Regulation of stem cell self-renewal versus differentiation is critical for embryonic development and adult tissue homeostasis. Drosophila larval neuroblasts divide asymmetrically to self-renew, and are a model system for studying stem cell self-renewal. This study identified three mutations showing increased brain neuroblast numbers that map to the aurora-A gene, which encodes a conserved kinase implicated in human cancer. Clonal analysis and time-lapse imaging in aurora-A mutants show single neuroblasts generate multiple neuroblasts (ectopic self-renewal). This phenotype is due to two independent neuroblast defects: abnormal atypical protein kinase C (aPKC)/Numb cortical polarity and failure to align the mitotic spindle with the cortical polarity axis. numb mutant clones have ectopic neuroblasts, and Numb overexpression partially suppresses aurora-A neuroblast overgrowth (but not spindle misalignment). Conversely, mutations that disrupt spindle alignment but not cortical polarity have increased neuroblasts. It is concluded that Aurora-A and Numb are novel inhibitors of neuroblast self-renewal and that spindle orientation regulates neuroblast self-renewal (Lee, 2006).
Mutations in aurA lead to a massive increase in larval brain neuroblasts. The major cause of this phenotype appears to be misregulation of neuroblast cortical polarity. One cortical polarity defect is increased basal localization of aPKC, which is sufficient to induce ectopic neuroblasts. Consistent with this hypothesis, aPKC aurA double mutants show strong suppression of the aurA supernumerary neuroblast phenotype, consistent with aPKC functioning downstream from AurA. While it is possible that loss of aPKC suppresses the phenotype in a nonspecific way (e.g., by arresting neuroblast cell proliferation or inducing neuroblast apoptosis), ni similarly strong suppression of the brat supernumerary neuroblast phenotype was observed in aPKC brat double mutants. This shows that aPKC functions more specifically in the AurA pathway than in the Brat pathway (Lee, 2006).
The only other detectable cortical polarity defect seen in aurA mutant neuroblasts is a delocalization of Numb from the basal cortex. A similar Numb defect is seen during asymmetric cell division of pupal SOPs in aurA mutants, perhaps reflecting a specific and direct regulation of Numb by AurA, although Numb is not phosphorylated by AurA in vitro. The importance of the Numb delocalization phenotype is revealed by the ability of Numb overexpression in neuroblasts to rescue most of the aurA mutant phenotype (all except the component due to spindle orientation defects). Thus, Numb acts downstream from AurA to inhibit neuroblast self-renewal. Numb joins Mira/Pros/Brat as proteins that are partitioned into the GMC during neuroblast asymmetric cell division, where they function to inhibit neuroblast self-renewal (Lee, 2006).
Where does AurA function to inhibit neuroblast self-renewal? AurA appears to be required in the neuroblast lineage, and not in surrounding glial cells or nonneuronal tissues of the larva, because neuroblast-specific expression of either AurA or the downstream component Numb can rescue most of the aurA supernumerary neuroblast phenotype. This shows that AurA is not required outside the neuroblast lineage to inhibit neuroblast self-renewal. Within the neuroblast, AurA appears to function in the cytoplasm and not at the centrosome, because cnn mutants lack all detectable AurA centrosomal localization yet do not match the aurA supernumerary neuroblast phenotype. It is concluded that AurA acts in the neuroblast cytoplasm to promote aPKC/Numb cortical polarity and spindle-to-cortex alignment (Lee, 2006).
How does Numb inhibit neuroblast self-renewal in the GMC? Numb is a well-characterized inhibitor of Notch signaling that is segregated into the GMC, and Notch signaling is active in larval neuroblasts but not in GMCs. Thus the most obvious model is that Numb blocks Notch receptor signaling in the GMC. However, Notch mutant clones generated in larval neuroblasts do not affect neuroblast survival or clone size. Similarly, no change has been seen in neuroblast number in two different Notch-ts mutants (although the expected small wing imaginal disc phenotype was observed. In addition, no supernumerary neuroblasts were observed in larval neuroblast clones overexpressing the constitutively active Notch intracellular domain, although the same Notch intracellular domain generates the expected sibling neuron phenotype when expressed in the embryonic CNS. Thus, Notch is an excellent candidate for promoting neuroblast self-renewal, but additional experiments will be needed to test this model more rigorously. In this context, it is interesting to note that Notch promotes stem cell self-renewal in mammals (Lee, 2006).
