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Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer.

van Breugel M, Drechsel D, Hyman A - J. Cell Biol. (2003)

Bottom Line: Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends.Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends.These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

ABSTRACT
The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

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Stu2p decreases microtubule length in vitro. (A) Microtubule asters at increasing Stu2p concentrations visualized by incorporation of rhodamine tubulin. Shown are representative examples of asters at 0, 0.1, 0.33, and 1 μM Stu2p. Bar, 4 μm. (B) Plot showing the distribution of aster radii at each Stu2p concentration. (C) Stu2p does not cause spontaneous tubulin nucleation in solution. Increasing amounts of Stu2p were incubated with 22 μM tubulin for 10 min at 29°C. Reactions were spun through a 40% glycerol cushion, and equivalent amounts of supernatants (S) and pellets (P) were analyzed by SDS-PAGE and Coomassie blue staining.
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fig3: Stu2p decreases microtubule length in vitro. (A) Microtubule asters at increasing Stu2p concentrations visualized by incorporation of rhodamine tubulin. Shown are representative examples of asters at 0, 0.1, 0.33, and 1 μM Stu2p. Bar, 4 μm. (B) Plot showing the distribution of aster radii at each Stu2p concentration. (C) Stu2p does not cause spontaneous tubulin nucleation in solution. Increasing amounts of Stu2p were incubated with 22 μM tubulin for 10 min at 29°C. Reactions were spun through a 40% glycerol cushion, and equivalent amounts of supernatants (S) and pellets (P) were analyzed by SDS-PAGE and Coomassie blue staining.

Mentions: We tested the effects of recombinant Stu2p on the length of microtubules growing in vitro from purified centrosomes using rhodamine tubulin to monitor microtubule length. Microtubule growth was initiated at 29°C at 26 μM tubulin in the presence of increasing amounts of Stu2p. After 10 min, the reactions were fixed, quenched, and analyzed microscopically. Fig. 3 A shows a representative aster for each tested Stu2p concentration, and Fig. 3 B is a plot of the aster size distribution at the different Stu2p concentrations. Stu2p addition was found to lead to a clear reduction in microtubule length. This effect starts at 0.1 μM Stu2p and saturates below 0.33 μM. Thus, we conclude that Stu2p acts to destabilize microtubules in vitro.


Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer.

van Breugel M, Drechsel D, Hyman A - J. Cell Biol. (2003)

Stu2p decreases microtubule length in vitro. (A) Microtubule asters at increasing Stu2p concentrations visualized by incorporation of rhodamine tubulin. Shown are representative examples of asters at 0, 0.1, 0.33, and 1 μM Stu2p. Bar, 4 μm. (B) Plot showing the distribution of aster radii at each Stu2p concentration. (C) Stu2p does not cause spontaneous tubulin nucleation in solution. Increasing amounts of Stu2p were incubated with 22 μM tubulin for 10 min at 29°C. Reactions were spun through a 40% glycerol cushion, and equivalent amounts of supernatants (S) and pellets (P) were analyzed by SDS-PAGE and Coomassie blue staining.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172899&req=5

fig3: Stu2p decreases microtubule length in vitro. (A) Microtubule asters at increasing Stu2p concentrations visualized by incorporation of rhodamine tubulin. Shown are representative examples of asters at 0, 0.1, 0.33, and 1 μM Stu2p. Bar, 4 μm. (B) Plot showing the distribution of aster radii at each Stu2p concentration. (C) Stu2p does not cause spontaneous tubulin nucleation in solution. Increasing amounts of Stu2p were incubated with 22 μM tubulin for 10 min at 29°C. Reactions were spun through a 40% glycerol cushion, and equivalent amounts of supernatants (S) and pellets (P) were analyzed by SDS-PAGE and Coomassie blue staining.
Mentions: We tested the effects of recombinant Stu2p on the length of microtubules growing in vitro from purified centrosomes using rhodamine tubulin to monitor microtubule length. Microtubule growth was initiated at 29°C at 26 μM tubulin in the presence of increasing amounts of Stu2p. After 10 min, the reactions were fixed, quenched, and analyzed microscopically. Fig. 3 A shows a representative aster for each tested Stu2p concentration, and Fig. 3 B is a plot of the aster size distribution at the different Stu2p concentrations. Stu2p addition was found to lead to a clear reduction in microtubule length. This effect starts at 0.1 μM Stu2p and saturates below 0.33 μM. Thus, we conclude that Stu2p acts to destabilize microtubules in vitro.

Bottom Line: Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends.Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends.These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

ABSTRACT
The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

Show MeSH