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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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Spindle defects in bud6Δ dyn1Δ cells. (A) Spindle elongation and translocation of the SPBd into the bud are coupled in early anaphase of wild-type cells. Representative time-lapse series showing correct insertion of the spindle at the bud neck (0 min, 6.0 min) and initiation of spindle elongation across the bud neck (2.5 min, 8.5 min). For quantitative information see Table II. (B and C) Initiation of spindle elongation in dyn1Δ GFP:TUB1 cells occurred along the mother-bud axis but translocation of the SPBd into the bud was delayed. (B) SPBd translocation into the bud followed elongation of the spindle within the mother cell (9.0–12.0 min) without significant delay. (C) At anaphase the spindle rotated to become misaligned (25.5–43.0 min). (D–F) Spindle defects in bud6Δ dyn1Δ GFP:TUB1 cells. (D) After initiation of spindle elongation (1.5–2.5 min) the spindle became oriented along the mother-bud axis and continued elongation past the bud neck (5.0–9.0 min). (E) Spindle morphologies underscoring the genetic interaction between dyn1Δ and bud6Δ observed in asynchronous cells. Pairs of DIC and fluorescence images are shown. (a) Initiation of anaphase in the mother cell as in dyn1Δ cells. (b–d) Disrupted spindle polarity in anaphase. (d) Spindle elongation continued within the mother cell causing the spindle to curve. (e) Spindle disassembly in a mother cell followed by rebudding. (f) Cells containing multiple SPBs reached 10% in asynchronous populations. (F) Time-lapse series showing spindle disassembly (7.5–10.0 min) within the mother cell. Numbers indicate time elapsed in minutes. Bars, 2 μm.
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fig8: Spindle defects in bud6Δ dyn1Δ cells. (A) Spindle elongation and translocation of the SPBd into the bud are coupled in early anaphase of wild-type cells. Representative time-lapse series showing correct insertion of the spindle at the bud neck (0 min, 6.0 min) and initiation of spindle elongation across the bud neck (2.5 min, 8.5 min). For quantitative information see Table II. (B and C) Initiation of spindle elongation in dyn1Δ GFP:TUB1 cells occurred along the mother-bud axis but translocation of the SPBd into the bud was delayed. (B) SPBd translocation into the bud followed elongation of the spindle within the mother cell (9.0–12.0 min) without significant delay. (C) At anaphase the spindle rotated to become misaligned (25.5–43.0 min). (D–F) Spindle defects in bud6Δ dyn1Δ GFP:TUB1 cells. (D) After initiation of spindle elongation (1.5–2.5 min) the spindle became oriented along the mother-bud axis and continued elongation past the bud neck (5.0–9.0 min). (E) Spindle morphologies underscoring the genetic interaction between dyn1Δ and bud6Δ observed in asynchronous cells. Pairs of DIC and fluorescence images are shown. (a) Initiation of anaphase in the mother cell as in dyn1Δ cells. (b–d) Disrupted spindle polarity in anaphase. (d) Spindle elongation continued within the mother cell causing the spindle to curve. (e) Spindle disassembly in a mother cell followed by rebudding. (f) Cells containing multiple SPBs reached 10% in asynchronous populations. (F) Time-lapse series showing spindle disassembly (7.5–10.0 min) within the mother cell. Numbers indicate time elapsed in minutes. Bars, 2 μm.

Mentions: We also determined the effects of deleting BUD6 on spindle phenotypes of dyn1Δ cells, with particular focus on anaphase. In wild-type cells, the “fast phase” of spindle elongation (Fig. 8 A, 0–2.5 min and 6.0–10 min) is typically coupled to translocation of the SPBd into the bud (Yeh et al., 1995). SPB translocation is delayed in dyn1Δ mutants, although spindle elongation still begins along the mother-bud axis (Table II and Fig. 8 B). Spindles only became overtly misaligned during anaphase in 9% of cells (n = 35) after initial elongation along the mother-bud axis (Fig. 8 C).