aurA mutant neuroblasts have essentially random orientation of the mitotic spindle relative to the apical/basal cortical polarity axis, resulting in a some neuroblasts dividing symmetrically (in size and cortical polarity markers). This phenotype may arise due to lack of astral microtubule interactions with the neuroblast cortex; aurA mutant neuroblasts have reduced astral microtubule length. Alternatively, AurA may affect spindle orientation by phosphorylating proteins required for spindle orientation, such as Cnn, Pins, or Mud. For example, Mud has a consensus AurA/Ipl1 phosphorylation site within its microtubule-binding domain, and it will be interesting to determine if this site needs to be phosphorylated for Mud to bind microtubules. Spindle orientation defects only generate part of the supernumerary neuroblast phenotype in aurA mutant brains, however, because overexpression of Numb can rescue most of the phenotype without rescuing spindle alignment, and cnn or mud mutants have nearly random spindle alignment but only a modest increase in neuroblast number. Thus, it is proposed that spindle orientation defects and cortical polarity defects combine to generate the dramatic supernumerary neuroblast phenotype seen in aurA mutants (Lee, 2006).
Mammalian aurA has been termed an oncogene due to its overexpression in several cancers, its ability to promote proliferation in certain cell lines, and the fact that reduced levels lead to multiple centrosomes, mitotic delay, and apoptosis. However, an in vivo aurA mutant phenotype has not yet been reported. In contrast, aurA loss-of-function mutations result in a neuroblast 'brain tumor' phenotype, including prolonged neuroblast proliferation during pupal stages when wild-type neuroblasts have stopped proliferating. aurA mutants do not, however, have the imaginal disc epithelial overgrowth seen in other Drosophila tumor suppressor mutants, and aurA mutant neuroblasts have a delay in cell cycle progression. It is proposed that the aurA supernumerary neuroblast phenotype is not due to loss of growth control or a faster cell cycle time, but rather due to a cell fate transformation from a differentiating cell type (GMC) to a proliferating cell type (neuroblast) (Lee, 2006).
It is concluded that AurA restrains neuroblast numbers using two pathways: first by promoting Numb localization into the GMC, and second by promoting alignment of the mitotic spindle with the cortical polarity axis. Absence of the first pathway leads to increased neuroblasts at the expense of GMCs, whereas absence of the second pathway leads to increased neuroblasts due to symmetric cell division. It will be interesting to determine whether mammalian AurA uses one or both pathways to regulate stem cell asymmetric division and self-renewal (Lee, 2006).
The Aurora A-dependent localization of D-TACC to centrosomes prompted an examination of whether the two proteins show any physical interactions. To this end anti-Aurora A antibodies were used to immunoprecipitate Aurora A protein from Drosophila embryo extracts and the precipitate was analyzed for the presence of both Aurora A and D-TACC proteins by Western blotting. The presence of D-TACC in the Aurora A immunoprecipitate indeed suggests that the proteins interact either directly or via intermediate proteins. The reciprocal experiment was performed and D-TACC was immunoprecipitated from the extract, no Aurora A was detected in the precipitate. One possible interpretation of this result is that the anti-D-TACC antibody might interfere with the D-TACC-Aurora A interaction. Therefore, the experiment was repeated using a Drosophila line expressing a GFP-D-TACC fusion protein under the control of the polyubiquitin promoter and to perform immunoprecipitation using anti-GFP antibodies in an attempt to avoid possible antibody interference. GFP-tagged D-TACC protein can be immunoprecipitated from the extracts of such embryos, and Aurora A kinase is present in this immunoprecipitate. However, Aurora A was not found in the immunoprecipitate obtained using the same anti-GFP antibody on the extract from embryos expressing a Tau-GFP fusion protein. Together, these experiments demonstrate an interaction between the Aurora A and D-TACC proteins in extracts of wild-type embryos (Giet, 2002).
If this interaction has functional significance, it might be expected to have been conserved through evolution. Therefore, whether the human counterpart of Aurora A shows association with HsTACC3, the closest human counterpart to D-TACC, was examined. Just as in Drosophila cells, an immunoprecipitate of Hs Aurora A contained HsTACC3. Moreover, immunostaining has revealed both proteins to colocalize around centrosomes in cultured human cells (Giet, 2002).
The syncytial Drosophila embryo is provided with a rich dowry of maternal proteins that enable it to undertake the 13 rapid cycles of nuclear division before cellularization. Therefore, any collection of embryos will have not only representation of different cell cycle stages, but also of differing extents of development to which the maternally provided mitotic proteins make an increasing contribution. Thus, it was necessary to determine whether the interaction between Aurora A and D-TACC was mirrored by a major multisubunit complex that could represent either a maternal store or some other functional unit. Therefore, an extract was fractionated from a 2-h collection of predominantly syncytial embryos by sedimentation on a 5%-40% sucrose gradient. Western blotting of the fractions revealed that D-TACC sediments at 6-8 s, suggesting it is present in a complex with MSPS as shown by Lee (2001). The small, previously described complex of gamma-tubulin [thought to be a heterotetramer of two molecules of gamma-tubulin, and one molecule each of Dgrip84 and Dgrip91], by comparison sediments slightly ahead at 7 s. Aurora A, in contrast, sediments predominantly at 3-4 s, showing only a small proportion cosedimenting with the D-TACC complex. Thus, it seems that there are substantial maternal pools of both proteins, but they are not stockpiled in the same multiprotein complex, suggesting that only a small fraction of Aurora A interacts with the D-TACC complex (Giet, 2002).
