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. 2009 Apr 28;106(17):6998-7003.
doi: 10.1073/pnas.0901587106. Epub 2009 Apr 14.

The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells

Ryota Uehara  1 , Ryu-suke Nozawa, Akiko Tomioka, Sabine Petry, Ronald D Vale, Chikashi Obuse, Gohta Goshima
Affiliations

The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells

Ryota Uehara et al. Proc Natl Acad Sci U S A. .

Abstract

The mitotic spindle is constructed from microtubules (MTs) nucleated from centrosomes, chromosome proximal regions, and preexisting spindle MTs. Augmin, a recently identified protein complex, is a critical factor in spindle MT-based MT generation in Drosophila S2 cells. Previously, we identified one subunit of human augmin. Here, by using mass spectrometry, we identified the full human augmin complex of 8 subunits and show that it interacts with the gamma-tubulin ring complex (gamma-TuRC). Unlike augmin-depleted S2 cells, in which the defect in spindle-mediated MT generation is mostly compensated by centrosomal MTs, augmin knockdown alone in HeLa cells triggers the spindle checkpoint, reduces tension on sister kinetochores, and severely impairs metaphase progression. Human augmin knockdown also reduces the number of central spindle MTs during anaphase and causes late-stage cytokinesis failure. A link between augmin and gamma-TuRC is likely critical for these functions, because a gamma-TuRC mutant that attenuates interaction with augmin does not restore function in vivo. These results demonstrate that MT generation mediated by augmin and gamma-TuRC is critical for chromosome segregation and cytokinesis in human cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of Drosophila Dgt7, Dgt8, and Dgt9 as augmin subunits. (A) Dim γ-tubulin and MT intensities within the spindle after RNAi knockdown of Dgt7/CG2213, Dgt8/CG13879, and Dgt9/CG13914. The density of γ-tubulin or MTs inside the spindle decreased after RNAi of Dgt6–9 (n ≥ 5 each). The ratio of signal intensity in spindle (location at “b” in the lower image) to that in centrosome (“a”), which was normalized to control, is shown (±SEM). The normalized ratio for γ-tubulin and MT after Dgt7–9 knockdowns were significantly (P < 0.004) lower than in control. (B) Dgt7-HA, Flag-Dgt8, and Dgt9-GFP proteins coimmunoprecipitate with endogenous Dgt6. The supernatant (S) and precipitated (P: 40× volume of S) fractions after immunoprecipitation with anti-HA, anti-Flag, and anti-GFP antibodies were immunoblotted. (C) Dgt7-GFP, Dgt9-GFP, and Flag-Dgt8 were localized to spindle MTs during metaphase. Flag-Dgt8 was stained by anti-Flag antibody. Blue, DAPI. (Scale bars, 5 μm.)
Fig. 2.
Fig. 2.
Identification of human augmin as the 8-subunit complex. (A) Immunoblotting of hDgt6 for the control, siRNA-treated, and GFP-hDgt6-expressing cells. GFP-hDgt6 was expressed 2-fold more than endogenous hDgt6. (B) Coimmunoprecipitation of 7 HA-tagged hDgt6-associated proteins with endogenous hDgt6. The supernatant (S) and precipitated (P: 50× volume of S) fractions after anti-HA incubation were immunoblotted with ati-hDgt6 antibody. The asterisks in A and B indicate cross-reaction of anti-hDgt6 antibody to other proteins. (C) Localization of GFP-tagged C14orf94 and C4orf15 proteins (green) in metaphase (Left) and interphase (Right). The proteins were abundant at centrosomes during interphase, whereas spindle localization with slight enrichment near the poles was detected during metaphase. Chromosomes (blue) and γ-tubulin (red) were counterstained. (Scale bar, 5 μm.) (D) Two-hybrid interaction between full-length Hice1 and hDgt6. Colonies were visible on the 3-aminotriazole (3-AT)-containing plate only when both proteins were expressed. (E) Gel filtration analysis of human augmin subunits. A similar peak can be observed between hDgt6 and the hDgt6-interacting proteins at a Stokes radius of ≈9.5 nm, suggesting that they form a stable complex. The peak of KIAA0841 was shifted by one fraction to the left, probably because of GFP tagging of this protein (others were tagged with HA). The reason for the shift of Hice1 to higher molecular mass fractions is unclear but might be aggregation of a subpopulation of this protein in the extracts.
Fig. 3.
Fig. 3.
