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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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Loss of kinetochore-associated Nup358 after siRNA treatment. Double indirect immunofluorescence microscopy of mitotic HeLa cells using antibodies against Nup358 in combination either with a human anticentromere autoimmune serum (ACA) or an antibody against CENP-F. Cells were also labeled with Hoechst dye (shown in blue in A and B) to reveal the mitotic chromosomes. Untreated HeLa cells are shown in A and B. The insets reveal that Nup358 lies distal to the centromere and instead largely colocalizes with CENP-F, an outer kinetochore protein. Nup358 siRNA–treated cells are shown in C and reveal a loss of both kinetochore and spindle-associated Nup358, although ACA labeling is unaffected. Chromosome congression is clearly abnormal in these cells. In A and B, merged images are shown both with (right) and without (left) superimposition of the chromosomes. In C, the merged images show only the antibody labeling. In each set, Nup358 labeling is shown in red.
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fig6: Loss of kinetochore-associated Nup358 after siRNA treatment. Double indirect immunofluorescence microscopy of mitotic HeLa cells using antibodies against Nup358 in combination either with a human anticentromere autoimmune serum (ACA) or an antibody against CENP-F. Cells were also labeled with Hoechst dye (shown in blue in A and B) to reveal the mitotic chromosomes. Untreated HeLa cells are shown in A and B. The insets reveal that Nup358 lies distal to the centromere and instead largely colocalizes with CENP-F, an outer kinetochore protein. Nup358 siRNA–treated cells are shown in C and reveal a loss of both kinetochore and spindle-associated Nup358, although ACA labeling is unaffected. Chromosome congression is clearly abnormal in these cells. In A and B, merged images are shown both with (right) and without (left) superimposition of the chromosomes. In C, the merged images show only the antibody labeling. In each set, Nup358 labeling is shown in red.

Mentions: Further analyses of arrested cells after 96 h of siRNA treatment suggest that congression failure is due, at least partially, to defects at the kinetochore. A role for Nup358 in kinetochore function is suggested by work from Joseph et al. (2002), who have shown that a population of Nup358 is localized at kinetochores during mitosis. We have been able to confirm this observation, as well as show that Nup358 is associated, at least in part, with the outer portion of the kinetochore (Fig. 6). This localization was concluded from double label experiments using anti-Nup358 in combination with either an anticentromere human autoimmune serum (ACA; Fig. 6 A) or an antibody against CENP-F, a protein of the kinetochore fibrous corona (Fig. 6 B). Anti-Nup358 kinetochore labeling is clearly reduced in siRNA-treated cells (Fig. 6 C). We therefore examined the distribution, in arrested cells, of a number of components that normally associate transiently with mitotic kinetochores. These include dynein, CENP-E, CENP-F, the mitotic checkpoint proteins Mad1 and Mad2, and Zw10. None of these proteins show their normal mitotic distribution. Instead, they exhibit aberrant kinetochore targeting and many are partially or completely mislocalized to the cytoplasm (Fig. 7, A and B). Fluorescence intensity measurements indicate a reduction in kinetochore-associated CENP-E, CENP-F, Mad-1, and dynein of ∼60% using ACA labeling as an internal reference (Fig. 7 B). When these transient proteins do associate with the kinetochore in the Nup358-depleted prometaphase cells, they are almost invariably associated with chromosome clusters at the spindle poles and not with the clusters near the spindle equator (Fig. 7 A). Cell cycle invariant kinetochore components, including those that are recognized by the ACA serum, do show an appropriate localization (Fig. 7 A) and are found on both polar and equatorial clusters. The implication of these observations is that Nup358 is required for the normal assembly of proteins at the kinetochore and hence for kinetochore function. This view is reinforced and expanded by ultrastructural analysis of kinetochores in prometaphase cells that accumulate during 4 d of siRNA treatment.


Nup358 integrates nuclear envelope breakdown with kinetochore assembly.