Nevertheless, since immunoprecipitation experiments have suggested that the two proteins can associate, it was of interest to see whether Aurora A is also able to phosphorylate D-TACC. This was tested in two ways. In the first, GFP-tagged D-TACC protein was immunoprecipitated from Drosophila embryos and then the fusion protein was subjected to a heat treatment, to inactivate any endogenous protein kinases before incubating it with a preparation of active recombinant Aurora A-(His)6 protein kinase and radiolabeled ATP. The products of the kinase reaction were analyzed by SDS-PAGE on either a 10% or a 6% gel, followed by autoradiography. Western blotting was performed on the same membrane where the kinase reaction products were analyzed by autoradiography. When Aurora A kinase is added, the appearance of several phosphorylated bands was detected. These bands comigrate exactly with the GFP-D-TACC protein and its degradation products when observed by autoradiography. In a second test of whether D-TACC is an Aurora A substrate, bacterially expressed and purified fusion proteins between Maltose Binding protein and different domains of the D-TACC protein were used. It was found that Aurora A kinase phosphorylates the NH2- or the COOH-terminal domains of D-TACC only very poorly. In contrast, the middle fragment of the protein is a highly favored substrate. Thus, it appears that not only does D-TACC require active Aurora A kinase for its localization to the centrosome, but it is also a good in vitro substrate of the enzyme (Giet, 2002).
A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. This study shows that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, Centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN (Terada, 2003).
In animal cells, microtubules are organized from the centrosome/microtubule-organizing center (MTOC), composed of a pair of centrioles and the surrounding pericentriolar material. Individual microtubules are nucleated from an ~25-nm γ-tubulin–containing ring complex (γ-TuRC). At the onset of M phase, the centrosome becomes 'mature' and organizes more microtubules, which is accompanied with an increased level of γ-tubulin accumulation at each spindle pole. One of the molecules that has been implicated in the mechanism of centrosome maturation is Aurora-A, a mitosis-specific Ser/Thr kinase located at mitotic poles and spindle microtubules. The kinase, originally identified as a gene product important in spindle assembly and function in Drosophila, has recently been shown to be in the Ran-signaling pathway and to play an important role in efficient transmission of Ran-GTP gradient established by the condensed chromosomes for the control of spindle assembly and dynamics. Aurora-A binds to spindle components, such as TACC/XMAP215 and TPX2. Although possible functions of those molecules and their interaction with Aurora-A in bipolar spindle formation have been elucidated, mechanisms of how Aurora-A stimulates the recruitment of γ-tubulin to the centrosome at spindle poles have not yet been evaluated. To address this question, centrosomal proteins were sought that interact with Aurora-A and regulate the process of microtubule nucleation onto the centrosome (Terada, 2003).
By screening of a Drosophila two-hybrid library, two clones were isolated encoding a molecule capable of interaction with Aurora-A. The sequence corresponds to the COOH-terminal domain of centrosomin (CNN), a core component of the centrosome important for assembly of mitotic centrosomes in Drosophila. Although the truncated polypeptide covered by clone CNN-C1 appears to be sufficient for interaction with Aurora-A, the binding intensity was weaker than CNN-C. Endogenous Aurora-A, but not Aurora-B, immunoprecipitates with HA-tagged CNN expressed in S2 cells. Specificity of the COOH-terminal domain of CNN for interaction with Aurora-A was further confirmed by in vitro binding assays (Terada, 2003).
To investigate the role of protein interaction in the centrosome, S2 cells were prepared from which Aurora-A or CNN was depleted by RNA interference (RNAi). In cells lacking Aurora-A, not only CNN, but also γ-tubulin, were absent at each spindle pole. When CNN was depleted, neither γ-tubulin nor Aurora-A was seen at the spindle pole. In cells with partially depleted Aurora-A or CNN, comparable amounts of γ-tubulin and CNN or Aurora-A were detected at each pole. Besides γ-tubulin, other centrosome proteins, CP190 and CP60 , become dislocated from the spindle poles in RNAi cells. Therefore, it is concluded that CNN and Aurora-A are mutually dependent for localization at spindle poles, which is required for proper targeting of other centrosomal proteins to the centrosome. This is consistent with previous observations (Barbosa, 2000) that the centrosomal association of CNN is not dependent on the presence of γ-tubulin/γ-TuRC (Terada, 2003).
