EMBO/soda-nel huggingface dataset:
Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting (2019)
Figure 6

Figure 6. Galactose signaling regulates transcription levels by modulating burst frequency but not burst duration.

(A) Example trace of PP7-GAL10 transcription in 2% galactose.

(B) Boxplot of the onset of the first transcription event in different doses of galactose. The box indicates quartiles, the horizontal line inside the box indicates the median, the whiskers indicates 1.5 times the interquartile range of the box. Data points outside the whiskers are indicated with circles (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells).

(C) The autocorrelation was used to interpret the burst duration and burst frequency changes. Burst duration is measured by the intersect of the two linear fits. When burst duration is constant, the amplitude is inversely related to the burst frequency.

(D) Burst duration from autocorrelation is constant across 4 different galactose concentrations (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM.

(E) Amplitude of autocorrelation decreases (burst frequency increases) at higher doses of galactose. (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM.

(F) Example traces of two single cells exposed to two different doses of galactose.

(G, H) Same-cell dose response shows same burst duration (G) but lower amplitude (H) at higher galactose concentration, indicating that galactose levels regulate burst frequency (n = 13 cells). Error bars indicate SEM.
Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis. (2016)
Figure 1

Fig. 1 Autophagy is inhibited upon depletion of free fatty acids. (A, B) Wild-type (WT) (BY4741) cells expressing GFP‒Atg8 were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A), or were visualized by fluorescence microscopy (B). Scale bar, 5 μm. (C) Cells (TOS038) expressing Atg1-GFP were grown to mid-log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min. Cells were washed, shifted to SD-N for 4 h, and then visualized by fluorescence microscopy. Scale bar, 5 μm. (D) WT (BY4741) and pep4∆ (TOS015) strains were grown to mid-log phase and preincubated in YPD with cerulenin or DMSO for 30 min. Cells were pulse-labeled for 10 min with [35S] methionine and cysteine and chased for the indicated time periods. Cell lysates were subjected to immunoprecipitation with anti-Ape1 antibodies followed by SDS‒PAGE and X-ray film to detect radioactive signals. prApe1, premature Ape1; mApe1, mature Ape1. (E) Cells of the fas1∆ (TOS029) strain expressing GFP‒Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min. Cells were then shifted to SD-N for the indicated times. Cell lysates were subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies. (F) WT (BY4741) cells were grown to mid-log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin (25, 50) or with DMSO (-) in rich medium for 30 min. Cells were washed and shifted to SD-N in the presence of cerulenin (25, 50) or DMSO (-). Cells were lysed and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type YPD, complete medium.
Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis. (2016)
Figure 2

Fig. 2 Lipid droplets are important for autophagy. (A) Cells (TN124 strain) were grown to mid-log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods (Cer preincubation) or incubated with DMSO without preincubation] (-). Cells were then shifted to SD-N for 3 h with the addition of DMSO or 50 µM cerulenin (cer). Autophagic activity was measured by alkaline phosphatase assay. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student’s t-test). (B) Cells were grown as in (A), stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm. (C) WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 3 h. Cells were stained with Nile red and visualized by fluorescence microscopy. Scale bar, 5 μm. (D) WT (SCY62) and tag∆ste∆ (H1246) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS−PAGE, followed by western blot analysis using anti-GFP, anti-Ape1 (prApe1, premature Ape1; mApe1, mature Ape1), and anti-Fas1 and anti-Pgk1 antibodies. (E) WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type, YPD, complete medium.
Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis. (2016)
Figure 3

Fig. 3 Induction of STE and TAG enzymes rescues autophagy. (A) GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118 strain) was grown on SC (synthetic minimal medium without dextrose) + raffinose overnight, diluted to OD 0.4, and grown to mid-log phase in either SC + glucose or SC + galactose medium. Cells were shifted to SD-N for 2 h, stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm. (B) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 were grown as in A. Cells were lysed at the indicated times and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies. (C, D) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 (C) or GFP−Atg1 (D) were grown as in A and visualized by fluorescence microscopy after 2 h in SD-N. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild-type.
Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis. (2016)
Figure 5

