Aurora-A kinase is found at centrosomes and on microtubules of the mitotic spindle and is essential for setting up a functional mitotic spindle. Drosophila Aurora-A, the founding member of this kinase family, is also required for centrosome separation, and its complete absence leads to the formation of monopolar spindles with a characteristic circular chromosome arrangement (Glover, 1995). Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC (Transforming acidic coiled-coil protein). A subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. It is proposed that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein (mini spindles) may be recruited, and thus modulate the behavior of astral microtubules (Giet, 2002).

In addition to the Aurora-A function in centrosome maturation, Aurora A also functions in asymmetric protein localization during mitosis (see Effects of Mutation). Using photobleaching of a GFP-Aurora fusion protein, it has been show that two rapidly exchanging pools of Aurora-A are present, one in the cytoplasm and a second at the centrosome. These pools might carry out the two functions. Activation of the Aurora-A kinase at the onset of mitosis is required for the actin-dependent asymmetric localization of Numb. Aurora-A is, as described in this section, involved in centrosome maturation and spindle assembly, indicating that Aurora A regulates both actin- and microtubule-dependent processes in mitotic cells (Berdnik, 2002).

Correct regulation of the organization and dynamics of microtubules is an essential aspect of entry into M-phase. Microtubules are nucleated by the gamma-tubulin ring complex, the amount of which increases markedly at the centrosome upon entry into mitosis. Microtubule nucleation at the centrosome requires the cooperation of other microtubule-associated proteins (MAPs), notably the Abnormal spindle (Asp) in Drosophila. MAPs also play a central role in regulating microtubule dynamics. For example, Xenopus XMAP215 promotes the elongation rate of microtubules at their plus ends (less so at the minus ends), and appears to counteract the catastrophe-promoting activity (the transition from polymerization to a depolymerization) promoted by XKCM1. The counterpart of XMAP215 in Drosophila is encoded by the gene minispindles, mutations that appear to destabilize spindle microtubules which become small and associated with single chromosomes. The MSPS protein forms a complex with the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, the centrosomally associated protein D-TACC. Injections of antibodies against D-TACC or mutations in the d-tacc gene result in centrosomal microtubules that are abnormally short, as well as in the accumulation of mitotic defects. The D-TACC protein is found at the spindle poles and its recruitment of MSPS protein has been postulated to stabilize centrosomal microtubules. In the acentriolar spindles of female meiosis, both the motor protein Ncd and D-TACC are required for the proper localization of MSPS (Giet, 2002 and references therein).

The Aurora-related enzymes constitute a major family of mitotic kinases (Giet, 1999a). Most is known about the B-type subfamily, which first localizes to condensing chromosomes and centromeres, and subsequently to the central spindle and the midbody in anaphase and telophase, respectively. The Aurora B protein kinase is the functional subunit of a complex containing INCENP, and the BIR-1/survivin protein is required for its localization; it is required in chromosome segregation and it phosphorylates histone H3, which correlates with recruitment of the condensin complex. In contrast, less is known of the exact function of the Aurora A–type kinases, although ectopic expression of the human enzyme leads to aneuploidy, centrosome amplification, and transformation (Bischoff, 1998; Zhou, 1998). Mutations in the aurora A gene of Drosophila melanogaster lead to formation of spindles with abnormally organized poles, including characteristic monopolar structures (Glover, 1995). Bipolar spindles having abnormally organized centrosomes, and microtubules have been observed after double-stranded RNA mediated interference directed against the air1 gene of Caenorhabditis elegans (Schumacher, 1998). A catalytically inactive or truncated version of the pEg2 Aurora A-like kinase promotes the collapse of spindles assembled in Xenopus egg extracts (Roghi, 1998). This would be consistent with the known ability of Aurora A kinase to phosphorylate the kinesin-like protein XlEg5 that is also required for spindle assembly and stability (Giet, 1999b). To gain a better understanding of the role of Aurora A kinase, the requirement for the enzyme in organizing spindle poles in different mutant alleles was examined. In mutant cells or after RNA interference to eliminate the enzyme, the spindle poles have abnormal organization and abnormally short arrays of astral microtubules. The latter defect correlates with the loss of D-TACC from centrosomes. D-TACC interacts with a subpopulation of Aurora A in vivo, and D-TACC is a substrate of the kinase. A model in which one of the centrosomal functions of Aurora A kinase is to control microtubule dynamics at the spindle poles by regulating the recruitment of D-TACC and its associated MAP, the minispindles/XMAP215 protein (Giet, 2002).

A role for the Drosophila Aurora A in regulating centrosome behavior has suggested undertaking an analysis of the mutant phenotype, a characteristic of which is the generation of circular arrays of mitotic chromosomes around a single body of apparently duplicated centrosomes (Glover, 1995). It is possible that such a phenotype could arise through two different routes. One possibility, that Aurora A function is required to maintain spindle pole separation, receives support from the observation that bipolar mitotic spindles formed in frog egg extracts collapse following the addition of dominant negative mutant forms of Eg2, the Xenopus A-type Aurora kinase (Giet, 2000). However, the present observations of multiple centrioles at the spindle poles in aurora A mutant cells suggests that at least in these cells there has been a failure of centriole segregation at the onset of mitosis. An allelic series of mutations offers the possibility of studying multiple functions of a protein and its role at different developmental stages where the cell cycle may be under differing modes of regulation. This study concentrated on two mutants, one of which displays a mitotic phenotype in syncytial embryos that undertake rapidly alternating S and M phases, and the other in larval neuroblasts, cells that undergo conventional cell cycles with active checkpoints. In both situations focus was placed on events at the spindle poles that provide a common aspect of mutant phenotype in these differing cell cycles. In the first case, the centrosomes and astral microtubules were examined in syncytial embryos derived from mothers homozygous for aurA²⁸⁷, a weak hypomorphic aurA mutant that produces poorly functional protein that allows repeated, but increasingly abnormal mitoses in the syncytium. In the second case, the spindle poles were examined in aurA^e209, a strongly hypomorphic mutant that shows an equally elevated mitotic index, whether the mutation is homozygous or hemizygous. In each mutant a diminution of the length and number of astral microtubules was observed even though the ultimate consequences for mitotic progression differ markedly in these two circumstances; the embryonic mitotic cycles can continue whereas the larval cycles are blocked at metaphase. The origins of the monopolar spindles seen in larval neuroblasts are still unclear. The increase in proportion of such structures seen in the presence of two copies rather than a single copy of the aurA^e209 allele (in both cases in the absence of wild-type protein) points toward a neomorphic function for the mutant protein. Consistent with this is the finding that no monopolar spindles were seen following air-1 RNAi in C. elegans (Schumacher, 1998). The interphase-like arrays of microtubules seen in the air-1-depleted embryos resemble those seen in the cytoplasm of aurA²⁸⁷-derived embryos. However, depletion of Aurora A by RNAi does not block mitotic progression in the embryonic cell line S2, but nevertheless it does lead to defects at the spindle poles similar to those in the aurA mutants (Giet, 2002).

Examination of the ultrastructure of the spindle poles in aurA^e209 mutant larval neuroblasts has revealed that when centrosomes are present they are comprised of multiple centrioles surrounded by electron-dense material having a distribution similar to centrosomal antigens revealed by light microscopy. These abnormal centrosomes contain core centrosomal antigens such as centrosomin, proteins required for the nucleation of microtubules such as gamma-tubulin and Asp and which can be removed by salt washes from the core centrosome, and proteins such as CP-190 whose association with the centrosome may be mediated through an interaction with gamma-tubulin. However, a characteristic of the aurA^e209 mutant centrosomes, also seen in the aurA²⁸⁷-derived embryos and after aurA RNAi in S2 cells, is the reduced amounts of the D-TACC protein and the MSPS proteins, known to be required to maintain the length and/or number of astral microtubules (Cullen, 1999; Gergely, 2000a; Lee, 2001). Thus, the failure of D-TACC-MSPS complex to be recruited to the centrosome when Aurora A kinase function is compromised provides an explanation for the correlation of the apparent diminution of length and/or number of astral microtubules in such circumstances (Giet, 2002).

D-TACC is not only associated with centrosomes, but also with spindle microtubules. This latter association, which appears not to require Aurora A kinase, has been shown to depend on binding to the MSPS MAP, a member of the ch-TOG/XMAP 215 family of proteins (Cullen, 2001; Lee, 2001). Aurora A kinase can bind to the D-TACC protein in Drosophila, as can the orthologs of these proteins in human cells. However, in extracts of Drosophila embryos, only a minor proportion of each protein appears to be associated in the same complex, probably reflecting the fact that both Aurora A and D-TACC proteins are supplied as an abundant maternal dowry that is only used for mitosis as development proceeds. Aurora A is capable of phosphorylating D-TACC, that this phosphorylation is required to recruit D-TACC to the centrosome early in mitosis. However, it is equally possible that Aurora A phosphorylates other centrosomal proteins in such a way as to facilitate the recruitment of D-TACC. The association of D-TACC with the centrosome could occur either independently or when it is already complexed with MSPS. In either case, the docking of the D-TACC-MSPS complex to the centrosome could then allow the complex access to microtubules nucleated by the gamma-tubulin ring complex in concert with the Asp protein. Association of MSPS may then promote the growth of the microtubules at both minus and plus ends as is normally seen in mitotic asters. This is suggested by the known properties of the Xenopus counterpart of MSPS, XMAP215, which promotes microtubule elongation rates strongly at the plus end but also at the minus end. Thus, the complex may also be carried on the extending microtubule giving it the appearance of accumulating near the plus ends as well as on the centrosome. Such localization of D-TACC has been observed near to the putative plus ends of microtubules on the spindles of S2 cells. It is also possible that the D-TACC-MSPS complex acts to prevent ejection of microtubules from the centrosome (Giet, 2002).

Although both hypomorphic and null alleles of d-tacc lead to female sterility, the gene is not essential for the larval division cycles. Thus, homozygote mutants can transit the earliest stages of development using wild-type protein from their heterozygous mothers and develop to adulthood. One possible explanation is that a need for D-TACC to target MSPS to centrosomes and thereby provide an efficient means of stabilizing astral microtubules is of particular importance in the rapid division cycles of the syncytial embryo. The longer cell cycle of larval cells coupled with their strong metaphase checkpoint could permit time to correctly assemble spindle poles in the absence of D-TACC protein. However, strong hypomorphic aurora A mutants do arrest at metaphase, pointing toward additional functions of the Aurora A enzyme beyond D-TACC recruitment, possibly in aspects of the metaphase-anaphase transition itself (Giet, 2002).

Ultrastructural studies have shown that bipolar spindles in aurA^e209 neuroblasts are missing centrioles from one of their poles and have multiple centrioles and pericentriolar material at the other pole. These observations correlate well with observations made by immunostaining with the light microscope. Such studies reveal many mitotic figures in which multiple centrosomal antigens were missing from one of the poles and multiple bodies containing centrosomal antigens were present at the other. The ability to make a stabilized and focused spindle pole in the absence of centrosomes is well known. Such focused spindle poles have been shown to form in the absence of centrosomes both in Xenopus extracts and in Drosophila through the concerted action of microtubule motors and MAPS to organize and stabilize focused microtubule minus ends. Such acentriolar spindle poles are also seen in female meiosis in Drosophila where the minus end directed motor Ncd is essential to organize the poles. Cullen (2001) has proposed that complexes of D-TACC and MSPS at the acentriolar poles of the spindles of female meiosis could stabilize the bipolar structure, thus accounting for its loss of bipolarity in MSPS or d-tacc mutants. Such a function is unlikely to be essential to maintain the mitotic spindle in larval neuroblasts, which can adopt a stable bipolar structure in the absence of Aurora A function and hence D-TACC accumulation, and indeed in the absence of centrosomes (Giet, 2002).

Could the failure to recruit the D-TACC protein to the centrosome also explain the accumulation of replicated centrioles at the spindle poles and their failure to segregate? The dispersed distribution of centrosomal antigens at the spindle pole is explained at the ultrastructural level by the finding of multiple centrioles surrounded by electron-dense pericentriolar material. It is possible that the presence of D-TACC is required to maintain aspects of the structural integrity of the centrosome, since the molecule is endowed with a coiled coil region. It has also been demonstrated to form polymers that could be of structural importance (Gergely, 2000a). Overexpression of the TACC domain of D-TACC alone results in the formation of TACC aggregates that bind MSPS and nucleate asters of microtubules (Lee, 2001). Overexpression of the human counterpart of D-TACC, HsTACC3, in mammalian cells also leads to the formation of aggregates to bind ch-TOG and appears to increase the numbers of microtubules (Gergely, 2000a). This apparent tendency of the TACC proteins to form large polymers could perhaps account for the cytoplasmic accumulation of D-TACC aggregates in aurA mutant neuroblasts. These show some tendency to cluster over the spindle microtubules. However, their failure to affect spindle structure might reflect the lower levels of MSPS that are found in this tissue compared with the syncytial embryo. It may be particularly important during mitosis that this tendency of D-TACC to aggregate is controlled. Aurora A kinase could fulfil such a role by permitting recruitment of D-TACC once mitosis is underway and when the centriole pairs have separated before prophase. Regulation of the behavior of astral microtubules may be important at the time that centrosomes are migrating around the nuclear envelope before the nuclear lamins depolymerize. The length, density, and dynamics of these microtubules may be essential for the migration of the centrosome not only around the nuclear envelope, but in other developmental processes. Indeed, one developmental failure seen in d-tacc mutant embryos is the failure of centrosomes to migrate to the cortex of the syncytial embryo in cycles 9 and 10 (Gergely, 2000b), a process known to be microtubule dependent. Failure of centrosomes to migrate to opposite sides of the nucleus could explain the origins of monoastral biploar spindles. It is proposed that accumulation of microtubule nucleating centers at one pole could in its extreme disrupt the inherent tendency to form a bipolar structure leading to formation of monopolar spindles. The failure of duplicated centrioles to segregate to both ends of the mitotic spindle as it forms raises the possibility that D-TACC/MSPS recruitment may also be required to stably associate the replicated centriole pair (Giet, 2002).

D-TACC is only the second substrate of the A-type Aurora kinases to be identified, the first being the Eg5 kinesin-like protein (Giet, 1999a). Undoubtedly there exist many others, and indeed it is possible that D-TACC is only one of several centrosomal substrates of the Aurora A kinase that may play a role in facilitating the equitable segregation of centrioles to the spindle poles. Indeed as d-tacc does not appear to be essential for viability it may be functionally redundant and so the larval lethality shown by strongly hypomorphic alleles of aurA may reflect a role for the kinase in modifying other mitotic targets. Identifying other substrates of the Aurora A kinases and evaluating their roles in mitotic progression remains a future challenge (Giet, 2002).

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).

Epithelial cells undergo cortical rounding at the onset of mitosis to enable spindle orientation in the plane of the epithelium. In cuboidal epithelia in culture, the adherens junction protein E-cadherin recruits Pins/LGN/GPSM2 and Mud/NuMA to orient the mitotic spindle. In the pseudostratified columnar epithelial cells of Drosophila, septate junctions recruit Mud/NuMA to orient the spindle, while Pins/LGN/GPSM2 is surprisingly dispensable. This study shows that these pseudostratified epithelial cells downregulate E-cadherin as they round up for mitosis. Preventing cortical rounding by inhibiting Rho-kinase-mediated actomyosin contractility blocks downregulation of E-cadherin during mitosis. Mitotic activation of Rho-kinase depends on the RhoGEF ECT2/Pebble and its binding partners RacGAP1/MgcRacGAP/CYK4/Tum and MKLP1/KIF23/ZEN4/Pav. Cell cycle control of these Rho activators is mediated by the Aurora A and B kinases, which act redundantly during mitotic rounding. Thus, in Drosophila pseudostratified epithelia, disruption of adherens junctions during mitosis necessitates planar spindle orientation by septate junctions to maintain epithelial integrity (Aguilar-Aragon, 2020).

Adherens junctions have long been thought to be continuously essential for maintaining epithelial form and function. The current findings demonstrate transient loss of adherens junctions during division of pseudostratified epithelial cells, an event that involves adherens junction remodelling during the extensive rounding up of cell shape in mitosis. This study has furthermore shown that loss of adherens junctions is a direct consequence of the increased Rho activity and actomyosin contractility that drives mitotic rounding, which is both necessary and sufficient to regulate the level of junctional E-cadherin in the pseudostratified wing imaginal disc epithelium of Drosophila. These findings are consistent with previous observations that adherens junctions can be removed via E-cadherin endocytosis upon planar polarised Rho activation and actomyosin-driven junctional shrinkage generated during cell-cell rearrangements in the Drosophila embryo and recent optogenetic experiments in human cells. However, the global loss of E-cadherin observed during mitosis of pseudostratified epithelial cells is unprecedented and may be uniquely required by the rapid transformation of these cells from their highly columnar shape to a rounded sphere at mitosis, which involves a rapid increase in apical area and junctional length. This change in cell chape, driven by global actomyosin contractility during mitotic rounding, may both spread out junctions and disrupt cadherin-cadherin contacts between neighbouring cells to favour endocytosis (Aguilar-Aragon, 2020).