To confirm the role of CNN in recruiting γ-tubulin, protein interaction was analyzed in vitro. Nickel beads conjugated with His-tagged CNN were mixed with cell extracts prepared from colcemid-treated S2 cells. γ-Tubulin was specifically sedimented by the full and NH2-terminal sequence, but not the COOH-terminal sequence of CNN. Because neither in vitro binding assays nor two-hybrid screens demonstrated direct binding between two molecules, CNN may interact with a γ-tubulin complex, rather than γ-tubulin directly. Further, HA-tagged CNN was expressed in S2 cells. Exogenous proteins caused formation of γ-tubulin–containing cytoplasmic aggregates capable of microtubule formation and association with microtubule asters. These results clearly indicate that the NH2-terminal domain of CNN interacts with γ-tubulin/γ-TuRC and plays an important role in assembly of MTOCs (Terada, 2003).
γ-Tubulin/γ-TuRC–mediated microtubule assembly is believed to be common among species. Thus, it is highly likely that an Aurora-A–binding molecule(s) equivalent to CNN is functioning in a variety of organisms. Although Drosophila CNN was unable to associate with mammalian Aurora-A in transfected mammalian cells as well as by two-hybrid screens, the NH2-terminal domain of CNN still interacts with γ-tubulin/γ-TuRC in mammalian cells as in S2 cells. To analyze a possible role of CNN–γ-tubulin interaction in initiation of microtubule assembly, Drosophila CNN was overexpressed in mammalian cells. HA-tagged CNN induces cytoplasmic foci in various sizes and numbers. Significantly, the pattern of microtubule distribution is profoundly affected as a result of microtubule association with virtually every dot containing CNN. These sites can initiate microtubule formation as evidently shown in cells where short microtubules are assembled during brief recovery from nocodazole treatment. All cells overexpressing CNN induced microtubule-organizing sites, which were associated with centrosome proteins, such as pericentrin and Cep135. Particularly prominent was γ-tubulin, which was probably recruited from a large cytoplasmic pool. In support of this view, GFP-tagged exogenous γ-tubulin became colocalized with HA-CNN to participate in the formation of microtubule-nucleating sites. This was in striking contrast with cells expressing γ-tubulin alone, where cytoplasmic aggregates induced by γ-tubulin expression could not contribute to microtubule formation. These results suggest that microtubules are directly nucleated from the CNN aggregates through the mechanism mediated by γ-tubulin/γ-TuRC (Terada, 2003).
To confirm the microtubule-nucleating activity of the CNN aggregates, microtubules were polymerized in vitro by incubating isolated GFP-tagged CNN dots with X-rhodamine–conjugated brain tubulin. There was always a dot positive in GFP fluorescence at the center of the microtubule asters. Although variable numbers of microtubules emanated from the center, more microtubules tended to polymerize onto the GFP dots in larger sizes. The process of aster formation was monitored by time-lapse microscopy. A fluorescence image taken 10 min after mounting the sample on a microscopic stage revealed several microtubules growing from a GFP-positive site. As time progressed, more microtubules appeared to emanate from the center, indicating that microtubules were formed by direct polymerization onto the CNN-containing foci, rather than that preformed microtubules were gathered around the center (Terada, 2003).
Microtubules are nucleated from the pericentriolar material that surrounds the centrioles of the centrosome. To compare ultrastructure of microtubule-initiating sites induced by CNN with that of the pericentriolar material/centrosome, CHO cells expressing GFP-tagged CNN were examined by EM. Two microtubule asters were seen formed in cells that were briefly extracted before fixation. Located at each focal point of microtubule asters was an electron-dense particle in various sizes and shapes. Unlike the pericentriolar material, which has been described as an ill-defined amorphous cloud, the entire structure induced by CNN was well delineated by electron-dense materials to which microtubules were attached. In favorable sections, microtubules could be seen penetrating to the interior region of the aggregates. Neither centrioles nor centrosomal substructures, such as satellites, appendages, and CHO cell–specific virus particles, were generally seen at the site induced by CNN expression. Because CNN is a coiled-coil structural protein (Heuer, 1995), the dense particles likely represent the aggregated form of overexpressed CNN proteins (Terada, 2003).
