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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.

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Nup98 interacts with MCAK through the C-terminal domain and can inhibit MCAK microtubule-depolymerizing activity in vitro. (A) Nup98 C-terminal fragments or buffer (control) were incubated with CSF extract, immunoprecipitated with anti-T7 antibodies, and immunoblotted with anti-XKCM1 (MCAK) or anti-His. Sample lanes contain the material isolated from 4 μl extract. Extract lane contains 0.1 μl extract. (B) Immunoprecipitations from CSF extract were performed with either control IgG or anti-Nup98 (αGLFG domain). IPs were immunoblotted with Nup98 antibody or anti-XKCM1. The Nup98 blot contains IP from 0.5 μl extract. The XKCM1 blot contains IP from 10 μl extract. The extract lane contains 0.1 μl extract. (C) Extracts were immunoblotted with either anti-Nup98 or anti-XKCM1 to determine the extent of XKCM1 codepleted. Each lane contains 0.2 μl extract. (D) In vitro MCAK microtubule depolymerization assays were performed, and equal fractions of the supernatant and pellet were run on PAGE and Coomassie stained. Left top, microtubule polymerization was dependent upon both MCAK and ATP. Left bottom, Nup98 aa 506–920, but not the shorter construct, inhibited microtubule depolymerization by MCAK. Right, microtubule-depolymerization activity is represented as % tubulin pelleted, calculated from spot densitometry of Coomassie-stained gels. Values represent the average of three independent experiments.
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Figure 5: Nup98 interacts with MCAK through the C-terminal domain and can inhibit MCAK microtubule-depolymerizing activity in vitro. (A) Nup98 C-terminal fragments or buffer (control) were incubated with CSF extract, immunoprecipitated with anti-T7 antibodies, and immunoblotted with anti-XKCM1 (MCAK) or anti-His. Sample lanes contain the material isolated from 4 μl extract. Extract lane contains 0.1 μl extract. (B) Immunoprecipitations from CSF extract were performed with either control IgG or anti-Nup98 (αGLFG domain). IPs were immunoblotted with Nup98 antibody or anti-XKCM1. The Nup98 blot contains IP from 0.5 μl extract. The XKCM1 blot contains IP from 10 μl extract. The extract lane contains 0.1 μl extract. (C) Extracts were immunoblotted with either anti-Nup98 or anti-XKCM1 to determine the extent of XKCM1 codepleted. Each lane contains 0.2 μl extract. (D) In vitro MCAK microtubule depolymerization assays were performed, and equal fractions of the supernatant and pellet were run on PAGE and Coomassie stained. Left top, microtubule polymerization was dependent upon both MCAK and ATP. Left bottom, Nup98 aa 506–920, but not the shorter construct, inhibited microtubule depolymerization by MCAK. Right, microtubule-depolymerization activity is represented as % tubulin pelleted, calculated from spot densitometry of Coomassie-stained gels. Values represent the average of three independent experiments.

Mentions: To investigate this possibility, we performed pull-down experiments using purified Nup98 C-terminal fragments. Recombinant proteins were incubated in CSF extract to allow for binding and then recovered on beads, washed, and eluted. Antibody to the T7 tag was used for recovery to avoid potential competition between the Nup98 antibody and Nup98-binding proteins. Bound proteins were then immunoblotted using an antibody to the Xenopus MCAK homolog XKCM1 (Figure 5A). We detected association of MCAK with the longer Nup98 C-terminus (aa 506–920), but binding was greatly reduced with the shorter C-terminal domain (aa 678–920), which does not induce the excess microtubule phenotype. The mutant C-terminus (S864A), which also induced excess polymerization, bound MCAK at levels similar to the wild-type fragment.


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

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

Nup98 interacts with MCAK through the C-terminal domain and can inhibit MCAK microtubule-depolymerizing activity in vitro. (A) Nup98 C-terminal fragments or buffer (control) were incubated with CSF extract, immunoprecipitated with anti-T7 antibodies, and immunoblotted with anti-XKCM1 (MCAK) or anti-His. Sample lanes contain the material isolated from 4 μl extract. Extract lane contains 0.1 μl extract. (B) Immunoprecipitations from CSF extract were performed with either control IgG or anti-Nup98 (αGLFG domain). IPs were immunoblotted with Nup98 antibody or anti-XKCM1. The Nup98 blot contains IP from 0.5 μl extract. The XKCM1 blot contains IP from 10 μl extract. The extract lane contains 0.1 μl extract. (C) Extracts were immunoblotted with either anti-Nup98 or anti-XKCM1 to determine the extent of XKCM1 codepleted. Each lane contains 0.2 μl extract. (D) In vitro MCAK microtubule depolymerization assays were performed, and equal fractions of the supernatant and pellet were run on PAGE and Coomassie stained. Left top, microtubule polymerization was dependent upon both MCAK and ATP. Left bottom, Nup98 aa 506–920, but not the shorter construct, inhibited microtubule depolymerization by MCAK. Right, microtubule-depolymerization activity is represented as % tubulin pelleted, calculated from spot densitometry of Coomassie-stained gels. Values represent the average of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Nup98 interacts with MCAK through the C-terminal domain and can inhibit MCAK microtubule-depolymerizing activity in vitro. (A) Nup98 C-terminal fragments or buffer (control) were incubated with CSF extract, immunoprecipitated with anti-T7 antibodies, and immunoblotted with anti-XKCM1 (MCAK) or anti-His. Sample lanes contain the material isolated from 4 μl extract. Extract lane contains 0.1 μl extract. (B) Immunoprecipitations from CSF extract were performed with either control IgG or anti-Nup98 (αGLFG domain). IPs were immunoblotted with Nup98 antibody or anti-XKCM1. The Nup98 blot contains IP from 0.5 μl extract. The XKCM1 blot contains IP from 10 μl extract. The extract lane contains 0.1 μl extract. (C) Extracts were immunoblotted with either anti-Nup98 or anti-XKCM1 to determine the extent of XKCM1 codepleted. Each lane contains 0.2 μl extract. (D) In vitro MCAK microtubule depolymerization assays were performed, and equal fractions of the supernatant and pellet were run on PAGE and Coomassie stained. Left top, microtubule polymerization was dependent upon both MCAK and ATP. Left bottom, Nup98 aa 506–920, but not the shorter construct, inhibited microtubule depolymerization by MCAK. Right, microtubule-depolymerization activity is represented as % tubulin pelleted, calculated from spot densitometry of Coomassie-stained gels. Values represent the average of three independent experiments.
Mentions: To investigate this possibility, we performed pull-down experiments using purified Nup98 C-terminal fragments. Recombinant proteins were incubated in CSF extract to allow for binding and then recovered on beads, washed, and eluted. Antibody to the T7 tag was used for recovery to avoid potential competition between the Nup98 antibody and Nup98-binding proteins. Bound proteins were then immunoblotted using an antibody to the Xenopus MCAK homolog XKCM1 (Figure 5A). We detected association of MCAK with the longer Nup98 C-terminus (aa 506–920), but binding was greatly reduced with the shorter C-terminal domain (aa 678–920), which does not induce the excess microtubule phenotype. The mutant C-terminus (S864A), which also induced excess polymerization, bound MCAK at levels similar to the wild-type fragment.

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
Related in: MedlinePlus