Microtubule Nucleation in Mitosis by a Rangtp-Dependent Protein Complex

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provided by Elsevier - Publisher Connector Current Biology 25, 131–140, January 19, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2014.11.025 Article Microtubule Nucleation in Mitosis by a RanGTP-Dependent Protein Complex

Jacopo Scrofani,1,2 Teresa Sardon,1,2 Sylvain Meunier,1,2,* the precise role of these factors in chromosomal-dependent and Isabelle Vernos1,2,3,* MT assembly is still in many cases ill defined, and we therefore 1Cell and Developmental Biology Programme, Centre for still do not understand the mechanism at play. Because these Genomic Regulation (CRG), Doctor Aiguader 88, MTs are essential for building a functional bipolar spindle 08003 Barcelona, Spain whether centrosomes are present or not [7], understanding 2Universitat Pompeu Fabra (UPF), Doctor Aiguader 88, this pathway is essential to fully understand cell division. 08003 Barcelona, Spain In higher eukaryotic cells, MT nucleation relies on the 3Institucio´ Catalana de Recerca i Estudis Avanç ats (ICREA), g-tubulin ring complex (gTuRC), a multisubunit complex con- Passeig de Lluis Companys 23, 08010 Barcelona, Spain stituted by multiple copies of g-tubulin and a number of associated proteins called gamma tubulin complex proteins (GCPs) [8, 9]. The gTuRC and its adaptor protein NEDD1 are required Summary for all the known pathways of MT nucleation in animal mitosis [10, 11]. These pathways are defined spatially in the mitotic Background: The g-tubulin ring complex (gTuRC) is a multisu- cell by occurring at the centrosomes, on preexisting MTs, and bunit complex responsible for microtubule (MT) nucleation around the chromosomes. Work from different groups has in eukaryotic cells. During mitosis, its spatial and temporal shown that an important target for gTuRC regulation in mitosis regulation promotes MT nucleation through different path- is NEDD1 through its phosphorylation by different kinases ways. One of them is triggered around the chromosomes by [12–15]. We recently showed that chromosomal MT assembly RanGTP. Chromosomal MTs are essential for functional spin- requires NEDD1 phosphorylation on S405 by Aurora A [16]. dle assembly, but the mechanism by which RanGTP activates Another factor that is essential for RanGTP-dependent MT MT nucleation has not yet been resolved. nucleation is TPX2 [17], but this RanGTP-regulated protein Results: We used a combination of Xenopus egg extracts was shown to play multiple roles during spindle assembly and in vitro experiments to dissect the mechanism by which [18, 19]. Interestingly, TPX2 is a RanGTP-dependent activator RanGTP triggers MT nucleation. In egg extracts, NEDD1- of Aurora A [20–23]. However, the link between this activation coated beads promote MT nucleation only in the presence of and the phosphorylation of NEDD1 by Aurora A has not been RanGTP. We show that RanGTP promotes a direct interaction established yet. Although TPX2 can nucleate MTs in vitro between one of its targets, TPX2, and XRHAMM that defines [24], experiments performed in Xenopus egg extracts showed a specific gTuRC subcomplex. Through depletion/add-back that RanGTP-dependent MT nucleation does not occur in experiments using mutant forms of TPX2 and NEDD1, we the absence of g-tubulin, suggesting that the mechanism at show that the activation of MT nucleation by RanGTP requires play is far more complex and involves more components both NEDD1 phosphorylation on S405 by the TPX2-activated in addition to TPX2 [25]. The mechanism by which RanGTP Aurora A and the recruitment of the complex through a triggers MT nucleation is therefore still unresolved [26–28]. TPX2-dependent mechanism. Here, we used the Xenopus laevis egg extract system in com- Conclusions: The XRHAMM-gTuRC complex is the target for bination with in vitro experiments to identify the components of activation by RanGTP that promotes an interaction between the MT nucleation machinery involved in the RanGTP-depen- TPX2 and XRHAMM. The resulting TPX2-RHAMM-gTuRC dent MT assembly pathway and the mechanism driving its supracomplex fulfills the two essential requirements for activation in the M-phase cytoplasm. the activation of MT nucleation by RanGTP: NEDD1 phosphorylation on S405 by the TPX2-activated Aurora A and the Results recruitment of the complex onto a TPX2-dependent scaffold. Our data identify TPX2 as the only direct RanGTP target RanGTP Activates the MT Nucleation Activity of and NEDD1 as the only Aurora A substrate essential for the NEDD1-gTuRC activation of the RanGTP-dependent MT nucleation pathway. To investigate the mechanism by which RanGTP drives MT nucleation in M phase, we used Xenopus laevis egg extracts Introduction in which the whole pathway can be triggered by addition of a GTP hydrolysis-deficient form of Ran (RanQ69L) bound to During mitosis, different microtubule (MT) nucleation path- GTP (RanGTP) [29, 30]. Previous data showed that the gTuRC ways drive the assembly of dynamic MTs to organize a bipolar adaptor protein NEDD1 is essential for RanGTP-dependent spindle [1]. One of them relies on a gradient of guanosine MT nucleation [13, 16]. To determine whether the associa- triphosphate (GTP)-bound Ran (RanGTP) centered on the tion of NEDD1 with the gTuRC is regulated by RanGTP, we chromosomes that promotes the dissociation of nuclear immunoprecipitated NEDD1 from extracts in the presence localization signal (NLS)-containing proteins from karyopher- or absence of RanGTP. In both conditions, NEDD1 coimmuno- ins and drives MT nucleation, stabilization, and organization precipitated the components of the gTuRC XGrip109 (GCP3), [2]. Several spindle assembly factors have been identified g-tubulin, and XGrip195 (GCP6; Figure 1A). These results sug- as direct targets of RanGTP during mitosis [3–6]. However, gest that, in extracts, NEDD1 associates with intact gTuRCs in a RanGTP-independent manner. We then addressed the functionality of this complex by examining whether the *Correspondence: [email protected] (S.M.), [email protected] NEDD1 beads were associated with MTs in the extract. (I.V.) Surprisingly, the NEDD1 beads only generated MT asters in 132

