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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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Depletion of Mad1 reverses the Nup358 siRNA–induced prometaphase arrest. Double indirect immunofluorescence microscopy of HeLa cell cultures treated with Nup358 and/or Mad1 siRNAs and labeled with antibodies against both Nup358 and Mad1 (A). In inter phase cells, Mad1, like Nup358, localizes almost exclusively to the nuclear periphery. Depletion of either protein in interphase cells has little or no effect on the distribution of the other. During mitosis, Nup358 depletion results in a significant (70%) increase in the number of premetaphase and metaphase cells over mock-treated cultures (B). This effect is reversed by codepletion of Mad1. Conversely, depletion of both proteins causes an increase in the number of multinucleate cells. 2–4,000 cells were scored in each category in four independent experiments. It should be noted that these counts represent percentages derived from total cell populations. To eliminate bias, no attempt was made to discriminate between transfected versus nontransfected cells. Bar, 10 μm.
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fig8: Depletion of Mad1 reverses the Nup358 siRNA–induced prometaphase arrest. Double indirect immunofluorescence microscopy of HeLa cell cultures treated with Nup358 and/or Mad1 siRNAs and labeled with antibodies against both Nup358 and Mad1 (A). In inter phase cells, Mad1, like Nup358, localizes almost exclusively to the nuclear periphery. Depletion of either protein in interphase cells has little or no effect on the distribution of the other. During mitosis, Nup358 depletion results in a significant (70%) increase in the number of premetaphase and metaphase cells over mock-treated cultures (B). This effect is reversed by codepletion of Mad1. Conversely, depletion of both proteins causes an increase in the number of multinucleate cells. 2–4,000 cells were scored in each category in four independent experiments. It should be noted that these counts represent percentages derived from total cell populations. To eliminate bias, no attempt was made to discriminate between transfected versus nontransfected cells. Bar, 10 μm.

Mentions: A final issue that arises is whether Nup358 itself might have a role as a spindle assembly checkpoint protein. To address this, we examined the effects of simultaneous depletion of Nup358 and the bona fide checkpoint protein Mad1 (Fig. 8). Depletion of Mad1 has previously been shown to result in premature anaphase and the appearance of lagging chromosomes (Luo et al., 2002; Martin-Lluesma et al., 2002). The prediction is that if the spindle assembly checkpoint remains functional in Nup358-depleted cells, then loss of Mad1 should result in a decline in the number of prometaphase/metaphase cells. At the same time, given the Nup358-associated congression defect, there should be an increase in the number of cells containing multiple micronuclei. As shown in Fig. 8, this is precisely what occurs. In cultures depleted only of Nup358, there is a 70% increase in the number metaphase and premetaphase cells over mock-treated cultures. It must be emphasized that this figure represents a minimum value, given the heterogeneity of the Nup358 siRNA–treated cells. If we had only counted cells overtly depleted of Nup358, this increase would be on the order of 150–300%. Simultaneous depletion of both Nup358 and Mad1 yielded a sevenfold decline in the number of metaphase and premetaphase cells (compared with Nup358 depletion alone). Taken together, these results indicate that Nup358 is unlikely to possess a checkpoint function.


Nup358 integrates nuclear envelope breakdown with kinetochore assembly.

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

Depletion of Mad1 reverses the Nup358 siRNA–induced prometaphase arrest. Double indirect immunofluorescence microscopy of HeLa cell cultures treated with Nup358 and/or Mad1 siRNAs and labeled with antibodies against both Nup358 and Mad1 (A). In inter phase cells, Mad1, like Nup358, localizes almost exclusively to the nuclear periphery. Depletion of either protein in interphase cells has little or no effect on the distribution of the other. During mitosis, Nup358 depletion results in a significant (70%) increase in the number of premetaphase and metaphase cells over mock-treated cultures (B). This effect is reversed by codepletion of Mad1. Conversely, depletion of both proteins causes an increase in the number of multinucleate cells. 2–4,000 cells were scored in each category in four independent experiments. It should be noted that these counts represent percentages derived from total cell populations. To eliminate bias, no attempt was made to discriminate between transfected versus nontransfected cells. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Depletion of Mad1 reverses the Nup358 siRNA–induced prometaphase arrest. Double indirect immunofluorescence microscopy of HeLa cell cultures treated with Nup358 and/or Mad1 siRNAs and labeled with antibodies against both Nup358 and Mad1 (A). In inter phase cells, Mad1, like Nup358, localizes almost exclusively to the nuclear periphery. Depletion of either protein in interphase cells has little or no effect on the distribution of the other. During mitosis, Nup358 depletion results in a significant (70%) increase in the number of premetaphase and metaphase cells over mock-treated cultures (B). This effect is reversed by codepletion of Mad1. Conversely, depletion of both proteins causes an increase in the number of multinucleate cells. 2–4,000 cells were scored in each category in four independent experiments. It should be noted that these counts represent percentages derived from total cell populations. To eliminate bias, no attempt was made to discriminate between transfected versus nontransfected cells. Bar, 10 μm.
Mentions: A final issue that arises is whether Nup358 itself might have a role as a spindle assembly checkpoint protein. To address this, we examined the effects of simultaneous depletion of Nup358 and the bona fide checkpoint protein Mad1 (Fig. 8). Depletion of Mad1 has previously been shown to result in premature anaphase and the appearance of lagging chromosomes (Luo et al., 2002; Martin-Lluesma et al., 2002). The prediction is that if the spindle assembly checkpoint remains functional in Nup358-depleted cells, then loss of Mad1 should result in a decline in the number of prometaphase/metaphase cells. At the same time, given the Nup358-associated congression defect, there should be an increase in the number of cells containing multiple micronuclei. As shown in Fig. 8, this is precisely what occurs. In cultures depleted only of Nup358, there is a 70% increase in the number metaphase and premetaphase cells over mock-treated cultures. It must be emphasized that this figure represents a minimum value, given the heterogeneity of the Nup358 siRNA–treated cells. If we had only counted cells overtly depleted of Nup358, this increase would be on the order of 150–300%. Simultaneous depletion of both Nup358 and Mad1 yielded a sevenfold decline in the number of metaphase and premetaphase cells (compared with Nup358 depletion alone). Taken together, these results indicate that Nup358 is unlikely to possess a checkpoint function.

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