The Nup107-160 complex and gamma-TuRC regulate microtubule polymerization at kinetochores - PubMed (original) (raw)

The Nup107-160 complex and gamma-TuRC regulate microtubule polymerization at kinetochores

Ram Kumar Mishra et al. Nat Cell Biol. 2010 Feb.

Abstract

The metazoan nuclear pore complex (NPC) disassembles during mitosis, and many of its constituents distribute onto spindles and kinetochores, including the Nup107-160 sub-complex. We have found that Nup107-160 interacts with the gamma-tubulin ring complex (gamma-TuRC), an essential and conserved microtubule nucleator, and recruits gamma-TuRC to unattached kinetochores. The unattached kinetochores nucleate microtubules in a manner that is regulated by Ran GTPase; such microtubules contribute to the formation of kinetochore fibres (k-fibres), microtubule bundles connecting kinetochores to spindle poles. Our data indicate that Nup107-160 and gamma-TuRC act cooperatively to promote spindle assembly through microtubule nucleation at kinetochores: HeLa cells lacking Nup107-160 or gamma-TuRC were profoundly deficient in kinetochore-associated microtubule nucleation. Moreover, co-precipitated Nup107-160- gamma-TuRC complexes nucleated microtubule formation in assays using purified tubulin. Although Ran did not regulate microtubule nucleation by gamma-TuRC alone, Nup107-160-gamma-TuRC complexes required Ran-GTP for microtubule nucleation. Collectively, our observations show that Nup107-160 promotes spindle assembly through Ran-GTP-regulated nucleation of microtubules by gamma-TuRC at kinetochores, and reveal a relationship between nucleoporins and the microtubule cytoskeleton.

PubMed Disclaimer

Figures

Figure 1. Nup107-160 binds γ-TuRC

(a) 293T cells were co-transfected with plasmids encoding HA-GCP3 and myc-Nup98, myc-Nup96 or myc-Nup107. Immunoprecipitations were performed with anti-myc antibodies or non-specific IgG, and precipitates were subjected to Western blotting with anti-HA antibodies (left panel). A Western blot of input extracts with anti-myc antibodies is shown in the right panel. (b) Anti-xGCP2 and -xNedd1 antibodies were used for immunoprecipitation from XEEs. In each case, untreated XEE is shown on the left, the precipitated fraction is shown on the right, and the center lane shows proteins eluted from control IgG-coated beads. Uncropped images of the blots are shown in Supplementary Information, Fig. S3. (c) XEEs were depleted of xNup107-160 using anti-xNup160 antibodies. Co-precipitating proteins (B) and the unbound fraction (U) were analyzed by Western blotting against xNup107-160 or against γ-TuRC components, as indicated. For comparison, similar fractions associated with IgG-coated beads are shown, as well as untreated extracts (Input). Uncropped images of the blots are shown in Supplementary Information, Fig. S3.

Figure 2. γ-TuRC is recruited to kinetochores by Nup107-160

(a) HeLa cells were depleted of GCP2 (second row) or Nup160 (third row) by siRNA. The cells were treated with nocodazole for 16 hrs and processed for indirect immunofluorescence with antibodies against GCP2 (second column, green) and kinetochore marker CREST serum (third column, red) and counterstained with Hoechst 33342 dye to visualize chromosomal DNA (left, blue). Inset shows enlarged, merged image of kinetochores. Top row shows HeLa cells transfected with a control siRNA, treated in an identical manner. Scale bar = 5 µm. (b) HeLa cells were treated as in (a), stained with antibodies against Nup107 (second row, green) and counterstained with Hoechst 33342 dye to visualize chromosomal DNA (top row, blue). (c) Whole cell extracts from control HeLa cells (left), cells depleted of Nup160 (center) or cells depleted of GCP2 (right) were analyzed by Western blotting with the indicated antibodies to monitor the overall level of depletion through siRNAs.