Fig. 5 TAG and STE are both essential for efficient autophagy. (A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. (B) Quantification of the GFP/GFP−Atg8 ratio. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student’s t-test). (C) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. (D) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells were grown as in (A). Lysates were subjected to SDS−PAGE in urea gel, followed by western blot analysis using anti-Atg8 and anti-Pgk1 antibodies. Atg8 I, non-lipidated Atg8; Atg8 II, lipidated Atg8. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.
Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis. (2016)
Figure 6

Fig. 6 Lack of lipid droplets inhibits starvation-induced formation of autophagosomes. (A, H) WT pep4∆ (pep4∆) (A,B), tag∆ste∆ pep4∆ (tag∆ste∆pep4∆)(C,D), ste∆ pep4∆ (ste∆pep4∆)(E,F) and tag∆ pep4∆ (tag∆pep4∆) (G,H) cells were grown to an exponential phase in YPD before being starved in SD-N for 2 h, and were then processed for electron microscopy. H is a magnification of the box in panel G. Magnification of proliferating ER in a tag∆ste∆ (H1246) cell is shown in (I) Asterisks indicate autophagic bodies. N, nucleus; PM, plasma membrane; CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole; #, trealose; F, ER proliferation. Scale bars: 1 µm (A-H) and 200 nm (I). (J) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles. Error bars represent standard deviations from counting of the three grids. ***P < 0.001 (Student’s t-test). SD-N, nitrogen-starvation medium; WT, wild type YPD, complete medium.
Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis. (2016)
Figure 7

Fig. 7 Lipolysis of lipid droplets is essential for autophagy. (A) WT (BY4741), tgl1∆, yeh1∆ ,and yeh2∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student’s t-test). (B) WT (BY4741) and tgl3∆tgl4∆tgl5∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student’s t-test). (C) WT (BY4741), ayr1∆ and ldh1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student’s t-test). (D) WT (BY4741), ldh1∆ayr1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. **P < 0.01 (Student’s-t test). (E) WT (BY4741), ice2∆ and ldb16∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 ***P < 0.001 (Student’s t-test). (F) WT (BY4741), ice2∆ and yeh1∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 4 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.
Long non-coding subgenomic flavivirus RNAs have extended 3D structures and are flexible in solution (2019)
Figure 6

Figure 6. Mutational analysis on the relevance of DB12 pseudoknots in ZIKV replication.

(A, B) Mutational schemes of the two PK3 mutants, ZIKV-PK3^ (A) and ZIKV-PK3*(B) mapping to the secondary structures of the DB12 region.

(C) E protein expression of ZIKV and the mutants showed by IFA.

(D) The progeny virus RNA in cultured supernatants of ZIKV and the mutants were detected by qRT-PCR in three technical replicates. Two-way ANOVA and Dunnett's multiple comparison test was used for statistical analysis. The error bars represent the standard deviation. *** p<0.001, **** p<0.0001.

(E) The luciferase activity of the ZIKV-Rep and the mutants were measured at different time points after transfection in three technical repelicates. The error bars represent the standard deviation.
A novel fidelity mechanism in DNA polymerase lambda promotes error-free bypass of 8-oxo-dG (2016)
Figure 7

Figure 7: Pol λ discriminates against extension past an 8-oxo-dG(syn):dA mispair.

