Correspondence to Kazuhisa Kinoshita: kinoshita{at}mpi-cbg.de
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Introduction |
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Xenopus laevis egg extracts have been particularly useful for studying the regulation of microtubule polymerization from centrosomes. Because meiotic spindles in Xenopus egg extracts can form without centrosomes by nucleation and stabilization of microtubules around chromosomes (Heald et al., 1996; Karsenti and Vernos, 2001; Sampath et al., 2004), it is possible to study separately the regulation of microtubule polymerization around mitotic centrosomes or chromosomes. In Xenopus egg extracts, the dynamic behavior of microtubules in the cytoplasm is regulated, in large part, by the activity of XMAP215, which is a member of a conserved family of microtubule-associated proteins (Kinoshita et al., 2002; Gard et al., 2004). XMAP215 both stimulates the growth rate and opposes catastrophe activity of XKCM1/XlMCAK, which is member of the Kin I/kinesin-13 family (Walczak et al., 1996; Tournebize et al., 2000; Noetzel et al., 2005). Physiological microtubule dynamics can be reconstituted in vitro by a mixture of XMAP215 and mitotic centromere-associated kinesin (MCAK) together with tubulin (Kinoshita et al., 2001). Although XMAP215 and MCAK clearly have a central role in determining microtubule dynamics in Xenopus egg extracts, we know little about how these proteins are regulated during cell cycle progression. However, these proteins are localized to centrosomes, suggesting that they could be involved in the regulation of microtubule growth from centrosomes (Kinoshita et al., 2002; Gard et al., 2004).
A clue as to the possible mechanisms regulating XMAP215 family proteins at centrosomes came from the discovery that the Drosophila melanogaster member of the XMAP215 family is associated with D-TACC (D. melanogaster transforming acidic coiled coil; Cullen and Ohkura, 2001; Lee et al., 2001). Mutants in TACC family members reduce microtubule number and prevent the localization of XMAP215 family proteins to centrosomes or spindles in D. melanogaster (Cullen and Ohkura, 2001; Lee et al., 2001), Caenorhabditis elegans (Bellanger and Gönczy, 2003; Le Bot et al., 2003; Srayko et al., 2003), yeasts (Usui et al., 2003; Sato et al., 2004), and human cells (Gergely et al., 2003). Beyond the fact that TACC family members are required to localize XMAP215 family members to centrosomes, we know little about the role of TACC in the modulation of microtubule dynamics and in regulating the activity of XMAP215. In this study, we investigated these issues by using purified proteins and Xenopus egg extracts. Maskin is a Xenopus TACC homologue (Stebbins-Boaz et al., 1999); in this paper, we call it Xenopus TACC3 because of its sequence similarity to the mammalian TACC3 subfamily (Still et al., 2004). We demonstrate that a Xenopus TACC3 is a cell cycledependent regulator of XMAP215.
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Results |
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In all systems in which localization has been examined, TACC and XMAP215 are found at centrosomes, suggesting that TACC3 could have a centrosome-specific role in mitosis. Accordingly, we looked specifically at the role of TACC3 in microtubule growth from mitotic centrosomes. Traditionally, centrosomal microtubule assembly has been examined in Xenopus egg extracts by adding human centrosomes directly to mitotic extracts (Belmont et al., 1990). However, the formation of a mitotic centrosome is likely to be a complex process involving cell cycle transition. To create mitotic centrosomes, we added isolated centrosomes to interphase extracts and drove the extracts into mitotic (M) phase by the addition of a nondegradable cyclin B (cyclin B 90; Glotzer et al., 1991). In mock-depleted extracts, microtubule assembly around centrosomes was activated by the induction of M phase (Fig. 3 A, left). In TACC3-depleted extracts, the microtubule number around centrosomes was decreased to 13% of that in control extracts (Fig. 3 A, right; and B). Importantly, adding back recombinant TACC3 protein to depleted extracts increased microtubule assembly from mitotic centrosomes, returning it to the levels that were found in control extracts (Fig. 3, CE). In contrast to M phaseinduced extracts, no detectable effect of TACC3 depletion was observed on microtubule assembly in interphase extracts (Fig. 3 A, top). We confirmed that TACC3 was localized to these mitotic centrosomes in mock-depleted and TACC3 add-back extracts but not in TACC3-depleted extracts (Fig. 3 D, insets). We conclude that TACC3 is required specifically for microtubule assembly around mitotic centrosomes in Xenopus egg extracts.
