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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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ipl1–321 mutants are defective in spindle disassembly. (A) Live microscopy was performed on wild-type (SBY130, left) and ipl1–321 mutant cells (SBY97, right) containing Tub1–GFP that were released from α-factor at 35°C. Eight z sections at 0.5-μm intervals were acquired every minute. Images of the spindle in a single cell are shown every 2 min after the initiation of anaphase (time 0'). An outline of the cell is shown at time 0'. Spindle disassembly is delayed in ipl1–321 cells, and the spindle orientation changes during the initial phases of anaphase. A hyperelongated spindle in an ipl1–321 mutant cell is shown in A*. See also videos 1 and 2 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1. Bar, 10 μm. (B) The spindle length at each time point was measured, and the averages of 10 cells for each strain are graphed. Spindles disassemble in wild-type cells (○) 14 min after anaphase B initiation, whereas ipl1–321 mutant cells (▪) take 20 min.
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fig3: ipl1–321 mutants are defective in spindle disassembly. (A) Live microscopy was performed on wild-type (SBY130, left) and ipl1–321 mutant cells (SBY97, right) containing Tub1–GFP that were released from α-factor at 35°C. Eight z sections at 0.5-μm intervals were acquired every minute. Images of the spindle in a single cell are shown every 2 min after the initiation of anaphase (time 0'). An outline of the cell is shown at time 0'. Spindle disassembly is delayed in ipl1–321 cells, and the spindle orientation changes during the initial phases of anaphase. A hyperelongated spindle in an ipl1–321 mutant cell is shown in A*. See also videos 1 and 2 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1. Bar, 10 μm. (B) The spindle length at each time point was measured, and the averages of 10 cells for each strain are graphed. Spindles disassemble in wild-type cells (○) 14 min after anaphase B initiation, whereas ipl1–321 mutant cells (▪) take 20 min.

Mentions: The dynamic localization of Ipl1p on spindles suggested that it might regulate spindle function. To test this, we performed live cell imaging of wild-type and ipl1–321 mutants containing Tub1–GFP. Cells were synchronized in G1 with α-factor and then released to 35°C to inactivate Ipl1–321p, and time-lapse images were captured every minute. The start of spindle elongation was used as a reference for anaphase initiation and examples of the time-lapse data for a wild-type (video 1 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1) and an ipl1–321 mutant cell (video 2 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1) are shown in Fig. 3 A. Spindle elongation was quantified by measuring the length of the spindle every minute after the initiation of anaphase B (Fig. 3 B). In wild-type cells, we observed biphasic spindle elongation (Yeh et al., 1995; Straight et al., 1997). Wild-type spindles reach ∼8.4 μm in length ∼14 min after the initiation of anaphase B and then disassemble. In ipl1–321 mutant cells, spindle elongation occurs with kinetics similar to wild-type cells. However, the spindle continues to grow to a length of ∼10.4 μm, delaying spindle disassembly for ∼6 min, a 42% increase in the duration of anaphase B. In two of the ten cells analyzed, the spindle is forced to bend when reaching the cell membrane (Fig. 3 A, A*). Since these phenotypes are not seen in wild-type cells and are similar to mutants in the motor protein Kip3 (Straight et al., 1998), we also analyzed spindle elongation and breakdown kinetics in ipl1–321, kip3Δ, and kip3Δ ipl1–321 mutants and found they exhibit similar delays in spindle breakdown (unpublished data). Therefore, Ipl1p and Kip3p may act in the same pathway to promote spindle disassembly.


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

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

ipl1–321 mutants are defective in spindle disassembly. (A) Live microscopy was performed on wild-type (SBY130, left) and ipl1–321 mutant cells (SBY97, right) containing Tub1–GFP that were released from α-factor at 35°C. Eight z sections at 0.5-μm intervals were acquired every minute. Images of the spindle in a single cell are shown every 2 min after the initiation of anaphase (time 0'). An outline of the cell is shown at time 0'. Spindle disassembly is delayed in ipl1–321 cells, and the spindle orientation changes during the initial phases of anaphase. A hyperelongated spindle in an ipl1–321 mutant cell is shown in A*. See also videos 1 and 2 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1. Bar, 10 μm. (B) The spindle length at each time point was measured, and the averages of 10 cells for each strain are graphed. Spindles disassemble in wild-type cells (○) 14 min after anaphase B initiation, whereas ipl1–321 mutant cells (▪) take 20 min.
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Related In: Results  -  Collection

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

fig3: ipl1–321 mutants are defective in spindle disassembly. (A) Live microscopy was performed on wild-type (SBY130, left) and ipl1–321 mutant cells (SBY97, right) containing Tub1–GFP that were released from α-factor at 35°C. Eight z sections at 0.5-μm intervals were acquired every minute. Images of the spindle in a single cell are shown every 2 min after the initiation of anaphase (time 0'). An outline of the cell is shown at time 0'. Spindle disassembly is delayed in ipl1–321 cells, and the spindle orientation changes during the initial phases of anaphase. A hyperelongated spindle in an ipl1–321 mutant cell is shown in A*. See also videos 1 and 2 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1. Bar, 10 μm. (B) The spindle length at each time point was measured, and the averages of 10 cells for each strain are graphed. Spindles disassemble in wild-type cells (○) 14 min after anaphase B initiation, whereas ipl1–321 mutant cells (▪) take 20 min.
Mentions: The dynamic localization of Ipl1p on spindles suggested that it might regulate spindle function. To test this, we performed live cell imaging of wild-type and ipl1–321 mutants containing Tub1–GFP. Cells were synchronized in G1 with α-factor and then released to 35°C to inactivate Ipl1–321p, and time-lapse images were captured every minute. The start of spindle elongation was used as a reference for anaphase initiation and examples of the time-lapse data for a wild-type (video 1 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1) and an ipl1–321 mutant cell (video 2 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1) are shown in Fig. 3 A. Spindle elongation was quantified by measuring the length of the spindle every minute after the initiation of anaphase B (Fig. 3 B). In wild-type cells, we observed biphasic spindle elongation (Yeh et al., 1995; Straight et al., 1997). Wild-type spindles reach ∼8.4 μm in length ∼14 min after the initiation of anaphase B and then disassemble. In ipl1–321 mutant cells, spindle elongation occurs with kinetics similar to wild-type cells. However, the spindle continues to grow to a length of ∼10.4 μm, delaying spindle disassembly for ∼6 min, a 42% increase in the duration of anaphase B. In two of the ten cells analyzed, the spindle is forced to bend when reaching the cell membrane (Fig. 3 A, A*). Since these phenotypes are not seen in wild-type cells and are similar to mutants in the motor protein Kip3 (Straight et al., 1998), we also analyzed spindle elongation and breakdown kinetics in ipl1–321, kip3Δ, and kip3Δ ipl1–321 mutants and found they exhibit similar delays in spindle breakdown (unpublished data). Therefore, Ipl1p and Kip3p may act in the same pathway to promote spindle disassembly.

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