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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 induces catastrophes by decreasing microtubule growth rate. Real-time VE-DIC analysis of microtubules in the presence of increasing amounts of Stu2p. (A) Three examples of traces of individual microtubules at 0, 62.5, and 500 nM Stu2p. (B) Stu2p inhibits microtubule growth rate. A plot showing the averaged growth rates of microtubules at different concentrations of Stu2p. Error bars represent the SEM (P ≤ 0.05). (C) Stu2p increases microtubule catastrophe frequency. A plot showing the microtubule catastrophe frequency at different concentrations of Stu2p. (D) Stu2p decreases microtubule shrinkage rate. A plot showing the averaged shrinkage rates of microtubules at different concentrations of Stu2p. Error bars represent the SEM (P ≤ 0.05). (E) The derived dynamic parameters of microtubule polymerization in the presence of increasing amounts of Stu2p. The rescue frequency could not be determined. (F) Stu2p does not directly stimulate microtubule catastrophes. A plot showing the microtubule growth rate catastrophe frequency relation in pure tubulin (varying tubulin). The growth rates and corresponding catastrophe frequencies with varying amounts of Stu2p (as summarized in E) were added to this plot. Growth rate and corresponding catastrophe frequency at increasing concentrations of pure tubulin were obtained by VE-DIC, individual microtubule traces were binned in 0.06 μm/min growth rate intervals, and the catastrophe frequency in each interval was calculated subsequently.
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fig5: Stu2p induces catastrophes by decreasing microtubule growth rate. Real-time VE-DIC analysis of microtubules in the presence of increasing amounts of Stu2p. (A) Three examples of traces of individual microtubules at 0, 62.5, and 500 nM Stu2p. (B) Stu2p inhibits microtubule growth rate. A plot showing the averaged growth rates of microtubules at different concentrations of Stu2p. Error bars represent the SEM (P ≤ 0.05). (C) Stu2p increases microtubule catastrophe frequency. A plot showing the microtubule catastrophe frequency at different concentrations of Stu2p. (D) Stu2p decreases microtubule shrinkage rate. A plot showing the averaged shrinkage rates of microtubules at different concentrations of Stu2p. Error bars represent the SEM (P ≤ 0.05). (E) The derived dynamic parameters of microtubule polymerization in the presence of increasing amounts of Stu2p. The rescue frequency could not be determined. (F) Stu2p does not directly stimulate microtubule catastrophes. A plot showing the microtubule growth rate catastrophe frequency relation in pure tubulin (varying tubulin). The growth rates and corresponding catastrophe frequencies with varying amounts of Stu2p (as summarized in E) were added to this plot. Growth rate and corresponding catastrophe frequency at increasing concentrations of pure tubulin were obtained by VE-DIC, individual microtubule traces were binned in 0.06 μm/min growth rate intervals, and the catastrophe frequency in each interval was calculated subsequently.

Mentions: We wanted to further understand the mechanism by which Stu2 destabilizes microtubules. Stu2p could destabilize microtubules by inhibition of growth rate, promotion of microtubule shrinkage rate, through induction of catastrophes or prevention of rescues. To distinguish between these possibilities, we used video-enhanced differential interference contrast microscopy (VE-DIC). Microtubules were nucleated from purified centrosomes adsorbed to the coverslips of small perfusion chambers. Fig. 5 A shows three typical microtubule traces for three different Stu2p concentrations. Fig. 5 B shows the average growth rate, Fig. 5 C shows the catastrophe rate, and Fig. 5 D displays the average shrinkage rate as a function of Stu2p concentration. These data are summarized in Fig. 5 E. Rescue events occurred too rarely to make a statement about Stu2p's influence on the rescue frequency. Stu2p has a very minor effect on microtubule shrinkage rate that is only seen at high Stu2p concentrations. In contrast, Stu2p shows a marked inhibition of microtubule growth rate that starts at very low concentrations (0.03 μM) and begins to saturate at ∼0.1 μM. Similarly, Stu2p shows a marked promotion of catastrophes that begins at 0.03 μM and saturates at ∼0.1 μM. Thus, Stu2p destabilizes microtubules by decreasing their growth rate and increasing their catastrophe rate. This is in direct contrast to its Xenopus homologue XMAP215 that under very similar buffer conditions in vitro acts as a microtubule stabilizer (Vasquez et al., 1994; Kinoshita et al., 2001).


