Giet R , McLean D , Descamps S , Lee MJ , Raff JW , Prigent C , Glover DM .

Wellcome Trust

	Figure 1. Aurora A localizes to the centrosomes and the spindle poles independently of microtubules. (A) An anti–Aurora A anti-serum recognizes the NH2-terminal recombinant histidine-tagged protein domain used for immunization (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 μm. 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 μm. Inset, α-tubulin (red); bar, 5 μm.
	Figure 3. Mutation in aurora A leads to metaphase arrest in neuroblasts and decreased length of astral microtubules in both neuroblasts and embryos. Squashed preparations of larval brains from wild-type (A and B) or aurAe209/Df(3R)T61 (C–G) were stained with aceto-orcein and the mitotic figures were scored. A and B wild-type metaphase and anaphase figures, respectively. (C and D) aurAe209Df(3R)T61 metaphase-like figures spindles and E and F circular mitotic figures. Note the high chromosome condensation in panels C–F. (G) An abnormal anaphase figure with lagging chromosomes. (H–S) aurA mutants have short astral microtubules in mitosis. (H and I) Spindles from wild- type and aurAe209/Df(3R)T61 mutant neuroblasts, respectively. Arrow point to a small aster at the mutant spindle poles. Bar, 10 μm. Wild-type embryos (J, prophase; L, early anaphase; N, telophase) and aurA287-derived embryos (K, prophase; M, early anaphase; O, telophase) that have been fixed and stained for tubulin (green) or DNA (blue). Note the decreased length of the astral microtubules in the mutant embryos (arrows). (P–S) Microtubule behavior monitored in live wild type (P, early anaphase; R, telophase) and aurA287-derived embryos (Q, early anaphase; S, telophase) expressing a tau–GFP transgene under the control of the polyubiquitin promoter. Mutant embryos show a reduced number of apparently shorter astral microtubules (arrows). Bar, 5 μm.
	Figure 2. aurA287 encodes an enzyme of reduced activity that localizes to the centrosome. aurA e209 and aurA287 encode full-length Aurora A kinases with the indicated amino acid changes (underlined). The amino acid changes in aurAe209 were previously reported (Glover et al., 1995). Such a mutation was described to be essential for kinase activity (Hunter and Sefton, 1981). (B) The aurA287 mutant shows reduced protein kinase activity. Extracts of wild-type or aurA287-derived embryos (lanes 2 and 3, respectively) were submitted to immunoprecipitation using anti–Aurora A antibodies. One aliquot of immunoprecipitate was subjected to Western blotting (top panels) and a second (bottom panels) assayed for its ability to phosphorylate myelin basic protein (Materials and methods). Quantification using a phosphoimager identified the mutant Aurora A287 kinase had 35% of the activity of wild-type. (C) Mutations in aurA do not prevent its centrosome localization. Wild-type (top left) or auroraAe209 (top right) neuroblasts showing localization of Aurora A (red) to centrosomes of a bipolar and monopolar spindle respectively. Embryos derived from wild-type (bottom left) or homozygous aurora A287 mothers (bottom right) showing Aurora A (red) at centrosomes. The arrow shows a centrosome pair that has dissociated from the nucleus. DNA is stained blue and tubulin, green. Bars, 5 μM.
	Figure 4. aurAe209/Df(3R)T61 neuroblasts show abnormal mitotic spindle poles. Localization of γ-tubulin (A–C), centrosomin (D–F) and CP190 (G–I) in wild-type Canton S (A, D, and G) or aurAe209/Df(3R)T61 metaphase spindles (B, C, E, F, H, and I). Note the absence of centrosomal antigens at the poles indicated by arrowheads (C and I) and the presence of additional bodies of staining (B, E, H, arrows). Cnn, γ-tubulin and CP190 are shown in red, microtubules in green and DNA in blue. Bar is 10 μm. (J and K) ultrastructure of an aurAe209/Df(3R)T61 spindle pole by electron microscopy (one section). Chromosomes are indicated Ch. Note the presence of 5 centrioles on the spindle pole, boxed and shown enlarged in K. Bars: (J) 5 μm; (K) 1 μm.
	Figure 5. D-TACC and MSPS proteins are mislocalized in aurA mutant cells. D-TACC (A–F) and MSPS (G–L) localization in wild-type (A, prophase; C and I, telophase; G, metaphase) or AurA287 mutant embryos (B, prophase; H, metaphase; D and J, telophase). Staining was also performed in wild-type (E and K) or aurAe209/Df neuroblasts (F and L). In all color panels, D-TACC and MSPS are red, microtubules are green and DNA is blue. Lower panels show D-TACC or MSPS staining alone in monochrome. Note that D-TACC and MSPS disappears from the poles of aurA mutant cells (arrows) but remains associated with spindle microtubules. In aurAe209 neuroblasts, both D-TACC and MSPS tends to generate aggregates in the cytoplasm or that stick on the mitotic spindles. Bars, 5 μm.
