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FEAR-mediated activation of Cdc14 is the limiting step for spindle elongation and anaphase progression

Nature Cell Biology volume 17, pages 251–261 (2015)Cite this article

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Abstract

Cleavage of cohesins and cyclin-dependent kinase (CDK) inhibition are thought to be sufficient for triggering chromosome segregation. Here we identify an essential requirement for anaphase chromosome movement. We show that, at anaphase onset, the phosphatase Cdc14 and the polo-like kinase Cdc5 are redundantly required to drive spindle elongation. This role of Cdc14 is mediated by the FEAR network, a group of proteins that activates Cdc14 at anaphase onset, and we suggest that Cdc5 facilitates both Cdc14 activation and CDK inhibition. We further identify the kinesin-5 motor protein Cin8 as a key target of Cdc14. Indeed, Cin8 mutants lacking critical CDK phosphorylation sites suppress the requirement for Cdc14 and Cdc5 in anaphase spindle elongation. Our results indicate that cohesin dissolution and CDK inhibition per se are not sufficient to drive sister chromatid segregation but that the motor protein Cin8 must be activated to elongate the spindle.

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Figure 1: Cells lacking Cdc14 and Cdc5 arrest with undivided nuclei and short bipolar spindles.
Figure 2: The separase Esp1 is active in cdc14 cdc5 mutant cells.
Figure 3: Cohesin is cleaved in cdc14 cdc5 mutant cells.
Figure 4: Cdc14 and Cdc5 activities are required to promote spindle elongation.
Figure 5: FEAR is essential for spindle elongation.
Figure 6: Lowering mitotic CDK allows spindle elongation in cdc14 cdc5 cells.
Figure 7: A non-phosphorylatable allele of Cin8 is sufficient to rescue the double-mutant phenotype.
Figure 8: A model for anaphase spindle elongation.

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Acknowledgements

We thank F. Uhlmann (LRI, UK), L. Gheber (Ben Gurion University, Israel), U. Surana (IMCB, Singapore), A. Amon (MIT, USA), S. Gasser (FMI, Switzerland), A. Rudner (Ottawa Institute of Systems Biology, Canada), E. Schiebel (ZMBH, Germany) and A. Marston (Wellcome Trust Centre for Cell Biology, UK) for strains and reagents; L. Fornasari for helping with statistical analyses; S. Piatti for critical discussion and A. Amon, P. De Wulf, M. Foiani, A. Rudner and members of our laboratory for critical reading of the manuscript. Work in the R.V. laboratory was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute, by a grant from the Associazione Italiana Ricerca sul Cancro (AIRC-IG-12878) to R.V. and by an ‘Armanda e Enrico Mirto’ FIRC Fellowship to F.T.

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  1. Michela Roccuzzo & Federico Tili

    Present address: Present addresses: Center for Integrative Biology (CIBIO), University of Trento, Trento 38123, Italy (M.R.); Department of Biosciences and Center for Stem Cell Research, Università degli Studi di Milano, Milan 20133, Italy (F.T.).,

Authors and Affiliations

  1. Department of Experimental Oncology, European Institute of Oncology, Milan 20139, Italy

    Michela Roccuzzo, Clara Visintin, Federico Tili & Rosella Visintin

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Contributions

M.R. and C.V. performed all the experiments. F.T. prepared the strains and made the initial observation for the experiment shown in Fig. 5c and Supplementary Fig. 4c. R.V. conceived the experiments with the person(s) performing them. M.R. and R.V. wrote the paper. All authors have approved of the manuscript.

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Correspondence to Rosella Visintin.

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Integrated supplementary information

Supplementary Figure 1 cdc14 cdc5 loss-of-function mutants arrest with undivided nuclei and short bipolar spindles, related to Fig. 1.

(a) Growth of serially diluted wild type, cdc14-3, cdc5-1, cdc5-2, cdc14-3 cdc5-1, cdc14-3 cdc5-2, cdc5-as1, cdc14-1 cdc5-as1, cdc14-3 cdc5-as1 and cdc14-1 cdc5-1 strains are shown. Serial dilutions (1:5) of yeast cell suspensions starting from OD600 = 1 were spotted onto YEPD plate and incubated at 23 °C, 25 °C, 28 °C and 30 °C for 48 h. (b) Wild type, cdc5-as1, cdc14-1 and cdc14-1 cdc5-as1 cells were arrested in G1 byα-factor in YEPD at 23 °C. When more than 90% of cells was unbudded, cells were released in fresh YEPD media supplemented with the CMK inhibitor23 and incubated at 37 °C to inactivate the cdc5-as1 and cdc14-1 alleles, respectively. At the indicated time-points, cells were collected to determine the DNA content by FACS analysis. (c) cdc14-1 cdc5-as1 (open circles), cdc14-3 cdc5-as1 (closed circles), cdc14-1 cdc5-1 (closed diamonds), cdc14-3 cdc5-1 (closed triangles) and cdc14-3 cdc5-2 (closed squares) cells were treated as in (b). Samples were taken at the indicated times to determine the percentage of cells with metaphase spindles. N = 100 cells were scored for each data point. The cell drawn in each graph is representative of the terminal phenotype of the analysed strain. Representative experiments are shown (see Methods).

Supplementary Figure 2 The cdc14 cdc5 arrest is checkpoint independent, related to Fig. 2.