XRHAMM Defines a Subset of gTuRCs Specifically Involved in RanGTP-Dependent MT Nucleation In addition to NEDD1 and g-tubulin, two proteins, TPX2 and XRHAMM, have been reported to be essential for RanGTP- induced MT assembly [17, 31, 32]. TPX2 has been extensively characterized in the egg extract system, where it was shown that it does not stabilize MTs, thereby suggesting that its role is at the level of MT nucleation [27, 28]. The precise role of RHAMM in the chromosomal pathway is less clear [33, 34], although recent works also suggested that it plays a role in MT nucleation [32, 35]. To better define this role, we investi- gated XRHAMM function in RanGTP-dependent MT assembly using the Xenopus egg extract system. As previously shown, the gTuRC components XGrip109 and g-tubulin coimmunoprecipitated with XRHAMM in egg extract (Figure 2A) [32]. In addition, we also detected NEDD1 and XGrip195 in the immunoprecipitation, suggesting that XRHAMM interacts with the full gTuRC (Figure 2A). Interest- ingly, these interactions were not regulated by RanGTP (Figure 2A), suggesting that the mitotic Xenopus egg extract contains a stable XRHAMM-NEDD1-gTuRC complex. To determine whether this complex is in fact a general MT nucleation complex in egg extracts, we monitored MT nucleation by the sperm-associated immature centrosome upon incubation in egg extracts depleted for either NEDD1, TPX2, or XRHAMM (Figures 2B and S2)[36]. As expected, MT nucleation at the centrosome was strongly impaired in NEDD1-depleted extract. In contrast, centrosomal MT nucleation was as efficient in XRHAMM or TPX2-depleted extracts as in control extracts (Figure 2B). These results strongly indicated that XRHAMM and TPX2 are not required for MT nucleation at the centrosome, suggesting that their function is specific for the RanGTP pathway. Figure 1. RanGTP Activates gTuRC MT Nucleation Activity Altogether, our data show that XRHAMM forms a complex (A) Western blot of control IgG and anti-NEDD1 immunoprecipitation from egg extract with or without RanGTP to detect the gTuRC components Xgrip195 with NEDD1-gTuRC not directly regulated by RanGTP that is and Xgrip109 and g-tubulin as well as NEDD1, as indicated. IP, immuno- not involved in centrosomal-regulated MT nucleation. precipitation. (B) Experimental design for the experiments shown in (C) and (D). EE, egg RanGTP Promotes the Interaction between TPX2 extract. and the RHAMM-NEDD1-gTuRC Complex (C) MT assembly around anti-NEDD1 beads incubated in egg extract It was previously reported that XRHAMM coimmunoprecipi- containing rhodamin-tubulin. The graph shows the percentage of beads associated with MTs. Quantifications were performed on squashes. Repre- tates with TPX2 in a RanGTP-independent manner [32]. Using sentative images are shown (right). Beads are autofluorescent. Data our anti-XRHAMM or anti-TPX2 antibodies, we found that obtained from three independent experiments counting more than 500 XRHAMM and TPX2 coimmunoprecipitate in a RanGTP- beads for each condition. Error bars: SD. ***p < 0.001 using X-squared dependent way in egg extracts (Figure 3A). To determine test. The scale bars represent 10 mm. whether the two proteins interact directly, we performed pull- (D) MT nucleation assay around anti-NEDD1 beads retrieved from egg downs in vitro using recombinant GFP-TPX2 and a His-tagged extract and incubated in pure tubulin. Beads were spun onto coverslips and processed for immunofluorescence to visualize MTs. The graph shows C-terminal fragment of XRHAMM (137 last amino acids: 1,038– the percentage of beads associated with MTs. Representative images are 1,175). As shown in Figure 3B, anti-GFP antibodies pulled down shown (right). Beads are autofluorescent. Data obtained from four indepen- both proteins, indicating that TPX2 interacts directly with the dent experiments counting more than 500 beads for each condition. Error C-terminal region of XRHAMM (Figures 3B and S3). These re- bars: SD. ***p < 0.001 using X-squared test. The scale bars represent 10 mm. sults suggested that RanGTP promotes the direct interaction between its target TPX2 and XRHAMM. Because XRHAMM interacts with the gTuRC (Figure 2A) [32], we then tested extracts containing RanGTP (Figures 1B and 1C). To discrimi- whether TPX2 coimmunoprecipitated with the gTuRC. Indeed, nate between an activation of MT nucleation and an increase of we found that TPX2 coimmunoprecipitated with NEDD1 and MT stabilization, we retrieved the NEDD1-gTuRC beads from g-tubulin in a RanGTP-dependent manner (Figure 3C). We the egg extract and incubated them in pure tubulin (Figure 1B). then tested whether this interaction was XRHAMM dependent. Consistently with our previous result, beads retrieved from We found that in XRHAMM-depleted extracts, TPX2 did not a RanGTP-containing egg extract promoted MT assembly coimmunoprecipitate with NEDD1 (Figure 3D). Therefore, our whereas those retrieved from a control extract did not (Fig- data strongly suggest that RanGTP induces a specific direct ure 1D and Figure S1 available online). Altogether, these interaction between TPX2 and XRHAMM, triggering the forma- results indicate that the M-phase egg cytoplasm contains tion of a TPX2-XRHAMM-NEDD1-gTuRC supracomplex. inactive or poorly active NEDD1-gTuRC complexes that We then examined whether this supracomplex is specifically become activated through a RanGTP-dependent mechanism. required for the RanGTP-dependent MT nucleation pathway. 133

Figure 2. XRHAMM Deﬁnes a gTuRC Sub- set Involved in Chromosome-Dependent MT Nucleation (A) Western blot analysis of control IgG and anti-XRHAMM immunoprecipitations from egg extract with or without RanGTP to detect the gTuRC components Xgrip109, Xgrip195, and g-tubulin as well as NEDD1, as indicated. (B) Centrosomal MT nucleation from sperm nuclei incubated in control or depleted extract containing rhodamine tubulin. Quantiﬁcations were done on squashes taken after 6, 8, and 10 min of incubation. The graph (left) shows the proportion of active centrosomes in three independent experiments. Representative images are shown (right). More than 100 nuclei were counted at each time point. Error bars: SD. The scale bars represent 10 mm.

