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Vol. 16, Issue 11, 5152-5162, November 2005
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* Section of Life Sciences, Södertörns University College, SE-141 89 Huddinge, Sweden;
Department of Biosciences at Novum, Karolinska Institute, SE-141 86 Huddinge, Sweden; and
Institute of Cytology and Genetics, 630090 Novosibirsk-90, Russia
Submitted July 18, 2005;
Revised August 16, 2005;
Accepted August 17, 2005
Monitoring Editor: Karsten Weis
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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30 different proteins called nucleoporins (Rout et al., 2000
; Cronshaw et al., 2002), most of which are conserved in higher eukaryotes (Mans et al., 2004). About a third of the nucleoporins contain phenylalanine-glycine (FG) repetitive domains, which are believed to be involved in the selective permeability of the NPC (Weis, 2003; Peters, 2005). In mammals the majority of the FG-nucleoporins are modified with O-linked N-acetyl-D-glucosamine residues (Snow et al., 1987), whose function is still unknown. In addition to NPCs some cell types, such as oocytes and early embryonic cells, also contain mimics of NPCs inserted in cytoplasmic annulate lamellae (AL) membranes (Kessel, 1992). The annulate lamellae pore complexes (ALPCs) have a similar structure and nucleoporin composition as the NPCs (Meier et al., 1995; Cordes et al., 1996; Imreh and Hallberg, 2000; Miller and Forbes, 2000).
During the open mitosis of higher eukaryotes the NPCs (as well as the ALPCs) disassemble into distinct soluble nucleoporin subcomplexes (Macaulay et al., 1995; Cordes et al., 1996; Belgareh et al., 2001), whereas the pore membrane proteins diffuse throughout the ER network (Yang et al., 1997; Imreh and Hallberg, 2000; Daigle et al., 2001). Disassembly of both types of pore complexes is accompanied by phosphorylation of a subset of the nucleoporins (Macaulay et al., 1995; Favreau et al., 1996; Ganeshan and Parnaik, 2000; Belgareh et al., 2001). At mitotic exit the NPCs sequentially reassemble in the reforming NEs of the daughter nuclei (Bodoor et al., 1999; Belgareh et al., 2001; Daigle et al., 2001).
Mitotic events are controlled by a family of evolutionary conserved cyclin-dependent kinases (Cdks), which are enzymatically active only in a complex with regulatory cyclin subunits (reviewed in Doree and Hunt, 2002). Cdk1, which forms an active kinase complex with B-type cyclins plays an essential and specific role in triggering the mitotic events (Riabowol et al., 1989; Beckhelling et al., 2003). The activity of Cdks is regulated by the synthesis and destruction of the cyclins as well as by activating and inhibitory phosphorylation (reviewed in Obaya and Sedivy, 2002). Interestingly, some of the nucleoporins phosphorylated during mitosis were shown to be substrates for Cdk1 (Macaulay et al., 1995; Favreau et al., 1996; Ganeshan and Parnaik, 2000). However, at the moment very little is known about how the mitotic machinery controls disassembly and reassembly of the NPCs.
After fertilization the Drosophila embryo represents a multinucleated cell (syncytium) where embryonic nuclei rapidly (every 10 min) and synchronously divide for 13 rounds without undergoing cytokinesis (Foe and Alberts, 1983). Syncytial embryos contain an excessive pool of maternally contributed nucleoporins, which assemble into NPCs and ALPCs during interphases and disassemble during mitosis (Stafstrom and Staehelin, 1984; Kiseleva et al., 2001; Onischenko et al., 2004). From the 9th division cycle the nuclei are aligned 10–20 µm beneath the embryonal periphery where mitotic divisions can be easily followed directly in living embryos (Foe and Alberts, 1983). Here we used early Drosophila embryos as a model system to dissect the mechanism regulating mitotic disassembly/reassembly of the NPCs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) obtained from Dr. Christian Lehner. The mutant fly stock png172 (Shamanski and Orr-Weaver, 1991; Fenger et al., 2000) was obtained from Dr. Terry Orr-Weaver. The embryos were collected at 22°C on agar plates, studded with dry yeast, and harvested according to (Allis et al., 1977). The embryos were batch-dechorionated with Clorox according to (Elgin and Hood, 1973). For time-lapse microscopy of live embryos they were manually dechorionated on a scotch tape.