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Aurora Aphosphorylated TACC3 is enriched at mitotic centrosomes
The results argue that TACC3 activity at centrosomes is specifically required for M phase microtubule growth and poses the question as to whether cell cyclespecific modulation of TACC3 activity could regulate the growth of microtubules from centrosomes. Experiments in D. melanogaster and C. elegans suggested a role of the mitotic kinase Aurora A in TACC3 localization to centrosomes (Giet et al., 2002; Bellanger and Gönczy, 2003; Le Bot et al., 2003) but could not distinguish between a specific role of the kinase in modulating TACC3 activity at centrosomes and a more general role in regulating centrosome assembly and maturation. Therefore, we decided to investigate the specific role of Aurora A in the regulation of TACC3 localization and activity. TACC3 has three consensus Aurora A sites (Cheeseman et al., 2002) that are conserved between Xenopus (Ser33, Ser620, and Ser626) and humans (Ser34, Ser552, and Ser558; Fig. 4 A). Recent experiments have shown that TACC3 is an in vitro substrate of Aurora A in Xenopus (at Ser626; Pascreau et al., 2005) and humans (Tien et al., 2004). We confirmed by mass spectrometry that Xenopus TACC3 is indeed phosphorylated by Aurora A at these three consensus sites (Fig. S2, available at http://jcb.org/cgi/content/full/jcb.200503023/DC1). Furthermore, we mutated these three conserved sites to alanine and showed that the incorporation of labeled 32P into the alanine-mutated protein (3A mutant; TACC3-3A) is reduced to 3% compared with the wild-type (WT) protein (TACC3-WT; Fig. 4 B). We raised phosphospecific antibodies to the three conserved Aurora A sites (Fig. 4 C). Because of the limitations of cytology in Xenopus systems and in order to use RNA interference as a control for antibody specificity, we examined the localization of phosphorylated TACC3 in human tissue culture cells. Interestingly, phospho-TACC3, which was stained by antibodies against P-Ser626 (in Xenopus; Ser558 in humans), localized specifically to mitotic centrosomes, whereas the general population of TACC3, which was stained by polyclonal antibodies against the fragment of TACC3 protein (73265 amino acids), localized throughout the mitotic spindle as previously reported (Fig. 5 A; Gergely et al., 2000, 2003). siRNA of TACC3 greatly reduced the staining of phospho-TACC3 as well as that of nonspecific TACC3 (Fig. 5 B). Phospho-TACC3 antibodies recognized a band of 130 kD molecular mass specifically in mitosis-arrested tissue culture cells (Fig. 5 C) as well as a band of the same molecular mass that disappeared after the immunodepletion of either TACC3 (see Fig. 6 D) or Aurora A (Fig. S3, available at http://jcb.org/cgi/content/full/jcb.200503023/DC1) from Xenopus egg extracts in mitosis. Therefore, we conclude that TACC3 protein on centrosomes is specifically phosphorylated during M phase by Aurora A.
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Discussion |
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Microtubule growth from centrosomes or chromosomes requires the presence of XMAP215 (Tournebize et al., 2000). Because Xenopus meiotic spindle assembly is apparently unaffected in the absence of TACC3, it appears that TACC3 is not required for the global activity of XMAP215. Why is TACC3 required specifically for XMAP215 activity at mitotic centrosomes? Our data suggest that targeting of the TACC3XMAP215 complex to mitotic centrosomes overcomes the high microtubule-destabilizing activity of MCAK at centrosomes. Consistent with this idea, MCAK is localized to centrosomes in many systems (Walczak et al., 1996; Oegema et al., 2001), and colonic and hepatic tumor overexpressed gene (TOG)/TOG protein, which is a human XMAP215 orthologue, is required to protect spindle microtubules from MCAK activity at centrosomes (Holmfeldt et al., 2004). We propose that in the absence of TACC3, XMAP215 is still able to counteract the activity of cytoplasmic MCAK. MCAK localization to centrosomes, however, generates an environment in which plus end growth is not favored, and XMAP215 cannot stabilize nascent nucleated plus ends. This could either be because MCAK activity at centrosomes is enhanced or because of an increased concentration of MCAK at centrosomes. Targeting of TACC3XMAP215 would enhance the activity of XMAP215 at centrosomes, stabilizing nascent plus ends and allowing them to grow from centrosomes (Fig. 7). This provides an explanation for the seemingly paradoxical observation that a plus end stabilizer localizes to centrosomes where microtubules are attached via their minus ends (Gard et al., 2004; Gräf et al., 2004).
