Correspondence to Jordan W. Raff: j.raff{at}gurdon.cam.ac.uk
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Introduction |
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It was recently shown that Aurora A can phosphorylate the transforming acidic coiled coil (TACC) family of centrosomal proteins in vitro (Giet et al., 2002; Pascreau et al., 2005). TACC proteins stabilize spindle MTs in flies (Gergely et al., 2000a; Lee et al., 2001), humans (Gergely et al., 2003), worms (Bellanger and Gonczy, 2003; Le Bot et al., 2003; Srayko et al., 2003), and frogs (O'Brien et al., 2005) apparently by recruiting the MT-stabilizing protein Minispindles (Msps)/XMAP215/ch-TOG (colonic and hepatic tumor overexpressing gene; hereafter referred to as Msps) to the centrosome. Msps proteins bind directly to MTs and regulate MT dynamics primarily by influencing events at MT plus ends (for review see Cassimeris, 1999; Ohkura et al., 2001; Kinoshita et al., 2002). In Xenopus laevis egg extracts, for example, the balance of activity between XMAP215 and the MT-destabilizing protein XKCM1/MCAK (mitotic centromere-associated kinesin) at MT plus ends seems to be the main parameter that determines the overall stability of MTs (Tournebize et al., 2000; Kinoshita et al., 2001).
These findings present something of a paradox; Msps proteins act mainly on MT plus ends, yet, in vivo, they are most strongly concentrated at centrosomes, where the minus ends of MTs are clustered. To explain this paradox, it has been proposed that TACC proteins recruit Msps to centrosomes to ensure either that Msps is efficiently "loaded" onto MT plus ends as they grow out from centrosomal nucleation sites or that Msps can stabilize the minus ends of centrosomal MTs after they have been released from their nucleating sites (Lee et al., 2001). The finding that a GFPD. melanogaster TACC (D-TACC) fusion protein appears to associate with both the plus and minus ends of MTs in living D. melanogaster embryos is consistent with both possibilities (Lee et al., 2001).
Ser626 of X. laevis TACC3/maskin has recently been identified as a major site of Aurora A phosphorylation in vitro (Kinoshita et al., 2005; Pascreau et al., 2005), and this site is conserved in humans (Ser558) and flies (Ser863). We have investigated the potential significance of the phosphorylation of this site in D-TACC in regulating MT behavior in D. melanogaster embryos. Our findings suggest that D-TACCMsps complexes can stabilize MTs in two ways: (1) when not phosphorylated on Ser863, they can stabilize MTs throughout the embryo, presumably through interactions with MT plus ends; (2) when D-TACC is phosphorylated on Ser863, the complexes can stabilize MTs by interactions with MT minus ends. This second mechanism appears to be activated by Aurora A specifically at centrosomes, which perhaps explains why centrosomes are such dominant sites of MT assembly during mitosis.
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Results |
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In immunofluorescence experiments, both antiD-TACC and antiphosphoD-TACC (PD-TACC) antibodies strongly stained centrosomes during mitosis; antiD-TACC antibodies also stained spindle MTs, but this staining was essentially undetectable with anti-PD-TACC antibodies. This demonstrates that these antibodies did not recognize all of the D-TACC in the embryo, presumably because not all of the D-TACC was phosphorylated on Ser863 (Fig. 1 A). In early embryos, in which rapid mitotic cycles lack G1 and G2 phases, both antibodies stained centrosomes throughout the cell cycle (not depicted), whereas in cellularized embryos (Fig. 1 B), they stained centrosomes only during mitosis. Thus, in somatic cells, D-TACC and PD-TACC are only detectable at centrosomes during mitosis.
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PD-TACC can only be detected at centrosomes
These immunofluorescence studies suggested that PD-TACC was more concentrated at centrosomes than the bulk of the D-TACC protein. To confirm this, we performed Western blotting experiments on whole embryo extracts and on purified centrosome fractions. AntiD-TACC antibodies recognized D-TACC in both preparations, and we calculated that 1% of total embryonic D-TACC is present in purified centrosome fractions (Fig. 2 B; see Centrosome purification). We obtained similar results with antibodies against other centrosomal proteins, including Aurora A,
-tubulin (Fig. 2 B), Msps, CP190, and CP60 (not depicted). In contrast, anti-PD-TACC antibodies did not recognize D-TACC in the embryo extract, again demonstrating that these antibodies do not recognize the majority of D-TACC in the embryo, presumably because it is not phosphorylated on Ser863 (Fig. 2 B, left lane). These antibodies did, however, recognize a protein of exactly the same size as D-TACC in purified centrosome fractions (Fig. 2 B, right lane). Together with our immunofluorescence studies (Fig. 1), these data indicate that our anti-PD-TACC antibodies recognize only PD-TACC, that only a small fraction of total D-TACC in the embryo is phosphorylated at Ser863, and that this fraction of PD-TACC appears to be exclusively found at centrosomes.
