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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 binds preferentially to microtubule plus ends. Visualization of Stu2p binding to microtubules using directly Cy3-labeled Stu2p and polarity-marked microtubules. Stu2p intensity was scanned in the rhodamine channel from the minus end along the length of microtubules, and signals higher than 10% of the median intensity were scored with their location. (A) Representative examples of polarity-marked microtubules in the absence or in the presence of 16 nM Stu2p. Polarity marked microtubules are in green, and Stu2p is in red. The minus end of microtubules is brighter than the plus end. Bar, 3 μm. (B) Histogram showing the number of Stu2p spots per microtubule. (C) Histogram showing the percentage of Stu2p signals found in 10% intervals from the minus ends of the microtubules to their plus ends.
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fig7: Stu2p binds preferentially to microtubule plus ends. Visualization of Stu2p binding to microtubules using directly Cy3-labeled Stu2p and polarity-marked microtubules. Stu2p intensity was scanned in the rhodamine channel from the minus end along the length of microtubules, and signals higher than 10% of the median intensity were scored with their location. (A) Representative examples of polarity-marked microtubules in the absence or in the presence of 16 nM Stu2p. Polarity marked microtubules are in green, and Stu2p is in red. The minus end of microtubules is brighter than the plus end. Bar, 3 μm. (B) Histogram showing the number of Stu2p spots per microtubule. (C) Histogram showing the percentage of Stu2p signals found in 10% intervals from the minus ends of the microtubules to their plus ends.

Mentions: Binding of Stu2p to sheared microtubules is an indirect measurement of the affinity of Stu2p to microtubule ends. To directly visualize Stu2p binding to microtubule ends, we labeled Stu2p with the fluorescent dye Cy3. Cy3-Stu2p was incubated with taxol-stabilized microtubules labeled with Oregon green. To mark the microtubule polarity, the microtubule minus ends were brighter than the plus ends (Fig. 7 A; see Materials and methods). Reactions were fixed, quenched, and analyzed microscopically. Fig. 7 A shows representative Stu2p-bound microtubules. Microtubules are in green, and Stu2p is in red. Stu2p can be seen preferentially at the microtubule plus end. The distribution of Stu2p binding to microtubules was quantified by scanning the intensities of the Stu2p signal along the length of individual microtubules. Signals higher than 10% of the median intensity along the given microtubule were scored with their location. Fig. 7 B shows the distribution of the number of Stu2p spots per microtubule. Fig. 7 C shows a histogram with the frequency at which Stu2p signals are found at 10% distance intervals from the minus end to the plus end of the microtubules. Almost 40% of the Stu2p signals were found at microtubule plus ends. The remainder of the signals are relatively uniformly distributed with a frequency of ∼7% per distance interval along the rest of the microtubule, arguing against any preference of Stu2p among these lateral sites. To eliminate the possibility that Stu2p binding to the microtubule minus ends was inhibited by the relatively high levels of labeling there, we made nonpolarity marked microtubules and examined the end binding of Stu2p. Under these conditions as with polarity marked microtubules, ∼40% of the microtubules had Stu2p bound to one end only (unpublished data).


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 binds preferentially to microtubule plus ends. Visualization of Stu2p binding to microtubules using directly Cy3-labeled Stu2p and polarity-marked microtubules. Stu2p intensity was scanned in the rhodamine channel from the minus end along the length of microtubules, and signals higher than 10% of the median intensity were scored with their location. (A) Representative examples of polarity-marked microtubules in the absence or in the presence of 16 nM Stu2p. Polarity marked microtubules are in green, and Stu2p is in red. The minus end of microtubules is brighter than the plus end. Bar, 3 μm. (B) Histogram showing the number of Stu2p spots per microtubule. (C) Histogram showing the percentage of Stu2p signals found in 10% intervals from the minus ends of the microtubules to their plus ends.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172899&req=5

fig7: Stu2p binds preferentially to microtubule plus ends. Visualization of Stu2p binding to microtubules using directly Cy3-labeled Stu2p and polarity-marked microtubules. Stu2p intensity was scanned in the rhodamine channel from the minus end along the length of microtubules, and signals higher than 10% of the median intensity were scored with their location. (A) Representative examples of polarity-marked microtubules in the absence or in the presence of 16 nM Stu2p. Polarity marked microtubules are in green, and Stu2p is in red. The minus end of microtubules is brighter than the plus end. Bar, 3 μm. (B) Histogram showing the number of Stu2p spots per microtubule. (C) Histogram showing the percentage of Stu2p signals found in 10% intervals from the minus ends of the microtubules to their plus ends.
Mentions: Binding of Stu2p to sheared microtubules is an indirect measurement of the affinity of Stu2p to microtubule ends. To directly visualize Stu2p binding to microtubule ends, we labeled Stu2p with the fluorescent dye Cy3. Cy3-Stu2p was incubated with taxol-stabilized microtubules labeled with Oregon green. To mark the microtubule polarity, the microtubule minus ends were brighter than the plus ends (Fig. 7 A; see Materials and methods). Reactions were fixed, quenched, and analyzed microscopically. Fig. 7 A shows representative Stu2p-bound microtubules. Microtubules are in green, and Stu2p is in red. Stu2p can be seen preferentially at the microtubule plus end. The distribution of Stu2p binding to microtubules was quantified by scanning the intensities of the Stu2p signal along the length of individual microtubules. Signals higher than 10% of the median intensity along the given microtubule were scored with their location. Fig. 7 B shows the distribution of the number of Stu2p spots per microtubule. Fig. 7 C shows a histogram with the frequency at which Stu2p signals are found at 10% distance intervals from the minus end to the plus end of the microtubules. Almost 40% of the Stu2p signals were found at microtubule plus ends. The remainder of the signals are relatively uniformly distributed with a frequency of ∼7% per distance interval along the rest of the microtubule, arguing against any preference of Stu2p among these lateral sites. To eliminate the possibility that Stu2p binding to the microtubule minus ends was inhibited by the relatively high levels of labeling there, we made nonpolarity marked microtubules and examined the end binding of Stu2p. Under these conditions as with polarity marked microtubules, ∼40% of the microtubules had Stu2p bound to one end only (unpublished data).

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
Related in: MedlinePlus