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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 containing multiple micronuclei are depleted of Nup358. A single high molecular weight band is recognized on a Western blot of a HeLa whole cell extract by the anti-Nup358 antibodies that are employed in this study (A). Total protein content of the cell extract is shown in the Coomassie blue–stained lane (gel). Molecular weight markers (MW, kD) are indicated on the left. A 50% reduction in total Nup358 levels in Nup358 siRNA–treated (RNAi) versus control cultures (Mock) is shown by quantitative Western blot analysis (B). For this experiment, twofold dilution series of HeLa cell extracts were analyzed by Western blot using antibodies against Nup358 (A) as well as β-tubulin as an internal standard. Two sample pairs (differing in concentration by a factor of two) from both control and siRNA-treated cultures are shown. Changes in Nup358 levels were determined from the ratios of the Nup358/β-tubulin band intensities. These experiments were performed in quadruplicate over a 16-fold sample concentration range. Immunofluorescence microscopy of Nup358 siRNA–treated cultures reveals a loss of NE-associated Nup358 (C). This is particularly evident in cells containing multiple micronuclei. A second nucleoporin, Nup153, shows no such decline. Fluorescence intensity measurements performed on randomly selected interphase cells (Total) indicate a 66% reduction in Nup358 levels (±5%; P < 0.001) in siRNA-treated versus control (Mock) cells. A larger average reduction in anti-Nup358 fluorescence intensity of 77% (±1%; P < 0.001) is observed when measurements are restricted to cells containing multiple micronuclei (MN). Bar, 10 μm.
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fig2: Cells containing multiple micronuclei are depleted of Nup358. A single high molecular weight band is recognized on a Western blot of a HeLa whole cell extract by the anti-Nup358 antibodies that are employed in this study (A). Total protein content of the cell extract is shown in the Coomassie blue–stained lane (gel). Molecular weight markers (MW, kD) are indicated on the left. A 50% reduction in total Nup358 levels in Nup358 siRNA–treated (RNAi) versus control cultures (Mock) is shown by quantitative Western blot analysis (B). For this experiment, twofold dilution series of HeLa cell extracts were analyzed by Western blot using antibodies against Nup358 (A) as well as β-tubulin as an internal standard. Two sample pairs (differing in concentration by a factor of two) from both control and siRNA-treated cultures are shown. Changes in Nup358 levels were determined from the ratios of the Nup358/β-tubulin band intensities. These experiments were performed in quadruplicate over a 16-fold sample concentration range. Immunofluorescence microscopy of Nup358 siRNA–treated cultures reveals a loss of NE-associated Nup358 (C). This is particularly evident in cells containing multiple micronuclei. A second nucleoporin, Nup153, shows no such decline. Fluorescence intensity measurements performed on randomly selected interphase cells (Total) indicate a 66% reduction in Nup358 levels (±5%; P < 0.001) in siRNA-treated versus control (Mock) cells. A larger average reduction in anti-Nup358 fluorescence intensity of 77% (±1%; P < 0.001) is observed when measurements are restricted to cells containing multiple micronuclei (MN). Bar, 10 μm.

Mentions: Immunoblot analysis of HeLa cultures subjected to Nup358 siRNA treatment for a period of 4 d reveals a decline in the level of Nup358 of ∼50% when compared with mock-treated cells (Fig. 2, A and B). As an independent means of verification of these immunoblot results, we performed fluorescence intensity measurements on cells labeled with affinity-purified anti-Nup358 antibodies. In both siRNA-treated and control cultures, measurements were performed on all cells in multiple randomly selected fields. These measurements revealed an average decline in Nup358-associated fluorescence of ∼60%, a value that is consistent with the immunoblot data (Fig. 2 C). At the single cell level, fluorescence intensity reductions of 80% or more could be observed. Cells containing multiple micronuclei exhibited an average reduction in anti-Nup358 fluorescence intensity of ∼77% when compared with mock-treated cells (Fig. 2 C).


Nup358 integrates nuclear envelope breakdown with kinetochore assembly.