Notably, loss of E-cadherin does not seem to occur during mitosis in cuboidal epithelial cells, which undergo a much milder cell shape change during mitotic rounding, such as the Drosophila follicle cell epithelium or in many human cultured epithelial cell lines. Indeed, E-cadherin was reported to play essential roles in planar spindle orientation in cultured human cells. Thus, the pseudostratified epithelia of Drosophila face a unique challenge of orienting the mitotic spindle in the plane of the epithelium without the use of adherens junctions as a cue, which may explain why these cells instead rely on septate junctions, while planar spindle orientation can occur normally in the cuboidal follicle cell epithelium before septate junctions form. In the absence of septate junctions, spindle-orienting factors such as Pins, Mud, Dlg and Scrib localise to lateral membranes, overlapping with E-cadherin, which can directly interact with Scrib in both Drosophila and human cells (Aguilar-Aragon, 2020).

The findings of this study also shed light on the molecular mechanisms linking the cell cycle with control of Rho activation during mitotic rounding. Downstream of the master mitotic kinase Cdk1, a key role for both Aurora A and B kinases (acting redundantly) was identified in initiating mitosis and maintaining cortical rounding, with Aurora B then acting alone to drive furrow formation during cytokinesis. Aurora kinases are known to activate the key cell cycle kinase Plk1/Polo, which can then activate RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl, possibly via the kinesin-like protein MKLP1/KIF23/ZEN4/Pav. One report suggested that Aurora B also acts to directly phosphorylate RacGAP1/MgcRacGAP/CYK4/Tum on S387, but this study found that a CRISPR-knockin mutation of this site (tum_S387A) is homozygous viable and fertile in Drosophila. In addition, Aurora B can also directly phosphorylate MKLP1/KIF23/ZEN4/Pav to oligomerise and activate RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl during cytokinesis, via a mechanism involving plasma membrane association of clustered C1 domains from the RacGAP1/MgcRacGAP/CYK4/Tum protein. Accordingly, it was found that a CRISPR-knockin mutation of this site (pavS734A/S735A) is homozygous lethal in Drosophila, suggesting that Aurora A/B could act via this complex during mitotic rounding. Indeed, MKLP1/KIF23/ZEN4/Pav undergoes cell cycle-dependent re-localisation from the nucleus (in interphase) to the cytoplasm (in mitosis), including a clear localisation to the entire plasma membrane during mitosis and then to the cleavage furrow during cytokinesis. The results show that mitotic activation of the MKLP1/KIF23/ZEN4/Pav binding partner RacGAP1/MgcRacGAP/CYK4/Tum also involves translocation from the nucleus to the cytoplasm, similar to ECT2/Pbl, where all three proteins are then available to bind the entire plasma membrane and generate global Rho activity to drive cortical contractility and loss of adherens junctions during mitotic rounding (Aguilar-Aragon, 2020).

The complex of MKLP1/KIF23/ZEN4/Pav and RacGAP1/MgcRacGAP/CYK4/Tum is often referred to as the 'centralspindlin' complex, due to its association with the spindle midzone at anaphase. However, the use of this term was avoided because association with the spindle midzone is not required for the function of this complex in activating ECT2/Pbl and Rho at the plasma membrane during cytokinesis. Furthermore, the results show that the same complex also functions prior to anaphase or cytokinesis in activating ECT2/Pbl and Rho for cortical rounding and downregulation of adherens junctions from the very onset of mitosis (Aguilar-Aragon, 2020).

Importantly, the function of the three cell cycle-regulated Rho activators discussed in this study - MKLP1/KIF23/ZEN4/Pav, RacGAP1/MgcRacGAP/CYK4/Tum and ECT2/Pbl - on the regulation of adherens junctions appears to be dose-dependent and cell shape change-dependent. The sudden re-localisation of these proteins to the cytoplasm at mitosis drives a global increase in Rho activation, cell shape change and loss of junctions. In contrast, the relatively low level of these proteins in the cytoplasm during interphase appears to contribute to the normal maintenance of Rho activity at the junctional actomyosin ring to maintain adherens junctions-at least in mammalian cells. In Drosophila, an interphase role of ECT2/Pbl in contributing to Rho activation and maintenance of adherens junctions is also plausible and should be most easily distinguished from the mitotic role in non-dividing epithelial cells. However, in the ovarian follicle cell epithelium, silencing of ECT2/Pbl by RNAi affected cytokinesis (leading to larger cells) but did not affect overall epithelial architecture after cells arrest their proliferation, suggesting that any interphase function may be obscured by redundancy with other RhoGEFs in this tissue. Note also that, in this cuboidal epithelium, mitotic rounding itself is more subtle and ECT2/Pbl is dispensable for planar spindle orientation and epithelial integrity (Aguilar-Aragon, 2020).

Finally, it is noted that ECT2/Pbl was initially reported to be capable of acting as GEF for another GTPase, Cdc42, in addition to Rho 38. This activity suggested a possible role in regulating the apical Cdc42-Par6-aPKC complex in epithelial polarity and in mitosis. In Drosophila, ECT2/Pbl was found to drive transient apical spreading of the Cdc42-Par6-aPKC complex during mitotic rounding of pupal notum epithelial cells. This study found evidence for a similar function of ECT2/Pbl in activating Cdc42 during interphase in the post-mitotic ovarian follicle cell epithelium, although the loss-of-function phenotype of ECT2/Pbl is obscured due to redundancy with other Cdc42 GEFs such as beta-PIX. Loss of both ECT2/Pbl and beta-PIX reduced the level of the Cdc42-Par6-aPKC complex localising to the apical domain of post-mitotic follicle cells and also reduced apical ZO-1 localisation in human Caco2 intestinal epithelial cells in culture. In contrast, the gain-of-function phenotype caused by ECT2/Pbl overexpression is a neoplastic tumour-like phenotype and clearly involves ectopic spreading of the apical Cdc42-Par6-aPKC complex, in addition to persistent cell rounding in both cuboidal follicle cells and pseudostratified wing epithelial cells (Aguilar-Aragon, 2020).

In conclusion, the findings provide new insights into the cell biology of mitotic rounding, identifying remodelling of adherens junctions as a key event in pseudostratified epithelia, where rounding is extensive, but not cuboidal epithelia, where rounding is more subtle. These results are consistent with the hypothesis that Rho activation and actomyosin contractility can stabilise adherens junctions in the absence of mechanical strain, but that Rho activation can induce E-cadherin endocytosis above a critical strain threshold, be it either junctional shrinkage or expansion, both of which may alter the geometry of the junctional actomyosin ring and disrupt cadherin-cadherin contacts between neighbouring cells to favour endocytic internalisation of E-cadherin. The results also clarify the molecular mechanisms linking cell cycle control machinery, particularly the Aurora A and B kinases, with Rho activation and mitotic rounding. Lastly, this work may have direct relevance to certain human epithelial cancers, such as lung cancer or glioma, where overexpression of ECT2/Pbl has been reported to correlate for poor prognosis, and where the findings suggest it could drive not only disruption of epithelial polarity via activation of Cdc42 or Rac but also loss of adherens junctions via sustained Rho activation to promote tumour progression (Aguilar-Aragon, 2020).

The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. This study used Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. It was further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA (Aurora A). Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly (Lim, 2017).

Genome-wide exome sequencing of microcephaly (MCPH) patients identified wd40-repeat protein 62 (wdr62) as the second most commonly mutated gene. WDR62 is a ubiquitously expressed cytoplasmic protein in interphase and localizes to the spindle pole in mitosis. A feature of many WDR62 MCPH-associated alleles is an inability to localize to the mitotic spindle pole, and wdr62 depletion is also associated with defects in spindle and centrosomal integrity, mitotic delay, and reduced brain growth in rodents. The neural stem cell (NSC) population gives rise to all neuronal cells in the adult brain. NSC behavior is governed by both cell intrinsic factors and extrinsic factors from the supporting stem cell niche, including the glial lineage, which acts non-autonomously to control stem cell renewal and differentiation of daughters (Lim, 2017).

The connection between NSCs and their niche, and the importance of spindle integrity to asymmetric division, has been best defined for Drosophila NSCs/neuroblasts (NB). WDR62 scaffolds kinases that are important mitotic regulators including c-Jun N-terminal kinase (JNK) members of the mitogen-activated protein kinase superfamily and Aurora kinase A (AURKA). In flies, AURKA regulates NB proliferation and is required for the localization of mitotic NB polarity complex protein Bazooka (mammalian Par3) to the apical Par complex (comprising the Par anchor, Inscuteable [Insc] adaptor protein, and Gαi/Pins/Mud complex). This establishes the apical-basal NB axis essential for self-renewal and differentiation. As a consequence, mutant aurka causes NB overproliferation and tissue overgrowth. The WDR62 ortholog in Drosophila (dWDR62) is required for brain growth (Nair, 2016), but whether signaling between WDR62 and AURKA modulates brain development has not been reported (Lim, 2017).

In addition to the NB lineage, Drosophila studies suggest that the glial lineage governs overall brain volume through regulation of cell-cycle re-entry and neuroepithelial expansion of NBs. However, potential contribution(s) of individual brain lineage(s) (NB or glia) to the defective brain growth associated with global depletion of wdr62 or aurka is currently unclear. This study confirmed that WDR62 is required for spindle orientation in NBs (Nair, 2016), however, wdr62 depletion specifically in NBs does not significantly retard brain growth. Rather, control of brain growth predominantly depends upon glial lineage function, as depletion of either aurka or wdr62 specifically in the glial lineage significantly reduces brain volume. Moreover, although wdr62 depletion suppressed brain overgrowth associated with aurka depletion in NBs, wdr62 knockdown specifically in the glial lineage enhanced the small brain phenotype associated with aurka depletion. Collectively, these data suggest that WDR62 function is negatively regulated by AURKA in NBs but positively regulated by AURKA in glia, and thus demonstrates that lineage-specific signaling functions of AURKA-WDR62 in Drosophila orchestrate larval brain growth and development (Lim, 2017).

In the mammalian brain, radial glia behave as NSCs that are supported by outer radial glia through cell-cell contact and secretion of growth factors required for maintenance of a stem cell niche. Another class of glial cells, the microglia population, regulates neural precursor cell numbers to govern final neuronal numbers residing in the cortex. However, whether MCPH genes such as wdr62 are important for glial cell fate is unclear. This study dissected the lineage-specific contribution of WDR62 to brain development and revealed that loss of WDR62 function specifically in the glial, but not the NB lineage, profoundly altered brain growth. Moreover, wdr62 depletion in glia likely impairs brain growth autonomously (i.e., through depletion of glia), and also results in NSC loss, suggesting that a focus on WDR62 function in glia will be integral to elucidating how wdr62 loss of function contributes to MCPH. That depletion of wdr62 in the NB lineage was not associated with reduced brain volume, despite reducing NB number, also provides a likely explanation for the recent observation that NB defects associated with global wdr62 depletion fail to account for reduced brain size (Lim, 2017).

Hypomorphic wdr62 mutant mice have reduced brain size, with associated mitotic defects and an overall decrease in neural progenitor cells. In Drosophila, spindle orientation defects following wdr62 loss of function likely underlie the G2 delay and increased mitotic figures in NBs. This phenotype is also reminiscent of the cleavage plane misorientation observed in NSCs in wdr62-depleted rat brains. In Drosophila NBs, WDR62 regulates the interphase localization of Centrosomin (CNN, mammalian CDK5RAP2) to the apical centrosome, and thus centrosomal maturation and positioning. Interestingly, CNN is also an AURKA target that governs spindle orientation independently from cortical polarity establishment during mitosis. Similar to the phenotype observed for wdr62 depletion in NBs, cnn loss of function is associated with spindle orientation defects and reduced NB number. Thus, it is tempting to speculate that WDR62 and CNN function in the same AURKA-dependent signaling complex during mitosis (Lim, 2017).

Ex vivo studies have demonstrated that AURKA phosphorylation of WDR62 promotes spindle pole localization during mitosis. Mouse models suggest AURKA and WDR62 interact in vivo to control brain growth. Compound heterozygous wdr62+/-;aurka+/- mice have a much smaller body size than single heterozygotes but, although the mitotic index of the cerebral cortex was significantly increased and NSCs were reduced, consistent with a mitotic delay radial glia, potential changes to brain volume were not measured. This study, demonstrates that the brain overgrowth associated with aurka depletion specifically in NBs was suppressed by co-depletion of wdr62, bringing brain volume to within the control range. In contrast, the small brain phenotypes, due to glial-specific depletion of either aurka or wdr62, were further reduced by co-knockdown. Thus, in the context of normal brain development, AURKA likely acts to promote WDR62-dependent glial proliferation, but antagonizes WDR62 function in the NB lineage (Lim, 2017).

These findings indicate that WDR62 likely functions in AURKA-mediated regulation of spindle orientation but not in the establishment of cortical polarity. One reason for the differential output of AURKA regulation of WDR62 (between NB and glia) could stem from the symmetrical nature of glial division, where there is no evidence for cortical polarization. In contrast to the in vivo mammalian studies and previous Drosophila studies, which employed global depletion strategies for wdr62, these studies have enabled dissection of the relative contribution of wdr62 loss-of-function from each of the major brain lineages. In particular, the observation that depletion in either the NB or glial lineage is associated with reduced cell number, but an overall reduction in brain size was only observed when wdr62 was reduced in glia, places great interest in examining the relative contribution of glial-specific depletion of wdr62 in mice to brain size. Moreover, future studies of the pathogenic wdr62 mutations, and identified AURKA phosphorylation sites on WDR62, in the glial lineage are likely to inform on the contribution of this lineage to impaired brain growth in microcephaly (Lim, 2017).

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? Lgl^3A, 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 numb^S52F or upon expression of Lgl^3A 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 numb^S52F mutant and Lgl^3A 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 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 (Pins^LINKER) 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 Pins^TPR domain has no function alone, but placing the Pins^TPR in cis to the Pins^LINKER 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 Pins^LINKER 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 Pins^LINKER to the plus ends of astral microtubules: (1) Aurora-A phosphorylates Pins^LINKER on a single amino acid, serine 436, (2) the phosphorylated Pins^LINKER 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-73^MBS domain binds the cortical Dlg^GK 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 Pins^LINKER pathway provide only partial spindle orientation function? Live imaging rules out several possible explanations, such as Pins^LINKER-induced spindle rocking variability, or that Pins^LINKER functions during only a narrow window during mitosis. Live imaging shows that in Pins^LINKER 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 Pins^TPR domain can improve the Pins^LINKER spindle orientation to a level matching wild-type neuroblasts. It is proposed that the Pins^TPR domain directly binds Mud and that Mud interacts with the dynein/dynactin/Lis1 complex to enhance Pins^LINKER spindle orientation. This model is based on five observations. First, the Pins^TPR 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 Pins^TPR and Pins^TPR+LINKER but not the Pins^LINKER can recruit Mud to the cortex of S2 cells. Third, Pins^TPR+LINKER-mediated spindle orientation requires the dynein complex proteins Dlc and Lis1. Fourth, Pins^TPR+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, Pins^TPR+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 Pins^TPR recruits Mud, and Mud recruits the dynein complex, then why doesn't Pins^TPR have spindle-orienting function on its own? The simplest model is that Pins^TPR/Mud alone is unable to recruit or activate the dynein complex. Alternatively, the Pins^LINKER pathway could be required for 'presenting' microtubule plus ends to an active Pins^TPR/Mud/Dynein complex, which fits with the requirement for Pins^TPR and Pins^LINKER acting in cis. In summary, these data show that the Pins^TPR and Pins^LINKER 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 Pins^TPR 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 Pins^TPR+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 Pins^LINKER pathway is not regulated by Galphai, because full-length Pins in the absence of Galphai provides equal spindle orientation to Pins^LINKER, suggesting that the Pins^LINKER 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 Pins^LINKER pathway. First, cortically tethered Dlg^GK 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 Pins^LINKER to recruit Dlg to the cortex (Johnston, 2009).

This study has shown that Aurora-A kinase activates the Pins^LINKER 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/Pins^LINKER 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 Pins^TPR+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).

Mitotic spindle orientation is essential to control cell-fate specification and epithelial architecture. The tumor suppressor Lgl localizes to the basolateral cortex of epithelial cells, where it acts together with Dlg and Scrib to organize apicobasal polarity. Dlg and Scrib also control planar spindle orientation but how the organization of polarity complexes is adjusted to control symmetric division is largely unknown. Lgl redistribution during epithelial mitosis is reminiscent of asymmetric cell division, where it is proposed that Aurora A promotes aPKC activation to control the localization of Lgl and cell-fate determinants. This study shows that the Dlg complex is remodeled during Drosophila follicular epithelium cell division, when Lgl is released to the cytoplasm. Aurora A controlled Lgl localization directly, triggering its cortical release at early prophase in both epithelial and S2 cells. This relied on double phosphorylation within the putative aPKC phosphorylation site, which was required and sufficient for Lgl cortical release during mitosis and could be achieved by a combination of aPKC and Aurora A activities. Cortical retention of Lgl disrupted planar spindle orientation, but only when Lgl mutants that could bind Dlg were expressed. Taken together, Lgl mitotic cortical release is not specifically linked to the asymmetric segregation of fate determinants, and the study proposes that Aurora A activation breaks the Dlg/Lgl interaction to allow planar spindle orientation during symmetric division via the Pins (LGN)/Dlg pathway (Carvalho, 2015).

