The S. pombe cytokinesis NDR kinase Sid2 activates Fin1 NIMA kinase to control mitotic commitment through Pom1/Wee1

LETTERS

The S. pombe cytokinesis NDR kinase Sid2 activates Fin1 NIMA kinase to control mitotic commitment through Pom1/Wee1 Agnes Grallert1,4 , Yvonne Connolly2 , Duncan L. Smith2 , Viesturs Simanis3 and Iain M. Hagan1,4 Mitotic exit integrates the reversal of the phosphorylation events initiated by mitotic kinases with a controlled cytokinesis event that cleaves the cell in two. The mitotic exit network (MEN) of budding yeast regulates both processes, whereas the fission yeast equivalent, the septum initiation network (SIN), controls only the execution of cytokinesis. The components and architecture of the SIN and MEN are highly conserved1 . At present, it is assumed that the functions of the core SIN–MEN components are restricted to their characterized roles at the end of mitosis. We now show that the NDR (nuclear Dbf2-related) kinase component of the fission yeast SIN, Sid2–Mob1, acts independently of the other known SIN components in G2 phase of the cell cycle to control the timing of mitotic commitment. Sid2–Mob1 promotes mitotic commitment by directly activating the NIMA (Never In Mitosis)-related kinase Fin1. Fin1’s activation promotes its own destruction, thereby making Fin1 activation a transient feature of G2 phase. This spike of Fin1 activation modulates the activity of the Pom1/Cdr1/Cdr2 geometry network towards Wee1. Schizosaccharomyces pombe contains a single NIMA kinase, Fin1 (ref. 2). Deletion of fin1+ (fin1.∆) delays mitotic commitment2 , indicating that Fin1 emulates its Aspergillus nidulans counterpart in regulating the G2 to M transition3 . We monitored Fin1 levels in cultures in which cell cycle progression had been synchronized by size selection of small, early G2 phase, cells. During mitosis, Fin1 levels paralleled the septation profile, dropping markedly as the septation index decreased (Fig. 1a, arrow C for cytokinetic decline). Fin1 levels rose sharply once more at the start of the following G2 phase before rapidly declining again midway through this G2 phase (Fig. 1a; arrow G2 for G2 phase decline). Fin1 accumulation/decline is a G2, rather than size-dependent, event

because it did not occur when cell cycle progression was arrested at START or early S phase (data not shown). Ablation of the APC/C had no impact on Fin1 protein levels2 (data not shown); however, Fin1 accumulated in size-selected skp1.A4 cultures following inactivation of the ubiquitous component of all Cullin-based E3 ubiquitin ligase complexes Skp1 (ref. 4; Fig. 1b). Substrate recognition by Cullin family E3 ligases is often contingent on phosphorylation to generate a phospho-degron recognition motif. Therefore, we investigated whether any kinases associated with either G2 or septation events influenced Fin1’s stability, starting with Fin1 itself. Fin1 levels were elevated in fin1.KD cells in which Fin1 is catalytically inactive (Fig. 1c; for characterization of fin1.KD, see Supplementary Fig. S2a–d) and did not oscillate as synchronized fin1.KD cultures transited the cell cycle (Fig. 1d). Expression of a fin1.GFP fusion gene from a heterologous locus (wild-type Fin1 kinase, 27 kDa larger than Fin1.KD) induced fluctuations in the stability of the Fin1.KD protein that paralleled those of the wildtype fusion protein in the same cells (Fig. 1e). This ability of Fin1 activity to promote Fin1 destruction in trans probably arises from direct phosphorylation because Fin1 phosphorylates recombinant Fin1.KD in vitro (Fig. 1f). We combined mutations in kinases associated with either G2 or septation events with cdc25.22. Inactivation of Cdc25 by incubation at 36 ◦ C arrests cell cycle progression at the G2/M boundary, after the point at which Fin1 destruction is normally triggered. Deficiency in any kinase that promotes Fin1 destruction during G2 phase would be expected to elevate Fin1 levels in these arrested cells. Fin1 levels were markedly elevated in G2-arrested sid2.250 cdc25.22 cells (Fig. 1g). We therefore used the temperature- and ATP-analogue-sensitive alleles sid2.250 and sid2.as4 (Supplementary Fig. S2h,i–l) to assess the impact of Sid2 function on Fin1 stability. In each case, Sid2 inactivation in early G2 abolished the sharp declines in Fin1 levels that normally occur

1

CRUK Cell Division Group, Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, UK. 2 MBCF Biological Mass Spectrometry Facility, Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, UK. 3 EPFL SV ISREC UPSIM, SV2.1830, Station 19, CH-1015 Lausanne, Switzerland. 4 Correspondence should be addressed to A.G. or I.M.H. (e-mail: [email protected] or [email protected]) Received 14 November 2011; accepted 8 May 2012; published online 10 June 2012; DOI: 10.1038/ncb2514

738

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 © 2012 Macmillan Publishers Limited. All rights reserved.

LETTERS

Fin1

d

200 300 Time (min)

0 500

400

Septation index

e

fin1.KD 5

40

Fin1

4

30

3

20

2

10

1 0

60 40 20 400 500 Time (min)

Fin1 Tetranucleate cells

20

1

10 0

600

0

j 80 60 40

1

20 250

350 450 Time (min)

Fin1 Tetranucleate cells

casein

Substrate

cdc25.22, G2 arrest cdc10.v50. G1 arrest orb2.34 orb6.25 pck1.Δ sts5.7 pom1.Δ wis1.DD wis1.Δ plo1.ts19 cut17.275 gsk3.Δ cdc7.A20 sid2.250

cdc25.22

100

2

fin1.K33R

Fin1 0

sid2.as4, 20 μM 3MB-PP1

3

Binucleate cells 8 nuclei cells

2 1

0 200 300 400 500 Time (min) Fin1.nGFP levels Fin1.KD levels Septation index

0 150

25 °C 36 °C, 210 min 36 °C, 240 min

3

100

4

4

32P

0 550

Binucleate cells

mob1.E9, shift to 36 °C 7 6 5 4 3 2 1 0 150 200 250 300 350 400 450 500 Time (min) Fin1 Tetranucleate cells

80 60 40 20

No. of cells (%)

80

300

2

120 100

40 30

i

sid2.250, shift to 36 °C

50

C

3

0

Septation index

g 60

G2

4

Fin1 Cdc2

No. of cells (%)

14 12 10 8 6 4 2 0 200

5

No. of cells (%)

Fin1

h

200 300 400 500 Time (min) Fin1 Septation index

fin1.KD leu1::nmt41fin1.nGFP C

6

0 100 200 300 400 500 600 Time (min) Fin1

100

Septation index (%)

50

Septation index (%)

6

0

0

His–Fin1.KD

Control

100

32P

cdc25.22

0

His–Fin1.KD Casein

0

assay

fin1.K33RN165A

10

1

f Fin1 kinase

Wild type

20

2

3 2 1 0

Fin1

30

G2

3

35 30 25 20 15 10 5 0

Fin1

4

Fin1

40

Fin1

Fin1

5

7 6 5 4 3 2 1 0

Septation index (%)

C

c

skp1.A4, shift to 36 °C

cdc25.22 orb2.34 orb6.25 pck1.Δ sts5.7 pom1.Δ wis1.DD wis1.Δ plo1.ts19 cut17.275 gsk3.Δ cdc7.A20 sid2.250

b 50

Septation index (%)