(A) Glu529 (purple) forms a hydrogen bond with the C2-amino group of the primer terminal templating 8-oxo-dG (orange) in the anti-conformation. (B) This interaction is abolished when 8-oxo-dG (orange) adopts the syn-conformation. An overlay with the 8-oxo-dG(anti)-containing structure reveals that Glu529 (black) would likely clash (red-dotted line) with the C8-carbonyl of 8-oxo-dG(syn). Repositioning of Glu529 (purple) places the side chain 4.5 Å away from the C8-carbonyl. (C) Comparison of catalytic efficiencies for extension past an 8-oxo-dG(anti):dC, 8-oxo-dG(syn):dA and dG:dC base pairs at the primer terminus using WT and E529A Pol λ.
A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling (2020)
Figure 1

Figure 1. NLxxxL motif promotes S-CDK-mediated phosphorylation and degradation of N-Far1.

(A) Scheme showing the major cyclin-substrate interactions of Cdk1 complex.

(B) Scheme showing the CDK phosphorylation sites and the potential Clb5 docking region in the disordered N terminus of Far1 (residues 1-150).

(C) Co-immunoprecipitation of Cdc4 using either unphosphorylated or Clb5-Cdk1-phosphorylated Far1(85-150) as bait. The experiment was repeated twice, a representative example is shown.

(D) Time-lapse fluorescence images showing the expression and degradation of wild-type and S87A S91A mutant Far1(1-150)-GFP and nuclear-cytoplasmic shuttling of Whi5-mCherry. Nuclear export of Whi5-mCherry takes place at 0 min (Start point) and was used to synchronize the cells for quantitative analysis.

(E, F) Mean±SEM fluorescence intensities of the indicated Far1(1-150)-GFP mutants over the cell cycle. The number of cells analyzed for each construct is noted in Table EV2.

(G) Time from Start to degradation of 50% of the indicated Far1-GFP sensor in single cells. The bars show median ±95% confidence intervals. The numbers above the plot show median values. **** indicates p-value <0.0001, *** <0.001 and ns >0.05 for pair-wise comparisons with wild-type using Mann Whitney U test. The number of cells analyzed for each construct is noted in Table EV2.

(H) In vitro kinase assay showing the effect of N130A, L131A, L135A and L136A mutations on Far1(1-150)-GFP phosphorylation by Clb5-Cdk1. WT/mut shows the decrease in phosphorylation rate of the mutant compared to wild-type. The experiment was repeated twice, a representative example is shown.
A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling (2020)
Figure 2

Figure 2. NLxxxL is a specific SLiM for docking by S-phase Clb5/6-Cdk1.

(A) Autoradiograph of a cyclin specificity analysis of the indicated substrates with wild-type or docking site mutant cyclin-Cdk1 complexes. Far1(1-150 mut) is L131A L135A L136A mutant, Sic1ΔC is the non-inhibitory mutant of Sic1 (deletion of 216-284). The experiment was repeated twice, a representative example is shown.

(B) Mean±SEM fluorescence intensities of wild-type and NLxxxL motif mutant Far1(1-150)-GFP in either wild-type or clb5(hpm) clb6(hpm) strain over the cell cycle. The number of cells analyzed is noted in Table EV2.
A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling (2020)
Figure 3

Figure 3. NLxxxL motif is more effective than RxL motif in directing Clb5-Cdk1 activity.

(A) Schemes of Far1(1-150)-GFP constructs, either wild-type or mutant where NLxxxL motif is replaced with RxL motif (VKRTLF), that are used in time-lapse microscopy experiments in panel 'C'.

(B) Sequence alignment showing the introduction of wild-type or mutated RxL motif to Far1.

(C) Plot showing the mean±SEM fluorescence intensities of Far1(1-150)-GFP constructs with the indicated docking site. In rxl mutant, the NLxxxL motif is replaced with a mutated non-functional RxL motif (VKATAF). The number of cells analyzed is noted in Table EV2.

(D) Kinetic characterization of Far1(1-150 WT)-GFP and Far1(1-150 RxL)-GFP phosphorylation by Clb5-Cdk1. The data is from two experimental replicates.

(E) Mean±SEM fluorescence intensities of wild-type or RxL-dependent Far1(1-150)-GFP in wild-type or the indicated cyclin hpm mutant strains. The number of cells analyzed is noted in Table EV2.

