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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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Abnormal chromosome congression and defective mitosis after Nup358 siRNA treatment. Indirect immunofluorescence microscopy of Nup358 siRNA–treated (RNAi) versus mock-treated HeLa cells. Prometaphase and metaphase cells labeled with anti–β-tubulin and Hoechst dye to reveal the condensed chromosomes (A). Up to 75% of premetaphase cells in siRNA-treated cultures exhibit abnormal or elongated spindle morphology associated with abnormal chromosome congression (A) featuring irregular chromosome clusters at the spindle equator as well as smaller clusters over each pole. This unusual morphology is rarely observed in control cells. Fluorescence intensity measurements on prometaphase cells labeled with an anti-Nup358 antibody (B) reveal an average reduction of 61% when compared with mock-treated cells (±6%; P < 0.001). Telophase and early G1 siRNA-treated cells exhibit a high frequency of multiple micronuclei and abnormal cytokinesis (C) in which lagging chromosomes and chromatin bridges connecting the daughters are observed (D).
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fig5: Abnormal chromosome congression and defective mitosis after Nup358 siRNA treatment. Indirect immunofluorescence microscopy of Nup358 siRNA–treated (RNAi) versus mock-treated HeLa cells. Prometaphase and metaphase cells labeled with anti–β-tubulin and Hoechst dye to reveal the condensed chromosomes (A). Up to 75% of premetaphase cells in siRNA-treated cultures exhibit abnormal or elongated spindle morphology associated with abnormal chromosome congression (A) featuring irregular chromosome clusters at the spindle equator as well as smaller clusters over each pole. This unusual morphology is rarely observed in control cells. Fluorescence intensity measurements on prometaphase cells labeled with an anti-Nup358 antibody (B) reveal an average reduction of 61% when compared with mock-treated cells (±6%; P < 0.001). Telophase and early G1 siRNA-treated cells exhibit a high frequency of multiple micronuclei and abnormal cytokinesis (C) in which lagging chromosomes and chromatin bridges connecting the daughters are observed (D).

Mentions: Until at least the fourth day of Nup358 siRNA treatment, some members of the unusual prometaphase population display the ability to escape mitotic arrest. In these cells, an NE reforms around individual chromosomes and groups of chromosomes, giving rise to the multiple micronuclei described above. These cells invariably show reduced labeling with anti-Nup358 antibodies (Fig. 2 C and Fig. 3 D). Surprisingly, many of these cells form an intracellular bridge and undergo cytokinesis (Fig. 5 C). Indeed at the 96-h time point, ∼34% of “telophase” or early G1 cells (defined by the presence of an intracellular bridge) were found to contain multiple micronuclei. Few such cells were observed in corresponding mock-treated populations. The ultimate fate of these unusual cells seems to be death, because, as pointed out above, the frequency of apoptosis increases steadily up to 5 d after siRNA treatment. After this time point, the occurrence of cells containing multiple micronuclei generally declines (Fig. 1 B). Remarkably, a virtually identical effect, including micronuclear formation and aberrant cytokinesis, has recently been reported in cells depleted of CENP-A and hMis12, a human kinetochore protein (Goshima et al., 2003).


Nup358 integrates nuclear envelope breakdown with kinetochore assembly.

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

Abnormal chromosome congression and defective mitosis after Nup358 siRNA treatment. Indirect immunofluorescence microscopy of Nup358 siRNA–treated (RNAi) versus mock-treated HeLa cells. Prometaphase and metaphase cells labeled with anti–β-tubulin and Hoechst dye to reveal the condensed chromosomes (A). Up to 75% of premetaphase cells in siRNA-treated cultures exhibit abnormal or elongated spindle morphology associated with abnormal chromosome congression (A) featuring irregular chromosome clusters at the spindle equator as well as smaller clusters over each pole. This unusual morphology is rarely observed in control cells. Fluorescence intensity measurements on prometaphase cells labeled with an anti-Nup358 antibody (B) reveal an average reduction of 61% when compared with mock-treated cells (±6%; P < 0.001). Telophase and early G1 siRNA-treated cells exhibit a high frequency of multiple micronuclei and abnormal cytokinesis (C) in which lagging chromosomes and chromatin bridges connecting the daughters are observed (D).
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Related In: Results  -  Collection

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

fig5: Abnormal chromosome congression and defective mitosis after Nup358 siRNA treatment. Indirect immunofluorescence microscopy of Nup358 siRNA–treated (RNAi) versus mock-treated HeLa cells. Prometaphase and metaphase cells labeled with anti–β-tubulin and Hoechst dye to reveal the condensed chromosomes (A). Up to 75% of premetaphase cells in siRNA-treated cultures exhibit abnormal or elongated spindle morphology associated with abnormal chromosome congression (A) featuring irregular chromosome clusters at the spindle equator as well as smaller clusters over each pole. This unusual morphology is rarely observed in control cells. Fluorescence intensity measurements on prometaphase cells labeled with an anti-Nup358 antibody (B) reveal an average reduction of 61% when compared with mock-treated cells (±6%; P < 0.001). Telophase and early G1 siRNA-treated cells exhibit a high frequency of multiple micronuclei and abnormal cytokinesis (C) in which lagging chromosomes and chromatin bridges connecting the daughters are observed (D).
Mentions: Until at least the fourth day of Nup358 siRNA treatment, some members of the unusual prometaphase population display the ability to escape mitotic arrest. In these cells, an NE reforms around individual chromosomes and groups of chromosomes, giving rise to the multiple micronuclei described above. These cells invariably show reduced labeling with anti-Nup358 antibodies (Fig. 2 C and Fig. 3 D). Surprisingly, many of these cells form an intracellular bridge and undergo cytokinesis (Fig. 5 C). Indeed at the 96-h time point, ∼34% of “telophase” or early G1 cells (defined by the presence of an intracellular bridge) were found to contain multiple micronuclei. Few such cells were observed in corresponding mock-treated populations. The ultimate fate of these unusual cells seems to be death, because, as pointed out above, the frequency of apoptosis increases steadily up to 5 d after siRNA treatment. After this time point, the occurrence of cells containing multiple micronuclei generally declines (Fig. 1 B). Remarkably, a virtually identical effect, including micronuclear formation and aberrant cytokinesis, has recently been reported in cells depleted of CENP-A and hMis12, a human kinetochore protein (Goshima et al., 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.

Show MeSH
Related in: MedlinePlus