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Differential contribution of Bud6p and Kar9p to microtubule capture and spindle orientation in S. cerevisiae.

Huisman SM, Bales OA, Bertrand M, Smeets MF, Reed SI, Segal M - J. Cell Biol. (2004)

Bottom Line: Here, we show that Kar9p does not mediate Bud6p functions in spindle orientation.Thus, Kar9p-independent capture at Bud6p sites can effect spindle orientation provided MT turnover is reduced.Together, these results demonstrate Bud6p function in MT capture at the cell cortex, independent of Kar9p-mediated MT delivery along actin cables.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, CB2 3EH UK.

ABSTRACT
In Saccharomyces cerevisiae, spindle orientation is controlled by a temporal and spatial program of microtubule (MT)-cortex interactions. This program requires Bud6p/Aip3p to direct the old pole to the bud and confine the new pole to the mother cell. Bud6p function has been linked to Kar9p, a protein guiding MTs along actin cables. Here, we show that Kar9p does not mediate Bud6p functions in spindle orientation. Based on live microscopy analysis, kar9Delta cells maintained Bud6p-dependent MT capture. Conversely, bud6Delta cells supported Kar9p-associated MT delivery to the bud. Moreover, additive phenotypes in bud6Delta kar9Delta or bud6Delta dyn1Delta mutants underscored the separate contributions of Bud6p, Kar9p, and dynein to spindle positioning. Finally, tub2C354S, a mutation decreasing MT dynamics, suppressed a kar9Delta mutation in a BUD6-dependent manner. Thus, Kar9p-independent capture at Bud6p sites can effect spindle orientation provided MT turnover is reduced. Together, these results demonstrate Bud6p function in MT capture at the cell cortex, independent of Kar9p-mediated MT delivery along actin cables.

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Effect of decreased MT turnover on orientation of MT–cortex interactions in kar9Δ cells. (A) Astral MT–cortex interactions in the indicated strains expressing GFP-Tub1 (number of cells recorded: 81 wild type, 77 kar9Δ, 64 kar9Δ tub2C354S, 52 kar9Δ tub2C354S bud6Δ, 65 kar9Δ bud6Δ, 51 bud6Δ tub2C354S, and 45 bud6Δ) were scored by cell compartment (mother, bud neck, or bud) from bud emergence to preanaphase spindle assembly. n = total number of interactions. Error bars indicate 95% confidence limits. (B–E) Selected frames from representative time-lapse series showing orientation of MT–cortex interactions in kar9Δ tub2C354S GFP:TUB1 cells. (B) Early orientation of astral MT–cortex interactions toward the prebud site occurred after mitotic exit (9.0 min). Orientation was maintained throughout bud emergence (16.0–34.5 min). DIC images corresponding to 0, 9.0, 16.5, and 34.5 min are shown. (C) A small budded cell showing an astral MT oriented toward the bud (0 min) maintained its orientation throughout spindle assembly and alignment (18.0–23.0 min) of the preanaphase spindle along the mother-bud axis. DIC images correspond to the first and last frame of the series. (D) Selected frames from a time-lapse series showing preanaphase spindle orientation. A cell completed spindle assembly (0–7.5 min), maintaining MT–bud cortex interactions during spindle alignment. (E) Selected frames from a time-lapse series showing correct SPB orientation before spindle assembly (0–6 min). MT–cortex interactions within the bud continued throughout spindle assembly and orientation (56 min). The spindle was correctly aligned as the cell proceeded through anaphase (62–66 min). (F and G) Time-lapse series showing failure to orient MTs toward the emerging bud in a kar9Δ bud6Δ tub2C354S GFP:TUB1 cell. (F) Astral MTs interacted with the cell cortex away from the bud. DIC images correspond to the first and last frame. (G) Defective preanaphase spindle orientation (0–15.0 min) and misaligned spindle elongation (20.0–29.0 min) followed by spindle orientation through dynein-dependent interactions (44.5–53.0 min) in mid-anaphase. Numbers indicate time elapsed in minutes. Bars, 2 μm.
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fig9: Effect of decreased MT turnover on orientation of MT–cortex interactions in kar9Δ cells. (A) Astral MT–cortex interactions in the indicated strains expressing GFP-Tub1 (number of cells recorded: 81 wild type, 77 kar9Δ, 64 kar9Δ tub2C354S, 52 kar9Δ tub2C354S bud6Δ, 65 kar9Δ bud6Δ, 51 bud6Δ tub2C354S, and 45 bud6Δ) were scored by cell compartment (mother, bud neck, or bud) from bud emergence to preanaphase spindle assembly. n = total number of interactions. Error bars indicate 95% confidence limits. (B–E) Selected frames from representative time-lapse series showing orientation of MT–cortex interactions in kar9Δ tub2C354S GFP:TUB1 cells. (B) Early orientation of astral MT–cortex interactions toward the prebud site occurred after mitotic exit (9.0 min). Orientation was maintained throughout bud emergence (16.0–34.5 min). DIC images corresponding to 0, 9.0, 16.5, and 34.5 min are shown. (C) A small budded cell showing an astral MT oriented toward the bud (0 min) maintained its orientation throughout spindle assembly and alignment (18.0–23.0 min) of the preanaphase spindle along the mother-bud axis. DIC images correspond to the first and last frame of the series. (D) Selected frames from a time-lapse series showing preanaphase spindle orientation. A cell completed spindle assembly (0–7.5 min), maintaining MT–bud cortex interactions during spindle alignment. (E) Selected frames from a time-lapse series showing correct SPB orientation before spindle assembly (0–6 min). MT–cortex interactions within the bud continued throughout spindle assembly and orientation (56 min). The spindle was correctly aligned as the cell proceeded through anaphase (62–66 min). (F and G) Time-lapse series showing failure to orient MTs toward the emerging bud in a kar9Δ bud6Δ tub2C354S GFP:TUB1 cell. (F) Astral MTs interacted with the cell cortex away from the bud. DIC images correspond to the first and last frame. (G) Defective preanaphase spindle orientation (0–15.0 min) and misaligned spindle elongation (20.0–29.0 min) followed by spindle orientation through dynein-dependent interactions (44.5–53.0 min) in mid-anaphase. Numbers indicate time elapsed in minutes. Bars, 2 μm.