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Aurora A regulates centrosomal microtubule assembly in mitosis
The targeting of TACC3 to centrosomes defines a potential mechanism for regulating the polymerization of microtubules from centrosomes in mitosis. Previous data in other systems has shown that Aurora A is required to target TACC to centrosomes (Giet et al., 2002; Bellanger and Gönczy, 2003; Le Bot et al., 2003). However, the specific role of TACC3 phosphorylation has been unclear, as Aurora A has general roles in both centrosome assembly and cell cycle progression (Hannak et al., 2001; Giet et al., 2002; Hirota et al., 2003). Our data show that targeting of TACC3 to centrosomes specifically requires Aurora A phosphorylation. Furthermore, targeting is essential for TACC activity at centrosomes. Because Aurora A activity increases as cells enter M phase, this would account for the increase in microtubule polymerization that was observed as centrosomes mature during the cell cycle. Consistent with this idea, the protein level of both TACC3 and Aurora A as well as the kinase activity of Aurora A are all highly cell cycle regulated, reaching a peak during mitosis in human tissue culture cells (Bischoff et al., 1998; Gergely et al., 2003). In Xenopus egg extracts, the increase of Aurora A kinase activity may be sufficient to trigger the activation of centrosomal microtubule polymerization in mitosis. Thus, the phosphorylation of TACC3 provides a mechanism for Aurora A to specifically modulate the growth of microtubules from centrosomes. However, Aurora A regulates processes other than microtubule assembly at centrosomes. Identification of the other Aurora A substrates will be essential for understanding the mechanisms of mitotic spindle assembly.
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Materials and methods |
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Immunoprecipitation, sucrose density gradient centrifugation, and gel filtration
For immunoprecipitation using insect cell lysates, protein ASepharose beads that bound control rabbit IgG or anti-TACC3 antibodies were incubated with lysate from insect cells that were infected either with TACC3 baculovirus or with both TACC3 and XMAP215 baculovirus. Eluted immunoprecipitates were separated by SDS-PAGE and stained by Coomassie brilliant blue.
Sucrose gradients (315%) were poured as step gradients (five steps of equal volume) in 1x PBS, 0.1% Tween 20, 1 mM DTT, and 0.2x protease inhibitor mix. Gradients were incubated at 4°C for 12 h to allow diffusion into a continuous gradient. Equal concentrations of TACC3 and XMAP215 were mixed in vitro, incubated for 10 min on ice, diluted into gradient buffer to a final concentration of 1% glycerol, loaded onto the gradient, and spun for 6.5 h at 4°C and 120,000 g. Gradients were fractionated from the top by hand with a cut pipette tip. Fractions were analyzed by 412% SDS-PAGE (NuPage; Invitrogen) and stained by Coomassie brilliant blue. Standard proteins with known sedimentation values were run in parallel and scanned, band intensities were quantified, and peak fractions were assigned. Standard curves of peak fractions versus sedimentation coefficient were used to estimate the S value of proteins. Standard proteins that were used in sucrose gradients are listed as follows (sedimentation values are indicated in parentheses): chymotrypsinogen (2.6 S); ovalbumin (3.5 S); BSA (4.6 S); aldolase (7.3 S); and catalase (11.3 S).
Gel filtration chromatography was performed using a column (model G5000PWXL; Tosoh) in 1x PBS, 0.1% Tween 20, 1 mM DTT, 5% glycerol, and 0.2x protease inhibitor mix. The column was calibrated with standards of known Stokes radii. Standard curves of peak fractions versus the logarithm of the Stokes radii were used to determine the Stokes radii of proteins. Fractions were separated by 412% SDS-PAGE (NuPage; Invitrogen) and stained by Coomassie brilliant blue. Standard proteins that were used in gel filtration chromatography are listed as follows (Stokes radii are indicated in parentheses): ribonuclease A (1.64 nm); chymotrypsinogen (2.09 nm); ovalbumin (3.05 nm); BSA (3.55 nm); aldolase (4.81 nm); catalase (5.22 nm); ferritin (6.1 nm); and thyroglobin (8.5 nm)
Depolymerization assay and sedimentation analysis of stabilized microtubules
Sedimentation analysis of the depolymerization of GMPCPP-stabilized microtubules was performed as described previously (Desai et al., 1999a). 2.5 µM of prepolymerized GMPCPP microtubules were added to the reaction mix that was supplemented with 125 nM MCAK or control buffer in 1x BRB80 (80 mM Pipes, pH 6.8, 1 mM MgCl2, and 1 mM EGTA) containing 125 mM KCl and 1.5 mM Mg-ATP. 500 nM XMAP215 and increasing amounts of TACC3 or control buffer were added to reactions. Reactions were sedimentated after 38 min at 30°C through a glycerol cushion. Equivalent amounts of the supernatant and pellets were analyzed by 412% SDS-PAGE followed by Coomassie brilliant blue staining. For sedimentation assays to monitor the affinity of XMAP215 to microtubules, 2.5 µm of prepolymerized GMPCPP microtubules were incubated with 500 nM XMAP215 or control buffer in 1x BRB80 containing 125 nM KCl in the presence of TACC3 or control buffer. In each condition, we confirmed that all proteins were in the supernatant after sedimentation without GMPCPP microtubules.