Evolutionarily conserved polarity complexes establish distinct membrane domains and the polarized assembly of junctions along the apicobasal axis has been extensively characterized. One general feature is that it relies on mutual antagonism between apical atypical protein kinase C (aPKC) and Crumbs complexes and a basolateral complex formed by Scribble (Scrib), Lethal giant larvae (Lgl), and Discs large (Dlg). This study used the Drosophila follicular epithelium as an epithelial polarity model to address how polarity is coordinated during symmetric division. Dlg and Scrib have been shown to provide a lateral cue for planar spindle orientation. Accordingly, Scrib and Dlg remain at the cortex during follicle cell division. In contrast, Lgl is released from the lateral cortex to the cytoplasm during mitosis. This subcellular reallocation begins during early prophase, since Lgl starts to be excluded from the cortex prior to cell rounding, one of the earliest mitotic events, and is completely cytoplasmic before nuclear envelope breakdown (NEB). Thus, the Dlg complex is remodeled at mitosis onset in epithelia (Carvalho, 2015).

The subcellular localization of Lgl is controlled by aPKC-mediated phosphorylation of a conserved motif, which blocks Lgl interaction with the apical cortex. To address the mechanism of cortical release during mitosis, nonphosphorytable form Lgl^3A-GFP was expressed in the follicular epithelium. Lgl^3A-GFP remains at the cortex throughout mitosis indicating that Lgl dynamics during epithelial mitosis also rely on the aPKC phosphorylation motif. Although the apical aPKC complex depolarizes during follicle cell division, Lgl cortical release precedes aPKC depolarization. Using Par-6-GFP as a marker for the aPKC complex and the Lgl cytoplasmic accumulation as readout of its cortical release, it was found that maximum cytoplasmic accumulation of Lgl occurs when most Par-6 is still apically localized (~70% relative to interphase levels). Thus, Lgl cortical release is the first event of the depolarization that characterizes follicle cell division, indicating that Lgl reallocation does not require extension of aPKC along the lateral cortex (Carvalho, 2015).

Although the major pools of Lgl and aPKC are segregated during interphase, Lgl has a dynamic cytoplasmic pool that rapidly exchanges with the cortex. Thus, further activation of aPKC at mitosis onset would be expected to shift the equilibrium toward cytoplasmic localization. Lgl dynamic redistribution in epithelia is similar to the neuroblast, where activation of Aurora A (AurA) leads to Par-6 phosphorylation and subsequent aPKC activation. To test whether a similar mechanism induced Lgl cortical release during epithelial mitosis, Lgl subcellular localization was analyzed in aPKC mutants and in par-6 mutants unphosphorylatable by AurA. Lgl cytoplasmic accumulation is unaffected in par-6; par-6^S34A mutant cells. Temperature-sensitive aPKC^ts/aPKC^k06403 mutants display strong cytoplasmic accumulation of Lgl during prophase, with a minor delay relatively to the wild-type). Moreover, homozygous mutant clones for null (aPKC^k06403) and kinase-defective (aPKC^psu141) alleles also display Lgl cortical release during mitosis. These results implicate that although aPKC activity may contribute for Lgl mitotic dynamics, the putative aPKC phosphorylation motif is under the control of a different kinase, which triggers Lgl cortical release in the absence of aPKC (Carvalho, 2015).

AurA is a good candidate to induce Lgl cortical release as it controls polarity during asymmetric division. Furthermore, Drosophila AurA is activated at the beginning of prophase, which coincides with the timing of Lgl cytoplasmic reallocation. To examine whether AurA controls Lgl dynamics in the follicular epithelium, homozygous mutant clones were generated for the kinase-defective allele aurA³⁷. In contrast to wild-type cells, only low amounts of cytoplasmic Lgl were detected during prophase in aurA³⁷ mutants, which display a pronounced delay in the cytoplasmic reallocation of Lgl during mitosis. This delayed cortical release of Lgl has been previously reported during asymmetric cell division in aurA³⁷ mutants, possibly resulting from residual kinase activity. Thus, AurA is essential to trigger Lgl cortical exclusion at epithelial mitosis onset (Carvalho, 2015).

The idea that Lgl mitotic reallocation is directly controlled by a mitotic kinase implies that Lgl should display similar dynamics regardless of the polarized status of the cell. Consistently, Lgl-GFP is also released from the cortex before NEB in nonpolarized Drosophila S2 cells. Furthermore, Lgl^3A-GFP is retained in the cortex during mitosis, revealing that Lgl cortical release is also phosphorylation dependent in S2 cells. Treatment with a specific AurA inhibitor (MLN8237), or with aurA RNAi, strongly impairs Lgl cortical release during prophase, as Lgl is present in the cortex at NEB. However, inhibition of AurA still allows later cortical exclusion, which could result from the activity of another kinase. Despite their distinct roles, AurA and Aurora B (AurB) phosphorylate common substrates in vitro. Therefore, whether AurB could act redundantly with AurA was analyzed. Inactivation of AurB with a specific inhibitor, Binucleine 2, enables normal Lgl cytoplasmic accumulation before NEB and still allows later cortical exclusion in cells treated simultaneously with the AurA inhibitor As AurB does not seem to participate on Lgl mitotic dynamics, RNAi directed against aPKC was used to examine whether it could act redundantly with AurA. aPKC depletion did not block Lgl cortical exclusion, but it was slightly delayed. However, simultaneous AurA inhibition and aPKC RNAi produced almost complete cortical retention of Lgl during mitosis. Thus, AurA induces Lgl release during early prophase, but aPKC retains its ability to phosphorylate Lgl during mitosis (Carvalho, 2015).

To address which serine(s) within the phosphorylation motif of Lgl control its dynamics during mitosis, individual and double mutants were enerated. As complete cortical release occurs before NEB, the ratio of cytoplasmic to cortical mean intensity of Lgl-GFP at NEB was quantified to compare each different mutant. All the single mutants displayed similar dynamics to Lgl^WT, exiting to the cytoplasm prior to NEB. In contrast, all double mutants were cortically retained during mitosis, indicating that double phosphorylation is both sufficient and required to efficiently block Lgl cortical localization (Carvalho, 2015).

The ability to doubly phosphorylate Lgl would explain how AurA drives Lgl cortical release. Accordingly, the sequence surrounding S656 perfectly matches AurA phosphorylation consensus, whereas the S664 surrounding sequence shows an exception in the -3 position. In contrast, the sequence surrounding S660 does not resemble AurA phosphorylation consensus, and AurA does not directly phosphorylate S660 in vitro as detected by phosphospecific antibodies against S660. That S656 is directly phosphorylated by recombinant AurA was confirmed in vitro using a phosphospecific antibody for S656. Moreover, AurA inhibition or aurA RNAi results in a similar cortical retention at NEB to Lgl^S656A,S664A, suggesting that AurA also controls S664 phosphorylation during mitosis, whereas aPKC would be the only kinase active on S660. Consistent with this, aPKC RNAi increases the cortical retention of Lgl^S656A,S664A, mimicking the localization of Lgl^3A. Furthermore, whereas S660A mutation does not significantly affect the cytoplasmic accumulation of Lgl in aPKC RNAi, S656A and S664A mutations disrupt Lgl cortical release in aPKC-depleted cells, leading to the degree of cortical retention of Lgl^S656A,S660A and Lgl^S660A,S664A, respectively. Altogether, these results support that AurA controls S656 and S664 and that these phosphorylations are partially redundant with aPKC phosphorylation to produce doubly phosphorylated Lgl, which is released from the cortex (Carvalho, 2015).

RNAi-mediated knockdown of Lgl in vertebrate HEK293 cells results in defective chromosome segregation. Furthermore, overexpressed Lgl-GFP shows a slight enrichment on the mitotic spindle suggesting that relocalization of Lgl could be important to control chromosome segregation. However, lgl mutant follicle cells assemble normal bipolar spindles, and although it was possible to detect minor defects on chromosome segregation, the mitotic timing (time between NEB and anaphase) is indistinguishable between lgl and wild-type cells. Additionally, loss of Lgl activity allows proper chromosome segregation in both Drosophila S2 cells and syncytial embryos. Thus, Lgl does not seem to have a general role in the control of faithful chromosome segregation in Drosophila (Carvalho, 2015).

Nevertheless, Lgl cortical release could per se play a mitotic function, as key mitotic events are controlled at the cortex. In fact, the orientation of cell division requires the precise connection between cortical attachment sites and astral microtubules, which relies on the plasma membrane associated protein Pins (vertebrate LGN). Pins uses its TPR repeat domain to bind Mud (vertebrate NUMA), which recruits the dynein complex to pull on astral microtubules, and its linker domain to interact with Dlg, which participates on the capture of microtubule plus ends. Notably, Pins/LGN localizes apically during interphase in Drosophila and vertebrate epithelia, being reallocated to the lateral cortex to orient cell division. Pins relocalization relies on aPKC in some epithelial tissues, but not in chick neuroepithelium and in the Drosophila follicular epithelium, where Dlg provides a polarity cue to restrict Pins to the lateral cortex. Dlg controls Pins localization during both asymmetric and symmetric division, and a recent study has shown that vertebrate Dlg1 recruits LGN to cortex via a direct interaction. However, Dlg uses the same phosphoserine binding region within its guanylate kinase (GUK) domain to interact with Pins/LGN and Lgl. Thus, maintenance of a cortical Dlg/Lgl complex during mitosis is expected to impair the ability of Dlg to bind Pins and control spindle orientation (Carvalho, 2015).

Interaction between the Dlg's GUK domain and Lgl requires phosphorylation of at least one serine within the aPKC phosphorylation site. Although the phosphorylation-dependent binding of Lgl to Dlg remains to be shown in Drosophila, crystallographic studies revealed that all residues directly involved in the interaction with p-Lgl are evolutionarily conserved from C. elegans to humans. Thus, whereas Lgl^3A does not form a fully functional Dlg/Lgl polarity complex, double mutants should bind Dlg's GUK domain and are significantly retained at the cortex during mitosis due to the inability to be double phosphorylated. This led to an examination of their ability to support epithelial polarization during interphase and to interfere with mitotic spindle orientation. Rescue experiments were performed in mosaic egg chambers containing lgl^27S3 null follicle cell clones. lgl mutant clones display multilayered cells with delocalization of aPKC. This phenotype is rescued by Lgl-GFP, but not by Lgl^3A-GFP. More importantly, in contrast to Lgl^S660A,S664A, which extends to the apical domain in wild-type cells and fails to rescue epithelial polarity in lgl mutant cells, Lgl^S656A,S660A and Lgl^S656A,S664A can rescue epithelial polarity, localizing with Dlg at the lateral cortex and below aPKC. Hence, aPKC-mediated phosphorylation of S660 or S664 is sufficient on its own to control epithelial polarity and to confine Lgl to the lateral cortex (Carvalho, 2015).

Whether exclusion of Lgl from the cortex and the consequent release from Dlg would be functionally relevant for oriented cell division was examined. Expression of Lgl-GFP or Lgl^3A-GFP does not affect planar spindle orientation during follicle cell division. In contrast, Lgl double mutants display metaphasic cells in which the spindle axis, determined by centrosome position, is nearly perpendicular to the epithelial layer. Live imaging revealed that these spindle orientation defects were maintained throughout division as it was possible to follow daughter cells separating along oblique and perpendicular angles to the epithelia. Moreover, equivalent defects on planar spindle orientation were detected upon expression of Lgl^S656A,S664A in the lgl or wild-type background, indicating that cortical retention of Lgl exerts a dominant effect. Interestingly, Lgl^S656A,S660A and Lgl^S656A,S664A induce higher randomization of angles, whereas Lgl^S660A,S664A, which is less efficiently restricted to the lateral cortex, produces a milder phenotype. Altogether, these results indicate that retention of Lgl at the lateral cortex disrupts planar spindle orientation only if Lgl can interact with Dlg (Carvalho, 2015).

Despite the ability of Lgl^S656A,S660A-GFP to rescue epithelial polarity in lgl mutants, strong overexpression of Lgl^S656A,S660A-GFP, but not of other Lgl double mutants, can dominantly disrupt epithelial polarity during the proliferative stages of oogenesis. One interpretation is that Lgl^S656A,S660A forms the most active lateral complex of the mutant transgenes, disrupting the balance between apical and lateral domains. Therefore whether the dominant effect of Lgl cortical retention on spindle orientation could solely result from Dlg mislocalization was assessed. Dlg is properly localized at the lateral cortex in Lgl^S656A,S660A-expressing cells presenting misoriented spindles, but this position does not correlate with the orientation of the centrosomes. Thus, cortical retention of Lgl interferes with Dlg's ability to transmit its lateral cue to instruct spindle orientation, which may result from an impairment of the Dlg/Pins interaction (Carvalho, 2015).

In conclusion, these findings outline a mechanism that explains how the lateral domain is remodeled to accomplish oriented epithelial cell division, unveiling that AurA has a central role in controlling the subcellular distribution of Lgl. AurA regulates the activity of aPKC at mitotic entry during asymmetric division, and these results are consistent with the ability of aPKC to phosphorylate and collaborate in Lgl cortical release. However, in epithelia, aPKC accumulates in the apical side during interphase, where it induces apical exclusion of Lgl, in part by generating a phosphorylated form that binds Dlg. Consequently, aPKC has a reduced access to the cortical pool of Lgl at mitotic entry and would be unable to rapidly induce Lgl cortical exclusion. These data show that cell-cycle-dependent activation of AurA removes Lgl from the lateral cortex through AurA's ability to control Lgl phosphorylation on S656 and S664 independently of aPKC. Thus, AurA and aPKC exert the spatiotemporal control of Lgl distribution to achieve unique cell polarity roles in distinct cell types (Carvalho, 2015).

It is proposed that release of Lgl from the cortex allows Dlg interaction with Pins to promote planar cell division in Drosophila epithelia. Lgl cortical release requires double phosphorylation, indicating that whereas Lgl-Dlg association involves aPKC phosphorylation, multiple phosphorylations break this interaction, acting as an off switch on Lgl-Dlg binding. Triple phosphomimetic Lgl mutants display weak interactions with Dlg, suggesting that multiple phosphorylations could directly block Lgl-Dlg interaction. Alternatively, the negative charge of two phosphate groups may suffice to induce association between the N- and C-terminal domains of Lgl, impairing its ability to interact with the cytoskeleton and plasma membrane as previously proposed. This would reduce the local concentration of Lgl available to interact with Dlg, enabling the interaction of Dlg's GUK domain with the pool of Pins phosphorylated by AurA. Therefore, AurA converts the Lgl/Dlg polarity complex generated upon aPKC phosphorylation into the Pins/Dlg spindle orientation complex. This study, underlines the critical requirement of synchronizing the cell cycle with the reorganization of polarity complexes to achieve precise control of spindle orientation in epithelia (Carvalho, 2015).

The Lethal giant larvae (Lgl) protein was discovered in Drosophila as a tumor suppressor in both neural stem cells (neuroblasts) and epithelia. In neuroblasts, Lgl relocalizes to the cytoplasm at mitosis, an event attributed to phosphorylation by mitotically activated aPKC kinase and thought to promote asymmetric cell division. This study shows that Lgl also relocalizes to the cytoplasm at mitosis in epithelial cells, which divide symmetrically. The Aurora A and Aurora B kinases directly phosphorylate Lgl to promote its mitotic relocalization, whereas aPKC kinase activity is required only for polarization of Lgl. A form of Lgl that is a substrate for aPKC, but not Aurora kinases, can restore cell polarity in lgl mutants but reveals defects in mitotic spindle orientation in epithelia. It is proposed that removal of Lgl from the plasma membrane at mitosis allows Pins/LGN to bind Dlg and thus orient the spindle in the plane of the epithelium. These findings suggest a revised model for Lgl regulation and function in both symmetric and asymmetric cell divisions (Bell, 2014).

To examine the subcellular localization of Aurora A kinase in Drosophila cells, a polyclonal antibody was raised that was specifically directed against the NH₂-terminal domain of the enzyme expressed in E. coli, and which recognized a single 47-kD protein in an extract of 2h Drosophila embryos. The antibody strongly decorates the centrosomes of S2 cells in prophase, and anaphase. At cytokinesis, there occurs a decrease in the intensity of staining at the centrosome that is apparent and which may be a consequence of anaphase-promoting complex-mediated degradation of the enzyme (Walter, 2000). To determine if the localization of Aurora A to centrosomes requires microtubules, S2 cells were treated with the microtubule-depolymerizing drug colchicine or the microtubule-stabilizing drug taxol. Neither treatment disrupts the centrosomal association of the enzyme, and although taxol treatment induces the formation of ectopic asters, Aurora A only remains associated with asters nucleated by centrosomes. Thus, the Aurora A kinase appears to associate with centrosomes independently of microtubules, as previously described (Gopalan, 1997; Roghi, 1998) for Aurora A orthologs (Giet, 2002 and references therein).

Female sterile mutations of aurora are allelic to mutations in the lethal complementation group ck10. Syncytial embryos derived from aur mothers display closely paired centrosomes at inappropriate mitotic stages and develop interconnected spindles in which the poles are shared. Amorphic alleles result in pupal lethality and in mitotic arrest in which condensed chromosomes are arranged on circular monopolar spindles. The size of the single centrosomal body in these circular figures suggests that loss of function of the serine-threonine protein kinase encoded by aur leads to a failure of the centrosomes to separate and form a bipolar spindle (Glover, 1995).

An allelic series of mutations at the aurora A locus shows disruption to mitotic progression at differing developmental stages (Glover, 1995). All of these mutations can be fully rescued by a transgene carrying an aurora A gene (Glover, 1995). The allele aurA²⁸⁷ exemplifies a weak hypomorph that displays no apparent mitotic defects in larval brains. Females homozygous for aurA²⁸⁷ survive to adulthood, but show a maternal effect and produce embryos that cannot complete the 13 rapid cycles of syncytial mitosis as a consequence of accumulating defects that include asynchronous mitotic cycles, loss of centrosomes, and fusion of mitotic spindles (Glover, 1995). The aurA^e209 allele is a strong hypomorph; the aurA^e209 larval central nervous system shows a 5-6-fold elevated mitotic index with the majority of cells delayed in a metaphase-like state. In most of these cells, metaphase chromosomes were arranged in a configuration consistent with their association with bipolar spindles, but a substantial proportion were in circular arrays that correspond to monopolar spindles (Giet, 2002).