Ser863 is essential for the function of D-TACC but not for its localization to centrosomes
To test the functional significance of the phosphorylation of D-TACC on Ser863, we examined the behavior of GFPD-TACC and GFP-S863L embryos. We first tested the viability of these embryos. Most d-tacc mutant embryos die early in embryonic development as a result of either a failure in pronuclear fusion or an accumulation of mitotic defects; both phenotypes appear to be caused by a destabilization of MTs (Gergely et al., 2000b; Lee et al., 2001). Less than 1% of d-tacc mutant embryos were viable (Fig. 3 A), and those that did develop had a high incidence of mitotic defects (Fig. 1 B), which were quantified in this study as the percentage of embryos with "extra centrosomes" at the cortex during nuclear cycle 14 (these extra centrosomes are indicative of earlier failures in mitosis; Fig. 3 B; for review see Raff, 2003). In contrast, 90% of GFPD-TACC embryos were viable (Fig. 3 A), and these embryos had very few mitotic defects (Fig. 3 B), suggesting that GFPD-TACC was almost fully functional. The viability of GFP-S863L embryos, however, was only
30% (Fig. 3 A), and those embryos that did develop had a high incidence of mitotic defects (Fig. 3 B), suggesting that GFP-S863L was only partially functional.
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To distinguish between these possibilities, we fixed GFPD-TACC and GFP-S863L embryos and stained them with antitubulin antibodies to examine the distribution of MTs (Fig. 6). In GFPD-TACC embryos, astral MTs were readily detectable at all stages of the cell cycle and were particularly prominent during anaphase (Fig. 6 A). In GFP-S863L embryos, few astral MTs were detectable at any stage of the cell cycle, including anaphase, although spindle MTs appeared to be relatively normal (Fig. 6 B). To confirm that spindle MTs were relatively unaffected in GFP-S863L embryos, we compared the lengths of mitotic spindles in living GFPD-TACC and GFP-S863L embryos at the same stage of mitosis (10 s before the initiation of anaphase) during nuclear cycle 10. The mean spindle length was 14.7 ± 0.35 µm (mean ± SD) in GFPD-TACC embryos (22 spindles were measured from three different embryos) and 13.2 ± 0.5 µm in GFP-S863L embryos (20 spindles were measured from three different embryos), which is a difference of only 10%. In contrast, when we injected antiD-TACC antibodies into wild-type embryos to perturb global D-TACC function, the mitotic spindles shortened by
25% (Gergely et al., 2000b; unpublished data). Thus, although GFP-S863L is apparently unable to stabilize astral MTs, it can at least partially stabilize spindle MTs. It is presumably this lack of astral MTs that explains the high frequency of mitotic defects in GFP-S863L embryos (de Saint Phalle and Sullivan, 1998).
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Discussion |
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It has previously been reported that Aurora A is required to recruit D-TACC to centrosomes (Giet et al., 2002). We find, however, that GFP-S863L still concentrates at centrosomes, although this concentration is somewhat weaker than that seen with GFPD-TACC, demonstrating that phosphorylation on Ser863 plays some part in recruiting D-TACC to centrosomes but is not absolutely essential. In the accompanying paper (Kinoshita et al., 2005), we show that the X. laevis TACC (X-TACC) protein X-TACC3 is phosphorylated by Aurora A in vitro on three sites that are conserved between frogs and humans, only one of which (Ser863) is conserved in flies. A form of X-TACC3 that mutated at all three serines localizes to centrosomes very weakly. It is possible, therefore, that there are other, nonconserved, Aurora A phosphorylation sites in D-TACC that have a more important role in recruiting the protein to centrosomes. Importantly, GFPD-TACC and GFP-S863L interact equally well with Msps in immunoprecipitation experiments, and the localization of Msps to centrosomes appears largely unperturbed in GFP-S863L embryos. Thus, we conclude that the defects in centrosome/MT behavior that we observe in GFP-S863L embryos are unlikely to arise simply from a failure to recruit D-TACCMsps complexes to centrosomes.
