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Stu2 promotes mitotic spindle elongation in anaphase.

Severin F, Habermann B, Huffaker T, Hyman T - J. Cell Biol. (2001)

Bottom Line: We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3.Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I).We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse, 01307 Dresden, Germany.

ABSTRACT
During anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

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stu2-10/kip3Δ/mad2Δ cells do elongate their spindles and do not delay in metaphase. stu2-10/kip3Δ/mad2Δ l cells were grown at 25°C, and then elutriated and released at 34°C. (A) Photomicrographs taken 135 min after the release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and is shown in blue. (B) A FACS® profile shows that there were many dead cells that failed to replicate. The dead cells obscure the FACS® profile and do not allow the determination of the exact time point of cytokinesis. (C) Sister chromatids separate without any delay. (Only those cells that showed GFP–CEN signal were scored for budding. This allowed us to exclude dead cells from the count.) stu2-10/kip3Δ/mad2Δ cells elongate their spindles similar to the wild type. (D) The kinetics of long spindles accumulation in all of the analyzed strains. Bar, 5 μm.
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Figure 5: stu2-10/kip3Δ/mad2Δ cells do elongate their spindles and do not delay in metaphase. stu2-10/kip3Δ/mad2Δ l cells were grown at 25°C, and then elutriated and released at 34°C. (A) Photomicrographs taken 135 min after the release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and is shown in blue. (B) A FACS® profile shows that there were many dead cells that failed to replicate. The dead cells obscure the FACS® profile and do not allow the determination of the exact time point of cytokinesis. (C) Sister chromatids separate without any delay. (Only those cells that showed GFP–CEN signal were scored for budding. This allowed us to exclude dead cells from the count.) stu2-10/kip3Δ/mad2Δ cells elongate their spindles similar to the wild type. (D) The kinetics of long spindles accumulation in all of the analyzed strains. Bar, 5 μm.

Mentions: Homologues of Stu2 are required for microtubule stabilization in other eukaryotic species. Therefore, it seemed likely that stu2 mutants are unable to elongate their spindles because they cannot stabilize spindle microtubules. However, other possibilities exist, such as a role for Stu2 in spindle pole body structure or kinetochore function. In Xenopus egg extracts, it has been shown that activity of the Stu2 homologue XMAP215 is opposed by XKCM1, a member of the Kin I subfamily of microtubule-destabilizing kinesins. Removal of XMAP215 from a Xenopus extract results in microtubule destabilization, but this microtubule destabilization can be rescued by concomitant removal of XKCM1 (Tournebize et al. 2000). Extrapolating from the Xenopus experiments, we reasoned that if anaphase fails in stu2-10 because of a defect in microtubule stabilization, then removal of a microtubule-destabilizing factor in stu2-10 would stabilize spindles during anaphase. No orthologue of XKCM1 has been identified in S. cerevisiae. However, two kinesins, KAR3 and KIP3, have been genetically implicated in microtubule destabilization in S. cerevisiae (Cottingham et al. 1999). A double mutant kar3Δ /stu2-10 was synthetically lethal (data not shown). However, we were able to construct a kip3Δ/stu2-10 double mutant. To test whether kip3Δ/stu2-10 cells elongate their spindles at the restrictive temperature (34°C), we analyzed the double mutant by centrifugal elutriation and found that it arrests with short spindles (Fig. 4), as is seen for the stu2-10 single mutant. This arrest corresponds to metaphase: the DNA content is 2C, and the sister chromatids are not separated (Fig. 4B and Fig. C). Interestingly, the arrest of the double mutant is tighter than that of the single stu2-10 mutant, whereas spindle structure is indistinguishable by light microscopy. To examine anaphase spindle structure in the stu2-10/kip3Δ double mutant, we deleted MAD2 and analyzed the release of G1 cells of a triple mutant stu2-10/kip3Δ/mad2Δ at 34°C. Fig. 5 shows that the stu2-10/kip3Δ/mad2Δ triple mutant does not delay in metaphase, and sister chromatids separate with similar timing as wild-type cells. Analysis of spindle length in the triple mutant showed that many long spindles are seen during anaphase. The morphology of the elongated spindles appears to be similar to that of wild-type spindles as judged by light microscopy (Fig. 5 A). To illustrate these data, we reploted spindle lengths of all the elutriation–release experiments in this paper on the same graph (Fig. 5 D). This analysis shows that the percentage of long spindles in the triple mutant reaches 75% of the wild-type level (Fig. 5 C).