A weak hypomorphic allele, aurA²⁸⁷, encodes a protein with the amino acid substitutions H3Y, D47A, T90P, and R162C. The amino acid at position 47 represents polymorphism of the wild-type sequence. This modified enzyme is likely to have a reduced kinase activity because R162 is a conserved arginine in the ATP binding pocket in subdomain I of the catalytic domain of all protein kinases. Consistent with the sequencing data, the full-length protein is expressed in embryos derived from homozygous aurA^e287 mothers, but an Aurora A immunoprecipitate has kinase activity <35% of a wild-type immunoprecipitate. This reduced activity is consistent with the very weak hypomorphic nature of the allele. aurA^e209 encodes a full-length protein with two amino acid substitutions at other sites, D47A and E202K (Glover, 1995). The latter mutation changes a conserved acidic amino acid to a basic one in subdomain III of the catalytic domain, known to be required for the binding of the Mg-ATP complex. Consequently, it is expected that the aurA^e209 gene encodes a kinase that is likely to be catalytically inactive, thus accounting for the strongly hypomorphic nature of the mutation. It is noteworthy that brains from hemizygous aurA^e209 larvae show no significant increase in mitotic index over homozygotes, but do show a reduction of circular figures and a concomitant increase in bipolar spindles. Thus, although by the criterion of mitotic index alone aurA^e209 appears to exhibit extreme loss of function, it remains possible that the increase in circular mitotic figures seen in homozygous rather than hemizygous aurA^e209 larvae are favored by the increase in dose of such a catalytically inactive protein that can behave as a neomorphic mutant with respect to the formation of circular figures (Giet, 2002).

To determine whether centrosomal localization is affected in either mutant form, immunostaining of the brains of wild-type and hemizygous aurA^e209 larvae (heterozygous for the recessive aurA^e209 allele and a deletion of the locus and surrounding genes) was performed with anti-Aurora A antibodies. The mutant kinase localizes to centrosomes of both bipolar and monopolar spindles found in the mutant brains. Similarly, the mutant kinase in aurA²⁸⁷-derived embryos was also found on centrosomes. Thus, the mutations present in neither of these two mutant enzymes affect their ability to localize to centrosomes (Giet, 2002).

Although the ultimate consequences of the two mutations differ at the two developmental stages they affect, their phenotypes suggest that aurora A is required for aspects of centrosome function. Therefore, the bipolar spindles produced either in syncytial embryos derived from aurA²⁸⁷ mothers, or in the central nervous systems of aurA^e209 larvae were examined to determine whether there are any common defects affecting the spindle poles. In addition to the previously described characteristics of aurA²⁸⁷-derived embryos (Glover, 1995), in contrast to wild-type embryos, the astral microtubules at the spindle poles appear to be very short and/or reduced in density. This observation was made by introducing a gene encoding a Tau-GFP fusion protein into the aurA²⁸⁷ line to enable centrosome-associated microtubules to be observed in live embryos. This process also revealed that astral microtubules seem short and apparently reduced in number throughout mitosis compared with wild-type embryos carrying the same Tau-GFP transgene. Examination of the poles of bipolar spindles in neuroblasts from aurA^e209 larvae also revealed a similar apparent reduction in length and/or number of the astral microtubules relative to wild-type. Taken together, this suggests that one role of the Aurora A protein kinase is to regulate microtubule dynamics at the spindle poles (Giet, 2002).

The reduction in length and/or density of microtubules at the spindle poles after mutation of aurA led to an examination of whether this might correlate with changes in the association of any known centrosomal antigen with the poles. This was assessed by immunostaining neuroblasts from hemizygous mutant larvae of genotype aurA^e209/Df(3R)T61. An elevated frequency of mitotic cells is found in whole-mount preparations, the majority of which contain bipolar metaphase spindles. Many of these spindles appear to be bipolar but monoastral. The asters are diminutive and are nucleated around centrosomal antigens frequently only present at one pole. However, when centrosomal bodies are present, the intensity of staining given by antibodies to detect either gamma-tubulin, Centrosomin, CP190, or Asp is comparable to that observed at the poles of wild-type spindles. These centrosomal antigens are often not present as a single body at the poles, but as multiple punctate bodies. Such punctate bodies could arise either by fragmentation of centrosomes or by the accumulation of multiple centrosomes that have failed to segregate during mitosis. To attempt to distinguish between these possibilities, preparations of aurA^e209/Df(3R)T61 brains were sectioned for electron microscopy. Poles of bipolar spindles in such mutant brains either show focused microtubules with no evidence of an organized centrosome, or spindle microtubules nucleated from bodies containing multiple centrioles. Thus mutation in aurA appears to result, at least in part, in a block to centrosome separation resulting in the accumulation of multiple centrioles. These are surrounded by electron-dense pericentriolar material that is likely to correspond to the multiple regions of punctate staining of centrosomal antigens observed at these spindle poles by light microscopy. Similar defects were observed in ten cells that were examined in this way (Giet, 2002).

A reduction in the length of astral microtubules comparable to that seen after reduction in Aurora A kinase function has also been described in embryos derived from mothers carrying mutations in the d-tacc gene and in brains derived from flies having mutations in the MSPS gene. Therefore, whether the distribution of D-TACC might be affected in Aurora A-deficient cells was examined. The distribution of D-TACC was examined in wild-type and mutant embryos derived from homozygous aurA²⁸⁷ mothers. Anti-D-TACC antibodies strongly decorate the centrosomes and weakly stain the spindle microtubules of wild-type embryos. In contrast, in aurA²⁸⁷-derived embryos, D-TACC protein is poorly localized to centrosomes. However, D-TACC protein is clearly present and can be seen through increased cytoplasmic staining and in association with the spindle microtubules, indicating that this latter aspect of its localization is not dependent on Aurora A kinase. The localization of D-TACC was examined in larval neuroblasts hemizygous for the aurA^e209 mutation. In wild-type cells from this larval tissue, D-TACC is found to have a centrosomal association. However, in the aurA mutant cells, D-TACC is absent from the poles of the mitotic spindle, but is distributed in the region occupied by the spindle microtubules and throughout the cytoplasm in a punctate array. Thus, the altered distribution of D-TACC after perturbation of Aurora A function with different mutations suggests that Aurora A kinase function is necessary for localization of D-TACC to the centrosomes, but not the spindle microtubules in two cell types (Giet, 2002).

The product of the MSPS gene encodes a MAP that stabilizes microtubules and is found in a complex with the D-TACC protein (Cullen, 1999; Lee, 2001). To determine whether MSPS localization is also disrupted when Aurora A kinase function is compromised, embryos derived from wild-type and homozygous aurA²⁸⁷ mothers were examined. The MSPS staining is highly reduced from spindle poles, but the protein remains on spindle microtubules. In aurA^e209 neuroblasts, MSPS is also absent from the centrosome, but it remains associated with the region of the spindle compared with wild-type cells where it was associated with both. Thus, in aurora A mutant cells, neither MSPS nor D-TACC properly localize to the centrosome (Giet, 2002).

Since the mutant Aurora A kinases still localize to the centrosome, and because the stronger mutant allele seems to display some neomorphic function in the absence of zygotically expressed wild-type protein, it was of interest to determine whether there are similar spindle pole defects upon depletion of Aurora A protein. In the absence of protein-null mutants, RNAi was used as a means of reducing levels of the enzyme. Three days after transfection of S2 cells with ds aurA RNA, the protein kinase has disappeared from the centrosome and is reduced in levels by >95% as judged by Western blotting. Examination of Aurora A-depleted cells indicates a high frequency of metaphase abnormalities in which chromosomes are frequently misaligned on the metaphase plate. However, cells appear to undertake anaphase with such defects and in contrast to the aurA mutant neuroblasts do not arrest at metaphase. At present, the reason for this difference is not clear, but it is likely to be a consequence of different checkpoint responses in these two cell types. Such differences are known in Drosophila, where for example meiocytes only show a transient checkpoint response evident by a lengthening of prometaphase in response to spindle damage. It is noted that there is a significant increase in the number of cells with fragmented DNA after aurA RNAi, indicating that defective mitotic cells may be eliminated by an apoptotic pathway (Giet, 2002).

In spite of these differences, striking similarities are observed between defects at the spindle poles of aurA RNAi-treated cells that compare with those seen in the mutants. (1) Both the length and number of astral microtubules are reduced in Aurora A-depleted cells. (2) Most of the aurA RNAi mitotic spindles had abnormal accumulations of gamma-tubulin, frequently as multiple bodies at the spindle poles similar to those seen in aurA^e209 neuroblasts. (3) Finally, although D-TACC is associated with centrosomes and the mitotic spindle in S2 cells, its levels on centrosomes are greatly reduced. This also correlates with a reduction of MSPS protein at the spindle poles of aurA RNAi cells (Giet, 2002).

Together, these experiments show that when Aurora A kinase activity is reduced, either as a consequence of mutation or depletion of protein, there are similar effects upon centrosome behavior in three different division cycles: in syncytial embryos, neuroblasts, and cultured embryonic cells. In each case the astral microtubules appear short and reduced in number and centrosomal levels of D-TACC are reduced. D-TACC remains strongly associated with the centrosome in the absence of microtubules after nocodazole treatment. Thus, the decrease microtubule length and density with reduced Aurora A activity is likely to be due to reduced D-TACC-MSPS complex on the centrosome. To determine whether the association of Aurora A kinase with the centrosome is itself dependent on D-TACC, Aurora A localization was monitored in embryos derived from wild-type or homozygous d-tacc mothers. It was found that Aurora A remains associated with the centrosome in mutant embryos containing either ~10% (d-tacc1-derived) or null levels (d-tacc^stella-derived) of D-TACC protein. This indicates that Aurora A localization to the centrosome is independent of the D-TACC protein. It strengthens the previous result that even when D-TACC protein is mislocalized in Aurora A mutants, the mutant Aurora A protein is always found on the centrosome and never in association with D-TACC. These experiments suggest that Aurora A kinase might phosphorylate centrosomal proteins, D-TACC itself, or both in such a way as to target D-TACC to the centrosome and thereby stabilize microtubules (Giet, 2002).

To characterize the cellular defects that cause the bristle phenotypes in aurA³⁷ mutants, the SOP lineage in these mutants was analyzed. The four cell types in Drosophila ES organs can be distinguished by their characteristic size and marker gene expression: of the two larger outer cells, only the socket cell expresses the transcription factor Suppressor of Hairless (Su[H]). The two smaller inner cells can be distinguished based on expression of Prospero only in the sheath cell. One of each of these four cell types is found in control ES organs. Most aurA37 mutant ES organs, in contrast, consist of four large cells, two of which express Su(H), which indicates that the two inner cells are transformed into additional outer cells. Thus, instead of dividing asymmetrically, aurA³⁷ mutant SOP cells divide symmetrically into two pIIa cells that then each generate one hair and one socket. The Numb protein is crucial for asymmetric SOP cell division, and, therefore, whether this lineage defect is due to missegregation of Numb was tested by staining homozygous aurA³⁷ mutant pupae for Numb and DNA. While the Numb protein accumulates at the anterior SOP cell cortex during late prophase in wild-type and segregates into the anterior pIIb cell, no asymmetric localization of Numb is observed in aurA³⁷ mutants. The protein is uniformly distributed around the cell cortex throughout mitosis and segregates into both daughter cells. Asymmetric localization of Gαi to the anterior cell cortex and localization of Bazooka to the posterior cell cortex is unaffected in aurA³⁷ mutants, indicating that cell polarity is set up correctly. Thus, aurora-A is required for the asymmetric localization of Numb during mitosis (Berdnik, 2002).

A mitotic function for Drosophila Aurora-A has been described before (Glover, 1995). In strong alleles of aurora-A, centrosomes fail to separate, leading to the generation of abnormal monopolar mitotic spindles, defects in chromosome segregation, and the formation of polyploid cells. aurA³⁷ mutant cells, in contrast, complete mitosis and divide into two daughter cells and can therefore be used to characterize other aspects of Aurora-A function. To analyze mitotic spindles in aurA³⁷ mutants, control and mutant pupae were stained for DNA and alpha-Tubulin. Bipolar mitotic spindles are formed in aurA³⁷ mutants, but while microtubule minus ends converge on the centrosome in wild-type, they are less focused in aurA³⁷ mutants. This spindle morphology phenotype could reflect a defect in centrosome function, and therefore aurA³⁷ mutants were stained for the centrosomal marker gamma-Tubulin. In control SOP cells, gamma-Tubulin staining is weak during interphase, but two strong dots appear during mitosis, indicating that gamma-Tubulin is recruited to centrosomes. In 67% of the aurA³⁷ mutant mitotic SOP cells, no strong dots of gamma-Tubulin staining were visible, indicating a failure to recruit the protein to centrosomes. This defect is not completely penetrant, since one dot was observed in 17% of the cells, and, in another 17%, two closely spaced dots were seen. Defects in mitotic recruitment of gamma-Tubulin have been described before in flies mutant for cnn, a centrosomal core component that is dispersed in interphase but localized to centrosomes during mitosis. aurA³⁷ mutant pupae were therefore double stained for Cnn and gamma-Tubulin. In contrast to wild-type, where Cnn is detected in two strong dots at either spindle pole, the protein is undetectable on centrosomes of most aurA³⁷ mutant SOP cells. Thus, Aurora-A is required for recruiting both gamma-Tubulin and Cnn to centrosomes during mitosis, and these defects in centrosome maturation might be the cause of the abnormal spindle morphology. Despite the spindle defects in aurA³⁷ mutants, however, the two daughter cells of SOPs are still preferentially arranged along the anterior-posterior axis, indicating that spindle orientation is unaffected (Berdnik, 2002).

Cnn has been shown to be required for localization of gamma-Tubulin to centrosomes during mitosis. The defects in gamma-Tubulin localization in aurA³⁷ mutants could therefore be an indirect consequence of the failure to recruit Cnn. To test whether the defects in Numb localization are also caused by the failure to recruit Cnn, cnn null mutant Drosophila pupae were stained for Numb, gamma-Tubulin, and DNA. Even though no Cnn protein could be detected, these flies are viable. As in wild-type, Numb localizes into a cortical crescent during late prophase in all cnn mutant SOP cells even though, in 9% of the mitotic SOP cells (n = 22), the Numb crescent is mispositioned and does not correlate with the orientation of the metaphase plate. Since gamma-Tubulin is not recruited to centrosomes in these mutants, it is concluded that neither Cnn nor gamma-Tubulin recruitment to mitotic centrosomes is required for the asymmetric localization of Numb (Berdnik, 2002).

The failure to localize Numb asymmetrically in aurA³⁷ mutants could still be caused by the spindle defects. In neuroblasts, Numb localization still occurs after complete disruption of the mitotic spindle. To test the requirement of a mitotic spindle for Numb localization in SOP cells, wild-type pupae were incubated in 20 µg/ml colcemid for 1 or 2 hr and stained for Numb and gamma-Tubulin. After 1 hr of treatment, on average, nine SOP cells per pupal notum showed the mitotic arrest phenotype typical of microtubule inhibitors: chromosomes were no longer aligned in the metaphase plate, and centrosomes were distributed at random positions. Numb was still asymmetrically localized in 78% of these colcemid-arrested SOP cells. After 2 hr of treatment, the average number of metaphase-arrested SOP cells per notum increased to 27, and Numb was asymmetrically localized in 81% of them, indicating that new Numb crescents can be formed in the absence of a functional mitotic spindle. No effect on centrosome position was observed in a control experiment in which colcemid was omitted and Numb crescents were observed in 71% of the mitotic SOP cells. Thus, neither a functional mitotic spindle nor recruitment of Centrosomin and gamma-Tubulin to centrosomes are required for asymmetric localization of Numb. It is concluded that the defects in Numb localization observed in aurA³⁷ mutants are not indirect consequences of the spindle or centrosome defects. Rather, they indicate an independent role for Aurora-A in asymmetric protein localization during mitosis (Berdnik, 2002).

In C. elegans and vertebrates, Aurora-A proteins localize to centrosomes and the mitotic spindle (Nigg, 2001). The subcellular localization of Aurora-A in Drosophila has not been described, but if Aurora-A is exclusively localized to centrosomes, a function in asymmetric protein localization at the cell cortex is hard to imagine. Antibody was therefore generated against Aurora-A and it was used to stain wild-type and aurA³⁷ mutant Drosophila pupae. As in other organisms, Aurora-A is concentrated at centrosomes and the mitotic spindle in prophase and metaphase of wild-type Drosophila cells. In contrast, no centrosomal staining is detected in aurA³⁷ mutant cells. In addition to the centrosomal staining, significant staining was detected in the cytoplasm of both wild-type and aurA³⁷ mutant cells that can be blocked by preincubation of the Aurora-A antibody with the immunogenic peptide. To better analyze the dynamics of Aurora-A localization in living cells, transgenic flies were generated expressing a GFP-Aurora-A fusion protein (GFP-AurA). Using the UAS/Gal4 system, GFP-AurA was expressed in pupal SOP cells, and its distribution during mitosis was followed using confocal time-lapse microscopy. Like the endogenous protein, GFP-AurA is concentrated at centrosomes and the mitotic spindle during mitosis, but a fraction of the protein was detected in the cytoplasm. To determine the exchange rate between the centrosomal and the cytoplasmic pool of Aurora-A, photobleaching experiments were performed. When one of the two centrosomes was bleached by intense laser light, centrosomal staining was completely recovered within 10 s. No recovery was observed, however, when both the cytoplasm and the centrosomes were bleached using the same bleaching protocol. Thus, centrosomal localization of Aurora-A is transient, and the centrosomal pool rapidly interchanges with Aurora-A in the cytoplasm (Berdnik, 2002).