Anti-NEDD1-coated beads were incubated in control or To gain additional support for this idea, we monitored MT XRHAMM or TPX2-depleted extracts with or without RanGTP, aster formation in TPX2 and NEDD1-depleted egg extracts retrieved from the extracts, washed, and their MT nucleation upon addition of another form of TPX2DNLS lacking the first activity tested in pure tubulin (Figure 3E). As determined by 39 amino acids (D39TPX2DNLS) (Figure 4B) and therefore western blot analysis, the NEDD1 beads carried g-tubulin in unable to activate Aurora A [20]. MT assembly did not occur all conditions (Figure 3D). In agreement with our previous re- in depleted extracts complemented with D39TPX2DNLS and sults, the NEDD1 beads retrieved from extract containing either NEDD1-WT or NEDD1-S405A. However, addition of the RanGTP triggered MT nucleation in vitro. However, NEDD1 phospho-mimicking NEDD1-S405D restored MT assembly (Fig- beads retrieved from XRHAMM- or TPX2-depleted extracts ures 4D, S4A, and S4B). These data strongly suggested that containing RanGTP did not trigger MT assembly in pure tubulin MT nucleation involves the TPX2-dependent Aurora A activa- (Figure 3E). tion that promotes the essential phosphorylation of NEDD1 on We conclude that, by promoting a direct interaction between S405. Because NEDD1-S405D is sufficient for MT nucleation TPX2 and XRHAMM, RanGTP induces the formation of an in the absence of active Aurora A, our results also indicate active MT nucleation supracomplex. However, the specific that NEDD1 S405 is the only Aurora A target essential for the function of TPX2 in the activation of MT nucleation is unclear. RanGTP-dependent MT nucleation. Moreover, this critical phosphorylation is also specific for the RanGTP pathway, as it does The Essential Phosphorylation of NEDD1 on S405 Requires not interfere with MT nucleation at the centrosome (Figure S5A). TPX2-Dependent Aurora A Activation To explore the role of NEDD1 S405 phosphorylation by RanGTP promotes the interaction of TPX2 with Aurora A, Aurora A, we then examined whether it could regulate the leading to its activation [20–23], and we recently showed interaction of NEDD1 with the gTuRC. Pull-down experiments that NEDD1 phosphorylation by Aurora A is essential for using the three phospho-variants of NEDD1 in egg extract did RanGTP-dependent MT assembly [16]. Consistently, we found not provide any evidence for a differential phospho-dependent that NEDD1 interaction with Aurora A is strongly enhanced by binding activity of NEDD1 to the gTuRC components (Fig- RanGTP (Figure 4A). This suggested that, by promoting the ure S5B). We then decided to explore the possibility that interaction of TPX2 with the gTuRC, RanGTP could indirectly NEDD1 phosphorylation could promote the recruitment of favor the phosphorylation of NEDD1 by Aurora A on S405. To one or more additional factors required for the activation of explore this possibility, we took advantage of the RanGTP- the MT nucleation complex. NEDD1 beads were retrieved independent activity of a recombinant TPX2 protein mutated from egg extract in the absence of RanGTP. As reported above, in its NLS (TPX2-DNLS) (Figures 4B and S4A). This protein pro- these beads do not carry TPX2 or Aurora A and they do not motes MT nucleation and activates Aurora A in the absence trigger MT nucleation in pure tubulin. The beads were incu- of RanGTP [24, 37]. Egg extracts were codepleted for TPX2 bated in vitro with Aurora A in the presence or absence of and NEDD1. Addition of either NEDD1 wild-type (WT) or any ATP and placed in pure tubulin to test their activity (Figure 4E). of its phospho-variants, NEDD1-S405A (unphosphorylable) or Strikingly, the NEDD1 beads incubated with Aurora A and ATP NEDD1-S405D (phospho-mimicking) [16] did not promote MT efficiently promoted MT nucleation in pure tubulin (Figure 4E), nucleation (Figure S4B). As previously described, TPX2-DNLS whereas those incubated without ATP did not (data not shown). did promote MT aster formation in egg extracts in a RanGTP- These results indicate that all the proteins required for MT independent way [24]. Interestingly, this activity was strictly nucleation are recruited on the NEDD1 beads retrieved from dependent on NEDD1 and, in agreement with our previous egg extract in the absence of RanGTP. However, to be active data, on its phosphorylation on S405 (Figure 4C) [16]. These in MT nucleation, this complex requires the phosphorylation results show that TPX2 is the only factor that needs to be of one or more of its components by Aurora A. Our data strongly directly regulated by RanGTP to trigger MT assembly. They suggest that the critical substrate of Aurora A is NEDD1. also show that TPX2 cannot induce MT nucleation directly in egg extract as it does in vitro [24] but functions through a In Addition to Aurora A Activation, TPX2 Provides Another mechanism that involves NEDD1 and its phosphorylation Essential Function for RanGTP-Dependent MT Nucleation on S405. Because TPX2-DNLS interacts with and activates through the Recruitment of gTuRCs Aurora A in a RanGTP-independent manner, this suggests Our results suggested that the activation of Aurora A by TPX2 that the main mechanism by which TPX2 triggers MT nucle- could be sufficient for triggering MT nucleation in the egg ation is by promoting NEDD1 phosphorylation by Aurora A. extract. We therefore decided to test experimentally this 134

