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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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The Nup98 C-terminal domain increases microtubule polymerization in Ran asters and in preformed spindles. (A) Left, Nup98 C-terminus or BSA was added along with 25 μM RanQ69L to Xenopus extract, and Ran aster formation was monitored. Microtubules are labeled with X-rhodamine tubulin (red). Scale bar is 20 μm. Right, Ran asters formed in the presence or absence of the Nup98 C-terminus were assayed by tubulin spin-down. (B) Spindles were preformed in Xenopus egg extract for 60 min. Either BSA or Nup98 C-terminus was then added (T0), and spindles were monitored over time. Scale bar is 20 μm. (C) Spindle length was measured as pole-to-pole distance (indicated by white dots). Percentage of change was calculated as (average spindle length in sample) – (average T0 length) / (average T0 length) × 100. An average of 10 spindles was quantified for each point except the 15-min Nup98 sample, in which only 6 intact spindles could be identified. (D) Average spindle area in μm2 for each time point was measured using MetaMorph.
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Figure 2: The Nup98 C-terminal domain increases microtubule polymerization in Ran asters and in preformed spindles. (A) Left, Nup98 C-terminus or BSA was added along with 25 μM RanQ69L to Xenopus extract, and Ran aster formation was monitored. Microtubules are labeled with X-rhodamine tubulin (red). Scale bar is 20 μm. Right, Ran asters formed in the presence or absence of the Nup98 C-terminus were assayed by tubulin spin-down. (B) Spindles were preformed in Xenopus egg extract for 60 min. Either BSA or Nup98 C-terminus was then added (T0), and spindles were monitored over time. Scale bar is 20 μm. (C) Spindle length was measured as pole-to-pole distance (indicated by white dots). Percentage of change was calculated as (average spindle length in sample) – (average T0 length) / (average T0 length) × 100. An average of 10 spindles was quantified for each point except the 15-min Nup98 sample, in which only 6 intact spindles could be identified. (D) Average spindle area in μm2 for each time point was measured using MetaMorph.

Mentions: There are multiple distinct pathways that can work in conjunction to build a mitotic spindle (O’Connell and Khodjakov, 2007; Kalab and Heald, 2008; Walczak and Heald, 2008). In cultured cells, the predominant pathway is thought to be the centrosome-driven or search-and-capture pathway, in which microtubules extend dynamically from the centrosome and search for the kinetochore complex found on the centromeric region of the chromosome. However, there is also a second, chromatin-driven pathway, regulated by the Ran GTPase, in which microtubules polymerize adjacent to chromatin and are then organized by molecular motors and other factors to form a spindle pole. The chromatin-driven pathway is thought to be the major spindle assembly pathway in Xenopus egg extracts. Spindle-like Ran asters form in CSF extract upon addition of a nonhydrolyzing variant of Ran-GTP, even in the absence of centrosomes (Carazo-Salas et al., 1999; Kalab et al., 1999; Ohba et al., 1999; Wilde and Zheng, 1999; Zhang et al., 1999). We found that addition of purified Nup98 C-terminus to Ran aster assays led to excess polymerization of microtubules (Figure 2A). Asters were larger, with more unfocused microtubules. This result was confirmed biochemically by the tubulin spin-down assay (Figure 2A).


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

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

The Nup98 C-terminal domain increases microtubule polymerization in Ran asters and in preformed spindles. (A) Left, Nup98 C-terminus or BSA was added along with 25 μM RanQ69L to Xenopus extract, and Ran aster formation was monitored. Microtubules are labeled with X-rhodamine tubulin (red). Scale bar is 20 μm. Right, Ran asters formed in the presence or absence of the Nup98 C-terminus were assayed by tubulin spin-down. (B) Spindles were preformed in Xenopus egg extract for 60 min. Either BSA or Nup98 C-terminus was then added (T0), and spindles were monitored over time. Scale bar is 20 μm. (C) Spindle length was measured as pole-to-pole distance (indicated by white dots). Percentage of change was calculated as (average spindle length in sample) – (average T0 length) / (average T0 length) × 100. An average of 10 spindles was quantified for each point except the 15-min Nup98 sample, in which only 6 intact spindles could be identified. (D) Average spindle area in μm2 for each time point was measured using MetaMorph.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: The Nup98 C-terminal domain increases microtubule polymerization in Ran asters and in preformed spindles. (A) Left, Nup98 C-terminus or BSA was added along with 25 μM RanQ69L to Xenopus extract, and Ran aster formation was monitored. Microtubules are labeled with X-rhodamine tubulin (red). Scale bar is 20 μm. Right, Ran asters formed in the presence or absence of the Nup98 C-terminus were assayed by tubulin spin-down. (B) Spindles were preformed in Xenopus egg extract for 60 min. Either BSA or Nup98 C-terminus was then added (T0), and spindles were monitored over time. Scale bar is 20 μm. (C) Spindle length was measured as pole-to-pole distance (indicated by white dots). Percentage of change was calculated as (average spindle length in sample) – (average T0 length) / (average T0 length) × 100. An average of 10 spindles was quantified for each point except the 15-min Nup98 sample, in which only 6 intact spindles could be identified. (D) Average spindle area in μm2 for each time point was measured using MetaMorph.
Mentions: There are multiple distinct pathways that can work in conjunction to build a mitotic spindle (O’Connell and Khodjakov, 2007; Kalab and Heald, 2008; Walczak and Heald, 2008). In cultured cells, the predominant pathway is thought to be the centrosome-driven or search-and-capture pathway, in which microtubules extend dynamically from the centrosome and search for the kinetochore complex found on the centromeric region of the chromosome. However, there is also a second, chromatin-driven pathway, regulated by the Ran GTPase, in which microtubules polymerize adjacent to chromatin and are then organized by molecular motors and other factors to form a spindle pole. The chromatin-driven pathway is thought to be the major spindle assembly pathway in Xenopus egg extracts. Spindle-like Ran asters form in CSF extract upon addition of a nonhydrolyzing variant of Ran-GTP, even in the absence of centrosomes (Carazo-Salas et al., 1999; Kalab et al., 1999; Ohba et al., 1999; Wilde and Zheng, 1999; Zhang et al., 1999). We found that addition of purified Nup98 C-terminus to Ran aster assays led to excess polymerization of microtubules (Figure 2A). Asters were larger, with more unfocused microtubules. This result was confirmed biochemically by the tubulin spin-down assay (Figure 2A).

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