Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization - PubMed (original) (raw)

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

C L Lemos et al. EMBO J. 2000.

Abstract

Through mutational analysis in Drosopjila 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.

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Figures

Fig. 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 _mast_P4 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.

Fig. 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 _mast_P1 (P1) and a P{EP}-element in _mast_P2 (P2) or _mast_P3 (P3), and black boxes represent the exons. The bar under the map corresponds to the deleted region in _mast_P4 (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).

Fig. 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.

Fig. 4. 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.

Fig. 5. 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.

Fig. 6. 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).

Fig. 7. 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.

Fig. 8. Centrosomes and mitotic spindles in mast mutant cells. Squashed preparations of brains isolated from either wild-type (A) or various mast mutant allelic combinations (B–G) were incubated with an anti-CNN antibody to reveal the centrosome, anti-α-tubulin antibody to visualize the microtubules and DAPI to stain DNA. The first column shows a magnified view of the CNN staining of centrosomes shown in the second column (A–F). In the last column, individual images of CNN (red), α-tubulin (green) and DNA (blue) have been merged. (A) During metaphase, wild-type neuroblasts show a normal bipolar spindle organized from ring-like CNN-positive structures. (B) Most mast mutant cells show abnormal spindle organization even if a polyploid cell is able to organize a bipolar structure. The spindle poles are highly unequal in size and show many ring-like structures tightly associated. (C) Monopolar spindle in a mast mutant cell organized from two associated ring-like structures. (D) Monopolar spindle in a mast mutant cell organized by an aggregate of CNN-positive centrosomal material that shows no clear internal organization. (E) Mutant cell with highly condensed chromosomes organized into a ball-like structure around a mass of centrosomal material. Note the extensive microtubule network emanating from the single large centrosome. (F) Mutant cell with highly condensed chromosomes organized around several CNN-positive aggregates from which asters are irradiated. Note, in the magnified view of the centrosome, two pairs of ring-like structures resembling normal centrosomes. (G) Highly polyploid cell exhibiting a large number of centrosomal aggregates within the chromosome mass, as well as multiple cytoplasmatic CNN-positive aggregates. Note that all CNN-positive aggregates are associated with microtubule asters. Bar = 2 µm in the ‘CNN zoom 3.5×’ column and 5 µm in all other figures.

Fig. 9. Immunolocalization of a spindle checkpoint protein in mast mutant cells. Wild-type (A–C) or mast mutant (D–F) neuroblasts were stained with antibodies against Bub1 (red) and α-tubulin (green). DNA is shown in blue. In wild-type cells, Bub1 accumulates at the centromeres during prophase (A) and prometaphase (B) and is severely reduced as the chromosomes align in the metaphase plate (C). In mast mutant cells, Bub1 shows strong accumulation at the centromeres of chromosomes in monopolar (D and E) and polyploid (F) cells. Bar = 5 µm.

References

1. Aizawa H., Emori,Y., Mori,A., Murofushi,H., Sakai,H. and Suzuli,K. (1991) Functional analysis of the domain structure of microtubule-associated protein-4 (MAP-U). J. Biol. Chem., 266, 9841–9846. - PubMed
1. Altschul S.F., Madden,T.L., Schäffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., 25, 3389–3402. - PMC - PubMed
1. Andrade M.A. and Bork,P. (1995) HEAT repeats in the Huntington’s disease protein. Nature Genet., 11, 115–116. - PubMed
1. Basu J., Bousbaa,H., Logarinho,E., Li,Z., Williams,B.C., Lopes,C., Sunkel,C.E. and Goldberg,M.L. (1999) Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila. J. Cell Biol., 146, 13–28. - PMC - PubMed
1. Bonaccorsi S., Giansanti,M.G. and Gatti,M. (2000) Spindle assembly in Drosophila neuroblasts and ganglion mother cells. Nature Cell Biol., 2, 54–56. - PubMed

Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization - PubMed (original) (raw)