Although GFP-S863L concentrates at centrosomes, it is only partially functional. Whereas spindle MTs are relatively unperturbed in GFP-S863L embryos, astral MTs are dramatically destabilized. In addition, unlike GFPD-TACC, GFP-S863L appears unable to interact with the minus ends of spindle MTs, suggesting that this interaction requires the Aurora Adependent phosphorylation of D-TACC. If this were so, we might expect to detect PD-TACC on the minus ends of spindle MTs. Although this is not usually the case (Fig. 1 A), we can detect such a staining with anti-PD-TACC antibodies in favorable preparations of fixed embryos (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200504097/DC1). We suspect, therefore, that PD-TACC generated at the centrosome can interact with the minus ends of spindle MTs, but this is difficult to visualize in fixed preparations. In addition, we speculate that PD-TACC can bind to the minus ends of all centrosomal MTs (not just those in the spindle), but this interaction can only be visualized in the spindle, where large numbers of minus ends are tightly clustered in a region that is slightly separated from the centrosome.
Altogether, our observations suggest a model for how Aurora A, D-TACC, and Msps may cooperate to stabilize MTs during mitosis in D. melanogaster embryos (Fig. 7). We propose that D-TACCMsps complexes normally stabilize MTs in two ways. First, when D-TACC is not phosphorylated on Ser863, the complexes are present throughout the embryo and can potentially stabilize all MTs through either lateral interactions with MTs or interactions with MT plus ends (Fig. 7, mechanism 1). We favor the latter possibility because both D-TACC and Msps appear to concentrate at MT plus ends (Lee et al., 2001), and Msps family members primarily influence MT dynamics through interactions with plus ends. As this stabilization is independent of phosphorylation on Ser863, GFP-S863L can fulfill this function, which would explain why the expression of GFP-S863L significantly rescues the viability of d-tacc mutant embryos (from <1% to 30%). In support of this possibility, we show in an accompanying paper that nonphosphorylated X-TACC3 can enhance the ability of XMAP215 to stabilize MTs in vitro (Kinoshita et al., 2005).
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Although it is unclear how the phosphorylation of D-TACC on Ser863 leads to MT stabilization at centrosomes, we propose that phosphorylation allows D-TACC to interact with MT minus ends and stabilize them (Fig. 7, mechanism 2). This proposal will be controversial, as Msps proteins appear to stabilize MTs mainly through interactions with MT plus ends (Cassimeris, 1999; Ohkura et al., 2001; Kinoshita et al., 2002). Msps proteins are thought to have such a dramatic effect on MT plus end stability because they specifically counteract the MT destabilizing activity of Kin I kinesins at plus ends (Tournebize et al., 2000; Kinoshita et al., 2001, 2002; Ohkura et al., 2001; Popov et al., 2001; Usui et al., 2003; van Breugel et al., 2003). Several Kin I kinesins, however, are also concentrated at centrosomes (for review see Moore and Wordeman, 2004). In D. melanogaster embryos, the Kin I kinesin Klp10A has been reported to destabilize the minus ends of centrosomal MTs (Rogers et al., 2004). Like D-TACC, Klp10A is concentrated both at centrosomes and on the minus ends of spindle MTs that are clustered close to centrosomes (Rogers et al., 2004), and we find that Klp10A remains clustered at these MT minus ends in GFP-S863L embryos (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200504097/DC1). Perhaps the phosphorylation of D-TACC on Ser863 allows D-TACCMsps complexes to counteract the destabilizing activity of Klp10A at MT minus ends. If so, then a balance between the activities of Msps/XMAP215 and a Kin I kinesin seems to regulate the stability of MTs at both plus and minus ends.
Finally, our findings provide important insight into why centrosomes are the dominant sites of MT assembly during mitosis. As cells enter mitosis, centrioles recruit pericentriolar material in the Aurora Adependent process of centrosome maturation, which increases the MT nucleating capacity of centrosomes (Hannak et al., 2001; Berdnik and Knoblich, 2002). Our results suggest that this increase in nucleating capacity is insufficient on its own to generate large centrosomal arrays of MTs during mitosis; Aurora A must also phosphorylate D-TACC to activate D-TACCMsps complexes at centrosomes, which can then stabilize these centrosomal MTs. In this new model, Aurora A ensures that centrosomes are the major site of MT assembly during mitosis both by increasing the MT nucleating capacity of centrosomes and by stabilizing centrosomal MTs. As Aurora A, TACC, and ch-TOG (the human homologue of Msps) have all been implicated in human cancer (Raff, 2002; Meraldi et al., 2004), it will be interesting to determine whether their common role in stabilizing centrosomal MTs is linked to their roles in oncogenesis.