Thus the mitotic kinase Aurora-A is required for the asymmetric localization of Numb. Aurora-A is also involved in centrosome maturation and spindle assembly. However, neither functional centrosomes nor the mitotic spindle are required for Numb localization. Numb localization is an actin/myosin-dependent process, suggesting that Aurora-A regulates both actin- and microtubule-dependent processes during mitosis. Two rapidly interchanging pools of Aurora-A are found in the cytoplasm and at centrosomes and might carry out these two functions (Berdnik, 2002).

The results are consistent with the functions of Aurora-A described in other systems. In C. elegans, inactivation of the Aurora-A homolog air-1 by RNAi leads to defects in spindle morphology and failure to recruit the proteins CeGrip, ZYG-9, and gamma-Tubulin to centrosomes in mitosis (Schumacher, 1998; Hannak, 2001). Interestingly, air-1(RNAi) embryos also have defects in asymmetric distribution of cellular determinants: P-granules and the cytoplasmic protein Pie-1, both markers for the C. elegans germline, fail to segregate into one of the two daughter cells and are frequently found in both cells instead (Schumacker, 1998). Since Aurora-A is localized to centrosomes, this was thought to indicate a role for microtubules in asymmetric protein segregation (Schumacher, 1998). However, the asymmetric localization of both P-granules and the Pie-1 protein are actin dependent and microtubule independent. The results presented in this paper indicate that centrosomal localization of Aurora-A is transient and the protein is rapidly exchanging with a cytoplasmic pool. Assuming that Aurora-A localization is similarly dynamic in C. elegans, the defects in P-granule and Pie-1 localization could actually indicate an evolutionarily conserved function of Aurora-A in actin-dependent asymmetric protein localization during mitosis (Berdnik, 2002).

Mutations in Drosophila aurora-A were shown before to have defects in centrosome separation and chromosome segregation (Glover, 1995), leading to the formation of polyploid cells. Defects in centrosome separation are seen in a fraction of aurA³⁷ mutant cells, but monopolar mitotic spindles or polyploid cells are not observed. aurA³⁷ could be a hypomorphic allele that affects some Aurora-A-dependent processes more than others. aurA³⁷ affects an arginine in kinase subdomain III that is found in all Aurora- and MAP-kinases but is not generally conserved between all protein kinases. In MAP kinases, the equivalent residue is predicted to make contact with threonine 183, a residue in the activation loop that needs to be phosphorylated to activate the kinase. Phosphorylation of the residue equivalent to threonine 183 can also activate Aurora-A kinase (Walter, 2000), and aurA³⁷ could prevent the conformational change needed for full activation of the kinase (Berdnik, 2002).

How could Aurora-A function in asymmetric cell division? Aurora-A is not required for setting up polarity during interphase since both Galphai and Bazooka are asymmetrically localized in aurA³⁷ mutants. Instead, it is needed for interpreting this polarity to initiate the asymmetric localization of Numb at the onset of mitosis. In vertebrates, Aurora-A activity peaks at the G2/M transition (Bischoff, 1998), and phosphorylation of either Numb itself or a component of the Numb localization machinery could be required for Numb localization. So far, Lgl (Lethal [2] giant larvae) is the only other protein required for Numb localization but not polarity establishment. Since phosphorylation of Numb or Lgl by Aurora-A in vitro could not be detected, another, yet to be identified, component of the Numb localization machinery seems to be phosphorylated by Aurora-A at the onset of mitosis (Berdnik, 2002).

Numb localization also requires activation of the Cdc2 kinase, and, in hypomorphic cdc2 mutants, cells enter mitosis but have defects in asymmetric protein localization, a phenotype that is remarkably similar to aurA³⁷. How the two kinases act together is unclear. In vertebrates, Aurora-A activity peaks before activation of Cdc2, suggesting that Cdc2 is not required for Aurora-A activation. Conversely, Cdc2 activation is Aurora-A independent, since many Cdc2-dependent events do not require Aurora-A. Thus, it is likely that activation of the Numb localization machinery requires both Cdc2 and Aurora-A-dependent phosphorylation events (Berdnik, 2002).

The human homolog of Aurora-A, Aurora2, is amplified in colorectal cancer, suggesting that it is involved in carcinogenesis. How Aurora2 causes cancer is unclear. Overexpression of Aurora-A could force quiescent cells to reenter the cell cycle or cause defects in chromosome segregation and aneuploidy. Assuming that Aurora-A is also involved in asymmetric cell division in vertebrates, cell lineage defects are an interesting alternative possibility (Berdnik, 2002).

The choice of self-renewal versus differentiation is a fundamental issue in stem cell and cancer biology. Neural progenitors of the Drosophila post-embryonic brain, larval neuroblasts (NBs), divide asymmetrically in a stem cell-like fashion to generate a self-renewing NB and a ganglion mother cell (GMC), which divides terminally to produce two differentiating neuronal/glial daughters. Aurora-A (AurA) acts as a tumor suppressor by suppressing NB self-renewal and promoting neuronal differentiation. In aurA loss-of-function mutants, supernumerary NBs are produced at the expense of neurons. AurA suppresses tumor formation by asymmetrically localizing atypical protein kinase C (aPKC), an NB proliferation factor. Numb, which also acts as a tumor suppressor in larval brains, is a major downstream target of AurA and aPKC. Notch activity is up-regulated in aurA and numb larval brains, and Notch signaling is necessary and sufficient to promote NB self-renewal and suppress differentiation in larval brains. These data suggest that AurA, aPKC, Numb, and Notch function in a pathway that involved a series of negative genetic interactions. This study has identified a novel mechanism for controlling the balance between self-renewal and neuronal differentiation during the asymmetric division of Drosophila larval NBs (Wang, 2006).

When aurA function is compromised, mutant NBs acquire some features of cancer stem cells. They divide to generate a large number of daughter cells capable of self-renewal. This excessive self-renewal occurs at the expense of neuronal differentiation, suggesting that the normally asymmetric NB divisions have been altered such that the mutant NBs can divide symmetrically to generate two NB-like daughters. Cell cycle regulator CycE and cell growth factor dMyc are expressed in most of these tumor-like cells. Up-regulation of CycE is required for aurA overgrowth phenotype. AurA also regulates proper orientation of the mitotic spindle probably by controlling asymmetric localization of Mud. Both proteins are localized to centrosomes and are required for centrosome function. Centrosome abnormality and chromosome segregation defects in aurA could lead to aneuploidy, and many cancer cells exhibit centrosome defects and chromosome instability. Mammalian AurA when overexpressed can be oncogenic. However, future studies on its possible role as a tumor suppressor will be particularly interesting (Wang, 2006).

The data suggest that aurA negatively regulates aPKC function to regulate NB self-renewal. aPKC appears to act as a NB proliferation factor since overexpression of a modified membrane-targeted version, aPKC-CAAX, which exhibits ectopic cortical localization throughout the NB cortex, leads to overproliferation and tumor formation, similar to loss of aurA. AurA is required for the asymmetric localization of aPKC and restrict aPKC to the cortical region associated with the future NB daughter and loss of aurA results in delocalization of aPKC to the entire cortex. Consistent with and supporting this notion, loss of aPKC can suppress, albeit partially, the aurA mutant overgrowth phenotype (Wang, 2006).

In contrast to the well-studied role of Numb as a cell fate determinant during asymmetric divisions of embryonic GMCs, SOPs, or muscle progenitors, a role for Numb during NB asymmetric divisions has not been described. This study shows that Numb also acts as a tumor suppressor in Drosophila larval brains, and that Numb is a key downstream target of AurA and aPKC in the regulation of NB self-renewal. In both aurA mutant NBs or NBs overexpressing aPKC-CAAX, the asymmetric localization of Numb is compromised and the resultant overgrowth phenotype is consistent with that of numb loss-of-function. numb and aurA mutant NBs also share several common features including excessive self-renewal at the expense of neuronal differentiation as well as the membrane enrichment of Spdo, a positive regulator of Notch signaling. These data suggest that AurA positively regulates Numb function. Genetic analysis is consistent with the notion that this is achieved through the negative regulation of aPKC that in turn negatively regulates Numb (Wang, 2006).

Numb is known to be a negative regulator of Notch signaling. The current findings indicate that Notch is necessary and sufficient for promoting larval NB proliferation and suppressing neuronal differentiation. Genetic epistasis studies suggest that an AurA-aPKC-Numb-Notch genetic hierarchy acts to regulate self-renewal of Drosophila neural progenitor cells. During a wild-type larval NB asymmetric division, aurA acts to negatively regulate aPKC and restrict its localization to the cortical region associated with the future NB daughter; aPKC negatively regulates Numb and ensures that its localization/activity is restricted to the future GMC where Numb acts to antagonize Notch. The net effect is that Notch is asymmetrically activated in the NB daughter where it acts to promote self-renewal and suppress differentiation. Although these data suggest that aurA acts through the aPKC/Numb/Notch pathway, given the partial suppression seen in the double mutants aPKC;aurA and Notch^ts-¹;aurA, the possibility that additional mechanisms may be involved cannot be excluded (Wang, 2006).

S. cerevisiae Ipl1, Drosophila Aurora, and the mammalian centrosomal protein IAK-1 define a new subfamily of serine/threonine kinases that regulate chromosome segregation and mitotic spindle dynamics. Mutations in ipl1 and aurora result in the generation of severely aneuploid cells and, in the case of aurora, monopolar spindles arising from a failure in centrosome separation. A related, essential protein from C. elegans, AIR-1 (Aurora/Ipl1 related), is localized to mitotic centrosomes. Disruption of AIR-1 protein expression in C. elegans embryos results in severe aneuploidy and embryonic lethality. Unlike aurora mutants, this aneuploidy does not arise from a failure in centrosome separation. Bipolar spindles are formed in the absence of AIR-1, but they appear to be disorganized and are nucleated by abnormal-looking centrosomes. In addition to its requirement during mitosis, AIR-1 may regulate microtubule-based developmental processes as well. The data suggests AIR-1 plays a role in P-granule segregation and the association of the germline factor PIE-1 with centrosomes (Schumacher, 1998).

The Aurora kinase Ipl1p plays a crucial role in regulating kinetochore-microtubule attachments in budding yeast, but the underlying basis for this regulation is not known. To identify Ipl1p targets, 28 kinetochore proteins were purified from yeast protein extracts. These studies identified five previously uncharacterized kinetochore proteins and defined two additional kinetochore subcomplexes. Mass spectrometry was used to identify 18 phosphorylation sites in 7 of these 28 proteins. Ten of these phosphorylation sites are targeted directly by Ipl1p, allowing the identification of a consensus phosphorylation site for an Aurora kinase. This systematic mutational analysis of the Ipl1p phosphorylation sites demonstrates that the essential microtubule binding protein Dam1p is a key Ipl1p target for regulating kinetochore-microtubule attachments in vivo (Cheeseman, 2002).

The spindle checkpoint inhibits anaphase until all chromosomes have established bipolar attachment. Two kinetochore states trigger this checkpoint. The absence of microtubules activates the attachment response, while the inability of attached microtubules to generate tension triggers the tension/orientation response. The processes regulated by the single aurora kinase of fission yeast, Ark1, represent a combination of the events that are regulated by aurora-A and aurora-B kinases in higher systems. The aurora kinase of budding yeast, Ipl1, is required for the tension/orientation, but not attachment, response. In contrast, the single aurora kinase of fission yeast, Ark1, is required for the attachment response. Having established that the initiator codon assigned to ark1⁺ was incorrect and that Ark1-associated kinase activity depends upon survivin function and phosphorylation, it was found that the loss of Ark1 from kinetochores by either depletion or use of a survivin mutant overides the checkpoint response to microtubule depolymerization. Ark1/survivin function is not required for the association of Bub1 or Mad3 with the kinetochores. However, it is required for two aspects of Mad2 function that accompany checkpoint activation: full-scale association with kinetochores and formation of a complex with Mad3. Neither the phosphorylation of histone H3 that accompanies chromosome condensation nor condensin recruitment to mitotic chromatin is seen when Ark1 function is compromised. Cytokinesis is not affected by Ark1 depletion or expression of the 'kinase dead' ark1.K118R mutant (Petersen, 2003).

Chromosome segregation depends on kinetochore bi-orientation so that sister kinetochores attach to microtubules from opposite poles and come under tension. The budding yeast Ipl1/Aurora protein kinase allows the absence of tension to activate the spindle checkpoint. Checkpoint activation in the mtw1-1 kinetochore mutant requires Ipl1p, suggesting that Mtw1p promotes tension. mtw1-1 dosage suppressors were isolated and Dsn1, a kinetochore protein that immunoprecipitates with the Mif2/CENP-C and Cse4/CENP-A proteins, was identified, as well as the Mtw1, Nnf1, and Nsl1 kinetochore proteins. mtw1 and dsn1 mutant strains exhibit similar phenotypes, suggesting that Mtw1p and Dsn1p act together. Although mtw1 mutant cells contain unattached chromosomes, attachment is restored by impairing Ipl1p function. These results suggest that mtw1 mutant kinetochores are competent to bind microtubules but Ipl1p generates unattached chromosomes. It is therefore proposed that an Mtw1 complex is required for kinetochore bi-orientation that is monitored by the Ipl1p kinase (Pinsky, 2003).

An understanding of kinetochore function has been greatly aided by studies in the budding yeast where over 40 kinetochore proteins have been identified, many of which are conserved. The kinetochore is organized into distinct subcomplexes that have been placed into inner, central, and outer domains based on two criteria: (1) in vitro centromere or microtubule binding activities and (2) in vivo dependency relationships for centromere association. The inner kinetochore CBF3 complex binds centromeric DNA and is required for the centromere association of all other kinetochore proteins. The inner kinetochore also contains Cse4/CENP-A, the conserved centromeric histone H3 variant, as well as the Cbf1 and Mif2/CENP-C proteins. The central kinetochore includes the conserved Ndc80/HEC-1 complex, implicated in the stabilization of kinetochore-microtubule attachments, and the large Ctf19 complex, which can be divided into at least three subcomplexes, the only one known to require microtubules for its kinetochore association, as well as several microtubule motors and nonmotor microtubule-associated proteins (Pinsky, 2003 and references therein).

Although a number of kinetochore proteins have been identified, little is known about how their assembly and activity is regulated. The Ipl1/Aurora protein kinase is a key regulator of kinetochore function because it is required to generate bi-oriented kinetochore-microtubule attachments. In ipl1 mutant cells, kinetochores are competent to bind to microtubules, but 85% of the time sister chromatids are segregated to the same spindle pole. A similar increase in mono-oriented attachments is observed in cultured vertebrate cells when Aurora B function is compromised using inhibitory antibodies or small-molecule inhibitors, suggesting conservation of function. Because the biased segregation of chromosomes to the bud in ipl1 mutants was eliminated by the transient disruption of microtubules, it was proposed that Ipl1p promotes the turnover of mono-oriented kinetochore-microtubule interactions. A crucial in vivo target of Ipl1p is the Dam1/DASH/DDD complex. The phenotypes of a dam1 mutant thought to mimic constitutive Ipl1p phosphorylation are consistent with this complex acting as a microtubule release factor (Pinsky, 2003 and references therein).

It is not known whether there are kinetochore proteins in addition to Ipl1 and Dam1 that are required for bi-orientation. The essential Mtw1 (mis twelve-like) protein, which copurifies with the largely nonessential Ctf19 complex, is a particularly intriguing candidate for a mediator of kinetochore bi-orientation. In mtw1 mutants and cells depleted of Mtw1 protein, DNA segregates into two unequal masses, and sister centromeres frequently lose bi-orientation, suggesting that kinetochore-microtubule attachments are made but not properly regulated. Mutations in the fission yeast mis12 result in sister chromatids that are initially pulled toward the same pole, indicating mono-oriented attachments. Furthermore, when protein levels of the human homolog, hMis12, are reduced by siRNA in cell culture, chromosomes are misaligned in a manner consistent with -ientation defects. MTW1 (DSN3, NSL2) was also identified along with the essential genes DSN1 (dosage suppressor of nnf1-17) and NSL1 (nnf1-17 synthetic lethal) based on genetic interactions with NNF1 (necessary for nuclear function), an essential gene with a role in chromosome segregation. The genetic interactions and similar localization patterns suggested that these proteins might physically interact at the kinetochore (Pinsky, 2003 and references therein).

Mtw1p does associate with Dsn1, Nnf1, and Nsl1, as well as the Mif2 and Cse4 inner kinetochore proteins. Defects in Mtw1p require Ipl1p function to activate the spindle checkpoint, suggesting that Mtw1p is required for kinetochore tension. Mutations in MTW1 and DSN1 have similar phenotypes leading to spindle checkpoint activation with chromosomes unattached to the spindle. Strikingly, chromosome attachments to the spindle are completely restored in mtw1 mutants when Ipl1p function is absent, suggesting that Mtw1p is required to generate kinetochore bi-orientation but not microtubule attachments. Taken together, these data demonstrate that the Ipl1p kinase detects improper kinetochore-microtubule attachments and is required to generate unattached chromosomes in response to these defects (Pinsky, 2003).