Dgt phenotype after RNAi knockdown of human augmin subunits. (A) Reduced spindle localization of γ-tubulin or MT after knockdowns of human augmin subunits. A hDgt3-knockdown cell is shown as a representative. (B) Quantification of spindle localization of γ-tubulin or MT. The ratio of spindle to centrosome signal intensity after knockdowns of human augmin subunits, which was normalized to control (±SEM), was significantly (P < 0.01) lower than that in the control (luciferase RNAi) (n ≥ 9). (C) Astral MTs (arrowheads) emanated from the centrosome (green) in metaphase-arrested cells depleted of hDgt6. GFP-tubulin was imaged in a living cell, and the region around one spindle pole is presented. (Scale bars, 5 μm.)
Fig. 4.
Fig. 4.
Reduced metaphase kinetochore tension in the absence of human augmin. (A) Mitotic cells (rounded cells in the phase-contrast microscopy image) were accumulated after hDgt6 RNAi. (B) The average intensity of the kinetochore-localized Mad2 signals normalized to that of CENP-A (n ≥ 41 kinetochores from ≥3 cells each). The Mad2 signals diminished on the aligned kinetochores in both the control and hDgt6-depleted cells. The brightest kinetochore Mad2 signal in each cell was also comparable between RNAi and control samples (Fig. S3C). (C) Reduced tension on sister kinetochores after hDgt6 RNAi. (Lower) The interkinetochore distance of the congressed chromosomes (blue) was quantified based on immunostaining of CENP-B (green) and CENP-C (red) (≥20 pairs of kinetochores from ≥6 cells each). (Upper) An enlarged image of a pair of sister kinetochores. Treatment with the proteasome inhibitor MG132 (10 μM, 90 min), which prolongs metaphase, did not alter the interkinetochore distance of hDgt6-depleted cells. (Scale bars, 5 μm.)
Fig. 5.
Fig. 5.
Cytokinesis failure in the absence of human augmin. (A) Binucleated cells were increased after hDgt6 RNAi. (B) Cleavage furrow regression was observed after hDgt6 RNAi (40–45 min) (phase-contrast image). Images were acquired every 5 min. (C) Lowered central spindle MT density (green) after hDgt6 RNAi in late anaphase. Blue, DNA. (D) Localization of GFP-hDgt6 and γ-tubulin on the central spindle MTs during late anaphase. (Scale bars, 5 μm.)
Fig. 6.
Fig. 6.
Augmin–γ-TuRC interaction is necessary for spindle MT generation. (A) hDgt6 was efficiently coimmunoprecipitated with NEDD1 but not with a point-mutant NEDD1 (S411A). HA-tagged NEDD1 or NEDD1 (S411A) expressed in HeLa mitotic cells was immunoprecipitated. (B) NEDD1 was efficiently coimmunoprecipitated with N-terminal truncated hDgt6(480–955) but not with C-terminal truncated hDgt6(1–438). Mitotic HeLa cells expressing Myc-NEDD1 with or without HA-hDgt6(1–438) or HA-hDgt6(480–955) mutant were used. (C) hDgt6(1–438), but not hDgt6(480–955), associates with augmin subunit Cep27. Myc-hDgt6(1–438) or Myc-hDgt6(480–955) with or without HA-Cep27 was immunoprecipitated. A 20-fold higher amount of the sample was loaded in the pellet lane in A–C. (D) The dgt phenotype was rescued by full-length GFP-hDgt6 but not by GFP-hDgt6(1–438). (Scale bar, 5 μm.) (E) Quantification of γ-tubulin (Left) or MT (Center) intensity within the spindle. The normalized ratio of spindle to centrosome signal intensity (±SEM) after knockdowns of hDgt6 was significantly (P < 0.0007) restored when siRNA-insensitive, full-length GFP-hDgt6 was expressed, but less efficiently by GFP-hDgt6(1–438) (n ≥ 7). (Right) Percentage of binucleated cells after depletion of endogenous hDgt6 was lowered by transfection of siRNA-insensitive full-length GFP-hDgt6, but not by a truncated GFP-hDgt6(1–438) or GFP alone (n ≥ 77).
Fig. 7.
Fig. 7.
Model for the augmin- and γ-TuRC-dependent MT amplification in the mitotic spindle in human cells. (A) The augmin/γ-TuRC machinery generates MTs within the spindle. The new MTs contribute to kinetochore MT formation and net kinetochore capture by the search-and-capture mechanism. This model was originally proposed in ref. . A similar mechanism may work within the central spindle during anaphase. (B) A speculative molecular model of the augmin/γ-TuRC-dependent MT generation. Human and Drosophila augmin were identified as 8-subunit complexes (human augmin subunits are described in this diagram). Hice1 is known to bind to MTs directly in vitro (14). Hice1-hDgt6 interaction was detected in the yeast 2-hybrid assay, whereas NEDD1-containing γ-TuRC was efficiently coprecipitated with the C-terminal fragment of hDgt6. Attenuation of this interaction caused defects in spindle MT generation. Thus, we suggest that augmin recruits γ-TuRC to spindle MTs, where γ-TuRC nucleates new MTs. This MT amplification process is critical for generating kinetochore tension and also completion of cytokinesis in human tissue culture cells.
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