Since the discovery of γ-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. Efficient assembly of a mitotic spindle and stable attachment of microtubules (k-fibers) to kinetochores are essential for the high fidelity of chromosome segregation. Both spindle assembly and k-fiber formation require robust nucleation and polymerization of microtubules mediated by the γ-tubulin ring complex (γTuRC). However, mislocalization of γ-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila, suggesting that a critical function for γ-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2–6) required for localizing γ-tubulin to spindle microtubules. This study has shown that the Dgt proteins interact, forming a stable complex. Spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex, named “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. A functional human homologue of Dgt6 was identified. These results suggest that an important mitotic function for γ-tubulin may lie within the spindle, where augmin and γ-tubulin function cooperatively to amplify the number of microtubules (Goshima, 2008).

Microtubule (MT) nucleation from centrosomes requires γ-tubulin and its several associated proteins (γ-tubulin ring complex [γ-TuRC]), which are docked onto the pericentriolar material. It has been suggested that MT growth and shrinkage from the centrosome allows chromosomes to capture and stabilize MTs and thus build a mitotic spindle. However, centrosome-mediated MT nucleation is dispensable for functional mitotic spindle formation and centrosome-based “search and capture” may be insufficient to capture all the kinetochores in the rapid time frame observed in living cells (Goshima, 2008).

Recent observations have revealed that spindle MTs can also be generated by noncentrosomal pathways. The best studied is the chromatin-mediated pathway, in which high concentration of Ran-GTP on chromatin stimulates γ-tubulin–dependent MT nucleation proximal to chromatin in meiotic and somatic cells. Another recently suggested mechanism for MT generation involves nucleation from existing MTs in the spindle by docking γ-tubulin onto those MTs. This idea stemmed from observations that growing MT plus ends visualized by EB1-GFP emerge throughout the body of the spindle, not solely at centrosomes and chromosomes, and that γ-tubulin is localized throughout the mitotic spindle. Imaging of EB1-GFP also revealed that MT growth within the spindle is preferentially directed toward the chromosomes. Interestingly, directional growth of MTs from existing MTs has been observed in interphase Schizosaccharomyces pombe and plant cells, although it is unclear whether this mechanism is the same as the one that operates within the mitotic spindle (Goshima, 2008).

>Using a genome-wide RNAi screen for mitotic spindle morphology in the Drosophila melanogaster S2 cell line, several genes were identified involved in γ-tubulin localization in metaphase (Goshima, 2007). Among them are the outer γ-TuRC subunits (Dgrip71, 75, 128, and 163) and five uncharacterized proteins (called Dgt2–6, with Dgt an acronym for dim γ-tubulin) that are necessary for localizing γ-tubulin to spindle MTs but not to the centrosomes. Dgt proteins do not have any known motifs from which their molecular activity can be predicted. This paper demonstrates that the Dgt proteins form an MT binding complex involved in MT generation within the spindle and that this process plays a vital role in mitotic spindle assembly. Human Dgt6 was identified, indicating that the Dgt-dependent MT generation pathway is conserved in human mitosis.

Dgt2–6 proteins in S2 extracts comigrated during sucrose gradient centrifugation and gel filtration chromatography. γ-TuRC was detected as a much bigger complex in both assays. It was also found that reduction of one Dgt reduced the levels of other Dgts (an exception was Dgt2-HA). Multisubunit complexes are often unstable when one component is absent, thus likely explaining this result. Knockdown of γ-TuRC components, in contrast, did not significantly affect the levels of Dgt proteins. Collectively, these experiments suggest that Dgt2–6 proteins coassemble into a stable complex. The size of the complex was estimated from the size-based fractionation as 340 kD, which is a little larger than that predicted from the sum of the individual molecular masses of the five proteins (265 kD) (Goshima, 2008).

