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mini spindles: A gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila.

Cullen CF, Deák P, Glover DM, Ohkura H - J. Cell Biol. (1999)

Bottom Line: Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells.The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast.In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell and Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.

ABSTRACT
We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.

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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.
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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.

Mentions: To characterize the msps gene product, we raised a polyclonal antibody against Msps protein (Materials and Methods). The affinity-purified antibody recognizes mainly one polypeptide of ∼220-kD in immnoblots. To confirm that the antibody specifically recognizes Msps protein, protein samples were prepared from wild-type and mspsP homozygous late third instar larvae (Fig. 5 a, left). The 220-kD band and other minor bands are greatly reduced in the mspsP homozygote. It can be argued, however, that as the sizes of imaginal discs are reduced in the mutant, any proteins highly expressed in discs can be reduced. To eliminate this possibility we used a semi-lethal allele, mspsMJ15 which we created by P-element remobilization. Adult males from this allele are morphologically normal except for weak rough eyes and a few missing bristles, and have normal testis with no spermatogenesis defects. The quantity of 220-kD protein and smaller proteins recognized by the antibody is significantly reduced (Fig. 5 a, right). These minor smaller proteins sometimes recognized by this antibody are likely to be degradation products as their quantity is greatly reduced in msps mutants and is variable in one preparation to another. These results indicate that the affinity-purified antibody specifically recognizes Msps protein.


mini spindles: A gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila.

Cullen CF, Deák P, Glover DM, Ohkura H - J. Cell Biol. (1999)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169485&req=5

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.
Mentions: To characterize the msps gene product, we raised a polyclonal antibody against Msps protein (Materials and Methods). The affinity-purified antibody recognizes mainly one polypeptide of ∼220-kD in immnoblots. To confirm that the antibody specifically recognizes Msps protein, protein samples were prepared from wild-type and mspsP homozygous late third instar larvae (Fig. 5 a, left). The 220-kD band and other minor bands are greatly reduced in the mspsP homozygote. It can be argued, however, that as the sizes of imaginal discs are reduced in the mutant, any proteins highly expressed in discs can be reduced. To eliminate this possibility we used a semi-lethal allele, mspsMJ15 which we created by P-element remobilization. Adult males from this allele are morphologically normal except for weak rough eyes and a few missing bristles, and have normal testis with no spermatogenesis defects. The quantity of 220-kD protein and smaller proteins recognized by the antibody is significantly reduced (Fig. 5 a, right). These minor smaller proteins sometimes recognized by this antibody are likely to be degradation products as their quantity is greatly reduced in msps mutants and is variable in one preparation to another. These results indicate that the affinity-purified antibody specifically recognizes Msps protein.

Bottom Line: Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells.The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast.In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell and Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.

ABSTRACT
We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.

Show MeSH
Related in: MedlinePlus