Cullen CF , Deák P , Glover DM , Ohkura H .

Wellcome Trust

	Figure 1. Chromosome segregation is disrupted in the mspsP mutant. Aceto-orcein was used to stain chromosomes in cells of squashed preparations of the central nervous system from late third instar larvae of wild-type (a and c) and mspsP homozygotes (b and d). The mspsP mutant shows overcondensation of mitotic chromosomes (b). A quarter of anaphases show V-shaped alignment of chromosomes (d). (e and f) Open bars and solid lines represent mean and standard deviation calculated from the quantitation of mitotic figures in five individuals of wild-type or the mspsP mutant. This corresponds to roughly five to ten thousand cells from each individual. (e) Mitotic index is calculated as number of mitotic cells per microscope field using a 100× objective (a field area of 0.05 mm2 containing typically 200–400 cells). (f) % Anaphases is calculated as 100 × (number of anaphases)/(number of all mitotic cells). Bar, 10 μm.
	Figure 2. Structural integrity of mitotic spindle is disrupted in the mspsP mutant. Whole mount preparations of larval central nervous system were prepared from mspsP and stained to reveal tubulin (green), DNA (red), and a centrosomal antigen, CP190 (blue). The focal planes shown display the overall spindle structure and CP190 staining for each mitotic figure. (a) Category I. Apparently normal bipolar spindle with CP190 at both poles. (b) Category II. One bipolar spindle and one smaller bipolar spindle (arrowhead). CP190 is found at both poles of larger spindle and is missing from one pole of the smaller one. (c) Category II. Two bipolar spindles with one shared pole. CP190 is found at both poles of one spindle, while it is absent from the unshared pole of the other spindle (arrowhead). (d) Category II. At least three chromosomes are associated with each of small bipolar spindles. One spindle has CP190 at both poles, another has the protein at one pole, and the other shows no CP190 at the poles. (e) Category IV. One bipolar spindle and a monopolar spindle. The bipolar spindle has CP190 at both poles. (f) Category III. Short disorganized microtubule bundles associated with a chromosome mass. No discrete CP190 staining is seen. (g) Frequencies of each category of spindle structure observed. Bar, 5 μm.
	Figure 3. Molecular cloning and identification of the msps gene. (a) Genomic region at 89B around the P-lacW insertion site in the mspsP mutant. The arrows represent four transcription units in this region. Introns are represented by kinked lines. A question mark on the far left indicates uncertainty of the location of the second exon of this transcript that lies beyond the sequenced region. The first exon of this particular transcript does not contain an open reading frame. The lower lines represent the genomic regions used for transgenic constructs (HN267, HN276). HN267 rescued both lethality and mitotic defects of mspsP mutation, while HN276 rescued neither, indicating that the shaded transcript corresponds to msps gene. (b) Rescue of mitotic defects in mspsP by transgenic constructs. Transgenes were tested for rescue of mitotic defects in mspsP. Squashed preparation of central nervous systems was prepared for orcein staining from wild-type, mspsP homozygotes, mspsP homozygotes carrying the HN267 transgenic construct and mspsP homozygotes carrying HN276. Means and standard deviations of the frequencies of anaphases are represented by open bars and solid lines, respectively.
	Figure 4. The Msps protein belongs to the dis1-TOG family. (a) Sequence comparison between Msps and human TOGp. Only identical residues are marked. Five putative cdc2 phosphorylation sites (S/TPXK/R) were identified in the COOH-terminal portion of the Msps protein (amino acids 1,564, 1,568, 1,803, 1,859, and 2,029). (b) Domain structure of members of the dis1-TOG family. From the top, human TOGp, D. melanogaster Msps, C. elegans ZYG-9, S. pombe Dis1, and S. cerevisiae STU2. Shaded boxes represent repeats (see c) common to all the dis1-TOG family. The COOH-terminal portions of the human and Drosophila proteins can be divided into two domains, one of which is also conserved in C. elegans protein, and the other found in Drosophila and vertebrates. (c) Amino acid sequences of the NH2-terminal repeats from the different proteins are aligned. Numbers in parentheses indicate the length of intervening amino acid sequences. The sequence of msps gene is available from GenBank/EMBL/DDBJ under accession number AJ249115.