Preparation of Xenopus egg extracts and immunodepletion
Xenopus egg extracts were prepared as described previously (Murray, 1991; Desai et al., 1999b). Interphase extracts were prepared by the addition of calcium to cytostatic factor extracts. Cycloheximide was added to 100 µg/ml to avoid translation of cyclin B messenger in extracts and to arrest them in interphase. To generate mitotic extracts, a purified, nondegradable cyclin B fragment (cyclin B 90; a gift from H. Funabiki, The Rockefeller University, New York, NY; Glotzer et al., 1991) was added to 25 µg/ml to interphase extracts (Vorlaufer and Peters, 1998; Funabiki and Murray, 2000). Immunodepletion in Xenopus egg extracts was performed as described previously (Funabiki and Murray, 2000). All of the immunodepletion experiments were performed in the presence of 100 µg/ml cycloheximde in extracts to block translation, as maskin has been reported to be a negative regulator of translational machinery during oocyte maturation (Stebbins-Boaz et al., 1999).
Antibodies and immunoblotting
Phosphospecific antibodies were raised and purified against phosphopeptides, including phosphoserine in the consensus target sites of Aurora A phosphorylation in Xenopus TACC3 (P-Ser33; QTTGRPphospho-SILRPSQ and P-Ser620; CNSFKEphopho-SVLRKQ and P-Ser626; VLRKQphopho-SLYLKFC). AntiXenopus TACC3 antibodies were raised and purified against the GST fusion protein that contained an NH2-terminal fragment of TACC3 (aa 7208). Antihuman TACC3 antibodies were previously described (Gergely et al., 2000). AntiAurora A and -XMAP215 antibodies were raised and purified against COOH-terminal peptides (Aurora A, CKNSQLKKKDEPLPGAQ; and XMAP215, CNIDDLKKRLERIKSSRK) as described previously (Field et al., 1998). Anti-tubulin antibodies (DM1A) were purchased from Sigma-Aldrich. AntiNH2-terminal fragments of XlMCAK (XKCM1-NT) antibodies were gifts from C. Walczak (Indiana University, Bloomington, IN; Walczak et al., 1996). Immunoblotting was performed by using ECL (GE Healthcare). For quantification of phospho-TACC3 signal, the signal intensities on the blot were measured by image software (Scion Corp.).
Microtubule assembly and localization assays using Xenopus egg extracts
Recycled and fluorescently labeled tubulins (rhodamine- or Cy3-labeled) were prepared from porcine instead of bovine brain as previously described (Hyman et al., 1991; Ashford et al., 1998). Centrosomes were purified from human KE37 cells as described previously (Moudjou and Bornens, 1998). Xenopus interphase egg extracts that were supplemented with centrosomes (5 x 106/ml final) and fluorescently labeled tubulin (0.51 µM final) to visualize microtubules were incubated with or without cyclin B 90 for 2030 min at RT. A part of the reactions was saved in SDS-PAGE sample buffer for immunoblotting analysis. For microtubule assembly assay, the extracts were fixed with extract fix (60% glycerol, 1x Marc's modified Ringer's solution, 1 µg/ml Hoechst 33342, and 10% formaldehyde; Desai et al., 1999b) and squashed onto coverslips. For the immunofluorescence of TACC3 on centrosomes, extracts were treated with 20 µM nocodazole, fixed with fixation buffer (10% glycerol, 1x BRB80, 0.1% Triton X-100, and 5% formaldehyde), and spun down through glycerol cusion (30% glycerol in 1x BRB80) onto coverslips. The samples on coverslips were postfixed in 20°C methanol and processed for immunofluorescence as described previously (Desai et al., 1999b). The images were collected using a wide-field microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.) that was equipped with plan-Apochromat 100x NA 1.40 objective lens and a digital camera (model C4742-95; Hamamatsu). For quantification of fluorescence intensity, images were acquired by using a plan-Neofluor 16x NA 0.50 for the fluorescence signal of tubulin and a plan-Apochromat 100x NA 1.40 for the signal of TACC3 staining. The integrated pixel intensities within the circle (21.079 µm for tubulin; 1.079 µm for centrosomal TACC3) around centrosomes were calculated by using Metamorph software (Universal Imaging Corp.). All of the measured values were corrected by the subtraction of background signal in the same field.
