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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-deleted cells undergo delayed mitosis due to activation of the spindle assembly checkpoint. (A) Time course images of GFP-tubulin to follow mitotic spindle formation in germlings with four nuclei during G2-M-G1. ∆nup2 (from heterokaryon SM96) and ∆nupA (from heterokaryon SM98) cells display a prolonged mitosis only when the SAC is functional. Bar, ∼5 μm. (B) Bar graph representing average time spent in mitosis with mitotic spindles present for each indicated genotype. The number on each bar represents the average time in seconds. ∆nup2 and ∆nupA cells spend significantly longer time in mitosis than wild type (strain HA375) (with p < 0.01, highly significant when comparing the means). Δmad2 + ∆nup2 (from heterokaryon SM129) and Δmad2 + ∆nupA (from heterokaryon SM131) mutants show no statistical difference compared with the wild type or Δmad2 mutant (strain CDS864). (C) Time course images of wild-type and nupA- and nup2- cells during first mitosis following Mad1-GFP in combination with the kinetochore marker Ndc80-ChRFP in strain CDS831 and spores from heterokaryon SM154 and SM152, respectively. In the first mitosis of ∆nupA and ∆nup2 nuclei, Mad1-GFP remains at kinetochores during the delayed mitosis. Bar, ∼5 μm.
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Figure 6: nup2- and nupA-deleted cells undergo delayed mitosis due to activation of the spindle assembly checkpoint. (A) Time course images of GFP-tubulin to follow mitotic spindle formation in germlings with four nuclei during G2-M-G1. ∆nup2 (from heterokaryon SM96) and ∆nupA (from heterokaryon SM98) cells display a prolonged mitosis only when the SAC is functional. Bar, ∼5 μm. (B) Bar graph representing average time spent in mitosis with mitotic spindles present for each indicated genotype. The number on each bar represents the average time in seconds. ∆nup2 and ∆nupA cells spend significantly longer time in mitosis than wild type (strain HA375) (with p < 0.01, highly significant when comparing the means). Δmad2 + ∆nup2 (from heterokaryon SM129) and Δmad2 + ∆nupA (from heterokaryon SM131) mutants show no statistical difference compared with the wild type or Δmad2 mutant (strain CDS864). (C) Time course images of wild-type and nupA- and nup2- cells during first mitosis following Mad1-GFP in combination with the kinetochore marker Ndc80-ChRFP in strain CDS831 and spores from heterokaryon SM154 and SM152, respectively. In the first mitosis of ∆nupA and ∆nup2 nuclei, Mad1-GFP remains at kinetochores during the delayed mitosis. Bar, ∼5 μm.

Mentions: To investigate mitotic progression without Nup2 or NupA, we used GFP-tagged tubulin to monitor the dynamics of spindle formation (Ovechkina et al., 2003). We found that the time to complete mitosis was longer and overall more variable in nup2- and nupA-deleted cells, taking 10–11 min on average to complete versus 5.5 min for wild-type cells (Figure 6, A and B, and Supplemental Figure S3). To see whether the mitotic defects caused by the absence of Nup2 and NupA are monitored by the SAC, we asked whether the mitotic delays in these mutants are dependent on a functional SAC response (Li and Murray, 1991; Musacchio and Salmon, 2007). We found that in the absence of Mad2, the mitotic delays caused by nup2 or nupA deletion were abolished, and the average time of mitosis was similar to that of wild-type controls, with the variability in the duration of mitosis no longer apparent (Figure 6, A and B, and Supplemental Figure S3). We also followed Mad1-GFP in nup2- and nupA- mutants to monitor SAC activation, which causes the transition of Mad1-GFP from NPCs to mitotic kinetochores. As occurs in the wild type, in the mutant Mad1-GFP translocates to kinetochores during the first mitosis during spore germination. Mad1-GFP then stays associated with the kinetochore region, as defined by Ndc80-ChRFP, during the extended mitotic period (Figure 6C). Absence of Nup2 or NupA therefore causes defects in mitosis, which are monitored by the SAC.