Immunostaining
Except otherwise mentioned all the procedures were performed at RT. Untreated png embryos (see Figure 3) were fixed in the equal volumes of n-heptane and –70°C methanol. Microinjected png embryos (see Figure 6) were fixed for 20 min in 1:1 mixture of n-heptane and 3.7% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.4, and subsequently freed from vitelline membranes with thin needles. Fixed embryos were permeabilized for 1 h with a blocking solution (0.5% bovine serum albumin [BSA] in PBS containing 0.1% Tween-20) + 0.1% Triton X-100 and subsequently incubated overnight at +4° in the blocking solution containing RL1 antibodies (Affinity Bioreagents, Golden, CO) diluted 1:5000 or anti-cyclin B antibodies F2F4 (Hybridoma Bank, Iowa City, IA) diluted 1:100, used as primary antibodies. The embryos were subsequently washed and incubated with 0.1 µg/ml WGA-Alexa594 (Molecular Probes, Eugene, OR) and 1:2000 dilution of FITC donkey anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) or FITC goat anti-mouse antibodies (Molecular Probes), used as secondary antibodies. For visualization of nuclei the embryos were treated for 1 h with 1 mg/ml RNase-A in PBS-T (0.1% Tween-20 in PBS), followed by staining with 15 µg/ml propidium iodide. The embryos were mounted in Vectashield media (Vector Laboratories, Burlingame, CA) and imaged as described in Microinjections and Time-lapse Microscopy but using Leica PL APO 635/1.32 oil objective (Deerfield, IL).
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1–5% of the total embryonal volume, using inverted microscope with air-driven Microinjector 5242 (Eppendorf, Fremont, CA) under halocarbon oil. For experiments performed at 22°C embryos were covered with 1:1 mixture of Halocarbon oil 700 and Halocarbon oil 27 (Sigma), whereas for the experiments performed at 30°C they were covered with pure Halocarbon oil 27 to increase the oxygen supply. For studies of living embryos the slides with the injected embryos were placed directly under the microscope equipped with The Cube conditioning system (Life Imaging Services, Reinach, Switzerland). Images were acquired with Leica TCS-SP confocal microscope equipped with Leica Cofocal Software 2.61 and Leica PL APO 405/1.25 oil objective and processed with Adobe PhotoShop 7.0 software (Adobe Systems). For immunostaining and EM studies the injected embryos were incubated in a humid chamber at the appropriate temperature, released from the slides with n-heptane, and transferred either into glutaraldehyde-based fixative (for EM), or into formaldehyde fixative (for immunostaining).
Cycloheximide and colchicine (Sigma-Aldrich) were added at final concentration of 1 mg/ml to injection buffer (0.5x PBS, pH 7.4). R-roscovitine (Calbiochem, San Diego, CA) and okadaic acid (Sigma-Aldrich) were injected at final concentrations of 5 mM and 150 µM, respectively. In the case of roscovitine we also added dimethyl sulfoxide to the injection solutions up to 20% to increase the solubility. Drosophila GST-cyclin B purified according to Carroll et al. (1998) from the bacterial expression construct (a gift of Dr. Douglas Kellogg) was added to the microinjection solutions at concentrations up to 7.5 mg/ml. For visualization of the NPCs and ALPCs in live embryos we microinjected WGA-Alexa594 (0.1 mg/ml), resulting in a final concentration of WGA inside the embryo of 0001–0005 mg/ml, which is far below the concentrations reported to affect nuclear import and pore complex assembly (Finlay et al., 1987; Dabauvalle et al., 1990).
Electron Microscopy and EM-Morphometry
The embryos, fixed and stained en bloc with uranyl acetate essentially according to Zalokar and Erk (1977), were dehydrated with ethanol and embedded individually into Agar-100 according to the standard protocol. The ultrathin sections were stained with uranyl acetate followed by lead citrate and examined with a JEM-100 electron microscope (JEOL, Peabody, MA).
For morphometric analysis, individual embryos were sectioned in the equatorial plane. To determine the total number of ALPCs per embryo, we counted the numbers of ALPCs directly under the electron microscope in 10 randomly selected areas (504 µm2) of the embryo section. These data and the diameter of the ALPC (0.12 µm) were used to calculate the average volume density (VD) of ALPCs in each embryo by standard stereologic equations (Gruzdev, 1974). The volume (V) of the embryo was determined through its horizontal and vertical dimensions (measured under the light microscope in the semithin sections), assuming an elliptical shape. The total number of ALPCs in the embryos was calculated by multiplying V with VD.