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)

Spindle defects in bud6Δ dyn1Δ cells. (A) Spindle elongation and translocation of the SPBd into the bud are coupled in early anaphase of wild-type cells. Representative time-lapse series showing correct insertion of the spindle at the bud neck (0 min, 6.0 min) and initiation of spindle elongation across the bud neck (2.5 min, 8.5 min). For quantitative information see Table II. (B and C) Initiation of spindle elongation in dyn1Δ GFP:TUB1 cells occurred along the mother-bud axis but translocation of the SPBd into the bud was delayed. (B) SPBd translocation into the bud followed elongation of the spindle within the mother cell (9.0–12.0 min) without significant delay. (C) At anaphase the spindle rotated to become misaligned (25.5–43.0 min). (D–F) Spindle defects in bud6Δ dyn1Δ GFP:TUB1 cells. (D) After initiation of spindle elongation (1.5–2.5 min) the spindle became oriented along the mother-bud axis and continued elongation past the bud neck (5.0–9.0 min). (E) Spindle morphologies underscoring the genetic interaction between dyn1Δ and bud6Δ observed in asynchronous cells. Pairs of DIC and fluorescence images are shown. (a) Initiation of anaphase in the mother cell as in dyn1Δ cells. (b–d) Disrupted spindle polarity in anaphase. (d) Spindle elongation continued within the mother cell causing the spindle to curve. (e) Spindle disassembly in a mother cell followed by rebudding. (f) Cells containing multiple SPBs reached 10% in asynchronous populations. (F) Time-lapse series showing spindle disassembly (7.5–10.0 min) within the mother cell. Numbers indicate time elapsed in minutes. Bars, 2 μm.
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fig8: Spindle defects in bud6Δ dyn1Δ cells. (A) Spindle elongation and translocation of the SPBd into the bud are coupled in early anaphase of wild-type cells. Representative time-lapse series showing correct insertion of the spindle at the bud neck (0 min, 6.0 min) and initiation of spindle elongation across the bud neck (2.5 min, 8.5 min). For quantitative information see Table II. (B and C) Initiation of spindle elongation in dyn1Δ GFP:TUB1 cells occurred along the mother-bud axis but translocation of the SPBd into the bud was delayed. (B) SPBd translocation into the bud followed elongation of the spindle within the mother cell (9.0–12.0 min) without significant delay. (C) At anaphase the spindle rotated to become misaligned (25.5–43.0 min). (D–F) Spindle defects in bud6Δ dyn1Δ GFP:TUB1 cells. (D) After initiation of spindle elongation (1.5–2.5 min) the spindle became oriented along the mother-bud axis and continued elongation past the bud neck (5.0–9.0 min). (E) Spindle morphologies underscoring the genetic interaction between dyn1Δ and bud6Δ observed in asynchronous cells. Pairs of DIC and fluorescence images are shown. (a) Initiation of anaphase in the mother cell as in dyn1Δ cells. (b–d) Disrupted spindle polarity in anaphase. (d) Spindle elongation continued within the mother cell causing the spindle to curve. (e) Spindle disassembly in a mother cell followed by rebudding. (f) Cells containing multiple SPBs reached 10% in asynchronous populations. (F) Time-lapse series showing spindle disassembly (7.5–10.0 min) within the mother cell. Numbers indicate time elapsed in minutes. Bars, 2 μm.
Mentions: We also determined the effects of deleting BUD6 on spindle phenotypes of dyn1Δ cells, with particular focus on anaphase. In wild-type cells, the “fast phase” of spindle elongation (Fig. 8 A, 0–2.5 min and 6.0–10 min) is typically coupled to translocation of the SPBd into the bud (Yeh et al., 1995). SPB translocation is delayed in dyn1Δ mutants, although spindle elongation still begins along the mother-bud axis (Table II and Fig. 8 B). Spindles only became overtly misaligned during anaphase in 9% of cells (n = 35) after initial elongation along the mother-bud axis (Fig. 8 C).

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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