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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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Analysis of SPB movement coupled to Kar9p-bound MTs. (A) SPB movement associated with Kar9p return to the pole in wild-type or bud6Δ cells was categorized as described in Materials and methods. Movement coupled to all Kar9p returns to the pole by either MT shrinkage or translocation was scored in time-lapse recordings described in Fig. 4. Error bars indicate 95% confidence limits. (B and C) Selected frames from a time-lapse series showing orientation of an MT decorated by Kar9-GFP at the plus end in a wild-type cell. Overlays of fluorescence images of CFP-Tub1 (red) and Kar9-GFP (green), and the DIC image corresponding to the first frame are shown. (B) A small-budded cell with the SPB already positioned near the bud neck showed Kar9-GFP localization at the plus end of a growing MT (15 s). After orientation toward the bud (30–75 s) the MT shrunk (arrows), bringing Kar9-GFP toward the SPB. Shrinkage was not coupled to SPB movement toward the bud (105–120 s). (C) Absence of coupling between shrinkage of an MT bound to Kar9-GFP (arrows) and SPB movement in a cell exhibiting an oriented preanaphase spindle. Numbers indicate time elapsed in seconds. Bars, 2 μm.
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fig5: Analysis of SPB movement coupled to Kar9p-bound MTs. (A) SPB movement associated with Kar9p return to the pole in wild-type or bud6Δ cells was categorized as described in Materials and methods. Movement coupled to all Kar9p returns to the pole by either MT shrinkage or translocation was scored in time-lapse recordings described in Fig. 4. Error bars indicate 95% confidence limits. (B and C) Selected frames from a time-lapse series showing orientation of an MT decorated by Kar9-GFP at the plus end in a wild-type cell. Overlays of fluorescence images of CFP-Tub1 (red) and Kar9-GFP (green), and the DIC image corresponding to the first frame are shown. (B) A small-budded cell with the SPB already positioned near the bud neck showed Kar9-GFP localization at the plus end of a growing MT (15 s). After orientation toward the bud (30–75 s) the MT shrunk (arrows), bringing Kar9-GFP toward the SPB. Shrinkage was not coupled to SPB movement toward the bud (105–120 s). (C) Absence of coupling between shrinkage of an MT bound to Kar9-GFP (arrows) and SPB movement in a cell exhibiting an oriented preanaphase spindle. Numbers indicate time elapsed in seconds. Bars, 2 μm.

Mentions: To correlate the direction of movement with Kar9p dynamic behavior, all events involving Kar9p return to the spindle pole were scored for coupled SPB movement toward the cortex. As shown in Fig. 5 A, there was no correlation between the direction of SPB movement and shrinkage of Kar9p-bound MTs in wild-type or bud6Δ cells (< 7%, n > 120). In fact, the SPB remained stationary relative to the cortex in the majority of Kar9p returns to the pole.


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)

Analysis of SPB movement coupled to Kar9p-bound MTs. (A) SPB movement associated with Kar9p return to the pole in wild-type or bud6Δ cells was categorized as described in Materials and methods. Movement coupled to all Kar9p returns to the pole by either MT shrinkage or translocation was scored in time-lapse recordings described in Fig. 4. Error bars indicate 95% confidence limits. (B and C) Selected frames from a time-lapse series showing orientation of an MT decorated by Kar9-GFP at the plus end in a wild-type cell. Overlays of fluorescence images of CFP-Tub1 (red) and Kar9-GFP (green), and the DIC image corresponding to the first frame are shown. (B) A small-budded cell with the SPB already positioned near the bud neck showed Kar9-GFP localization at the plus end of a growing MT (15 s). After orientation toward the bud (30–75 s) the MT shrunk (arrows), bringing Kar9-GFP toward the SPB. Shrinkage was not coupled to SPB movement toward the bud (105–120 s). (C) Absence of coupling between shrinkage of an MT bound to Kar9-GFP (arrows) and SPB movement in a cell exhibiting an oriented preanaphase spindle. Numbers indicate time elapsed in seconds. Bars, 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172562&req=5

fig5: Analysis of SPB movement coupled to Kar9p-bound MTs. (A) SPB movement associated with Kar9p return to the pole in wild-type or bud6Δ cells was categorized as described in Materials and methods. Movement coupled to all Kar9p returns to the pole by either MT shrinkage or translocation was scored in time-lapse recordings described in Fig. 4. Error bars indicate 95% confidence limits. (B and C) Selected frames from a time-lapse series showing orientation of an MT decorated by Kar9-GFP at the plus end in a wild-type cell. Overlays of fluorescence images of CFP-Tub1 (red) and Kar9-GFP (green), and the DIC image corresponding to the first frame are shown. (B) A small-budded cell with the SPB already positioned near the bud neck showed Kar9-GFP localization at the plus end of a growing MT (15 s). After orientation toward the bud (30–75 s) the MT shrunk (arrows), bringing Kar9-GFP toward the SPB. Shrinkage was not coupled to SPB movement toward the bud (105–120 s). (C) Absence of coupling between shrinkage of an MT bound to Kar9-GFP (arrows) and SPB movement in a cell exhibiting an oriented preanaphase spindle. Numbers indicate time elapsed in seconds. Bars, 2 μm.
Mentions: To correlate the direction of movement with Kar9p dynamic behavior, all events involving Kar9p return to the spindle pole were scored for coupled SPB movement toward the cortex. As shown in Fig. 5 A, there was no correlation between the direction of SPB movement and shrinkage of Kar9p-bound MTs in wild-type or bud6Δ cells (< 7%, n > 120). In fact, the SPB remained stationary relative to the cortex in the majority of Kar9p returns to the pole.

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