In Vitro Assay for NPC Disassembly
The embryonic nuclei were purified from fresh 3–7-h old embryos essentially as described in Fisher et al. (1982), except that for homogenization of embryos and washes of nuclei we used 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 50 mM NaCl, 250 mM sucrose, 1 mM dithiothreitol (DTT). The nuclei were washed an additional two times with the same buffer, resuspended in 1 volume (initial volume of embryos) of 20 mM Tris-HCl, pH 7.4, and 5 mM MgCl2 and kept on ice. Two microliters of the freshly prepared nuclear suspension was mixed with 30 µl of the reaction buffer (50 mM KCl, 40 mM 
-glycerophosphate, 10 mM EGTA, 5 mM MgCl2, 2 mM DTT, 0.2% BSA, 20 mM Pipes, pH 7.4, with the addition of 1/20 volume of ATP-regenerating system). The disassembly reaction was performed for 1 h at RT in the presence of 1 µg (10 µl) of active recombinant human Cdc2-cyclin B complex (Upstate Biotechnology, Lake Placid, NY). The reactions were centrifuged at 100,000 x g in a fixed-angle rotor for 10 min at 4°C. The supernatants were mixed 1:1 with sample buffer. The pellets were resuspended and diluted in the reaction buffer to the equal volumes as supernatants and mixed 1:1 with sample buffer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) that wheat germ agglutinin (WGA) specifically bound to only one Drosophila NPC protein, NUP58, and perfectly colocalized with two well-established monoclonal nucleoporin marker antibodies (mAb414; Davis and Blobel, 1986 and RL1; Snow et al., 1987). In addition, microinjected gold-labeled WGA specifically decorated the central portion of the NPCs and ALPCs in syncytial embryos analyzed by EM. We wanted to exploit the use of WGA as marker for NPCs and ALPCs in live Drosophila embryos. For this, we microinjected 0.1 mg/ml fluorescently labeled WGA (WGA-Alexa594) into live early embryos and followed its distribution with time by confocal laser scanning microscopy. During interphase syncytial embryos displayed brightly labeled NEs and cytoplasmic foci (Figure 1A and Supplementary Video 1) in agreement with the distribution of WGA to the NPCs and the ALPCs. The nuclear rim staining and the dotty pattern in the cytoplasm synchronously disappeared during mitosis and reappeared in telophase, consistent with the timing of disassembly and reassembly of NPCs and ALPCs (Stafstrom and Staehelin, 1984; Onischenko et al., 2004). Usually the staining of the NEs was more intense than the staining of the cytoplasmic foci either because of lower levels of NUP58 or lower accessibility for WGA in the AL membrane stacks. Microinjection buffers containing 0.1 mg/ml WGA-Alexa can be safely used for getting a quick qualitative assessment of disassembly/reassembly of NPC and ALPC in live Drosophila embryos, since we did not observe any abnormalities in nuclear import (Figure 1B), timing of mitotic divisions, or embryonic development (unpublished data). At 0.2 mg/ml some effects on nuclear import and nuclear growth were observed locally at the area of injection.
