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Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization

2000; Springer Nature; Volume: 19; Issue: 14 Linguagem: Inglês

10.1093/emboj/19.14.3668

1460-2075

Catarina Lemos, Paula Sampaio, Hélder Maiato, Madalena Costa, L. V. Omelyanchuk, Vasco Liberal, Cláudio E. Sunkel,

Cellular transport and secretion

Article17 July 2000free access Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization Catarina L. Lemos Catarina L. Lemos Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Paula Sampaio Paula Sampaio Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Helder Maiato Helder Maiato Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Madalena Costa Madalena Costa Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Leonid V. Omel'yanchuk Leonid V. Omel'yanchuk Laboratory of Genetics of Cell Cycle, Institute of Cytology and Genetics, Lavrentyeva, Russian Federation Search for more papers by this author Vasco Liberal Vasco Liberal Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Claudio E. Sunkel Corresponding Author Claudio E. Sunkel Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Instituto de Ciências Biomédicas de Abel Salazar, Porto, Portugal Search for more papers by this author Catarina L. Lemos Catarina L. Lemos Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Paula Sampaio Paula Sampaio Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Helder Maiato Helder Maiato Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Madalena Costa Madalena Costa Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Leonid V. Omel'yanchuk Leonid V. Omel'yanchuk Laboratory of Genetics of Cell Cycle, Institute of Cytology and Genetics, Lavrentyeva, Russian Federation Search for more papers by this author Vasco Liberal Vasco Liberal Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Search for more papers by this author Claudio E. Sunkel Corresponding Author Claudio E. Sunkel Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal Instituto de Ciências Biomédicas de Abel Salazar, Porto, Portugal Search for more papers by this author Author Information Catarina L. Lemos1, Paula Sampaio1, Helder Maiato1, Madalena Costa1, Leonid V. Omel'yanchuk2, Vasco Liberal1 and Claudio E. Sunkel 1,3 1Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular, Universidade do Porto, R.Campo Alegre, 823, 4150-180 Porto, Portugal 2Laboratory of Genetics of Cell Cycle, Institute of Cytology and Genetics, Lavrentyeva, Russian Federation 3Instituto de Ciências Biomédicas de Abel Salazar, Porto, Portugal *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:3668-3682https://doi.org/10.1093/emboj/19.14.3668 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Through mutational analysis in Drosophila, we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein–Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle. Introduction The mitotic spindle is a specialized structure required during mitosis for chromosome segregation, made up of microtubules (MTs) organized from microtubule-organizing centres (MTOCs) that in most animal cells correspond to centrosomes (reviewed in Zimmerman et al., 1999). Assembly of the mitotic spindle starts during late G2, after centrosome replication. This transition is marked by a dramatic change in MT behaviour thought to be triggered by activation of maturation-promoting factor (MPF) (Verde et al., 1992). At this stage, centrosomes begin to nucleate more and highly dynamic MTs from well defined asters (Saxton et al., 1984) that are involved in centrosome separation and migration to opposite poles of the cell (Saunders and Hoyt, 1992). It is now thought that the formation and maintenance of a bipolar spindle involves at least three families of molecular motors. These include the bipolar kinesins, C-terminal kinesins and cytoplasmic dynein (Robinson et al., 1999; Sharp et al., 1999, 2000). Recent studies on a new family of non-motor microtubule-associated proteins (MAPs), the dis1-TOG family, have suggested that these proteins may also play important roles in the organization of the bipolar spindle. This family includes the human ch-TOG (Charrasse et al., 1998), Xenopus XMAP215 (Vasquez et al., 1994; Tournebize et al., 2000), Drosophila melanogaster Msps (Cullen et al., 1999), Caenorhabditis elegans ZYG-9 (Kemphues et al., 1986; Matthews et al., 1998), Schizosaccharomyces pombe p93dis1 (Nabeshima et al., 1995), Saccharomyces cerevisiae Stu2p (Wang and Huffaker, 1997) and Dictyostelium discoideum