(F) Pheromone sensitivity halo assay with PGAL1-3HA-CLB5 sic1Δ strains carrying either wild-type FAR1, far1Δ, far1(L135A) or far1(RxL). Different concentrations of α-factor were pipetted on the paper discs. On glucose plates, α-factor triggers cell cycle arrest. CLB5 overexpression from PGAL1 in sic1Δ strain causes lethality presumably by inhibition of replication licensing, which is rescued by the presence of α-factor, which leads to Far1-depedent inhibition of excess Clb5-Cdk1 activity. The experiment was repeated twice, a representative example is shown.
A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling (2020)
Figure 4

Figure 4. The Cks1-dependency is affected by the affinity of the cyclin docking motif.

(A) Schemes of the Far1(1-150)-GFP constructs used in 'B'. The constructs contain either wild-type phosphorylation sites or have all TP sites mutated to SP sites (SP mutant). The predicted Cks1 docking connections are shown with the arrows below the scheme.

(B) The degradation of Far1(1-150)-GFP with either wild-type or all serine-based phosphorylation sites and the indicated docking motifs was measured in time-lapse microscopy. Plot shows mean±SEM of GFP fluorescence intensities from Start. The number of cells is shown in Table EV2.

(C) Phos-tag SDS-PAGE autoradiographs showing the Cks1-dependency of Clb5-Cdk1-mediated multisite phosphorylation of Far1(1-150) with either NLxxxL (WT) motif or RxL motif. The experiment was performed twice, a representative example is shown.

(D) Plot showing the kinetics of Clb5-Cdk1-mediated phosphorylation of Far1(85-150) with either wild-type (NLxxxL) or RxL docking motif. The data is from two replicates.

(E) Scheme of Far1(85-150)-GFP with either NLxxxL, RxL or mutated cyclin docking motif and with or without T3 primer site introduced upstream of the S87 S91 degron.

(F) Mean±SEM GFP fluorescence intensities of the indicated Far1(1-85) constructs, synchronized at the time of Start. The number of cells is noted in Table EV2.

(G) Plot showing the timing of 50% degradation of the indicated Far1(85-150) constructs in single cells. The numbers above the plot show median values. The bars show median ±95% CI. **** and ns indicate p-values <0.0001 and >0.05 by Mann-Whitney test for pair-wise comparisons with WT. The number of cells is noted in Table EV2.
A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling (2020)
Figure 5

Figure 5. NLxxxL motif can substitute RxL in targeting Clb5-Cdk1 activity for degradation of Sic1.

(A) Schemes of the CDK threshold sensors based on Sic1 used in panels 'C' and 'D'. The sensors contain the non-inhibitory domain of Sic1 (1-215), have mutated RxL and LP docking sites and insertion of 4 residues between T33 and T45. The docking sites are inserted to the position of Sic1 RxL motif.

(B) Alignment of the Sic1 segment with the different introduced docking motifs.

(C) Plot showing the mean±SEM fluorescence intensities of the Sic1-based threshold sensors shown in 'A' containing the indicated cyclin docking motif. The sample size for each strain is noted in Table EV2.

(D) The time from Start to the fluorescence levels of the threshold sensors with indicated docking motifs decreasing to 50% of peak level was measured in single cells. The black lines show median±95% CI. The numbers above the plot show median degradation timings of the indicated sensors. The number of cells is noted in Table EV2.

(E) In vitro phosphorylation kinetics of Sic1(T33+16T45 rxl llpp ΔC) with the indicated cyclin docking motif by Clb5-Cdk1. The data is from two replicate experiments.

(F) Analysis of multisite phosphorylation of Sic1(T33+16T45 rxl llpp ΔC) with different docking motifs using Phos-tag SDS-PAGE autoradiography. The experiment was performed twice, a representative example is shown.