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-deleted cells undergo delayed mitosis due to activation of the spindle assembly checkpoint. (A) Time course images of GFP-tubulin to follow mitotic spindle formation in germlings with four nuclei during G2-M-G1. ∆nup2 (from heterokaryon SM96) and ∆nupA (from heterokaryon SM98) cells display a prolonged mitosis only when the SAC is functional. Bar, ∼5 μm. (B) Bar graph representing average time spent in mitosis with mitotic spindles present for each indicated genotype. The number on each bar represents the average time in seconds. ∆nup2 and ∆nupA cells spend significantly longer time in mitosis than wild type (strain HA375) (with p < 0.01, highly significant when comparing the means). Δmad2 + ∆nup2 (from heterokaryon SM129) and Δmad2 + ∆nupA (from heterokaryon SM131) mutants show no statistical difference compared with the wild type or Δmad2 mutant (strain CDS864). (C) Time course images of wild-type and nupA- and nup2- cells during first mitosis following Mad1-GFP in combination with the kinetochore marker Ndc80-ChRFP in strain CDS831 and spores from heterokaryon SM154 and SM152, respectively. In the first mitosis of ∆nupA and ∆nup2 nuclei, Mad1-GFP remains at kinetochores during the delayed mitosis. Bar, ∼5 μm.
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Figure 6: nup2- and nupA-deleted cells undergo delayed mitosis due to activation of the spindle assembly checkpoint. (A) Time course images of GFP-tubulin to follow mitotic spindle formation in germlings with four nuclei during G2-M-G1. ∆nup2 (from heterokaryon SM96) and ∆nupA (from heterokaryon SM98) cells display a prolonged mitosis only when the SAC is functional. Bar, ∼5 μm. (B) Bar graph representing average time spent in mitosis with mitotic spindles present for each indicated genotype. The number on each bar represents the average time in seconds. ∆nup2 and ∆nupA cells spend significantly longer time in mitosis than wild type (strain HA375) (with p < 0.01, highly significant when comparing the means). Δmad2 + ∆nup2 (from heterokaryon SM129) and Δmad2 + ∆nupA (from heterokaryon SM131) mutants show no statistical difference compared with the wild type or Δmad2 mutant (strain CDS864). (C) Time course images of wild-type and nupA- and nup2- cells during first mitosis following Mad1-GFP in combination with the kinetochore marker Ndc80-ChRFP in strain CDS831 and spores from heterokaryon SM154 and SM152, respectively. In the first mitosis of ∆nupA and ∆nup2 nuclei, Mad1-GFP remains at kinetochores during the delayed mitosis. Bar, ∼5 μm.
Mentions: To investigate mitotic progression without Nup2 or NupA, we used GFP-tagged tubulin to monitor the dynamics of spindle formation (Ovechkina et al., 2003). We found that the time to complete mitosis was longer and overall more variable in nup2- and nupA-deleted cells, taking 10–11 min on average to complete versus 5.5 min for wild-type cells (Figure 6, A and B, and Supplemental Figure S3). To see whether the mitotic defects caused by the absence of Nup2 and NupA are monitored by the SAC, we asked whether the mitotic delays in these mutants are dependent on a functional SAC response (Li and Murray, 1991; Musacchio and Salmon, 2007). We found that in the absence of Mad2, the mitotic delays caused by nup2 or nupA deletion were abolished, and the average time of mitosis was similar to that of wild-type controls, with the variability in the duration of mitosis no longer apparent (Figure 6, A and B, and Supplemental Figure S3). We also followed Mad1-GFP in nup2- and nupA- mutants to monitor SAC activation, which causes the transition of Mad1-GFP from NPCs to mitotic kinetochores. As occurs in the wild type, in the mutant Mad1-GFP translocates to kinetochores during the first mitosis during spore germination. Mad1-GFP then stays associated with the kinetochore region, as defined by Ndc80-ChRFP, during the extended mitotic period (Figure 6C). Absence of Nup2 or NupA therefore causes defects in mitosis, which are monitored by the SAC.

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