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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 can destabilize cytoplasmic microtubules in vivo. Wild-type or stu2–10 cells were HU arrested at the permissive temperature, shifted to the restrictive temperature, and the cytoplasmic microtubule length was determined by indirect immunofluorescence and three dimensional tracking. (A) FACS® profiles of cycling wild-type or stu2–10 cells at 25°C (top) that were arrested at 25°C with HU before (middle) or after shifting the cells to 37°C (bottom). (B) Projected indirect antitubulin immunofluorescence of wild-type or stu2–10 cells arrested at 25°C with HU before (top) or after shifting the cells to 37°C (bottom). Green, microtubules; blue, DNA. Bar, 5 μm. (C) Projected indirect antitubulin immunofluorescence of wild-type (left) or stu2–10 cells (right) arrested at 25°C with HU after shifting the cells to 37°C. Close-ups of four representative cells. Green, microtubules; blue, DNA. Bar, 5 μm. (D) Length distribution (in 1-μm intervals) of cytoplasmic microtubules of wild-type and stu2–10 cells arrested at 25°C with HU before (top) or after shifting the cells to 37°C (bottom). Green, wild-type microtubules; red, stu2–10 microtubules. (E) Average length of cytoplasmic microtubules of wild-type or stu2–10 cells arrested at 25°C with HU before and after shifting the cells to 37°C. Error bars represent SEM (P < 0.05).
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fig4: Stu2p can destabilize cytoplasmic microtubules in vivo. Wild-type or stu2–10 cells were HU arrested at the permissive temperature, shifted to the restrictive temperature, and the cytoplasmic microtubule length was determined by indirect immunofluorescence and three dimensional tracking. (A) FACS® profiles of cycling wild-type or stu2–10 cells at 25°C (top) that were arrested at 25°C with HU before (middle) or after shifting the cells to 37°C (bottom). (B) Projected indirect antitubulin immunofluorescence of wild-type or stu2–10 cells arrested at 25°C with HU before (top) or after shifting the cells to 37°C (bottom). Green, microtubules; blue, DNA. Bar, 5 μm. (C) Projected indirect antitubulin immunofluorescence of wild-type (left) or stu2–10 cells (right) arrested at 25°C with HU after shifting the cells to 37°C. Close-ups of four representative cells. Green, microtubules; blue, DNA. Bar, 5 μm. (D) Length distribution (in 1-μm intervals) of cytoplasmic microtubules of wild-type and stu2–10 cells arrested at 25°C with HU before (top) or after shifting the cells to 37°C (bottom). Green, wild-type microtubules; red, stu2–10 microtubules. (E) Average length of cytoplasmic microtubules of wild-type or stu2–10 cells arrested at 25°C with HU before and after shifting the cells to 37°C. Error bars represent SEM (P < 0.05).

Mentions: To further confirm that Stu2p can act in vivo as a destabilizer of cytoplasmic microtubules, we used the temperature-sensitive mutant stu2–10 to abolish Stu2p function (Severin et al., 2001). To avoid complications from a stu2–10–dependent cell cycle arrest (Severin et al., 2001), we arrested both wild-type and stu2–10 cells in S phase at the permissive temperature with hydroxyurea (HU). After HU arrest, the cultures were shifted to 37°C, and the length of cytoplasmic microtubules were determined. Fig. 4 A shows a FACS® analysis of the cells throughout the experiment, demonstrating a complete cell cycle block at S phase. Fig. 4 B shows a projected antitubulin immunofluorescence of wild-type or stu2–10 cells arrested in HU at the permissive temperature before (top) or after the shift to the restrictive temperature (bottom and representative close ups in Fig. 4 C). Fig. 4 D shows the respective length distribution of cytoplasmic microtubules, and Fig. 4 E shows the average cytoplasmic microtubule lengths. Very similar in length before the temperature shift (wild type, 1.03 ± 0.07 [SEM; p < 0.05]) μm; stu2–10, 1.07 ± 0.07 μm), the length of cytoplasmic microtubules of stu2–10 and wild-type cells increases after the shift to the restrictive temperature (Fig. 4, D and E), reflecting the effect of the higher temperature and the larger cell size (see Materials and methods). However, after the temperature shift cytoplasmic microtubules of stu2–10 cells are on average ∼30% longer than the ones of wild-type cells (wild-type, 1.70 ± 0.13 μm; stu2–10, 2.29 ± 0.20 μm) (Fig. 4, D and E). Thus, Stu2p can act as a microtubule destabilizer in vivo and 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 