Wild type, 25 °C C

6

Fin1

a

0

Binucleate cells

Figure 1 Fin1 kinase is destroyed twice in each cell cycle in a Cullin-, Fin1and Sid2-dependent manner. (a,b,d,e,h–j) Fin1 levels were normalized to those of Cdc2 kinase in the same lane on the same blot and plotted against time as cells transit the cell cycle (for images of blots see Supplementary Fig. S1b). (a) Fin1 levels declined at two points in wild type cultures; mid-G2 (grey arrow G2) and during septation (open arrow C). Destruction was seen irrespective of whether the culture was maintained at 25 ◦ C throughout the experiment, or shifted to 36 ◦ C immediately after size selection (Supplementary Fig. S4c). (b) Oscillations in Fin1 levels were not seen after synchronized skp1.A4 cultures were shifted to 36 ◦ C immediately after size selection at 25 ◦ C to inactivate Skp1. (c,g) Normalized Fin1 levels in blots of asynchronous or cdc25.22 arrested double-mutant cultures reveal threefold increases in Fin1 levels in the fin1.K33RN165A kinase-dead and sid2.250 backgrounds. (d) Fin1 levels did not fluctuate as fin1.K33RN165A cultures transited a synchronized cell cycle. (e) Strikingly the levels of both the inactive fin1.K33RN165A protein and the GFP-tagged

wild-type protein oscillate as cells transit the cell cycle when a wild-type Fin1.GFP fusion protein was constitutively expressed within the same cells. (f) Fin1 immunoprecipitates from asynchronous cells were employed in kinase assays that used recombinant Fin1 or casein as substrates. (g) Left: 210 and 240 min refers to the duration of incubation at 36 ◦ C to inactivate and arrest cell cycle progression at the G2/M boundary. Right: fluorescence-activated cell sorting profiles of DNA content demonstrate G2 arrest in all strains. (h–j) Assessing the impact of Sid2–Mob1 function on Fin1 levels in size-selected synchronized cultures. (h,j) sid2.250 and mob1.E9 cultures were maintained at 25 ◦ C during transit through the first cell division before a portion of the culture was shifted to 36 ◦ C to inactivate the kinase/regulatory subunit. (i) A sid2.as4 culture was split into three after the first wave of septation was complete (Supplementary Fig. S1c) and either nothing, methanol or 3-MB-PP1 in methanol was added to a final concentration of 20 µM at time point 190. Images of blots/gels are shown in Supplementary Fig. S1.

during G2 phase and mitotic exit (Fig. 1h,i). The sharp increase in Fin1 levels in early G2 phase was also absent in the sid2.250 culture (Fig. 1h), suggesting that Sid2 activity may also promote Fin1 production/stability at this point in the cell cycle. The levels of Fin1 in fin1.KD skp1.A4, and sid2.as skp1.A4 were identical to those seen in the fin1.KD or skp1.A4 or sid2.as single mutant backgrounds (Supplementary Fig. S3a,b). As Sid2 function in the SIN requires association with Mob1, we assessed Fin1 behaviour in synchronized mob1.E9 mutant cultures. Fin1 behaviour in mob1.E9 mirrored that seen in the sid2.250 mutant background (Fig. 1j).

We studied the changes in Fin1 kinase specific activity in size-selected synchronized cultures (Fig. 2a and Supplementary Fig. S2b,d). Fin1 activity increased midway through G2 phase, and increased further after mitotic commitment (40–60 min before septation). Addition of the ATP analogue 3-MB-PP1 to size-selected sid2.as4 skp1.A4 cultures abolished both the G2 and mitotic enhancement of Fin1 activity, supporting the view that Sid2 activity in G2 phase of the cell cycle promotes Fin1 activation (Fig. 2b). Similarly to its budding yeast counterpart Dbf2, Sid2 targets the consensus sequence RXXS/T (refs 5,6). The two RXXS/T sequences in

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 © 2012 Macmillan Publishers Limited. All rights reserved.

739

LETTERS

1.2

40 30

0.8

20

0.4 0

10 0

0 200 300 400 500 Time (min) Kinase activity Septation index

Fin1 kinase activity

Untreated, binucleate Methanol, binucleate 3MB-PP1, binucleate 3MB-PP1, tetranucleate

80 60

Specific kinase activity

50 G2

No. of cells (%)

1.6

100

40 20 0

100

0

100

Methanol

200 300 Time (min)

400

+ +

+ λ phosph – Na3VO4

Asyn 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405

180 187 194 200 207 214 220 227 234 240 247 253 260 267 274 280 290 300 320 340 360 380 400 420 440

– –

c

d

Activity Fin1 protein

e Sid2 kinase

F1S377P

f

1 0 150

1

assay

F1S377P

2

200

3MB-PP1

Activity Fin1 protein

3MB-PP1 Methanol

3

250 300 Time (min)

350

2

3

4

5

6

7

Fin1

F1S377P F1S526P F1S698P Sid2 Fin1

F1S526P F1S526P

2

G2

1 0

20 10

0

32P

Wild type sid2.250 36 °C 180 200 220 240 260 280 300 320 340 360 380 400 420 440

4

40

3

30

2

20

1

10

0

100

200 300 Time (min)

Kinase activity

400

500

0

sid2.as4 no 3MB-PP1

Wild type, no ATP

Wild type, no substrate

Wild type, control beads

Wild type

Septation index F1S698P Sid2

G2

Figure 2 Sid2 phosphorylation of Fin1 on Ser 377, 526 and 698 promotes Fin1 activity in G2 phase before a peak of each kinase activity accompanies mitotic progression. (a,b) Fin1 kinase assays from size-selected cultures in which His-tagged Fin1.KD was labelled with [γ −32 P]ATP to quantify activity that is plotted alongside the septation profile. Wild-type (a) and small G2 sid2.as4 skp1.A4 (b) cells were isolated from a culture grown at 25 ◦ C and immediately shifted to 36 ◦ C to inactivate Skp1 (and so preserve activated Fin1) at t = 0. The culture was split in two and 20 µM 3-MB-PP1 (left kinase assay) or solvent alone (right kinase assay) was added after the first division at t = 180. (c,d) Fin1 immunoprecipitates from asynchronous skp1.A4 (c) or cell-size-selected sid2.as4 skp1.A4 (d) cultures were split in two and probed with antibodies that recognize the indicated phosphorylation sites or polyclonal antibodies that recognize the non-catalytic domain of Fin1. See Supplementary Fig. S3g for details of the scheme used for each of the three identical cultures used to generate the samples and Supplementary Fig. 3h for the phenotypic characterization

740

0

sid2.as4 + 20 μM 3MB-PP1

50

casein

Sid2

G2

Casein

Methanol

0 200 400 Time (min) Kinase activity Septation index

Specific kinase activity

30

Sid2 kinase activity 5

Wild type sid2.250 25 °C sid2.250 36 °C 140 160 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 340 360 380 400 420 440 460 480 sid2.250 36 °C sid2.250 36 °C sid2.250 25 °C

50 40

3

Substrate

h

Septation index (%)

4

3MB-PP1

Asyn

Sid2 kinase activity C C

Septation index (%)

Specific kinase activity

g

Fin1

His– Fin1.KD

F1S698P Fin1

sid2.250, 25 °C

Casein F1S698P

sid2.250, 36 °C

Fin1

Fin1

Activity Fin1 protein

8

His–Fin1.KD Sid2

Fin1

400

Asyn 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405

b

Fin1 kinase activity C Septation index (%)

Specific kinase activity

a

of one of the three cultures. Samples from asynchronous cultures are run in the left lane in each case to provide a reference standard. (e) Sid2 immunoprecipitates were isolated from asynchronous cultures and employed in in vitro kinase assays using [γ −32 P]ATP and either recombinant Fin1.KD or casein as indicated. (f) Sid2 was isolated from the indicated strains and combined with recombinant Fin1.KDnHis before blotting with the indicated antibodies. (g) The incorporation of 32 P into casein from [γ −32 P]ATP was used to monitor Sid2 activity in size-selected wild-type cultures. (h) Sid2 immunoprecipitates were processed as for g with the exception that the shorter Fin1.FP1 (non-catalytic carboxy-terminal domain20 ) was used as a substrate and the F1S698P antibody was used to develop the assay with the secondary reagent BCIP. The loading of the Sid2.250 36 ◦ C sample in the second to last lane was four times that in other lanes to ensure that the basal level dictated by the reduced level of Sid2.250 protein in the 36 ◦ C sample was representative of the reference point for normalization. (g,h) Plots show the activity per unit protein (that is, specific activity).

