Figure S1, related to Figure 1. Characterization of biosensor behavior in vivo.

Transcription

1 Developmental Cell, Volume 23 Supplemental Information Separase Biosensor Reveals that Cohesin Cleavage Timing Depends on Phosphatase PP2A Cdc55 Regulation Gilad Yaakov, Kurt Thorn, and David O. Morgan Inventory of Supplemental Information Supplemental Figures and Table Figure S1, related to Figure 1. Characterization of biosensor behavior in vivo. Figure S2, related to Figure 2. Development of algorithm for quantitation of biosensor intensity. Figure S3, related to Figure 2. Characterization of polo-kinase role in biosensor cleavage. Figure S4, related to Figure 4. Characterization of timing of biosensor cleavage and sister separation. Figure S5, related to Figure 5. Activities of biosensor-phosphatase fusions in vivo. Table S1, related to Figure 2. Summary of all biosensor cleavage rates. Supplemental References 1

2 Figure S1, related to Figure 1. (A) The biosensor does not functionally complement endogenous Scc1. The GAL promoter was integrated in the 5 UTR of the endogenous SCC1 gene, and the indicated plasmid, or no plasmid, was introduced. A wild-type (wt) strain with the endogenous SCC1 promoter is shown on top. 3-fold serial dilutions of the indicated strains were plated on galactose or dextrose plates. The Scc1 TEV plasmid is a control that encodes a version of Scc1 in which the R268 separase cleavage site is replaced with a TEV site (Uhlmann et al., 2000). The GFP-LacI plasmid encodes the conventional fusion protein that generates a splitting 2

3 dot, and the GFP-intScc1-LacI plasmid encodes the separase reporter fusion that generates the disappearing dot. (B) The reporter fusion protein and endogenous Scc1 are cleaved with similar timing during mitosis. Cells with C-terminally tagged endogenous Scc1 (Scc1-Flag) or an N- terminally tagged GFP-intScc1-LacI reporter (Flag-Reporter) were arrested in metaphase (meta) with the spindle poison benomyl. Cells were released from the arrest, and full-length (FL) and separase cleavage fragments (frag) of the tagged proteins were detected by Western blot analysis. Note that in the reporter, the flag-tagged fragment is the N-terminal fragment, which is not degraded by the N-end rule (Rao et al., 2001) and therefore appears stable as compared to the endogenous fragment. Note also that there is a non-specific band in the whole cell extract detected by the flag antibody (*) that comigrates with the FL Scc1-Flag. Nearly all of this crossreactive protein is removed from the chromatin preparations in Fig. 1D. 3

4 Figure S2, related to Figure 2. (A) Automated MATLAB analysis tool used to detect and quantify the disappearing dot. The MATLAB quantification algorithm that was used to quantify disappearing dots in all strains analyzed in this study was developed to detect and quantify dots 4

5 that split as they disappeared (see Experimental Procedures for details). Raw data of a dot from the SSAA reporter (top rows) was modeled to a Gaussian-fitted dot for each 10-second timepoint. The Gaussian fit was performed for either one dot (middle rows) or two dots (bottom rows). Note that as the dot disappears, the raw data becomes noisier and nuclear fluorescence increases. (B) The algorithm then tested the quality of the fit to a single Gaussian dot versus two Gaussian dots, and assigned the preferred fit for each time-point. The ratio of the errors of the fits (right axis) is shown in the blue solid curve, and the preferred model (1 vs 2 dot) in the red dashed line. (C) The time required for dot disappearance in all wild-type cells analyzed (n=116) was plotted as a function of the intensity of the GFP dot in the time-point directly before it began to disappear. The R 2 value for a linear regression is given. This lack of correlation was observed for all strains analyzed in this study. 5

