Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk
- Universidade do Porto, Portugal
- University of Massachusetts, United States
Figures
Figure 1 with 3 supplements
Cyclin B1 continues to be degraded during anaphase.
(a) Drosophila S2 cell from nuclear envelope breakdown to NER showing the different pools of Cyclin B1 in the mitotic apparatus. Scale bar is 5 μm. (b) Two neighbor HeLa cells (* indicates a cell …
Figure 1—figure supplement 1
Endogenous Cyclin B1 is detectable on centrosomes in metaphase and anaphase and can localize to spindle midzone microtubules in Drosophila S2 cells.
(a) Images of fixed Drosophila S2 cells co-stained for endogenous Cyclin B1 and α-tubulin. (a’) Highlight of Cyclin B1 levels with the LUT ‘fire’. Note the global decrease in endogenous Cyclin B1 …
Figure 1—figure supplement 2
Cyclin B1 degradation during anaphase correlates with DNA decondensation.
(a) Control Drosophila S2 cell stably expressing Cyclin B1-GFP/mCherry-α-tubulin and stained with SiR-DNA to follow mitotic chromosomes. Cyclin B1-GFP localization and degradation on centrosomes …
Figure 1—figure supplement 3
Aurora B localization at the spindle midzone impacts Cyclin B1 degradation during anaphase in hTERT-RPE1 cells.
(a) and (b) Representative examples of a control and Mklp2-depleted hTERT-RPE1 cells expressing endogenous Cyclin B1-Venus and co-stained with SiR-DNA. Cyclin B1-Venus localization and levels are …
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Figure 1—figure supplement 3—source data 1
Cyclin B1-Venus half-life in control and Mklp2-depleted cells.
- https://doi.org/10.7554/eLife.47646.006
Figure 2 with 1 supplement
Cdk1 inhibition at anaphase onset accelerates NER.
(a) and (b) Control and Cdk1-inhibited Drosophila S2 cells at anaphase onset (A.O.) stably expressing Lamin B-GFP/mCherry-α-tubulin. Scale bars are 5 μm. Time is in min:sec. Panels on the right side …
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Figure 2—source data 1
Calculation of anaphase duration after Cdk1 inhibition at anaphase onset.
- https://doi.org/10.7554/eLife.47646.014
Figure 2—figure supplement 1
Cdk1 inhibition during anaphase is required for mitotic exit.
(a) Control Drosophila S2 cell stably expressing H2B-GFP/mCherry-α-tubulin transiently expressing ΔCycB1-GFP, showing the expected anaphase arrest. (b) The same experimental set-up was used for Cdk1 …
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Figure 2—figure supplement 1—source data 1
Calculation of half-spindle elongation velocity in non-degradable Cyclins.
- https://doi.org/10.7554/eLife.47646.013
Figure 3
Cdk1 inactivation during anaphase licenses mitotic exit and requires proteasome-mediated proteolysis.
(a) Representative Drosophila S2 cell stably expressing Cyclin B1-GFP/mCherry-α-tubulin and stained with SiR-DNA to follow mitotic chromosomes, showing a strong anaphase arrest after treatment with …
Figure 4
Proteasome inhibition at anaphase onset arrests human cells in anaphase in a Cdk1-dependent manner.
(a) Representative U2OS cell stably expressing H2B-GFP/mCherry-α-tubulin treated with MG132 at anaphase onset (time 00:00). The cell became arrested in anaphase, after full chromosome separation and …
Figure 5 with 1 supplement
APC/CCdc20 and APC/CCdh1 are required for Cyclin B1 degradation during anaphase and timely mitotic exit.
(a) and (b) Drosophila S2 cells stably expressing WT Cyclin B1 or a KEN-box mutant version co-expressing mCherry-α-tubulin. (c) Sequence alignment showing the conservation of the Drosophila Cyclin …
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Figure 5—source data 1
Anaphase duration after KEN-box mutation or FZR RNAi.
- https://doi.org/10.7554/eLife.47646.026
Figure 5—figure supplement 1
APC/C inhibition at anaphase onset shows a synergistic effect with expression of KEN-box Cyclin B1 mutant.
(a) and (b) Drosophila S2 cells stably expressing WT Cyclin B1 or a KEN-box mutant version and co-expressing mCherry-α-tubulin with Apcin addition at anaphase onset. Cyclin B1-GFP localization is …
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Figure 5—figure supplement 1—source data 1
Cyclin B1- GFP half-life after KEN-box mutation and/or APCin treatment at anaphase onset.