Previous study showed that GFP-tagged Dgt2 and 4–6 localize to spindle MTs. Spindle localization of endogenous Dgt4 was confirmed by immunofluorescence using a specific polyclonal antibody. GFP-Dgt5 signal was clearly detected on kinetochore MTs (kMTs) but was greatly reduced at the spindle equator where only non-kMTs are present, suggesting that the Dgt complex may preferentially localize to stable MTs within the spindle. FRAP showed a fast turnover of GFP-Dgt5 on the metaphase spindle, suggesting a rapid binding and dissociation of the Dgt complex with MTs in vivo. Whether the Dgt complex interacts with exogenous MTs added to S2 cell extracts was tested, and it was found that all of the Dgts cosedimented with taxol-stabilized MTs, whereas expressed GFP (as a control) remained in the supernatant. However, additional work will be required to establish whether the Dgt–MT interaction is direct or mediated by other proteins (Goshima, 2008).

A previous study reported live imaging of Dgt5-depleted cells and showed an increased number of monopolar spindles, reduced density of MTs within bipolar spindles, and chromosome misalignment (Goshima, 2007). This paper extend these studies of Dgt-depleted cells to better understand the mechanisms that underlie these phenotypes. In the experiments described in the subsequent sections, a single Dgt was depleted. However, it was also found that knockdown of any of the five Dgts generates identical phenotypes (Goshima, 2008). <

MT staining intensity within the spindle was significantly reduced after Dgt RNAi, but the spindle phenotype was not as severe as that seen after knockdown of γ-tubulin. This phenotypic difference may be caused by γ-tubulin continuing to nucleate MTs from centrosomes and chromosomes after Dgt RNAi. Indeed, Dgt knockdown did not diminish MT nucleation at centrosomes, and centrosome-nucleated astral MTs were, on average, much longer in Dgt5-depleted cells than in control cells, which might be because of an increase in the pool of soluble tubulin resulting from impaired nucleation and decreased MT polymer mass within the spindle. It was therefore reasoned that the Dgt phenotype might become more severe if γ-tubulin–mediated nucleation at the centrosome was simultaneously inhibited (Goshima, 2008).

To test this idea, double RNAi of Dgt and Cnn (centrosomin), the protein responsible for docking γ-tubulin to the centrosomes was performed. Cnn depletion alone has little effect on spindle morphology and function. However, after depletion of both Cnn and Dgt, fixed cell analysis and time-lapse imaging (1–4 h) revealed that spindle morphology was dramatically impaired. MTs were longer, sparse, and wavy, spindle poles were frayed, and the spindle displayed an overall disorganized morphology. This is one of the most severe mitotic defects reported in S2 cells, virtually phenocopying RNAi of γ-tubulin itself. These results suggest that Dgt and Cnn, as γ-tubulin–localizing factors, both contribute to making the MTs that comprise the spindle but that the Dgt–γ-tubulin activity in the spindle plays a dominant role (Goshima, 2008).

To better understand the function of the Dgt complex in the metaphase spindle, FRAP analysis was performed of GFP-tubulin in the spindle. In control cells, the fluorescence recovered after photobleaching of a half spindle with a t_1/2 = 30 s. Further analyses revealed that the recovery took place everywhere within the half spindle, including the spindle pole regions, although the recovery was faster near the spindle equator than spindle poles, likely reflecting polymerization at the plus ends of kMTs. MT turnover throughout the spindle also has been reported in mammalian cells and sea urchin embryos. Interestingly, very similar recovery curves were found for Cnn-depleted cells, indicating that centrosomes play a relatively minor role in generating new MTs during metaphase. In contrast, fluorescence recovery of the half spindle was slower in the absence of Dgt5, particularly near the spindle pole. Relatively fast recovery took place near the spindle equator, suggesting that kMT polymerization is not impaired in cells lacking Dgt5. However, the recovery was slower than that of control cells also at this region, implicating that Dgt5 is also involved in chromatin-proximal MT generation. FRAP analysis in Cnn/Dgt5-codepleted cells showed slow recovery of GFP-labeled spindle MTs, which was similar to single Dgt5 RNAi. Collectively, these results indicate that the Dgt complex, more prominently than the centrosome, is required for the MT generation taking place within the metaphase spindle (Goshima, 2008).