(a) cdc14-1 cdc5-as1 (open circles) and cdc14-1 cdc5-as1 mec1Δ sml1Δ (closed circles), cdc14-1 cdc5-as1 rad53K227A (closed triangles) and cdc14-1 cdc5-as1 rad9Δ (closed squares) cells were arrested in G1 byα-factor in YEPD at 23 °C. When arrest was complete, cells were released in fresh YEPD medium containing the CMK inhibitor23 and incubated at 37 °C to inactivate the cdc5-as1 and cdc14-1 alleles, respectively. Samples were taken at the indicated times to determine the percentage of cells with metaphase spindles. (b) cdc14-1 cdc5-as1 (open circles) and cdc14-1 cdc5-as1 mad2Δ (closed circles), cdc14-1 cdc5-as1 mad1Δ (closed triangles) and cdc14-1 cdc5-as1 mad1Δ mad2Δ (closed squares) cells were treated as described in (a). Samples were taken at the indicated times to determine the percentage of cells with metaphase spindles. One hundred cells were scored for each data point. The cell drawn in each graph is representative of the terminal phenotype of the analysed strain. Representative experiments are shown (see Methods).

Supplementary Figure 3 Characterization of the spindle elongation defect exhibited by cdc14 cdc5 cells, related to Fig. 4.

(a) pMET-CDC20, cdc23-1 and cdc14-1 cdc5-as1 cells were arrested in G1 byα-factor in YEPD at 23 °C. When more then 90% of cells were in G1, cells were released in fresh YEPD media supplemented with the CMK inhibitor, methionine and/or incubated at 37 °C to inactivate the cdc5-as1, pMET-CDC20 and/or cdc23-1 and cdc14-1 alleles. At the terminal phenotype (240 min after G1 release spindle length measurements were taken. There is not a significant difference between the frequency of spindles >4 μm in cdc14-1 cdc5-as1 cells compared with both cdc23-1 (on average 3.3% versus 7.7%; unpaired samples t = 1.3, df = 4, p = 0.2562), and pMET-CDC20 (on average 3.3% versus 1.7%; unpaired samples t = 1.4, df = 4, p = 0.2327) cells. (b,c) cdc14-1 cdc5-as1 and pGAL-CDC6 cdc6Δmad1Δ cdc14-1 cdc5-as1 cells were treated as in Fig. 4a. Spindle lengths were measured at the 120 min (b) and 240 min (c) time-points. Mean and s.e.m. (error bars) deriving from n = three independent experiments are shown, 100 cells counted for each time-point in each experiment (a,b).

Supplementary Figure 4 Lack of FEAR network activity arrest cells at anaphase entry, related to Fig. 5.

(a) Schematic representation of different models for the FEAR network. Albeit discovered a decade ago the organization of the FEAR network remains unclear. One model proposes that the Esp1–Slk19 branch act in parallel to the Spo12/Bns1 one12. A second model places all FEAR network components in the same branch36. Given the central role of Cdc5 in the release of Cdc14 (readout for FEAR network activity) it has been difficult so far to place the kinase within the network. Data in the literature are consistent with Cdc5 acting in parallel to the Spo12/Bns1 branch of the network15. Our data are consistent with a new model indicating that the network is likely composed of three branches, the Spo12/Bns1, the Esp1–Slk19 and finally the one controlled by Cdc5. (b,c) esp1-1 mcd1-1 mad1Δ and spo12Δ bns1Δ mad1Δcells (b) and cdc5-as1 mad1Δ, cdc5-as1 mad1Δesp1-1 mcd1-1, cdc5-as1 mad1Δ spo12Δbns1Δ and cdc5-as1 mad1Δ esp1-1 mcd1-1 spo12Δ bns1Δ cells (c) were arrested in G1 in YEPD at 23 °C. When more than 90% of cells reached the G1 block, cells were released in fresh YEPD medium supplemented with the CMK inhibitor and incubated at 37 °C, to inactivate esp1-1 and mcd1-1 alleles. Samples were taken at the indicated times to determine the percentage of cells with metaphase (closed circles) and anaphase (open squares) spindles (b). Clb2, Pds1 and Pgk1 protein levels were assessed by western blot analyses. Pgk1 was used as an internal loading control in immunoblots (c). n = 100 cells counted from each time-point in each experiment. Representative experiments are shown (see Methods).

Supplementary Figure 5 Deletion of CLB5 does not allow cdc14 cdc5 cells to elongate their spindles, related to Fig. 6.

(a,b) cdc5-as1 clb5Δ, cdc14-1 clb5Δ and cdc14-1 cdc5-as1 clb5Δ cells (a) and cdc5-as1 swe1Δ, cdc14-1 swe1Δ and cdc14-1 cdc5-as1 swe1Δ cells (b) were arrested in G1 byα-factor in YEPD at 23 °C. When arrest was complete, cells were released in fresh YEPD medium containing the CMK inhibitor and incubated at 37 °C to inactivate the cdc5-as1 and cdc14-1 allele, respectively. Samples were taken at the indicated times to determine the percentage of cells with metaphase (closed circles) and anaphase (open squares) spindles. n = 100 cells counted from each time-point in each experiment. The cell drawn in each graph is representative of the terminal phenotype of the analysed strain. Representative experiments are shown (see Methods).

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Roccuzzo, M., Visintin, C., Tili, F. et al. FEAR-mediated activation of Cdc14 is the limiting step for spindle elongation and anaphase progression. Nat Cell Biol 17, 251–261 (2015). https://doi.org/10.1038/ncb3105

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