Multiple centrosomes/MTOCs have been detected in cells in which the mechanism of centrosome duplication coupled with the cell cycle control becomes deregulated. In the case of CNN-containing MTOCs, their number and size formed during relatively short periods (8–12 h) varied greatly according to the level of protein expression. Moreover, no centrioles were found at ectopic MTOCs by EM and immunostaining with centriole-specific centrin-2 antibodies. Therefore, it is plausible that CNN expression causes the formation of protein aggregates that acquire the microtubule-nucleating capacity by recruiting γ-tubulin/γ-TuRC. This unique property of CNN to generate microtubule-nucleating sites by interacting with γ-tubulin/γ-TuRC suggested CNN may function as an adaptor for connecting γ-tubulin to the centrosome (Terada, 2003).
By expressing truncated polypeptides, it was concluded that CNN's ability to interact with γ-tubulin/γ-TuRC and induce ectopic microtubule-nucleating sites resides in the NH2-terminal sequence of CNN from which the Aurora-A–binding domain is omitted. In contrast, cytoplasmic aggregates formed in cells expressing the COOH-terminal domain failed to initiate microtubule formation in both S2 and mammalian cells. These results lead to the conclusion that CNN consists of two functionally distinct subdomains: the Aurora-A–binding site is at the COOH terminus capable of formation of the protein complex to be recruited to the spindle pole, and the NH2-terminal sequence is involved in assembling centrosomes/MTOCs by recruiting γ-tubulin/γ-TuRC. Although no CNN homologues have yet been identified outside Drosophila, Aurora-A would likely be involved in the control of microtubule nucleation through its association with the COOH terminus of a CNN-related molecule(s) in mammalian cells (Terada, 2003). Control of mitotic spindle assembly onto the centrosome could be achieved by several mechanisms, including nucleation of individual microtubules onto γ-tubulin–containing protein complexes, stimulation of microtubule nucleation and stabilization of polymerized microtubules by MAPs, and recruitment of minus ends of preexisting microtubules by the action of motor activity to the centrosome. Aurora-A binds not only CNN but also the D-TACC/MSPS/XMAP215 complex. These components appear to be required for microtubule assembly on mitotic centrosomes/poles controlled through the distinct mechanisms from that of γ-tubulin recruitment. Therefore, it is reasonable that Aurora-A plays a role in regulating the overall process of centrosome maturation by orchestrating multiple pathways of microtubule assembly during mitosis. It is worth mentioning that individual mechanisms of microtubule assembly may show a distinct requirement for protein phosphorylation and the Aurora-A kinase activity; although both Aurora-A and CNN are still able to locate at the centrosome, D-TACC/MSPS complex failed to be recruited to spindle poles in the absence of enzymatic activity of Aurora-A kinase (Terada, 2003).
Aurora kinases are highly expressed in cells derived from many human tumor cell types, which frequently contain multiple centrosomes. Because defects in the number, structures, and function of centrosomes are closely associated with the genetic instability in transformed cells, Aurora-A might be involved in tumorigenesis by inducing abnormal numbers of MTOCs as a result of inappropriate distribution of CNN-like molecule(s) (Terada, 2003).
Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. Drosophila melanogaster TACC (D-TACC) has been shown to be phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A-dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. It is proposed that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC-Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis (Barros, 2005).
The centrosome is the main microtubule (MT) organizing center in animal cells, and it plays an important part in organizing many processes in the cell, including cell polarity, intracellular transport, and cell division. Aurora A protein kinases are centrosomal proteins that are essential for mitosis and have been widely implicated in human cancer. They have several functions in mitosis, and they appear to play a particularly important part in regulating centrosome behavior. They are, for example, required for the dramatic recruitment of pericentriolar material to the centrosome, which occurs as cells enter mitosis. This centrosome 'maturation' is thought to ensure that centrosomes are the dominant sites of MT assembly during mitosis (Barros, 2005 and references therein).
It was recently shown that Aurora A can phosphorylate the transforming acidic coiled coil (TACC) family of centrosomal proteins in vitro. TACC proteins stabilize spindle MTs in flies, humans, worms, and frogs 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. 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 (Barros, 2005).
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. The finding that a GFP-D. 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 (Barros, 2005).
Ser626 of X. laevis TACC3/maskin has recently been identified as a major site of Aurora A phosphorylation in vitro, and this site is conserved in humans (Ser558) and flies (Ser863). The potential significance of the phosphorylation of this site in D-TACC in regulating MT behavior was investigated in D. melanogaster embryos. The findings suggest that D-TACC-Msps 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 (Barros, 2005).