can destabilize cytoplasmic microtubules in vivo. Wild-type or stu2–10 cells were HU arrested at the permissive temperature, shifted to the restrictive temperature, and the cytoplasmic microtubule length was determined by indirect immunofluorescence and three dimensional tracking. (A) FACS® profiles of cycling wild-type or stu2–10 cells at 25°C (top) that were arrested at 25°C with HU before (middle) or after shifting the cells to 37°C (bottom). (B) Projected indirect antitubulin immunofluorescence of wild-type or stu2–10 cells arrested at 25°C with HU before (top) or after shifting the cells to 37°C (bottom). Green, microtubules; blue, DNA. Bar, 5 μm. (C) Projected indirect antitubulin immunofluorescence of wild-type (left) or stu2–10 cells (right) arrested at 25°C with HU after shifting the cells to 37°C. Close-ups of four representative cells. Green, microtubules; blue, DNA. Bar, 5 μm. (D) Length distribution (in 1-μm intervals) of cytoplasmic microtubules of wild-type and stu2–10 cells arrested at 25°C with HU before (top) or after shifting the cells to 37°C (bottom). Green, wild-type microtubules; red, stu2–10 microtubules. (E) Average length of cytoplasmic microtubules of wild-type or stu2–10 cells arrested at 25°C with HU before and after shifting the cells to 37°C. Error bars represent SEM (P < 0.05).
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Related In: Results  -  Collection

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fig4: Stu2p can destabilize cytoplasmic microtubules in vivo. Wild-type or stu2–10 cells were HU arrested at the permissive temperature, shifted to the restrictive temperature, and the cytoplasmic microtubule length was determined by indirect immunofluorescence and three dimensional tracking. (A) FACS® profiles of cycling wild-type or stu2–10 cells at 25°C (top) that were arrested at 25°C with HU before (middle) or after shifting the cells to 37°C (bottom). (B) Projected indirect antitubulin immunofluorescence of wild-type or stu2–10 cells arrested at 25°C with HU before (top) or after shifting the cells to 37°C (bottom). Green, microtubules; blue, DNA. Bar, 5 μm. (C) Projected indirect antitubulin immunofluorescence of wild-type (left) or stu2–10 cells (right) arrested at 25°C with HU after shifting the cells to 37°C. Close-ups of four representative cells. Green, microtubules; blue, DNA. Bar, 5 μm. (D) Length distribution (in 1-μm intervals) of cytoplasmic microtubules of wild-type and stu2–10 cells arrested at 25°C with HU before (top) or after shifting the cells to 37°C (bottom). Green, wild-type microtubules; red, stu2–10 microtubules. (E) Average length of cytoplasmic microtubules of wild-type or stu2–10 cells arrested at 25°C with HU before and after shifting the cells to 37°C. Error bars represent SEM (P < 0.05).
Mentions: To further confirm that Stu2p can act in vivo as a destabilizer of cytoplasmic microtubules, we used the temperature-sensitive mutant stu2–10 to abolish Stu2p function (Severin et al., 2001). To avoid complications from a stu2–10–dependent cell cycle arrest (Severin et al., 2001), we arrested both wild-type and stu2–10 cells in S phase at the permissive temperature with hydroxyurea (HU). After HU arrest, the cultures were shifted to 37°C, and the length of cytoplasmic microtubules were determined. Fig. 4 A shows a FACS® analysis of the cells throughout the experiment, demonstrating a complete cell cycle block at S phase. Fig. 4 B shows a projected antitubulin immunofluorescence of wild-type or stu2–10 cells arrested in HU at the permissive temperature before (top) or after the shift to the restrictive temperature (bottom and representative close ups in Fig. 4 C). Fig. 4 D shows the respective length distribution of cytoplasmic microtubules, and Fig. 4 E shows the average cytoplasmic microtubule lengths. Very similar in length before the temperature shift (wild type, 1.03 ± 0.07 [SEM; p < 0.05]) μm; stu2–10, 1.07 ± 0.07 μm), the length of cytoplasmic microtubules of stu2–10 and wild-type cells increases after the shift to the restrictive temperature (Fig. 4, D and E), reflecting the effect of the higher temperature and the larger cell size (see Materials and methods). However, after the temperature shift cytoplasmic microtubules of stu2–10 cells are on average ∼30% longer than the ones of wild-type cells (wild-type, 1.70 ± 0.13 μm; stu2–10, 2.29 ± 0.20 μm) (Fig. 4, D and E). Thus, Stu2p can act as a microtubule destabilizer in vivo and 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