Mitotic Cyclins Are Required for Disassembly of NPCs and ALPCs in Syncytial Drosophila Embryos
In a previous study we reported that the major fraction of excess nucleoporins in syncytial Drosophila embryos remains soluble and that one of the nucleoporins (p150) is hyperphosphorylated (Onischenko et al., 2004). Because syncytial embryos are known to display high levels of mitotic cyclins and Cdk1 activity (Edgar et al., 1994), we were interested in investigating how mitotic factors influence disassembly/reassembly of pore complexes. We microinjected syncytial embryos with the protein synthesis inhibitor cycloheximide, reported to cause depletion of mitotic cyclins (Edgar et al., 1994). As expected, cycloheximide quickly reduced the level of cyclin B (Figure 2A). Cycloheximide also completely blocked disassembly of NPCs and ALPCs in the injected embryos as shown using time-lapse microscopy (Figure 2B and Supplementary Video 2). This conclusion was supported by EM analysis because all cycloheximide-injected embryos (n = 6) contained typical interphase nuclei with intact NPCs (Figure 2, C and D) and abundant ALPCs (Figure 2, E and F). Quantification of ALPCs using EM-morphometry showed that cycloheximide-injected embryos actually contained more ALPCs (about twofold) than interphase control embryos, injected with buffer (Figure 2G). The effect of cycloheximide was primarily due to depletion of mitotic cyclins, since it could be completely reversed by coinjection with nondegradable recombinant Drosophila GST-cyclin B (Su et al., 1998) resulting in persistently disassembled NPCs and ALPCs as judged by time-lapse microscopy of live embryos (Figure 2H and Supplementary Video 3), by EM analysis (Figure 2, I and J) and morphometric quantification of ALPCs (Figure 2K). Furthermore, cycloheximide injection also caused massive dephosphorylation of the nucleoporin p150, which is normally dephosphorylated when assembled into NPCs and ALPCs during interphase (Onischenko et al., 2004). The cycloheximide-dependent dephosphorylation of p150 was completely abolished by coinjection of GST-cyclin B (Figure 2L). The results indicate that in normal syncytial embryos disassembly of NPCs and ALPCs is dependent on mitotic cyclins.
If disassembly of pore complexes requires mitotic cyclins one would expect impaired disassembly of pore complexes in syncytial embryos obtained from png mutant flies, which have low endogenous levels of the major mitotic cyclins A and B (Fenger et al., 2000). Indeed, in all png embryos immunostaining with WGA and RL1 antibodies revealed intense labeling of the NEs of a few giant nuclei unable to divide (Lee et al., 2001), as well as abundant cytoplasmic structures resembling AL (Figure 3, A and B). This was confirmed by EM analysis, which revealed assembled NPCs (Figure 3, C and D) as well as unusually large stacks of AL with tightly packed ALPCs (Figure 3, E and F). The number of ALPCs in png mutants was even higher than in the cycloheximide-injected embryos (for EM-morphometry; see Figure 4C). The large quantity of persistently assembled ALPCs in cyclin-deficient png mutants supports the idea that disassembly of NPCs and ALPCs requires mitotic cyclins.
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15-fold lower in png embryos injected with GST-cyclin B (mean value, 0.73 x 106 ALPCs/embryo) than that of buffer-injected png control embryos (mean value, 12.1 x 106 ALPCs/embryo). The cyclin B induced disassembly of ALPCs was completely blocked by coinjection with the specific Cdk inhibitor roscovitine (Figure 4D), indicating that in cyclin-deficient embryos exogenous cyclin B is able to promote disassembly of pore complexes by activating Cdk1 (Campbell et al., 1995; Jacobs et al., 1998).
This led us to investigate the role of Cdk1 in regulation of pore complex disassembly/reassembly. We took advantage of embryos, bearing a point mutation in Cdk1, which makes the enzyme thermosensitive (Sigrist et al., 1995). The activity of Cdk1 can be efficiently blocked in the Cdk1ts embryos by raising the temperature from 22 to 30°C. Time-lapse movies of WGA-Alexa injected Cdk1ts embryos showed that at 30°C NPCs and ALPCs did not disassemble (Figure 4E, top row, and Supplementary Video 6). The inability to disassemble was specifically due to inactivation of Cdk1 because the pore complexes disassembled and reassembled normally in Cdk1ts embryos incubated at 22°C (Figure 4E, middle row and Supplementary Video 7) and wild-type embryos incubated at 30°C (Figure 4E, bottom row). At the EM level all of the Cdk1ts embryos at 30°C (n = 6) had intact NEs with assembled NPCs (Figure 4, F and G) and ALPCs (Figure 4H). The quantity of ALPCs in all Cdk1ts embryos at 30°C (n = 5) was substantially higher than in any of the wild-type embryos at 30°C (n = 5) or Cdk1ts mutants at 22°C (n = 5; Figure 4I). On the basis of our findings we conclude that Cdk1 activity is critically required for disassembly of pore complexes in Drosophila embryos.
