204-07-21369c petersen and hagan, supplementary figure 1 cdc2 plo1 -p-s402 0306090120150180 wt time...
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204-07-21369C Petersen and Hagan, Supplementary Figure 1
Cdc2
Cdc2
Plo1
-P-S402
0 30 60 90 120 150 180
wt
Time (min)
C
0 60 0 30 60 90 120 150 180
wt sty1::ura4+
Time (min)
D 25ºC 37 ºC
-P-S402
-tubulin
Cdc2
0 15 60 15 60
Sorbitol 1M
Heat 37C
0 15 60
H2O2
25 mM E
116.2
97.4
78.0
55.042.0
A 25ºC 37 ºCWCEM
-P-
S402
-Pl
o1
-P-
S402
+
-Plo
1
B
Asynch. cdc25.22 25ºC 37 ºCB
0 30 60 90 120 150 180
Cdc2
P-S402
P-S402
25ºC 37 ºC
0
10
20
30
40
0 30 60 90 120 150 180 210 240
0 60 120 180 240
cdc2
Time (min)
% S
ep
tatio
n
A
-P-Y
-P-TY
0 10 20 30 45 60 90 120 150 180
Sty1.myc
25ºC 37 ºCC
0 30 60 90 120 150 180-AB 37°C
Plo1
Sty1.myc
Plo1(Input)
-m
yc I
.P’s
0 30 60 90 120 150 180-AB 37°C
Plo1
Sty1.myc
Plo1(Input)
-m
yc I
.P’s
D
E
204-07-21369C Petersen and Hagan, Supplementary Figure 2
Before centrifugation After centrifugation
204-07-21369C Petersen and Hagan, Supplementary Figure 3
I.P. of HA-Plo1 after Heatshift
Total 0 10 20 30 60 90
Anti HA
0 10 20 30 60 90
Anti Plo1
Total
M
M
204-07-21369C Petersen and Hagan, Supplementary Figure 4
CyclinB
Cdc2Cdc25
Wee1
P P
Plo1
Spc1/Sty1
Mitotic commitment
+
Cut12
G2 M
P
Re-initiationof tip growth
Plo1
Spc1/Sty1
Recovery from stress
P
Re-entryinto cycle
Re-organisation of actin at the cell tips
nuclear envelope
SPB
P phosphateActin
Heat / Centrifugation
Response to deal with the stress incl. stop division, de-polarise actin
Return to normal growth
CyclinB
Cdc2
A
B
?
?
204-07-21369C Petersen and Hagan, Supplementary Figure 5
Strains used in this study
IH163 h- 972 Lab stockIH367 h- ura4.d18 leu1.32 his2 Lab stockIH634 h+ cdc25.22 ura4.d18 leu1- his- Lab stockIH635 h- cdc25.22 ura4.d18 Lab stockIH565 h+ cdc25.22 ura4.d18 leu1 Lab stockIH666 h+ cut12.s11 leu1.32 Bridge et al. 1998IH740 h+ cdc25.22 cut12.s11 ura4.d18 leu1.32 Bridge et al. 1998!H1314 h- leu1::pint5(ura4+)- plo1.NHA ura4.d18 Tanaka et al. 2001IH1624 h+ plo1.ts2 ura4.d18 leu1.32 his2 ade6.M210 MacIver et al. 2003IH2420 h- sty1::ura4+ Millar et al. 1995IH2783 h- wis1.DD::ura4+ ura4.d18 leu1.32 Shiozaki et al. 1998IH2873 h+ wis1::ura4 ura4.d18 Millar et al. 1995IH2986 h90 atf1::ura4 ura4.d18 leu1.32 Quinn et al. 2002IH3124 h+ plo1.S402A ura4.d18 leu1.32 his2 This studyIH3125 h+ plo1.S402E ura4.d18 leu1.32 his2 This studyIH3224 h+ cdc25.22 wis1.DD::ura4+ leu1.32 his2 This studyIH3225 h+ cdc25.22 cut12.s11 plo1.S402A ura4.d18 leu1.32 his2 This studyIH3226 h- cdc25.22 cut12.s11 plo1S402E ura4.d18 leu1.32 This studyIH3270 h+ cdc25.22 wis1,DD plo1.S402A ura4.d18 leu1.32 his2 This studyIH3272 h+ cdc25.22 wis1.DD plo1.S402E ura4.d18 leu1.32 his2 This studyIH3275 h- cdc25.22 plo1.S402A ura4.d18 leu1.32 This studyIH3277 h- cdc25.22 plo1.S402E ura4.d18 leu1.32 This studyIH3278 h- wis1.DD plo1.S402A ura4.d18 leu1.32 This studyIH3279 h+ wis1.DD plo1.S402E ura4.d18 leu1.32 his2 This studyIH3282 h- cut12.s11 plo1.S402A ura4.d18 leu1.32 ade6 This studyIH3283 h- cut12.s11 plo1.S402E ura4.d18 leu1.32 ade6 This studyIH3286 h+ sty1::ura4+ plo1.S402A ura4.d18 leu1.32 his2 This studyIH3287 h+ sty1::ura4+ plo1.S402E ura4.d18 leu1.32 his2 This studyIH3306 h- cdc25.22 leu1::plo1.S402con:ura4+ ura4.d18 This studyIH3307 h- cdc25.22 leu1::plo1.S402Acon:ura4+ ura4.d18 This studyIH3308 h- cdc25.22 leu1::plo1.S402Econ:ura4+ ura4.d18 This studyIH3380 h+ wis1.DD::ura4+ plo1.ts2 leu1.32 his2 ura4.d18 This study IH3724 h- plo1.ts2 sty1::ura4 This studyIH3758 h- plo1.S402A This studyIH3759 h- plo1.S402E This study IH3789 h- spc1-12 myc (ura4+) Gaits et al. 1995IH3988 h- spc1-12 myc (ura4+) leu1.32 p81NPkPlo1 This study
Petersen and Hagan, Supplementary Table 1
ReferencesBridge, A. J., Morphew, M., Bartlett, R., and Hagan, I. M. (1998). The fission yeast SPB component Cut12 links bipolar spindle formation to mitotic control. Genes and Development 12, 927-942.
Gaits F, Degols G, Shiozaki K, Russell P. (1998) Phosphorylation and association with the transcription factor Atf1 regulate localization of Spc1/Sty1 stress-activated kinase in fission yeast. Genes Dev. 12, 1464-73.
MacIver, F. H., Tanaka, K., Robertson, A. M., and Hagan, I. M. (2003). Physical and functional interactions between polo kinase and the spindle pole component Cut12 regulate mitotic commitment in S. pombe. Genes Dev 17, 1507-1523.
Millar, J. B., Buck, V., and Wilkinson, M. G. (1995). Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast. Genes Dev 9, 2117-2130.
Quinn, J., Findlay, V. J., Dawson, K., Millar, J. B., Jones, N., Morgan, B. A., and Toone, W. M. (2002). Distinct regulatory proteins control the graded transcriptional response to increasing H2O2 levels in fission yeast Schizosaccharomyces pombe. Mol Biol Cell 13, 805-816.
Shiozaki, K., Shiozaki, M., and Russell, P. (1998). Heat stress activates fission yeast Spc1/StyI MAPK by a MEKK-independent mechanism. Mol Biol Cell 9, 1339-1349
Tanaka, K., Petersen, J., MacIver, F., Mulvihill, D. P., Glover, D.M. and Hagan, I.M. (2001). The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe. EMBO J. 20 1259-1270