JCBArticle Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules Régis Giet,1 Doris McLean,2 Simon Descamps,3 Michael J. Lee,4 Jordan W. Raff,4 Claude Prigent,3 and David M. Glover1 Downloaded from http://rupress.org/jcb/article-pdf/156/3/437/904547/jcb1563437.pdf by California Institute Of Technology user on 31 July 2020 1Department of Genetics, Cancer Research Campaign Cell Cycle Genetics Group, University of Cambridge, Cambridge CB2 3EH, UK 2Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK 3UMR 6061 Génétique et Développement, Centre National de la Recherche Scientifique, Faculté de Médecine, Université de Rennes 1, 35043 Rennes cedex, France 4Wellcome/CRC Institute and Department of Genetics, Cambridge CB2 1QR, UK isruption of the function of the A-type Aurora kinase antigens but are missing the Drosophila counterpart of of Drosophila by mutation or RNAi leads to a reduc- mammalian transforming acidic coiled coil (TACC) proteins, Dtion in the length of astral microtubules in syncytial D-TACC. We show that a subpopulation of the total Aurora embryos, larval neuroblasts, and cultured S2 cells. In neuro- A is present in a complex with D-TACC, which is a substrate blasts, it can also lead to loss of an organized centrosome for the kinase. We propose that one of the functions of and its associated aster from one of the spindle poles, Aurora A kinase is to direct centrosomal organization such whereas the centrosome at the other pole has multiple that D-TACC complexed to the MSPS/XMAP215 microtubule- centrioles. When centrosomes are present at the poles of associated protein may be recruited, and thus modulate the aurA mutants or aurA RNAi spindles, they retain many behavior of astral microtubules. Introduction Correct regulation of the organization and dynamics of XMAP215 promotes the elongation rate of microtubules at microtubules is an essential aspect of entry into M-phase. their plus ends (less so at the minus ends), and appears to Microtubules are nucleated by the ␥-tubulin ring complex counteract the catastrophe-promoting activity (the transition (Pereira and Schiebel, 1997), the amount of which increases from polymerization to a depolymerization) promoted by markedly at the centrosome upon entry into mitosis. Micro- XKCM1 (Vasquez et al., 1994; Tournebize et al., 2000). tubule nucleation at the centrosome requires the cooperation The counterpart of XMAP215 in Drosophila is encoded by of other microtubule-associated proteins (MAPs),* notably the gene minispindles (MSPS), mutations that appear to the abnormal spindle protein (Asp) in Drosophila (do Carmo destabilize spindle microtubules which become small and Avides and Glover, 1999). MAPs also play a central role associated with single chromosomes (Cullen et al., 1999). in regulating microtubule dynamics (Hyman and Kar- The MSPS protein was recently shown to form a complex senti, 1996; Shirasu et al., 1999). For example, Xenopus with the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, the centrosomally asso- ciated protein D-TACC (Gergely et al., 2000b; Cullen and Address correspondence to David M. Glover, Dept. of Genetics, Uni- Ohkura, 2001; Lee et al., 2001). Injections of antibodies versity of Cambridge, Downing St., Cambridge CB2 3EH, United Kingdom. Tel.: 44-1223-333-988. Fax: 44-1223-333-968. E-mail: against D-TACC or mutations in the d-tacc gene result in [email protected] centrosomal microtubules that are abnormally short, as well *Abbreviations used in this paper: Asp, abnormal spindle protein; MAP, as in the accumulation of mitotic defects. The D-TACC microtubule-associated protein; MSPS, minispindles; TACC, transform- protein is found at the spindle poles and its recruitment of ing acidic coiled coil. MSPS protein has been postulated to stabilize centrosomal Key words: Aurora A; D-TACC; mitosis; centrosomes; microtubule microtubules (Lee et al., 2001). In the acentriolar spindles of The Rockefeller University Press, 0021-9525/2002/2/437/15 $5.00 The Journal of Cell Biology, Volume 156, Number 3, February 4, 2002 437–451 http://www.jcb.org/cgi/doi/10.1083/jcb.200108135 437 438 The Journal of Cell Biology | Volume 156, Number 3, 2002 female meiosis, both the motor protein Ncd and D-TACC ing chromosomes and centromeres, and subsequently to the are required for the proper localization of MSPS (Cullen and central spindle and the midbody in anaphase and telophase, Ohkura, 2001). respectively. The Aurora B protein kinase is the functional A number of protein kinases are known to regulate the be- subunit of a complex containing INCENP (Adams et al., havior of microtubules upon mitotic entry by phosphorylat- 2000; Kaitna et al., 2000), and the BIR-1/survivin protein is ing MAPs. For example, MAP4, which induces the forma- required for its localization (Speliotes et al., 2000); it is re- tion of long and stable microtubules by increasing the rescue quired in chromosome segregation and it phosphorylates