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) or GST-cyclin B (Su et al., 1998). After 15 min of incubation, when nuclear divisions were arrested in mitosis and both NPCs and ALPCs were disassembled, we raised the temperature to 30°C and monitored pore complex reassembly by time-lapse microscopy of live embryos. In both cases we observed massive reassembly of the NPCs and ALPCs in Cdk1ts embryos 5–10 min after shifting to the restrictive temperature, whereas wild-type embryos remained unaffected (Figure 5 and Supplementary Video 8). The results show that Cdk1 activity is continuously required in order to keep NPCs and ALPCs disassembled during mitosis.
Purified Recombinant Cdk1-Cyclin B Complex Is Able to Dissociate Nucleoporins from Purified Embryonic Nuclei In Vitro
To further dissect the mechanism behind Cdk1-dependent disassembly of pore complexes, we injected one pole of the cyclin-deficient png mutant embryos with GST-cyclin B followed by immediate fixation and immunostaining to study the relation between distribution of cyclin B, ALPCs, and NPCs by immunofluorescence microscopy. The propagation of the cyclin B diffusion zone strictly correlated with the sharp border between diffuse WGA staining and fluorescing nuclear rims and cytoplasmic foci (Figure 6), demonstrating that local cyclin B-induced Cdk1 activity is able to promote efficient disassembly of both NPCs and ALPCs.
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; Bialojan and Takai, 1988). Reassembly of NPCs and ALPCs was prevented in OA-injected Cdk1ts mutant embryos incubated at 22°C (Figure 8A and Supplementary Video 9) as well as in OA-injected wild type embryos (unpublished data). To exclude the possibility that OA prevents reassembly of the pore complexes by affecting Cdk1 activity, we pretreated Cdk1ts embryos with OA for 15 min at 22°C to allow complete disassembly and then inactivated Cdk1 at 30°C. This did not induce reassembly of ALPCs and NPCs (Figure 8B and Supplementary Video 10), demonstrating that reassembly of NPCs and ALPCs requires OA-sensitive protein phosphatase activity. If the OA-sensitive protein phosphatases continuously drive reassembly of pore complexes during mitosis, one would expect OA treatment to induce disassembly of pore complexes when limited Cdk1 activity is normally insufficient to drive this process. This was indeed the case in png mutant embryos (which are deficient in Cdk1 activity compared with normal embryos) where microinjection of OA immediately induced massive disassembly of ALPCs (Figure 8C, top row). As expected this effect was suppressed by roscovitine (Figure 8C, bottom row). Taken together our findings suggest a model where the activities of Cdk1 and OA-sensitive phosphatase(s) antagonistically regulate disassembly and reassembly of pore complexes during mitosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). However, complete understanding of this process requires complementary in vivo experimental system(s). Such systems should preferentially be based on one of the available genetic model organisms and should be easy to monitor and manipulate. Developing early Drosophila embryos very well fit these requirements. From an experimental perspective syncytial embryos can be regarded as a sample in a test tube containing a pool of excessive maternally contributed nucleoporins, which rapidly and synchronously oscillate between a disassembled state and a state of assembly in NPC and ALPC structures, respectively. Even during interphase in normal syncytial embryos the nucleoporins are only incompletely assembled (Onischenko et al., 2004), making it possible to affect the system in the direction of increased or decreased assembly by manipulation of factors controlling the assembly and disassembly processes. The extent of pore complex assembly can be directly monitored in live embryos injected with fluorochrome-labeled WGA using time-lapse microscopy or visualized by immunofluorescence microscopy, by EM or quantified using EM-morphometric analysis. Assembly of NPCs and ALPCs in this system was sensitive to classical inhibitors of NE formation and pore complex assembly, such as BAPTA, GTP
S, and NEM (unpublished data). Thus, the syncytial Drosophila embryo model appears to be compatible with other higher eukaryotic models and, as demonstrated in this article, allows design of experiments to "push" the process of NPC disassembly/reassembly in either direction by microinjection of inhibitors or recombinant proteins or by using mutant fly strains.
