstrom and Staehelin, 1984; Kiseleva et al., 2001). Drosophila NPC disassembly was completed by metaphase and preceded lamina depolymerization, a property shared by C. elegans embryos (Lee et al., 2000). Compared with Nup107, ketelGFP (which, as discussed above, likely interacts with peripheral FG-Nups), as well as Nup153 and Mtor (the Drosophila orthologue of Tpr; Qi et al., 2004), two asymmetrically localized nuclear basket nucleoporins, were released slightly earlier, in prophase. Previous studies performed in Drosophila embryos, starfish oocytes, and in vitro assembled Xenopus nuclei (Kiseleva et al., 2001; Lenart et al., 2003; Cotter et al., 2007), also found an earlier release of peripheral NPC structures.
In contrast, the WGA signal, which was demonstrated previously to reflect the localization of Drosophila Nup58 (Onischenko et al., 2004), persisted longer than mRFP-Nup107 at the NE periphery. Although Nup58 forms part of the central channel of the NPC in mammals (Guan et al., 1995), a previous electron microscopy study suggested that the release of the central transporter or central material occurs at an early stage of NPC disassembly in Drosophila embryos (Kiseleva et al., 2001). Our unexpected result was however corroborated by a study that came out while this manuscript was under revision, revealing that human Nup58 remains longer than Nup133 (another constituent of the Nup107 complex) in fragments of the NE during disassembly (Dultz et al., 2008). During the closed mitosis of the fungus Aspergillus nidulans, however, FG-nups, including those making up the central channel, disperse throughout the cell, whereas constituents of the Nup107 subcomplex persist at NPCs (De Souza et al., 2004; Osmani et al., 2006). Accordingly, the fate of NPC components in Drosophila and human cells, in which all NPC constituents disassemble, seems to be distinct from that occurring during the closed mitosis of A. nidulans.
The various steps of NPC reassembly at the end of mitosis have been extensively investigated previously both in vivo and in vitro (for reviews, see Hetzer et al., 2005; Prunuske and Ullman, 2006). Our study, revealing the early recruitment of Nup107 to the reforming NE in late anaphase, the slightly delayed recruitment of ketelGFP and WGA-Nup, and the late recruitment of Mtor, is consistent with previous studies in other organisms (Bodoor et al., 1999; Burke and Ellenberg, 2002; Dultz et al., 2008). Along with data recently gathered in human cells (Dultz et al., 2008), it further indicates that NPC reassembly is not simply reversing the sequence of its disassembly.
Mad1 and Mad2 during Drosophila Mitosis
We found that a fraction of Drosophila Mad1 and Mad2 associates with the NE in syncytial embryos, which probably reflects their binding to the nuclear side of the NPCs as demonstrated in yeast and vertebrates (Campbell et al., 2001; Iouk et al., 2002). The release of GFP-Mad2 at an early stage of Drosophila NPC disassembly in prophase and the late recruitment of Mad1 and Mad2 at the NE corroborate previous observations in Xenopus and human cells (Chen et al., 1996; Shah et al., 2004). However, we show in addition that relocalization of Drosophila Mad1 and Mad2 to the NE occurs in two steps, with their nuclear import preceding their NE association. This shared behavior suggests that both proteins might be reimported into the nucleus as a complex.
The coincident NPC recruitment of Mad1 and Mad2 with Mtor indicates that Drosophila Megator likely represents their NPC-anchoring determinant as are the yeast Mtor orthologues (Mlp1/2) (Scott et al., 2005). This seems to be an evolutionarily conserved interaction, because small interfering RNA-induced depletion of Tpr (the vertebrate orthologue of yeast Mlp1/2-Drosophila Mtor) in HeLa cells significantly reduces Mad2 labeling at the NE (our unpublished data). Our in vivo study further revealed that in syncytial embryos, a fraction of GFP-Mad2 is found within the spindle area in metaphase and subsequently localizes between the two reforming nuclei during telophase (Figure 7), localizations similar to that of Mtor (Qi et al., 2004). It is thus conceivable that an interaction between Mad1-Mad2 and Mtor may also occur at this stage of mitosis.
Specific Features of Drosophila Mitosis
Although a spindle envelope was initially identified in syncytial Drosophila embryos (Stafstrom and Staehelin, 1984; Harel et al., 1989; Bobinnec et al., 2003; Frescas et al., 2006; Wagner et al., 2006) and embryo-derived Drosophila cell lines (Debec and Marcaillou, 1997; Maiato et al., 2006), it was unclear whether all Drosophila cells had one. In the S2R+ cell line for example, NE remnants were observed in cells fixed shortly after NE breakdown, but not at later stages of metaphase (Maiato et al., 2006). Our in vivo studies of larval neuroblasts, revealing the presence of a membrane-like structure stained by GFP-PDI and the persistence of a fraction of lamins around the mitotic spindle up to anaphase, indicate that the spindle envelope is not restricted to embryonic cells and thus seems to be a general feature of Drosophila mitosis. By confining the prometaphase chromosomes as well as a subset of proteins to a small region of the cell, the persistence of an envelope and/or the Mtor-defined spindle matrix (Qi et al., 2004) might explain the remarkable efficiency of fly mitosis, in which kinetochores capture spindle fibers so rapidly that chromosomes can segregate properly even in the absence of the spindle checkpoint (Buffin et al., 2007).
Another specific feature of Drosophila mitosis uncovered by our study was the absence of detectable Nup107 at kinetochores. In embryos and to a lesser extent larval neuroblasts, however, a fraction of Nup107 remained confined within the area defined by the spindle envelope up to late metaphase. Unlike in Xenopus extracts, in which the localization of the Nup107-160 complex throughout the mitotic spindle requires microtubules (Orjalo et al., 2006), this signal persists upon colchicine treatment in Drosophila embryos (Figure 10A). Accordingly, it may reflect either a weak interaction of the Nup107-160 complex with the Mtor-defined spindle, or the confinement of this huge complex within the spindle envelope.
Because the Nup107 complex is also absent from kinetochores in fission yeast, which undergoes a closed mitosis (Bai et al., 2004), the lack of Nup107 at kinetochores could potentially be related to the presence of a spindle envelope in flies. However, the localization of Nup107 at kinetochores of early C. elegans embryos in which NPCs and NE disassembly only take place in anaphase does not support this hypothesis (Lee et al., 2000; Galy et al., 2006). Moreover, Nup107 was not found at kinetochores during the open mitosis of the fungus Ustilago maydis in which Nup107 is first dispersed throughout the cytoplasm before being recruited to chromatin in metaphase (Theisen et al., 2008). Understanding how various spatial and temporal localization of the Nup107-160 complex occurring in distinct organisms may underlie species-specific properties of mitotic spindle assembly and chromosome segregation will be a fruitful avenue of future studies.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present addresses: 
Department of Molecular Biology and Genetics, Democritus University of Thrace, 68100 Alexandroupolis, Greece; 
# Institut Jacques Monod, Unité Mixte de Recherche 7592, 75251 Paris, France.
Address correspondence to: Valérie Doye (vdoye{at}curie.fr)
Abbreviations used: NE, nuclear envelope; NPC, nuclear pore complex; Nup, nucleoporin; SE, spindle envelope; TLSCM, time-lapse scanning confocal microscopy; WGA, wheat germ agglutinin.
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