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 aurAe209 allele is a strong hypomorph; the aurAe209 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 aurAe287 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. aurAe209 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 aurAe209 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 aurAe209 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 aurAe209 appears to exhibit extreme loss of function, it remains possible that the increase in circular mitotic figures seen in homozygous rather than hemizygous aurAe209 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 aurAe209 larvae (heterozygous for the recessive aurAe209 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 aurAe209 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 aurAe209 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 aurAe209/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 aurAe209/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 aurAe209 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 aurAe209 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 aurAe209 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-taccstella-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).
During asymmetric cell division in the Drosophila nervous system, Numb segregates into one of two daughter cells where it is required for the establishment of the correct cell fate. Numb is uniformly cortical in interphase, but in late prophase, the protein concentrates in the cortical area overlying one of two centrosomes in an actin/myosin-dependent manner. What triggers the asymmetric localization of Numb at the onset of mitosis is unclear. The mitotic kinase Aurora-A is required for the asymmetric localization of Numb. In Drosophila sensory organ precursor (SOP) cells mutant for the aurora-A allele aurA37, Numb is uniformly localized around the cell cortex during mitosis and segregates into both daughter cells, leading to cell fate transformations in the SOP lineage. aurA37 mutant cells also fail to recruit Centrosomin (Cnn) and gamma-Tubulin to centrosomes during mitosis, leading to spindle morphology defects. However, Numb still localizes asymmetrically in cnn mutants or after disruption of microtubules, indicating that there are two independent functions for Aurora-A in centrosome maturation and asymmetric protein localization during mitosis. Using photobleaching of a GFP-Aurora fusion protein, it has been shown that two rapidly exchanging pools of Aurora-A are present in the cytoplasm and at the centrosome and might carry out these two functions. These results suggest that activation of the Aurora-A kinase at the onset of mitosis is required for the actin-dependent asymmetric localization of Numb. Aurora-A is also involved in centrosome maturation and spindle assembly, indicating that it regulates both actin- and microtubule-dependent processes in mitotic cells (Berdnik, 2002).
In a screen for mutations that affect asymmetric localization of Numb, aurA37, a mutant in aurora-A in which bipolar mitotic spindles are formed and chromosomes segregate, but Numb fails to localize asymmetrically, was identified. aurA37 mutants also have defects in centrosome maturation and fail to recruit the proteins Cnn and gamma-Tubulin to centrosomes during mitosis. However, Numb still localizes asymmetrically in cnn mutants that were reported to lack functional mitotic centrosomes, suggesting that the centrosome maturation defects are not responsible for the failure to localize Numb asymmetrically. Aurora-A is concentrated at centrosomes, but photobleaching experiments reveal rapid exchange with a cytoplasmic pool of the protein. These results suggest that two rapidly interchanging pools of Aurora-A kinase are involved in spindle assembly and asymmetric protein localization during mitosis (Berdnik, 2002).
To identify mutations that cause defects in Numb localization, it was predicted that such mutations would lead to cell fate transformations in the SOP lineage and cause morphological abnormalities in fly bristles. In a genetic screen, the eyeless-Flp/FRT system was used to generate heterozygous flies that become homozygous by mitotic recombination in all tissues that are derived from the eye imaginal disk. These include most of the head cuticle, and therefore mutant phenotypes can be analyzed in bristles on the head. One of the mutations identified (termed aurA37) causes the frequent formation of morphologically abnormal bristles that contain two hairs and two sockets, indicating that inner cells are transformed into additional outer cells. The mutation was mapped to the cytological interval 87A7-9 based on lethality over Df(3R)P-58. Df(3R)P-58 includes the Drosophila aurora-A gene, and, indeed, aurA37 is lethal over known alleles of aurora-A. Flies homozygous for aurA37, transheterozygous for aurA37 and the strong allele aur87Ac-3 (Glover, 1995), or transheterozygous for aurA37 and the deficiency Df(3R)M-Kx1 (which includes aurora-A) die during pupal stages with bristle abnormalities similar to the ones observed in aurA37 mutant clones. aurA37 has no dominant phenotype, indicating that the protein has not acquired a novel activity. aurA37 mutants carry a single G-to-A nucleotide exchange that is predicted to exchange a conserved arginine (amino acid 201) into histidine. The affected residue is located in the catalytic domain and is conserved between all kinases of the Aurora-family but not in all other protein kinases. It is concluded that aurA37 is a new allele of Drosophila aurora-A (Berdnik, 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 Notchts-1;aurA, the possibility that additional mechanisms may be involved cannot be excluded (Wang, 2006).
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date revised: 10 February 2008
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