	Figure 6. S2 cells display spindle pole abnormalities following aurA RNAi. Control (A, D, and G) and aurA RNAi treated (B, C, E, F, H, and I) S2 cells immunostained to reveal either Aurora A (A-C), gamma-tubulin (D–F), or D-TACC (G–I) in red. DNA is stained blue and microtubules, green. Monochrome images show the red channel alone and the scale bar represents 10 μm. Arrows in panel B indicate reduced levels of Aurora A staining and reduced length and density of astral microtubules following aurA RNAi. Arrows in panels E and F indicate multiple gamma-tubulin containing bodies at the poles and reduced length and density of astral microtubules in aurA RNAi cells. J, aurora A and γ-tubulin Western blot of S2 cells 3 d after aurA RNAi treatment. Aurora A levels were reduced by >95%, whereas Aurora B, or D-TACC protein levels were unchanged (unpublished data).
	Figure 7. Aurora A is found on centrosomes in d-tacc mutants. Embryos derived from wild-type (A) d-tacc1, (B) or d-taccstella, (C) mothers stained to reveal microtubules (green), DNA (red) and aurora A (blue). Bar, 10 μm.
	Figure 8. Aurora A interacts with and phosphorylates D-TACC. (A) Drosophila Aurora A (lane 2) and GFP–D-TACC coimmunoprecipitate (lane 4) and human (Hs) Aurora A precipitates with Hs TACC3 (lane 6). Lanes 1 and 2 show control preimmune and anti–Aurora A immunoprecipitates blotted to reveal D-TACC (top) or Aurora A (bottom). Lanes 3 and 4 show anti-GFP immunoprecipitates from transgenic lines expressing Tau–GFP and D-TACC–GFP fusion proteins, respectively and blotted with anti–D-TACC (top) or anti–Aurora A (bottom). Lanes 5 and 6 show control preimmune and anti-human Aurora A immunoprecipitates blotted to reveal human TACC3 (top) or human Aurora A (bottom). (B) Hs aurora (green) colocalizes with Hs TACC3 (red) to the centrosomes and the spindle poles. DNA is blue. (C) Extracts of a 2-h collection of Drosophila embryos were fractionated on a 5–40% sucrose gradient. Aliquots from each fraction were subjected to Western blotting with anti-TACC antibodies (top), anti-minispindle (second panel from the top), anti–γ-tubulin (second panel from the bottom) and anti–Aurora A (bottom). Molecular mass marker (4.3, 7.4, and 11 S are indicated). (D) GFP–D-TACC (lanes 2, 3, 5, 6, 8, and 9) or GFP-Tau (lanes 1, 4, and 7) were immunoprecipitated from Drosophila embryos, heat treated to inactivate potential endogenous kinases, and incubated with recombinant aurA-(His)6 kinase (lanes labeled +) and [γ-32P] ATP. Enzyme was not added to the reaction mixtures analyzed on lanes marked −. Left and middle panels show the radioactive products of the same kinase assay analyzed by 10 and 6% SDS-PAGE, respectively. The right panel shows an equivalent aliquot of material from a 6% gel analyzed by Western blotting with an anti-GFP antibody. Note the correspondence between the phosphorylated bands in the middle pannel and the presence of the GFP-D-TACC protein and its degradation products in the right panel. (E) Bacterially expressed chimeras of Maltose Binding Protein fused to the NH2-terminal (MBP–Nt amino acids 2–433), middle (MBP-Mid, amino acids 433–889), or COOH-terminal domain (MBP–Ct, amino acids 853–1189) of D-TACC were incubated in the presence (+, lanes 2, 4, and 6) or absence (−, lanes 1, 3, and 5) of purified aurora A-(His) 6 kinase and [γ-32P] ATP. Note the strong phosphorylation of the middle fragment of D-TACC (lane 4) and the very weak phosphorylation of the MBP–Nt and MBP–Ct fusion proteins (lanes 2 and 6).