(G) The fraction of maximally phosphorylated form from total phosphorylation signal was quantified from Phos-tag gels as shown in panel 'F'.
A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling (2020)
Figure 6

Figure 6. NLxxxL motif is present in other S-CDK targets and is homologous with RxL motif.

(A) An alignment of predicted NLxxxL motifs from Far1, Lif1, Slx4, Fin1 and Spc110. In case of Fin1 and Spc110, the motif overlaps with an RxL motif.

(B) Autoradiograph showing the cyclin specificity and docking dependency of Lif1 and Slx4 phosphorylation. The experiments were repeated twice, representative examples are shown.

(C) NLxxxL is necessary for efficient phosphorylation of Lif1 and Slx4 by Clb5-Cdk1, as shown by the in vitro kinase assay. The experiments were repeated twice, representative examples are shown.

(D) Autoradiograph of Clb5-Cdk1-mediated phosphorylation of indicated Fin1 mutants. The experiments were repeated twice, representative examples are shown.

(E) Alignment showing the introduction of RxL motif determinants to Far1 NLxxxL motif in mutants analyzed in time-lapse microscopy in panels 'G-H'.

(F) Scheme of Far1(1-150 SP)-GFP sensors used for time-lapse microscopy-based analysis of different docking motif variants. All TP sites have been mutated to SP sites.

(G) The effect of different docking motifs on the degradation of Far1(1-150 SP)-GFP, where all TP sites are mutated to SP. Plot shows mean±SEM of fluorescence intensities in the cell cycle. For the sample sizes, please see Table EV2.

(H) The time from Start to the degradation of 50% of Far1(1-150 SP)-GFP with the indicated docking motif was measured in single cells. The numbers above the plot show median values for the sensors, the black lines show median±95% CI. **** and ns denote p-value <0.0001 and >0.05, respectively, for pair-wise comparisons with SP WT using Mann-Whitney U test. For the sample sizes, please see Table EV2.
A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling (2020)
Figure 7

Figure 7. Exclusively Clb5-specific docking motifs improve substrate dephosphorylation in anaphase. (A) Time-lapse microscopy was used to measure cyclin-Citrine levels during mitotic exit in unperturbed cell cycles. The cells were synchronized by the nuclear import of 50% of Whi5-mCherry at mitotic exit. The plot shows mean ±SEM fluorescence intensities averaged over a population of cells. The vertical line at -14 minutes marks the estimated time of anaphase entry. Sample sizes are shown in Table EV2.

(B) Cyclin-Citrine degradation relative to the nuclear import of 50% Whi5-mCherry was followed in single cells. Plot shows the time from nuclear import of 50% Whi5-mCherry to the degradation of 50% of the indicated cyclin in individual cells. The error bars show 95% CI of the mean. The number of cells is noted in Table EV2.

(C) Simplified cell cycle model showing the expression profiles of 4 cyclins and the activation and inactivation of 3 CDK-dependent switches A, B, C. On the left plot, the switches are controlled in the last-in-first-out order and on the right, the first-in-first-out order.

(D) Sequence alignment of predicted Clb5-specific motifs in Fin1, Spc110 and Cnn1.

(E) The effect of cyclin specificity on the phosphorylation of Fin1, Cnn1 and Spc110 was studied in a kinase assay. The plots show the phosphorylation by Clb5-, Clb3- and Clb2-Cdk1, the error bars show standard deviation (n=2).

(F) Images from a time-lapse microscopy experiment of an exemplar cell expressing Spc42-mCherry for detection of SPBs and EGFP-Fin1. Cells were imaged every 2 minutes, the white arrow indicates onset of anaphase.

(G) The accumulation of EGFP-Fin1 to SPBs in anaphase was measured in experiments described in 'F'. The plot shows mean EGFP fluorescence intensities at SPBs around the time of anaphase onset. The error bars are ±SEM. The number of cells analyzed is shown in Table EV2.