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 © 2012 Macmillan Publishers Limited. All rights reserved.

LETTERS d

sid2.as4

9 7

Binucleate cells (%)

Mitotic index (%)

8 6 5 4 3 2

Wild type sid2.as4 fin1.as3

1 0

b

0

1

2 3 Time (h)

4

5

100 90 80 70 60 50 40 30 20 10 0 260 300 340 380 420 460 500 Time (min)

30

Mitotic index (%)

20 µM 3MB-PP1

a

10

0 260 300 340 380 420 Time (min)

60 50 40 30 20

25

15

Mitotic index (%)

Histone H3 positive cells (%)

10

5

Histone H3 positive cells (%)

40 30 20 10

Untreated

10

15 25

10

5

20 15 10 5

0 260 300 340 380 420 Time (min)

0 260 300 340 380 420 460 500 Time (min)

15

0 260 300 340 380 420 Time (min)

0 260 300 340 380 420 Time (min)

0 260 300 340 380 420 460 500 Time (min) fin1.as3 50

20

5

Mitotic index (%)

Septation index (%)

15

30

10

Septation index (%)

20

5

Wild type 70

c

25

Methanol

0 260 300 340 380 420 Time (min)

3MB-PP1

Figure 3 Inhibition of Sid2 or Fin1 delays mitotic commitment. (a) 3-MB-PP1 was added to asynchronous cultures of wild-type, fin1.as3 and sid2.as4 cells, and the mitotic index monitored by anti-α-tubulin immunofluorescence at the indicated times. The analogue transiently inhibited mitotic commitment of fin1.as3 and sid2.as4 but not wild-type cells. (b–d) Wild-type, fin1.as3 and sid2.as4 cultures were synchronized with respect to cell cycle progression by size selection and split into three equal cultures after the first round of

septation. Methanol or 3-MB-PP1 (to a final concentration of 20 µM) in methanol was added to two of these sub-cultures at 160 min. Commitment to mitosis was monitored by the spindle index or phospho-histone H3 reactivity, as indicated. Addition of solvent alone had no impact on cell cycle progression whereas addition of analogue in solvent delayed mitotic commitment in fin1.as3 and sid2.as4 cells, but had no impact on wild-type cells.

Fin1 (Ser 377 (RVTS) and Ser 526 (RKVS)) are phosphorylated in vivo (Supplementary Fig. S3c–e). Antibodies that specifically recognize each site when phosphorylated (F1S377P, F1S526P) recognized Fin1 that had been immunoprecipitated from skp1.A4 cells grown at 36 ◦ C, but failed to do so when the precipitate had been treated with λ phosphatase (Fig. 2c). Their ability to recognize Fin1 precipitated from mid/late G2 phase sid2.as4 skp1.A4 cells was dependent on Sid2 activity (Fig. 2d and Supplementary Fig. S3g,h). Polyclonal antibodies against the non-catalytic domain of Sid2 (Supplementary Fig. S2m) were used for in vitro Sid2 kinase assays using either casein or His-tagged Fin1.KD as the in vitro substrate (Fig. 2e

and Supplementary Fig. S2k,n). Both Ser 377 and Ser 526 of Fin1.KD (Fig. 2f, lanes 3–6) were phosphorylated by Sid2 immunoprecipitates; this activity was temperature dependent when Sid2 had been isolated from sid2.250 cultures (Fig. 2f, lanes 1 and 2). Addition of 3-MB-PP1 to the analogue-sensitive kinase precipitated from sid2.as4 cells also abolished activity towards each site (Fig. 2f, lanes 7 and 8). The continued fluctuation of Fin1.S377AS526A levels during cell cycle progression (Supplementary Fig. S3i) suggested that Sid2 might phosphorylate additional sites on Fin1. Consistently, Ser 698 was phosphorylated in mass spectra of recombinant Fin1.KD that had been incubated with Sid2 in vitro and in spectra of Fin1 precipitated from

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 © 2012 Macmillan Publishers Limited. All rights reserved.

741

LETTERS Table 1 Cell length measurements support the model depicted in Fig. 4h. Cell length at division

Cell length at division following treatment with ATP analogues Cell length in micrometres

Cell length in micrometres

Anaphase cells Wild type, 36 ◦ C, 2 h fin1.∆, 36 ◦ C, 2 h sid2.250, 36 ◦ C, 2 h

Septating cells 13.0 ± 1.7 14.0 ± 2.1 15.6 ± 2.2

Wild type

Septating cells Wild type fin1.∆ fin1.K33R fin1.KD fin1.S698A fin1.S698E fin1.2A fin1.2E fin1.3A fin1.3E cdc2.1w fin1.∆ cdc2.1w fin1.KD cdc2.1w cdc2.3w fin1.∆ cdc2.3w fin1.KD cdc2.3w pom1.∆ pom1.∆fin1.KD pom1.∆fin1.3A pom1.∆fin1.3E cdr1.∆ cdr1.∆fin1.KD cdr2.∆ cdr2.∆fin1.KD fin1.3A cdr2.∆ fin1.3E cdr2.∆

12.5 ± 1.8 13.3 ± 2.0 15.0 ± 2.2 15.9 ± 1.6 13.9 ± 1.3 12.5 ± 1.3 12.9 ± 1.4 12.8 ± 1.2 14.9 ± 1.9 11.7 ± 1.7 7.9 ± 0.9 8.1 ± 1.5 8.1 ± 0.7 8.0 ± 1.4 10.7 ± 2.8 11.3 ± 4.0 11.1 ± 1.0 11.4 ± 1.9 11.0 ± 1.4 10.9 ± 1.8 18.4 ± 1.3 18.0 ± 1.3 19.1 ± 1.5 19.3 ± 1.3 18.9 ± 1.9 18.4 ± 2.3