D-TACC phosphorylated on Ser863 (P-D-TACC) is detectable only at centrosomes, whereas nonphosphorylated D-TACC, like most other centrosomal proteins (including γ-tubulin, Aurora A, Msps, CP190, CP60, and centrosomin), has large pools of protein that are present in the cytoplasm of Drosophila embryos. It is concluded that Aurora A stimulates the phosphorylation of D-TACC only at centrosomes and that, once phosphorylated, P-D-TACC is either unable to exchange with the soluble pool of D-TACC or is rapidly dephosphorylated when it leaves the centrosome. Since Ser863 is a conserved site of Aurora A phosphorylation in vitro, it seems likely that Aurora A directly phosphorylates Ser863 in vivo, although the possibility cannot be excluded that Aurora A indirectly stimulates the phosphorylation of Ser863 at centrosomes by activating another kinase. It is unclear why Aurora A stimulates the phosphorylation of D-TACC only at centrosomes, but it is noted that two activators of Aurora A kinase, TPX2 and Ajuba, are themselves concentrated at centrosomes (Barros, 2005).
It has been reported that Aurora A is required to recruit D-TACC to centrosomes. This study found, however, that GFP-S863L still concentrates at centrosomes, although this concentration is somewhat weaker than that seen with GFP-D-TACC, demonstrating that phosphorylation on Ser863 plays some part in recruiting D-TACC to centrosomes but is not absolutely essential. An accompanying paper (Kinoshita, 2005), shows 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 was 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, GFP-D-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, it is concluded that the defects in centrosome/MT behavior that were observe in GFP-S863L embryos are unlikely to arise simply from a failure to recruit D-TACC-Msps complexes to centrosomes (Barros, 2005).
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 GFP-D-TACC, GFP-S863L appears unable to interact with the minus ends of spindle MTs, suggesting that this interaction requires the Aurora A-dependent phosphorylation of D-TACC. If this were so, one might expect to detect P-D-TACC on the minus ends of spindle MTs. Although this is not usually the case, such a staining can be detected with anti-P-D-TACC antibodies in favorable preparations of fixed embryos. It is suspected, therefore, that P-D-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, it is speculated that P-D-TACC can bind to the minus ends of all centrosomal MTs (not just those in the spindle), but this interaction can be visualized only in the spindle, where large numbers of minus ends are tightly clustered in a region that is slightly separated from the centrosome (Barros, 2005).
Altogether, these observations suggest a model for how Aurora A, D-TACC, and Msps may cooperate to stabilize MTs during mitosis in D. melanogaster embryos. It is proposed that D-TACC-Msps 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 (see Mechanism 1 of A schematic model of how the D-TACC-Msps complex stabilizes MTs in Drosophila embryos; Barros, 2005). The latter possibility is favored because both D-TACC and Msps appear to concentrate at MT plus ends, and Msps family members primarily influence MT dynamics through interactions with plus ends. Since 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, it has been shown (Kinoshita, 2005) that nonphosphorylated X-TACC3 can enhance the ability of XMAP215 to stabilize MTs in vitro (Barros, 2005).
The Aurora A-dependent phosphorylation of D-TACC on Ser863 at centrosomes, however, activates a second MT stabilization mechanism that acts exclusively on MTs associated with the centrosome. This mechanism cannot operate in GFP-S863L embryos, and, as a result, astral MTs are dramatically destabilized. The lack of this stabilization mechanism in GFP-S863L embryos, however, appears to have only a limited effect on spindle MTs. It is speculated that this is because there is a chromatin-based acentrosomal pathway of spindle assembly that can compensate for the instability of centrosomal MTs. Such a pathway exists in many cell types and is especially robust in Drosophila. Because centrosomes still nucleate many MTs in GFP-S863L embryos (centrosomal MTs are simply less stable than normal), these centrosomal MTs can interact with the MTs that assembled around the chromatin to form relatively normal spindles. In contrast, astral MTs, which are exclusively nucleated by centrosomes and do not interact with MTs nucleated around the chromosomes, are dramatically destabilized in GFP-S863L embryos. Kinoshita (2005) shows that the Aurora A-dependent phosphorylation of X-TACC3 is also required to stabilize centrosomal (but not spindle) MTs in X. laevis egg extracts, suggesting that this mechanism is conserved at least in frogs and flies (Barros, 2005).
Although it is unclear how the phosphorylation of D-TACC on Ser863 leads to MT stabilization at centrosomes, it is proposes that phosphorylation allows D-TACC to interact with MT minus ends and stabilize them (see Mechanism 2 of A schematic model of how the D-TACC-Msps complex stabilizes MTs in Drosophila embryos; Barros, 2005). This proposal will be controversial, since Msps proteins appear to stabilize MTs mainly through interactions with MT plus ends. 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. Several Kin I kinesins, however, are also concentrated at centrosomes. In D. melanogaster embryos, the Kin I kinesin Klp10A has been reported to destabilize the minus ends of centrosomal MTs. Like D-TACC, Klp10A is concentrated both at centrosomes and on the minus ends of spindle MTs that are clustered close to centrosomes, and this study finds that Klp10A remains clustered at these MT minus ends in GFP-S863L embryos. Perhaps the phosphorylation of D-TACC on Ser863 allows D-TACC-Msps 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 (Barros, 2005).
