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Address correspondence to Christos Samakovlis, Dept. of Developmental Biology, Wenner-Gren Institute, Arrhenius Labs E3, Sv. Arrhenius 16-18, Stockholm University, S-10691 Stockholm, Sweden. Tel.: 46-8-161564. Fax: 46-8- 6126127. email: christos{at}devbio.su.se
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Abstract |
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Key Words: NFB; mbo; CAN; NPC; nucleocytoplasmic transport
The online version of this article includes supplemental material.
Abbreviations used in this paper: emb, embargoed; LMB, leptomycin B; mbo, members only; NES, nuclear export signal; NPC, nuclear pore complex.
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Introduction |
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The function of individual nucleoporins is often difficult to study in vivo, as their genetic ablations may result in the rapid accumulation of several indirect transport defects. Drosophila development may provide an advantageous system for the analysis of nuclear pore components. Females often deposit into the egg a sufficient amount of maternal gene product, which supports embryonic development and gradually decreases during larval life, enabling the phenotypic analysis of transport events at different concentrations of the gene product in zygotic null mutants.
Mutants in members only (mbo), encoding the Drosophila nucleoporin DNup88, fail to accumulate the Rel proteins Dif and Dorsal in the nucleus after bacterial infection and also fail to fully activate their immune response (Uv et al., 2000). The nuclear translocation of several other proteins and RNA export are not affected in mbo mutants (Uv et al., 2000). Here, we investigate the mechanism of Nup88 function in nucleocytoplasmic transport.
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Results and discussion |
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If the increased amount of cytoplasmic EGFP-NES observed in mbo mutants results from hyperactivated protein export, then treatment of the mutants with the CRM1 inhibitor leptomycin B (LMB) (Kudo et al., 1998) should revert this phenotype. In salivary glands of wild-type larvae, inducible EGFP-NES expression results in both nuclear and cytoplasmic localization of EGFP-NES (Fig. 1 B). Culture of wild-type glands with 10 nM LMB for 2 h after the induction of EGFP-NES expression did not have any detectable effect on EGFP amount or localization. However, in glands of mbo mutants, the same concentration of the drug was able to revert the decreased nuclear accumulation of the CRM1 cargo (Fig. 1 B). These results indicate that the exclusion of EGFP-NES from the nucleus in mbo mutants is due to an overactivation of the CRM1-mediated export pathway.
Dorsal is exported from the nucleus by CRM1
Is the defect in Rel protein nuclear accumulation and the lack of a fully active immune response in mbo mutants due to the increased levels of protein export? Previous extensive mutagenesis and functional analysis of Dorsal identified a short leucine-rich segment in the COOH end of the protein required for its cytoplasmic retention (Rushlow et al., 1989; Isoda et al., 1992; Bergmann et al., 1996). This segment is outside the Cactus-IBbinding region of the protein and is strikingly similar to the CRM1-binding motif, suggesting that Dorsal nuclear concentration may be controlled by DCRM1. CRM1-mediated protein export has also been implicated in the control of Rel protein localization in mammalian cells (Carlotti et al., 2000). First, we tested whether the localization of the Drosophila Dorsal protein is sensitive to the CRM1 inhibitor LMB. Drosophila S2 cell lines expressing Dorsal fused to EGFP or an EGFP protein carrying the PKI export signal were generated, and both fusion proteins were found in the nucleus and the cytoplasm under normal culture conditions. LMB treatment of the cells resulted in stronger nuclear accumulation of both reporters, suggesting that their cytoplasmic localization requires CRM1 for export (Fig. 2 A).
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DNup88 anchors DNup214 at the nuclear envelope
How, then, does DNup88 affect the levels of CRM1-mediated nuclear export? Human Nup88 is localized at the cytoplasmic side of the NPC (Cronshaw et al., 2002), where it binds to another nucleoporin, CAN/Nup214 (Fornerod et al., 1997b). The two nucleoporins are in complex with CRM1 because anti-Nup214 antibodies can coprecipitate Nup88 and CRM1 (Fornerod et al., 1997b). First, we tested whether the Drosophila Nup214 homologue binds to Nup88 directly by GST pull-down experiments and the yeast two-hybrid system (Belgareh et al., 1998). As expected from previous experiments in other systems, the two proteins were found to bind directly to each other in both assays (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200304046/DC1).
To assess the fate of Nup214 in mbo larvae, we generated a polyclonal antiserum against an NH2-terminal fragment of the protein. On Western blot of Schneider cell extracts, the antiserum recognized a band of 210 kD, whose intensity became severely reduced after treatment of the cells with double-stranded RNA deriving from the DNup214 cDNA (Fig. 3 C). Staining of wild-type larvae with the anti-DNup214 antiserum and analysis by confocal microscopy showed that DNup214 is localized at the nuclear rim (Fig. 3 A). This localization was disturbed in mbo mutants, DNup214 was found predominantly inside the nucleus, and later when the mutants were arrested at a prolonged 3rd instar stage (Uv et al., 2000), the signal became weaker, suggesting that DNup214 became degraded. The mislocalization of DNup214 in mbo mutants does not reflect a general breakdown in NPC structure because overexpression of DNup88 in mbo mutants restored the localization of DNup214 at the nuclear envelope, and analysis of mbo mutants with an antibody recognizing several phenylalanine-glycine repeatcontaining nucleoporins (mAb414) or a serum raised against the nuclear basket component Nup153 did not reveal any alterations in the amount or distribution of these proteins (Fig. 3 B). These results indicate that DNup88 binds to DNup214 and is selectively required for its localization at the nuclear envelope.