Figure 3. TPX2-RHAMM Interaction Is Essential for RanGTP-Dependent MT Nucleation (A) Western blot analysis of control IgG, anti- XRHAMM, and anti-TPX2 immunoprecipitations from egg extracts with or without RanGTP to detect XRHAMM and TPX2, as indicated. (B) In vitro TPX2-RHAMM interaction. Colloidal blue staining of anti-GFP in vitro pull-down of control or recombinant GFP-TPX2, as indicated, incubated with XRHAMM C-terminal fragment. (C) Western blot analysis of control IgG and anti- NEDD1 immunoprecipitation from egg extracts with or without RanGTP to detect TPX2, NEDD1, and g-tubulin, as indicated. (D) Western blot analysis of anti-NEDD1 immunoprecipitation from mock (control)-, TPX2-, or XRHAMM- depleted extracts, as indicated, with or without RanGTP to detect TPX2, NEDD1, and g-tubulin, as indicated. (E) MT nucleation assay in pure tubulin of anti-NEDD1 beads retrieved from control, XRHAMM-, or TPX2-depleted extracts with or without RanGTP. The experimental design is shown (top). The graph shows the proportion of beads associated with MTs. Representative immunoﬂuorescence images of MTs and beads are shown (right). Beads are autoﬂuorescent. Three independent experiments were performed, counting more than 500 beads in each condition. Error bars: SD. ***p < 0.001% using X-squared test. The scale bars represent 10 mm.

idea. Addition of the phospho-mimicking NEDD1-S405D to support of this idea, NEDD1 beads retrieved from a TPX2- a NEDD1-depleted extract in the absence of RanGTP did not depleted extract containing the Aurora-A-activating fragment promote MT aster formation (Figure 5A). Consistently, TPX2-N39 triggered MT nucleation in pure tubulin indepen- the addition of TPX2-N39 that is sufficient to fully activate dently of RanGTP (Figure 5D). Altogether, our data show Aurora A in egg extract did not promote MT assembly either that, in addition to Aurora A activation, TPX2 provides another (Figure 5B). These results indicated that, although the phos- essential activity that can be recapitulated by recruiting the phorylation of NEDD1 S405 by Aurora A is essential, it is not nucleation complex on beads. sufficient to trigger MT nucleation, suggesting that TPX2 has additional functions in the pathway that go beyond Aurora TPX2 Has Oligomerization Properties that May Be A activation. Regulated by RanGTP All our previous experiments showing that NEDD1 phos- To gain some insights into how TPX2 may provide the putative phorylation was essential and sufficient to trigger MT nucle- scaffolding activity, we first explored whether TPX2 may form ation in extract without RanGTP and in vitro were performed oligomers in vitro. Pull-downs performed in vitro with anti-GFP using NEDD1 beads. We therefore hypothesized that, under antibodies on a mixture of GFP-TPX2 (GFP-TPX2) and His- physiological conditions, TPX2 could provide a similar recruit- D39-TPX2 showed that both proteins interact, indicating that ment or scaffolding activity. To test this idea, we repeated the TPX2 can oligomerize in vitro (Figure 6A). To test whether same experiments as above (Figures 5A and 5B) but using the TPX2 may also oligomerize under physiological conditions, NEDD1 phospho-variants loaded on beads. The beads were we performed pull-downs with anti-GFP antibodies in egg ex- retrieved from the extract and their MT nucleation capacity tracts containing either GFP-TPX2 (GFP-TPX2) or a shorter tested in pure tubulin. As described above, the NEDD1-WT C-terminal fragment (GFP-TPX2-CT). In addition, the extracts beads nucleated MTs only when retrieved from an extract con- were supplemented or not with RanGTP. Both recombinant taining RanGTP (Figures 1D and 5C) whereas the NEDD1- TPX2 proteins pulled down endogenous TPX2 but strikingly S405A beads did not promote MT nucleation in any condition only in extracts supplemented with RanGTP (Figure 6B). These (Figure 5C). In contrast, the NEDD1-S405D beads did trigger results indicated that TPX2 may oligomerize in the M-phase MT nucleation in a RanGTP-independent manner (Figure 5C). egg extract, and interestingly, this property is regulated by Western blot analysis showed that the NEDD1-S405D beads RanGTP. Moreover, our results indicate that this property is associated with gTuRC components, but not with TPX2 and mediated by TPX2 C-terminal region (Figure 6B). These data Aurora A in absence of RanGTP (Figure S5B). Therefore, these suggest that RanGTP-dependent TPX2 oligomerization may results suggest that NEDD1 phosphorylation on S405 alone facilitate the recruitment of the MT nucleation complexes is sufficient for triggering MT nucleation in the absence of required for efficient MT nucleation. RanGTP as long as the nucleation complex is recruited onto Altogether, our data support a model in which the release of a scaffolding element (here provided by beads). In further TPX2 from the importins triggered by RanGTP promotes two 135

