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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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The role of Bud6p and Kar9p in cortical capture. A model summarizing astral MT dynamic behavior associated with Bud6p or Kar9p during MT capture and establishment of spindle polarity. (1) As a cell proceeds through G1, the old SPB moves toward the incipient bud site by shrinking of astral MTs (thin black lines) at cortical Bud6p sites. (2) After bud emergence and SPB duplication, Kar9p or Bud6p-associated capture coexist and can separately direct astral MTs to the bud cortex. For example, in 2a, an MT reaching cortical Bud6p undergoes shrinkage coupled with SPB movement toward the cortex (without Kar9p being present at the MT plus end); in 2b, Kar9p translocates to an MT plus end and binds to Myo2p to guide the MT to the bud via an actin cable. In addition, Kar9p can travel along astral MTs already contacting the cortex (at Bud6p). Kar9p may also prevent MTs in the bud from undergoing catastrophe past the bud neck. As a result of these combined contributions of Bud6p and Kar9p, dynamic MT–cortex interactions are maintained as the bud grows and SPB separation begins. (3) During early spindle assembly, Bud6p associated capture and Kar9p-dependent delivery continue to focus MTs toward the bud. (4) As new astral MTs are organized by the respective SPB outer plaques (thick black lines) cortical capture begins at newly defined Bud6p sites at the bud neck. These interactions effectively restrict the new pole to the mother cell (irrespective of Kar9p initial presence on both SPBs; Fig. 4 C). (5) Progressively, Kar9p is recruited solely at the SPBd (spindle > 1 μm). (6) Coupled to continued SPB separation, MTs generated by the SPBm are no longer directed toward the bud and the spindle becomes aligned as it reaches final preanaphase length (∼2 μm).
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fig10: The role of Bud6p and Kar9p in cortical capture. A model summarizing astral MT dynamic behavior associated with Bud6p or Kar9p during MT capture and establishment of spindle polarity. (1) As a cell proceeds through G1, the old SPB moves toward the incipient bud site by shrinking of astral MTs (thin black lines) at cortical Bud6p sites. (2) After bud emergence and SPB duplication, Kar9p or Bud6p-associated capture coexist and can separately direct astral MTs to the bud cortex. For example, in 2a, an MT reaching cortical Bud6p undergoes shrinkage coupled with SPB movement toward the cortex (without Kar9p being present at the MT plus end); in 2b, Kar9p translocates to an MT plus end and binds to Myo2p to guide the MT to the bud via an actin cable. In addition, Kar9p can travel along astral MTs already contacting the cortex (at Bud6p). Kar9p may also prevent MTs in the bud from undergoing catastrophe past the bud neck. As a result of these combined contributions of Bud6p and Kar9p, dynamic MT–cortex interactions are maintained as the bud grows and SPB separation begins. (3) During early spindle assembly, Bud6p associated capture and Kar9p-dependent delivery continue to focus MTs toward the bud. (4) As new astral MTs are organized by the respective SPB outer plaques (thick black lines) cortical capture begins at newly defined Bud6p sites at the bud neck. These interactions effectively restrict the new pole to the mother cell (irrespective of Kar9p initial presence on both SPBs; Fig. 4 C). (5) Progressively, Kar9p is recruited solely at the SPBd (spindle > 1 μm). (6) Coupled to continued SPB separation, MTs generated by the SPBm are no longer directed toward the bud and the spindle becomes aligned as it reaches final preanaphase length (∼2 μm).

Mentions: Finally, Kar9p-bound MTs did not support shrinkage coupled to SPB movement toward the cortex (Fig. 5 A). Interestingly, although the SPBd is associated with, on average, three MTs in wild-type cells only one is usually decorated by Kar9p at any given time. Thus, MT shrinkage coincident with Bud6p or Kar9p-associated transport may take place on different MT subpopulations (Fig. 10). Indeed, contrary to the high frequency of MT interactions with cortical Bud6p, Kar9-GFP rarely colocalized with CFP-Bud6 (unpublished data).


