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Nup2 requires a highly divergent partner, NupA, to fulfill functions at nuclear pore complexes and the mitotic chromatin region.

Markossian S, Suresh S, Osmani AH, Osmani SA - Mol. Biol. Cell (2014)

Bottom Line: These mitotic problems are not caused by overall defects in mitotic NPC disassembly-reassembly or general nuclear import.However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis.Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Gene Regulation and Development, National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.

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Nup2 and NupA are dispensable for nuclear NLS-DsRed nuclear import. (A) NLS-dsRed is actively imported to the nucleoplasm in Δnup2 and ΔnupA nuclei. Bright-field and confocal images of ΔnupA or Δnup2 germinated spores from heterokaryons SM117 and SM118, respectively, and wild-type spores grown on selective media. Bar, ∼5 μm. (B) Time course images of NLS-DsRed nuclear dispersal and import during mitosis of wild-type (strain SM112), ∆nup2 (from heterokaryon SM118), and ∆nupA (from heterokaryon SM117) cells. Bar, ∼5 μm. (C) Rate of mitotic entry dispersal and mitotic exit import of NLS-DsRed in wild-type, ∆nup2, and ∆nupA cells, showing average normalized nuclear pixel intensity vs. time (N = 10; error bars ± SD). The pixel intensity of the red signal was quantified in a defined area within the nucleus every 10 s and the highest pixel intensity normalized to 100%. Time 0 represents the time at which 50% loss during mitotic entry, or gain during mitotic exit, of the normalized fluorescence intensity of the NLS-DsRed signal occurred.
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Figure 8: Nup2 and NupA are dispensable for nuclear NLS-DsRed nuclear import. (A) NLS-dsRed is actively imported to the nucleoplasm in Δnup2 and ΔnupA nuclei. Bright-field and confocal images of ΔnupA or Δnup2 germinated spores from heterokaryons SM117 and SM118, respectively, and wild-type spores grown on selective media. Bar, ∼5 μm. (B) Time course images of NLS-DsRed nuclear dispersal and import during mitosis of wild-type (strain SM112), ∆nup2 (from heterokaryon SM118), and ∆nupA (from heterokaryon SM117) cells. Bar, ∼5 μm. (C) Rate of mitotic entry dispersal and mitotic exit import of NLS-DsRed in wild-type, ∆nup2, and ∆nupA cells, showing average normalized nuclear pixel intensity vs. time (N = 10; error bars ± SD). The pixel intensity of the red signal was quantified in a defined area within the nucleus every 10 s and the highest pixel intensity normalized to 100%. Time 0 represents the time at which 50% loss during mitotic entry, or gain during mitotic exit, of the normalized fluorescence intensity of the NLS-DsRed signal occurred.

Mentions: Nup2 and its mammalian orthologue Nup50 facilitate importin-α/β–mediated nuclear transport (Solsbacher et al., 2000; Gilchrist et al., 2002; Lindsay et al., 2002; Matsuura and Stewart, 2005; Stewart, 2007; Makise et al., 2012). Consistent with A. nidulans Nup2 playing a similar role, it purified with importins α and β (Figure 1C). To investigate their roles in nuclear transport, we monitored NLS-DsRed to detect whether Δnup2 and ΔnupA mutant nuclei are defective in nuclear protein import. NLS-DsRed was transported into Δnup2 and ΔnupA nuclei during interphase, with little to no cytoplasmic signal apparent (Figure 8, A and B, 0′). This indicates that nuclear transport is active in the absence of Nup2 or NupA. To determine whether the rate of NLS-DsRed nuclear transport is modified without Nup2 and NupA, we followed the rate of dispersal of NLS-DsRed during mitotic entry and then its rate of import during mitotic exit. As cells entered mitosis, NLS-DsRed dispersed from nuclei and was rapidly transported back into daughter G1 nuclei during mitotic exit in wild-type as well as Δnup2 and ΔnupA nuclei (Figure 8B). Note that NLS-DsRed remained mitotically dispersed longer in the mutants than the wild type because they have prolonged SAC-mediated delayed mitosis (Figure 6, A and B, and Supplemental Figure S3). We quantified the fluorescence pixel intensity of the nuclear NLS-DsRed signal at 10-s intervals in comparison to wild type (Figure 8C). Dispersal and import rates of NLS-DsRed in the mutants were comparable to wild type, with the kinetics of both being essentially superimposable between the mutants and wild type. This suggests there are no overall defects in nuclear protein import in the absence of Nup2 or NupA. In addition, because the disassembly and reassembly of NPCs drive release and reimport of NLS-DsRed, respectively, the results further indicate that the rates of mitotic NPC disassembly and reassembly are not affected by the absence of Nup2 or NupA.