Figure 4. NEDD1 Phosphorylation by TPX2-Acti- vated Aurora A Is Needed for MT Nucleation (A) Western blot analysis of control IgG and anti- Aurora-A immunoprecipitation from egg extracts with or without RanGTP to detect TPX2, NEDD1, and Aurora A, as indicated. (B) Schematic representation of TPX2 showing its Aurora A (N39)- and importin (NLS)-binding domains. Below, the two NLS-mutated TPX2 proteins used in (C) and (D) are shown. (C) MT assembly in the absence of RanGTP in TPX2- and NEDD1-depleted extracts containing TPX2DNLS and Flag-NEDD1 phospho-variants, as indicated. The experimental design is shown at the top. The graph shows the number of asters per microliter of extract in one representative out of three independent experiments. (D) MT assembly in the absence of RanGTP in TPX2- and NEDD1-depleted extracts containing D39TPX2DNLS and Flag-NEDD1 phospho-variants, as indicated. The experimental design is shown at the top. The graph shows the number of asters per microliter of extract in one representative out of four independent experiments. (E) MT nucleation assay around anti-NEDD1 beads retrieved from egg extract with or without RanGTP and incubated or not in vitro with Aurora A, as indicated. Beads were then incubated in pure tubulin and their MT assembly activity quan- tiﬁed. The experimental design is shown at the top. The graph shows the percentage of beads associated with MTs in one representative exper- iment out of three. More than 500 beads were counted for each condition.

and organization. The chromosome/ RanGTP-dependent MTs are essential for spindle formation and function both when centrosomes are present or not. Here, we uncovered the mechanism by which RanGTP triggers the activation of MT nucleation by the gTuRC. Previous reports suggested that the gTuRCs obtained from cells or egg extracts have an intrinsic MT nucleation activity [38, 39]. However, here, we showed that the gTuRCs pulled down from Xenopus egg extracts through essential events: (1) the recruitment of RHAMM-gTuRC com- NEDD1 did not promote MT nucleation in extract or in vitro un- plexes through the direct interaction between TPX2 and less activated by a RanGTP-dependent mechanism. These XRHAMM and TPX2 oligomerization and (2) a TPX2-depen- apparently contradictory results may be due to differences in dent activation of Aurora A that promotes the phosphorylation the composition of the complexes as a result of the precise of NEDD1 on S405. We propose that this series of connected experimental conditions that could either alter their composi- events is at the basis of the mechanism that drives RanGTP- tion or regulation. Our data in fact suggest that the M-phase dependent MT nucleation in M phase (Figure 6C). cytoplasm of the egg contains different gTuRC subcomplexes having a common set of components and additional associ- Discussion ated protein(s). One of them, including RHAMM, is the target for regulation by RanGTP. The gTuRC is the multiprotein complex that drives MT nucle- Previous work reported that g-tubulin and GCP3 coimmu- ation in higher animal cells. Its spatial and temporal regulation noprecipitated with XRHAMM in egg extract [32]. Here, we determines where and when this important process occurs in showed that this complex also includes GCP6, suggesting the cell, determining MT organization and cell function. During that it corresponds to the full gTuRC. Because depletion of cell division, MT nucleation occurs through different path- XRHAMM leaves almost intact levels of g-tubulin and gTuRC ways, all of them depending on the gTuRC. One of them is components in the egg extract (Figures 3D and S2), this triggered by the chromosomes that generate a gradient of indicates that a large pool of gTuRCs is in fact not associ- RanGTP that promotes local MT nucleation, stabilization, ated with XRHAMM. Accordingly, we found that depleting 136

Figure 5. TPX2 Essential Function in Promoting gTuRC Recruitment (A) MT assembly in NEDD1-depleted extracts complemented with NEDD1 phospho-variants (as indicated), with or without RanGTP. The experimental design is shown at the top. The graph shows the number of asters per microliter of extract in three independent experiments. Error bars: SD. ***p < 0.001% using X-squared test. (B) MT assembly in TPX2-depleted extracts complemented with TPX2 full length (control) or TPX2-N39 (as indicated) with or without RanGTP. The experimental design is shown at the top. The graph shows the number of asters per microliter of extract in three independent experiments. Er- ror bars: SD. ***p < 0.001% using X-squared test. ns, nonsigniﬁcant. (C) MT nucleation in pure tubulin around beads coated with Flag-NEDD1 phospho-variants retrieved from NEDD1-depleted extract containing or not RanGTP. The experimental design is shown at the top. The graph shows the percentage of beads associated with MTs. Data from three independent experiments. More than 500 beads were counted for each condition. Error bars: SD. ***p < 0.001% using X-squared test. (D) MT nucleation in pure tubulin around NEDD1- coated beads retrieved from control or TPX2- depleted extract containing or not RanGTP. The depleted extract was supplemented or not withTPX2 full length (control) and TPX2-N39, as indicated. The experimental design is shown at the top. The graph shows the percentage of beads associated with MTs. Data from three independent experiments. More than 500 beads were counted for each condition. Error bars: SD. ***p < 0.001% using X-squared test.