It is proposed that Mtw1p is required for the physical tension that results from bi-orienting sister kinetochores. Although unattached chromosomes were detected in the mtw1-1 mutant, two lines of evidence are provided consistent with this being due to a bi-orientation defect that leads to Ipl1p-mediated release of microtubule attachments. First, the checkpoint arrest induced by the mtw1-1 mutant requires Ipl1p function. Because Ipl1p is required to activate the spindle checkpoint in response to defects in kinetochore tension but not attachment, this suggests that mtw1-1 mutant kinetochores are not under sufficient tension and that Ipl1p does directly monitor the state of kinetochores. This result is not necessarily specific to mtw1-1, and future work will be required to determine if defects in other kinetochore components also require Ipl1p to engage the spindle checkpoint. This tension deficiency is thought to be a direct consequence of defects in Mtw1p function and not a failure in kinetochore assembly, because kinetochore proteins that were tested remain centromere associated in mtw1-1 mutant cells. In addition, Mtw1p is required to maintain bi-orientation that is established at metaphase. Second, it was found that removing Ipl1p function in mtw1-1 mutants restores the attachment of sister chromatids to the spindle. Because both chromosome attachment and segregation in mtw1-1 ipl1-321 cells was identical to the ipl1-321 cells, the mtw1-1 attachment defect is completely suppressed by loss of Ipl1p function. It is therefore unlikely that the elimination of Ipl1p function simply strengthens weak kinetochore-microtubule interactions. In addition, this data strongly argues that mtw1-1 mutants have kinetochores that are competent to attach to microtubules. In support of this, the lack of a cell cycle delay when Mis12 is depleted by siRNA in human cells or mutated in fission yeast is consistent with microtubules attaching to kinetochores, thus satisfying the spindle checkpoint. Therefore the interpretation is favored that mtw1-1 mutant kinetochores bind to microtubules but are defective in bi-orientation so that Ipl1p generates unattached kinetochores (Pinsky, 2003).

There are several ways that Mtw1p could facilitate the bi-orientation of sister kinetochores. First, Mtw1p might contribute to the rigidity of the kinetochore and sterically inhibit mono-orientation. The placement of Mtw1p at the inner kinetochore due to interactions with the Mif2 and Cse4 proteins might reflect a role for centromeric chromatin structure in mediating proper kinetochore orientation. However, if Mtw1p facilitated sister kinetochore orientation, mtw1-1 ipl1-321 double mutants would likely be more defective for bi-orientation than either single mutant. Since the double mutant phenotype is the same as the ipl1-321 phenotype, it suggests that they act in the same pathway to ensure bi-orientation. Mtw1p cannot be required to signal Ipl1p that there is a tension defect, because Ipl1p can detach chromosomes in the absence of Mtw1p function. Therefore, one possibility is that Mtw1p acts downstream of Ipl1p to stabilize bi-oriented attachments. For example, Mtw1p could convert lateral microtubule attachments into end-on attachments that are more stable. Alternatively, Mtw1p might be important for generating tension on kinetochores that have already made bipolar attachments (Pinsky, 2003).

The data presented here suggest that Mtw1p is involved in bi-orientation and not in microtubule attachment. In addition, this work directly demonstrates that Ipl1p promotes the turnover of improper kinetochore-microtubule interactions. Evidence is provided that Mtw1p is part of an inner kinetochore complex, and in the future it will be important to elucidate the mechanism that this complex uses to promote bi-orientation. The molecular basis for the difference between mtw1 and mtw1 ipl1 mutant kinetochores is being actively pursued to understand how Ipl1p detects and responds to defects in Mtw1p function (Pinsky, 2003).

Cilia and flagella play key roles in development and sensory transduction, and several human disorders, including polycystic kidney disease, are associated with the failure to assemble cilia. The aurora protein kinase CALK in the biflagellated alga Chlamydomonas has a central role in two pathways for eliminating flagella. Cells rendered deficient in CALK are defective in regulated flagellar excision and regulated flagellar disassembly. Exposure of cells to altered ionic conditions, the absence of a centriole/basal body for nucleating flagellar assembly, cessation of delivery of flagellar components to their tip assembly site, and formation of zygotes all lead to activation of the regulated disassembly pathway as indicated by phosphorylation of CALK and the absence of flagella. It is proposed that cells have a sensory pathway that detects conditions that are inappropriate for possession of a flagellum, and that CALK is a key effector of flagellar disassembly in that pathway (Pan, 2004).

One model to explain these data is that the distal ends of the flagellar microtubules are monitored by a surveillance mechanism. A sensor ensures that flagellar components are successfully being added onto the tip of the growing flagellar axoneme. In a newly divided cell, the initial assembly would occur on the distal (plus) ends of the basal body triplet microtubules. If the proper assembly is not detected, because for example the machinery for transporting flagellar components to their site of assembly (IFT) is nonfunctional, because the intracellular ionic or osmotic conditions are inappropriate, or because the site of flagellar assembly itself (the basal body) is defective, then the sensing system is activated, CALK is phosphorylated, and flagella do not form or existing flagella are removed. Many cilia-related diseases, including some caused by aberrant cell proliferation, are associated with the absence of cilia. A further understanding of the molecular mechanisms that underlie regulation of the possession of cilia and flagella has the potential to offer important new insights into normal and pathological processes (Pan, 2004 and references therein).

Centrosomes mature as cells enter mitosis, accumulating gamma-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). RNA-mediated interference (RNAi) was used to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal alpha-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, AIR-1 is found to be required for the increase in centrosomal gamma-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1-dependent increase in centrosomal gamma-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation (Hannak, 2001).

Aurora A kinase localizes to centrosomes and is required for centrosome maturation and spindle assembly. This study describes a microtubule-independent role for Aurora A and centrosomes in nuclear envelope breakdown (NEBD) during the first mitotic division of the C. elegans embryo. Aurora A depletion does not alter the onset or kinetics of chromosome condensation, but dramatically lengthens the interval between the completion of condensation and NEBD. Inhibiting centrosome assembly by other means also lengthens this interval, albeit to a lesser extent than Aurora A depletion. By contrast, centrosomally nucleated microtubules and the nuclear envelope-associated motor dynein are not required for timely NEBD. These results indicate that mitotic centrosomes generate a diffusible factor, which is proposed is activated Aurora A, that promotes NEBD. A positive feedback loop, in which an Aurora A-dependent increase in centrosome size promotes Aurora A activation, may temporally couple centrosome maturation to NEBD during mitotic entry (Portier, 2007).

In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. TPXL-1, a C. elegans protein is the first characterized invertebrate ortholog of TPX2. An essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. These data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly (Ozlu, 2005).

The data suggest that the most likely reason that spindles collapse in the absence of TPXL-1 is destabilization of kinetochore microtubules. One possibility is that the kinetochore-attached plus ends are unstable in the absence of TPXL-1. Unfortunately, the high-turnover rate of microtubules in C. elegans prophase precludes testing this model by FRAP. The fact that TPXL-1 operates together with Aurora A and Aurora A is localized to spindle poles suggests that the primary defect of tpxl-1(RNAi) could be in organization of the spindle pole. Supporting evidence for this is that the number of microtubules at centrosomes is quite reduced, whereas the nucleation rate seems to be the same as wild-type, and a substantial number of polymerizing microtubules exhibit freedom of movement (visible as retrograde plus-end movement) inconsistent with a stable attachment to centrosomes. Perhaps the minus ends of microtubules are unstable, and this allows the centrosomes to be pulled toward the chromosomes (Ozlu, 2005).

Therefore, it is proposed that in C. elegans mitosis, the homolog of TPX2 -- TPXL-1 -- targets Aurora A to the spindle, where Aurora A phosphorylates downstream targets required for kinetochore microtubule stabilization. These could be centrosome, spindle, or kinetochore components. One possible candidate is TAC-1, a protein implicated in microtubule stability whose homologs are known to be an Aurora A substrate. TPX2 itself is a microtubule binding protein, so it is possible that phosphorylation of TPXL-1 by Aurora A may also contribute to the stabilization activity. The other known substrate of Aurora A, the motor protein Eg5, is not required for C. elegans spindle assembly. Identification of other Aurora A substrates would provide important insights into the mechanisms of spindle assembly (Ozlu, 2005).

C. elegans Aurora A has a number of different roles in the first embryonic cell division: (1) cell polarity, (2) centrosome maturation, (3) spindle assembly, and (4) spindle elongation at anaphase. In contrast, TPXL-1 is required only for spindle assembly, and the rescue experiments with wild-type or mutant TPXL-1 indicate that all of the functions of TPXL-1 are contained within a subset of Aurora A functions. Thus, it seems that a key role for Aurora A in spindle assembly is to stabilize microtubules nucleated from centrosomes that are bound to kinetochores. Because RNAi is a run-down technique, it is possible that the difference between Aurora A phenotypes and TPX2 phenotypes represents a difference in sensitivity of different Aurora A functions to TPX2 levels. However, it is more likely that other Aurora A activation and targeting subunits exist that are responsible for the various functions of Aurora A. In this way, the cell can ensure that Aurora A is targeted at the correct time and to the correct place during the cell cycle to perform its various roles (Ozlu, 2005).

By differential screening of a Xenopus laevis egg cDNA library, a 2,111 bp cDNA has been isolated that corresponds to a maternal mRNA specifically deadenylated after fertilization. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family that includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, that corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 'invades' the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. pEg2 binds directly to taxol stabilized microtubules in vitro. In addition, the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. A catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts (Roghi, 1998).

XlEg5 is a Xenopus laevis kinesin-related protein from the bimC family. pEg2 is an Aurora-related serine/threonine kinase. Inhibition of either XlEg5 or pEg2 activity during mitosis in Xenopus egg extract leads to monopolar spindle formation. In Xenopus XL2 cells, pEg2 and XlEg5 are both confined to separated centrosomes in prophase, and then to the microtubule spindle poles. pEg2 co-immunoprecipitates with XlEg5 from egg extracts and XL2 cell lysates. Both proteins can directly interact in vitro, but also through the two-hybrid system. Furthermore immunoprecipitated pEg2 is found to remain active when bound to the beads and phosphorylates XlEg5 present in the precipitate. Two-dimensional mapping of XlEg5 tryptic peptides phosphorylated in vivo first confirms that XlEg5 is phosphorylated by p34(cdc2) and next reveals that in vitro pEg2 kinase phosphorylates XlEg5 on the same stalk domain serine residue that is phosphorylated in metabolically labeled XL2 cells. The kinesin-related XlEg5 is thought to be the first in vivo substrate ever reported for an Aurora-related kinase (Giet, 1999a).

The Xenopus laevis aurora/Ip11p-related kinase pEg2 is required for centrosome separation, which is a prerequisite for bipolar mitotic spindle formation. Inhibition of pEg2 by addition of either an inactive kinase or a monoclonal antibody destabilizes bipolar spindles previously assembled in Xenopus egg extracts. The bipolar spindles collapse to form structures such as microtubule asters with chromosome rosettes, monopolar spindles, and multipolar spindles. In collapsed spindles, chromosomes remain attached to the microtubules plus ends. The destabilization of the bipolar spindle is reminiscent of the destabilization observed after inhibition of cross-linking activities which maintain parallel and anti-parallel microtubules linked together. pEg2 phosphorylates the kinesin-related protein XlEg5 which is involved in centrosome separation but which was also reported to be involved in spindle stability. The collapse of the bipolar spindle observed after inhibition of pEg2 suggests that the kinase might regulate the cross-linking activity of XlEg5. pEg2 probably also regulates other microtubule-based motor proteins involved in bipolar spindle stability. This is the first evidence that aurora/Ip11p-related kinase activity actually participates not only in mitotic spindle formation by regulating centrosome separation but also in mitotic spindle stabilization (Giet, 2000).

The mitotic kinase Aurora A (Aur-A) is required for formation of a bipolar mitotic spindle and accurate chromosome segregation. In somatic cells, Aur-A protein and kinase activity levels peak during mitosis, and Aur-A is degraded during mitotic exit. This study has investigated how Aur-A protein and kinase activity levels are regulated, taking advantage of the rapid synchronous cell division cycles of Xenopus eggs and cell-free systems derived from them. Aur-A kinase activity oscillates in the early embryonic cell cycles, just as in somatic cells, but Aur-A protein levels are constant, indicating that regulated activation and inactivation, instead of periodic proteolysis, is the dominant mode of Aur-A regulation in these cell cycles. Cdh1, the APC/C activator that targets many mitotic proteins for ubiquitin-dependent proteolysis during late mitosis and G1 in somatic cells, is missing in Xenopus eggs and early embryos. Addition of Cdh1 to egg extracts undergoing M phase exit is sufficient to induce rapid degradation of Aur-A. Aur-A contains both of the two known APC/C recognition signals, (1) a C-terminal D box similar to those required for ubiquitin-dependent destruction of cyclin B and several other mitotic proteins, and (2) an N-terminal KEN box similar to that found on cdc20, which is ubiquitinated in response to APC/CCdh1. The D box is required for Cdh1-induced destruction of Aur-A but the KEN box is not. Destruction also requires a short region in the N terminus, which contains a newly identified recognition signal, the A box. The A box is conserved in vertebrate Aur-As and contains serine 53, which is phosphorylated during M phase. Mutation of serine 53 to aspartic acid, which can mimic the effect of phosphorylation, completely blocks Cdh1-dependent destruction of Aur-A. These results suggest that dephosphorylation of serine 53 during mitotic exit could control the timing of Aur-A destruction, allowing recognition of both the A box and D box by Cdh1-activated APC/C (Littlepage, 2002).

Aurora kinase indirectly regulates the translation of dormant maternal mRNAs. Full-grown Xenopus oocytes arrest at the G2/M border of meiosis I. Progesterone breaks this arrest, leading to the resumption of the meiotic cell cycles and maturation of the oocyte into a fertilizable egg. In these oocytes, progesterone interacts with an unidentified surface-associated receptor, which induces a non-transcriptional signaling pathway that stimulates the translation of dormant c-mos messenger RNA. Mos, a mitogen-activated protein (MAP) kinase kinase kinase, indirectly activates MAP kinase, which in turn leads to oocyte maturation. The translational recruitment of c-mos and several other mRNAs is regulated by cytoplasmic polyadenylation, a process that requires two 3' untranslated regions, the cytoplasmic polyadenylation element (CPE) and the polyadenylation hexanucleotide AAUAAA. Although the signaling events that trigger c-mos mRNA polyadenylation and translation are unclear, they probably involve the activation of CPEB, the CPE binding (Drosophila homolog: Orb). An early site-specific phosphorylation of CPEB is essential for the polyadenylation of c-mos mRNA and its subsequent translation, and for oocyte maturation. This selective, early phosphorylation of CPEB is catalysed by Eg2, a member of the Aurora family of serine/threonine protein kinases (Mendez, 2000a).

The release of Xenopus oocytes from prophase I arrest is largely driven by the cytoplasmic polyadenylation-induced translation of dormant maternal mRNAs. Cytoplasmic polyadenylation requires two cis-acting sequences and three core polyadenylation factors. The cis elements are the hexanucleotide AAUAAA, which is also essential for nuclear pre-mRNA cleavage and polyadenylation, and an upstream U-rich sequence called the cytoplasmic polyadenylation element (CPE). The core factors include CPEB, an RNA recognition motif (RRM), and zinc finger-containing protein that has a strong binding preference for the CPE, and a cytoplasmic form of cleavage and polyadenylation specificity factor (CPSF), which interacts with AAUAAA. In contrast to the nuclear form of CPSF, which consists of four subunits of 160 kDa, 100 kDa, 70 kDa, and 30 kDa, the cytoplasmic form contains the 160 kDa (or a functional equivalent thereof), the 100 kDa, and the 30 kDa subunits, but lacks the 70 kDa species. The third core factor is poly(A) polymerase (PAP). Based on the events associated with nuclear polyadenylation, the function of CPSF is probably to recruit PAP to the end of the mRNA. The molecular function of CPEB in cytoplasmic polyadenylation has not been elucidated. Although all the factors required for cytoplasmic polyadenylation are present in oocytes, they become active only at the onset of maturation. In response to progesterone-stimulated maturation, CPEB undergoes an early phosphorylation at a single residue that is essential for polyadenylation. This phosphorylation event is catalyzed by Eg2, a member of the Aurora family of Ser/Thr protein kinases. In this study the molecular function of this CPEB phosphorylation has been investigated (Mendez, 2000b and references therein).

The most proximal stimulus for polyadenylation is Eg2-catalyzed phosphorylation of CPEB serine 174. This phosphorylation event stimulates an interaction between CPEB and CPSF. This interaction is direct, does not require RNA tethering, and occurs through the 160 kDa subunit of CPSF. Eg2-stimulated and CPE-dependent polyadenylation is reconstituted in vitro using purified components. These results demonstrate that the molecular function of Eg2-phosphorylated CPEB is to recruit CPSF into an active cytoplasmic polyadenylation complex (Mendez, 2000b).