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The varying levels of DNup88 in different cell types during Drosophila development and mbo mutants show several cell typespecific developmental defects and fail to mount an effective immune response (Uv et al., 2000). The nuclear concentration of the EGFP-NES also shows a consistent spatial and temporal variation during larval development. Western blot analysis of the amounts of Nup88 in the gut of 2nd and 3rd instar larvae revealed a correlation between the amount of Nup88 and the effectiveness of EGFP-NES export during development (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200304046/DC1). We propose that DNup88 functions at the NPC to tether CRM1 and to attenuate its recycling back to the nucleus for another round of export. However, overexpression of Nup88 in wild-type larvae did not generate any obvious phenotype, suggesting that its CRM1-binding ability may be modulated by another factor or by posttranslational modification (Uv et al., 2000). To assess the proposed inhibitory role of Nup88 in the export of other endogenous CRM1 cargoes, we performed a genetic interaction experiment using mbo and the two emb alleles. emb larvae die at the 2nd instar stage (70 h after egg laying). Removal of one copy of the mbo gene from emb homozygous mutants prolonged the life span of 20% of the individuals to the 3rd instar larval stage (
120 h after egg laying; Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200304046/DC1). This prolonged survival is accompanied by eversion of the anterior spiracles, a morphological characteristic of progression through larval development, arguing that DNup88 is a general NES export attenuator. The surprising function of DNup88 in anchoring CRM1 at the nuclear envelope and down-regulating the levels of NES protein export suggests an additional level of control in the activation and duration of cellular responses to signaling.
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Materials and methods |
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Cell lines
S2 cells were transfected as described previously (Ausubel et al., 1993). Stable transformant lines were selected for 45 wk in supplemented cell medium containing 300 µg/ml hygromycin B (Invitrogen).
LMB treatment
Dorsal-EGFP and EGFP-NES expression was induced by addition of 0.3 mM CuSO4 to the culture medium for 13 h. LMB (Sigma-Aldrich) was used at a final concentration of 10 nM for 2 h. Salivary glands were dissected and incubated at RT in Ringer's solution with or without 10 nM LMB. After 2 h, the glands were fixed and stained.
Immunostaining of larvae and Western blots
Larvae were heat induced for 45 min at 37°C and analyzed after 3 h. Antibody stainings of larval tissues were performed as described previously (Patel, 1994). Dorsal translocation experiments were done as described previously (Uv et al., 2000). Primary antibodies were used at dilutions as follows: anti-DCRM1 (directed against aa 1306 of DCRM1) 1:1,000; anti-DNup214 (directed against aa 160620 of DNup214) 1:10,000; anti-NXF1 (Herold et al., 2001) 1:500; anti-Nup153 (Cordes et al., 1993) 1:300; anti-mAb414 (BabCO) 1:5,000; anti-GFP (Molecular Probes, Inc.) 1:1,000; anti-Dorsal (Gillespie and Wasserman, 1994) 1:1,000; anti-Lamin Dm1 (ADL 84; Stuurman et al., 1995) 1:500; anti-Ketel (Lippai et al., 2000) 1:1,000; and anti-DRanGAP (Merrill et al., 1999) 1:1,000. Stainings were viewed using a fluorescent microscope (Carl Zeiss MicroImaging, Inc.). Openlab v3.1.4 software (Improvision) was used for image acquisition. Laser-scanning microscopes (from Leica or Carl Zeiss MicroImaging, Inc.) were used for confocal imaging. Acquired images were processed with LSM510 software (Carl Zeiss MicroImaging, Inc.). For Western blots, antibody dilutions were used as follows: anti-DNup214 1:1,000; anti-ß-tubulin (Amersham Biosciences) 1:1,000; and anti-Hsp70 (Sigma-Aldrich) 1:1,000.
Binding assays
All constructs were generated by PCR amplification from cDNA clones. For the pull-down assay, fragments were cloned either in pGEX-5X or in pRSET vectors. Protein expression and binding reactions were performed as described previously (Uv et al., 1994). A protocol is available online at http://www.jcb.org/cgi/content/full/jcb.200304046/DC1.
Online supplemental material
Supplemental information contains a Materials and methods section and corresponding references, figures, and a table. Fig. S1 shows quantitation of the ratio of nuclear/cytoplasmic NLS-EGFP and EGFP-NES in wild-type and mbo mutants. Western blot illustrates the amounts of DNup88 in different wild-type larval stages. Fig. S2 shows DNup88DNup214 binding in an in vitro pull-down assay and the yeast two-hybrid system. Table S1 describes genetic interaction of emb and mbo mutants. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200304046/DC1.
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Acknowledgments |
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This work was supported by grants from the Swedish Research Council, Cancerfonden, and Wallenberg Consortium North to C. Samakovlis.
Submitted: 9 April 2003
Accepted: 22 September 2003
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