DdCP224 (Gräf et al., 2000) proteins. These MAPs localize to either the centrosome/spindle pole body, spindle MTs or both during mitosis or meiosis and have been implicated in the control of microtubule dynamics, stability of the mitotic apparatus, duplication of the centrosome and cytokinesis. Although all members of the dis1-TOG family have been shown to bind microtubules, the MT-binding domain has only been determined for Stu2p, p93dis1 and ch-TOG, and this region falls outside the conserved domains (Nakaseko et al., 1996; Wang and Huffaker, 1997; Charrasse et al., 1998). The MT-binding domain of ch-TOG is also found in other MAPs including tau2, MAP4 and MAP2b (Charrasse et al., 1998). Here we report the identification and characterization of a new Drosophila gene that we have named multiple asters (mast). Mutations in mast cause abnormal chromosome segregation associated with irregular centrosome separation and severely disrupted spindles. We show that the gene encodes a conserved 165 kDa MAP, defining a new conserved family of proteins that is related to the dis1-TOG family. We also show that the mast gene product localizes to centrosomes, interphase and spindle MTs, centromeres and the spindle midzone. Our data suggest that Mast is required for centrosome segregation and organization of the bipolar spindle. Results Identification and characterization of the multiple asters (mast) mutations The first multiple asters (mastP1) mutant allele was identified by Omel'yanchuk et al. (1997). Subsequently, we identified two other P-element-induced alleles, mastP2 and mastP3, from the Berkeley Drosophila Genome Project (BDGP) database. A fourth allele, mastP4, an imprecise excision allele, was obtained after remobilization of the P-element in mastP1. mastP1 and mastP4 cause late larval/pupal lethality when homozygous or hemizygous over Df(3L)31A. mastP3 causes early embryonic lethality of homozygous individuals, suggesting the presence of a second mutation, since mastP3/Df(3L)31A die during late larval/pupal stages. The mastP2 allele is semi-lethal, and viable adults homozygous, hemizygous or heterozygous over the other alleles are obtained. These adults are sterile; moreover, testes and ovaries of mastP2 and mastP2/Df(3L)31A adults are rudimentary. Neuroblasts of homozygous or hemizygous larvae carrying mast mutant alleles show severe mitotic abnormalities (Figure 1), including highly condensed chromosomes (Figure 1B–F, J and K) that frequently are organized in circular arrangements (Figure 1B and C), very few and irregular anaphases (Figure 1H and I) and highly polyploid cells (figure 1C–F, J> and K). Quantification of mitotic parameters shows that mastP1 and mastP2 do not cause a significant increase in the mitotic index. However, mastP3/Df(3L)31A shows an elevated mitotic index and mastP4 a severe mitotic arrest (Figure 1L). Quantification of mitotic figures with respect to mitotic progression indicates that all mutant alleles cause a decrease in the number of cells in prophase, a significant increase of cells in prometaphase/metaphase and a decrease in the proportion of cells in anaphase or telophase (Figure 1M). Quantification of the different types of mitotic abnormalities suggests that most alleles cause either a severe increase in the proportion of polyploid cells, metaphases with a circular chromosome configuration or abnormal anaphases (Figure 1N). The effects on viability, mitotic phenotype and mitotic progression allowed us to order the alleles from least affected to very severe according to the following series: mastP2 < mastP1 < mastP3 < mastP4. Taken together, these results indicate that mutations in mast cause severe abnormalities in chromosome segregation, leading cells to arrest at prometaphase/metaphase. However, the arrest can be overcome and cells undergo multiple rounds of proliferation since most of them are polyploid. Figure 1.Cytological analysis and quantification of mitotic phenotypes in mast mutant neuroblasts. Third instar larval brains were dissected from wild-type (A and G) or mast mutant (B–F and H–K) individuals. Wild-type cells in metaphase (A) or anaphase (G) are shown for comparison. mast mutant cells show either diploid (B) or polyploid (C) circular mitotic figures (CMF) with chromosomes organized with their centromeres facing a central region where the small fourth chromosomes are located. Most cells show highly condensed chromosomes (B–F, J and K). mast mutant cells at anaphase (H and I) can also be found and occasionally