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).
Drosophila neural precursor cells divide asymmetrically by segregating the Numb protein into one of the two daughter cells. Numb is uniformly cortical in interphase but assumes a polarized localization in mitosis. This study shows that a phosphorylation cascade triggered by the activation of Aurora-A is responsible for the asymmetric localization of Numb in mitosis. Aurora-A phosphorylates Par-6, a regulatory subunit of atypical protein kinase C (aPKC). This activates aPKC, which initially phosphorylates Lethal (2) giant larvae (Lgl), a cytoskeletal protein that binds and inhibits aPKC during interphase. Phosphorylated Lgl is released from aPKC and thereby allows the PDZ domain protein Bazooka to enter the complex. This changes substrate specificity and allows aPKC to phosphorylate Numb and release the protein from one side of the cell cortex. These data reveal a molecular mechanism for the asymmetric localization of Numb and show how cell polarity can be coupled to cell-cycle progression (Wirtz-Peitz, 2008).
Since the discovery of Numb asymmetry, several proteins required for Numb localization have been identified, but how they cooperate remained unclear. This paper describes a cascade of interactions among these proteins that culminates in the asymmetric localization of Numb in mitosis. In interphase, Lgl localizes to the cell cortex, where it forms a complex with Par-6 and aPKC. At the onset of mitosis, AurA phosphorylates Par-6 in this complex, thereby releasing aPKC from inhibition by Par-6. Activated aPKC phosphorylates Lgl, causing its release from the cell cortex. Since Baz competes with Lgl for entry into the Par complex, the disassembly of the Lgl/Par-6/aPKC complex allows for the assembly of the Baz/Par-6/aPKC complex. Baz is a specificity factor that allows aPKC to phosphorylate Numb on one side of the cell cortex. Since p-Numb is released from the cortex (Nishimura, 2007; Smith, 2007), these events restrict Numb into a cortical crescent on the opposite side (Wirtz-Peitz, 2008).
The data show that Lgl acts as an inhibitory subunit of the Par complex. Given that Par-6 inhibits aPKC activity until the onset of mitosis, why would an additional layer of regulation be required? Like all phosphoproteins Numb is in a dynamic equilibrium between the phosphorylated and unphosphorylated states. Too high a rate of phosphorylation shifts this equilibrium toward the phosphorylated state, mislocalizing Numb into the cytoplasm. Too low a rate shifts it toward the unphosphorylated state, mislocalizing Numb around the cell cortex. Importantly, these data show that only the Baz complex can phosphorylate Numb. Assuming an abundance of Lgl over cortical Par-6, an increase in aPKC activity would translate into a comparatively small increase in the levels of Baz complex. This is because assembly of the Baz complex requires free subunits of Par-6 and aPKC, which become available only once the pool of cortical Lgl has been completely phosphorylated. Therefore, it is proposed that Lgl acts as a molecular buffer for the activity of the Par complex toward Numb. This maintains Numb phosphorylation within a range that is sufficiently high to exclude Numb from one side of the cell cortex but sufficiently low to permit the cortical localization of Numb to the other side (Wirtz-Peitz, 2008).
What is the evidence for this model? Lgl3A, a nonphosphorylatable mutant of Lgl in which the three aPKC phosphorylation sites are mutated to Ala, infinite buffering capacity, induces the mislocalization of Numb around the cell cortex. Conversely, in lgl mutants, having no buffering capacity, Numb is mislocalized into the cytoplasm. Moreover, the model predicts the loss of buffering capacity in the lgl mutant to be offset by an increase in the amount of substrate, since this would render the excess activity of the Par complex limiting. Indeed, overexpression of Numb in lgl mutants restores the cortical localization of Numb as well as its cortical asymmetry (Wirtz-Peitz, 2008).
