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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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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.
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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.

Mentions: As the predicted sequence of the Msps protein showed high similarity to microtubule-associated proteins from other organisms, we wished to determine whether it was capable of binding to microtubules. We therefore prepared a soluble protein extract (S1 in Fig. 6) from embryos under conditions that depolymerize microtubules. Taxol (paclitaxel) and GTP were then added to repolymerize microtubules, and after 30 min incubation at room temperature, the microtubule fraction (P2) and the soluble fraction (S2) were separated by centrifugation. These protein fractions were run on an SDS–polyacrylamide gel and visualized by Coomassie blue staining. The protein profiles of soluble fractions before and after taxol addition are almost identical, indicating the specific effect of taxol. The microtubule fraction consisted predominantly of proteins of ∼55 kD corresponding to tubulins, together with a number of minor proteins that can be seen by silver staining (data not shown). Immunoblotting indicates that both α-tubulin and Msps are found exclusively in the microtubule fraction (P2), confirming that Msps has microtubule binding activity.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169485&req=5

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.
Mentions: As the predicted sequence of the Msps protein showed high similarity to microtubule-associated proteins from other organisms, we wished to determine whether it was capable of binding to microtubules. We therefore prepared a soluble protein extract (S1 in Fig. 6) from embryos under conditions that depolymerize microtubules. Taxol (paclitaxel) and GTP were then added to repolymerize microtubules, and after 30 min incubation at room temperature, the microtubule fraction (P2) and the soluble fraction (S2) were separated by centrifugation. These protein fractions were run on an SDS–polyacrylamide gel and visualized by Coomassie blue staining. The protein profiles of soluble fractions before and after taxol addition are almost identical, indicating the specific effect of taxol. The microtubule fraction consisted predominantly of proteins of ∼55 kD corresponding to tubulins, together with a number of minor proteins that can be seen by silver staining (data not shown). Immunoblotting indicates that both α-tubulin and Msps are found exclusively in the microtubule fraction (P2), confirming that Msps has microtubule binding activity.

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