show chromatin bridges and abnormal segregation. In the most severe mastP4 allele, cells show extensive polyploidy with most chromosomes organized in a sphere-like conformation (F). (L) Quantification of mitotic index. (M) Quantification of mitotic cells with respect to different stages of mitosis. (N) Quantification of the abnormal mitotic parameters in all alleles. Bar = 5 μm except in (J) and (K), 50 μm. Download figure Download PowerPoint Molecular cloning of the mast gene In order to identify the mutated gene, we characterized the locus at the molecular level. We mapped by in situ hybridization a single P-element insertion in the mastP1 allele to the 78C1–C2 cytological region (data not shown) and cloned both sides of the insertion by plasmid rescue and inverse PCR. DNA sequence analysis with the BDGP databases indicated that it was a new gene and identified a number of expressed sequence tags (ESTs) that had already been partially sequenced. We fully sequenced the largest cDNA (LD11488) of 5938 bp (these sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession No. AF250842). DNA sequence comparisons between the LD11488 cDNA and the corresponding genomic sequence, as well as sequences from cDNAs isolated from adult heads and larva/early pupa, allowed us to determine the intron–exon organization of the gene (Figure 2A). The results indicate that there are at least two types of transcripts: a 5.2 kb transcript, present in cDNA libraries from adult heads and larva/early pupa, and a second transcript of 5.9 kb present in cDNA libraries prepared from embryos. Since in situ hybridization to polytene chromosomes with the larger cDNA hybridizes to a single site (data not shown), the simplest interpretation is that the two transcripts are produced by tissue-specific alternative splicing. The cDNA sequence contains a single open reading frame of 1491 amino acids, coding for a protein with a predicted molecular mass of 165.5 kDa and a pI of 9.17 (Figure 2B). Figure 2.Molecular characterization and expression analysis of mast. (A) Molecular map of the mast locus. Wedges represent the insertion of a P{1ArB}-element in mastP1 (P1) and a P{EP}-element in mastP2 (P2) or mastP3 (P3), and black boxes represent the exons. The bar under the map corresponds to the deleted region in mastP4 (ΔP4). Arrows represent the two transcripts corresponding to the short cDNA from adult heads or larva/pupa, and the long cDNA from embryos. The open reading frame is represented by a grey box. Note that neither exon 1 or exon 1′ are coding exons. (B) Predicted amino acid sequence of Mast. Black boxes represent HEAT repeats, and predicted sites of phosphorylation by p34cdc2 are bold underlined. The grey region defines the conserved MAP-4 microtubule-binding domain. (C) Developmental expression of Mast. Protein samples prepared from successive developmental stages of the wild-type strain were loaded in equal amounts (see α-tubulin as control). P, rMast1; E0-2, 0–2 h embryos; E2-24, 2–24 h embryos; B, third instar larval brains; T, adult testes; O, adult ovaries. The anti-Mast antibody specifically recognizes the recombinant protein and a band of 165 kDa, corresponding to Mast, in all extracts. Note that in early embryo extracts the antibody recognizes an additional band of higher molecular weight. (D) Expression of Mast in different mutant alleles. Protein samples from brains of wild-type or homozygous mutant third instar larvae were loaded in equal amounts (see α-tubulin as control). Download figure Download PowerPoint We also characterized the molecular lesions in other mast alleles. The mastP1 and mastP3 alleles were obtained in different screens; however, they carry a P insertion at the same genomic position, 2317 bp upstream of the predicted ATG. The P-element in mastP2 is inserted 1679 bp from the ATG. The fourth allele, mastP4, was obtained after remobilization of the P-element in mastP1. Southern analysis of mastP4 shows that it has lost part of the P-element (including the ry+ gene). However, all genomic restriction fragments upstream from the insertion are unchanged, and it has a deletion of up to 1 kb of genomic sequence downstream from the insertion site (Figure 2A). Furthermore, western analysis shows that a residual amount of full-size protein is expressed in mastP4 homozygotes, confirming that the coding region of mast is not affected (see Figure 2D). As a result of the remobilization of the insertion in mastP1, we also obtained 39 independent lines that had lost the