There are some key similarities between cytoplasmic and nuclear polyadenylation. In the nucleus, the affinity of CPSF for the AAUAAA is very low and must be stabilized by other RNA binding proteins. These other factors include CstF, which binds the GU-rich element downstream of the AAUAAA element, and U1A protein, which binds an upstream U-rich element present in at least one viral mRNA. While cytoplasmic polyadenylation has no downstream element, the upstream element is certainly equivalent to the CPE, and the function of CPEB is analogous to that shown for U1A, to stabilize the binding of CPSF to the AAUAAA. However, while there is no evidence that the CPSF-stabilizing activity of U1A or CstF can be modulated by phosphorylation, that of CPEB is regulated by Eg2-catalyzed phosphorylation (Mendez, 2000b and references therein).

In spite of these similarities, there are also some key differences that distinguish nuclear from cytoplasmic polyadenylation. (1) While both processes require CPSF, the cytoplasmic form of this multiprotein complex lacks the 70 kDa subunit. While the function of the 70 kDa protein in nuclear polyadenylation is unclear, it is possible that cytoplasmic CPSF contains a functional substitute, or that cytoplasmic polyadenylation has dispensed altogether with any need for a 70 kDa protein-like activity. (2) While nuclear polyadenylation is strongly coupled to pre-mRNA cleavage, the cytoplasmic process is not associated with any cleavage event. (3) Nuclear pre-mRNA polyadenylation occurs on virtually all mRNAs whereas cytoplasmic polyadenylation takes place on just those few species that contain a CPE. (4) cdc2-catalyzed phosphorylations of PAP inhibit nuclear pre-mRNA polyadenylation as cells enter M phase; cytoplasmic polyadenylation, in contrast, is induced as cells enter M phase (i.e., maturation) (Mendez, 2000b and references therein).

While Eg2-catalyzed phosphorylation of CPEB probably occurs on all CPE-containing mRNAs, there are additional processes that control the timing of polyadenylation. That is, the mRNA encoding the serine/threonine kinase Mos is the first mRNA known to undergo polyadenylation-induced translation following progesterone stimulation. Mos synthesis activates the M phase promoting factor (cyclinB/cdc2), which in turn induces the polyadenylation of other mRNAs later during maturation. What distinguishes early from late adenylating mRNAs? While additional mRNA-specific regulatory proteins could certainly affect the timing of polyadenylation, the number of CPEs appears to have an important influence. For example, the early adenylating c-mos mRNA has a single CPE while the late adenylating cyclin B1, histone B4, and cyclin A1 mRNAs have two CPEs. If one of the cyclin B1 CPEs is removed, the message adenylates early and is Mos (and cdc2) independent. It is possible that two CPEs, and thus two CPEBs, could actually inhibit the formation of an active polyadenylation complex by, for example, dimerizing or interacting with another factor that bridges the two CPEBs. While such a configuration of factors could preclude a CPEB-CPSF interaction early during maturation, a late cdc2-mediated partial destruction of CPEB could liberate one of the molecules to interact with CPSF and promote polyadenylation (Mendez, 2000b).

The polyadenylation complex includes other factors, which, while not directly influencing polyadenylation, mediate translational regulatory events associated with polyadenylation. The translational repression (masking) of CPE-containing mRNAs prior to progesterone stimulation appears to be mediated by maskin, a CPEB-associated protein that also binds the cap binding protein eIF4E. The maskin-eIF4E interaction prevents the association of eIF4G with eIF4E, thereby inhibiting translation. The dissociation of the maskin-eIF4E complex, which presumably allows eIF4G to bind eIF4E, is coincident with, and possibly the result of, cytoplasmic polyadenylation (Mendez, 2000b and references therein).

The synthesis and destruction of cyclin B drives mitosis in eukaryotic cells. Cell cycle progression is also regulated at the level of cyclin B translation. In cycling extracts from Xenopus embryos, progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells (Groisman, 2002).

Xenopus oocytes are arrested in meiotic prophase I and resume meiotic divisions in response to progesterone. Progesterone triggers activation of M-phase promoting factor (MPF) or Cdc2-cyclin B complex and neosynthesis of Mos kinase, responsible for MAPK activation. Both Cdc2 and MAPK activities are required for the success of meiotic maturation. However, the signaling pathway induced by progesterone and leading to MPF activation is poorly understood, and most of the targets of both Cdc2 and MAPK in the oocyte remain to be determined. Aurora-A is a Ser/Thr kinase involved in separation of centrosomes and in spindle assembly during mitosis. It has been proposed that in Xenopus oocytes Aurora-A could be an early component of the progesterone-transduction pathway, acting through the regulation of Mos synthesis upstream Cdc2 activation. This study addressed the question of Aurora-A regulation during meiotic maturation by using new in vitro and in vivo experimental approaches. Cdc2 kinase activity was found to be necessary and sufficient to trigger both Aurora-A phosphorylation and kinase activation in Xenopus oocyte. In contrast, these events are independent of the Mos/MAPK pathway. Aurora-A is phosphorylated in vivo at least on three residues that regulate differentially its kinase activity. Therefore, Aurora-A is under the control of Cdc2 in the Xenopus oocyte and could be involved in meiotic spindle establishment (Maton, 2003).

The success of cell division relies on the activation of its master regulator Cdc2-cyclin B, and many other kinases controlling cellular organization, such as Aurora-A. Most of these kinase activities are regulated by phosphorylation. Despite numerous studies showing that okadaic acid-sensitive phosphatases regulate both Cdc2 and Aurora-A activation, their identity has not yet been established in Xenopus oocytes and the importance of their regulation has not been evaluated. Using an oocyte cell-free system, it has been demonstrated that PP2A depletion is sufficient to lead to Cdc2 activation, whereas Aurora-A activation depends on Cdc2 activity. The activity level of PP1 does not affect Cdc2 kinase activation promoted by PP2A removal. PP1 inhibition is also not sufficient to lead to Aurora-A activation in the absence of active Cdc2. It is therefore conclude that in Xenopus oocytes, PP2A is the key phosphatase that negatively regulates Cdc2 activation. Once this negative regulator is removed, endogenous kinases are able to turn on the activator Cdc2 system without any additional stimulation. In contrast, Aurora-A activation is indirectly controlled by Cdc2 activity independently of either PP2A or PP1. This strongly suggests that in Xenopus oocytes, Aurora-A activation is mainly controlled by the specific stimulation of kinases under the control of Cdc2 and not by downregulation of phosphatase (Maton, 2005).

Segregation of chromosomes during mitosis requires interplay between several classes of protein on the spindle, including protein kinases, protein phosphatases, and microtubule binding motor proteins. Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation. Aurora A localization to the mitotic spindle depends on the motor binding protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2), but the protein(s) involved in Aurora A activation are unknown . An activator of Aurora A has been purified from Xenopus eggs and identified as TPX2. Remarkably, Aurora A that has been fully deactivated by Protein Phosphatase 2A (PP2A) becomes phosphorylated and reactivated by recombinant TPX2 in an ATP-dependent manner. Increased phosphorylation and activation of Aurora A requires its own kinase activity, suggesting that TPX2 stimulates autophosphorylation and autoactivation of the enzyme. Consistently, wild-type Aurora A, but not a kinase inactive mutant, becomes autophosphorylated on the regulatory T loop residue (Thr 295) after TPX2 treatment. Active Aurora A from bacteria is further activated at least 7-fold by recombinant TPX2, and TPX2 also impairs the ability of protein phosphatases to inactivate Aurora A in vitro. This concerted mechanism of stimulation of activation and inhibition of deactivation implies that TPX2 is the likely regulator of Aurora A activity at the mitotic spindle and may explain why loss of TPX2 in model systems perturbs spindle assembly. The finding that a known binding protein, and not a conventional protein kinase, is the relevant activator for Aurora A suggests a biochemical model in which the dynamic localization of TPX2 on mitotic structures directly modulates the activity of Aurora A for spindle assembly (Eyers, 2003)

The activated form of Ran (Ran-GTP; see Drosophila Ran) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. Since the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, it is hypothesized that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics (Tsai, 2003).

The oncogenic protein kinase Aurora A is a critical regulator of meiotic and mitotic cell cycles in eukaryotic cells. Aurora A autoactivation by autophosphorylation is promoted by specific non-catalytic binding proteins. One such protein is TPX2, a required spindle assembly factor in higher eukaryotes whose ability to activate Aurora A by direct binding to the kinase catalytic domain has been established by biochemical and structural analysis. This report clarifies the autoactivation mechanism of Aurora A by demonstrating that of seven amino acids which become autophosphorylated by Aurora A, only Thr-295 is required for activity. Association of Aurora A with TPX2 leads to activation of the kinase, in parallel with phosphorylation of TPX2. The sites are identified as three Ser residues in the N terminus of TPX2; however, mutation of these residues does not affect Aurora A activation by TPX2. In contrast, the mutation of a putative Aurora A-binding motif in TPX2 abolishes both phosphorylation of TPX2 and activation of Aurora A. The interaction between Xenopus p53 and Xenopus Aurora A was investigated. p53 blocks the activity of either full-length Aurora A or the isolated catalytic domain. Interestingly, inhibition is blocked by TPX2, suggesting that the ability of Aurora A to transform cells could be regulated by p53, TPX2, or other binding proteins (Eyers, 2004).

The Aurora A and B protein kinases are key players in mitotic control and the etiology of human cancer. Despite the near identity of amino acid sequence in the catalytic domain, monomeric Aurora B is 50 fold lower in activity than monomeric Aurora A, and previous studies have shown that TPX2 binding to the catalytic domain activates Aurora A but not Aurora B. This study identifies G205 in Xenopus Aurora A as a key determinant of both intrinsic activity and regulation by TPX2. Mutation of G205 in Aurora A to N, the equivalent residue in Aurora B, has no effect on autophosphorylation of the T-loop but leads to a 10-fold loss of specific activity, whereas mutation of N158 in Aurora B to G causes a 350-fold increase in specific activity. G205 N Aurora A is still activated by TPX2, but protection of pT295 from dephosphorylation by protein phosphatase 1 is abolished. Structural analysis of these effects suggests that the G205 forms a pivot point in the enzyme that results in movement of the N-terminal domain glycine-rich loop closer to the ATP binding site of the enzyme and also moves the C-helix slightly closer to the activation loop. Changes in these positions are comparable to those reported for other protein kinases and demonstrate that phosphorylation of the activation loop alone is not sufficient for enzyme activation. The generation of an activated mutant of Aurora B will be important for studying its role in cell cycle control and tumorigenesis (Eyers, 2005).

GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. This study identifies a complex that is required for the conversion of aster-like to spindle-like structures. The complex consists of two characterized MAPs (TPX2, XMAP215), a plus end-directed motor (Eg5), a mitotic kinase (Aurora A), and HURP, a protein associated with hepatocellular carcinoma. Formation and function of the complex is dependent on Aurora A activity. HURP protein was further characterized and shown to bind microtubules and affect their organization both in vitro and in vivo. In egg extract, anti-HURP antibodies disrupt the formation of both Ran-dependent and chromatin and centrosome-induced spindles. HURP is also required for the proper formation and function of mitotic spindles in HeLa cells. It is concluded that HURP is a new and essential component of the mitotic apparatus. HURP acts as part of a multicomponent complex that affects the growth or stability of spindle MTs and is required for spindle MT organization (Koffa, 2006).

A novel mammalian protein kinase is related to two newly identified yeast and fly kinases -- Ipl1 and aurora, respectively -- mutations in which cause disruption of chromosome segregation. This kinase has been designated Ipl1- and aurora-related kinase 1 (IAK1). IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle. Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis. The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase. IAK1 protein has a distinct subcellular and temporal pattern of localization. It is first identified on the centrosomes immediately after the duplicated centrosomes have separated. The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis. In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers. A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast. Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells. It is suggested that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily. The members of this subfamily may be important regulators of chromosome segregation (Gopalan, 1997).

Genetic and biochemical studies in lower eukaryotes have identified several proteins that ensure accurate segregation of chromosomes. These include the Drosophila aurora and yeast Ipl1 kinases that are required for centrosome maturation and chromosome segregation. Two human homologs of these genes, termed aurora1 and aurora2, have been identified; these encode cell-cycle-regulated serine/threonine kinases. The aurora2 gene maps to chromosome 20q13, a region amplified in a variety of human cancers, including a significant number of colorectal malignancies. It is proposed that aurora2 may be a target of this amplicon since its DNA is amplified and its RNA overexpressed, in more than 50% of primary colorectal cancers. Furthermore, overexpression of aurora2 transforms rodent fibroblasts. These observations implicate aurora2 as a potential oncogene in many colon, breast and other solid tumors, and identify centrosome-associated proteins as novel targets for cancer therapy (Bischoff, 1998).

The centrosomes are thought to maintain genomic stability through the establishment of bipolar spindles during cell division, ensuring equal segregation of replicated chromosomes to two daughter cells. Deregulated duplication and distribution of centrosomes have been implicated in chromosome segregation abnormalities, leading to aneuploidy seen in many cancer cell types. STK15 (also known as BTAK and aurora2), encoding a centrosome-associated kinase, is amplified and overexpressed in multiple human tumor cell types, and is involved in the induction of centrosome duplication-distribution abnormalities and aneuploidy in mammalian cells. STK15 amplification has been previously detected in breast tumor cell lines and in colon tumors; it is also amplified in approximately 12% of primary breast tumors, as well as in breast, ovarian, colon, prostate, neuroblastoma and cervical cancer cell lines. Additionally, high expression of STK15 mRNA was detected in tumor cell lines without evidence of gene amplification. Ectopic expression of STK15 in mouse NIH 3T3 cells leads to the appearance of abnormal centrosome number (amplification) and transformation in vitro. Finally, overexpression of STK15 in near diploid human breast epithelial cells has revealed similar centrosome abnormality, as well as induction of aneuploidy. These findings suggest that STK15 is a critical kinase-encoding gene, whose overexpression leads to centrosome amplification, chromosomal instability and transformation in mammalian cells (Zhou, 1998).

Aurora2 is a cell cycle regulated serine/threonine protein kinase that is overexpressed in many tumor cell lines. Aurora2 is regulated by phosphorylation in a cell cycle dependent manner. This phosphorylation occurs on a conserved residue, Threonine 288, within the activation loop of the catalytic domain of the kinase and results in a significant increase in the enzymatic activity. Threonine 288 resides within a consensus motif for the cAMP dependent kinase and can be phosphorylated by PKA in vitro. The protein phosphatase 1 is shown to dephosphorylate this site in vitro, and in vivo the phosphorylation of T288 is induced by okadaic acid treatment. Aurora2 kinase is regulated by proteasome dependent degradation and Aurora2 phosphorylated on T288 may be targeted for degradation during mitosis. These experiments suggest that phosphorylation of T288 is important for regulation of the Aurora2 kinase both for its activity and its stability (Walter, 2000).

Activity-dependent local translation of dendritic mRNAs is one process that underlies synaptic plasticity. Several of the factors known to control polyadenylation-induced translation in early vertebrate development [cytoplasmic polyadenylation element-binding protein (CPEB), maskin, poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF) and Aurora] also reside at synaptic sites of rat hippocampal neurons. The induction of polyadenylation at synapses is mediated by the N-methyl-D-aspartate (NMDA) receptor, which transduces a signal that results in the activation of Aurora kinase. This kinase in turn phosphorylates CPEB, an essential RNA-binding protein, on a critical residue that is necessary for polyadenylation-induced translation. These data demonstrate a remarkable conservation of the regulatory machinery that controls signal-induced mRNA translation, and elucidate an axis connecting the NMDA receptor to localized protein synthesis at synapses (Huang, 2002).

STK15 is an Aurora/Ipl-1 related serine/threonine kinase that is associated with centrosomes and induces aneuploidy when overexpressed in mammalian cells. Mechanisms by which STK15 activity is regulated have not been elucidated. STK15 contains two functional binding sites for protein phosphatase type 1 (PP1), and the binding of these proteins is cell cycle-regulated peaking at mitosis. Activated STK15 at mitosis phosphorylates PP1 and inhibits PP1 activity in vitro. In vivo, PP1 activity co-immunoprecipitated with STK15 is also reduced. These data indicate that STK15 inhibits PP1 activity during mitosis. Also, PP1 is shown to dephosphorylate active STK15 and abolish its activity in vitro. Furthermore, non-binding mutants of STK15 for PP1 are superphosphorylated, but their kinase activities are markedly reduced. Cells transfected with these non-binding mutants manifest aberrant chromosome alignment during mitosis. These results suggest that a feedback regulation through phosphorylation/dephosphorylation events between STK15 kinase and PP1 phosphatase operates through the cell cycle. Deregulation of this balance may contribute to anomalous segregation of chromosomes during mitotic progression of cancer cells (Katayama, 2001).

Human Aurora-A is related to a protein kinase originally identified by its close homology to Ipl1p from Saccharomyces cerevisiae and aurora from Drosophila melanogaster, both of which are key regulators of the structure and function of the mitotic spindle. Human Aurora-A is turned over through the anaphase promoting complex/cyclosome (APC/C)-ubiquitin-proteasome pathway. The association of two distinct WD40 repeat proteins known as Cdc20 and Cdh1, respectively, sequentially activates the APC/C. The present study shows that Aurora-A degradation is dependent on hCdh1 in vivo, not on hCdc20, and that Aurora-A is targeted for proteolysis through distinct structural features of the destruction box, the KEN box motifs and its kinase activity (Taguchi, 2002).

