Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle - PubMed (original) (raw)
Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle
Gohta Goshima et al. J Cell Biol. 2008.
Abstract
Since the discovery of gamma-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of gamma-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for gamma-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2-6) required for localizing gamma-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for gamma-tubulin may lie within the spindle, where augmin and gamma-tubulin function cooperatively to amplify the number of microtubules.
Figures
Figure 1.
Dgt2–6 proteins form a complex and associate with MTs. (A) Dgt2–6 proteins coimmunoprecipitate with one another but not with γ-TuRC subunits. Supernatant (S) and precipitated (P) fractions after extract incubation with anti-HA beads were immunoblotted for specific subunits. White lines indicate that intervening lanes have been spliced out. (B and C) Sucrose gradient sedimentation (B) and gel filtration chromatography (C) of Dgt2–6 proteins and γ-tubulin. Dgt proteins had a common peak at 11S and 7.5 nm, whereas the majority of γ-tubulin was in the larger γ-TuRC complex. The positions of standard markers are indicated. (D) FRAP analysis of GFP-Dgt5 on the spindle (n = 17). Bleached area is indicated by a white circle. Bar, 5 μm. Error bars show SD. See also Video 1 (available at
http://www.jcb.org/cgi/content/full/jcb.200711053/DC1
). (E) MT cosedimentation assay. Significant portions of Dgt2–6 proteins, but not control GFP, cosedimentated with MTs (pellet). WCE; whole cell extract.
Figure 2.
Dgt2–6 proteins are important for MT generation within the spindle and bipolar spindle formation. (A) MT density specifically inside the spindle decreased in the absence of Dgt2–6 or Dgrip71. Signal intensities at the centrosome and within the spindle (red boxes) were measured. Ratio of spindle and centrosome signal intensity after Dgt2–6 and Dgrip71 knockdowns (0.43–0.55; n = 5 each) were significantly (P < 0.0001) lower than in control (1.81 ± 0.15 SEM; n = 10). (B) A high-resolution still image of MT (green) and kinetochore marker CENP-ACid (red) after double Cnn/Dgt5 RNAi. Spindle morphology and chromosome alignment were severely impaired. (C–F) FRAP analysis of GFP-tubulin for control (n = 13), Dgt5 (n = 14), and Cnn (n = 17) RNAi spindles. Cells were arrested in metaphase by Cdc16 RNAi, entire half spindles (excluding the centrosome) were photobleached (time 0), and relative GFP intensities were plotted (with SEM) for the half spindles (D, yellow), spindle equators (E, yellow), and pole regions (F, yellow). Only partial fluorescence recovery, mostly from the kinetochores (C, arrows), was observed in the absence of Dgt5, whereas recovery was seen in the whole bleached area in control or Cnn spindles (_t_1/2 = 30 s). The initial steep recovery (0–5 s) is likely because of diffusion of GFP-tubulin in the cytoplasm because it was also detected for the cells in which MTs were depolymerized by colcemid (not depicted). See also Video 4 (available at
http://www.jcb.org/cgi/content/full/jcb.200711053/DC1
). Bars, 5 μm.
Figure 3.
Dgt is not involved in chromatin-mediated MT nucleation but is needed for chromosome alignment and K-fiber formation. (A) MT regrowth assay after control Cnn (top) or double Cnn/Dgt5 (bottom) RNAi. MTs (green) initially appeared around chromatin (blue) in both samples, whereas γ-tubulin (red) failed to localize to the MTs in the absence of Dgt5. The double RNAi cells failed to form robust bipolar spindles. (B) Delayed chromosome alignment and anaphase onset in the bipolar spindle in the absence of Dgt6 (mCherry-tubulin [red] and Mis12-GFP [green]). See also Video 5 (available at
http://www.jcb.org/cgi/content/full/jcb.200711053/DC1
). Mean duration (and SEM) from NEBD to metaphase and from metaphase to anaphase onset is plotted (control; RNAi against pBluescript sequences). (C) kMTs visualized after 10 min of colcemid treatment. Some kinetochores (Mis12-mCherry) do not associate with K-fibers (GFP-tubulin) in the absence of Cnn/Dgt5. See text for quantitation. Boxes on the left indicate the enlarged areas shown on the right. Bars, 5 μm.
Figure 4.
Characterization of human Dgt6 in HeLa cells. (A) Alignment of N-terminal regions of Dgt6 proteins from D. melanogaster, Xenopus laevis, and Homo sapiens. Identical amino acids are boxed and the similar ones are hatched. (B) Uniform spindle localization of GFP-tagged human Dgt6 in metaphase (green). (C) Dim γ-tubulin and sparse MT phenotypes after RNAi knockdown of hDgt6 in HeLa cells (72 h). (D) Quantitation of γ-tubulin or MT level at centrosomes and spindles. Ratio of spindle and centrosome signal intensity in hDgt6 RNAi cells (γ-tubulin, 0.17 ± 0.02 SEM [n = 14]; MT, 0.46 ± 0.02 SEM [n = 19]) was significantly (P < 0.0005) lower than in control (γ-tubulin, 0.33 ± 0.04 SEM [n = 7]; MT, 0.75 ± 0.02 SEM [n = 15]). γ-tubulin (red) and DNA (blue) were counterstained. Bars, 10 μm.
Figure 5.
Model for augmin-dependent MT amplification in the spindle. (A) MTs are initially nucleated from centrosomes (brown) and near chromosomes/kinetochores (blue) in early prometaphase. γ-TuRC (red) is the major nucleator of these MTs. (B) The augmin/γ-TuRC machinery (yellow/red) then binds to those MTs, preferably to stable ones such as kMTs, and nucleates new MTs. The new MTs, which are oriented parallel or slightly angled to the template MTs, contribute to K-fiber generation and net kinetochore capture by the search-and-capture mechanism.
References
- Gadde, S., and R. Heald. 2004. Mechanisms and molecules of the mitotic spindle. Curr. Biol. 14:R797–R805. - PubMed
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