Finally, these 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 A-dependent process of centrosome maturation, which increases the MT nucleating capacity of centrosomes. The 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-TACC-Msps 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. Since Aurora A, TACC, and ch-TOG (the human homologue of Msps) have all been implicated in human cancer, it will be interesting to determine whether their common role in stabilizing centrosomal MTs is linked to their roles in oncogenesis (Barros, 2005).
The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. Perturbation of the Ran pathway disrupts multiple steps of mitosis in syncytial Drosophila embryos and new mitotic processes have been uncovered that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles (Silverman-Gavrila, 2006).
Perturbing the Ran pathway in vivo has dramatic effects on spindle assembly and function. Ran could achieve this by directly regulating the activity of a large number of inhibiting spindle assembly factors (SAF)s, by regulating signal transduction pathways, or both. One candidate target is the Aurora A kinase, which in vitro is suggested to be downstream of Ran in the Ran spindle assembly pathway. Aurora A is recruited to centrosomes in interphase by Centrosomin where it is activated by ajuba and HEF-1. During mitosis in somatic mammalian cells Aurora A relocates to spindle MTs in a TPX2-dependent manner (Silverman-Gavrila, 2006 and references therein).
To address whether Aurora A could be affected by Ran, Aurora A localization was assessed in control and RanT24N-injected embryos. In control embryos, Aurora A localized to centrosomes in interphase and prophase, but in prometaphase it began to redistribute along spindle MTs. On injection of RanT24N, 83.3% of spindles showed mislocalization of Aurora A, which now concentrated at centrosomes and did not localize to spindle MTs (Silverman-Gavrila, 2006).
To determine whether Aurora A targeting to MTs is regulated by NTRs as demonstrated in vitro, DeltaN importin ß was injected and Aurora A localization was examined. DeltaN importin ß injection also prevented Aurora A redistribution to spindle MTs in 92% of spindles. This strongly suggests that in vivo Aurora A function is regulated by a NTR sensitive SAF (Silverman-Gavrila, 2006).
The protein kinase Aurora-A is required for centrosome maturation, spindle assembly, and asymmetric protein localization during mitosis. Borealis (Bora, so named for aurora borealis to indicate its similarity with aurora-A) is a conserved protein that is required for the activation of Aurora-A at the onset of mitosis. In the Drosophila peripheral nervous system, bora mutants show defects during asymmetric cell division identical to those observed in aurora-A. Furthermore, overexpression of bora can rescue defects caused by mutations in aurora-A. Bora is conserved in vertebrates, and both Drosophila and human Bora can bind to Aurora-A and activate the kinase in vitro. In interphase cells, Bora is a nuclear protein, but upon entry into mitosis, Bora is excluded from the nucleus and translocates into the cytoplasm in a Cdc2-dependent manner. A model is presented here in which activation of Cdc2 initiates the release of Bora into the cytoplasm where it can bind and activate Aurora-A (Hutterer, 2006).
To test whether the genetic interaction reflects a physical interaction between Bora and Aurora-A, binding assays were performed in Drosophila tissue culture cells. Drosophila S2 cells were transfected with Aurora-A and Bora-GFP, and protein lysates were subjected to immunoprecipitation by anti-GFP. Since Aurora-A is specifically detected in the immunoprecipitate, it is concluded that Bora can bind to Aurora-A in vivo. To test whether this is due to a direct interaction, in vitro binding experiments were performed. In vitro translated Aurora-A binds to a GST-Bora fusion-protein but not to GST alone. While the nonconserved C terminus of Bora is dispensible for Aurora-A binding, the interaction is abrogated by deleting the conserved region (BoraΔ2) or a region N-terminal to the conserved part (BoraΔ1). Interestingly, the interaction is also observed between in vitro translated human Aurora-A and MBP-HsBora. Human Aurora-A can even bind to Drosophila MBP-Bora in vitro. The interaction with Aurora-A seems to be essential for Bora function since the N-terminal 404 amino acids of Bora (almost identical to BoraΔ3) can rescue the bora and aurA37 mutant phenotypes, while the C terminus (amino acids 404–539) does not. Thus, Bora and its homologs act as binding partners of Aurora-A (Hutterer, 2006).
