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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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A C-terminal domain fragment of Nup98 causes excess microtubule polymerization during assembly of meiotic spindles. (A) Nup98 C-terminal domain fragments or BSA were added at 6 μM at the start of assembly assays. The excess microtubule phenotype could be observed at concentrations as low as 2.5 μM; by immunoblotting, we estimate the endogenous Nup98 concentration at ∼0.6 μM in the extract. Samples were examined at the indicated time points. Microtubules are labeled with X-rhodamine tubulin (red), and DNA is labeled with Hoechst (blue). Scale bar represents 20 μm. (B) Left, polymerized tubulin was isolated and immunoblotted for α-tubulin. Both γ-tubulin and RCC1 were blotted as loading controls. Right, quantitation of tubulin polymerization using either γ-tubulin (left) or RCC1 (right) for normalization.
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Figure 1: A C-terminal domain fragment of Nup98 causes excess microtubule polymerization during assembly of meiotic spindles. (A) Nup98 C-terminal domain fragments or BSA were added at 6 μM at the start of assembly assays. The excess microtubule phenotype could be observed at concentrations as low as 2.5 μM; by immunoblotting, we estimate the endogenous Nup98 concentration at ∼0.6 μM in the extract. Samples were examined at the indicated time points. Microtubules are labeled with X-rhodamine tubulin (red), and DNA is labeled with Hoechst (blue). Scale bar represents 20 μm. (B) Left, polymerized tubulin was isolated and immunoblotted for α-tubulin. Both γ-tubulin and RCC1 were blotted as loading controls. Right, quantitation of tubulin polymerization using either γ-tubulin (left) or RCC1 (right) for normalization.

Mentions: To investigate the potential mitotic function of Nup98, we added a bacterially expressed Nup98 C-terminal fragment (His-tagged amino acids [aa] 506–920; Supplemental Figure 1) to Xenopus laevis spindle assembly assays in vitro. The purified fragment was added to Xenopus cytostatic factor (CSF) egg extracts to a final concentration of 6 μM along with sperm chromatin, and, at time points during spindle assembly, samples were fixed and analyzed by fluorescence microscopy. As expected, after 15 min, microtubule asters had formed in control samples (Figure 1Aa). Surprisingly, in the presence of the Nup98 C-terminal fragment, asters contained longer microtubules (Figure 1Ab). This microtubule phenotype persisted throughout the time course of spindle formation and resulted in highly perturbed bipolar spindle structures. In contrast, spindles formed in the presence of the same concentration of a control protein (bovine serum albumin [BSA]) displayed normal bipolar spindle morphology (Figure 1A, compare panels i and m to panels j and n). Protein obtained by purification of control bacterial lysate over a nickel chromatography column had no effect when added to the spindle assembly assay (unpublished data).


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

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

A C-terminal domain fragment of Nup98 causes excess microtubule polymerization during assembly of meiotic spindles. (A) Nup98 C-terminal domain fragments or BSA were added at 6 μM at the start of assembly assays. The excess microtubule phenotype could be observed at concentrations as low as 2.5 μM; by immunoblotting, we estimate the endogenous Nup98 concentration at ∼0.6 μM in the extract. Samples were examined at the indicated time points. Microtubules are labeled with X-rhodamine tubulin (red), and DNA is labeled with Hoechst (blue). Scale bar represents 20 μm. (B) Left, polymerized tubulin was isolated and immunoblotted for α-tubulin. Both γ-tubulin and RCC1 were blotted as loading controls. Right, quantitation of tubulin polymerization using either γ-tubulin (left) or RCC1 (right) for normalization.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: A C-terminal domain fragment of Nup98 causes excess microtubule polymerization during assembly of meiotic spindles. (A) Nup98 C-terminal domain fragments or BSA were added at 6 μM at the start of assembly assays. The excess microtubule phenotype could be observed at concentrations as low as 2.5 μM; by immunoblotting, we estimate the endogenous Nup98 concentration at ∼0.6 μM in the extract. Samples were examined at the indicated time points. Microtubules are labeled with X-rhodamine tubulin (red), and DNA is labeled with Hoechst (blue). Scale bar represents 20 μm. (B) Left, polymerized tubulin was isolated and immunoblotted for α-tubulin. Both γ-tubulin and RCC1 were blotted as loading controls. Right, quantitation of tubulin polymerization using either γ-tubulin (left) or RCC1 (right) for normalization.
Mentions: To investigate the potential mitotic function of Nup98, we added a bacterially expressed Nup98 C-terminal fragment (His-tagged amino acids [aa] 506–920; Supplemental Figure 1) to Xenopus laevis spindle assembly assays in vitro. The purified fragment was added to Xenopus cytostatic factor (CSF) egg extracts to a final concentration of 6 μM along with sperm chromatin, and, at time points during spindle assembly, samples were fixed and analyzed by fluorescence microscopy. As expected, after 15 min, microtubule asters had formed in control samples (Figure 1Aa). Surprisingly, in the presence of the Nup98 C-terminal fragment, asters contained longer microtubules (Figure 1Ab). This microtubule phenotype persisted throughout the time course of spindle formation and resulted in highly perturbed bipolar spindle structures. In contrast, spindles formed in the presence of the same concentration of a control protein (bovine serum albumin [BSA]) displayed normal bipolar spindle morphology (Figure 1A, compare panels i and m to panels j and n). Protein obtained by purification of control bacterial lysate over a nickel chromatography column had no effect when added to the spindle assembly assay (unpublished data).

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