SUPPLEMENTARY INFORMATION

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3 C Wild type cdc14-3 cdc14-3 23 C 25 C 28 C 3 C Wild type cdc5-1 cdc5-1 b Wild type 24 24 24 24 12 12 12

cdc5-1 cdc14-3 cdc5-2 12 18 24 Supplementary Figure 1 cdc14 cdc5 loss-of-function mutants arrest with

(a) Growth of serially diluted wild type, cdc14-3, cdc5-1, cdc5-2, cdc14-3 cdc5-1, cdc14-3 cdc5-2,,,

Serial dilutions (1:5) of yeast cell suspensions starting from OD = 1 were spotted onto YED plate and

(b) Wild type,, and cells were arrested in G1 by a-factor in YED at 23 C.

inhibitor 23 and incubated at 37 C to inactivate the and alleles, respectively.

(c) (open circles), cdc14-3 (closed circles), cdc5-1 (closed diamonds), cdc14-3 cdc5-1 (closed triangles)

1 with metaphase spindles DOI: 1.1/ncb315 a 23 C 25 C 28 C 3 C Wild type cdc14-3 cdc5-1 cdc5-2 cdc14-3 cdc5-1 cdc14-3 cdc C 25 C 28 C 3 C Wild type cdc14-3 cdc C 25 C 28 C 3 C Wild type cdc5-1 cdc5-1 b Wild type Time (min) 1C 2C Time (min) 1C 2C Time (min) 1C 2C Time (min) 1C 2C c cdc14-3 cdc5-1 cdc14-3 cdc5-1 cdc14-3 cdc Supplementary Figure 1 cdc14 cdc5 loss-of-function mutants arrest with undivided nuclei and short bipolar spindles, related to Figure 1. (a) Growth of serially diluted wild type, cdc14-3, cdc5-1, cdc5-2, cdc14-3 cdc5-1, cdc14-3 cdc5-2,,, cdc14-3 and cdc5-1 strains are shown. Serial dilutions (1:5) of yeast cell suspensions starting from OD = 1 were spotted onto YED plate and incubated at 23 C, 25 C, 28 C and 3 C for 48 hours. (b) Wild type,, and cells were arrested in G1 by a-factor in YED at 23 C. When more than 9% of cells was unbudded, cells were released in fresh YED media supplemented with the CMK inhibitor 23 and incubated at 37 C to inactivate the and alleles, respectively. At the indicated time-points, cells were collected to determine the DNA content by FACS analysis. (c) (open circles), cdc14-3 (closed circles), 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=1 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) Macmillan ublishers Limited. All rights reserved

2 with metaphase spindles a mec1δ sml1δ rad53k227a rad9δ b with metaphase spindles mad2δ mad1δ mad1δ mad2δ Supplementary Figure 2 The cdc14 cdc5 arrest is checkpoint independent, related to Figure 2. (a) (open circles) and mec1δ sml1δ (closed circles), rad53k227a (closed triangles) and rad9δ (closed squares) cells were arrested in G1 by a-factor in YED at 23 C. When arrest was complete, cells were released in fresh YED medium containing the CMK inhibitor 23 and incubated at 37 C to inactivate the and alleles, respectively. Samples were taken at the indicated times to determine the percentage of cells with metaphase spindles. (b) (open circles) and mad2δ (closed circles), mad1δ (closed triangles) and 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) Macmillan ublishers Limited. All rights reserved

3 a 12 b 1 c cdc2 cdc23 cdc14 cdc5 spindle length shorter than 2 μm spindle length between 2-4 μm spindle length between 4-6 μm spindle length longer than 6 μm x 2 2<x 4 4<x 6 x>6 Spindle lenght (μm) 12 min. after G1 release pgal-cdc6 cdc6δ mad1δ x 2 2<x 4 4<x 6 x>6 Spindle lenght (μm) 24 min. after G1 release Supplementary Figure 3 Characterization of the spindle elongation defect exhibited by cdc14 cdc5 cells, related to Figure 4. (a) pmet-cdc2, cdc23-1 and cells were arrested in G1 by a-factor in YED at 23 C. When more then 9% of cells were in G1, cells were released in fresh YED media supplemented with the CMK inhibitor, methionine and/or incubated at 37 C to inactivate the, pmet-cdc2 and/or cdc23-1 and alleles. At the terminal phenotype (24 min. after G1 release spindle length measurements were taken. There is not a significant difference between the frequency of spindles >4 mm in cells compared with both cdc23-1 (on average 3.3% vs 7.7%; unpaired samples t=1.3, df=4, p=.2562), and pmet-cdc2 (on average 3.3% vs 1.7%; unpaired samples t=1.4, df=4, p=.2327) cells. (b-c) and pgal-cdc6 cdc6δ mad1δ cells were treated as in Figure 4a. Spindle lengths were measured at the 12 min. (b) and 24 min. (c) time-points. Mean and S.E.M. (error bars) deriving from n= three independent experiments are shown, 1 cells counted for each time-point in each experiment (a, b) Macmillan ublishers Limited. All rights reserved