Untreated 3-MB-PP1, 2 h 3-MB-PP1, 4 h

12.5 ± 1.8 12.4 ± 1.3 12.8 ± 1.3

3-MB-PP1, 8 h

12.3 ± 1.0

Untreated 3-MB-PP1, 2 h 3-MB-PP1, 4 h 3-MB-PP1, 8 h

13.8 ± 1.4 13.2 ± 1.0 15.2 ± 1.8 15.7 ± 1.0

Untreated 3-MB-PP1, 2 h 3-MB-PP1, 4 h 3-MB-PP1, 8 h

7.7 ± 0.9 7.8 ± 0.8 8.2 ± 0.9 8.0 ± 0.9

Untreated 3-MB-PP1, 2 h 3-MB-PP1, 4 h 3-MB-PP1, 8 h

10.8 ± 1.1 11.0 ± 1.2 11.9 ± 1.4 12.0 ± 1.5

Untreated 3-MB-PP1, 2 h 3-MB-PP1, 4 h 3-MB-PP1, 8 h

11.5 ± 1.4 11.6 ± 1.6 12.0 ± 1.6 12.0 ± 1.6

Untreated 3-MB-PP1, 2 h 3-MB-PP1, 4 h 3-MB-PP1, 8 h

17.3 ± 1.1 17.4 ± 1.6 18.3 ± 1.5 18.2 ± 1.6

Untreated 3-MB-PP1, 2 h 3-MB-PP1, 4 h 3-MB-PP1, 8 h

19.4 ± 1.4 19.9 ± 1.2 21.1 ± 1.6 20.2 ± 1.6

420 min 460 min 580 min 620 min

16.1 ± 1.1 17.5 ± 1.1 18.8 ± 1.4 18.9 ± 1.2

fin1.as3

fin1.as3 cdc2.1w

fin1.as3 cdc2.3w

fin1.as3 pom1.∆

fin1.as3 cdr1.∆

fin1.as3 cdr2.∆

fin1.as3 syncnhronous

For cell length at division, cells were grown in EMM2 medium to an attenuance of 0.1 at 595 nm at 25 ◦ C unless otherwise indicated and stained with either DAPI/calcofluor to identify anaphase cells, or calcofluor alone to identify septating cells, as indicated. For cell length at division following treatment with 20 µM 3MB-PP1, cells were grown in EMM2 medium to an attenuance of 0.1 at 595 nm at 25 ◦ C and stained with calcofluor to identify septating cells.

skp1.A4 cells (Supplementary Fig. S3f). Although the context of Ser 698 (LPGS) does not conform to the RXXS/T consensus5 , we note that the analogous budding yeast kinase, Dbf2, also phosphorylates sites that deviate from this in vitro-defined consensus7 . The recognition of Fin1 by antibodies that specifically recognize phosphorylated Ser 698 (F1S698P) mirrored the in vitro and in vivo dependency on Sid2 function exhibited by F1S377P and F1S526P (Fig. 2c,d,f), indicating that Ser 698 is directly phosphorylated by Sid2 in G2 phase. Integration of the triply mutated fin1.S377AS526AS698A (fin1.3A) allele at the fin1 locus generated a protein whose levels remained constant as cells transited the cell cycle (Supplementary Fig. S3j), supporting the view that direct phosphorylation of all three sites by Sid2 regulates Fin1 activity in vivo. We quantified the cell-cycle-dependent variation in Sid2 activity by using either 32 P incorporation into casein or F1698P reactivity of Fin1.KD (Fig. 2g,h). The signal from Sid2 precipitated from sid2.250 cells that had been incubated at the restrictive temperature of 36 ◦ C defined the baseline level of activity in each assay. Sid2 specific activity

742

in size-selected synchronous cultures increased significantly following mitotic commitment. Importantly, whereas the activity did not exceed that of the Sid2.250 control early in G2 (Fig. 2g, t = 180 and 200; Fig. 2h, t = 220 and t = 230), it did in the later stages (Fig. 2g,h; compare sid2.250 lanes with: 220–280 (Fig. 2g) and 240–280 (Fig. 2h)). We conclude that Sid2 activity towards Fin1 appears in G2 phase before being considerably enhanced during mitosis. We addressed the functional significance of Fin1 activation by Sid2 in G2 phase by exploiting our ability to inhibit Fin1.as3 (Supplementary Fig. S2e–g) and Sid2.as4 (Supplementary Fig. S2h–l) function with the ATP analogue 3-MB-PP1. Analogue addition to asynchronous sid2.as4 or fin1.as3 cultures prompted an immediate, but transient, decline in mitotic index (Fig. 3a). Cell size at division increased during the recovery from this transient G2 arrest (Table 1), indicating that inhibition of Sid2 or Fin1 delayed the timing of mitotic commitment until a new size threshold for division was met. Addition of analogue to size-selected wild-type cultures immediately after completion of the first synchronous division had little impact on the timing of the

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 © 2012 Macmillan Publishers Limited. All rights reserved.

LETTERS

Cell cycle delay, cells accumulate in G2

Inhibition of the kinase S

G2

M

Activation of the kinase S

M

e

20 15 10 5 0

0

50 100 Time (min)

g

15 10 5 0

50

100 Time (min)

20

f

10 5

10 5

50 100 Time (min)

0

50 100 Time (min)

150

150

fin1.as3 cdr2.Δ sid2.as4 cdr2.Δ fin1.as3 cdr1.Δ sid2.as4 cdr1.Δ

25 20 15 10 5 0

50 100 Time (min)

150

Box Skp1 FF box

pom1.cGFP

Cullin

pom1.cGFP fin1.KD

15

0 0

h

P P P

P P P

Fin1

Fin1

Fin1

Ub Ub Ub

Ub P P P P Fin1

P

Fin1

?

P P P

Pom1 Nif1 Cdr1 Cdr2

Fin1

Sid2 pom1.cGFP fin1.Δ

20

0

15

sid2.as4 fin1.2A sid2.as4 fin1.3A sid2.as4 fin1.S698A

25

150

fin1.as3 pom1.Δ sid2.as4 pom1.Δ fin1.as3 pom1.2 sid2.as4 pom1.2

25

0

150

Wild type fin1.as3 sid2.as4 sid2.as4 fin1.KD

20

0

G1

fin1.as3 cdc2.3w fin1.as3 cdc2.1w sid2.as4 cdc2.3w sid2.as4 cdc2.1w

25 Mitotic index (%)

G1

c

25

Burst in mitosis G2

d

b

G1

Mitotic index (%)

M

Mitotic index (%)

G2

Mitotic index (%)

S

Mitotic index (%)

a

Wee1

Sid2

P

Sid1/Cdc14

Cdc2

Cdc2

Cyclin B

Cyclin B

? G2

10 μM

M

Figure 4 Sid2 and Fin1 target Cdr1/Cdr2/Pom1 to control mitotic commitment through Wee1. (a) Schematic detailing the analogue wash-out approach. (b–f) 5 h after the addition of 3-MB-PP1 to early log-phase cultures of the indicated strains cells were filtered from the culture and re-suspended at the same density in growth medium that contained no inhibitor. Restoration of Sid2 and Fin1 function induced a burst of mitosis. Importantly, restoration of Sid2 function failed to induce mitotic commitment when Fin1 kinase was inactivated by the fin1.KD mutations (b). (c) Restoring Sid2 activity in strains harbouring mutation of the candidate Sid2 phosphorylation sites in Fin1 suggests that Sid2 can activate Fin1 by phosphorylating the serine at either 377, 526 or 698. Both Sid2 and Fin1 were able to induce mitosis when the cdc2.3w mutation compromised sensitivity to Cdc25, but not when the cdc2.1w mutation compromised Wee1 inhibition of Cdc2 (d), or the functions of Pom1, Cdr1 or Cdr2 are ablated (e,f). (g) Pom1.GFP signals in the indicated

strain backgrounds. (h) A schematic depicting the model for G2/M control by Sid2/Fin1. P represents phosphorylation, and Ub represents ubiquitin conjugation. Fin1 is activated in G2 by phosphorylation by Sid2. This promotes mitotic commitment through modulation of the geometry network; however, the exact mechanism remains to be determined. Activated Fin1 promotes its own destruction, thereby limiting its activity temporally. Our present lack of understanding of the means by which Fin1 regulates the Pom1/Cdr1/Cdr2/Wee1 cell geometry network is represented by incorporating all members of this pathway that are required for Fin1 to regulate mitotic commitment within a single rectangle. The question mark to the left of this rectangle reflects our lack of knowledge as to whether it is the Sid2 or the auto-phosphorylated form of Fin1 that is responsible for the control of the cell geometry network. The question mark beneath Sid1–Cdc14 reflects our ignorance as to the nature of the cue in G2 phase that triggers this pathway. See main text for further details.

subsequent mitosis (Fig. 3b). In contrast it delayed the appearance of mitotic spindles in both fin1.as3 and sid2.as4 cells (Fig. 3c,d). Importantly, the length of septating fin1.as3 cells increased following

analogue addition (Table 1), confirming that the delayed appearance of mitotic cells represented a true delay in mitotic commitment rather than an impact on growth rate.