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 induces catastrophes by decreasing microtubule growth rate. Real-time VE-DIC analysis of microtubules in the presence of increasing amounts of Stu2p. (A) Three examples of traces of individual microtubules at 0, 62.5, and 500 nM Stu2p. (B) Stu2p inhibits microtubule growth rate. A plot showing the averaged growth rates of microtubules at different concentrations of Stu2p. Error bars represent the SEM (P ≤ 0.05). (C) Stu2p increases microtubule catastrophe frequency. A plot showing the microtubule catastrophe frequency at different concentrations of Stu2p. (D) Stu2p decreases microtubule shrinkage rate. A plot showing the averaged shrinkage rates of microtubules at different concentrations of Stu2p. Error bars represent the SEM (P ≤ 0.05). (E) The derived dynamic parameters of microtubule polymerization in the presence of increasing amounts of Stu2p. The rescue frequency could not be determined. (F) Stu2p does not directly stimulate microtubule catastrophes. A plot showing the microtubule growth rate catastrophe frequency relation in pure tubulin (varying tubulin). The growth rates and corresponding catastrophe frequencies with varying amounts of Stu2p (as summarized in E) were added to this plot. Growth rate and corresponding catastrophe frequency at increasing concentrations of pure tubulin were obtained by VE-DIC, individual microtubule traces were binned in 0.06 μm/min growth rate intervals, and the catastrophe frequency in each interval was calculated subsequently.
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Related In: Results  -  Collection

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fig5: Stu2p induces catastrophes by decreasing microtubule growth rate. Real-time VE-DIC analysis of microtubules in the presence of increasing amounts of Stu2p. (A) Three examples of traces of individual microtubules at 0, 62.5, and 500 nM Stu2p. (B) Stu2p inhibits microtubule growth rate. A plot showing the averaged growth rates of microtubules at different concentrations of Stu2p. Error bars represent the SEM (P ≤ 0.05). (C) Stu2p increases microtubule catastrophe frequency. A plot showing the microtubule catastrophe frequency at different concentrations of Stu2p. (D) Stu2p decreases microtubule shrinkage rate. A plot showing the averaged shrinkage rates of microtubules at different concentrations of Stu2p. Error bars represent the SEM (P ≤ 0.05). (E) The derived dynamic parameters of microtubule polymerization in the presence of increasing amounts of Stu2p. The rescue frequency could not be determined. (F) Stu2p does not directly stimulate microtubule catastrophes. A plot showing the microtubule growth rate catastrophe frequency relation in pure tubulin (varying tubulin). The growth rates and corresponding catastrophe frequencies with varying amounts of Stu2p (as summarized in E) were added to this plot. Growth rate and corresponding catastrophe frequency at increasing concentrations of pure tubulin were obtained by VE-DIC, individual microtubule traces were binned in 0.06 μm/min growth rate intervals, and the catastrophe frequency in each interval was calculated subsequently.
Mentions: We wanted to further understand the mechanism by which Stu2 destabilizes microtubules. Stu2p could destabilize microtubules by inhibition of growth rate, promotion of microtubule shrinkage rate, through induction of catastrophes or prevention of rescues. To distinguish between these possibilities, we used video-enhanced differential interference contrast microscopy (VE-DIC). Microtubules were nucleated from purified centrosomes adsorbed to the coverslips of small perfusion chambers. Fig. 5 A shows three typical microtubule traces for three different Stu2p concentrations. Fig. 5 B shows the average growth rate, Fig. 5 C shows the catastrophe rate, and Fig. 5 D displays the average shrinkage rate as a function of Stu2p concentration. These data are summarized in Fig. 5 E. Rescue events occurred too rarely to make a statement about Stu2p's influence on the rescue frequency. Stu2p has a very minor effect on microtubule shrinkage rate that is only seen at high Stu2p concentrations. In contrast, Stu2p shows a marked inhibition of microtubule growth rate that starts at very low concentrations (0.03 μM) and begins to saturate at ∼0.1 μM. Similarly, Stu2p shows a marked promotion of catastrophes that begins at 0.03 μM and saturates at ∼0.1 μM. Thus, Stu2p destabilizes microtubules by decreasing their growth rate and increasing their catastrophe rate. This is in direct contrast to its Xenopus homologue XMAP215 that under very similar buffer conditions in vitro acts as a microtubule stabilizer (Vasquez et al., 1994; Kinoshita et al., 2001).

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