Mentions: Second, onset of spindle elongation along the mother-bud axis was markedly reduced (33%, n = 39), possibly due to the additional absence of MT interactions with the bud neck (Fig. 7 B and see last section of Results describing Fig. 9 A). This contrasted with kar9Δ cells (Table I), in which MT interactions with the bud neck still contribute toward orientation (Segal et al., 2000b; and see last section of Results describing Fig. 9 A). Initial spindle elongation within the mother cell in kar9Δ bud6Δ cells was followed by dynein-driven positioning of the spindle part way through anaphase (Fig. 7 B) as in kar9Δ cells (Segal et al., 2000b; Yeh et al., 2000).


Differential contribution of Bud6p and Kar9p to microtubule capture and spindle orientation in S. cerevisiae.

Huisman SM, Bales OA, Bertrand M, Smeets MF, Reed SI, Segal M - J. Cell Biol. (2004)

Effect of decreased MT turnover on orientation of MT–cortex interactions in kar9Δ cells. (A) Astral MT–cortex interactions in the indicated strains expressing GFP-Tub1 (number of cells recorded: 81 wild type, 77 kar9Δ, 64 kar9Δ tub2C354S, 52 kar9Δ tub2C354S bud6Δ, 65 kar9Δ bud6Δ, 51 bud6Δ tub2C354S, and 45 bud6Δ) were scored by cell compartment (mother, bud neck, or bud) from bud emergence to preanaphase spindle assembly. n = total number of interactions. Error bars indicate 95% confidence limits. (B–E) Selected frames from representative time-lapse series showing orientation of MT–cortex interactions in kar9Δ tub2C354S GFP:TUB1 cells. (B) Early orientation of astral MT–cortex interactions toward the prebud site occurred after mitotic exit (9.0 min). Orientation was maintained throughout bud emergence (16.0–34.5 min). DIC images corresponding to 0, 9.0, 16.5, and 34.5 min are shown. (C) A small budded cell showing an astral MT oriented toward the bud (0 min) maintained its orientation throughout spindle assembly and alignment (18.0–23.0 min) of the preanaphase spindle along the mother-bud axis. DIC images correspond to the first and last frame of the series. (D) Selected frames from a time-lapse series showing preanaphase spindle orientation. A cell completed spindle assembly (0–7.5 min), maintaining MT–bud cortex interactions during spindle alignment. (E) Selected frames from a time-lapse series showing correct SPB orientation before spindle assembly (0–6 min). MT–cortex interactions within the bud continued throughout spindle assembly and orientation (56 min). The spindle was correctly aligned as the cell proceeded through anaphase (62–66 min). (F and G) Time-lapse series showing failure to orient MTs toward the emerging bud in a kar9Δ bud6Δ tub2C354S GFP:TUB1 cell. (F) Astral MTs interacted with the cell cortex away from the bud. DIC images correspond to the first and last frame. (G) Defective preanaphase spindle orientation (0–15.0 min) and misaligned spindle elongation (20.0–29.0 min) followed by spindle orientation through dynein-dependent interactions (44.5–53.0 min) in mid-anaphase. Numbers indicate time elapsed in minutes. Bars, 2 μm.
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Related In: Results  -  Collection