insertion, were viable and show normal mitotic parameters. These results, together with the genetic complementation data, suggest that the late larval lethality and mitotic phenotypes are the result of mutations in the locus mast. Analysis of the protein sequence shows that Mast contains a 170 amino acid domain that shares limited homology with the proline-rich domain of MAP4 (Figure 2B), which is thought to be involved in the high efficiency binding to MTs (Aizawa et al., 1991). It also contains two regions with significant homology to the HEAT repeat (Andrade and Bork, 1995), at positions 169–207 and 1414–1452 (Figure 2B). This motif was identified in Huntingtin, a protein associated with Huntington's disease, and is also present in the 65 kDa regulatory subunit of protein phosphatase 2A (Hemmings et al., 1990) and in proteins of the dis1-TOG family (Tournebize et al., 2000). The Mast protein also contains two cyclin-dependent kinase p34cdc2 consensus phosphorylation sites (Kennelly and Krebs, 1991) (Figure 2B). Expression of the mast gene in wild-type and mutant tissues In order to analyse the expression of the mast gene, we raised antibodies (Rb726) against a fragment of the Mast protein. The immunopurified antibody (IP726α) recognizes the bacterially expressed protein and, in extracts from embryos, larvae and adult tissues, a large protein of 165 kDa (Figure 2C). The protein is present during early embryogenesis, but it is highly expressed during late embryogenesis, in larval brains and ovaries, and significantly reduced in testes. In early embryos, we also find a slightly larger protein that is recognized specifically by IP726α. We have also analysed the level of the Mast protein in homozygous mutant individuals and found that it is reduced in both mastP1 and mastP2 and barely detectable in mastP4 when compared with the wild-type control (Figure 2D). Evolutionary conservation of Mast In order to determine whether the Mast protein is evolutionarily conserved, we performed BLAST searches against current databases (see Materials and methods). Mast shares significant identity with proteins encoded by two human cDNAs (KIAA0622 and KIAA0627; Ishikawa et al., 1998), three putative proteins in C.elegans (C07H6.3, R107.6 and ZC84.3; Wilson et al., 1994) and also limited identity with Stu1p from S.cerevisiae (Pasqualone et al., 1994) and its putative homologue in S.pombe, which we have called SpStu1p. Multiple alignment of the Mast sequence with those most closely related from other species (Figure 3A) shows that all the proteins share identity throughout their sequence; however, three regions (CR-1, CR-2 and CR-3) are more highly conserved (Figure 3A and B). These results suggest that Mast and its homologues define a new evolutionarily conserved protein family that we have named Stu1-Mast. Figure 3.Protein sequence alignment and phylogenetic analysis. (A) Multiple sequence alignment of the predicted protein sequences closely related to Mast, including two from human (KIAA0622 and KIAA0627), three from C.elegans (CeC07H6.3, CeR107.6 and CeZC84.3), one from S.pombe (SpStu1p) and one from S.cerevisiae (Stu1p), revealed three regions of more significant identity. (B) Conserved regions are represented in grey boxes and the percentage identity and similarity (in parentheses) of the most conserved proteins are indicated below. Additionally, a small domain of 18 amino acids that is highly conserved between Mast and members of the dis1-TOG family is represented. (C) Phylogenetic unrooted tree with all proteins that share significant sequence identity with Mast. Download figure Download PowerPoint Figure 4. Download figure Download PowerPoint Figure 5. Download figure Download PowerPoint Database searches also indicate that Mast shares identity with proteins from the dis1-TOG family, especially at the N-terminal half of the protein (amino acids 1–494), where they are 20–25% identical and 40–45% similar. Inside this region, there is a small domain of 18 amino acid residues that is highly conserved among these proteins and falls inside the first HEAT repeat of Mast (Figure 3B). Phylogenetic analysis including all sequences from the two groups suggests that they are evolutionarily close, but distinct, since they are positioned in different branches of the dendrogram (Figure 3C). Localization of Mast during the cell cycle To determine the intracellular localization of Mast during different stages of the cell cycle, we used IP726α for indirect immunofluorescence in S2 Drosophila