The results indicate that Lgl gain- and loss-of-function phenotypes are entirely accounted for by the role of Lgl in inhibiting the assembly of the Baz complex. Previously, however, it was thought that the asymmetric phosphorylation of Lgl by aPKC restricts an activity of Lgl to the opposite side of the cell cortex. Based on this model, it was subsequently proposed that Lgl mediates the asymmetric localization of cell fate determinants by inhibiting the cortical localization of myosin-II. In addition, the role of the yeast orthologs of Lgl in exocytosis led to speculation that Lgl establishes an asymmetric binding site for cell fate determinants by promoting targeted vesicle fusion. However, the data show that Lgl asymmetry is extremely transient, and that the protein is completely cytoplasmic from NEBD onward. Lgl cannot therefore interact with any cortical proteins in prometaphase or metaphase, when myosin-II was reported to localize asymmetrically, or establish a stable landmark for vesicle fusion. Interestingly, a recent study demonstrated that yeast Lgl inhibits the assembly of SNARE complexes by sequestering a plasma membrane SNARE (Hattendorf, 2007). This mechanism is reminiscent of fly Lgl sequestering Par-6 and aPKC from interaction with Baz, suggesting that the defining property of Lgl-family members is not a specific role in exocytosis, but a more generic role in regulating the assembly of protein complexes (Wirtz-Peitz, 2008).
The data identify Numb as a key target of aPKC in tumor formation and suggest that Lgl acts as a tumor suppressor in the larval brain by inhibiting the aPKC-dependent phosphorylation of Numb. Although it is tempting to conclude that tumor formation in lgl mutants results from the missegregation of Numb, missegregation of Numb in numbS52F or upon expression of Lgl3A does not cause neuroblast tumors. How might this be explained? During mitosis, unphosphorylated cortical Numb is inherited by the differentiating daughter. At the same time, Baz and aPKC are excluded from this daughter, which limits Numb phosphorylation after exit from mitosis. In the subsequent interphase, some differentiating daughters reexpress members of the Baz complex (Bowman, 2008), but Numb continues to be protected from phosphorylation since cortical Lgl prevents the reassembly of the Baz complex. Thus, Lgl acts both in mitosis and interphase to maximize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).
In lgl mutants, Numb phosphorylation is increased in mitosis, and less unphosphorylated Numb is segregated into the basal daughter cell. Moreover, the assembly of the Baz complex is unrestrained in the subsequent interphase, which is exacerbated by the missegregation of aPKC into both daughter cells. Together, these defects minimize the amount of unphosphorylated Numb in the differentiating daughter cell (Wirtz-Peitz, 2008).
Why is the amount of unphosphorylated Numb critical for differentiation? Recently, it was shown that aPKC-dependent phosphorylation of Numb inhibits not only its cortical localization, but also its activity, owing to the reduced affinity of p-Numb for its endocytic targets (Nishimura, 2007). Therefore, ectopic phosphorylation of Numb leads to its inactivation, transforming the basal daughter cell into a neuroblast in a manner similar to mutation of numb. Consistent with this model, studies in SOP cells have documented ectopic Notch signaling in lgl mutants. Although the numbS52F mutant and Lgl3A overexpression also lead to missegregation of Numb, the levels of active unphosphorylated Numb are increased rather than decreased in these cases and are sufficient to support differentiation (Wirtz-Peitz, 2008).
The data also provide additional insight into the mechanism of tumor formation in aurA mutants. In aurA mutants, the differentiating daughter cell inherits less Numb because Numb is mislocalized around the cell cortex. At the same time, aPKC is missegregated into the differentiating daughter cell, where it promotes Numb phosphorylation in the subsequent interphase. Together, these events result in subthreshold amounts of unphosphorylated Numb in some basal daughter cells, transforming these into neuroblasts. This model explains why aurA mutants are characterized by reduced aPKC activity in mitosis, but are nonetheless suppressed by aPKC mutations, since a lack of aPKC in the differentiating daughter cell restores threshold amounts of unphosphorylated Numb (Wirtz-Peitz, 2008).
The data reveal that Lgl inhibits Numb phosphorylation to maintain Numb activity, whereas AurA promotes Numb phosphorylation in mitosis to ensure its asymmetric segregation. It is concluded that Lgl and AurA act on opposite ends of a regulatory network that maintains appropriate levels of Numb phosphorylation at the appropriate time in the cell cycle (Wirtz-Peitz, 2008).