To better understand the monopolar phenotype, long time-lapse imaging of monopolar spindles was performed and it was found that all remained in a monopolar state for >150 min and did not convert to monastral bipolar spindles. This is quite different from wild-type S2 cells, which undergo monopolar to monastral bipolar conversion within 30 min, a process that involves MT generation around the chromatin and then focusing of these MTs to create an acentrosomal pole. Klp61F/Kinesin-5 is critical for this conversion process. However, because this kinesin localized normally to spindle MTs and the centrosome after Dgt5 RNAi, the lowered bipolar conversion cannot be explained by mislocalization of this kinesin in the absence of Dgt (Goshima, 2008).

Other potential explanations for the severe defect in monopolar to monastral bipolar conversion are that Dgt depletion interferes either with chromosome-mediated MT nucleation or the subsequent stabilization/amplification of chromatin-nucleated MTs. To test these possibilities, an assay was established for observing the nucleation of MTs from chromatin. First, Cnn-depleted cells (to eliminate centrosome-based nucleation) were arrested in prometaphase by colcemid-induced MT depolymerization. After colcemid washout, cells were fixed and stained for MT and γ-tubulin every 15 min. In the single Cnn RNAi cells, MTs appeared around chromatin after 45 min, followed by formation of robust bipolar spindles by 75 min. γ-Tubulin was localized to the newly formed MTs. In cells depleted of both Dgt5 and Cnn, MTs appeared around chromatin with the same timing and in a manner indistinguishable from the single Cnn RNAi cells. However, γ-tubulin did not colocalize with these MTs, and cells did not form robust bipolar spindles. Identical results were obtained after RNAi of other Dgt proteins. These data strongly suggest that the Dgt complex is dispensable for the initial nucleation of MTs around chromatin but is important in the subsequent amplification and/or stabilization of MTs, thereby producing sufficient numbers of MTs for robust chromatin-mediated bipolar spindle formation (Goshima, 2008).

High-throughput long-term live-cell microscopy was applied on a cell line stably expressing both mCherry-tubulin and Mis12-GFP (a kinetochore marker) to follow the time course of chromosome positioning immediately after nuclear envelope breakdown. In control cells, Mis12-GFP congressed to the metaphase plate at 18 ± 2 min after NEBD. Sister chromatid separation took place at 11 ± 2 min after congression. In contrast, Dgt6 knockdown cells took 35 ± 4 min for congression and a further 19 ± 2 min for separation. However, despite this significant delay, the majority of the Dgt6-depleted cells eventually achieved metaphase congression and successful sister chromatid separation, suggesting that kinetochore–MT interactions were still present. The moderate misalignment phenotype was also found in other Dgt RNAi. However, in the absence of both centrosomes and Dgt, chromosomes were much more severely misaligned and rarely congressed to form a stable metaphase plate (six of seven Cnn/Dgt6 double RNAi cells observed never formed a clear metaphase plate within a 4-h span of observation) (Goshima, 2008).

Defects were observed in chromosome congression after Dgt depletion may be the consequence of decreased numbers of kinetochore-MT associations. To explore this idea, a MT depolymerization assay was applied, in which colcemid treatment rapidly and preferentially destabilizes dynamic non-kMTs while stable kMT bundles (K-fibers) remain intact for several minutes. In control Cnn-depleted cells, K-fibers (visualized by GFP-tubulin) were detected for all the kinetochores (Mis12-mCherry) in 17/20 cells, whereas only 3 cells had a kinetochore that was not associated with MTs (3/1024, 800)33 C). However, in 14/20 cells after Cnn/Dgt5 depletion, a subset of kinetochores (2 ± 0.9) did not associate with K-fibers. Even when K-fibers were still present, they were disorganized and the kinetochores were not aligned in the middle of the spindle. This assay revealed that Dgt5-depleted spindles lack robust K-fibers, which may account for the chromosome alignment defects found in these cells (Goshima, 2008).