tissue culture cells (Figure 4). At interphase, Mast is focused on MTOCs and shows a punctuate pattern co-localizing with α-tubulin. At prophase, Mast accumulates at the MTOCs, as shown by α-tubulin co-staining (Figure 4). Double immunostaining with either anti-centrosomin (CNN) or γ-tubulin antibodies also shows co-localization with Mast (data not shown). In some cells, Mast also associates with a rod-like structure present in interphase or mitotic cells (Figure 4). This structure is stained by IP726α when cells are prepared by different fixation methods and in different cell types. At metaphase, Mast localizes to centrosomes, the mitotic spindle and centromeres, maintaining this localization during anaphase A. Later, at anaphase B, Mast appears concentrated at the spindle midzone, associated with polar MTs, and shows faint centrosomal localization until late telophase. During very late telophase, it localizes at either side of the midbody, and centrosomal localization is barely detectable. After cytokinesis, Mast can be seen associated again with the rod-like structure (data not shown). This structure might correspond to the remains of the midbody. Figure 6.Immunolocalization of Mast in S2 Drosophila culture cells. Individual images for DNA, Mast and α-tubulin are shown. In the merged images, DNA is in blue, Mast in red and α-tubulin in green. Cells in interphase show Mast localized in a punctuate cytoplasmic pattern. At prophase, Mast is found at the centrosomes and most of the time is also associated with an unidentified rod-like structure (arrowhead). During metaphase, Mast associates with spindle microtubules and it is also concentrated at the centromeres. During anaphase, Mast is found at the spindle poles, microtubules and at the spindle midzone. At early telophase, the whole spindle midzone is labelled and some Mast is still present at the spindle poles, and at later stages Mast localizes at either side of the midbody even after cytokinesis is almost complete. Bar = 10 μm. Download figure Download PowerPoint To confirm the specificity of the immunofluorescence labelling, the coding region of mast was cloned in both Drosophila and mammalian transfection vectors to express an enhanced green fluorescent protein (EGFP)–Mast fusion protein in S2 and HeLa cells. As a control, we expressed EGFP alone and verified that it has a homogeneous distribution in S2 or HeLa cells (data not shown). However, the EGFP–Mast fusion protein follows a pattern of localization in both cell types similar to that described in the previous section (Figure 5). In interphase, EGFP–Mast signal is strongly associated with MTOCs and with a fibrillar network that resembles MT bundles. This extensive fibrillar network is not observed in transfected cells treated with colchicine (data not shown). At prophase, the protein localizes to the cytoplasm and shows accumulation at the centrosome. Later, during prometaphase/metaphase, spindle association is clearly evident, as well as localization to the centrosomes and centromeres. During early anaphase, EGFP–Mast appears more diffuse, although, at later stages, centrosomes, spindle MTs and the spindle midzone show accumulation of the protein. In telophase, both S2 and HeLa cells show EGFP signal at the midbody and centrosomes. These results indicate that Mast associates with MTs and centrosomes during most of the cell cycle but undergoes accumulation in additional structures during mitosis. Figure 7.Transfection of EGFP–Mast in Drosophila (S2) and human (HeLa) culture cells. DNA is shown in red and EGFP–Mast in green. During interphase, both S2 and HeLa cells show strong EGFP–Mast signal associated with a fibrillar network that resembles microtubule bundles. In prophase, EGFP–Mast is restricted to the centrosomes. At prometaphase and metaphase, EGFP–Mast signal accumulates at the spindle poles, spindle microtubules and the centromeres. During anaphase, spindle poles, microtubules and a more diffuse cytoplasmic signal are observed. Finally, at telophase, EGFP–Mast localizes to the centrosomes and spindle midzone. Bar = 10 μm. Download figure Download PowerPoint Microtubule-binding assays Indirect and direct localization show that Mast associates with MTs throughout the cell cycle. To determine whether Mast is a MAP, we performed subcellular fractionation and followed the protein using IP726α. The results show that during sequential purification of MTs from embryo extracts, Mast remains tightly bound to the insoluble fraction of polymerized