The LIM-domain protein Ajuba localizes at sites of epithelial cell-cell adhesion and has also been implicated in the activation of Aurora-A (Aur-A). Despite the expected importance of Ajuba, Ajuba-deficient mice are viable, which has been attributed to functional redundancy with the related LIM-domain protein LIMD1. To gain insights into the function of Ajuba, this study investigated its role in Drosophila, where a single gene (jub) encodes a protein closely related to Ajuba and LIMD1. A key function were identified in neural stem cells, where Jub localizes to the centrosome. In these cells, mutation in jub leads to centrosome separation defects and aberrant mitotic spindles, which is a phenotype similar to that of aur-A mutants. In jub mutants Aur-A activity is not perturbed, but that recruitment and maintenance at the centrosome is affected. As a consequence the active kinase is displaced from the centrosome. On the basis of studies in Drosophila neuroblasts, it is proposed that a key function of Ajuba, in these cells, is to maintain active Aur-A at the centrosome during mitosis (Sabino, 2010).
The regulation of kinase activity in time and space is crucial for the coordination of cellular events. Aurora-A (Aur-A), one of the three members of the Aurora family of kinases in mammals, is a serine/threonine kinase that functions as a key regulator of several events. The kinase Aur-A was first identified in Drosophila as a mitotic kinase. In flies, mutations in aur-a cause severe developmental defects and pleiotropic phenotypes, which include abnormal centrosome and spindle behavior, lack of astral microtubules (MTs), defects in chromosome segregation, spindle positioning, cortical targeting of cell fate determinants and neural stem-cell self-renewal (Sabino, 2010).
In vertebrate cells, Aur-A also plays a major role in mitosis, and recently an unexpected role for this kinase has been described in non-mitotic cells. Aur-A phosphorylates and activates the tubulin deacetylase HDAC-6 to promote disassembly of cilia and cell cycle re-entry (Pugacheva, 2007). The large spectrum of functions attributed to the kinase Aur-A is thought to be, at least in part, regulated by different cofactors or activators (Carmena, 2009). TPX2, a MT-associated protein (MAP), binds Aur-A, thereby promoting Aur-A autophosphorylation and targeting it to the mitotic spindle (Wittmann, 2000). Hef-1 (also known as Nedd9) binding and activation of Aur-A is required for HDAC-6 phosphorylation (Pugacheva et al., 2007). In Drosophila, a single Aur-A activator, Bora, has been described so far. In bora mutants, defects in centrosome behavior and spindle assembly, together with defects in the asymmetric cell division of sensory organ precursors (SOPs), have been identified (Sabino, 2010).
Ajuba (Jub) is a LIM-domain protein that localizes at the sites of cell-cell adhesion in epithelial cells and has also been implicated in the activation of Aur-A (Hirota, 2003). Surprisingly, however, Jub-deficient mice are viable (Pratt, 2005); this has been attributed to functional redundancy with the related LIM-domain protein LIMD1. To gain insight into the function of Jub, its role was investigated in Drosophila, where a single gene encodes a protein closely related to mouse Jub and LIMD1. A mutation was generated in ajuba (jub) and jub mutants were found to die at the larval-pupal transition. No defects were detected in cell adhesion or epithelial polarity. However, a key function was detected in neural stem cells, where Jub localized to the centrosome. In these cells, mutation of jub led to centrosome separation defects and abnormal mitotic spindles. Surprisingly, It was found that in jub mutants Aur-A activity was not perturbed, but that Aur-A recruitment and maintenance at the centrosome was affected. As a consequence the active kinase was ectopically displaced into the cytoplasm, which resulted in abnormalities of the mitotic spindle. On the basis of these studies, it is proposed that a major function of Jub in Drosophila neuroblasts is to restrict active Aur-A to the centrosome during mitosis, but that Jub does not function as an Aur-A activator (Sabino, 2010).
