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The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly.

Buvelot S, Tatsutani SY, Vermaak D, Biggins S - J. Cell Biol. (2003)

Bottom Line: As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules.Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly.We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

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Ipl1p localizes to metaphase kinetochores that are under tension. (A) Microscopy was performed on cells containing Ipl1–YFP (green) and Ndc10–CFP (red) (SBY1246). DIC pictures are shown on the far left. The merged image (yellow, far right) shows that Ipl1p and Ndc10p colocalize in metaphase cells where kinetochores are precociously separated (top). Some cells also show Ipl1p and Ndc10p costaining on the short spindle (bottom). (B) pGAL-CDC20 cells containing Ipl1–YFP and Ndc10–CFP (SBY1246) were arrested in metaphase by shifting the cells to glucose medium. The merged microscopy images show that Ipl1p and Ndc10p also colocalize in metaphase-arrested cells. Bar, 10 μm.
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fig2: Ipl1p localizes to metaphase kinetochores that are under tension. (A) Microscopy was performed on cells containing Ipl1–YFP (green) and Ndc10–CFP (red) (SBY1246). DIC pictures are shown on the far left. The merged image (yellow, far right) shows that Ipl1p and Ndc10p colocalize in metaphase cells where kinetochores are precociously separated (top). Some cells also show Ipl1p and Ndc10p costaining on the short spindle (bottom). (B) pGAL-CDC20 cells containing Ipl1–YFP and Ndc10–CFP (SBY1246) were arrested in metaphase by shifting the cells to glucose medium. The merged microscopy images show that Ipl1p and Ndc10p also colocalize in metaphase-arrested cells. Bar, 10 μm.

Mentions: We found previously that Ipl1p localizes to kinetochores that are not under tension and that it is required for the spindle checkpoint at this time (Biggins and Murray, 2001). Once tension is established, Ipl1p likely needs to be inactivated to allow cell cycle progression. Although it was reported that Ipl1p no longer colocalizes with the kinetochore protein Ndc10p at metaphase (Tanaka et al., 2002), we always found Ipl1p localized in discrete dots. Therefore, we repeated their Ipl1–YFP and Ndc10–CFP colocalization experiment. In asynchronously growing cells, Ipl1–YFP and Ndc10–CFP always colocalized as characteristic kinetochore dots in metaphase cells (Fig. 2 A, top). In addition, they also colocalized as a line that looks like a short spindle in some cells (Fig. 2 A, bottom). Since the pole to pole distance is longer than a metaphase spindle, it likely represents cells that are initiating anaphase. We also analyzed Ipl1p localization in a population of metaphase-arrested cells by depleting the Cdc20 protein that activates the APC and found that Ipl1–YFP and Ndc10–CFP always colocalize (Fig. 2 B). Although the majority of cells show two distinct dots that represent the kinetochores, approximately one third of the cells exhibit colocalization in a line similar to the asynchronous population (unpublished data). This may mean that Ipl1p can transfer to the spindle in this arrest or that these cells do not have clustered kinetochores. Therefore, although Ipl1p leaves kinetochores after metaphase, it is still bound to kinetochores that are under tension.


The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly.

Buvelot S, Tatsutani SY, Vermaak D, Biggins S - J. Cell Biol. (2003)

Ipl1p localizes to metaphase kinetochores that are under tension. (A) Microscopy was performed on cells containing Ipl1–YFP (green) and Ndc10–CFP (red) (SBY1246). DIC pictures are shown on the far left. The merged image (yellow, far right) shows that Ipl1p and Ndc10p colocalize in metaphase cells where kinetochores are precociously separated (top). Some cells also show Ipl1p and Ndc10p costaining on the short spindle (bottom). (B) pGAL-CDC20 cells containing Ipl1–YFP and Ndc10–CFP (SBY1246) were arrested in metaphase by shifting the cells to glucose medium. The merged microscopy images show that Ipl1p and Ndc10p also colocalize in metaphase-arrested cells. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172676&req=5

fig2: Ipl1p localizes to metaphase kinetochores that are under tension. (A) Microscopy was performed on cells containing Ipl1–YFP (green) and Ndc10–CFP (red) (SBY1246). DIC pictures are shown on the far left. The merged image (yellow, far right) shows that Ipl1p and Ndc10p colocalize in metaphase cells where kinetochores are precociously separated (top). Some cells also show Ipl1p and Ndc10p costaining on the short spindle (bottom). (B) pGAL-CDC20 cells containing Ipl1–YFP and Ndc10–CFP (SBY1246) were arrested in metaphase by shifting the cells to glucose medium. The merged microscopy images show that Ipl1p and Ndc10p also colocalize in metaphase-arrested cells. Bar, 10 μm.
Mentions: We found previously that Ipl1p localizes to kinetochores that are not under tension and that it is required for the spindle checkpoint at this time (Biggins and Murray, 2001). Once tension is established, Ipl1p likely needs to be inactivated to allow cell cycle progression. Although it was reported that Ipl1p no longer colocalizes with the kinetochore protein Ndc10p at metaphase (Tanaka et al., 2002), we always found Ipl1p localized in discrete dots. Therefore, we repeated their Ipl1–YFP and Ndc10–CFP colocalization experiment. In asynchronously growing cells, Ipl1–YFP and Ndc10–CFP always colocalized as characteristic kinetochore dots in metaphase cells (Fig. 2 A, top). In addition, they also colocalized as a line that looks like a short spindle in some cells (Fig. 2 A, bottom). Since the pole to pole distance is longer than a metaphase spindle, it likely represents cells that are initiating anaphase. We also analyzed Ipl1p localization in a population of metaphase-arrested cells by depleting the Cdc20 protein that activates the APC and found that Ipl1–YFP and Ndc10–CFP always colocalize (Fig. 2 B). Although the majority of cells show two distinct dots that represent the kinetochores, approximately one third of the cells exhibit colocalization in a line similar to the asynchronous population (unpublished data). This may mean that Ipl1p can transfer to the spindle in this arrest or that these cells do not have clustered kinetochores. Therefore, although Ipl1p leaves kinetochores after metaphase, it is still bound to kinetochores that are under tension.

Bottom Line: As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules.Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly.We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

Show MeSH
Related in: MedlinePlus