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First published online 10 October 2006
doi: 10.1242/jcs.03201

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The nucleoporin Nup214 sequesters CRM1 at the nuclear rim and modulates NFB activation in Drosophila

Nikos Xylourgidis^*, Peggy Roth^*, Nafiseh Sabri, Vasilios Tsarouhas and Christos Samakovlis

Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, S-10691, Stockholm, Sweden

View larger version (40K): [in a new window]   Fig. 1. Drosophila Nup214 is in complex with Nup88 and CRM1. The amounts of CRM1 bound to the Nup214-Nup88 complex vary during fly development. Nup214 antiserum was used to coimmunoprecipitate protein complexes of 0-5, 5-10 and 10-15 hour wild-type embryo extracts. Extracts, unbound fractions and precipitates (IP) were analyzed by labeling the western blots with Nup214, CRM1 and Nup88 antibodies. Lamin serves as a loading control. Total amounts of Nup214, Nup88 and CRM1 appear unchanged in the extracts from different embryonic stages. The amount of co-immunoprecipitated CRM1 relative to Nup214 does not change in 0-5 hour and 5-10 hour embryos whereas three times less CRM1 is brought down in 10-15 hour embryos.

View larger version (61K): [in a new window]   Fig. 2. Nup88 is degraded in nup214 mutants. (A) Nup214, mAb414, lamin and Nup88 localization (red) in fat body cells of wild-type and nup214 mutant larvae. Nuclei are visualized by DAPI in the adjacent panel. Confocal sections are shown in the insets. Bars, 35 µm. (B) Western blot of protein extract from wild-type, heterozygotes and nup214 mutant larvae probed with Nup88 antibody. ß-tubulin provides a loading control. Nup88 is reduced in heterozygote animals and totally abolished in the homozygous mutant. (C) RT-PCR for mbo on poly(A)+-purified larval extracts of wild-type (lanes 1-4), P-element (l(2)10444) (lanes 5-8) and nup214 mutants (lanes 9-12). (Fourfold serial dilutions of mRNA from each genotype were used in the RT-PCR step.) rp49 provides a quantitative control. The mRNA levels of mbo remain the same in wild-type and mutant larvae.

View larger version (63K): [in a new window]   Fig. 3. Nup214 and Nup88 are interdependent. (A) Nup88 degradation in nup214 RNAi cells is prevented by epoxomicin. Drosophila S2 cells were transfected with dsRNA against nup214. On day 3 post transfection, cells were treated with epoxomicin (or DMSO) for 0, 8 or 16 hours. Cell lysates were analyzed by western blot with Nup214 or Nup88 antibodies. ß-tubulin served as a loading control. The arrow indicates the Nup88-specific band. The graph shows the levels of Nup88 (grey) and Nup214 (black) normalized against ß-tubulin. The western blot to the right represents a control for the specificity of the Nup88 antibody. Drosophila S2 cells were exposed for 4 days to nup88 RNAi. The Nup88-specific band (arrow) is absent in the RNAi cells. (B) Nup88 anchors Nup214 at the nuclear rim. Either Nup214 or Nup88 was expressed by the hsp70 promoter in wild-type, mbo or nup214 mutant larvae. The images show confocal sections of malpighian tube nuclei stained with anti-Nup214 and anti-Nup88. Bars, 5 µm.

View larger version (60K): [in a new window]   Fig. 4. Drosophila Nup214 attenuates CRM1-mediated protein export. (A) GFP-NES was expressed under the control of the hsp70 promoter in wild-type, emb3 and nup214 mutant larvae and detected with a GFP antibody (red). All panels show larval gut cells. Nuclei were visualized by DAPI staining (middle panels). Bar, 35 µm. (B) Nuclear export rates of GFP-NES are enhanced in nup214 mutants. The ratios of nuclear:cytoplasmic GFP-NES intensities of early second (eL2) and early third (eL3) instar wild-type, nup214 and emb3 larval gut cells are shown in a log2 graph. The nuclear accumulation of the GFP-NES reporter is decreased 30% in nup214 mutants, whereas in emb3 mutants the nuclear accumulation is increased by 40% (P<0.0001 by pair-wise t-test). Error bars represent s.e.m. (C) Confocal sections of fat body cells from wild-type and nup214 mutant larvae stained with anti-CRM1, anti-lamin and anti-RanGAP antibodies. Error bars, 5 µm. (D) Western blot of protein extract from wild-type and nup214 mutant larvae probed with anti-CRM1. ß-tubulin provides a loading control. (E) Quantification of RanGAP levels along the nuclear rim in wild-type and nup214 mutants. The nuclear rim staining in the mutants is reduced by 35%.

View larger version (13K): [in a new window]   Fig. 5. Prolonged development of emb2 mutants lacking one copy of nup214. Bars represent survival (days) of the indicated genotypes. All values are significantly different (P<0.0001; pair-wise t-test) when compared with survival of the emb2/emb2 mutants. Percentage values within bars represent the number of survivors through the developmental stages. A, adult stage; L1, first instar larvae; L2, second instar larvae; L3, third instar larvae; P, pupal stage.

View larger version (48K): [in a new window]   Fig. 6. Co-expression of Nup214 and Nup88 in the wild type causes formation of cytoplasmic foci and mislocalizes CRM1. (A) Nup214 and Nup88 (HS214+HS88) were expressed with the hsp70 promoter in wild-type third-instar larvae. Heat-treated wild type (wt) served as a control. All panels show confocal sections of malphigian tube nuclei stained either with Nup88 and Nup214 or with Nup214 and CRM1 antisera. Bar, 5 µm. (B) Overexpression of both nucleoporins affects the localization of GFP-NES. GFP-NES was expressed together with Nup88, Nup214 or Nup214 and Nup88 by heat shock induction of third-instar larvae. The ratios of nuclear:cytoplasmic GFP-NES intensities are illustrated in a log2 graph. Nuclear accumulation of the GFP-NES is increased in cells co-expressing both Nup214 and Nup88 (P<0.0001 by pair-wise t-test). Error bars represent s.e.m.

View larger version (73K): [in a new window]   Fig. 7. The nuclear accumulation of Dorsal and Dif is impaired in nup214 mutants. All panels show fat bodies of wild-type (wt) and nup214 mutant larvae, before (not challenged) and after bacterial infection (bact. challenged) stained with specific antibodies for Dorsal, Dif, ß-galactosidase for cecropin-lacZ or GFP for drosomycin-GFP expression. Nuclei are visualized with DAPI (blue). Bars, 35 µm.

View larger version (89K): [in a new window]   Fig. 8. Co-expression of Nup214 and Nup88 in the wild type is sufficient to mislocalize endogenous targets. Nup214 and Nup88 (HS214+HS88) expression was driven by the hsp70 promoter in wild-type third instar larvae. Heat-treated wild-type (wt) served as a control. (A) Overexpression of Nup214 and Nup88 interferes with the expression of a drosomycin-GFP (dromGFP) reporter. All panels show fat bodies, before (not challenged) and after bacterial infection (bact. challenged) stained with GFP antibody. The images for HS214+HS88 unchallenged and infected larval fat bodies were acquired at three times lower intensity for Nup214 labeling compared with the wild type. (B) Confocal sections of malpighian tube nuclei stained with Nup88, Nup214, Dorsal, CRM1 or Grainyhead antiserum. Co-expression of Nup214 and Nup88 can mislocalize Dorsal but not Grainyhead. Bars, 35 µm (A); 5 µm (B).