- https://doi.org/10.7554/eLife.47646.025
Figure 6
Proteasome and APC/C inhibition at anaphase onset induces an anaphase arrest in human hTERT-RPE1 cells.
(a) Control hTERT-RPE1 cell expressing endogenous Cyclin B1-Venus and co-stained with SiR-DNA to visualize mitotic chromosomes. (b) MG132 addition just before or at anaphase onset (time 00:00), …
Figure 7 with 4 supplements
Cyclin B1 localization at the spindle midzone depends on Aurora B localization and activity during anaphase.
(a) Control Drosophila S2 cell stably expressing Cyclin B1-GFP/mCherry-α-tubulin showing a faint pool of Cyclin B1-GFP at the spindle midzone/midbody. (a’) Collapsed kymograph of the cell in a where …
Figure 7—figure supplement 1
Cyclin B1 co-localization with Aurora B at the spindle midzone after Cdk1 inhibition in metaphase.
(a) Untreated control Drosophila S2 cell or (b) treated with Cdk1 inhibitor in metaphase and expressing GFP-Aurora B/Cyclin B1-mCherry, showing a clear co-localization between both signals upon Cdk1 …
Figure 7—figure supplement 2
Cyclin B1 localization with midzone microtubules is not dependent on Polo kinase activity.
Drosophila S2 cells treated with Cdk1 inhibitor during metaphase and Polo inhibitor 4 min after Cdk1 inhibition. Note that the microtubule localization of Cyclin B1 is not lost after Polo …
Figure 7—figure supplement 3
Cdk1 is enriched in the central spindle and midbody in human cells.
(a) Sum projection of a 3D data set from a human HeLa cell transiently expressing hCdk1-GFP as cells enter and progress through anaphase and cytokinesis. Spindle and central spindle enrichment is …
Figure 7—figure supplement 4
Aurora B overexpression induces a Cdk1-dependent anaphase delay.
(a) Control Drosophila S2 cell stably expressing GFP-Aurora B/mCherry-α-tubulin. (b) Drosophila S2 cell overexpressing GFP-Aurora B where a delay in anaphase progression could be observed. (c) Cdk1 …
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Figure 7—figure supplement 4—source data 1
Anaphase duration after Aurora B overexpression.
- https://doi.org/10.7554/eLife.47646.033
Figure 8
Preventing Aurora B localization at the spindle midzone delays Cyclin B1 degradation during anaphase.
(a) and (b) Control and Subito/Mklp2-depleted Drosophila S2 cells stably expressing Cyclin B1-GFP/mCherry-α-tubulin. Cyclin B1-GFP localization is highlighted with the LUT ‘fire’ and dashed white …
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Figure 8—source data 1
Anaphase duration after Subito RNAi.
- https://doi.org/10.7554/eLife.47646.035
Figure 9
Visualization of a CyclinB1-Cdk1 activity gradient in the vicinity of the midzone during anaphase.
(a) The chromatin targeted reporter exhibits a longer lifetime and lower FRET efficiency in interphase versus prometaphase. The sensor on lagging chromosomes in the vicinity of the midzone MTs, …
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Figure 9—source data 1
Mean lifetimes, percentage of fluorophores exhibiting short and long lifetimes, and the short lifetimes for the chromatin-targeted Cyclin B1-Cdk1 activity reporter.
Mitotic cells included prophase, prometaphase, and metaphase, but excluded anaphase and telophase/cytokinesis. * indicates p<0.05 relative to the control, non-phosphorylatable FRET reporter in interphase. # indicates p<0.05 relative to the phosphorylatable FRET reporter in interphase. Two-tailed P-values from a Student’s t-test are reported.
- https://doi.org/10.7554/eLife.47646.037
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Figure 9—source data 2
Mean FRET efficiency statistics of chromatin-targeted Cyclin B1-Cdk1 FRET sensors.
Analysis of mitotic cells includes prophase, prometaphase, and metaphase, but excludes anaphase and telophase/cytokinesis. The active sensor reported increased FRET in mitosis relative to the non-phosphorylatable control in interphase (p<0.001). P-values calculated using the PlotsOfDifferences web app (Goedhart, 2019). N-values reported in the table apply to Figure 9—source data 1.
- https://doi.org/10.7554/eLife.47646.038
Figure 10 with 2 supplements
Cyclin B1 degradation during anaphase responds to slow chromosome separation in an Aurora B-dependent manner.