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fig9: Effect of decreased MT turnover on orientation of MT–cortex interactions in kar9Δ cells. (A) Astral MT–cortex interactions in the indicated strains expressing GFP-Tub1 (number of cells recorded: 81 wild type, 77 kar9Δ, 64 kar9Δ tub2C354S, 52 kar9Δ tub2C354S bud6Δ, 65 kar9Δ bud6Δ, 51 bud6Δ tub2C354S, and 45 bud6Δ) were scored by cell compartment (mother, bud neck, or bud) from bud emergence to preanaphase spindle assembly. n = total number of interactions. Error bars indicate 95% confidence limits. (B–E) Selected frames from representative time-lapse series showing orientation of MT–cortex interactions in kar9Δ tub2C354S GFP:TUB1 cells. (B) Early orientation of astral MT–cortex interactions toward the prebud site occurred after mitotic exit (9.0 min). Orientation was maintained throughout bud emergence (16.0–34.5 min). DIC images corresponding to 0, 9.0, 16.5, and 34.5 min are shown. (C) A small budded cell showing an astral MT oriented toward the bud (0 min) maintained its orientation throughout spindle assembly and alignment (18.0–23.0 min) of the preanaphase spindle along the mother-bud axis. DIC images correspond to the first and last frame of the series. (D) Selected frames from a time-lapse series showing preanaphase spindle orientation. A cell completed spindle assembly (0–7.5 min), maintaining MT–bud cortex interactions during spindle alignment. (E) Selected frames from a time-lapse series showing correct SPB orientation before spindle assembly (0–6 min). MT–cortex interactions within the bud continued throughout spindle assembly and orientation (56 min). The spindle was correctly aligned as the cell proceeded through anaphase (62–66 min). (F and G) Time-lapse series showing failure to orient MTs toward the emerging bud in a kar9Δ bud6Δ tub2C354S GFP:TUB1 cell. (F) Astral MTs interacted with the cell cortex away from the bud. DIC images correspond to the first and last frame. (G) Defective preanaphase spindle orientation (0–15.0 min) and misaligned spindle elongation (20.0–29.0 min) followed by spindle orientation through dynein-dependent interactions (44.5–53.0 min) in mid-anaphase. Numbers indicate time elapsed in minutes. Bars, 2 μm.
Mentions: Second, onset of spindle elongation along the mother-bud axis was markedly reduced (33%, n = 39), possibly due to the additional absence of MT interactions with the bud neck (Fig. 7 B and see last section of Results describing Fig. 9 A). This contrasted with kar9Δ cells (Table I), in which MT interactions with the bud neck still contribute toward orientation (Segal et al., 2000b; and see last section of Results describing Fig. 9 A). Initial spindle elongation within the mother cell in kar9Δ bud6Δ cells was followed by dynein-driven positioning of the spindle part way through anaphase (Fig. 7 B) as in kar9Δ cells (Segal et al., 2000b; Yeh et al., 2000).

Bottom Line: Here, we show that Kar9p does not mediate Bud6p functions in spindle orientation.Thus, Kar9p-independent capture at Bud6p sites can effect spindle orientation provided MT turnover is reduced.Together, these results demonstrate Bud6p function in MT capture at the cell cortex, independent of Kar9p-mediated MT delivery along actin cables.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, CB2 3EH UK.

ABSTRACT
In Saccharomyces cerevisiae, spindle orientation is controlled by a temporal and spatial program of microtubule (MT)-cortex interactions. This program requires Bud6p/Aip3p to direct the old pole to the bud and confine the new pole to the mother cell. Bud6p function has been linked to Kar9p, a protein guiding MTs along actin cables. Here, we show that Kar9p does not mediate Bud6p functions in spindle orientation. Based on live microscopy analysis, kar9Delta cells maintained Bud6p-dependent MT capture. Conversely, bud6Delta cells supported Kar9p-associated MT delivery to the bud. Moreover, additive phenotypes in bud6Delta kar9Delta or bud6Delta dyn1Delta mutants underscored the separate contributions of Bud6p, Kar9p, and dynein to spindle positioning. Finally, tub2C354S, a mutation decreasing MT dynamics, suppressed a kar9Delta mutation in a BUD6-dependent manner. Thus, Kar9p-independent capture at Bud6p sites can effect spindle orientation provided MT turnover is reduced. Together, these results demonstrate Bud6p function in MT capture at the cell cortex, independent of Kar9p-mediated MT delivery along actin cables.

Show MeSH