This study generated mutations in the jub gene in Drosophila in order to examine its functions within an intact animal without the complications of potential redundancy with closely related genes. Unexpectedly, it was discovered that Jub had an essential role in just a subset of cells within the animal, namely the neural stem cells. Although not all cell types were exhaustively examined, cell cycles that are normally very sensitive to centrosome or MT perturbation, such as the nuclear divisions of the early embryo and the meiotic divisions of the male germline, occurred normally in the absence of Jub. Thus, Nbs are especially dependent on Jub to generate normal centrosomes and spindles, consistent with the clearly detectable levels of Jub-GFP on the centrosomes in these cells, but not in other cell types (Sabino, 2010).
Within the Nbs lacking Jub, three related, but distinct, phenotypes were detected: defects in the separation of centrosomes following mitosis, defects in spindle assembly, and defects in cortical targeting of determinants and orientation of the mitotic spindle. These phenotypes are shared by Nbs lacking Aur-A, consistent with previous work demonstrating that Jub and Aur-A proteins bind to each other and function together (Hirota, 2003). However, it was not possible to detect a biochemical interaction between these two proteins in Drosophila brains. In addition, the loss of Aur-A causes a number of additional defects that were not observed in jub mutant Nbs, such as defects in centrosome maturation and increased levels of genomic instability, demonstrating that Jub is not required for the majority of Aur-A functions (Sabino, 2010).
It is worth mentioning that no Nbs were obserged with supernumerary centrosomes in jub mutants. Defects in centrosome separation should result in the generation of daughter cells without centrosomes (which was see in 10% of the cells in jub mutant brain cells) and in those with two centrosomes, which should undergo duplication during the following cell cycle to produce extra centrosomes. Future work will be required to explain the absence of Nbs with supernumerary centrosomes (Sabino, 2010).
The results show that, in the absence of Jub, Aur-A is not as concentrated at the centrosome, and hence Tacc recruitment is affected. However, even in the absence of Jub, Tacc (transforming acidic coiled-coil protein), a MT-associated protein, can be phosphorylated by Aur-A, which further supports the idea that Jub is not an Aur-A activator, at least in Drosophila. Furthermore, it appears that loss of Jub results in a displacement of Aur-A from the centrosome. Thus, the key question is whether the defects caused by loss of Jub are due to diminished Aur-A activity on the centrosome, elevation of activity in the cytoplasm or both. The defects in centrosome separation, just after cell division, might be due to diminished levels of Aur-A on the centrosome, whereas the loss of astral MTs might be explained by diminished levels of P-Tacc or other MAPs. The manipulation of Aur-A levels in the presence or absence of Jub suggests that it is the elevated cytoplasmic Aur-A activity that is causing the defects in spindle assembly. Elevated cytoplasmic Aur-A is also likely to account for the defects in spindle positioning during asymmetric cell division (Sabino, 2010).
One possible explanation for the Nb-specific requirement for Jub is that the centrosome cycle is significantly different in these cells. In Nbs, just after centrosome duplication and migration to the apical cortex, one of the centrosomes moves away from the other. This dynamic centrosome continues to move throughout the S and G2 phases, which means that centrosome separation in Nbs takes place substantially before mitosis, in contrast with the timing in other cell types. It is therefore possible that Jub is only required for centrosome separation in cells where centrosomes separate earlier in the cell cycle, substantially before the following mitosis, when Aur-A activity is still present (Sabino, 2010).
Finally, no evidence has bee obtained to support a role for Jub as an Aur-A activator, since no reduction was seen in the phosphorylation of the Aur-A substrate Tacc. Many cell types require Aur-A function, including embryos and male spermatocytes. No Jub-GFP was observed at the centrosome in these cells and no jub mutant phenotypes were seen in the early embryo or male germline. In addition, no co-immunoprecipitate was seem of Jub and Aur-A in brain extracts. It is therefore proposed that the main function of Jub is to bind Aur-A at the centrosome, not to activate the kinase, but rather to restrict its activity in time and space. Too much active Aur-A in the cytoplasm during mitosis seems to perturb astral MT nucleation and centrosomal spindle assembly. Alternatively, Jub might also help to recruit and/or maintain Aur-A at the centrosome, so that it can be activated by another protein concentrated there, and, most crucially in Nbs, ‘hold’ the active Aur-A away from the cytoplasm. Unfortunately, such a candidate protein has not yet been identified in flies. Flies do not have an obvious TPX2 orthologue and the only Aur-A activator identified so far lacks a function in the fly brain. The failure of Jub to regulate Aur-A in Drosophila could also just reflect differences in the way Aur-A is regulated between vertebrates and invertebrates. In human cells, Jub is also associated with kinetochores and spindle MTs, and it has been shown that Jub, together with BubR1 and Aurora B, plays a role in the regulation of the metaphase-to-anaphase transition. However, the lack of a mitotic phenotype in Jub-knockout mice also strongly suggests that it might not play an essential role in Aur-A activation (Sabino, 2010).
