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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 orientation phenotypes in bud6Δ kar9Δ cells. Selected frames from representative time-lapse series showing spindle orientation defects in bud6Δ kar9Δ GFP:TUB1 cells. For quantitative information see Table I. (A) A misoriented preanaphase spindle (0–11.0 min) became aligned as an astral MT interacted with the bud cortex (12.0 min). The interaction, however, resulted in the translocation of the spindle past the bud neck (17.0–18.0 min). At onset of anaphase (18.5 min) an MT interacting with the mother cell cortex repositioned one pole past the bud neck within the mother cell as the spindle elongated. (B) A misaligned spindle began elongation within the mother cell in the absence of MT interactions with the bud neck (2.5–20.5 min). The spindle became oriented by dynein-driven sliding that pulled the mid-anaphase spindle into the bud (27.5–29.0 min). (C) A spindle that oriented at onset of anaphase along the mother-bud axis (4.5 min) and initiated spindle elongation across the bud neck (6.0–7.0 min) became misaligned part way through anaphase (8.0–8.5 min) but rapidly repositioned (9.0–10.0 min) to proceed with anaphase. Numbers indicate time elapsed in minutes. Bars, 2 μm.
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fig7: Spindle orientation phenotypes in bud6Δ kar9Δ cells. Selected frames from representative time-lapse series showing spindle orientation defects in bud6Δ kar9Δ GFP:TUB1 cells. For quantitative information see Table I. (A) A misoriented preanaphase spindle (0–11.0 min) became aligned as an astral MT interacted with the bud cortex (12.0 min). The interaction, however, resulted in the translocation of the spindle past the bud neck (17.0–18.0 min). At onset of anaphase (18.5 min) an MT interacting with the mother cell cortex repositioned one pole past the bud neck within the mother cell as the spindle elongated. (B) A misaligned spindle began elongation within the mother cell in the absence of MT interactions with the bud neck (2.5–20.5 min). The spindle became oriented by dynein-driven sliding that pulled the mid-anaphase spindle into the bud (27.5–29.0 min). (C) A spindle that oriented at onset of anaphase along the mother-bud axis (4.5 min) and initiated spindle elongation across the bud neck (6.0–7.0 min) became misaligned part way through anaphase (8.0–8.5 min) but rapidly repositioned (9.0–10.0 min) to proceed with anaphase. Numbers indicate time elapsed in minutes. Bars, 2 μm.

Mentions: Astral MT behavior and spindle orientation defects observed in bud6Δ kar9Δ cells demonstrated the additive impact of deleting BUD6 over a single kar9Δ mutation. Early orientation of astral MTs toward the emerging bud was abolished as in single bud6Δ mutants (not depicted; Segal et al., 2002), whereas spindle positioning in the double mutant was markedly impaired relative to either single mutant (Table I and Fig. 7).


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 orientation phenotypes in bud6Δ kar9Δ cells. Selected frames from representative time-lapse series showing spindle orientation defects in bud6Δ kar9Δ GFP:TUB1 cells. For quantitative information see Table I. (A) A misoriented preanaphase spindle (0–11.0 min) became aligned as an astral MT interacted with the bud cortex (12.0 min). The interaction, however, resulted in the translocation of the spindle past the bud neck (17.0–18.0 min). At onset of anaphase (18.5 min) an MT interacting with the mother cell cortex repositioned one pole past the bud neck within the mother cell as the spindle elongated. (B) A misaligned spindle began elongation within the mother cell in the absence of MT interactions with the bud neck (2.5–20.5 min). The spindle became oriented by dynein-driven sliding that pulled the mid-anaphase spindle into the bud (27.5–29.0 min). (C) A spindle that oriented at onset of anaphase along the mother-bud axis (4.5 min) and initiated spindle elongation across the bud neck (6.0–7.0 min) became misaligned part way through anaphase (8.0–8.5 min) but rapidly repositioned (9.0–10.0 min) to proceed with anaphase. Numbers indicate time elapsed in minutes. Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172562&req=5

fig7: Spindle orientation phenotypes in bud6Δ kar9Δ cells. Selected frames from representative time-lapse series showing spindle orientation defects in bud6Δ kar9Δ GFP:TUB1 cells. For quantitative information see Table I. (A) A misoriented preanaphase spindle (0–11.0 min) became aligned as an astral MT interacted with the bud cortex (12.0 min). The interaction, however, resulted in the translocation of the spindle past the bud neck (17.0–18.0 min). At onset of anaphase (18.5 min) an MT interacting with the mother cell cortex repositioned one pole past the bud neck within the mother cell as the spindle elongated. (B) A misaligned spindle began elongation within the mother cell in the absence of MT interactions with the bud neck (2.5–20.5 min). The spindle became oriented by dynein-driven sliding that pulled the mid-anaphase spindle into the bud (27.5–29.0 min). (C) A spindle that oriented at onset of anaphase along the mother-bud axis (4.5 min) and initiated spindle elongation across the bud neck (6.0–7.0 min) became misaligned part way through anaphase (8.0–8.5 min) but rapidly repositioned (9.0–10.0 min) to proceed with anaphase. Numbers indicate time elapsed in minutes. Bars, 2 μm.
Mentions: Astral MT behavior and spindle orientation defects observed in bud6Δ kar9Δ cells demonstrated the additive impact of deleting BUD6 over a single kar9Δ mutation. Early orientation of astral MTs toward the emerging bud was abolished as in single bud6Δ mutants (not depicted; Segal et al., 2002), whereas spindle positioning in the double mutant was markedly impaired relative to either single mutant (Table I and Fig. 7).

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
Related in: MedlinePlus