Limits...
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.

Show MeSH
Stu2p binds preferentially to microtubule ends. (A) Antitubulin immunofluorescence of taxol stabilized microtubules before and after shearing with a tip sonicator. Bar, 5 μm. (B) Stu2p binds to a higher extent to sheared microtubules. Increasing amounts of unsheared or sheared microtubules were incubated with 19 nM Stu2p, and bound Stu2p separated from unbound Stu2p by centrifugation. Equivalent amounts of supernatants (S) and pellets (P) were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody (top) or a monoclonal tubulin antibody (bottom). (C) Plot showing the percentage of Stu2p bound to microtubules at different microtubule concentrations.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172899&req=5

fig6: Stu2p binds preferentially to microtubule ends. (A) Antitubulin immunofluorescence of taxol stabilized microtubules before and after shearing with a tip sonicator. Bar, 5 μm. (B) Stu2p binds to a higher extent to sheared microtubules. Increasing amounts of unsheared or sheared microtubules were incubated with 19 nM Stu2p, and bound Stu2p separated from unbound Stu2p by centrifugation. Equivalent amounts of supernatants (S) and pellets (P) were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody (top) or a monoclonal tubulin antibody (bottom). (C) Plot showing the percentage of Stu2p bound to microtubules at different microtubule concentrations.

Mentions: How does Stu2p inhibit microtubule growth? Interestingly, the inhibition of the microtubule growth rate by Stu2p saturated at relatively low Stu2p concentrations (∼0.1 μM Stu2p with 21.5 μM tubulin in the VE-DIC assay, below 0.33 μM Stu2p with 26 μM tubulin in a fixed time point assay). Mechanistically, that argues in favor of a model in which a limited number of microtubule-binding sites necessary for efficient growth become preferentially bound and saturated by Stu2p. An obvious location of such sites is the microtubule end. Indeed, there is evidence for Stu2p binding in vivo to MT ends (He et al., 2001; Kosco et al., 2001). Therefore, we determined the relative affinities of Stu2p for microtubule ends versus the lattice. We took advantage of the fact that taxol-stabilized microtubules can be sheared so that the concentration of polymeric tubulin remains constant with a significant increase in the concentration of microtubule ends. If Stu2p binds microtubule ends preferentially, more Stu2p should bind to sheared microtubules. Examples of sheared and unsheared microtubules are shown in Fig. 6 A. Sheared microtubules were on average fivefold shorter then the unsheared microtubules (see Materials and methods). Increasing amounts of microtubules were incubated with 19 nM Stu2p at room temperature. Microtubule-bound Stu2p was separated from unbound Stu2p by centrifugation, and the supernatant and pellet were analyzed by Western blotting. Fig. 6 B shows the Western blot probed for Stu2p and tubulin. Fig. 6 C shows a graph with the percent of microtubule-bound Stu2p plotted against the microtubule concentrations. At each given concentration of microtubules, more Stu2p bound to the sheared than to the nonsheared microtubules. Thus, Stu2p binds preferentially to microtubule ends as opposed to lateral microtubule binding sites. Under less stringent conditions, some end binding has been reported previously in vitro for the human Stu2p homologue ch-TOG (Spittle et al., 2000).


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 ends. (A) Antitubulin immunofluorescence of taxol stabilized microtubules before and after shearing with a tip sonicator. Bar, 5 μm. (B) Stu2p binds to a higher extent to sheared microtubules. Increasing amounts of unsheared or sheared microtubules were incubated with 19 nM Stu2p, and bound Stu2p separated from unbound Stu2p by centrifugation. Equivalent amounts of supernatants (S) and pellets (P) were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody (top) or a monoclonal tubulin antibody (bottom). (C) Plot showing the percentage of Stu2p bound to microtubules at different microtubule concentrations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172899&req=5

fig6: Stu2p binds preferentially to microtubule ends. (A) Antitubulin immunofluorescence of taxol stabilized microtubules before and after shearing with a tip sonicator. Bar, 5 μm. (B) Stu2p binds to a higher extent to sheared microtubules. Increasing amounts of unsheared or sheared microtubules were incubated with 19 nM Stu2p, and bound Stu2p separated from unbound Stu2p by centrifugation. Equivalent amounts of supernatants (S) and pellets (P) were analyzed by SDS-PAGE and Western blotting using a polyclonal Stu2p antibody (top) or a monoclonal tubulin antibody (bottom). (C) Plot showing the percentage of Stu2p bound to microtubules at different microtubule concentrations.
Mentions: How does Stu2p inhibit microtubule growth? Interestingly, the inhibition of the microtubule growth rate by Stu2p saturated at relatively low Stu2p concentrations (∼0.1 μM Stu2p with 21.5 μM tubulin in the VE-DIC assay, below 0.33 μM Stu2p with 26 μM tubulin in a fixed time point assay). Mechanistically, that argues in favor of a model in which a limited number of microtubule-binding sites necessary for efficient growth become preferentially bound and saturated by Stu2p. An obvious location of such sites is the microtubule end. Indeed, there is evidence for Stu2p binding in vivo to MT ends (He et al., 2001; Kosco et al., 2001). Therefore, we determined the relative affinities of Stu2p for microtubule ends versus the lattice. We took advantage of the fact that taxol-stabilized microtubules can be sheared so that the concentration of polymeric tubulin remains constant with a significant increase in the concentration of microtubule ends. If Stu2p binds microtubule ends preferentially, more Stu2p should bind to sheared microtubules. Examples of sheared and unsheared microtubules are shown in Fig. 6 A. Sheared microtubules were on average fivefold shorter then the unsheared microtubules (see Materials and methods). Increasing amounts of microtubules were incubated with 19 nM Stu2p at room temperature. Microtubule-bound Stu2p was separated from unbound Stu2p by centrifugation, and the supernatant and pellet were analyzed by Western blotting. Fig. 6 B shows the Western blot probed for Stu2p and tubulin. Fig. 6 C shows a graph with the percent of microtubule-bound Stu2p plotted against the microtubule concentrations. At each given concentration of microtubules, more Stu2p bound to the sheared than to the nonsheared microtubules. Thus, Stu2p binds preferentially to microtubule ends as opposed to lateral microtubule binding sites. Under less stringent conditions, some end binding has been reported previously in vitro for the human Stu2p homologue ch-TOG (Spittle et al., 2000).

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