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

Loss of kinetochore-associated Nup358 after siRNA treatment. Double indirect immunofluorescence microscopy of mitotic HeLa cells using antibodies against Nup358 in combination either with a human anticentromere autoimmune serum (ACA) or an antibody against CENP-F. Cells were also labeled with Hoechst dye (shown in blue in A and B) to reveal the mitotic chromosomes. Untreated HeLa cells are shown in A and B. The insets reveal that Nup358 lies distal to the centromere and instead largely colocalizes with CENP-F, an outer kinetochore protein. Nup358 siRNA–treated cells are shown in C and reveal a loss of both kinetochore and spindle-associated Nup358, although ACA labeling is unaffected. Chromosome congression is clearly abnormal in these cells. In A and B, merged images are shown both with (right) and without (left) superimposition of the chromosomes. In C, the merged images show only the antibody labeling. In each set, Nup358 labeling is shown in red.
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Related In: Results  -  Collection

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fig6: Loss of kinetochore-associated Nup358 after siRNA treatment. Double indirect immunofluorescence microscopy of mitotic HeLa cells using antibodies against Nup358 in combination either with a human anticentromere autoimmune serum (ACA) or an antibody against CENP-F. Cells were also labeled with Hoechst dye (shown in blue in A and B) to reveal the mitotic chromosomes. Untreated HeLa cells are shown in A and B. The insets reveal that Nup358 lies distal to the centromere and instead largely colocalizes with CENP-F, an outer kinetochore protein. Nup358 siRNA–treated cells are shown in C and reveal a loss of both kinetochore and spindle-associated Nup358, although ACA labeling is unaffected. Chromosome congression is clearly abnormal in these cells. In A and B, merged images are shown both with (right) and without (left) superimposition of the chromosomes. In C, the merged images show only the antibody labeling. In each set, Nup358 labeling is shown in red.
Mentions: Further analyses of arrested cells after 96 h of siRNA treatment suggest that congression failure is due, at least partially, to defects at the kinetochore. A role for Nup358 in kinetochore function is suggested by work from Joseph et al. (2002), who have shown that a population of Nup358 is localized at kinetochores during mitosis. We have been able to confirm this observation, as well as show that Nup358 is associated, at least in part, with the outer portion of the kinetochore (Fig. 6). This localization was concluded from double label experiments using anti-Nup358 in combination with either an anticentromere human autoimmune serum (ACA; Fig. 6 A) or an antibody against CENP-F, a protein of the kinetochore fibrous corona (Fig. 6 B). Anti-Nup358 kinetochore labeling is clearly reduced in siRNA-treated cells (Fig. 6 C). We therefore examined the distribution, in arrested cells, of a number of components that normally associate transiently with mitotic kinetochores. These include dynein, CENP-E, CENP-F, the mitotic checkpoint proteins Mad1 and Mad2, and Zw10. None of these proteins show their normal mitotic distribution. Instead, they exhibit aberrant kinetochore targeting and many are partially or completely mislocalized to the cytoplasm (Fig. 7, A and B). Fluorescence intensity measurements indicate a reduction in kinetochore-associated CENP-E, CENP-F, Mad-1, and dynein of ∼60% using ACA labeling as an internal reference (Fig. 7 B). When these transient proteins do associate with the kinetochore in the Nup358-depleted prometaphase cells, they are almost invariably associated with chromosome clusters at the spindle poles and not with the clusters near the spindle equator (Fig. 7 A). Cell cycle invariant kinetochore components, including those that are recognized by the ACA serum, do show an appropriate localization (Fig. 7 A) and are found on both polar and equatorial clusters. The implication of these observations is that Nup358 is required for the normal assembly of proteins at the kinetochore and hence for kinetochore function. This view is reinforced and expanded by ultrastructural analysis of kinetochores in prometaphase cells that accumulate during 4 d of siRNA treatment.

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