Our work shows that one of the essential functional implications of the RanGTP-dependent association of TPX2 with the RHAMM-gTuRC complex XRHAMM from the egg extract did not compromise the is the phosphorylation of NEDD1 on S405 by the TPX2-activated capacity of centrosomes to nucleate MTs (Figure 2B). These Aurora A. Indeed, all the different approaches we used unequiv- data therefore suggest that the M-phase cytoplasm contains ocally show that this phosphorylation event is essential for this different complexes of gTuRCs that may potentially have pathway. In particular, we found that the MT nucleation activity specific roles in the different MT nucleation pathways. of NEDD1 beads could be fully restored in the absence of TPX2 Because XRHAMM and TPX2 interact directly, this by promoting or mimicking NEDD1 phosphorylation. Moreover, strongly suggests that the RHAMM-associated gTuRC com- because we found that the requirement for active Aurora A plex is the specific target of RanGTP-dependent regulation. can be substituted by the phospho-mimicking form of NEDD1, Indeed, our data show that RanGTP promotes the interac- our data also suggest very strongly that NEDD1 is the only tion between its direct target TPX2 and XRHAMM, itself Aurora A substrate that is essential for MT nucleation in associated with the MT nucleation complex, suggesting this pathway. Consistently with our findings, a recent study that this is the primary mechanism for the RanGTP-depen- correlates RHAMM, TPX2, and Aurora A activity in mammalian dent activation of MT nucleation. Groen and collaborators cells [35]. had previously reported that TPX2 and XRHAMM coimmu- We still do not know the precise role of NEDD1 phosphory- noprecipitated in egg extract in a RanGTP-independent lation on S405 by Aurora A. One possibility is that phosphory- manner [32], although they discussed that this could be lation triggers a conformational change of the complex. This due to antibody effects. Here, we found that our anti- idea is based on previous work suggesting that a relative XRHAMM and anti-TPX2 antibodies were efficient in coim- flexibility of the gTuRC could define inactive versus active tem- munoprecipitating the two proteins but only in extracts plate gTuRC complexes [9]. Another nonexclusive possibility containing RanGTP. The identical results obtained with two is that phosphorylation regulates a protein-protein interaction. different antibodies strengthen our conclusion. Moreover, However, the pull-down experiments we performed with the in line with this conclusion, we showed that the RanGTP- various phospho forms of NEDD1 did not reveal any change dependent interaction of TPX2 with the NEDD1-gTuRC com- in the interactions with the gTuRC components. In addition, plex requires XRHAMM. because we found that the in vitro phosphorylation of the 137

Figure 6. RanGTP Triggers TPX2 Oligomeri- zation (A) In vitro TPX2-TPX2 interaction. Colloidal blue staining of anti-GFP pull-down of recombinant GFP-TPX2 or control, as indicated, incubated with His-D39TPX2. The two recombinant proteins alone are shown on the left and the immunoprecipitation on the right. Both panels are part of the same gel. (B) Western-blot analysis of control IgG and anti- GFP pull-downs from egg extracts containing GFP-TPX2 full length (FL) or GFP-TPX2 C-terminal (CT) with or without RanGTP. Top: GFP recombinant proteins detected with anti-GFP antibodies. Bottom: GFP-TPX2 and endogenous TPX2 (TPX2 endo) detected with a monoclonal anti-TPX2 antibody that recognizes an N-terminal epitope. (C) Model: RanGTP releases TPX2 from importins triggering MT nucleation. TPX2 provides the two essential requirements for gTuRC activation by RanGTP: (1) the phosphorylation of NEDD1 on S405 and (2) a scaffolding activity for the recruitment of the XRHAMM-NEDD1-gTuRC. complex by Aurora A is sufficient to activate it (Figure 4E), the other RanGTP targets in the complete pathway of MT assem- recruitment of an additional factor is very unlikely. At present, bly and organization [1]. Although other RanGTP-regulated we cannot rule out that NEDD1 phosphorylation may promote proteins have been proposed to participate in MT nucleation the release of an inhibitory factor. Additional work will be [42], it still remains to be established at which level these pro- required to elucidate the molecular consequences of NEDD1 teins may function. phosphorylation by Aurora A for the activation of RanGTP- Recently, it was proposed that augmin, g-tubulin, and TPX2 dependent MT nucleation. work in complex in branching MT nucleation, linking the It is interesting to note that NEDD1 is highly phosphory- RanGTP and the augmin-dependent MT nucleation pathways lated in mitosis [40] and that the three M-phase MT nucle- [43]. The augmin pathway is a MT-amplification pathway and ation pathways require each a specific phosphorylation of relies on the presence of preexisting MTs [44, 45]. Consis- NEDD1 by a different kinase on residues that are all in close tently, augmin depletion does not abolish MT assembly proximity (S377, 411, and 405) [13, 14, 41]. It is therefore induced by RanGTP or chromatin beads in egg extracts [46]. tempting to speculate that this region of NEDD1 has In contrast, TPX2 depletion prevents altogether RanGTP- or essential features for the activation of MT nucleation by chromatin-beads-induced MT assembly [26, 37], suggesting the