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 © 2012 Macmillan Publishers Limited. All rights reserved.

743

LETTERS Neither Fin1 nor Sid2 is essential for mitotic progression at 25 ◦ C. If Sid2 activation of Fin1 promotes mitotic commitment, inhibition of either kinase would create a population of late G2 cells that had grown beyond the point in G2 at which the kinase normally promotes mitotic commitment. Restoration of kinase activity to those cells that occupy this newly created ‘permissive’ window within the extended G2 would be expected to promote immediate entry of this cohort into mitosis, resulting in a synchronized wave of division (Fig. 4a). We therefore transiently inhibited each kinase by analogue addition before restoring activity by filtration and resuspension into fresh medium devoid of analogue one generation time later. In each case restoration of kinase activity was followed by a peak of mitotic cells (Fig. 4b) confirming that each kinase promotes mitotic commitment. Further analogue wash-out experiments enabled us to draw two important conclusions about the relationship between Sid2 and Fin1. First, Fin1 seems to be the main target for Sid2 kinase in mitotic commitment control because inactivation of Fin1 abolished the burst of mitosis in a sid2.as4 fin1.KD analogue wash-out experiment (Fig. 4b). Second, the Sid2/Fin1 switch is very responsive to Sid2 activity, because phosphorylation of either 698 alone or 377 and 526 promotes mitosis (Fig. 4c). Next, we examined whether Sid2/Fin1 signalling targeted the maturation-promoting factor regulators Wee1 and Cdc25, by exploiting two well-characterized cdc2 mutations; cdc2.3w mutants are largely insensitive to Cdc25 (ref. 8) whereas cdc2.1w cells are largely insensitive to Wee1 (refs 9,10). The peaks of mitosis arising from reactivation of either Sid2 or Fin1 were abolished by cdc2.1w but not cdc2.3w (Fig. 4d), suggesting an exclusive routing through Wee1. Consistently, wee1.∆ also blocked the mitotic induction in analogue wash-out experiments (data not shown). Pom1 kinase controls the timing of mitotic commitment by regulating the inhibitory impact of Cdr1/Cdr2 on Wee1 activity11–14 . pom1.∆, pom1.2, cdr1.∆ or cdr2.∆ (but not blt1.∆, data not shown) mutations mimicked cdc2.1w in blocking the wave of mitosis (Fig. 4e,f), indicating that the Sid2/Fin1 switch regulates Wee1 through Pom1. The means by which it does so awaits further analysis; however, we note that Pom1 remained at the tips of fin1.KD or fin1.∆ cells (Fig. 4g). In S. pombe, there is a direct correlation between cell length at septation and the timing of mitotic commitment15 . Blocking Fin1 function by mutational or chemical ablation of kinase activity, or gene deletion, increased cell length at septation, indicating a delay in mitotic commitment (Table 1). Blocking Sid2 function with the sid2.250 mutation similarly delayed mitotic commitment at 36 ◦ C (Table 1). Mutations that either blocked, or mimicked, phosphorylation of Fin1 by Sid2, increased or reduced cell length at division, respectively (Table 1). Importantly, loss of function of any of the proteins predicted by the analogue wash-out experiments to act after Fin1 either abolished the delay to mitotic commitment arising from deletion of fin1+ (pom1.∆,cdc2.1w) or were not additive with it (cdr1.∆,cdr2.∆), confirming that Fin1 relies on Pom1 control of Cdr1/Cdr2 to modulate Wee1 activity (Table 1). In summary, we believe that activation of Sid2 midway through G2 phase phosphorylates Ser 377, Ser 526 and Ser 698 of Fin1. Fin1 activation has two consequences: Fin1 destruction and the promotion of mitotic commitment through the Pom1/Cdr1/Cdr2/Wee1 cell geometry network (Fig. 4h).

744

Although Sid2–Mob1 kinase is a well-characterized SIN component1,16 , three arguments suggest that the population of Sid2/Mob1 that drives the G2 control of Fin1 is not operating within the context of the SIN. First, restoration of Sid2 activity in analogue wash-out experiments promoted a burst of mitosis when SIN function was ablated in sid4.SA1 and cdc11.136 cells at 36 ◦ C (Supplementary Fig. S4a,b). Second, cycles of Fin1 accumulation and destruction persisted in synchronized cultures when SIN activity was abolished by the mutations cdc11.136 and cdc7.A20 spg1.B8 (Supplementary Fig. S4c–e). In sid4.SA1 cultures, Fin1 destruction in G2 persisted; however, the mitotic accumulation of Fin1 was perturbed (Supplementary Fig. S4f). Finally, induction of SIN signalling by inactivation of Cdc16 (ref. 17) did not promote Fin1 destruction (Supplementary Fig. S4g). The impact of conditional mutations in Sid1–Cdc14 kinase were more complex as it is required for the accumulation and degradation of Fin1 in early G2 phase (Supplementary Fig. S4h,i). This would suggest that the tight relationship proposed for Sid1 activation of Sid2 in the SIN may also couple the functions of these kinases in the control of Sid2 in G2 (ref. 18). Whereas the Fin1 profile of sid1.c14 cells shows only a marginal decline in Fin levels, the data for the cdc14.332 strain, which is more easily synchronized by size selection, clearly show persistence of Fin1 destruction at the end of mitosis (Supplementary Fig. S4i). As the diffuse distribution of Fin1 during G2 phase contrasts with the association of Sid2.GFP with the spindle pole body16,19 (SPB) at this time, we used the polyclonal Sid2 antibodies to re-examine the distribution of Sid2. Unlike the Sid2.GFP fusion protein, wild-type, untagged, Sid2 did not associate with interphase SPBs (Supplementary Fig. S5a,b), even though Sid2 was present at this stage of the cell cycle (Supplementary Fig. S5c). Thus, it is a cytoplasmic population of Sid2 that is activated midway through G2 to regulate a cytoplasmic population of Fin1. We assume that Fin1 activity in an individual cell in G2 is transient because Fin1 activation promotes its own destruction. When this control is missing, G2 phase is extended by 50%. This role for Fin1 in promoting mitotic commitment through Pom1/Cdr1/Cdr2 is distinct from its role in regulating Plo1 recruitment to the SPB (ref. 20), as the plo1.S402A mutation that perturbs the latter21 had no impact on the ability of Fin1 or Sid2 to promote mitotic commitment in analogue wash-out experiments (data not shown). The timing of Sid2/Fin1 activation is strikingly reminiscent of the point in G2 phase proposed when an invariant timer is triggered by the completion of the variable sizer to commence preparations for mitotic commitment22 . Thus, mitotic commitment in fission yeast may be more similar to the staged antephase/mitotic commitment of mammalian somatic cells than the rapid transitions seen in Xenopus or sea urchin oocyte systems23 .  METHODS Methods and any associated references are available in the online version of the paper at www.nature.com/naturecellbiology Note: Supplementary Information is available on the Nature Cell Biology website ACKNOWLEDGEMENTS We thank T. Toda (CRUK London Research Institute, UK), S. Martin (University of Lausanne, Switzerland), P. Nurse (Crick Institute, London, UK), D. McCollum (University of Massachusetts, USA) for strains, K. Gull (Oxford University, UK) for TAT1 antibody, T. Carr (GDSC, UK) for plasmids and B. Hodgson (Paterson Insitute) for FACs analysis. This work was supported by Cancer Research UK (CRUK) grant number C147/A6058 and Swiss National Science Foundation support to V.S.