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Materials and methods |
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Centrosome purification
Centrosomes were purified from embryo extracts as described previously (Moritz and Alberts, 1999). On SDS gels, 0.1% of the total embryo extract fraction and 10% of the total centrosome fraction (which equated to approximately equal amounts of protein as judged from Coomassie-stained gels) were loaded in each lane, and these were processed for Western blotting as described above. We estimated the percentage of proteins that was present in the centrosome fraction by comparing the total signal in each lane. A 1:1 ratio of signal would mean that 1% of the total protein that was present in the embryo extract was present in the purified centrosome fraction.
Image acquisition and processing
Fixed embryos were examined with a microscope (Eclipse 800; Nikon) and a 60x NA 1.4 plan Apo objective by using a confocal system (Radiance; BioRad Laboratories) that was equipped with LaserSharp 2000 software (Bio-Rad Laboratories). Live embryos were aligned on coverslips as described previously (Huang and Raff, 1999) and were examined on a microscope (Axiovert 200M; Carl Zeiss MicroImaging, Inc.) with either a 63x NA 1.25 or 100x NA 1.3 plan Neofluor objective on a spinning disc confocal system (Ultraview RS; PerkinElmer) that was equipped with Ultraview RS software (PerkinElmer). Images of fixed embryos are all maximum intensity projections of five to eight image stacks that were taken at 0.5-µm intervals and made with the LaserSharp 2000 software. All images were imported into Adobe Photoshop 7.0 and adjusted to use the whole range of pixel intensities. An unsharp mask filter was applied to some images. In these images, the filter was applied to the whole image, and control and experimental images were treated in exactly the same way. Videos were made using Volocity 3.0 software (Improvision), and spindle length was measured with this software.
Quantitation of the centrosomal fluorescence of GFPD-TACC and GFP-S863L in living embryos was performed by making maximum intensity projections of 1012 image stacks that were taken at 0.5-µm intervals from cycle 1011 embryos in midinterphase. The projections were imported into Metamorph software (Universal Imaging Corp.), and mean fluorescence intensities were measured in a small area that was manually positioned around 30 centrosomes in each embryo. The mean pixel intensity per centrosome for each embryo was calculated.
Generation of GFPD-TACC and GFP-S863Lexpressing flies
A full-length D-TACC cDNA in pBluescript-SK (Stratagene) was modified by PCR to allow GFP to be inserted in frame upstream of the initiating ATG codon (generating pBS-GFPD-TACC). This plasmid was modified by using PCR to replace Ser863 with a leucine (generating pBSGFP-S863L). All PCR products were sequenced to confirm that they contained no sequence errors. GFPD-TACC and GFP-S863L were then subcloned into the pWR-pUbq transformation vector (Huang and Raff, 1999), and w,f flies were transformed using standard techniques (Roberts, 1986). Transformed flies containing the GFPD-TACC or GFP-S863L transgene were mated with d-taccstella592 flies to generate stocks that were homozygous for the d-tacc mutation and contained one copy of the transgene. All experiments were performed with two independent transformed lines for each construct, and results from both lines were pooled.
Online supplemental material
Videos of the embryos shown in Fig. 4 are included, as are additional videos showing the flaring behavior of GFPD-TACC and GFP-S863L. Four additional figures are also included that show (1) the quantitation of centrosomal levels of GFPD-TACC and GFP-S863L; (2) the interaction between Msps and GFPD-TACC or GFP-S863L in immunoprecipitation experiments; (3) the staining of PD-TACC on the minus ends of spindle MTs; and (4) the localization of Klp10A in GFP-S863L embryos. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200504097/DC1.
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Acknowledgments |
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This work was funded by a Senior Research Fellowship from Cancer Research UK (to J.W. Raff) with earlier support from a Wellcome Trust Senior Research Fellowship (to J.W. Raff), Fundação para a Ciencia e a Tecnologia of Portugal (to T.P. Barros), and a Uehara Memorial Foundation Fellowship (to K. Kinoshita).
Submitted: 18 April 2005
Accepted: 4 August 2005
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