Stu2 promotes mitotic spindle elongation in anaphase.

Severin F, Habermann B, Huffaker T, Hyman T - J. Cell Biol. (2001)

stu2-10/kip3Δ/mad2Δ cells do elongate their spindles and do not delay in metaphase. stu2-10/kip3Δ/mad2Δ l cells were grown at 25°C, and then elutriated and released at 34°C. (A) Photomicrographs taken 135 min after the release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and is shown in blue. (B) A FACS® profile shows that there were many dead cells that failed to replicate. The dead cells obscure the FACS® profile and do not allow the determination of the exact time point of cytokinesis. (C) Sister chromatids separate without any delay. (Only those cells that showed GFP–CEN signal were scored for budding. This allowed us to exclude dead cells from the count.) stu2-10/kip3Δ/mad2Δ cells elongate their spindles similar to the wild type. (D) The kinetics of long spindles accumulation in all of the analyzed strains. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 5: stu2-10/kip3Δ/mad2Δ cells do elongate their spindles and do not delay in metaphase. stu2-10/kip3Δ/mad2Δ l cells were grown at 25°C, and then elutriated and released at 34°C. (A) Photomicrographs taken 135 min after the release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and is shown in blue. (B) A FACS® profile shows that there were many dead cells that failed to replicate. The dead cells obscure the FACS® profile and do not allow the determination of the exact time point of cytokinesis. (C) Sister chromatids separate without any delay. (Only those cells that showed GFP–CEN signal were scored for budding. This allowed us to exclude dead cells from the count.) stu2-10/kip3Δ/mad2Δ cells elongate their spindles similar to the wild type. (D) The kinetics of long spindles accumulation in all of the analyzed strains. Bar, 5 μm.
Mentions: Homologues of Stu2 are required for microtubule stabilization in other eukaryotic species. Therefore, it seemed likely that stu2 mutants are unable to elongate their spindles because they cannot stabilize spindle microtubules. However, other possibilities exist, such as a role for Stu2 in spindle pole body structure or kinetochore function. In Xenopus egg extracts, it has been shown that activity of the Stu2 homologue XMAP215 is opposed by XKCM1, a member of the Kin I subfamily of microtubule-destabilizing kinesins. Removal of XMAP215 from a Xenopus extract results in microtubule destabilization, but this microtubule destabilization can be rescued by concomitant removal of XKCM1 (Tournebize et al. 2000). Extrapolating from the Xenopus experiments, we reasoned that if anaphase fails in stu2-10 because of a defect in microtubule stabilization, then removal of a microtubule-destabilizing factor in stu2-10 would stabilize spindles during anaphase. No orthologue of XKCM1 has been identified in S. cerevisiae. However, two kinesins, KAR3 and KIP3, have been genetically implicated in microtubule destabilization in S. cerevisiae (Cottingham et al. 1999). A double mutant kar3Δ /stu2-10 was synthetically lethal (data not shown). However, we were able to construct a kip3Δ/stu2-10 double mutant. To test whether kip3Δ/stu2-10 cells elongate their spindles at the restrictive temperature (34°C), we analyzed the double mutant by centrifugal elutriation and found that it arrests with short spindles (Fig. 4), as is seen for the stu2-10 single mutant. This arrest corresponds to metaphase: the DNA content is 2C, and the sister chromatids are not separated (Fig. 4B and Fig. C). Interestingly, the arrest of the double mutant is tighter than that of the single stu2-10 mutant, whereas spindle structure is indistinguishable by light microscopy. To examine anaphase spindle structure in the stu2-10/kip3Δ double mutant, we deleted MAD2 and analyzed the release of G1 cells of a triple mutant stu2-10/kip3Δ/mad2Δ at 34°C. Fig. 5 shows that the stu2-10/kip3Δ/mad2Δ triple mutant does not delay in metaphase, and sister chromatids separate with similar timing as wild-type cells. Analysis of spindle length in the triple mutant showed that many long spindles are seen during anaphase. The morphology of the elongated spindles appears to be similar to that of wild-type spindles as judged by light microscopy (Fig. 5 A). To illustrate these data, we reploted spindle lengths of all the elutriation–release experiments in this paper on the same graph (Fig. 5 D). This analysis shows that the percentage of long spindles in the triple mutant reaches 75% of the wild-type level (Fig. 5 C).

Bottom Line: We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3.Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I).We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse, 01307 Dresden, Germany.

ABSTRACT
During anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

Show MeSH