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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 dynamic behavior in wild-type versus bud6Δ cells. (A) Kar9p dynamic behavior in association with MTs in wild type (107 cells recorded) or bud6Δ (99 cells recorded). Transits along MTs or movement at the MT plus end were as described in Materials and methods. Open boxes in each category indicate percentage of events confined to the bud neck. Error bars indicate 95% confidence limits. (B–D) Representative time-lapse series of Kar9p dynamic behavior in association with MTs oriented toward the bud in wild-type cells. Overlays of CFP-Tub1 (red) and Kar9-GFP (green) images and the DIC image corresponding to the first frame are shown. (B) After bud emergence, a cell exhibited Kar9-GFP transits along MTs oriented toward the emerging bud. Arrows at 4 and 5 min point to an MT reaching the cortex in the absence of Kar9-GFP at the MT plus end. A second MT is decorated by Kar9-GFP at the plus end (4–5 min). (C) Kar9-GFP transits along MTs in a cell before spindle assembly. In the absence of Kar9-GFP association, MTs interacted with the cell cortex. Of two MTs directed toward the bud at 4 min (arrowheads), only one is decorated at the plus end by Kar9-GFP (arrows). A line scan for fluorescence intensity along the overlapping MTs (4 min) is shown below (arrows show position of MT plus ends). (D) After spindle assembly, Kar9-GFP was bound to the MT plus end (0 min) of a shrinking MT (0.5 min) followed by localization at the SPB in the presence of an MT interacting with the cell cortex (3 min, arrows). Numbers indicate time elapsed in minutes. Bars, 2 μm.
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fig4: Kar9p dynamic behavior in wild-type versus bud6Δ cells. (A) Kar9p dynamic behavior in association with MTs in wild type (107 cells recorded) or bud6Δ (99 cells recorded). Transits along MTs or movement at the MT plus end were as described in Materials and methods. Open boxes in each category indicate percentage of events confined to the bud neck. Error bars indicate 95% confidence limits. (B–D) Representative time-lapse series of Kar9p dynamic behavior in association with MTs oriented toward the bud in wild-type cells. Overlays of CFP-Tub1 (red) and Kar9-GFP (green) images and the DIC image corresponding to the first frame are shown. (B) After bud emergence, a cell exhibited Kar9-GFP transits along MTs oriented toward the emerging bud. Arrows at 4 and 5 min point to an MT reaching the cortex in the absence of Kar9-GFP at the MT plus end. A second MT is decorated by Kar9-GFP at the plus end (4–5 min). (C) Kar9-GFP transits along MTs in a cell before spindle assembly. In the absence of Kar9-GFP association, MTs interacted with the cell cortex. Of two MTs directed toward the bud at 4 min (arrowheads), only one is decorated at the plus end by Kar9-GFP (arrows). A line scan for fluorescence intensity along the overlapping MTs (4 min) is shown below (arrows show position of MT plus ends). (D) After spindle assembly, Kar9-GFP was bound to the MT plus end (0 min) of a shrinking MT (0.5 min) followed by localization at the SPB in the presence of an MT interacting with the cell cortex (3 min, arrows). Numbers indicate time elapsed in minutes. 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)

Kar9p dynamic behavior in wild-type versus bud6Δ cells. (A) Kar9p dynamic behavior in association with MTs in wild type (107 cells recorded) or bud6Δ (99 cells recorded). Transits along MTs or movement at the MT plus end were as described in Materials and methods. Open boxes in each category indicate percentage of events confined to the bud neck. Error bars indicate 95% confidence limits. (B–D) Representative time-lapse series of Kar9p dynamic behavior in association with MTs oriented toward the bud in wild-type cells. Overlays of CFP-Tub1 (red) and Kar9-GFP (green) images and the DIC image corresponding to the first frame are shown. (B) After bud emergence, a cell exhibited Kar9-GFP transits along MTs oriented toward the emerging bud. Arrows at 4 and 5 min point to an MT reaching the cortex in the absence of Kar9-GFP at the MT plus end. A second MT is decorated by Kar9-GFP at the plus end (4–5 min). (C) Kar9-GFP transits along MTs in a cell before spindle assembly. In the absence of Kar9-GFP association, MTs interacted with the cell cortex. Of two MTs directed toward the bud at 4 min (arrowheads), only one is decorated at the plus end by Kar9-GFP (arrows). A line scan for fluorescence intensity along the overlapping MTs (4 min) is shown below (arrows show position of MT plus ends). (D) After spindle assembly, Kar9-GFP was bound to the MT plus end (0 min) of a shrinking MT (0.5 min) followed by localization at the SPB in the presence of an MT interacting with the cell cortex (3 min, arrows). Numbers indicate time elapsed in minutes. Bars, 2 μm.
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fig4: Kar9p dynamic behavior in wild-type versus bud6Δ cells. (A) Kar9p dynamic behavior in association with MTs in wild type (107 cells recorded) or bud6Δ (99 cells recorded). Transits along MTs or movement at the MT plus end were as described in Materials and methods. Open boxes in each category indicate percentage of events confined to the bud neck. Error bars indicate 95% confidence limits. (B–D) Representative time-lapse series of Kar9p dynamic behavior in association with MTs oriented toward the bud in wild-type cells. Overlays of CFP-Tub1 (red) and Kar9-GFP (green) images and the DIC image corresponding to the first frame are shown. (B) After bud emergence, a cell exhibited Kar9-GFP transits along MTs oriented toward the emerging bud. Arrows at 4 and 5 min point to an MT reaching the cortex in the absence of Kar9-GFP at the MT plus end. A second MT is decorated by Kar9-GFP at the plus end (4–5 min). (C) Kar9-GFP transits along MTs in a cell before spindle assembly. In the absence of Kar9-GFP association, MTs interacted with the cell cortex. Of two MTs directed toward the bud at 4 min (arrowheads), only one is decorated at the plus end by Kar9-GFP (arrows). A line scan for fluorescence intensity along the overlapping MTs (4 min) is shown below (arrows show position of MT plus ends). (D) After spindle assembly, Kar9-GFP was bound to the MT plus end (0 min) of a shrinking MT (0.5 min) followed by localization at the SPB in the presence of an MT interacting with the cell cortex (3 min, arrows). Numbers indicate time elapsed in minutes. 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