Cdk1 and OA-sensitive Protein Phosphatases Are Mediators of Mitotic Pore Complex Disassembly/Reassembly
Cdk1 is a key mediator of pore complex disassembly, because selective inactivation of Cdk1 abolished disassembly of NPCs and ALPCs (Figure 4E), whereas activation of Cdk1 induced disassembly of NPCs and ALPCs (Figure 4B). According to our in vitro experiments recombinant Cdk1-cyclin B was able to induce hyperphosphorylation of the Drosophila nucleoporin p150 and dissociation of p150 together with at least four other nucleoporins from the NPCs of the purified nuclei. It is tempting to speculate that Cdk1-mediated phosphorylation of nucleoporins disrupts the interactions holding the structure together. This view is supported by the requirement for dephosphorylation of nucleoporins for assembly of pore complexes in vitro (Walther et al., 2003). Although our data do not prove that Cdk1 phosphorylates nucleoporins directly, this view is consistent with the recently reported localization of Cdk1-cyclin B complex at the ALPCs just before the NE breakdown in Xenopus oocytes (Beckhelling et al., 2003). Furthermore, in the filamentous fungus Aspergillus nidulans, which undergo closed mitosis, phosphorylation of a subset of nucleoporins was also shown to correlate with dissociation from the NPC, whereas the NPC remained functional (De Souza et al., 2004). In this case phosphorylation of nucleoporins is carried out by the downstream serine/threonine kinase NIMA, which is activated by Cdk1. Although overexpression of NIMA apparently can induce NPC disassembly in mammalian cells (Lu and Hunter, 1995), attempts to identify a NIMA homologue in higher eukaryotes have been unsuccessful. It is possible that NIMA has a specialized function in reorganization of pore complexes in organisms undergoing closed mitosis. It should be pointed out that our data do not exclude the possibility that other kinases along with Cdk1 may also be involved in pore complex disassembly.
The reversibility of the Drosophila embryo model also allowed us to investigate the role of protein phosphatases in pore complex reassembly. Microinjection of OA at a concentration that specifically inhibits two major protein phosphatases, PP1 and PP2A, induced rapid disassembly of NPCs and ALPCs, suggesting that either or both of these enzymes are involved in postmitotic reassembly of the pore complexes. The involvement of PP1 and/or PP2A in pore complex reassembly is consistent with a number of evidence of their role in promoting postmitotic changes in the cell. For instance, in cultured cells excess PP1 promoted mitotic exit, whereas microinjection of anti-PP1 antibodies led to mitotic arrest (Fernandez et al., 1992). Additionally, PP1 was implicated in postmitotic reassembly of the nuclear lamina (Thompson et al., 1997; Steen et al., 2000), whereas PP2A was shown to play an important role in postmitotic reassembly of Golgi (Lowe et al., 2000).
Taken together our studies define Cdk1 and OA-sensitive protein phosphatases as the key regulators of pore complex disassembly/reassembly. However this by no means excludes involvement of other factors. Two recent studies performed using the Xenopus in vitro assembly system identified Importin- as a negative regulator of pore complex assembly (Harel et al., 2003; Walther et al., 2003). The latter study also identified Ran-GTP as a positive regulator of this process possibly acting via release of Importin- from the nucleoporin subcomplexes (Walther et al., 2003). Consistent with this our preliminary studies indicated that RanT24N and the nucleoporin-binding domain of importin- both inhibited assembly of NPCs and ALPCs in live syncytial embryos (unpublished results).
Disassembly/Reassembly of Pore Complexes Is Regulated by a Dynamic Equilibrium between the Activities of Cdk1 and OA-sensitive Protein Phosphatases
The oscillation of pore complexes between a disassembled and an assembled state during syncytial nuclear divisions enables determination of dynamic aspects of pore complex disassembly/reassembly. Using this advantage, we were able to demonstrate that: 1) inactivation of Cdk1 during mitosis results in immediate reassembly of NPCs and ALPCs (Figure 5); 2) reassembly of NPCs and ALPCs is blocked by OA, a specific PP1/PP2A inhibitor (Figure 7, A and B); 3) in png mutant embryos, displaying persistently assembled NPCs and ALPCs (due to low endogenous Cdk1 activity, which is insufficient to induce pore complex disassembly), OA treatment induces immediate disassembly of NPCs and ALPCs (Figure 7C). Together these findings suggest a model where disassembly and reassembly of pore complexes during mitosis is regulated by a dynamic equilibrium of the antagonistic actions of Cdk1 and OA-sensitive phosphatases (Figure 9).
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: AL, annulate lamellae; ALPC, annulate lamellae pore complex; NE, nuclear envelope; NPC, nuclear pore complex; OA, okadaic acid; WGA, wheat germ agglutinin.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Einar Hallberg (einar.hallberg{at}sh.se).
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