Nup2 requires a highly divergent partner, NupA, to fulfill functions at nuclear pore complexes and the mitotic chromatin region.

Markossian S, Suresh S, Osmani AH, Osmani SA - Mol. Biol. Cell (2014)

Nup2 and NupA are dispensable for nuclear NLS-DsRed nuclear import. (A) NLS-dsRed is actively imported to the nucleoplasm in Δnup2 and ΔnupA nuclei. Bright-field and confocal images of ΔnupA or Δnup2 germinated spores from heterokaryons SM117 and SM118, respectively, and wild-type spores grown on selective media. Bar, ∼5 μm. (B) Time course images of NLS-DsRed nuclear dispersal and import during mitosis of wild-type (strain SM112), ∆nup2 (from heterokaryon SM118), and ∆nupA (from heterokaryon SM117) cells. Bar, ∼5 μm. (C) Rate of mitotic entry dispersal and mitotic exit import of NLS-DsRed in wild-type, ∆nup2, and ∆nupA cells, showing average normalized nuclear pixel intensity vs. time (N = 10; error bars ± SD). The pixel intensity of the red signal was quantified in a defined area within the nucleus every 10 s and the highest pixel intensity normalized to 100%. Time 0 represents the time at which 50% loss during mitotic entry, or gain during mitotic exit, of the normalized fluorescence intensity of the NLS-DsRed signal occurred.
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Figure 8: Nup2 and NupA are dispensable for nuclear NLS-DsRed nuclear import. (A) NLS-dsRed is actively imported to the nucleoplasm in Δnup2 and ΔnupA nuclei. Bright-field and confocal images of ΔnupA or Δnup2 germinated spores from heterokaryons SM117 and SM118, respectively, and wild-type spores grown on selective media. Bar, ∼5 μm. (B) Time course images of NLS-DsRed nuclear dispersal and import during mitosis of wild-type (strain SM112), ∆nup2 (from heterokaryon SM118), and ∆nupA (from heterokaryon SM117) cells. Bar, ∼5 μm. (C) Rate of mitotic entry dispersal and mitotic exit import of NLS-DsRed in wild-type, ∆nup2, and ∆nupA cells, showing average normalized nuclear pixel intensity vs. time (N = 10; error bars ± SD). The pixel intensity of the red signal was quantified in a defined area within the nucleus every 10 s and the highest pixel intensity normalized to 100%. Time 0 represents the time at which 50% loss during mitotic entry, or gain during mitotic exit, of the normalized fluorescence intensity of the NLS-DsRed signal occurred.
Mentions: Nup2 and its mammalian orthologue Nup50 facilitate importin-α/β–mediated nuclear transport (Solsbacher et al., 2000; Gilchrist et al., 2002; Lindsay et al., 2002; Matsuura and Stewart, 2005; Stewart, 2007; Makise et al., 2012). Consistent with A. nidulans Nup2 playing a similar role, it purified with importins α and β (Figure 1C). To investigate their roles in nuclear transport, we monitored NLS-DsRed to detect whether Δnup2 and ΔnupA mutant nuclei are defective in nuclear protein import. NLS-DsRed was transported into Δnup2 and ΔnupA nuclei during interphase, with little to no cytoplasmic signal apparent (Figure 8, A and B, 0′). This indicates that nuclear transport is active in the absence of Nup2 or NupA. To determine whether the rate of NLS-DsRed nuclear transport is modified without Nup2 and NupA, we followed the rate of dispersal of NLS-DsRed during mitotic entry and then its rate of import during mitotic exit. As cells entered mitosis, NLS-DsRed dispersed from nuclei and was rapidly transported back into daughter G1 nuclei during mitotic exit in wild-type as well as Δnup2 and ΔnupA nuclei (Figure 8B). Note that NLS-DsRed remained mitotically dispersed longer in the mutants than the wild type because they have prolonged SAC-mediated delayed mitosis (Figure 6, A and B, and Supplemental Figure S3). We quantified the fluorescence pixel intensity of the nuclear NLS-DsRed signal at 10-s intervals in comparison to wild type (Figure 8C). Dispersal and import rates of NLS-DsRed in the mutants were comparable to wild type, with the kinetics of both being essentially superimposable between the mutants and wild type. This suggests there are no overall defects in nuclear protein import in the absence of Nup2 or NupA. In addition, because the disassembly and reassembly of NPCs drive release and reimport of NLS-DsRed, respectively, the results further indicate that the rates of mitotic NPC disassembly and reassembly are not affected by the absence of Nup2 or NupA.

Bottom Line: These mitotic problems are not caused by overall defects in mitotic NPC disassembly-reassembly or general nuclear import.However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis.Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Gene Regulation and Development, National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.

Show MeSH
Related in: MedlinePlus