6 Figure S3, related to Figure 2. (A) Inhibition of polo kinase Cdc5 leads to slower cohesin cleavage. Cells bearing the cdc5-as1 analog-sensitive mutant (Snead et al., 2007) were released from α-factor arrest into media containing increasing amounts (0 to 15 µm) of the specific Cdc5- as CMK inhibitor as well as 20 µm of nocodazole. Two hours after release, cell extracts were prepared and subjected to western blotting for Scc1-HA. (B) Rates of biosensor cleavage in cdc5-as1 cells. Yeast were synchronized with α factor and released in the absence of inhibitor (DMSO) or in the presence of 15 µm CMK. Cells were imaged as they progressed through anaphase and the rates of dot disappearance were quantified. The cdc5-as1 cells treated with CMK arrested as large-budded post-anaphase cells, as previously described (Snead et al., 2007). The biosensor cleavage rate in the CMK-treated cells was significantly slower than that in the mock-treated cells (P value = 2.3E-16 in a two-tailed T-test). 6

7 Figure S4, related to Figure 4. Anaphase begins after completion of cohesin cleavage. Timelapse images of diploid cells with a LacO array at the TRP1 locus of one homolog, and a TetO array at the TRP1 locus of the other homolog. (A) A GFP-intScc1-LacI disappearing dot and an mcherry-tetr splitting dot were expressed. A representative cell of tens imaged is shown. (B) Two splitting dots, GFP-LacI and mcherry-tetr, were expressed. The concurrence of full dot disappearance with sister separation in (A) is not an artifact of having the dots on different homologs, as separation of the sisters of both homologs occurs almost simultaneously. A representative cell is show and the mean time between the separation of the two TRP1 loci is 24+/-4 sec (n=38 cells). 7

8 Figure S5, related to Figure 5. (A) The Rts1 and Cdc55 fusion reporters complement growth defects in their respective deletion strains. The Rts1 or Cdc55 fusion reporters were introduced into either rts1δ or cdc55δ cells, and 3-fold serial dilutions of the indicated strains were grown on YPD plates at 26 C and 37 C. All strains had an integrated LacO array at the TRP1 locus. (B) Histone H1 (HH1) was phosphorylated with Clb2-Cdk1 and 32 P-ATP (HH1-P), and used as a substrate for dephosphorylation by 3-fold increasing concentrations of PP2A Cdc55. Reaction products were detected by SDS-PAGE and autoradiography. 8

9 n (cells) Mean (sec) SEM (sec) Median (sec) SD (sec) CV T-test vs wt (p value) CV vs wt (p value) Wild type na na esp E-31*** 0.87 securin E-29*** 0.57 SSAA-reporter E-26*** 0.45 securin-2a E-04*** 0.10 Telomeric dot cdc E-03*** Cdc55-fusion reporter E-19*** 0.81 Rts1-fusion reporter Cdc55-fusion+ SSAA reporter E-24*** 0.90 spo E-03*** slk zds1/ E-08*** 0.02 zds1/2 cdc Table S1, related to Figure 2. Reporter cleavage rates and statistical analysis for all strains used in this study. The genetic modifications indicated in column 1 are in the yeast genome, unless specifically labeled reporter. The number of cells analyzed, mean, standard error of mean (SEM), median, standard of deviation (SD), and coefficient of variation (CV) are provided for all strains. The mean disappearance time for each strain was compared against wild type (row 1) in a two-tailed T-test using MATLAB (T-test vs wt), and the P value is shown (na: not applicable). In addition, the CV for each strain was tested against the CV of the wt strain in a bootstrap resampling test (CV vs wt) and the P value is given. Asterisks indicate a P value <0.01. In the securin-2a strain, the N-terminal Cdk1 phosphorylation sites in endogenous securin are mutated (T27A and S71A). 9

10 Supplemental References Rao, H., Uhlmann, F., Nasmyth, K., and Varshavsky, A. (2001). Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability. Nature 410, Snead, J.L., Sullivan, M., Lowery, D.M., Cohen, M.S., Zhang, C., Randle, D.H., Taunton, J., Yaffe, M.B., Morgan, D.O., and Shokat, K.M. (2007). A coupled chemical-genetic and bioinformatic approach to Polo-like kinase pathway exploration. Chem Biol 14, Uhlmann, F., Wernic, D., Poupart, M.A., Koonin, E.V., and Nasmyth, K. (2000). Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103,