MTs and can be partially released from the polymer after incubation in high salt (Figure 6). These in vitro results support the intracellular localization data, suggesting that Mast associates with MTs. Figure 8.Mast binds to microtubules in vitro. Microtubules were purified from 0- to 3-h-old embryos by sequential rounds of polymerization and depolymerization. Lane 1, crude extract; lane 2, low speed pellet; lane 3, supernatant. The supernatant was centrifuged at high speed and the resulting supernatant (lane 4) was incubated on ice to depolymerize microtubules, followed by incubation with taxol and GTP at 20°C to repolymerize microtubules. After saccharose gradient centrifugation, the soluble material (lane 5) was separated from microtubules and associated proteins (lane 6). MAPs (lane 7) were extracted from microtubules (lane 8) with 0.5 M NaCl. Samples (30 μg) from each purification stage were separated by SDS–PAGE and the gel stained with Coomassie Blue (top panel) or immunoblotted with IP726α (middle panel) and anti-α-tubulin antibodies (bottom panel). Download figure Download PowerPoint Localization of Mast in cells arrested with colchicine Since Mast is a MAP, we wanted to determine whether its localization to the various compartments of the mitotic apparatus depends upon active MT polymerization. Accordingly, S2 tissue culture cells were incubated in the presence of colchicine for various periods of time, fixed and immunostained with IP726α and with an α-tubulin (Figure 7A and B) or γ-tubulin (Figure 7D) antibody. The results show that after 8 or 16 h incubation, no significant spindle MTs are present in these cells (Figure 7B–D). In all cells analysed, Mast shows co-localization with γ-tubulin (Figure 7D), suggesting that microtubules are not required to maintain its localization at the centrosome. Furthermore, in the absence of MTs, Mast shows strong accumulation at the primary constriction of highly condensed chromosomes (Figure 7B–D). In order to determine whether this localization corresponds to the centromere, isolated mitotic chromosomes from S2 cells were stained for both Mast and the mitotic kinase Polo that was previously shown to accumulate at this site (Logarinho and Sunkel, 1998). The results obtained show that Mast and Polo co-localize at the centromere of isolated chromosomes (Figure 7E). Figure 9.Immunolocalization of Mast after microtubule depolymerization. S2 cells were grown for 0, 8 or 16 h in the presence of colchicine. Cells were stained to reveal Mast (red), and α-tubulin (A–C) or γ-tubulin (D) (green). Isolated chromosomes (E) were stained to reveal Mast (red) and Polo (green). DNA is shown in blue. Control cells (A) show a well-organized bipolar spindle and Mast localization to the spindle poles and the centromeres. In cells incubated in colchicine for short (8 h, B) or longer periods (16 h, C and D), microtubules depolymerize and Mast remains associated with both centrosomes (arrows) and centromeres. Mast staining co-localizes with γ-tubulin at the centrosomes (D) and with Polo at the centromeres (E). Bar = 5 μm. Download figure Download PowerPoint Organization of the mitotic apparatus in mast mutant neuroblasts To characterize the organization of the mitotic apparatus in mast mutant cells, neuroblasts from mutant larvae were immunostained to visualize the spindle (α-tubulin) and centrosomes (CNN, γ-tubulin or CP190). Since the results with all three centrosomal markers are very similar, only CNN staining is shown (Figure 8). The wild-type control cell at metaphase shows a typical neuroblast bipolar spindle (Figure 8A). CNN stains the centrosomes in a ring-like pattern that is seen clearly in the amplified image shown in the left panel. All other images are from mast mutant cells that display various degrees of disorganization of the mitotic apparatus. Extensive analysis of mutant neuroblasts stained with α-tubulin to reveal mitotic MTs suggests that the overall morphology of MTs is not the same as that of the wild-type controls. A comparison between control (Figure 8A) and mutant (Figure 8B) cells at metaphase highlights the differences. While in the wild-type cell MTs are generally straight and form tight bundles, in mutant cells MTs are generally irregular in shape and do not always appear straight. Occasionally, mutant cells are able to organize bipolar spindles; however, both poles appear associated with large asters (Figure 8B). CNN staining shows that spindle poles contain an irregular number of ring-like stru