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 © 2012 Macmillan Publishers Limited. All rights reserved.

LETTERS AUTHOR CONTRIBUTIONS A.G. and I.M.H. conceived the study. A.G. did all of the experimentation with the exception of the mass spectrometry. Y.C. and D.L.S. did the mass spectrometry. V.S. provided essential advice and strains for the analysis of SIN function. A.G. and I.M.H. wrote the manuscript with comments and advice from V.S. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at www.nature.com/naturecellbiology Reprints and permissions information is available online at www.nature.com/reprints 1. 2. 3.

4. 5.

6. 7. 8.

9.

Simanis, V. Events at the end of mitosis in the budding and fission yeasts. J. Cell Sci. 116, 4263–4275 (2003). Krien, M. et al. A NIMA homologue promotes chromatin condensation in fission yeast. J. Cell Sci. 111, 967–976 (1998). Ye, X. S. et al. The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2 /cyclin B: coordination of two mitosis promoting kinases. EMBO J. 14, 986–994 (1995). Lehmann, A. & Toda, T. Fission yeast Skp1 is required for spindle morphology and nuclear membrane segregation at anaphase. FEBS Lett. 566, 77–82 (2004). Mah, A. S. et al. Substrate specificity analysis of protein kinase complex Dbf2–Mob1 by peptide library and proteome array screening. BMC Biochem. 6 22, http://dx.doi. org/10.1186/1471-2091-6-22 (2005). Chen, C. T. et al. The SIN kinase Sid2 regulates cytoplasmic retention of the S. pombe Cdc14-like phosphatase Clp1. Curr. Biol. 18, 1594–1599 (2008). Meitinger, F. et al. Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis. Genes Dev. 25, 875–888 (2011). Fantes, P. A. Isolation of cell-size mutants of a fission yeast by a new selective method—characterization of mutants and implications for division control mechanisms. J. Bacteriol. 146, 746–754 (1981). Nurse, P. & Thuriaux, P. Regulatory genes-controlling mitosis in the fission yeast Schizosaccharomyces pombe. Genetics 96, 627–637 (1980).

10. Russell, P. & Nurse, P. Negative regulation of mitosis by wee1+ , a gene encoding a protein-kinase homolog. Cell 49, 559–567 (1987). 11. Kanoh, J. & Russell, P. The protein kinase Cdr2, related to Nim1/Cdr1 mitotic inducer, regulates the onset of mitosis in fission yeast. Mol. Biol. Cell 9, 3321–3334 (1998). 12. Breeding, C. S. et al. The cdr2+ gene encodes a regulator of G(2)/M progression and cytokinesis in Schizosaccharomyces pombe. Mol. Biol. Cell 9, 3399–3415 (1998). 13. Martin, S. G. & Berthelot-Grosjean, M. Polar gradients of the DYRK-family kinase Pom1 couple cell length with the cell cycle. Nature 459, 852–856 (2009). 14. Moseley, J. B., Mayeux, A., Paoletti, A. & Nurse, P. A spatial gradient coordinates cell size and mitotic entry in fission yeast. Nature 459, 857–860 (2009). 15. Mitchison, J. M. & Nurse, P. Growth in cell length in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 75, 357–376 (1985). 16. Sparks, C. A., Morphew, M. & McCollum, D. Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis. J. Cell Biol. 146, 777–790 (1999). 17. Cerutti, L. & Simanis, V. Asymmetry of the spindle pole bodies and Spg1p GAP segregation during mitosis in fission yeast. J. Cell Sci. 112, 2313–2321 (1999). 18. Guertin, D. A., Chang, L., Irshad, F., Gould, K. L. & McCollum, D. The role of Sid1p kinase and Cdc14p in regulating the onset of cytokinesis in fission yeast. EMBO J. 19, 1803–1815 (2000). 19. Grallert, A., Krapp, A., Bagley, S., Simanis, V. & Hagan, I. M. Recruitment of NIMA kinase shows that maturation of the S. pombe spindle-pole body occurs over consecutive cell cycles and reveals a role for NIMA in modulating SIN activity. Genes Dev. 18, 1007–1021 (2004). 20. Grallert, A. & Hagan, I. M. Schizosaccharomyces pombe NIMA-related kinase Fin1, regulates spindle formation and an affinity of Polo for the SPB. EMBO J. 21, 3096–3107 (2002). 21. Petersen, J. & Hagan, I. M. Polo kinase links the stress pathway to cell cycle control and tip growth in fission yeast. Nature 435, 507–512 (2005). 22. Sveiczer, A., Novak, B. & Mitchison, J. M. The size control of fission yeast revisited. J. Cell Sci. 109, 2947–2957 (1996). 23. Pines, J. & Rieder, C. L. Re-staging mitosis: a contemporary view of mitotic progression. Nature Cell Biol. 3, E3–E6 (2001).

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 7 | JULY 2012 © 2012 Macmillan Publishers Limited. All rights reserved.

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METHODS

DOI: 10.1038/ncb2514

METHODS Cell culture and growth. The strains used in this study are listed in Supplementary Table S1. Cells were grown and maintained according to ref.24 . Appropriately supplemented EMM2 synthetic medium was used for all experiments, except cell length measurements, which were conducted with prototrophic strains in unsupplemented EMM2. Centrifugal elutriation was used to isolate small G2 cells25 . ATP analogues (Toronto Research Chemicals) were dissolved in methanol to generate 50 mM stock solutions that were subsequently added to cultures. For all data presented outwith Supplementary Fig. S2, 3-MB-PP1 at a final concentration of 20 µM (0.04 % methanol of total culture volume) was used to inhibit Sid2.as4 and Fin1.as3 function.

Microscopy and fluorescence-activated cell sorting analysis. Antitubulin/Sad1, anti-Sid2 and anti-histone H3 phospho-serine 10 immunofluorescence and calcofluor staining were conducted using established procedures after antibody dilution: TAT1 1:80, AP9.2 Sad1 1:25, Sid2 1:100 and phospho-histone H3 1:100 (refs 15,26,27). For Sid2 fluorescence, fixation for 10 min with 3% formaldehyde was used for optimal SPB staining, and the duration of this fixation period was extended to 30 min to give optimal preservation of the signal on the cytokinetic ring28 . DNA content through fluorescence-activated cell sorting analysis used Cytox green (Invitrogen) according to published procedures29 . Genetic manipulation, fusion protein production and antibody generation. Mutant alleles were generated by QuikChange mutagenesis (Stratagene) and inserted into the genome with the marker switch approach using natMX6 as a marker30 (Supplementary Fig. S2). The Sid2 sheep polyclonal antibody was raised by the Scottish National Blood Transfusion Service against recombinant Sid21−205 that had been purified from Escherichia coli. Rabbit polyclonal antibodies against histone H3 Ser 10 and peptides corresponding to the sequences surrounding Ser 377, Ser 526 and Ser 698 in which these serines were phosphorylated were generated by Eurogentec. The generation and purification of Fin1 polyclonal antibodies has been described previously19 . Recombinant Fin1.KDn6His was purified from E. coli.