Adams, INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. 2000, Pubmed, Xenbase

Adams, INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. 2000, Pubmed , Xenbase
Adams, Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. 2001, Pubmed
Andersen, Mitotic chromatin regulates phosphorylation of Stathmin/Op18. 1997, Pubmed , Xenbase
Belmont, Catastrophic revelations about Op18/stathmin. 1996, Pubmed
Bischoff, A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers. 1998, Pubmed
Blangy, Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. 1995, Pubmed , Xenbase
Budde, Regulation of Op18 during spindle assembly in Xenopus egg extracts. 2001, Pubmed , Xenbase
Cullen, Msps protein is localized to acentrosomal poles to ensure bipolarity of Drosophila meiotic spindles. 2001, Pubmed
Cullen, mini spindles: A gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila. 1999, Pubmed , Xenbase
do Carmo Avides, Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes. 2001, Pubmed
do Carmo Avides, Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal microtubule organizing centers. 1999, Pubmed
Donaldson, Metaphase arrest with centromere separation in polo mutants of Drosophila. 2001, Pubmed
Endow, Spindle dynamics during meiosis in Drosophila oocytes. 1997, Pubmed
Gausz, Genetic characterization of the region between 86F1,2 and 87B15 on chromosome 3 of Drosophila melanogaster. 1981, Pubmed
Gergely, D-TACC: a novel centrosomal protein required for normal spindle function in the early Drosophila embryo. 2000, Pubmed
Gergely, The TACC domain identifies a family of centrosomal proteins that can interact with microtubules. 2000, Pubmed
Giet, The Xenopus laevis aurora-related protein kinase pEg2 associates with and phosphorylates the kinesin-related protein XlEg5. 1999, Pubmed , Xenbase
Giet, Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. 2001, Pubmed
Giet, Aurora/Ipl1p-related kinases, a new oncogenic family of mitotic serine-threonine kinases. 1999, Pubmed
Giet, The Xenopus laevis aurora/Ip11p-related kinase pEg2 participates in the stability of the bipolar mitotic spindle. 2000, Pubmed , Xenbase
Glover, Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. 1995, Pubmed
Gopalan, A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators. 1997, Pubmed
Heald, Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. 1996, Pubmed , Xenbase
Heuer, The Drosophila homeotic target gene centrosomin (cnn) encodes a novel centrosomal protein with leucine zippers and maps to a genomic region required for midgut morphogenesis. 1995, Pubmed
Hyman, Morphogenetic properties of microtubules and mitotic spindle assembly. 1996, Pubmed
Kaitna, Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis. 2000, Pubmed
Lane, Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. 1996, Pubmed
Lee, Msps/XMAP215 interacts with the centrosomal protein D-TACC to regulate microtubule behaviour. 2001, Pubmed , Xenbase
Megraw, Zygotic development without functional mitotic centrosomes. 2001, Pubmed
Moritz, Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. 1998, Pubmed , Xenbase
Moritz, Microtubule nucleation by gamma-tubulin-containing rings in the centrosome. 1995, Pubmed
NULL, Protein phosphorylation. Part A. Protein kinases: assays, purification, antibodies, functional analysis, cloning, and expression. 1991, Pubmed
Ookata, Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics. 1995, Pubmed
Pereira, Centrosome-microtubule nucleation. 1997, Pubmed , Xenbase
Popov, XMAP215 regulates microtubule dynamics through two distinct domains. 2001, Pubmed , Xenbase
Raff, Centrosomes, and not nuclei, initiate pole cell formation in Drosophila embryos. 1989, Pubmed
Rebollo, Visualizing the spindle checkpoint in Drosophila spermatocytes. 2000, Pubmed
Roghi, The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly. 1998, Pubmed , Xenbase
Savoian, The rate of poleward chromosome motion is attenuated in Drosophila zw10 and rod mutants. 2000, Pubmed
Sawin, Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle. 1995, Pubmed , Xenbase
Sawin, Mitotic spindle organization by a plus-end-directed microtubule motor. 1992, Pubmed , Xenbase
Schumacher, A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos. 1998, Pubmed
Shirasu, Microtubule dynamics in Xenopus egg extracts. 1999, Pubmed , Xenbase
Speliotes, The survivin-like C. elegans BIR-1 protein acts with the Aurora-like kinase AIR-2 to affect chromosomes and the spindle midzone. 2000, Pubmed
Tournebize, Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts. 2000, Pubmed , Xenbase
Vasquez, XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover. 1994, Pubmed , Xenbase
Walter, The mitotic serine/threonine kinase Aurora2/AIK is regulated by phosphorylation and degradation. 2000, Pubmed
Whitfield, The 190 kDa centrosome-associated protein of Drosophila melanogaster contains four zinc finger motifs and binds to specific sites on polytene chromosomes. 1995, Pubmed
Zhou, Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. 1998, Pubmed