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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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Association of Kar9-GFP to SPBs during spindle assembly in wild-type cells. Selected frames of time-lapse series of wild-type cells expressing Kar9-GFP (overlaid in green) and CFP-Tub1 (red) showing Kar9p recruitment at both SPBs during the assembly of the mitotic spindle. The DIC image corresponds to the first frame. (A) Kar9-GFP associated with MTs from both SPBs. In the cell on the left, the label decorated one pole by 90 s (arrow). The cell on the right exhibited Kar9-GFP label on both poles during SPB separation (0–105 s). The label was present on a single pole by 150 s (arrow). (B) Kar9-GFP decorated both poles during the early part of spindle assembly (0–46 s). The time lapse also shows that SPB movement toward the bud did not coincide with the shrinkage of an MT decorated by Kar9-GFP (72–108 s, arrows). Instead, movement toward the bud occurred as an MT grew (168–192 s, arrows) and then maintained a constant length (192–216 s, arrows). (C) Kar9-GFP initially localized to MTs emanating from both SPBs (0 s) but only one pole established interactions with the bud (60–120 s, arrows), whereas label on the second pole progressively disappeared. Numbers indicate time elapsed in seconds. Bars, 2 μm.
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fig3: Association of Kar9-GFP to SPBs during spindle assembly in wild-type cells. Selected frames of time-lapse series of wild-type cells expressing Kar9-GFP (overlaid in green) and CFP-Tub1 (red) showing Kar9p recruitment at both SPBs during the assembly of the mitotic spindle. The DIC image corresponds to the first frame. (A) Kar9-GFP associated with MTs from both SPBs. In the cell on the left, the label decorated one pole by 90 s (arrow). The cell on the right exhibited Kar9-GFP label on both poles during SPB separation (0–105 s). The label was present on a single pole by 150 s (arrow). (B) Kar9-GFP decorated both poles during the early part of spindle assembly (0–46 s). The time lapse also shows that SPB movement toward the bud did not coincide with the shrinkage of an MT decorated by Kar9-GFP (72–108 s, arrows). Instead, movement toward the bud occurred as an MT grew (168–192 s, arrows) and then maintained a constant length (192–216 s, arrows). (C) Kar9-GFP initially localized to MTs emanating from both SPBs (0 s) but only one pole established interactions with the bud (60–120 s, arrows), whereas label on the second pole progressively disappeared. Numbers indicate time elapsed in seconds. Bars, 2 μm.

Mentions: Decoration of MTs by Kar9-GFP was initiated by recruitment at the SPB in most cases, both in wild-type or bud6Δ cells (93.5%, n = 113 MTs and 96.2%, n = 104, respectively). Kar9p was detected at both SPBs at onset of spindle assembly (14 of 16 time-lapse series spanning spindle assembly) but was clearly asymmetric in spindles longer than 1.2 ± 0.2 μm (Fig. 3). Kar9p traveled along MTs toward the plus or minus end. In addition, Kar9p moved while fixed at the plus end of a growing or shrinking MT (Fig. 4 A). These modes of dynamic behavior occurred significantly in bud6Δ cells. However, the bud6Δ mutation slightly reduced Kar9p translocation along persistent MTs (Fig. 4 A, black and gray bars in wild type vs. bud6Δ).


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)

Association of Kar9-GFP to SPBs during spindle assembly in wild-type cells. Selected frames of time-lapse series of wild-type cells expressing Kar9-GFP (overlaid in green) and CFP-Tub1 (red) showing Kar9p recruitment at both SPBs during the assembly of the mitotic spindle. The DIC image corresponds to the first frame. (A) Kar9-GFP associated with MTs from both SPBs. In the cell on the left, the label decorated one pole by 90 s (arrow). The cell on the right exhibited Kar9-GFP label on both poles during SPB separation (0–105 s). The label was present on a single pole by 150 s (arrow). (B) Kar9-GFP decorated both poles during the early part of spindle assembly (0–46 s). The time lapse also shows that SPB movement toward the bud did not coincide with the shrinkage of an MT decorated by Kar9-GFP (72–108 s, arrows). Instead, movement toward the bud occurred as an MT grew (168–192 s, arrows) and then maintained a constant length (192–216 s, arrows). (C) Kar9-GFP initially localized to MTs emanating from both SPBs (0 s) but only one pole established interactions with the bud (60–120 s, arrows), whereas label on the second pole progressively disappeared. Numbers indicate time elapsed in seconds. Bars, 2 μm.
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Related In: Results  -  Collection

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fig3: Association of Kar9-GFP to SPBs during spindle assembly in wild-type cells. Selected frames of time-lapse series of wild-type cells expressing Kar9-GFP (overlaid in green) and CFP-Tub1 (red) showing Kar9p recruitment at both SPBs during the assembly of the mitotic spindle. The DIC image corresponds to the first frame. (A) Kar9-GFP associated with MTs from both SPBs. In the cell on the left, the label decorated one pole by 90 s (arrow). The cell on the right exhibited Kar9-GFP label on both poles during SPB separation (0–105 s). The label was present on a single pole by 150 s (arrow). (B) Kar9-GFP decorated both poles during the early part of spindle assembly (0–46 s). The time lapse also shows that SPB movement toward the bud did not coincide with the shrinkage of an MT decorated by Kar9-GFP (72–108 s, arrows). Instead, movement toward the bud occurred as an MT grew (168–192 s, arrows) and then maintained a constant length (192–216 s, arrows). (C) Kar9-GFP initially localized to MTs emanating from both SPBs (0 s) but only one pole established interactions with the bud (60–120 s, arrows), whereas label on the second pole progressively disappeared. Numbers indicate time elapsed in seconds. Bars, 2 μm.
Mentions: Decoration of MTs by Kar9-GFP was initiated by recruitment at the SPB in most cases, both in wild-type or bud6Δ cells (93.5%, n = 113 MTs and 96.2%, n = 104, respectively). Kar9p was detected at both SPBs at onset of spindle assembly (14 of 16 time-lapse series spanning spindle assembly) but was clearly asymmetric in spindles longer than 1.2 ± 0.2 μm (Fig. 3). Kar9p traveled along MTs toward the plus or minus end. In addition, Kar9p moved while fixed at the plus end of a growing or shrinking MT (Fig. 4 A). These modes of dynamic behavior occurred significantly in bud6Δ cells. However, the bud6Δ mutation slightly reduced Kar9p translocation along persistent MTs (Fig. 4 A, black and gray bars in wild type vs. bud6Δ).

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