Immunoblotting and kinase assays. To monitor Fin1 levels, all blots were cut in half, one half developed with anti-Fin1 antibodies and the other with PN24 antibodies that recognize Cdc219,31 . Blots were developed with BCIP and scanned or developed with ECF (Amersham) and imaged with a Pharos FX Molecular Imager (BioRad). Identical profiles were obtained when fluorescent antibodies were used rather than BCIP as a substrate for the secondary antibody (compare Fig. 1a with Supplementary Fig. S1a). Blots were quantified using ImageJ. A mask was drawn around each band on the blot. The total counts were recorded when the mask was both over the band and again immediately below it, in the blank region of the lane. Subtraction of the second value from the first gave the protein level. In all cases, Fin1 levels were normalized to those of Cdc2 in the same lane on the same blot. The level of Fin1 observed immediately after elution of wild-type cells was set at 1 and all other levels were quantified relative to this basal level. For western blotting, Fin1, Sid2 and Cdc2 antibodies were used at a dilution of 1:500; the three phosphospecific antibodies were used at a dilution of 1:20. For immunoprecipitations, 15 µl Dynabeads A (Invitrogen, 100.02D) was loaded with 1.5 µl Fin1 or Sid2 undiluted antibody and used as per the manufacturer’s instructions. For the kinase assays, Fin1/Sid2 was precipitated from 2×108 cells with polyclonal antibodies with Dynabeads A (Invitrogen) in KA buffer: 50 mM HEPES, 10 mM

EDTA, 40 mM Na-β-glycPhosh, 4 mM Na3 VO4 , 50 mM NaF, 0.6% NP40, 150 mM NaCl, protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulphonyl fluoride. The kinase reaction was carried out at 30 ◦ C for 30 min in KR buffer: 20 mM HEPES, 15 mM KCl, 1 mM EGTA, 10 mM MgCl2 , 10 mM MnCl2 , 0.125 nM ATP and 10 µg of either recombinant Fin1.KDn6His (Fig. 2a–d and Supplementary Fig. S2b,d,k,n) casein (Figs 1f and 2e,g) or Fin1FP1 (ref. 19) (Fig. 2h). For assessment through 32 P incorporation, 5 µCi[γ −32 P]ATP was also added to the reaction mix. The amount of 32 P incorporated into the substrate was determined with a Phosphorimager (BioRad). To identify in vivo phosphorylation sites on Fin1, the protein was precipitated from 2 × 1011 skp1.A4 cells with polyclonal α-Fin1 antibodies. To identify in vitro Sid2-phosphorylation sites on Fin1, a Sid2 kinase assay was carried out as described in the previous paragraph using 50 µg recombinant Fin1.KDn6His as a substrate having isolated Sid2 from 6 × 108 cells. The Fin1.KD protein was isolated from the reaction mix using Ni-magnetic beads (Qiagen) before mass spectrometry.

Mass spectrometry. Samples were run on 4–12% NuPAGE bis-Tris gel (Invitrogen); Fin1 bands were excised and digested with either 20 ng sequencing-grade trypsin (Sigma-Aldrich), 400 ng LysN (Associates of Cape Cod) or 350 ng elastase (Calbiochem) in 100 µl 40 mM ammonium bicarbonate with 9% (v/v) acetonitrile at 37 ◦ C for 18 h. The peptides were separated using a Nano-Acquity UPLC system (Waters) using a Waters NanoAcquity BEH C18 column (75 µm inner diameter, 1.7 µm, 25 cm) with a gradient of 1–25% (v/v) of acetonitrile with 0.1% formic acid over 30 min at a flow rate of 400 nl min−1 . The LTQ-Orbitrap XL mass spectrometer was operated in parallel datadependent mode where the mass spectrometry survey scan was performed at a nominal resolution of 60,000 (at m/z 400) in the Orbitrap analyser over an m/z range of 400–2,000. The top 6 precursors were selected for collision-induced dissociation in the LTQ at a normalized collision energy of 35% using multi-stage activation at m/z 98.0, 49.0 and 32.7 Da.

24. Moreno, S., Hayles, J. & Nurse, P. Regulation of p34cdc2 protein-kinase during mitosis. Cell 58, 361–372 (1989). 25. Creanor, J. & Mitchison, J. M. Reduction of perturbations in leucine incorporation in synchronous cultures of Schizosaccharomyces pombe made by elutriation. J. General Microbiol. 112, 385–388 (1979). 26. Hagan, I. & Yanagida, M. The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability. J. Cell Biol. 129, 1033–1047 (1995). 27. Petersen, J., Paris, J., Willer, M., Philippe, M. & Hagan, I. M. The S. pombe aurora related kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation. J. Cell Sci. 114, 4371–4384 (2001). 28. Hagan, I. M. & Asycough, K. R. in Protein localisation by fluorescence microscopy (ed. Allan, V. J.) 179–206 (Oxford Univ. Press, 2000). 29. Knutsen, J. H. et al. Cell-cycle analysis of fission yeast cells by flow cytometry. PLoS ONE 6, e17175 (2011). 30. Van Driessche, B., Tafforeau, L., Hentges, P., Carr, A. M. & Vandenhaute, J. Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance. Yeast 22, 1061–1068 (2005). 31. Simanis, V. & Nurse, P. The cell-cycle control gene cdc2+ of ssion yeast encodes a protein-kinase potentially regulated by phosphorylation. Cell 45, 261–268 (1986).

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S U P P L E M E N TA R Y I N F O R M AT I O N DOI: 10.1038/ncb2514

Grallert et al. Supplementary Figure 1.

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fin1.K33RN165A leu1::nmt41fin1.nGFP, Fin1.KD levels, Figure 1e Fin1 Cdc2 0 20 40 60 80 100 120 140 160 170 180 187 194 200 207 214 220 227 234 240 247 254 260 267 274 280 287 294 300 310 320 340 360 380 400 420 440 460 480 500 520 540

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cdc11.136, shift to 36°C, Supplementary Figure 4e Fin1 Cdc2 180 195 202 210 217 225 232 240 247 255 262 270 277 285 292 300 307 315 322 330 345 360 375 390 405 420 435 450 465 480 495 510 525

sid2.250, shift to 36°C, Figure 1h Fin1 Cdc2 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 435 450 465 480 495 510 525 540 555 570 585 600 615

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sid2.as4, 20µM 3MBPP1, Figure 1i!

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with the chromogenic substrate BCIP. The profile in a) is indistinguishable from the plot in Figure 1a in which BCIP was used to develop the blot. b) The blots that were quantified to generate the data shown in the indicated figures. (c) Fin1 levels and septation profiles of the portions of the culture shown in in Figure 1i that received no treatment (left) or to which an equivalent volume of methanol had been added to that bearing 3-MB-PP1 in Figure 1i.

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S U P P L E M E N TA R Y I N F O R M AT I O N

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8

42

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40

SacI

SacI

6

NM-PP1, 8h

Wild type

3

NM-PP1, 4h

sid2.M285A T348A

2

NA-PP1, 8h

sid2.as4

1

NA-PP1, 4h

Wild type

0 80

Sid2 protein

m

20

untreated, 8h

sid2.M285G

40

untreated, 4h

sid2.as1

Septating cells (%)

l Phenotype without ATP analogue

Kinase activity

1.! 2.! 3.! 4.! 5.! 6.! 7.!

wild type sid2.AS1 sid2.AS4

60

1

wild type, no 3MB-PP1 wild type, 3MB-PP1, 0.5h wild type, 3MB-PP1, 4h sid2.as4, no 3MB-PP1 sid2.as4, 3MB-PP1, 0.5h sid2.as4, 3MB-PP1, 4h

Genotype

XhoI

NdeI

Binucleate cells (%) Tetranucleate cells (%)

7.! 8.! 9.! 10.! 11.! 12.!