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).
Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. A method has been developed to construct cortical polarity in a normally unpolarized cell line and this method was used to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals) -dependent spindle orientation. A previously unrecognized evolutionarily conserved Pins domain (PinsLINKER) was identified that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized PinsTPR domain has no function alone, but placing the PinsTPR in cis to the PinsLINKER gives dynein-dependent precise spindle orientation. This 'induced cortical polarity' assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity (Johnston, 2009).
A surprising result of these studies is the importance of the PinsLINKER domain for spindle orientation in the S2 assay and within neuroblasts in vivo. Only this domain is sufficient for spindle orientation, and a single point mutation in the linker domain (S436A) results in spindle orientation defects in larval neuroblasts that closely mimic the pins null mutant phenotype. On the basis of domain mapping and epistasis analysis, a linear pathway has been identified from cortical PinsLINKER to the plus ends of astral microtubules: (1) Aurora-A phosphorylates PinsLINKER on a single amino acid, serine 436, (2) the phosphorylated PinsLINKER binds and recruits Dlg, (3) the kinesin Khc-73 moves to astral microtubule plus ends using its motor domain and may be anchored at the plus ends by its Cap-Gly domain (Siegrist, 2005), and (4) the Khc-73MBS domain binds the cortical DlgGK domain, thereby linking Khc-73+ astral microtubule plus ends to the Dlg cortical domain. Interestingly, this pathway is active in both directions during mitosis. Cortical Pins acts through Dlg and Khc-73 to regulate spindle orientation, and spindle-associated Khc-73 acts through Dlg and Pins to induce Pins/Galphai functional cortical polarity in neuroblasts (Johnston, 2009).
Why does the PinsLINKER pathway provide only partial spindle orientation function? Live imaging rules out several possible explanations, such as PinsLINKER-induced spindle rocking variability, or that PinsLINKER functions during only a narrow window during mitosis. Live imaging shows that in PinsLINKER cells, the spindle drifts until it is immobilized at the edge of the crescent. This is consistent with the fact that Khc-73 is a plus end-directed motor protein, and thus unable to generate pulling forces to bring the centrosome closer to the cell cortex; at best, it would provide a static link between astral microtubules and the cell cortex (Johnston, 2009).
The PinsTPR domain can improve the PinsLINKER spindle orientation to a level matching wild-type neuroblasts. It is proposed that the PinsTPR domain directly binds Mud and that Mud interacts with the dynein/dynactin/Lis1 complex to enhance PinsLINKER spindle orientation. This model is based on five observations. First, the PinsTPR domain binds Mud in vitro and the two proteins coimmunoprecipitate from in vivo lysates; this interaction is conserved in the related C. elegans and mammalian proteins. Second, the PinsTPR and PinsTPR+LINKER but not the PinsLINKER can recruit Mud to the cortex of S2 cells. Third, PinsTPR+LINKER-mediated spindle orientation requires the dynein complex proteins Dlc and Lis1. Fourth, PinsTPR+LINKER-mediated spindle orientation exhibits rapid, directional spindle movement toward the center of the Pins cortical crescent, similar to dynein-dependent spindle orientation in Drosophila neuroblasts. Fifth, PinsTPR+LINKER-mediated spindle orientation leads to dynein-dependent movement of the spindle pole close to the cell cortex, consistent with dynein minus end-directed pulling of astral microtubules, as observed in other cell types (Johnston, 2009).
