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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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Kar9p-mediated MT transport in a bud6Δ cell. Selected frames from a time-lapse series showing Kar9p-mediated orientation of the SPB in a bud6Δ cell expressing CFP-Tub1 and Kar9-GFP. Overlays of CFP-Tub1 (red) and Kar9-GFP (green) images are shown. The DIC images correspond to 0 and 9 min (arrowhead indicates the bud). Initially, the SPB was positioned far from the prebud site (0–1.5 min). As Kar9-GFP occupied the plus end of an MT (1.5–3.0 min, arrows), the SPB became rapidly oriented toward the budding site (5 min). Kar9-GFP then continued to direct MTs into the growing bud (5–9 min). Numbers indicate time elapsed in minutes. Bar, 2 μm.
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fig6: Kar9p-mediated MT transport in a bud6Δ cell. Selected frames from a time-lapse series showing Kar9p-mediated orientation of the SPB in a bud6Δ cell expressing CFP-Tub1 and Kar9-GFP. Overlays of CFP-Tub1 (red) and Kar9-GFP (green) images are shown. The DIC images correspond to 0 and 9 min (arrowhead indicates the bud). Initially, the SPB was positioned far from the prebud site (0–1.5 min). As Kar9-GFP occupied the plus end of an MT (1.5–3.0 min, arrows), the SPB became rapidly oriented toward the budding site (5 min). Kar9-GFP then continued to direct MTs into the growing bud (5–9 min). Numbers indicate time elapsed in minutes. Bar, 2 μm.

Mentions: In bud6Δ cells SPBs were initially present away from the bud and became quickly repositioned as a Kar9p-bound MT moved toward the bud without observable shrinkage, presumably, along an actin cable (Fig. 6, arrows). This type of processive MT movements toward the bud were absent in bud6Δ kar9Δ cells (n > 150 MTs). Thus, a bud6Δ mutant supported Kar9p-dependent MT orientation and relied on long-range Kar9p-bound transport to compensate for lack of early MT–cortex interactions with the new bud and the inability to mobilize SPBs by MT shrinkage (Segal et al., 2002).


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)

Kar9p-mediated MT transport in a bud6Δ cell. Selected frames from a time-lapse series showing Kar9p-mediated orientation of the SPB in a bud6Δ cell expressing CFP-Tub1 and Kar9-GFP. Overlays of CFP-Tub1 (red) and Kar9-GFP (green) images are shown. The DIC images correspond to 0 and 9 min (arrowhead indicates the bud). Initially, the SPB was positioned far from the prebud site (0–1.5 min). As Kar9-GFP occupied the plus end of an MT (1.5–3.0 min, arrows), the SPB became rapidly oriented toward the budding site (5 min). Kar9-GFP then continued to direct MTs into the growing bud (5–9 min). Numbers indicate time elapsed in minutes. Bar, 2 μm.
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Related In: Results  -  Collection

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fig6: Kar9p-mediated MT transport in a bud6Δ cell. Selected frames from a time-lapse series showing Kar9p-mediated orientation of the SPB in a bud6Δ cell expressing CFP-Tub1 and Kar9-GFP. Overlays of CFP-Tub1 (red) and Kar9-GFP (green) images are shown. The DIC images correspond to 0 and 9 min (arrowhead indicates the bud). Initially, the SPB was positioned far from the prebud site (0–1.5 min). As Kar9-GFP occupied the plus end of an MT (1.5–3.0 min, arrows), the SPB became rapidly oriented toward the budding site (5 min). Kar9-GFP then continued to direct MTs into the growing bud (5–9 min). Numbers indicate time elapsed in minutes. Bar, 2 μm.
Mentions: In bud6Δ cells SPBs were initially present away from the bud and became quickly repositioned as a Kar9p-bound MT moved toward the bud without observable shrinkage, presumably, along an actin cable (Fig. 6, arrows). This type of processive MT movements toward the bud were absent in bud6Δ kar9Δ cells (n > 150 MTs). Thus, a bud6Δ mutant supported Kar9p-dependent MT orientation and relied on long-range Kar9p-bound transport to compensate for lack of early MT–cortex interactions with the new bud and the inability to mobilize SPBs by MT shrinkage (Segal et al., 2002).

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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