(a) Control S2 cell stably expressing Cyclin B1-GFP/mCherry-α-tubulin. (b) KLP10A depleted S2 cell stably expressing Cyclin B1-GFP/mCherry-α-tubulin. (c) and (d) Examples of S2 cells treated with 10 …
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Figure 10—source data 1
Cyclin B1-GFP half-life after attenuation of chromosome separation velocity.
- https://doi.org/10.7554/eLife.47646.043
Figure 10—figure supplement 1
Aurora B or phosphatase inhibition at anaphase onset does not affect Cyclin B1 degradation kinetics.
(a) Quantification of Cyclin B1-GFP fluorescence intensity at centrosomes in Drosophila S2 without treatment (n = 12 cells) or after Aurora B inhibition at anaphase onset (n = 6 cells). (b) …
Figure 10—figure supplement 2
Short exposure to low doses of Taxol do not compromise Aurora B localization at the spindle midzone.
(a) and (b) Representative examples of a control and a 10 nM Taxol treated cell stably expressing GFP-Aurora B/mCherry-α-tubulin. Scale bar is 5 μm. Time is in min:sec. (c) Quantification of the …
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Figure 10—figure supplement 2—source data 1
Time of GFP-Aurora B localization at the midzone after Taxol treatment.
- https://doi.org/10.7554/eLife.47646.042
Figure 11
A crosstalk between molecular ‘rulers’ (Aurora B) and ‘clocks’ (Cdk1) licenses mitotic exit only after proper chromosome separation.
APC/CCdc20 mediates the initial degradation of Cyclin B1 as chromosome align at the spindle equator and cells enter anaphase under SAC control. The consequent decrease in Cdk1 activity as cells …
Author response image 1
Author response image 2
Videos
Video 1
Exogenous expression of Cyclin B1-GFP in Drosophila S2 cells shows continuous degradation during anaphase.
Mitotic progression from nuclear envelope breakdown to nuclear envelope reformation in a Drosophila S2 cell expressing Cyclin B1-GFP (green) and mCherry-α-tubulin (magenta). Cyclin B1 becomes …
Video 2
Endogenous Cyclin B1 is continuously degraded during anaphase in human HeLa cells.
Mitotic progression in two slightly asynchronous HeLa cells expressing endogenous Cyclin B1 tagged with Venus (green) and exogenous H2B-mRFP (magenta). Cyclin B1 becomes undetectable at the …
Video 3
Endogenous Cyclin B1 is continuously degraded during anaphase in human hTERT-RPE1 cells.
Mitotic progression of two slightly asynchronous hTERT-RPE1 cells expressing endogenous Cyclin B1 tagged with Venus (green) and SiR-DNA (magenta). Cyclin B1 becomes undetectable at the …
Video 4
Endogenous Cyclin B1 is continuously degraded during anaphase in the Drosophila adult follicular epithelium.
Mitotic progression of dividing follicle cells expressing endogenous Cyclin B1 tagged with GFP (green) and His2Av-mRFP (magenta). Note a pool of cytoplasmic Cyclin B1 that remains detectable until …
Video 5
Proteasome inhibition during anaphase in Drosophila cells.
Drosophila S2 cell stably expressing Cyclin B1-GFP/mCherry-α-tubulin (white/green) and treated with SiR-DNA (magenta) to label mitotic chromosomes. The cell becomes arrested in anaphase for several …
Video 6
Proteasome inhibition at anaphase onset arrests human cells in anaphase.
Human U2OS cell stably expressing H2B-GFP/mCherry-α-tubulin (magenta/green) treated with MG132 at anaphase onset. Proteasome inhibition induced a strong anaphase arrest for several hours. Time is …
Video 7
Aurora B inhibition in anaphase-arrested Drosophila cells treated with MG132.
Representative Drosophila S2 cell stably expressing Cyclin B1-GFP/mCherry-α-tubulin (white/green) and treated with SiR-DNA (magenta) to label mitotic chromosomes. The Aurora B inhibitor was added 35 …
Video 8
Aurora B inhibition in anaphase-arrested human cells treated with MG132.
Human U2OS cell stably expressing H2B-GFP/mCherry-α-tubulin (magenta/green) treated with MG132 at anaphase onset. The Aurora B inhibitor was added 40 min after anaphase onset. Similarly to the …
Video 9
Cdk1 inhibition in anaphase-arrested Drosophila cells treated with MG132.