In vitro protein kinase assay and mass spectometric analysis
0.4 mg/ml of bacterially expressed TACC3 was incubated with or without 0.02 mg/ml Aurora A in kinase buffer (25 mM Hepes, pH 7.7, 100 mM KCl, 5 mM MgCl2, 50 mM sucrose, and 0.1% ß-mercaptoethanol) in the presence of 0.1 mM ATP containing -[32P] ATP for 20 min at 30°C. The reactions were stopped by the addition of SDS-PAGE sample buffer, and the samples were analyzed by SDS-PAGE. The incorporation of 32P into the TACC3 in the gel was detected by autoradiography by using a phosphoimager (model BAS-1800II; Fujifilm). Mass spectometric analysis for identification of in vitro Aurora A phosphorylation sites of TACC3 was performed as described previously (Kraft et al., 2003).
Cell culture and RNA interference
HeLa cells were grown in DME containing 10% serum supplemented with nonessential amino acids (GIBCO BRL) and were grown using standard procedures. For RNA interference, HeLa cells were seeded onto glass coverslips 16 h before transfection. Cells that were grown to 40% confluency were transfected for 6 h using the OligofectAMINE reagent (Invitrogen) and to a final concentration of 20 nM siRNA against either TACC3 (purchased from Ambion; Gergely et al., 2003) or a control siRNA (control siRNA#1; Ambion) according to the manufacturer's recommendations. After transfection, cells were incubated in fresh media for 72 h before analysis by immunofluorescence.
HeLa S3 cells were grown in suspension in MEM that was modified for suspension cultures (Biochrom) containing 10% serum supplemented with nonessential amino acids (Biochrom) by using standard procedures. Cells were arrested in mitosis by the addition of 90 ng/ml nocodazole (Sigma-Aldrich) 20 h before harvesting and were shown to be >95% mitotic as judged by DAPI staining.
Immunofluorescence and microscopy for culture cells
Transfected HeLa cells that were grown on coverslips were fixed and permeabilized in methanol at 20°C for 8 min. Cells were washed with PBS and incubated 10 min in PBS containing 0.2% fish skin gelatin (PBS-FSG; Sigma-Aldrich) to prevent nonspecific binding of antibodies. Cells were incubated 20 min with primary antibody. Antibodies that were used were anti-tubulin (DM1A; Sigma-Aldrich), anti-TACC3 (Gergely et al., 2000), and antiphospho-TACC3 (antiP-Ser626 in this study) diluted to 1 µg ml1 in PBS-FSG. After washing in PBS, cells were incubated for 20 min at 37°C in 1 µg ml1 (in PBS-FSG) of donkey secondary antibodies that were labeled with either Texas red, FITC, or Cy5 (Jackson ImmunoResearch Laboratories) and were washed in PBS before mounting in the presence of 1 µg ml1 DAPI to visualize chromatin. Three-dimensional datasets were acquired on an imaging system (DeltaVision; Applied Precision) that was equipped with a microscope (model IX70; Olympus), a camera (CoolSNAP; Roper Scientific), and a 100x NA 1.4 plan-Apochromat objective. Images were computationally deconvolved by using the SoftWork software package (Applied Precision) and were shown as two-dimensional projections.
Online supplemental material
Fig. S1 shows spindles assembled in mock-depleted versus TACC3-depleted Xenopus egg extracts. Fig. S2 shows in vitro phosphorylation sites in TACC3/maskin as determined by mass spectrometry. Fig. S3 shows TACC3 phosphorylation in Aurora Adepleted extracts. Fig. S4 shows the characterization of phosphorylation mutant TACC3 in Xenopus egg extracts and in vitro. Fig. S5 shows centrosomal localization of XMAP215-GFP in Xenopus egg extracts. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200503023/DC1.
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Acknowledgments |
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K. Kinoshita was supported by a research fellowship from the Uehara Memorial Foundation and a grant from the Deutsche Forschungsgemeinschaft priority program Molecular Motors. L. Pelletier was supported by a postdoctoral fellowship from the Human Frontier Science Program.
Submitted: 4 March 2005
Accepted: 19 August 2005
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References |
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