gTuRC. that the augmin pathway acts downstream of the RanGTP- Our work also provides a compelling evidence for an addi- TPX2 pathway. Here, we focused on understanding how tional function of TPX2 in RanGTP-dependent MT nucleation RanGTP promotes MT nucleation and found that it functions in addition to the activation of Aurora A. Indeed, the essential by promoting a specific interaction of TPX2 with the gTuRC. phosphorylation of NEDD1 is not sufficient for triggering MT Further work is needed to establish how the two pathways nucleation in the absence of TPX2. We found, however, that re- may be functionally connected. cruiting phosphorylated NEDD1-gTuRC complexes on beads In conclusion, our work provides the first integrated picture was sufficient to substitute for TPX2. Because we found that for the mechanism by which RanGTP triggers MT nucleation TPX2 can form oligomers in vitro and that RanGTP promotes in M phase. RanGTP induces the interaction between a the formation of complexes with various TPX2 molecules, we preformed, dedicated RHAMM-gTuRC complex with TPX2. propose that TPX2 provides a scaffolding role to recruit Within this new complex, TPX2 provides two essential require- various nucleation complexes favoring their activation. The ments for MT nucleation: the activation of Aurora A promoting fact that NEDD1 beads are active suggests that the mecha- the NEDD1phosphorylation on S405 and a scaffolding activity nism does not require a very specific spatial configuration. In for the recruitment of the MT nucleation complex. TPX2 is any case, it is interesting to note that NEDD1 phosphorylation therefore the central player of the RanGTP-dependent gTuRC and gTuRC recruitment seem to represent two common activation mechanism, leading to MT nucleation around the events required for the activation of MT nucleation in the chromosomes in mitosis. different MT nucleation pathways. In agreement with previous reports [24, 37], we show that a Experimental Procedures mutated form of TPX2 that does not bind importins promotes MT nucleation in the absence of RanGTP. Under these condi- DNA Constructs, Expression, and Purification of Recombinant Proteins tions, TPX2 provides the two essential requirements that we All hNEDD1 constructs and protein expression and purification methods have identified in this work: it promotes NEDD1 phosphoryla- were previously described [16]. The constructs for TPX2DNLS, tion through Aurora A activation and it acts as a recruitment D39TPX2DNLS, GST-N39 TPX2, and TPX2-CT and the methods for protein factor for the MT nucleation complex. As this occurs in the expression and purification were previously described [20, 37]. His- D39TPX2 was prepared cloning 1–39 deleted TPX2 in pHAT-2 vector. absence of RanGTP, we conclude that TPX2 is the only direct His-XRHAMM CT was prepared cloning a C-terminal fragment (last 137 target of RanGTP required for MT nucleation through this amino acids) of the Xenopus laevis protein into pET-28a vector (Novagen). pathway, although this does not rule out essential roles for His-Aurora A was cloned and purified as described previously [21]. 138

Antibodies incubated for 15 min at 20C with RanGTP or CSF-XB buffer (10 mM The rabbit polyclonal antibodies against Xenopus TPX2 and Xenopus HEPES [pH 7.7], 50 mM sucrose, 100 mM KCl, 0.1 mM CaCl2, and 5 mM NEDD1 were previously described [14, 17]. The mouse monoclonal antibody EGTA). anti-Xenopus TPX2 was raised by Abyntek Biopharma using the full-length protein and recognizes an N-terminal epitope. The rabbit polyclonal In Vitro Pull-Downs antibody against Xenopus RHAMM was raised against a GST-RHAMM-CT For in vitro pull-down experiments, recombinant proteins were incubated (corresponding to the last 137 amino acids) and affinity purified. The mouse in PBS for 20 min at 20C. Anti-GFP immunoprecipitation was then per- 1C1 monoclonal antibody against Xenopus Eg2 (Aurora A) was a kind gift formed using protein A Dynabeads (Life Technologies). Beads were washed from Claude Prigent (CNRS Universite´ de Rennes). Polyclonal antibodies four times in PBS Triton X-100 0.1% and directly eluted in loading buffer. against XGrip195 and XGrip109 were kind gifts from Yixian Zheng (Carnegie For better detection of interaction, SDS-PAGE was stained using colloidal Institution for Science). blue (Invitrogen). The following commercial antibodies were used: a monoclonal mouse antibody against g-tubulin (GTU-88; Sigma-Aldrich); a monoclonal anti- Bead Experiments human NEDD1 (that cross-reacts with Xenopus laevis NEDD1; Abcam); a To assay the gTuRC MT nucleation activity, beads coated with anti-NEDD1 mouse monoclonal anti-tubulin (12G10; Developmental Studies Hybridoma antibodies were incubated in egg extract. For analyzing the MT nucleation Bank) [47]; and the monoclonal mouse antibody against tubulin (DM1A; activity of NEDD1 phospho-variants, NEDD1-depleted egg extracts were Sigma-Aldrich). complemented with the corresponding recombinant proteins for 20 min at Secondary antibodies were anti-rabbit or anti-mouse conjugated to 20 C. The NEDD1 variants and associated proteins were then recovered Alexa 488, Alexa Fluor 568, or Alexa Fluor 680 (Life Technologies) and with anti-NEDD1 antibody-coated beads. So that the role of NEDD1 S405 were used at 1:1,000 for immunofluorescence and 1:10,000 for western blot. phosphorylation in vitro could be studied, NEDD1 beads were retrieved from the egg extract and incubated in kinase buffer (8 mM 3-(N-morpho- Immunofluorescence and Microscopy lino)propanesulfonic acid, 20 mM MgCl2, 0.2 mM EDTA, and 5% glycerol) All samples were visualized with a 340 or 363 objective on an inverted with recombinant His-tagged Xenopus Aurora A (0.2 mM), TPX2-N39 DMI-6000 Leica wide-field fluorescent microscope equipped with a Leica (0.2 mM), and ATP (4 mM). DFC 350FX camera. Spins down of NEDD1 beads were processed for To evaluate MT nucleation in egg extract, beads were washed four times immunofluorescence with anti-tubulin 12G10. Coverslips were fixed in in CSF-XB buffer (10 mM HEPES [pH 7.7], 50 mM sucrose, 100 mM KCl, methanol and mounted in Mowiol (Calbiochem). 