Several Aurora-A regulators—like TPX2 also act as substrates for the kinase. To test whether Bora can be phosphorylated by Aurora-A, in vitro kinase assays were performed. Drosophila Aurora-A expressed and purified from E. coli can phosphorylate bacterially expressed myelin basic protein tagged Bora (MBP-Bora) but not MBP alone. Interestingly, the kinase activity of Aurora-A toward Bora is as potent as toward myelin basic protein, which is often used as a model substrate. Similarly, human Aurora-A can phosphorylate the human Bora homolog. To test which region of Bora is phosphorylated, Bora deletions were used in the kinase assay. Deletion of 125 amino acids from the N terminus of Bora (BoraΔ2) eliminates phosphorylation by Aurora-A, while deletion of the C terminus from amino acid 209 onward (BoraΔ5) does not affect it. Interestingly, Bora is still phosphorylated when the N-terminal 67 amino acids are deleted (BoraΔ1), suggesting that direct binding to Aurora-A is not necessary for Bora to act as a substrate. These experiments suggest that the N terminus of Bora is phosphorylated by Aurora-A (Hutterer, 2006).
To test whether Bora can influence the kinase activity of Aurora-A, recombinant human Bora was used in an in vitro kinase assay with myelin basic protein as a substrate. Addition of Bora increases Aurora-A activity in a dose-dependent manner, and a 2.5-fold maximum increase in kinase activity was observed. Aurora-A is regulated by phosphorylation in the activation loop of the kinase. Since Aurora-A can autophosphorylate, any kinase preparation may be partially active, and this might explain the modest degree of activation by recombinant Bora. Consistent with this, when Aurora-A is inactivated by pretreatment with protein phosphatase 1 (PP1), addition of Bora induces an over 7-fold increase in kinase activity. Analogous experiments with the Drosophila homologs reveal that Drosophila Bora similarly activates the Drosophila kinase, showing that it acts as a kinase activator as well. Taken together, these results demonstrate that Bora is an activator of Aurora-A (Hutterer, 2006).
Mutation of the autophosphorylation site of Aurora-A to alanine renders the kinase inactive, and an interesting question is whether the stimulation of Aurora-A by Bora bypasses the need for autophosphorylation. It was found that addition of Bora does not restore activity to the mutant kinase, suggesting that activation by Bora requires autophosphorylation of Aurora-A (Hutterer, 2006).
To determine the subcellular localization of Bora in SOP cells, live imaging was performed of a Bora-GFP fusion protein, which can rescue both bora and aurA37 mutant phenotypes. Histone-RFP is used to label chromosomes and indicates the cell-cycle stage. Constructs were specifically expressed by neuralized-Gal4 in SOP cells and dividing cells were imaged in whole living pupae. In interphase, Bora is a nuclear protein. When chromosomes condense, however, Bora is released from the nucleus. It is completely excluded from the nucleus by late prophase and is uniformly distributed in the cytoplasm after nuclear envelope breakdown. In telophase, Bora enters both daughter cells where it relocates into the nucleus. Bora does not have an obvious nuclear localization signal. However, it was found that the first 125 amino acids of the protein are sufficient for nuclear retention, suggesting that they contain the sequence that mediates nuclear import. Live imaging of GFP-Aurora-A together with Histone-RFP allows correlation of the localization of Aurora-A with Bora. In interphase, the two proteins are in distinct compartments. Nuclear release of Bora coincides with centrosome separation and strong recruitment of Aurora-A to the maturing centrosomes. Since both centrosome separation and maturation defects are observed in aurora-A mutants, these results suggest that release of Bora coincides with Aurora-A activation (Hutterer, 2006).
While Aurora-A is required for a subset of mitotic events, Cdc2 is essential for all steps of mitosis. How Cdc2 activates Aurora-A is unclear. To test whether Cdc2 regulates the release of Bora into the cytoplasm, Bora localization was examined in string mutants. String is the Drosophila homolog of the Cdc25 phosphatase, and in string mutants, Cdc2 is not activated. Antibody staining of Drosophila embryos reveals that endogenous Bora shows the same dynamic localization during the cell cycle as the functional GFP fusion protein. In string mutant embryos, however, Bora was never observed in the cytoplasm, indicating that Cdc2 activation is required for the release of Bora from the nucleus. To test whether Cdc2 might directly phosphorylate Bora, in vitro kinase assays were performed. Both Bora and HsBora are phosphorylated by recombinant Cdk1. Although the in vivo relevance of Cdk1 phosphorylation remains to be tested, these experiments show that Bora is released into the cytoplasm at the onset of mitosis in a Cdc2-dependent manner (Hutterer, 2006).
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