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)

The role of Bud6p and Kar9p in cortical capture. A model summarizing astral MT dynamic behavior associated with Bud6p or Kar9p during MT capture and establishment of spindle polarity. (1) As a cell proceeds through G1, the old SPB moves toward the incipient bud site by shrinking of astral MTs (thin black lines) at cortical Bud6p sites. (2) After bud emergence and SPB duplication, Kar9p or Bud6p-associated capture coexist and can separately direct astral MTs to the bud cortex. For example, in 2a, an MT reaching cortical Bud6p undergoes shrinkage coupled with SPB movement toward the cortex (without Kar9p being present at the MT plus end); in 2b, Kar9p translocates to an MT plus end and binds to Myo2p to guide the MT to the bud via an actin cable. In addition, Kar9p can travel along astral MTs already contacting the cortex (at Bud6p). Kar9p may also prevent MTs in the bud from undergoing catastrophe past the bud neck. As a result of these combined contributions of Bud6p and Kar9p, dynamic MT–cortex interactions are maintained as the bud grows and SPB separation begins. (3) During early spindle assembly, Bud6p associated capture and Kar9p-dependent delivery continue to focus MTs toward the bud. (4) As new astral MTs are organized by the respective SPB outer plaques (thick black lines) cortical capture begins at newly defined Bud6p sites at the bud neck. These interactions effectively restrict the new pole to the mother cell (irrespective of Kar9p initial presence on both SPBs; Fig. 4 C). (5) Progressively, Kar9p is recruited solely at the SPBd (spindle > 1 μm). (6) Coupled to continued SPB separation, MTs generated by the SPBm are no longer directed toward the bud and the spindle becomes aligned as it reaches final preanaphase length (∼2 μm).
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Related In: Results  -  Collection

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fig10: The role of Bud6p and Kar9p in cortical capture. A model summarizing astral MT dynamic behavior associated with Bud6p or Kar9p during MT capture and establishment of spindle polarity. (1) As a cell proceeds through G1, the old SPB moves toward the incipient bud site by shrinking of astral MTs (thin black lines) at cortical Bud6p sites. (2) After bud emergence and SPB duplication, Kar9p or Bud6p-associated capture coexist and can separately direct astral MTs to the bud cortex. For example, in 2a, an MT reaching cortical Bud6p undergoes shrinkage coupled with SPB movement toward the cortex (without Kar9p being present at the MT plus end); in 2b, Kar9p translocates to an MT plus end and binds to Myo2p to guide the MT to the bud via an actin cable. In addition, Kar9p can travel along astral MTs already contacting the cortex (at Bud6p). Kar9p may also prevent MTs in the bud from undergoing catastrophe past the bud neck. As a result of these combined contributions of Bud6p and Kar9p, dynamic MT–cortex interactions are maintained as the bud grows and SPB separation begins. (3) During early spindle assembly, Bud6p associated capture and Kar9p-dependent delivery continue to focus MTs toward the bud. (4) As new astral MTs are organized by the respective SPB outer plaques (thick black lines) cortical capture begins at newly defined Bud6p sites at the bud neck. These interactions effectively restrict the new pole to the mother cell (irrespective of Kar9p initial presence on both SPBs; Fig. 4 C). (5) Progressively, Kar9p is recruited solely at the SPBd (spindle > 1 μm). (6) Coupled to continued SPB separation, MTs generated by the SPBm are no longer directed toward the bud and the spindle becomes aligned as it reaches final preanaphase length (∼2 μm).
Mentions: Finally, Kar9p-bound MTs did not support shrinkage coupled to SPB movement toward the cortex (Fig. 5 A). Interestingly, although the SPBd is associated with, on average, three MTs in wild-type cells only one is usually decorated by Kar9p at any given time. Thus, MT shrinkage coincident with Bud6p or Kar9p-associated transport may take place on different MT subpopulations (Fig. 10). Indeed, contrary to the high frequency of MT interactions with cortical Bud6p, Kar9-GFP rarely colocalized with CFP-Bud6 (unpublished data).

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