Stu2 promotes mitotic spindle elongation in anaphase - PubMed (original) (raw)
Stu2 promotes mitotic spindle elongation in anaphase
F Severin et al. J Cell Biol. 2001.
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.
Figures
Figure 4
_stu2-10/kip3_Δ cells do not elongate their spindles and delay in metaphase. _stu2-10/kip3_Δ 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) FACS® profile shows that the cells delay with 2C DNA and start to undergo cytokinesis late in the release. (C) Sister chromatids separate 120 min after budding. Less than 1% of _stu2-10/kip3_Δ cells elongate their spindles. Bar, 5 μm.
Figure 1
The control G1 cells released at 34°C. (A) Photomicrographs taken 105 min after release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and shown in blue. (B) FACS® profile shows that for these cells the time between DNA replication and cytokinesis is ∼75 min. (C) Sister chromatids separate 30 min after budding. 22% of the cells have long spindles in anaphase. Bar, 5 μm.
Figure 2
stu2-10 cells do not elongate their spindles and delay in metaphase. stu2-10 cells were grown at 25°C, and then elutriated and released at 34°C. (A) Photomicrographs taken 135 min after release. Microtubules were detected by indirect immunofluorescence and are shown in red. DNA was visualized by DAPI and is shown in blue. (B) FACS® profile shows that the cells delay with 2C DNA and start to cytokinese with a 45-min delay relative to the wild type. (C) Sister chromatids separate 90 min after budding. Less than 1% of stu2-10 cells elongate their spindles. Bar, 5 μm.
Figure 3
_stu2-10/mad2_Δ cells do not elongate spindles but otherwise progress normally through the cell cycle at 34°C. _stu2-10/mad2_Δ cells were grown at 25°C, and then elutriated and released at 34°C. (A) Microphotographs 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) FACS® profile shows that cytokinesis happens with the wild-type timing. (C) Sister chromatids separate 90 min after budding. Less than 1% of _stu2-10/mad2_Δ cells elongate their spindles. Bar, 5 μm.
Figure 5
_stu2-10/kip3_Δ/_mad2_Δ cells do elongate their spindles and do not delay in metaphase. _stu2-10/kip3_Δ/_mad2_Δ 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/kip3_Δ/_mad2_Δ 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.
Figure 6
Real-time imaging of anaphase spindle elongation. (A) The kinetics of spindle elongation of five control and 10 _stu2-10/kip3_Δ/_mad2_Δ cells. (B) Anaphase video sequence of a _stu2-10/kip3_Δ/_mad2_Δ cell. Bar, 2 μm.
Figure 7
Unrooted phylogenetic tree of the kinesin motor domains. A multiple sequence alignment for the kinesin motor domain sequences was constructed using the program ClustalX, a bootstrapped phylogenetic tree (neighbor-joining tree), was calculated using the same program and displayed as a radial tree using the program TreeView. Congruent with biological data, the Kip3 subfamily is found on the same branch as the MCAK/Kif2 subfamily, suggesting close evolutionary relationship between the members of these subfamilies. Only the bootstrap values for the Kip3 and MCAK/Kif2 subbranches are shown (538 and 528 for the branch leading to Kip2, Kip3, MACK/Kif2, and Kip3, MACK/Kif2, respectively).
References
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