If PinsTPR recruits Mud, and Mud recruits the dynein complex, then why doesn't PinsTPR have spindle-orienting function on its own? The simplest model is that PinsTPR/Mud alone is unable to recruit or activate the dynein complex. Alternatively, the PinsLINKER pathway could be required for 'presenting' microtubule plus ends to an active PinsTPR/Mud/Dynein complex, which fits with the requirement for PinsTPR and PinsLINKER acting in cis. In summary, these data show that the PinsTPR and PinsLINKER domains provide distinct functions, both of which are required for optimal spindle orientation. Interestingly, spindle orientation in S2 cells does not show 'telophase rescue'a phenomenon whereby spindles that are partially oriented in metaphase/anaphase neuroblasts become aligned with the cell polarity axis by telophase -- consistent with the absence of redundant spindle orientation pathways in this assay (Johnston, 2009).
The PinsTPR pathway is regulated by Galphai binding to the GoLoco domain, relieving intramolecular TPR-GoLoco interactions, and making the TPR domain accessible for intermolecular interactions. In addition, Galphai is required to recruit Pins to the cell cortex, where it can interact with regulator and effector proteins. In the S2 spindle orientation assay, a requirement for Galphai can be bypassed by simply deleting the GoLoco domain (thereby freeing the TPR for intermolecular interactions) and tethering the PinsTPR+LINKER protein to the cortex by fusion with the Ed transmembrane protein. Thus, Galphai is important to activate and localize full-length Pins, but not as an effector of Pins-mediated spindle orientation (Johnston, 2009).
In contrast, the PinsLINKER pathway is not regulated by Galphai, because full-length Pins in the absence of Galphai provides equal spindle orientation to PinsLINKER, suggesting that the PinsLINKER is active when Pins is in the 'closed' form. The Khc-73 mammalian ortholog GAKIN transports hDlg to the cell cortex, but there are several reasons to think that this mechanism does not activate the PinsLINKER pathway. First, cortically tethered DlgGK domain requires Khc-73 for spindle orientation, which rules out a role for Khc-73 in merely transporting Dlg to the cortex; second, khc-73 RNAi does not block the ability of PinsLINKER to recruit Dlg to the cortex (Johnston, 2009).
This study has shown that Aurora-A kinase activates the PinsLINKER spindle orientation pathway by phosphorylating S436 in the linker domain and that this pathway is required for accurate spindle orientation in vivo for larval neuroblast asymmetric cell division. Neuroblasts expressing the nonphosphorylatable form of Pins (S436A) have a weaker spindle orientation phenotype than aurora-A null mutants, as expected because of Aurora-A regulation of multiple Pins-independent processes required for spindle orientation, such as centrosome maturation, cell-cycle progression, and cell polarity in flies. However, this study shows that a Pins phosphomimetic mutant (S436D) allows spindle orientation even after RNAi depletion of Aurora-A levels, suggesting that Aurora-A phosphorylation of PinsS436 is essential for Pins-dependent spindle orientation in the S2 cell assay. Furthermore, the finding that the PinsS436A protein has no spindle orientation activity in pins mutant larval neuroblasts, and has dominant-negative activity in the presence of endogenous Pins, shows that the Aurora-A/PinsLINKER pathway is required for spindle orientation in larval neuroblasts as well (Johnston, 2009).
The Pins spindle orientation pathway is cell-cycle regulated: interphase S2 cells that have polarized PinsTPR+LINKER do not capture centriole/centrosomal microtubules. There are at least two reasons for the lack of Pins interphase activity. First, the level of the Aurora-A kinase is low during interphase, and Aurora-A phosphorylation of Pins S436 has been shown to be is essential for Pins-mediated spindle orientation. Second, interphase centrosomes are immature, lacking Cnn and nucleating few microtubules. Expression of the Pins S436D protein, which is fully functional during mitosis even after Aurora-A depletion, still has no ability to capture centrioles during interphase. Thus, both centrosome maturation and Aurora-A activation are required for Pins-mediated spindle orientation in S2 cells (Johnston, 2009).
