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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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Cells depleted of Nup358 contain identifiable NPCs. Indirect immunofluorescence microscopy reveals little or no change in the levels or localizations of multiple nucleoporins, including Nup153 and Nup214, in multinucleate cells depleted of Nup358 (A and B). EM reveals the presence of abundant NPCs associated with micronuclei in cells subjected to Nup358 siRNA treatment (C). Immunofluorescence microscopy reveals that such cells are always depleted of Nup358 (D). Cells depleted of Nup358, including multinucleate cells, continue to incorporate A-type lamins (D) into the nuclear lamina over a period of 96 h. Bars: (A, B, and D) 10 μm; (C) 200 nm.
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fig3: Cells depleted of Nup358 contain identifiable NPCs. Indirect immunofluorescence microscopy reveals little or no change in the levels or localizations of multiple nucleoporins, including Nup153 and Nup214, in multinucleate cells depleted of Nup358 (A and B). EM reveals the presence of abundant NPCs associated with micronuclei in cells subjected to Nup358 siRNA treatment (C). Immunofluorescence microscopy reveals that such cells are always depleted of Nup358 (D). Cells depleted of Nup358, including multinucleate cells, continue to incorporate A-type lamins (D) into the nuclear lamina over a period of 96 h. Bars: (A, B, and D) 10 μm; (C) 200 nm.

Mentions: Loss of Nup358 had little, if any, effect on NPC number, as estimated in immunofluorescence experiments using a variety of antinucleoporin antibodies. Nup153 (Fig. 2 C and Fig. 3 A) and other FG-repeat nucleoporins, including the NPC membrane protein POM121, remained NE associated and retained their usual punctate distribution (unpublished data). Puncta per unit NE surface remained essentially unchanged after siRNA treatment (unpublished data). Distribution of another nucleoporin, Nup214, which resides on the cytoplasmic face of the NPC, appears largely unaffected in cells, including multinucleate cells, depleted of Nup358 (Fig. 2). Similar results were obtained for Nup98, a mobile nucleoporin that, like Nup153, is localized to the nuclear face of the NPC (unpublished data). EM analysis of multinucleate cells from siRNA-treated cultures (such cells are always depleted of Nup358; Fig. 2 C and Fig. 3 D) revealed numerous NPCs (Fig. 3 B). However, the presence or absence of NPC-associated cytoplasmic filaments could not be reliably ascertained in this type of thin section analysis. Taken together, these data indicate that NPCs remain substantially intact after Nup358 depletion in vivo.


Nup358 integrates nuclear envelope breakdown with kinetochore assembly.

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

Cells depleted of Nup358 contain identifiable NPCs. Indirect immunofluorescence microscopy reveals little or no change in the levels or localizations of multiple nucleoporins, including Nup153 and Nup214, in multinucleate cells depleted of Nup358 (A and B). EM reveals the presence of abundant NPCs associated with micronuclei in cells subjected to Nup358 siRNA treatment (C). Immunofluorescence microscopy reveals that such cells are always depleted of Nup358 (D). Cells depleted of Nup358, including multinucleate cells, continue to incorporate A-type lamins (D) into the nuclear lamina over a period of 96 h. Bars: (A, B, and D) 10 μm; (C) 200 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172838&req=5

fig3: Cells depleted of Nup358 contain identifiable NPCs. Indirect immunofluorescence microscopy reveals little or no change in the levels or localizations of multiple nucleoporins, including Nup153 and Nup214, in multinucleate cells depleted of Nup358 (A and B). EM reveals the presence of abundant NPCs associated with micronuclei in cells subjected to Nup358 siRNA treatment (C). Immunofluorescence microscopy reveals that such cells are always depleted of Nup358 (D). Cells depleted of Nup358, including multinucleate cells, continue to incorporate A-type lamins (D) into the nuclear lamina over a period of 96 h. Bars: (A, B, and D) 10 μm; (C) 200 nm.
Mentions: Loss of Nup358 had little, if any, effect on NPC number, as estimated in immunofluorescence experiments using a variety of antinucleoporin antibodies. Nup153 (Fig. 2 C and Fig. 3 A) and other FG-repeat nucleoporins, including the NPC membrane protein POM121, remained NE associated and retained their usual punctate distribution (unpublished data). Puncta per unit NE surface remained essentially unchanged after siRNA treatment (unpublished data). Distribution of another nucleoporin, Nup214, which resides on the cytoplasmic face of the NPC, appears largely unaffected in cells, including multinucleate cells, depleted of Nup358 (Fig. 2). Similar results were obtained for Nup98, a mobile nucleoporin that, like Nup153, is localized to the nuclear face of the NPC (unpublished data). EM analysis of multinucleate cells from siRNA-treated cultures (such cells are always depleted of Nup358; Fig. 2 C and Fig. 3 D) revealed numerous NPCs (Fig. 3 B). However, the presence or absence of NPC-associated cytoplasmic filaments could not be reliably ascertained in this type of thin section analysis. Taken together, these data indicate that NPCs remain substantially intact after Nup358 depletion in vivo.

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