The GTPase-activating protein, RasGAP, functions as both a negative regulator and an effector of Ras proteins. In tumor cells, RasGAP is no longer able to deactivate oncogenic Ras proteins, and its effector function becomes predominant. Since RasGAP itself has no obvious enzymatic function that may explain this effector function, downstream RasGAP effectors were sought that could fulfill this role. The existence of RasGAP SH3 domain partners were sought, since this domain is involved in the regulation of cell proliferation and has an anti-apoptotic effect. Aurora is a RasGAP SH3 domain-binding protein. Drosophila Aurora Ser/Thr kinase has three human orthologs called Aurora/Ipl1-related kinase or HsAIRK-1, -2 and 3. Coimmunoprecipitation experiments in COS cells have confirmed that HsAIRK-1 and HsAIRK-2 both interact with RasGAP. RasGAP pull-down experiments showed its interaction with HsAIRK-1 in G2/M HeLa cells. RasGAP binds to the kinase domain of Aurora, and this interaction inhibits the kinase activity HsAIRK-1 and HsAIRK-2. RasGAP forms a ternary complex with HsAIRK and survivin. This complex may be involved in the regulation of the balance between cell division and apoptosis (Gigoux, 2002).

Human aurora A is a serine-threonine kinase that controls various mitotic events. The transcription of aurora A mRNA varies throughout the cell cycle and peaks during G(2)/M. To clarify the transcriptional mechanism, the 1.8-kb 5'-flanking region of aurora A including the first exon was cloned. Transient expression of aurora A promoter-luciferase constructs containing a series of 5'-truncated sequences or site-directed mutations identified a 7-bp sequence (CTTCCGG) from -85 to -79 as a positive regulatory element. Electromobility shift assays identified the binding of positive regulatory proteins to the CTTCCGG element. Anti-E4TF1-60 antibody generated a supershifted complex. Furthermore, coexpression of E4TF1-60 and E4TF1-53 markedly increases aurora A promoter activity. Synchronized cells transfected with the aurora A promoter-luciferase constructs revealed that the promoter activity of aurora A increase in the S phase and peaked at G(2)/M. In addition, a tandem repressor element, CDE/CHR, was identified just downstream of the CTTCCGG element; mutation within this element led to a loss of cell cycle regulation. It is concluded that the transcription of aurora A is positively regulated by E4TF1, a ubiquitously expressed ETS family protein, and that the CDE/CHR element is essential for the G(2)/M-specific transcription of aurora A (Tanaka, 2002).

The roles of the kinase Aurora A (AurA) in centrosome function and spindle assembly have been established in Drosophila, C. elegans, and Xenopus egg extracts. AurA acts downstream of the RanGTPase signaling pathway to stimulate spindle assembly in mitosis . However, it is still not clear whether AurA can stimulate the formation of microtubule organizing centers (MTOC) on its own. Moreover, whether AurA is essential for spindle assembly in the absence of centrosomes has remained unclear. This study reports the development of functional assays that show that activation of AurA by TPX2 is essential for Ran-stimulated spindle assembly in the presence or absence of centrosomes. Furthermore, AurA-coated magnetic beads function as MTOCs in the presence of RanGTP in Xenopus egg extracts and RanGTP stimulates AurA to recruit activities responsible for both MT nucleation and organization to the beads. The MTOC function of AurA-coated beads require both MT nucleators and motors. Compared to XMAP215-coated beads, AurA-coated beads increase the rate of bipolar spindle assembly in the presence of RanGTP, and the kinase activity of AurA is essential for the beads to function as MTOCs (Tsai, 2005).

Aurora-A is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. This study has investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. Suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G2 cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When anti-Aurora-A antibodies were injected into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells (Marumoto, 2003).

Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffold protein with well defined roles in focal adhesions in integrin-mediated cell adhesion, spreading, migration, and signaling. Using mass spectrometry-based proteomic approaches, centrosomal and mitotic spindle proteins were identified as interactors of ILK. alpha- and beta-tubulin, ch-TOG (XMAP215), and RUVBL1 (Pontin 52) associate with ILK and colocalize with it to mitotic centrosomes. Inhibition of ILK activity or expression induces profound apoptosis-independent defects in the organization of the mitotic spindle and DNA segregation. ILK fails to localize to the centrosomes of abnormal spindles in RUVBL1-depleted cells. Additionally, depletion of ILK expression or inhibition of its activity inhibits Aurora A-TACC3/ch-TOG interactions, which are essential for spindle pole organization and mitosis. These data demonstrate a critical and unexpected function for ILK in the organization of centrosomal protein complexes during mitotic spindle assembly and DNA segregation (Fielding, 2008).

Aurora-A is a serine-threonine kinase implicated in the assembly and maintenance of the mitotic spindle. Human Aurora-A binds to TPX2, a prominent component of the spindle apparatus. TPX2 was identified by mass spectrometry as a major protein coimmunoprecipitating specifically with Aurora-A from mitotic HeLa cell extracts. Conversely, Aurora-A could be detected in TPX2 immunoprecipitates. This indicates that subpopulations of these two proteins undergo complex formation in vivo. Binding studies demonstrated that the NH2 terminus of TPX2 can directly interact with the COOH-terminal catalytic domain of Aurora-A. Although kinase activity is not required for this interaction, TPX2 is readily phosphorylated by Aurora-A. Upon siRNA-mediated elimination of TPX2 from cells, the association of Aurora-A with the spindle microtubules is abolished, although its association with spindle poles is unaffected. Conversely, depletion of Aurora-A by siRNA has no detectable influence on the localization of TPX2. It is proposed that human TPX2 is required for targeting Aurora-A kinase to the spindle apparatus. In turn, Aurora-A might regulate the function of TPX2 during spindle assembly (Kufer, 2002).

Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin beta, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 nuclear targeting information, microtubule-binding and Aurora A binding activities were identified. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevents Aurora A activation and recruitment to microtubules. In addition nuclear targeting information was identified in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly (Trieselmann, 2003).

The related protein kinase Aurora B, whose substrates and subcellular location differ from those of Aurora A, may also be directly regulated by interaction with substrates. The Aurora B substrate Survivin exists in a complex with Aurora B and the chromosomal passenger protein INCENP, and this complex may regulate kinetochore attachment to spindle microtubules. Both Survivin and INCENP have been reported to further stimulate Aurora B activity in vitro, although the mechanism of enhancement was not determined. It will be interesting to ascertain whether other proteins also activate Aurora A by using the assay conditions described in this study, and whether TPX2 is the sole mediator of Aurora A activation in purified fractions. This work significantly advances understanding of the role of TPX2 in regulating spindle assembly because it demonstrates that TPX2 regulates Aurora A activity by directly stimulating autophosphorylation and autoactivation. It is therefore likely that TPX2 not only targets Aurora A to the mitotic spindle, but also directly regulates its activity. This regulation is likely to occur by direct binding of the N terminus of TPX2 to the Aurora A catalytic domain, where the key phosphorylated residue Thr 295 is located. The fact that TPX2 can activate a completely inactive Aurora A implies a mechanism involving autophosphorylation and autoactivation in cis. Once targeted and activated, Aurora A mediates selective phosphorylation of substrates such as the motor protein Eg5 and thereby ensures faithful bipolar spindle formation and ordered mitosis. The identification of these substrates and the regulation of TPX2 and Aurora A binding by phosphorylation represent important avenues for further study (Eyers, 2003).

Aurora-A is an oncogenic kinase essential for mitotic spindle assembly. It is activated by phosphorylation and by the microtubule-associated protein TPX2, which also localizes the kinase to spindle microtubules. The molecular mechanism of Aurora-A activation has been uncovered by determining crystal structures of its phosphorylated form both with and without a 43 residue long domain of TPX2 that was identified as fully functional for kinase activation and protection from dephosphorylation. In the absence of TPX2, the Aurora-A activation segment is in an inactive conformation, with the crucial phosphothreonine exposed and accessible for deactivation. Binding of TPX2 triggers no global conformational changes in the kinase but pulls on the activation segment, swinging the phosphothreonine into a buried position and locking the active conformation. The recognition between Aurora-A and TPX2 resembles that between the cAPK catalytic core and its flanking regions, suggesting this molecular mechanism may be a recurring theme in kinase regulation (Bayliss, 2003).

Aurora-A kinase is necessary for centrosome maturation, for assembly and maintenance of a bipolar spindle, and for proper chromosome segregation during cell division. Aurora-A is an oncogene that is overexpressed in multiple human cancers. Regulation of kinase activity apparently depends on phosphorylation of Thr-288 in the T-loop. In addition, interactions with targeting protein for Xenopus kinesin-like protein 2 (TPX2) allosterically activate Aurora-A. The Thr-288 phosphorylation is reversed by type-1 protein phosphatase (PP1). Mutations in the yeast Aurora, Ipl1, are suppressed by overexpression of Glc8, the yeast homolog of phosphatase inhibitor-2 (I-2). Human I-2 directly and specifically stimulates recombinant human Aurora-A activity in vitro. The I-2 increase in kinase activity is not simply due to inhibition of PP1 because it was not mimicked by other phosphatase inhibitors. Furthermore, activation of Aurora-A is unaffected by deletion of the I-2 N-terminal PP1 binding motif but is eliminated by deletion of the I-2 C-terminal domain. Aurora-A and I-2 were recovered together from mitotic HeLa cells. Kinase activation by I-2 and TPX2 is not additive and occurs without a corresponding increase in T-loop phosphorylation. These results suggest that both I-2 and TPX2 function as allosteric activators of Aurora-A. This implies that I-2 is a bifunctional signaling protein with separate domains to inhibit PP1 and directly stimulate Aurora-A kinase (Satinover, 2004).

Aurora-A and Plk1 are centrosomal kinases involved in centrosome maturation and spindle assembly. The microtubule-binding protein TPX2 interacts with, and activates, Aurora-A. RNA interference-mediated inactivation was used to investigate whether Aurora-A, Plk1 and TPX2 act independently or are part of one signaling cascade in spindle formation in mammalian cells. Both specific, and over- lapping, roles of each single regulator has been identified in centrosome maturation and spindle formation: (1) Aurora-A and TPX2 are required for centriole cohesion and spindle bipolarity; (2) TPX2, besides its known role in microtubule organization, is also involved in centrosome maturation; (3) finally, Plk1 controls the localization of Aurora-A to centrosomes, as well as TPX2 recruitment to microtubules. Based on these results therefore a hierarchical functional relation between Plk1 and the Aurora-A/TPX2 pathway emerges (De Luca, 2005).

The Aurora (Ipl1)-related kinases are universal regulators of mitosis. Aurora-A, in addition to Aurora-B, regulates kinetochore function in human cells. A two-hybrid screen identified the kinetochore component CENP-A as a protein that interacts with Aurora-A. Aurora-A phosphorylates CENP-A in vitro on Ser-7, a residue also known to be targeted by Aurora-B. Depletion of Aurora-A or Aurora-B by RNA interference reveals that CENP-A (Drosophila homolog: Centromere identifer) is initially phosphorylated in prophase in a manner dependent on Aurora-A, and that this reaction appears to be required for the subsequent Aurora-B-dependent phosphorylation of CENP-A as well as for the restriction of Aurora-B to the inner centromere in prometaphase. Prevention of CENP-A phosphorylation also led to chromosome misalignment during mitosis as a result of a defect in kinetochore attachment to microtubules. These observations suggest that phosphorylation of CENP-A on Ser-7 by Aurora-A in prophase is essential for kinetochore function (Kunitoku, 2003).

The phosphorylation of CENP-A is involved in efficient occupancy of kinetochores with spindle fibers. Concurrent with CENP-A phosphorylation at early prophase, various proteins assemble at the outer domain of the kinetochore. Given that CENP-A is essential for this assembly process in several species, the phosphorylation of CENP-A on Ser-7 might be required to initiate it during prophase, before the kinetochores begin to attach to microtubules. Such protein recruitment triggered by CENP-A phosphorylation might be important for the establishment of kinetochore-microtubule connections. However, this modification does not appear to be necessary for generation of the spindle assembly checkpoint signal, because Mad2, BubR1, and CENP-E localizes normally to kinetochores in prometaphase cells expressing CENP-A(S7A) and these cells show a marked delay in prometaphase (Kunitoku, 2003).

Given that Aurora-B plays an important role in correcting kinetochore-microtubule attachment in mammalian cells, the mislocalization of Aurora-B might contribute to the defect in chromosome alignment in cells expressing CENP-A(S7A) or in those deficient in Aurora-A. However, because Aurora-A-mediated phosphorylation of CENP-A on Ser-7 during prophase appears to be important for microtubule attachment, it was not possible to assess the possible contribution of the attachment-correcting function of Aurora-B. The many misaligned chromosomes that were found in cells in which CENP-A phosphorylation was prevented possessed either unattached or monotelic kinetochores, whereas those in Aurora-B-depleted cells exhibited syntelic attachment. Further molecular dissection of the regulation of kinetochore function by Aurora kinases could be facilitated by identification of the proteins that are recruited to the kinetochore in a manner dependent on CENP-A phosphorylation on Ser-7 (Kunitoku, 2003).

Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, Aurora-A has been shown to be required for mitotic entry. Initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, these data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment (Hirota, 2003).

Although the focal adhesion scaffolding protein HEF1 has a well-defined role in integrin-dependent attachment signalling at focal adhesions, it relocalizes to the spindle asters at mitosis. Overexpression of HEF1 causes an increase in centrosome numbers and multipolar spindles, resembling defects induced by manipulation of the mitotic regulatory kinase Aurora-A (AurA). HEF1 associates with and controls activation of AurA, and HEF1 depletion causes centrosomal splitting, mono-astral spindles and hyperactivation of Nek2, implying additional action earlier in the cell cycle. These results provide new insight into the role of an adhesion protein in coordination of cell attachment and division (Pugacheva, 2005).

Cyclins B1 and B2 are frequently elevated in human cancers and are associated with tumour aggressiveness and poor clinical outcome; however, whether and how B-type cyclins drive tumorigenesis is unknown. This study shows that cyclin B1 and B2 transgenic mice are highly prone to tumours, including tumour types where B-type cyclins serve as prognosticators. Cyclins B1 and B2 both induce aneuploidy when overexpressed but through distinct mechanisms, with cyclin B1 inhibiting separase activation, leading to anaphase bridges, and cyclin B2 triggering aurora-A-mediated Plk1 hyperactivation, resulting in accelerated centrosome separation and lagging chromosomes. Complementary experiments revealed that cyclin B2 and p53 act antagonistically to control aurora-A-mediated centrosome splitting and accurate chromosome segregation in normal cells. These data demonstrate a causative link between B-type cyclin overexpression and tumour pathophysiology, and uncover previously unknown functions of cyclin B2 and p53 in centrosome separation that may be perturbed in many human cancers (Nam, 2014).

Polo-like kinase-1 (PLK1) is an essential mitotic kinase regulating multiple aspects of the cell division process1. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 kinase domain, but the kinase responsible for this has not yet been affirmatively identified. This study shows that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. Aurora A can directly phosphorylate PLK1 on Thr 210, and activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A. Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A (Macurek, 2008).

Control of mitotic cell cycles by the anaphase-promoting complex or cyclosome (APC/C) ubiquitin ligase depends on its coactivators Cdc20 and Cdh1. APC/C(Cdc20) is active during mitosis and promotes anaphase onset by targeting mitotic cyclins and securin. APC/C(Cdh1) becomes active during mitotic exit and has essential targets in G1 phase. It is not known whether targeting of substrates by APC/C(Cdh1) plays any role in the final stages of mitosis. This study has investigated the role of APC/C(Cdh1) at this time in the cell cycle by using siRNA-mediated depletion of Cdh1 in human cells. In contrast to the current view that Cdh1 takes over from Cdc20 at anaphase, it was shown that reduced Cdh1 levels have no effect on destruction of many APC/C substrates during mitotic exit but strongly and specifically stabilize Aurora kinases. APC/C(Cdh1) is required for assembly of a robust spindle midzone at anaphase and for normal timings of spindle elongation and cytokinesis. The effect of Cdh1 siRNA on anaphase spindle dynamics requires Aurora A, and its effect can be mimicked by nondegradable Aurora kinase. It is concluded that targeting of Aurora kinases at anaphase by APC/C(Cdh1) participates in the control of mitotic exit and cytokinesis (Floyd, 2008).

Aurora-A, a mitotic serine/threonine kinase with oncogene characteristics, has recently drawn intense attention because of its association with the development of human cancers and its relationship with mitotic progression. Using the gene expression profiles of Aurora-A as a template to search for and compare transcriptome expression profiles in publicly accessible microarray data sets, HURP (encodes hepatoma upregulated protein) was identified as one of the best Aurora-A-correlated genes. Empirical validation indicates that HURP has several characteristics in common with Aurora-A. These two genes have similar expression patterns in hepatocellular carcinoma, liver regeneration after partial hepatectomy, and cell cycle progression and across a variety of tissues and cell lines. Moreover, Aurora-A phosphorylates HURP in vitro and in vivo. Ectopic expression of either the catalytically inactive form of Aurora-A or the HURP-4P mutant, in which the Aurora-A phosphorylation sites were replaced with Ala, results in HURP instability and complex disassembly. In addition, HURP-wild-type stable transfectants are capable of growing in low-serum environments whereas HURP-4P grows poorly under low-serum conditions and fails to proliferate. These studies together support the view that the ability to integrate evidence derived from microarray studies into biochemical analyses may ultimately augment predictive power when analyzing the potential role of poorly characterized proteins. While this combined approach was simply an initial attempt to answer a range of complex biological questions, these findings do suggest that HURP is a potential oncogenic target of Aurora-A (Yu, 2005).

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. This paper reports a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. It is concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function (Wong, 2008).

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date revised: 2 January 2023

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