Limits...
Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK.

Cross MK, Powers MA - Mol. Biol. Cell (2011)

Bottom Line: We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere-associated kinesin (MCAK).Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro.These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Developmental Biology Graduate Program, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere-associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.

Show MeSH
Addition of Nup98-specific antibodies causes a shift to monopolar spindles. (A) Antibodies raised to either human or Xenopus Nup98 were added to spindle assays at 200 or 100 μg/ml, respectively. Nonspecific rabbit IgG was added at 200 μg/ml. Assays were incubated for 60 min. Microtubules are in red; DNA is in blue. Scale bar is 20 μm. (B) Spindle morphology was quantified with spindles scored as either bipolar, imperfect bipolar, or monopolar; 25–50 spindles were scored for each condition in three independent experiments for a total of 250 spindles. Error bars represent standard error of the proportion (SEP). (C) Polymerized tubulin was pelleted and analyzed by immunoblotting. The smaller half-spindles formed in Nup98 samples pelleted less efficiently, leading to decreases in both α- and γ-tubulin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3046062&req=5

Figure 3: Addition of Nup98-specific antibodies causes a shift to monopolar spindles. (A) Antibodies raised to either human or Xenopus Nup98 were added to spindle assays at 200 or 100 μg/ml, respectively. Nonspecific rabbit IgG was added at 200 μg/ml. Assays were incubated for 60 min. Microtubules are in red; DNA is in blue. Scale bar is 20 μm. (B) Spindle morphology was quantified with spindles scored as either bipolar, imperfect bipolar, or monopolar; 25–50 spindles were scored for each condition in three independent experiments for a total of 250 spindles. Error bars represent standard error of the proportion (SEP). (C) Polymerized tubulin was pelleted and analyzed by immunoblotting. The smaller half-spindles formed in Nup98 samples pelleted less efficiently, leading to decreases in both α- and γ-tubulin.

Mentions: To this point, experiments had been conducted by the addition of exogenous, recombinant Nup98 fragments to the extract. To test for a contribution from endogenous Nup98 in spindle assembly, we first added Nup98-specific antibodies to block endogenous protein function. If the Nup98 C-terminal fragment competed for binding of a factor to endogenous Nup98, we hypothesized that an antibody binding this same region of endogenous Nup98 might phenocopy the effect of the fragment. However, in contrast to excess tubulin polymerization, the addition of antibody raised against either Xenopus or human Nup98 C-terminal domain led to a reduction in bipolar spindles and a corresponding increase in monopolar spindles up to >50% monopoles (Figure 3, A and B). Control assays in which the antibody was preabsorbed with the Nup98 fragment showed no effect of antibody addition (unpublished data), demonstrating that the inhibition was due to specific Nup98 binding. We measured the size of each spindle type in these assays and found no significant differences in size (Supplemental Figure 4A). Independent immunoblot and immunofluorescence studies revealed that the human Nup98 C-terminal antibody is directed almost entirely toward epitopes within aa 506–677 (S. Xu and M. Powers, unpublished data); there is only minimal recognition of the region from aa 678–920, most likely due to the high structural conservation of the autoproteolytic domain (Robinson et al., 2005). This antibody thus proved to be an ideal reagent for inhibition of this Nup98 function.


Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK.

Cross MK, Powers MA - Mol. Biol. Cell (2011)

Addition of Nup98-specific antibodies causes a shift to monopolar spindles. (A) Antibodies raised to either human or Xenopus Nup98 were added to spindle assays at 200 or 100 μg/ml, respectively. Nonspecific rabbit IgG was added at 200 μg/ml. Assays were incubated for 60 min. Microtubules are in red; DNA is in blue. Scale bar is 20 μm. (B) Spindle morphology was quantified with spindles scored as either bipolar, imperfect bipolar, or monopolar; 25–50 spindles were scored for each condition in three independent experiments for a total of 250 spindles. Error bars represent standard error of the proportion (SEP). (C) Polymerized tubulin was pelleted and analyzed by immunoblotting. The smaller half-spindles formed in Nup98 samples pelleted less efficiently, leading to decreases in both α- and γ-tubulin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046062&req=5

Figure 3: Addition of Nup98-specific antibodies causes a shift to monopolar spindles. (A) Antibodies raised to either human or Xenopus Nup98 were added to spindle assays at 200 or 100 μg/ml, respectively. Nonspecific rabbit IgG was added at 200 μg/ml. Assays were incubated for 60 min. Microtubules are in red; DNA is in blue. Scale bar is 20 μm. (B) Spindle morphology was quantified with spindles scored as either bipolar, imperfect bipolar, or monopolar; 25–50 spindles were scored for each condition in three independent experiments for a total of 250 spindles. Error bars represent standard error of the proportion (SEP). (C) Polymerized tubulin was pelleted and analyzed by immunoblotting. The smaller half-spindles formed in Nup98 samples pelleted less efficiently, leading to decreases in both α- and γ-tubulin.
Mentions: To this point, experiments had been conducted by the addition of exogenous, recombinant Nup98 fragments to the extract. To test for a contribution from endogenous Nup98 in spindle assembly, we first added Nup98-specific antibodies to block endogenous protein function. If the Nup98 C-terminal fragment competed for binding of a factor to endogenous Nup98, we hypothesized that an antibody binding this same region of endogenous Nup98 might phenocopy the effect of the fragment. However, in contrast to excess tubulin polymerization, the addition of antibody raised against either Xenopus or human Nup98 C-terminal domain led to a reduction in bipolar spindles and a corresponding increase in monopolar spindles up to >50% monopoles (Figure 3, A and B). Control assays in which the antibody was preabsorbed with the Nup98 fragment showed no effect of antibody addition (unpublished data), demonstrating that the inhibition was due to specific Nup98 binding. We measured the size of each spindle type in these assays and found no significant differences in size (Supplemental Figure 4A). Independent immunoblot and immunofluorescence studies revealed that the human Nup98 C-terminal antibody is directed almost entirely toward epitopes within aa 506–677 (S. Xu and M. Powers, unpublished data); there is only minimal recognition of the region from aa 678–920, most likely due to the high structural conservation of the autoproteolytic domain (Robinson et al., 2005). This antibody thus proved to be an ideal reagent for inhibition of this Nup98 function.

Bottom Line: We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere-associated kinesin (MCAK).Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro.These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Developmental Biology Graduate Program, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere-associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.

Show MeSH