Figure 3. Nup107-160 is critical for MT nucleation at kinetochores in HeLa cells

(a) HeLa cells transfected with a control siRNA (top panels), a siRNA against Nup160 (middle panels) or an siRNA directed against GCP2 (bottom panels). The cells were analyzed in a MT re-growth assay at 2 minutes after being returned to 37°C, with staining for α-tubulin (red) and Nup107 (green). Mitotic cells were identified by chromosome morphology through counterstaining with Hoechst 33342 dye (blue). Scale bar= 5 µm (b) Whole cell extracts were made from control HeLa cells (left), or cells depleted of Nup107-160 (center) or of GCP2 (right). The extracts were analyzed by Western blotting with the indicated antibodies to monitor the overall level of depletion through siRNAs. ). Uncropped images of the blots are shown in Supplementary Information, Fig. S3.

Figure 4. Ran-GTP regulates MT nucleation by xNup107-160 / γ-TuRC complexes

(a) Magnetic beads coated with control IgG, anti-xNup160 or anti-xGCP2 antibodies were incubated in CSF XEEs for 90 minutes. The beads were re-isolated, washed and incubated with tubulin and GTP for 10 minutes. After fixation, the beads were pelleted onto coverslips and stained with antibodies against α-tubulin. Scale bar = 2.8 µm (b) Proteins associated with beads prepared as in (a) were eluted with low pH. Eluted fractions and untreated XEE (input) were analyzed by Western blotting for xNup160 and γ-TuRC components. Uncropped images of the blots are shown in Supplementary Information, Fig. S3. (c) Magnetic beads coated with control IgG, anti-xGCP2 or anti-xNup160 antibodies were incubated for 90 minutes in Control, Ran-T24N, Ran-Q69L or Importin-b-treated CSF XEEs. The beads were re-isolated, washed and incubated with tubulin and GTP for 10 minutes. After fixation, the beads were pelleted onto coverslips and stained with antibodies against α-tubulin. Scale bar = 2.8 µm (d) Proteins bound to beads as in (c) were eluted with low pH and analyzed along with untreated XEE (input) by western blotting for GCP2 and Nedd1 (γ-TuRC components). Uncropped images of the blots are shown in Supplementary Information, Fig. S3. (e) To quantitate the extent of MT nucleation, magnetic beads coated with control IgG, anti-xGCP2 or anti-xNup160 antibodies were in incubated in untreated, Ran-T24N-, Ran-Q69L or Importin-β-treated CSF XEEs, as in (c). In three independent experiments, we scored clusters containing three to six beads for associated MTs. The graph shows the mean percentage of clusters showing MTs in each case, +/− SD.

Figure 5. Model for Nup107-160 and γ-TuRC regulation at kinetochores

Step 1: Nup107-160 (dark blue) and active γ-TuRC (purple) bind to unattached kinetochores. We postulate that a transport cargo (plum) may be released in the high Ran-GTP environment that maintains γ-TuRC in an active state. Step 2: Nup107-160 and γ-TuRC nucleate MTs (green lines) at kinetochores, promoting the assembly of k-fibers. Step 3: RanGAP1 (magenta) is recruited along k-fibers. Its recruitment lowers Ran-GTP levels near kinetochores. The lower Ran-GTP levels in turn allow Importin-b or another import receptor to bind the cargo protein. Step 4: After the cargo molecule is sequestered, γ-TuRC becomes inactive (pale lilac), but k-fibers remain stable.

References

1. Orjalo AV, et al. The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly. Mol Biol Cell. 2006;17:3806–3818. - PMC - PubMed
1. Loiodice I, et al. The entire Nup107-160 complex, including three new members, is targeted as one entity to kinetochores in mitosis. Mol Biol Cell. 2004;15:3333–3344. - PMC - PubMed
1. Luders J, Stearns T. Microtubule-organizing centres: a re-evaluation. Nat Rev Mol Cell Biol. 2007;8:161–167. - PubMed
1. Wiese C, Zheng Y. Microtubule nucleation: gamma-tubulin and beyond. J Cell Sci. 2006;119:4143–4153. - PubMed
1. Torosantucci L, De Luca M, Guarguaglini G, Lavia P, Degrassi F. Localized RanGTP Accumulation Promotes Microtubule Nucleation at Kinetochores in Somatic Mammalian Cells. Mol Biol Cell. 2008 - PMC - PubMed

The Nup107-160 complex and gamma-TuRC regulate microtubule polymerization at kinetochores - PubMed (original) (raw)