4 a MODEL 1 MODEL 2 OUR MODEL ds1 Esp1 Slk19? ds1 Zds2 Zds1 ds1 Esp1 Slk19 2A Cdc55 Esp1 Slk19? Zds2 Zds1 Cdc5 Clb2 Cdc28 Cdc5 Zds2 Zds1 Cdc5 Cdc5 2A Cdc55 Spo12 Bns1 2A Cdc55 Spo12 Bns1 Cdc5? Spo12 Bns1? Clb2 Cdc28 Fob1 Clb2 Cdc28 Fob1 Fob1 Cdc14 Cfi1 Cdc14 Cfi1 Cdc14 Cfi1 Cdc14 Cfi1 Cdc14 Cfi1 Cdc14 Cfi1 b 1 esp1-1 mcd1-1 mad1δ 1 spo12δ bns1δ mad1δ c 8 mad1δ Metaphase spindles mad1δ esp1-1 mcd Anaphase spindles mad1δ spo12δ bns1δ mad1δ esp1-1 mcd1-1 spo12δ bns1δ Time (min) - ds1 - Clb2 - gk1 Supplementary Figure 4 Lack of FEAR network activity arrest cells at anaphase entry, related to Figure 5. (a) Schematic representation of different models for the FEAR network. Albeit discovered a decade ago the organisation of the FEAR network remains unclear. One model proposes that the Esp1-Slk19 branch act in parallel to the Spo12/Bns1 one 12. A second model places all FEAR network components in the same branch 36. 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 network 15. 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 mad1δ cells (b) and mad1δ, mad1δ esp1-1 mcd1-1, mad1δ spo12δ bns1δ and mad1δ esp1-1 mcd1-1 spo12δ bns1δ cells (c) were arrested in G1 in YED at 23 C. When more than 9% of cells reached the G1 block, cells were released in fresh YED 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, ds1 and gk1 protein levels were assessed by western blot analyses. gk1 was used as an internal loading control in immunoblots (c). n=1 cells counted from each time-point in each experiment. Representative experiments are shown (see Methods) Macmillan ublishers Limited. All rights reserved

5 a 1 clb5δ 1 clb5δ 1 clb5δ Metaphase spindles Anaphase spindles b 1 swe1δ 1 swe1δ 1 swe1δ Metaphase spindles Anaphase spindles Supplementary Figure 5 Deletion of CLB5 does not allow cdc14 cdc5 cells to elongate their spindles, related to Figure 6. (a-b) clb5δ, clb5δ and clb5δ cells (a) and swe1δ, swe1δ and swe1δ cells (b) were arrested in G1 by a-factor in YED at 23 C. When arrest was complete, cells were released in fresh YED medium containing the CMK inhibitor and incubated at 37 C to inactivate the and 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=1 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) Macmillan ublishers Limited. All rights reserved

6 gk1 gk1 gk1 Fig. 2a ds1-3ha ( -HA) Fig. 2d Slk19-13myc ( -myc) Slk19-13myc ( -myc) Supplementary Figure 6 Full scan of gels related to Figure 2a, 2d; Figure 3a, 3c; Supplementary Figure 4 and Figure 6a and 6e Macmillan ublishers Limited. All rights reserved

8 Supplementary Fig. 6c mad1 mad1 esp1-1 mcd1-1 mad1 spo12 bns1 mad1 esp1-1 mcd1-1 spo12 bns1 ds1 ( -ds1) gk1 Clb2( -Clb2) mad1 esp1-1 mcd1-1 mad1 esp1-1 mcd1-1 spo12 bns1 mad1 mad1 spo12 bns1 Supplementary Figure 6 continued Macmillan ublishers Limited. All rights reserved