	Figure 5. Expression of Msps protein. (a) Specific immunoidentification of Msps protein. (Left) Total protein samples were prepared from late third instar larvae of wild-type and mspsP, immunoblotted and probed with the Msps antibody. (Right) Protein samples were prepared from adult males of wild-type and mspsMJ15. The levels of 220-kD protein and putative degradation fragments were greatly reduced in the msps mutants. The amounts and profiles of total proteins were identical between wild-type and msps mutants judged by Coomassie blue staining. An α-tubulin antibody was used to give a loading and blotting control. (b) Levels of Msps protein during development. Protein samples were prepared from successive developmental stages of wild-type. E1, 0–4-h embryos; E2, 4–20-h embryos; L1, 1st instar larvae; L2, 2nd instar larvae; L3, 3rd instar larvae; EP, early pupae; LP, late pupae/pharate adult; M, adult males; F, adult females. In the upper panel, the immunoblot is probed with the Msps antibody. Msps protein is most abundant in embryos but a significant amount is also found in other developmental stages. In the middle panel, the immunoblot is probed with α-tubulin antibody. In the bottom panel, Coomassie blue staining shows that each lane has a comparable amount of protein except L2 which is underloaded. (c) Msps protein in adults. Adult females were dissected into three parts, abdomen, thorax, and head. One-tenth of each part from individual flies was loaded in each lane. In the top left panel, the immunoblot was probed with Msps antibody. In the lower left panel, the immunoblot was probed with an α-tubulin antibody. The right panel shows Coomassie blue staining.
	Figure 6. Msps protein binds to microtubules in vitro. Crude protein extract from 0–8-h-old embryos was incubated on ice to depolymerize microtubules. The high speed supernatant (S1) was incubated with taxol (paclitaxel) and GTP at 20°C to repolymerize the microtubules. Microtubules and associated proteins (P2) were separated from soluble proteins (S2) by centrifugation. (Left) Coomassie blue staining. (Top right) Immunoblot probed by Msps antibody. (Bottom right) Immunoblot probed by α-tubulin antibody. After taxol treatment, Msps protein of unaltered size is exclusively detected on the microtubule fraction.
	Figure 7. Msps protein localizes to mitotic spindles and the centrosomal regions. Wild-type syncytial embryos were immunostained with α-tubulin and Msps antibodies and stained with DAPI to reveal DNA. In merged images, DNA, tubulin, and Msps staining are represented as blue, green, and red, respectively. Bar, 10 μm.
	Figure 8. Msps protein changes its localization during mitotic progression in syncytial and cellularized embryos. Wild-type embryos were immunostained with α-tubulin and Msps antibodies and stained with DAPI to reveal DNA. In the merged images, DNA, tubulin, and Msps staining are represented as blue, green, and red, respectively. (a) Two syncytial embryos are shown to represent progression through mitosis from prophase to late anaphase. (b) Mitotic domains from cellularized embryos at cycle 14. p, prophase; m, metaphase; a, anaphase; t, telophase; I, interphase. Bars, 10 μm.
	Figure 9. Localization of Msps protein in colchicine or taxol treated embryos. Syncytial embryos were incubated with colchicine (a) or taxol (b–d). (a–c) DNA (left, blue in merge), tubulin (middle, green), and Msps (right, red) were visualized by immunostaining. Overlap between tubulin and Msps staining resulted in yellow color. (d) DNA (left, blue), CP190 (middle, green), and Msps (right, red) were visualized. (a) Microtubules were depolymerized except weak tubulin staining of centrosomes. Msps protein remains on centrosomes. (b) After taxol treatment, Msps protein remains in the centrosomal region during interphase. (c and d) In mitotic cells, Msps protein follows taxol induced microtubules, while CP190 stays on centrosomes. Bars, 10 μm.

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