frequency (the transition from depolymerization to poly- histone H3, which correlates with recruitment of the con- merization) has to be inactivated in mitosis. This is achieved densin complex (Adams et al., 2001; Giet and Glover, by cdk1–cyclin B, thus promoting the increase in the micro- 2001). In contrast, less is known of the exact function of the tubule dynamics when the cell enters mitosis (Ookata et al., Aurora A–type kinases, although ectopic expression of the 1995). Cdk1 also activates the microtubule binding proper- human enzyme leads to aneuploidy, centrosome amplifica- ties of the kinesin-related protein Eg5 and dynactin to pro- tion, and transformation (Bischoff et al., 1998; Zhou et al., Downloaded from http://rupress.org/jcb/article-pdf/156/3/437/904547/jcb1563437.pdf by California Institute Of Technology user on 31 July 2020 mote their interaction with spindle microtubules (Blangy et 1998). Mutations in the aurora A gene of Drosophila mela- al., 1995; Sawin and Mitchison, 1995). The mitotic Polo- nogaster lead to formation of spindles with abnormally orga- like kinases are also required to recruit ␥-tubulin to the cen- nized poles, including characteristic monopolar structures trosome and thus increase its nucleating activity (Lane and (Glover et al., 1995). Bipolar spindles having abnormally or- Nigg, 1996; Donaldson et al., 2001). Polo kinase also phos- ganized centrosomes, and microtubules were observed af- phorylates the Asp protein and so activates its role to in- ter double-stranded RNA mediated interference directed crease the nucleating activity of centrosomes (do Carmo against the air1 gene of Caenorhabditis elegans (Schumacher Avides et al., 2001). Recent studies suggest that a Polo-like et al., 1998). A catalytically inactive or truncated version of kinase also seems to phosphorylate and inactivate stathmin/ the pEg2 Aurora A–like kinase promotes the collapse of Op18, a microtubule-destabilizing protein, around chroma- spindles assembled in Xenopus egg extracts (Roghi et al., tin to promote local microtubule stabilization (Belmont et 1998). This would be consistent with the known ability of al., 1996; Andersen et al., 1997; Budde et al., 2001) Aurora A kinase to phosphorylate the kinesin-like protein Another major family of mitotic kinases is the Aurora- XlEg5 that is also required for spindle assembly and stability related enzymes (Giet and Prigent, 1999). Most is known (Sawin et al., 1992; Giet et al., 1999). To gain a better un- about the B-type subfamily, which first localizes to condens- derstanding of the role of Aurora A kinase, we have exam- Figure 1. Aurora A localizes to the centrosomes and the spindle poles inde- pendently of microtubules. (A) An anti–Aurora A anti-serum recognizes the NH2-terminal recombinant histidine- tagged protein domain used for immuni- zation (left) and the 47-kD endogenous Aurora A protein kinase in Drosophila embryo extracts (right) by Western blotting. Similar results were obtained using affinity purified antibodies. (B–E) The Aurora A antibody (green) decorates centrosomes and spindle poles in S2 cultured cells in prophase (B), metaphase (C), anaphase (D), and cytokinesis (E). Microtubules are in red, DNA in blue. Right hand panels show Aurora A staining alone. Note the decrease in the Aurora A staining during cytokinesis (E, right, arrow). Bar is 10 ␮m. Colchicine (F) and taxol (G) treatments have no effects on the Aurora A centrosome localization. Note that aurora remains on the asters containing the centrosome (G, inset). Microtubules are red, aurora is green and DNA is blue and the scale bar represents 10 ␮m. Inset, ␣-tubulin (red); bar, 5 ␮m. Centrosome recruitment of D-TACC requires Aurora A | Giet et al. 439 ined the requirement for the enzyme in organizing spindle a 5–6-fold elevated mitotic index with the majority of poles in different mutant alleles. We now show that in mu- cells delayed in a metaphase-like state (Fig. 3; Table I). In tant cells or after RNA interference to eliminate the enzyme, most of these cells, metaphase chromosomes were ar- the spindle poles have abnormal organization and abnor- ranged in a configuration consistent with their association mally short arrays of astral microtubules. The latter defect with bipolar spindles (Fig. 3, C and D), but a substantial correlates with the loss of D-TACC from centrosomes. We proportion were in circular arrays (Fig. 3, E and F) previ- show that D-TACC interacts with a subpopulation of Au- ously shown to correspond to monopolar spindles (Glover rora A in vivo, and that D-TACC is a substrate of the ki- et al., 1995). nase. We propose a model in which one of the centrosomal We have now determined that the weak hypomorphic al- functions of Aurora A kinase is to control microtubule dy- lele, aurA287, encodes a protein with the amino acid substi- namics at the spindle poles by regulating the recruitment of tutions H3Y, D47A, T90P, and R162C.