Cell polarity and spindle orientation has been induced in a cultured cell line in this study. This system was used to identify two pathways regulating spindle orientation, to establish molecular epistasis within each pathway, and to identify the target of the mitotic kinase Aurora-A that coordinates cell-cycle progression with spindle orientation. In the future, this system should be useful for characterizing spindle orientation pathways from other Drosophila cell types or from other organisms, identifying the mechanisms that control centrosome or spindle asymmetry, and characterizing the establishment and maintenance of cortical polarity. In each of these cases, the induced polarity system should be useful for rapid protein structure/function studies and high-throughput drug or RNAi loss-of-function studies (Johnston, 2009).
Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. This study identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. Phosphoproteins were functionally characterized and integrated into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aurora, as well as the kinase CkIIbeta. Using a combinatorial RNA interference (RNAi) strategy, novel functions were demonstrated for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, previously unknown centrosome and/or spindle localization was identified via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was Otefin (Ote), an NE protein that was found to localize to centrosomes. Furthermore, evidence is provided that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. It is proposed that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells (Habermann, 2012).
A striking observation from these results was that 6 out of the 27 MS identified candidate proteins (22%) were components of the NE. These were not simply contaminants of the centrosome preparations, as centrosome cycle related functions could be assigned to 4 of them, either directly or in kinase-depleted backgrounds. In addition, localization studies of FLAG/GFP tagged Ote and Lam in SL2 cells support the notion that these proteins have cell cycle dependent functions for the centrosome and spindle despite their main role in assembling the nuclear membrane. There is accumulating evidence for an interaction between centrosomal and NE components from various studies. For example, it has been shown that nuclear pore sub-complexes relocate to kinetochores upon NEBD, where they interact with the γ-TuRC and promote mitotic spindle assembly. C. elegans ZYG-12 localizes to both centrosomes and the NE and is essential for their attachment. Centrin 2, a core component of the centriole, also associates with nuclear pore complexes in Xenopus and human cells. A microtubule-independent role for the centrosome and Aurora-A for NEBD was also demonstrated. In this study, several lines of evidence indicate that the nuclear inner membrane protein Ote is also a genuine component of centrosomes. It binds to Lam and is found in a complex with both γ- and α-Tub suggesting that it may facilitate bridging of the centrosome to the NE in interphase via microtubules. At the onset of mitosis prior to NEBD, invaginations were observed in the nuclear membrane in close proximity to centrosomes. In mammalian cells it has been shown that such invaginations are generated by dynein-mediated microtubule-dependent forces, which create mechanical tension in the nuclear membrane and thereby trigger NEBD. In support of this model, the minus-end directed microtubule motor dynein is required for nuclear attachment of centrosomes during mitosis in Drosophila. However, an interaction partner for dynein at the NE has so far been elusive. Interestingly, Ote has been identified as an in vitro binding partner of dynein light chain Dlc90F in a two hybrid study, a finding that may provide the missing link for centrosome-NE attachment and tearing of the NE in Drosophila. Ote was also shown to be involved in centrosome maturation and cell cycle progression downstream of aur. The results of this study strongly suggest Ote as a substrate for Aurora-A in vitro. Whether Ote is an in vivo substrate of Aur remains to be elucidated. However, Ote-phosphomutant analysis revealed that the Aurora-A consensus site threonine 63 is critical for progression through mitosis, supporting the results of the combinatorial RNAi study. A functional interdependency was also observed between Nup98 and polo. While depletion of polo leads to severe centrosome aberrations, a simultaneous knockdown of Nup98 significantly weakened the polo induced phenotype indicating that Nup98 is a downstream target in a pathway that maintains centrosome structure. Yet another interdependency of the centrosome kinase polo and the NE component Lam was revealed in the functional analysis. While polo is known to be required for mitotic exit and hence depletion leads to mitotic arrest, a role for Lam in a polo-dependent pathway of mitotic progression is not described. Based on the observation that parallel inhibition of polo and Lam partially rescues the polo induced phenotype while depletion of Lam alone has no significant effect on mitotic progression, it is suggestd that negative regulation of Lam in a parallel signaling pathway downstream of polo is required for mitotic exit. The connection of polo and NE proteins is consistent with previous studies identifying several nuclear pore components as well as Lamins as Plk1 binding partners and potential substrates, respectively (Habermann, 2012).
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