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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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Bud6p–astral MT interactions in kar9Δ cells. Selected frames from representative time-lapse series analyzed in Fig. 1 B, illustrating dynamic Bud6p-astral MT interactions in kar9Δ GFP:BUD6 GFP:TUB1 cells. (A) After mitotic exit and cytokinesis (0:00), astral MTs from each SPB progressively reached for the recent division site decorated by a GFP-Bud6 ring (arrowheads). In the mother cell, these interactions shifted to the prebud site newly marked by GFP-Bud6 (19:45–22:15, arrow). (B) MT interactions with the cortex of a small bud (0:00–9:30, arrowheads) were progressively lost as the spindle began to assemble. MT contacts within the mother cell became then randomized (12:15–20:15). (C) MT interactions with cortical Bud6p within the proximal region of the bud were coupled to MT growth and shrinkage (arrows). (D) MT–Bud6p interactions at the bud neck cortex after spindle assembly. These interactions led to movement of one SPB toward the bud neck (13:30–14:25, arrows). (E) MT–Bud6p interactions during anaphase. A budded cell initiating spindle elongation within the mother exhibited MT interactions with cortical Bud6p within the bud (arrows), which accompanied orientation of the spindle in mid-anaphase. Time elapsed is indicated in min:s. Bars, 2 μm.
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fig2: Bud6p–astral MT interactions in kar9Δ cells. Selected frames from representative time-lapse series analyzed in Fig. 1 B, illustrating dynamic Bud6p-astral MT interactions in kar9Δ GFP:BUD6 GFP:TUB1 cells. (A) After mitotic exit and cytokinesis (0:00), astral MTs from each SPB progressively reached for the recent division site decorated by a GFP-Bud6 ring (arrowheads). In the mother cell, these interactions shifted to the prebud site newly marked by GFP-Bud6 (19:45–22:15, arrow). (B) MT interactions with the cortex of a small bud (0:00–9:30, arrowheads) were progressively lost as the spindle began to assemble. MT contacts within the mother cell became then randomized (12:15–20:15). (C) MT interactions with cortical Bud6p within the proximal region of the bud were coupled to MT growth and shrinkage (arrows). (D) MT–Bud6p interactions at the bud neck cortex after spindle assembly. These interactions led to movement of one SPB toward the bud neck (13:30–14:25, arrows). (E) MT–Bud6p interactions during anaphase. A budded cell initiating spindle elongation within the mother exhibited MT interactions with cortical Bud6p within the bud (arrows), which accompanied orientation of the spindle in mid-anaphase. Time elapsed is indicated in min:s. Bars, 2 μm.

Mentions: The kar9Δ mutation did not affect MT–Bud6p contacts from mitotic exit to generation of a new budding site (ME to BE: 54.7%, n = 1105 in kar9Δ vs. 53.8%, n = 557 in wild type, Fig. 1 B). Once the Bud6p ring at the previous division site disassembled and accumulation began at the prebud site, MTs reoriented to this new area of capture (Fig. 2 A). In addition, interactions at Bud6p sites occurred at wild-type frequencies from onset of anaphase to mitotic exit in kar9Δ cells (Fig. 1 B, SE to ME: 76.1%, n = 822 in kar9Δ vs. 78.1%, n = 420 in wild type) and MT shrinkage at Bud6p sites was unperturbed (Fig. 1 B, open boxes within black bars).


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)

Bud6p–astral MT interactions in kar9Δ cells. Selected frames from representative time-lapse series analyzed in Fig. 1 B, illustrating dynamic Bud6p-astral MT interactions in kar9Δ GFP:BUD6 GFP:TUB1 cells. (A) After mitotic exit and cytokinesis (0:00), astral MTs from each SPB progressively reached for the recent division site decorated by a GFP-Bud6 ring (arrowheads). In the mother cell, these interactions shifted to the prebud site newly marked by GFP-Bud6 (19:45–22:15, arrow). (B) MT interactions with the cortex of a small bud (0:00–9:30, arrowheads) were progressively lost as the spindle began to assemble. MT contacts within the mother cell became then randomized (12:15–20:15). (C) MT interactions with cortical Bud6p within the proximal region of the bud were coupled to MT growth and shrinkage (arrows). (D) MT–Bud6p interactions at the bud neck cortex after spindle assembly. These interactions led to movement of one SPB toward the bud neck (13:30–14:25, arrows). (E) MT–Bud6p interactions during anaphase. A budded cell initiating spindle elongation within the mother exhibited MT interactions with cortical Bud6p within the bud (arrows), which accompanied orientation of the spindle in mid-anaphase. Time elapsed is indicated in min:s. Bars, 2 μm.
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fig2: Bud6p–astral MT interactions in kar9Δ cells. Selected frames from representative time-lapse series analyzed in Fig. 1 B, illustrating dynamic Bud6p-astral MT interactions in kar9Δ GFP:BUD6 GFP:TUB1 cells. (A) After mitotic exit and cytokinesis (0:00), astral MTs from each SPB progressively reached for the recent division site decorated by a GFP-Bud6 ring (arrowheads). In the mother cell, these interactions shifted to the prebud site newly marked by GFP-Bud6 (19:45–22:15, arrow). (B) MT interactions with the cortex of a small bud (0:00–9:30, arrowheads) were progressively lost as the spindle began to assemble. MT contacts within the mother cell became then randomized (12:15–20:15). (C) MT interactions with cortical Bud6p within the proximal region of the bud were coupled to MT growth and shrinkage (arrows). (D) MT–Bud6p interactions at the bud neck cortex after spindle assembly. These interactions led to movement of one SPB toward the bud neck (13:30–14:25, arrows). (E) MT–Bud6p interactions during anaphase. A budded cell initiating spindle elongation within the mother exhibited MT interactions with cortical Bud6p within the bud (arrows), which accompanied orientation of the spindle in mid-anaphase. Time elapsed is indicated in min:s. Bars, 2 μm.
Mentions: The kar9Δ mutation did not affect MT–Bud6p contacts from mitotic exit to generation of a new budding site (ME to BE: 54.7%, n = 1105 in kar9Δ vs. 53.8%, n = 557 in wild type, Fig. 1 B). Once the Bud6p ring at the previous division site disassembled and accumulation began at the prebud site, MTs reoriented to this new area of capture (Fig. 2 A). In addition, interactions at Bud6p sites occurred at wild-type frequencies from onset of anaphase to mitotic exit in kar9Δ cells (Fig. 1 B, SE to ME: 76.1%, n = 822 in kar9Δ vs. 78.1%, n = 420 in wild type) and MT shrinkage at Bud6p sites was unperturbed (Fig. 1 B, open boxes within black bars).

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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