Cross MK and Powers MA (2011), Nup98 regulates bipolar spindle assembly throug...

XB-ART-42554

Mol Biol Cell 2011 Mar 01;225:661-72. doi: 10.1091/mbc.E10-06-0478.

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

Cross MK , Powers MA .

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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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Species referenced: Xenopus
Genes referenced: isyna1 kif2c nup98 ran rcc1 stmn1

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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.
	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.
	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.
	FIGURE 4:. Depletion of Nup98 from Xenopus extracts causes disruption in bipolar spindle assembly, which can be restored by the Nup98 C-terminal domain. Extracts were depleted with either nonspecific IgG (mock) or anti-Nup98, and either BSA or purified Nup98 C-terminus was added to a final concentration of 1.5 μM. Spindles were assembled for 60 min. (A) Spindles assembled following Nup98 depletion and addback of the recombinant C-terminus. Microtubules are in red, and DNA is in blue. Scale bar is 20 μm. (B) Depletion was assessed by blotting 0.3 μl depleted extracts relative to serial twofold dilutions of Xenopus extract. (C) Spindle morphology was scored as bipolar, imperfect bipolar, or monopolar. Imperfect spindles typically resembled that of panel Ab, in which DNA was somewhat misaligned. Left, spindle morphology following mock depletion or depletion with anti-Nup98. Right, spindle morphology following depletion and addback of BSA or Nup98 C-terminus. Three or more independent experiments were scored for each condition, with 25–50 spindles per sample, for a total of 1429 spindles analyzed. Error bars represent SEP. (D) Summary of phenotypes observed after manipulation of Nup98 levels in Xenopus egg extract. *In depleted extracts, formation of Ran asters required an extra 15 min, but asters appeared normal. nd, not done.
	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.
	FIGURE 6:. Model of Nup98 function in spindle assembly. (A) The Ran-GTP gradient generated by RCCI promotes spindle assembly factor release in the vicinity of chromatin. Bipolar spindle assembly also requires down-regulation of oncoprotein 18/stathmin and MCAK by the CPC. We propose that Nup98 also negatively regulates MCAK activity to promote bipolar spindle assembly. (B) Potential mechanisms for Nup98 regulation of MCAK function. Top, Nup98 binds to microtubules and prevents lateral diffusion of MCAK. Middle, Nup98 binds to microtubule plus ends and competes for MCAK binding and depolymerization of plus ends. Bottom, Nup98 binds microtubules and stabilizes them against MCAK depolymerization.

References [+] :

Andrews, Aurora B regulates MCAK at the mitotic centromere. 2004, Pubmed