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

Cells containing multiple micronuclei are depleted of Nup358. A single high molecular weight band is recognized on a Western blot of a HeLa whole cell extract by the anti-Nup358 antibodies that are employed in this study (A). Total protein content of the cell extract is shown in the Coomassie blue–stained lane (gel). Molecular weight markers (MW, kD) are indicated on the left. A 50% reduction in total Nup358 levels in Nup358 siRNA–treated (RNAi) versus control cultures (Mock) is shown by quantitative Western blot analysis (B). For this experiment, twofold dilution series of HeLa cell extracts were analyzed by Western blot using antibodies against Nup358 (A) as well as β-tubulin as an internal standard. Two sample pairs (differing in concentration by a factor of two) from both control and siRNA-treated cultures are shown. Changes in Nup358 levels were determined from the ratios of the Nup358/β-tubulin band intensities. These experiments were performed in quadruplicate over a 16-fold sample concentration range. Immunofluorescence microscopy of Nup358 siRNA–treated cultures reveals a loss of NE-associated Nup358 (C). This is particularly evident in cells containing multiple micronuclei. A second nucleoporin, Nup153, shows no such decline. Fluorescence intensity measurements performed on randomly selected interphase cells (Total) indicate a 66% reduction in Nup358 levels (±5%; P < 0.001) in siRNA-treated versus control (Mock) cells. A larger average reduction in anti-Nup358 fluorescence intensity of 77% (±1%; P < 0.001) is observed when measurements are restricted to cells containing multiple micronuclei (MN). Bar, 10 μm.
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Related In: Results  -  Collection

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fig2: Cells containing multiple micronuclei are depleted of Nup358. A single high molecular weight band is recognized on a Western blot of a HeLa whole cell extract by the anti-Nup358 antibodies that are employed in this study (A). Total protein content of the cell extract is shown in the Coomassie blue–stained lane (gel). Molecular weight markers (MW, kD) are indicated on the left. A 50% reduction in total Nup358 levels in Nup358 siRNA–treated (RNAi) versus control cultures (Mock) is shown by quantitative Western blot analysis (B). For this experiment, twofold dilution series of HeLa cell extracts were analyzed by Western blot using antibodies against Nup358 (A) as well as β-tubulin as an internal standard. Two sample pairs (differing in concentration by a factor of two) from both control and siRNA-treated cultures are shown. Changes in Nup358 levels were determined from the ratios of the Nup358/β-tubulin band intensities. These experiments were performed in quadruplicate over a 16-fold sample concentration range. Immunofluorescence microscopy of Nup358 siRNA–treated cultures reveals a loss of NE-associated Nup358 (C). This is particularly evident in cells containing multiple micronuclei. A second nucleoporin, Nup153, shows no such decline. Fluorescence intensity measurements performed on randomly selected interphase cells (Total) indicate a 66% reduction in Nup358 levels (±5%; P < 0.001) in siRNA-treated versus control (Mock) cells. A larger average reduction in anti-Nup358 fluorescence intensity of 77% (±1%; P < 0.001) is observed when measurements are restricted to cells containing multiple micronuclei (MN). Bar, 10 μm.
Mentions: Immunoblot analysis of HeLa cultures subjected to Nup358 siRNA treatment for a period of 4 d reveals a decline in the level of Nup358 of ∼50% when compared with mock-treated cells (Fig. 2, A and B). As an independent means of verification of these immunoblot results, we performed fluorescence intensity measurements on cells labeled with affinity-purified anti-Nup358 antibodies. In both siRNA-treated and control cultures, measurements were performed on all cells in multiple randomly selected fields. These measurements revealed an average decline in Nup358-associated fluorescence of ∼60%, a value that is consistent with the immunoblot data (Fig. 2 C). At the single cell level, fluorescence intensity reductions of 80% or more could be observed. Cells containing multiple micronuclei exhibited an average reduction in anti-Nup358 fluorescence intensity of ∼77% when compared with mock-treated cells (Fig. 2 C).

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