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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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Dynamic behavior of cortical Bud6p–astral MT interactions in kar9Δ cells. (A) Landmark events in Bud6p-mediated spindle orientation in wild-type cells. Solid black arrows show operational definition of cell cycle stages used in this work. In an unbudded cell (top left), astral MTs interact with Bud6p (black circles) at the recent division site. Accumulation of Bud6p at the prebud drives MT interactions toward the new budding site with minimal probing of the mother cortex. After bud emergence (BE), interactions continue with the bud cortex. As the spindle assembles, Bud6p accumulates at the bud neck. This causes the daughter-bound pole (SPBd) to interact with the bud and bud neck cortex whereas the SPBm is prevented from interacting with the bud. As a result, the preanaphase spindle orients along the mother-bud axis. During spindle elongation (SE), the SPBd translocates into the bud and MT interactions with Bud6p continue until spindle disassembly at mitotic exit (ME). After spindle disassembly, MT interactions focus on the Bud6p ring directing SPBs toward the recent division site. (B) Distribution of astral MT–cortex interactions in wild-type versus kar9Δ cells expressing GFP-Bud6 and GFP-Tub1. MT–cortex interactions were scored in time-lapse recordings of 54 wild-type and 172 kar9Δ cells as described in Materials and methods. The distribution shows interactions coinciding with cortical GFP-Bud6 (black bars) or away from GFP-Bud6 (gray bars). Open boxes in each category indicate percentage of shrinkage at the cell cortex as an indicator of a Bud6p-dependent mode of interaction (Segal et al., 2002). n = total number of cortical interactions scored. At least data from 320 (wild type) or 690 (kar9Δ) MTs were collected. Error bars indicate 95% confidence limits.
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fig1: Dynamic behavior of cortical Bud6p–astral MT interactions in kar9Δ cells. (A) Landmark events in Bud6p-mediated spindle orientation in wild-type cells. Solid black arrows show operational definition of cell cycle stages used in this work. In an unbudded cell (top left), astral MTs interact with Bud6p (black circles) at the recent division site. Accumulation of Bud6p at the prebud drives MT interactions toward the new budding site with minimal probing of the mother cortex. After bud emergence (BE), interactions continue with the bud cortex. As the spindle assembles, Bud6p accumulates at the bud neck. This causes the daughter-bound pole (SPBd) to interact with the bud and bud neck cortex whereas the SPBm is prevented from interacting with the bud. As a result, the preanaphase spindle orients along the mother-bud axis. During spindle elongation (SE), the SPBd translocates into the bud and MT interactions with Bud6p continue until spindle disassembly at mitotic exit (ME). After spindle disassembly, MT interactions focus on the Bud6p ring directing SPBs toward the recent division site. (B) Distribution of astral MT–cortex interactions in wild-type versus kar9Δ cells expressing GFP-Bud6 and GFP-Tub1. MT–cortex interactions were scored in time-lapse recordings of 54 wild-type and 172 kar9Δ cells as described in Materials and methods. The distribution shows interactions coinciding with cortical GFP-Bud6 (black bars) or away from GFP-Bud6 (gray bars). Open boxes in each category indicate percentage of shrinkage at the cell cortex as an indicator of a Bud6p-dependent mode of interaction (Segal et al., 2002). n = total number of cortical interactions scored. At least data from 320 (wild type) or 690 (kar9Δ) MTs were collected. Error bars indicate 95% confidence limits.

Mentions: To evaluate the involvement of Kar9p in Bud6p-associated MT capture, we undertook a comparative analysis of wild-type versus kar9Δ cells coexpressing GFP-Bud6 and GFP-α tubulin (Tub1p) fusions. Interactions were studied along the cell cycle divided arbitrarily into three stages based on spindle pathway landmarks and the program of Bud6p localization (Fig. 1 A).


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)

Dynamic behavior of cortical Bud6p–astral MT interactions in kar9Δ cells. (A) Landmark events in Bud6p-mediated spindle orientation in wild-type cells. Solid black arrows show operational definition of cell cycle stages used in this work. In an unbudded cell (top left), astral MTs interact with Bud6p (black circles) at the recent division site. Accumulation of Bud6p at the prebud drives MT interactions toward the new budding site with minimal probing of the mother cortex. After bud emergence (BE), interactions continue with the bud cortex. As the spindle assembles, Bud6p accumulates at the bud neck. This causes the daughter-bound pole (SPBd) to interact with the bud and bud neck cortex whereas the SPBm is prevented from interacting with the bud. As a result, the preanaphase spindle orients along the mother-bud axis. During spindle elongation (SE), the SPBd translocates into the bud and MT interactions with Bud6p continue until spindle disassembly at mitotic exit (ME). After spindle disassembly, MT interactions focus on the Bud6p ring directing SPBs toward the recent division site. (B) Distribution of astral MT–cortex interactions in wild-type versus kar9Δ cells expressing GFP-Bud6 and GFP-Tub1. MT–cortex interactions were scored in time-lapse recordings of 54 wild-type and 172 kar9Δ cells as described in Materials and methods. The distribution shows interactions coinciding with cortical GFP-Bud6 (black bars) or away from GFP-Bud6 (gray bars). Open boxes in each category indicate percentage of shrinkage at the cell cortex as an indicator of a Bud6p-dependent mode of interaction (Segal et al., 2002). n = total number of cortical interactions scored. At least data from 320 (wild type) or 690 (kar9Δ) MTs were collected. Error bars indicate 95% confidence limits.
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Related In: Results  -  Collection

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fig1: Dynamic behavior of cortical Bud6p–astral MT interactions in kar9Δ cells. (A) Landmark events in Bud6p-mediated spindle orientation in wild-type cells. Solid black arrows show operational definition of cell cycle stages used in this work. In an unbudded cell (top left), astral MTs interact with Bud6p (black circles) at the recent division site. Accumulation of Bud6p at the prebud drives MT interactions toward the new budding site with minimal probing of the mother cortex. After bud emergence (BE), interactions continue with the bud cortex. As the spindle assembles, Bud6p accumulates at the bud neck. This causes the daughter-bound pole (SPBd) to interact with the bud and bud neck cortex whereas the SPBm is prevented from interacting with the bud. As a result, the preanaphase spindle orients along the mother-bud axis. During spindle elongation (SE), the SPBd translocates into the bud and MT interactions with Bud6p continue until spindle disassembly at mitotic exit (ME). After spindle disassembly, MT interactions focus on the Bud6p ring directing SPBs toward the recent division site. (B) Distribution of astral MT–cortex interactions in wild-type versus kar9Δ cells expressing GFP-Bud6 and GFP-Tub1. MT–cortex interactions were scored in time-lapse recordings of 54 wild-type and 172 kar9Δ cells as described in Materials and methods. The distribution shows interactions coinciding with cortical GFP-Bud6 (black bars) or away from GFP-Bud6 (gray bars). Open boxes in each category indicate percentage of shrinkage at the cell cortex as an indicator of a Bud6p-dependent mode of interaction (Segal et al., 2002). n = total number of cortical interactions scored. At least data from 320 (wild type) or 690 (kar9Δ) MTs were collected. Error bars indicate 95% confidence limits.
Mentions: To evaluate the involvement of Kar9p in Bud6p-associated MT capture, we undertook a comparative analysis of wild-type versus kar9Δ cells coexpressing GFP-Bud6 and GFP-α tubulin (Tub1p) fusions. Interactions were studied along the cell cycle divided arbitrarily into three stages based on spindle pathway landmarks and the program of Bud6p localization (Fig. 1 A).

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