Representative Drosophila S2 cell stably expressing Cyclin B1-GFP/mCherry-α-tubulin (white/green) and treated with SiR-DNA (magenta) to label mitotic chromosomes. The Cdk1 inhibitor was added 50 min …
Video 10
Cdk1 inhibition in anaphase-arrested human cells treated with MG132.
Human U2OS cell stably expressing H2B-GFP/mCherry-α-tubulin (magenta/green) treated with MG132 at anaphase onset. The Cdk1 inhibitor was added 90 min after anaphase onset and induced immediate …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Drosophila) | CycB1-GFP | Buszczak et al., 2007 | genotype: w; P{PTT-GC}ycBCC01846; P{His2Av-mRFP}/+ | |
| Biological sample (mice) | Oocytes from CD-1 mice | Charles River Laboratories | RRID:MGI:5652464 | |
| Cell line (Drosophila) | S2-U | Gohta Goshima | ||
| Cell line (Drosophila) | S2 H2B-GFP/mCherry-α-tubulin | Afonso et al., 2014 | ||
| Cell line (Drosophila) | S2 Lamin B-GFP/mCherry-α-tubulin | Afonso et al., 2014 | ||
| Cell line (Drosophila) | S2 KEN-Cyclin B1-GFP/mCherry-α-tubulin | This work | ||
| Cell line (Drosophila) | S2 GFP-Aurora B/WT-Cyclin B1-mCherry | This work | ||
| Cell line (Human) | HeLa Cyclin B1-venus | Jonathon Pines | ||
| Cell line (Human) | U2OS | Afonso et al., 2014 | ||
| Cell line (Human) | hTERT-RPE1 Cyclin B1-venus | Jonathon Pines | ||
| Plasmid for mRNA synthesis (mouse) | Cyclin B1-mCherry | Pasternak et al., 2016 | ||
| Transfected construct (Drosophila) | pMT-GFP-Aurora B | Afonso et al., 2014 | ||
| Transfected construct (Drosophila) | non-degradable Cyclin B1-GFP | Afonso et al., 2014 | ||
| Transfected construct (Drosophila) | WT Cyclin B1-GFP | Mathieu et al., 2013 | ||
| Transfected construct (Drosophila) | KEN Cyclin B1-GFP | This work | ||
| Antibody | rabbit polyclonal anti Drosophila Cyclin B1 | James Wakefield | 1:2500 | |
| Antibody | mouse monoclonal anti Human Aurora B | Aim1, BD Biosciences | RRID:AB_2227708 | 1:500 |
| Antibody | rabbit polyclonal anti Drosophila Cenp C | Claudio Sunkel | 1:10000 | |
| Antibody | mouse monoclonal anti-α-tubulin (B-512 clone) | Sigma | 1:2000 | |
| Antibody | Alexa-Fluor secondary antibodies | Thermo Fisher | 1:2000 | |
| Commercial assay or kit | site-directed mutagenesis kit | Agilent | ||
| Chemical compound, drug | RO3306 | Sigma | 10 μM | |
| Chemical compound, drug | Binucleine-2 | Sigma | 40 μM | |
| Chemical compound, drug | ZM447439 | Tocris Bioscience | 4–5 μM | |
| Chemical compound, drug | MG132 | Merck | 20 μM | |
| Chemical compound, drug | Apcin | Tocris Bioscience | 200 μM | |
| Chemical compound, drug | pro-TAME | Boston Biochem | 6.3 μM | |
| Chemical compound, drug | SiR-DNA | Spirochrome | 50 nM (Human cells) and 80 nM (Drosophila) | |
| Chemical compound, drug | Sir-TUB | Spirochrome | 20 nM | |
| Software, algorithm | Prism V8 | Graphpad | RRID:SCR_002798 | |
| Software, algorithm | Fiji | ImageJ/Fiji | RRID:SCR_002285 | |
| Software, algorithm | Matlab | The MathWorks | RRID:SCR_001622 |
Additional files
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Source code 1
Kymograph generation.
- https://doi.org/10.7554/eLife.47646.045
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Supplementary file 1
Conservation of D-box, KEN boxes and Aurora B phosphorylation sites on Drosophila Cyclin B1 and human Cyclins B1 and B2.
- https://doi.org/10.7554/eLife.47646.046
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Transparent reporting form
- https://doi.org/10.7554/eLife.47646.047