BLAST identified human FAM29A (family with sequence similarity, member 29A), an uncharacterized gene that has an 80-aa domain with 41% identity to D. melanogaster Dgt6. Similar to D. melanogaster Dgt6, it was found that GFP-tagged FAM29A localized throughout the metaphase spindle of HeLa cells. siRNA treatment of FAM29A was performed, and it was found that γ-tubulin and MT signals in the spindle, but not on the centrosomes, were significantly diminished. RNAi of FAM29A also caused a mitotic delay (two- to threefold higher mitotic index than control cells). Collectively, it is concluded that FAM29A is the human orthologue of Dgt6 (referring to it as hDgt6), and it is suggested that Dgt-dependent MT generation also plays an important role in human mitosis (Goshima, 2008).

This study shows that Dgt proteins associate to form a stable complex. Based upon its role in increasing MT numbers in the spindle, it is proposed that this complex be called “augmin” from the Latin verb augmentare, which means to increase. The role of augmin in increasing spindle MT density appears to be very important for building K-fibers and enabling chromatin-mediated MT nucleation to proceed toward the formation of a bipolar-shaped spindle, particularly when centrosome function is attenuated (Goshima, 2008).

The results suggest a model for how centrosome-, chromosome-, and spindle-based MT nucleation processes may cooperate to build mitotic spindles. After NEBD in somatic cells, the most obvious generation of new MTs occurs at centrosomes, producing astral MT arrays. MTs are also nucleated in the vicinity of chromosomes immediately after NEBD. These processes are critical for generating the first set of mitotic MTs, enabling the process of spindle assembly to begin. However, after the first sets of MTs are formed, it is proposed that augmin–γ-TuRC nucleates MT growth from existing MTs, thus providing a powerful mechanism for rapidly amplifying the number of MTs within the spindle to facilitate chromosome capture and K-fiber formation) (Goshima, 2008).

It is postulated that kMTs, initially generated via the centrosome or chromatin pathway, are used as templates for binding augmin, which in turn recruits and activates γ-TuRC for MT nucleation. The outer γ-TuRC subunits appear to be critical for this specific spindle function of γ-tubulin because knockdown of these subunits produces a very similar phenotype to augmin knockdown, although evidence for a direct interaction between augmin and γ-TuRC has yet to be obtained. An intriguing possibility is that augmin may dock onto an MT in a manner that positions γ-TuRC to preferentially nucleate new MT growth with the same polarity as the parent MT (Goshima, 2008).

As an alternative to this model, augmin might increase the number of spindle MTs by activating an MT-severing enzyme, although this is thought to be unlikely because RNAi of katanin and other AAA ATPases (e.g., spastin) does not give rise to the same mitotic phenotype as Dgt or γ-TuRC RNAi. Another class of models is that augmin increases spindle MT density by either stabilizing, elongating, or transporting MTs that are nucleated at the kinetochore/chromatin and that augmin–γ-tubulin are not involved in MT nucleation within the spindle. Although this possibility cannot be ruled out, the FRAP results showing the concomitant recovery of fluorescence at the center of the spindle and the spindle poles suggest that MTs are being formed throughout the spindle and not just near the chromatin. The notion of an MT amplification process is also supported by in vitro studies of the assembly of MT asters in X. laevis extract and accompanying computational simulations. The exact mechanism by which augmin increases MT density awaits future work, most decisively by in vitro reconstitution of the process with purified proteins (Goshima, 2008).

In addition to the mitotic spindle, there are other cellular MT networks in animals that are unlikely to be exclusively built by centrosome-based nucleation and assembly processes, such as axons or the meiotic spindle in oocytes. An MT-templated MT nucleation reaction could constitute an excellent means for generating new MTs while preserving the polarity of an existing MT array. Augmin is a candidate to play a role in this and numerous other cases where polarized noncentrosomal MTs are generated (Goshima, 2008).