0.1 mM CaCl2, and 5 mM EGTA) and then resuspended in the same initial All pictures were acquired with the Leica Application Suite software. volume of beads. Beads were then diluted 200–400 times in 20 ml CSF- Images were processed with ImageJ or Photoshop (Adobe) and mounted arrested extract and incubated at 20C for 20–30 min, depending on the using Photoshop (Adobe). extract. To study MT nucleation in vitro, beads were washed twice in CSF-XB, Xenopus laevis Egg Extract twice in BRB80 (80 mM PIPES [pH 6.8], 1 mM EGTA, and 1 mM MgCl2), All work involving animals was done according to standard protocols and resuspended in the same initial volume in BRB80. One to two microliters approved by the Centre for Genomic Regulation Ethics Committee. Cyto- of beads were then added to a 30 mM pure tubulin solution and incubated static-factor-arrested egg extracts from Xenopus laevis were prepared as for 10 min at 37C. previously described [48]. Recombinant RanQ69LGTP was expressed and For both approaches, the reaction mixture was fixed in 1% glutaraldehyde purified as previously described [37]. (in BRB80), spun down through a 10% glycerol in BRB80 cushion onto a RanGTP asters were induced by adding 15 mM of RanQ69L-GTP to egg coverslip, and fixed in methanol for 10 min at 220C. MTs were then visual- extracts containing 0.2 mg/ml of rhodamine-labeled tubulin. Extracts were ized by immunofluorescence using anti-tubulin antibody. The quantifica- incubated at 20C, and squashes of 3 ml were collected at the indicated tions were performed by evaluating the proportion of beads associated time points. Quantifications were made counting the total number of with MTs in 30 random fields for each condition. MT asters in ten random lines for each coverslip (403 magnification with Leica DMI6000B microscope equipped with a Leica DFC 350FX camera). Supplemental Information Data were normalized to obtain the number of aster per microliter of extract. Supplemental Information includes five figures and can be found with this Centrosome MT assembly was studied adding demembranated Xenopus article online at http://dx.doi.org/10.1016/j.cub.2014.11.025. sperm nuclei at a concentration of w500 nuclei/ml to egg extracts containing 0.2 mg/ml of rhodamine-labeled tubulin. Extracts were incubated at 20C, and squashes of 3 ml were collected after 6, 8, and 10 min of incubation. Author Contributions MT nucleation from sperm centrosomes was evaluated counting 100 nuclei for each condition. I.V., S.M., and J.S. designed the experiments. J.S. performed all the experiments except the TPX2-TPX2 interaction in egg extract, done by T.S. I.V., S.M., and J.S. analyzed the data and wrote the manuscript. Immunodepletions and Immunoprecipitations For NEDD1, TPX2, and XRHAMM immunodepletions, one volume of antibody-coated protein A Dynabeads (Life Technologies) was incubated in Acknowledgments 2.5 volumes of egg extract. Dynabeads were prepared following manu- ´ facturer’s recommendations. Two rounds of immunodepletion of 30 min J.S. was supported by Fundacion ‘‘La Caixa.’’ This work was supported each at 4C completely depleted the endogenous proteins from the by the Spanish Ministry of Economy and Competitiveness grants egg extract. Mock control depletions were performed in the same con- BFU2009-10202 and BFU2012-37163. We thank Y. Zheng for the anti- ´ ditions using unspecific anti-rabbit immunoglobulin G (IgG)-coated protein XGrip195 and XGrip109 antibodies. We thank N. Mallol, L. Avila, and A Dynabeads. After immunodepletions, RanGTP or buffer was added J. Cela for excellent technical support and members of the I.V. lab for their to the extract for 15 min at 20C. Add backs of NEDD1, NEDD1 phospho- useful comments. variants, TPX2-DNLS, and D39TPX2DNLS were performed as previously described [16, 20, 37]. TPX2-N39 was added to the egg extract as previously Received: August 5, 2014 described [21]. Revised: October 23, 2014 For immunoprecipitations, one volume of protein A Dynabeads (Life Accepted: November 7, 2014 Technologies) was coated with the indicated antibodies (6 mg for 20 ml Published: December 18, 2014 of beads) and incubated for 1 hr on ice in 2.5 volumes of colony-stimu- lating factor (CSF)-arrested extract. For detection of NEDD1-XRHAMM, References NEDD1-TPX2, and XRHAMM-TPX2 interactions, beads were washed without detergent (four times in Tris-buffered saline [TBS]). For all the 1. Meunier, S., and Vernos, I. (2012). Microtubule assembly during mitosis other immunoprecipitations, beads were washed in TBS 0.1% Triton X- - from distinct origins to distinct functions? J. Cell Sci. 125, 2805–2814. 100. 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