10 Supplementary Table 1 Strain Ry1 Ry Ry133 Ry129 Ry1223 Ry17 Ry1574 Ry1575 Ry12 Ry1821 Ry1932 Ry2169 Ry2446 Ry258 Ry2597 Ry2612 Ry2626 Ry2629 Ry2633 Ry2725 Ry2743 Ry2747 Ry2774 Ry27 Ry278 Ry2785 Ry279 Ry2795 Ry2875 Ry2882 Ry315 Ry32 Ry325 Ry318 Ry3136 Ry3141 Ry3145 Ry3166 Ry3186 Ry323 Ry324 Ry329 Ry3212 Ry3225 Ry3541 Ry36 Ry3675 Ry3726 Ry3729 Ry3732 Ry3771 Ry479 Ry4712 Ry4715 Ry4745 Ry4796 Ry481 Ry4815 Ry4863 Ry4988 Ry4931 Ry4928 Ry4948 Ry587 Ry588 Ry5113 Ry56 Relevant genotype MATa, ade2-1, leu2-3, ura3, trp1-1, his3-11,15, can1-1, GAL, psi+ MATa, cdc14-3 MATa, cdc5-1 MATa,, cdc5::pgal-url-3ha-cdc5::kanmx MATa, pmet3-cdc2::ura3, CDC14-3HA MATa, pgal-clb2dbδ::ura3 MATa, MATa,, pgal-clb2dbδ::ura3 MATa,, (L158G) MATa, spo12::his3, bns1::kanmx, mad1::ura3 MATa, cdc23-1 MATa,, (L158G), rad9::ura3 MATa, (L158G) MATa,, (L158G), rad53-k227a::kanmx MATa,, (L158G), mad1::ura3 MATa,, (L158G), pds1::ura3 MATa,, (L158G), DS1-3HA-LEU2::pds1 MATa, (L158G), DS1-3HA-LEU2::pds1 MATa,, DS1-3HA-LEU2::pds1 MATa,, (L158G), SCC1-18MYC::TR1, DS1-3HA-LEU2::pds1 MATa,, SCC1-18MYC::TR1, DS1-3HA- LEU2::pds1 MATa, (L158G), SCC1-18MYC::TR1, DS1-3HA- LEU2::pds1, CDC14-3HA MATa, cdc14-3, cdc5-2(msd2-1)::ura3 MATa, cdc14-3, (L158G) MATa,, (L158G), slk19::slk19-13myc::kanmx6 MATa,, slk19::slk19-13myc::kanmx6 MATa, (L158G), slk19::slk19-13myc::kanmx6 MATa,, (L158G), scc1::his3, scc1-tev268- HA::LEU2, pgal-nls-9myc-tev rotease-nls::tr1 MATa, cdc14-3, cdc5-1 MATa,, cdc5-1 MATa,, (L158G), swe1::leu2 MATa,, swe1::leu2 MATa, (L158G), swe1::leu2 MATa,, (L158G), ndc1-1 MATa,, (L158G), cdc6::hisg, URA3::pGALubiR-CDC6, mad1::ura3 MATa,, cdc6::hisg, URA3::pGAL-ubiR-CDC6, mad1::ura3 MATa, (L158G), cdc6::hisg, URA3::pGAL-ubiR- CDC6, mad1::ura3 MATa,, (L158G), mad1::ura3, mad2::kanmx6 MATa,, (L158G), mad2::kanmx6 MATa,, (L158G), pmet3-cdc2::ura3 MATa,, pmet3-cdc2::ura3 MATa, (L158G), pmet3-cdc2::ura MATa,, (L158G), scc1::his3, scc1-tev268- HA::LEU2, pgal-nls-9myc-tev rotease-nls::tr1, pmet3-cdc2::ura3 MATa, scc1::his3, scc1-tev268-ha::leu2, pgal-nls- 9MYC-TEV rotease-nls::tr1, pmet3-cdc2::ura3 MATa,, (L158G), sml1::tr1, mec1::ura3 MATa,, (L158G), ase1::his3mx6, trp1::gf-tub1::tr1, ura3::ase1-7a-6ha-nt2::ura3 MATa,, (L158G), sli15-6a-6ha-his3 ura3::pafs125-tub1p-gftub1::ura3 MATa,, (L158G), scc1::scc1-gf- KanMX6, SC11-mcherry::hphMX3 MATa,, scc1::scc1-gf-kanmx6, SC11- mcherry::hphmx3 MATa, (L158G), scc1::scc1-gf-kanmx6, SC11- mcherry::hphmx3 MATa,, (L158G), mad2::ura3, rad9::leu2 MATa,, (L158G), clb5::ura3 MATa,, clb5::ura3 MATa, (L158G), clb5::ura3 MATa, cdc5-2 (msd2-1)::ura3 MATa, esp1-1, mcd1-1, mad1::ura3 MATa, (L158G), spo12::his3, bns1::kanmx, mad1::ura3 MATa, (L158G), mad1::ura3 MATa, (L158G), esp1-1, mcd1-1, mad1::ura3 MATa, (L158G), esp1-1, mcd1-1, spo12::his3, bns1::kanmx, mad1::ura3 MATa, cdc2-aid::kanmx, ura3::padh-ostir1-9myc::ura3, scc1::his3, scc1-tev268-ha::leu2, pgal- NLS-9MYC-TEV rotease-nls::tr1 MATa, (L158G), cdc2-aid::kanmx, ura3::padh- OsTIR1-9MYC::URA3, scc1::his3, scc1-tev268-ha::leu2, pgal-nls-9myc-tev rotease-nls::tr1 MATa,, (L158G), his3::fin1-5a-gf::his3 MATa,, (L158G), cin8::leu2, CEN-CIN8-13MYC-URA3 MATa,, (L158G), cin8::leu2, CEN-cin8-3A- 13MYC-URA3 MATa,, (L158G), clb2-aid::kanmx, ura3::padh-ostir1-9myc::ura3 MATa,, (L158G), ask1-2a-9myc::leu2 Supplementary Table 1 List of yeast strains used in the manuscript Macmillan ublishers Limited. All rights reserved

Supplementary table 1

Supplementary table 1 S. pombe strain list Fig. 1A JX38 h + ade6-m216 nda3-km311 PX476 PW775 PX545 PX546 h- ade6-m216 sgo2::ura4 + nda3-km311 h 9 mad2::ura4 + nda3-km311 h + ade6-m21 nda3-km311 rad21 +