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Nup358 integrates nuclear envelope breakdown with kinetochore assembly.

Salina D, Enarson P, Rattner JB, Burke B - J. Cell Biol. (2003)

Bottom Line: After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation.The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function.Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610-0235, USA.

ABSTRACT
Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.

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Nuclei in cells depleted of Nup358 are import competent. Nuclear import of GRβ is unaffected in Nup358 siRNA–treated (RNAi) versus mock-treated cells. In the absence of dexamethasone (−Dex), GRβ remains almost exclusively cytoplasmic (A). After exposure to dexamethasone for 10 min (+Dex), GRβ relocates to the nucleus (or micronuclei). To establish a time course of GRβ import (B), cells were scored for nuclear accumulation of GRβ. This was defined as average nuclear fluorescence intensity greater than average cytoplasmic intensity. The graph (B) represents the mean of four experiments in which 200 cells were scored at each time point. For the RNAi samples, only cells clearly depleted of Nup358 were included in the counts. The overall distribution of the nuclear import receptor importin-β is also largely unaffected by depletion of Nup358 (C). Compare the large multinucleate cell in the RNAi panels with the mock-treated cells. Bars, 10 μm.
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fig4: Nuclei in cells depleted of Nup358 are import competent. Nuclear import of GRβ is unaffected in Nup358 siRNA–treated (RNAi) versus mock-treated cells. In the absence of dexamethasone (−Dex), GRβ remains almost exclusively cytoplasmic (A). After exposure to dexamethasone for 10 min (+Dex), GRβ relocates to the nucleus (or micronuclei). To establish a time course of GRβ import (B), cells were scored for nuclear accumulation of GRβ. This was defined as average nuclear fluorescence intensity greater than average cytoplasmic intensity. The graph (B) represents the mean of four experiments in which 200 cells were scored at each time point. For the RNAi samples, only cells clearly depleted of Nup358 were included in the counts. The overall distribution of the nuclear import receptor importin-β is also largely unaffected by depletion of Nup358 (C). Compare the large multinucleate cell in the RNAi panels with the mock-treated cells. Bars, 10 μm.

Mentions: Nup358-depleted nuclei, including micronuclei, retained the capacity to import both A-type lamins (Fig. 3 D) and an inducible (by dexamethasone) nuclear transport substrate, a glucocorticoid receptor–β-galactosidase fusion protein (GRβ; Fig. 4, A and B). In the case of the latter, import was followed over time in both mock-treated cells and in cells depleted of Nup358. For this experiment, cells were double labeled with antibodies against both Nup358 and β-galactosidase. In this way, cells specifically depleted of Nup358 could be scored for nuclear accumulation of GRβ. As shown in Fig. 4 B, Nup358 depletion had little or no effect on the nuclear uptake of GRβ. In addition, there were no obvious changes in the gross distribution of the import receptor importin/karyopherin-β (Fig. 4 C) in mock-treated versus Nup358-depleted multinucleate cells. These observations are consistent with recent findings that nuclei assembled in vitro in the absence of Nup358 contain functional NPCs and are import competent (Walther et al., 2002).


Nup358 integrates nuclear envelope breakdown with kinetochore assembly.

Salina D, Enarson P, Rattner JB, Burke B - J. Cell Biol. (2003)

Nuclei in cells depleted of Nup358 are import competent. Nuclear import of GRβ is unaffected in Nup358 siRNA–treated (RNAi) versus mock-treated cells. In the absence of dexamethasone (−Dex), GRβ remains almost exclusively cytoplasmic (A). After exposure to dexamethasone for 10 min (+Dex), GRβ relocates to the nucleus (or micronuclei). To establish a time course of GRβ import (B), cells were scored for nuclear accumulation of GRβ. This was defined as average nuclear fluorescence intensity greater than average cytoplasmic intensity. The graph (B) represents the mean of four experiments in which 200 cells were scored at each time point. For the RNAi samples, only cells clearly depleted of Nup358 were included in the counts. The overall distribution of the nuclear import receptor importin-β is also largely unaffected by depletion of Nup358 (C). Compare the large multinucleate cell in the RNAi panels with the mock-treated cells. Bars, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172838&req=5

fig4: Nuclei in cells depleted of Nup358 are import competent. Nuclear import of GRβ is unaffected in Nup358 siRNA–treated (RNAi) versus mock-treated cells. In the absence of dexamethasone (−Dex), GRβ remains almost exclusively cytoplasmic (A). After exposure to dexamethasone for 10 min (+Dex), GRβ relocates to the nucleus (or micronuclei). To establish a time course of GRβ import (B), cells were scored for nuclear accumulation of GRβ. This was defined as average nuclear fluorescence intensity greater than average cytoplasmic intensity. The graph (B) represents the mean of four experiments in which 200 cells were scored at each time point. For the RNAi samples, only cells clearly depleted of Nup358 were included in the counts. The overall distribution of the nuclear import receptor importin-β is also largely unaffected by depletion of Nup358 (C). Compare the large multinucleate cell in the RNAi panels with the mock-treated cells. Bars, 10 μm.
Mentions: Nup358-depleted nuclei, including micronuclei, retained the capacity to import both A-type lamins (Fig. 3 D) and an inducible (by dexamethasone) nuclear transport substrate, a glucocorticoid receptor–β-galactosidase fusion protein (GRβ; Fig. 4, A and B). In the case of the latter, import was followed over time in both mock-treated cells and in cells depleted of Nup358. For this experiment, cells were double labeled with antibodies against both Nup358 and β-galactosidase. In this way, cells specifically depleted of Nup358 could be scored for nuclear accumulation of GRβ. As shown in Fig. 4 B, Nup358 depletion had little or no effect on the nuclear uptake of GRβ. In addition, there were no obvious changes in the gross distribution of the import receptor importin/karyopherin-β (Fig. 4 C) in mock-treated versus Nup358-depleted multinucleate cells. These observations are consistent with recent findings that nuclei assembled in vitro in the absence of Nup358 contain functional NPCs and are import competent (Walther et al., 2002).

Bottom Line: After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation.The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function.Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610-0235, USA.

ABSTRACT
Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.

Show MeSH
Related in: MedlinePlus