80

0h

NM-PP1

3MB-PP1

36°C

25°C

NA-PP1

j

wild type, 25C wild type, 36C, 0.5 wild type, 36C, 2.5 sid2.250, 25C sid2.250, 36C, 0.5 sid2.250, 36C, 2.5 wild type, no MB wild type, 3MBwild type, 3MBsid2.AS4, no MB sid2.AS4, 3MBsid2.AS4, 3MBwild type, 25°C wild type, 36°C, 0.5h wild type, 36°C, 2.5h sid2.250, 25°C sid2.250, 36°C, 0.5h sid2.250, 36°C, 2.5h

M285T348

1.0 kb

1 2 3 4 5 6 7

1 2 3 4 5 6 7 8 9 10 11 12

1.! 2.! 3.! 4.! 5.! 6.!

k

terminator + natMX6

0.89 kb

Sid2 protein Sid2 protein

1 0.8 0.6 0.4 0.2 0

MeOH

1

sid2 NdeI

wild type wild type fin1.as3 fin1.A S3 fin1.as4 fin1.A S4

i 1.2

Septation index (%)

fin1.K33R

Genotype

12 10 8 6 4 2 0

2

0

o

fin1.K33R N165A

wild type

g

wild type wild type fin1.as1 fin1.AS1 fin1.as2 fin1.AS2 fin1.as3 fin1.AS3 fin1.as4 fin1.AS4

40

f Fin1 protein

MluI

60

NA-PP1 NA-PP1

His-Fin1.KD Fin1

80

3MB-PP1 3MB-PP1

Kinase activity

SnabI

SnabI

Fin1+ Fin1.KD

36C 36°C

-

MeOH MeOH

+ -

25C 25°C

e

Abnormal mitosis / no. of mitotic cells

NdeI

XhoI

M82 G168

IP

+

0.86 kb

Kinase activity Fin1 protein

fin1.K33R fin1.K33RN165A N165A

0.54 kb

1.2 kb

d

c

natMX6

wildtype type wild

fin1

fin1.K33R fin1.K33R

b

K33 N165

fin1.!"

a

Abnormal mitosis/no. of mitotic cells (%) Abnormal mitosis/

Grallert et al. Supplementary Figure 2.

500

Untreated MeOH 5 µM 10 µM 20 µM

30 20 10 0 200

Time (min)

2

300

400

500

Time (min)

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S U P P L E M E N TA R Y I N F O R M AT I O N

Figure S2 Fin1and Sid2 reagents and the impact of ATP analogues upon cell cycle progression. a-g) Fin1 reagents. a-d) fin1.K33RN165A encodes a catalytically inactive protein. a) A cartoon of the construct used to integrate fin1 mutant alleles at the native locus indicating the location of the residues within the catalytic domain that were mutated to abolish kinase activity (K33, N165) or generate analogue sensitive alleles (M82, G168). The selection of the two sites for kinase inactivation was based upon the previous work in many labs that has established that point mutation of either the conserved lysine in domain II to arginine or the asparagine of domain VIB to alanine severely compromises kinase activity1. b) Fin1 kinase assays in which the activity of wild type Fin1 and Fin1.K33R or Fin1.K33RN165A mutant proteins against casein is assessed. c) The spindle formation defect after 3 hours at 37°C of the double fin1.K33RN165A mutant was greater than that of the single fin1.K33R. Similarly Fin1 protein accumulated to a greater extent in fin1.K33RN165A than in fin1.K33R cells (Figure 1c). Finally, cell length measurements indicate a greater defect in the double fin1.K33RN165A than single fin1.K33R mutant (Table 1). We conclude that the fin1.K33RN165A allele encodes a catalytically inactive molecule and is therefore referred to as fin1.KD (for kinase dead) throughout the manuscript. d) Fin1 kinase assays in which the catalytically inactivated Fin1 substrate is mixed with immunoprecipitates from the indicated strains; the substrate on it’s own possesses no intrinsic kinase activity. e) A graph scoring the frequency of abnormal mitoses 3h following addition of analogue to the fin1.asx alleles indicated. f) Fin1 levels following normalisation to control Cdc2 protein levels in the indicated strains 3 hours after the addition of the indicated ATP analogues to early log phase cultures at 25°C and 36°C. g) A table showing the mutation sites and phenotypes of the different fin1.as alleles. (h-n) Sid2 reagents (h) A cartoon of the construct used to integrate sid2 alleles at the native locus indicating the location of the residues within the catalytic domain (M285, T348) that were selected for mutation to generate analogue sensitive alleles according to the published data from the Shokat lab2,3. (i) Sid2 protein levels (normalised to those of Cdc2 in the same lane on the same blots) in the indicated strains after the indicated manipulations. (j) Plots of the indicated phenotypes at the

times shown after the addition of the indicated ATP analogues or solvent alone to the indicated sid2.as alleles. (k) in vitro Sid2 kinase assays in which Sid2 immunoprecipitates from the indicated strains were used to phosphorylate casein with 32PgATP demonstrate that this assay specifically detects Sid2 kinase and no other activity. (l) A table showing the mutation sites and phenotypes of the different sid2.as alleles. (m) Characterisation of Sid2 antibodies. The slower migration of the band recognised by affinity purified Sid2 antibodies in wild type cells in a sid2.13myc background and the enhancement of the band upon induction of the expression of an integrated version of the sid2+ gene at the leu1 locus (leu1::nmt41sid2) indicates that these antibodies recognise the Sid2 protein. n) Sid2 kinase assays in which the catalytically inactivated Fin1 substrate is mixed with immunoprecipitates from the indicated strains. The right hand lane indicates that the substrate on it’s own possesses no intrinsic kinase activity under the reaction conditions used to monitor Sid2 activity. o) Impact of ATP analogue addition upon the septation profile of wild type cells. A wild type culture was synchronised with respect to cell cycle progression by size selection and split into equal portions to which the indicated analogues were added at the indicated concentrations. Septation was used as a read out of cell cycle progression. While the timing of septation was perturbed by the addition of the indicated concentrations of 1-NA-PP1, 1-NM-PP1 and Bn-PP1 in this assay, 3-MB-PP1 had the least impact. The modest impact of 20 mM MB upon the septation profile was not reproducible (see Figure 3) and probably arose because of the batch analysis approach in this extensive experiment. We therefore believe that in the assays used in this study had no impact upon the timing of mitotic commitment or the rate of mitotic progression. These specific conditions for which we believe this assertion holds true were the addition of 20 mM MB to asynchronous or size selected cultures grown to early log phase in the minimal medium EMM2 to which the supplements Leucine, Uracil, Adenine and Histidine had all been added to a final concentration of 200 mg ml-1. It is likely that in other conditions such as live cell imaging where there are considerable levels of UV induced stress and conditions in many cases are anaerobic, different levels of sensitivity may apply.

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S U P P L E M E N TA R Y I N F O R M AT I O N

b

90 80 70 60 50 40 30 20 10 0

2 Fin1

1.5 1

0.5 0 150

250 350 Time (min)

fin1.KD skp1.A4, 36°C

Fin1

2.5

No. of cells (%)

sid2.as4 skp1.A4, 3MB-PP1

Fin1 Binucleate cells Tetranucleate cells

6

30

5

25

4

20

3

15

2

10

1

5

0

0 0

450

c

100

200

300

Septation index (%)

a

Grallert et al. Supplementary Figure 3.

400

Time (min)

d pS377 (Elastase)

S526 LysN

G E G L P N R K V pS K L S b8

b8

e

b10 b11

b10

b11

f pS377 (Elastase)

pS698 LysN

a

* Indicates loss of H3PO4

h

100

Untreated, binucleate

80

3MB-PP1, binucleate

60

3MB-PP1

MeOH mitosis

40 20

3MB-PP1 delayed mitosis

0 0

j

fin1.2A

sample

No. of cells (%)

MeOH, binucleate

mid/late G2

20µM 3MB-PP1 / MeOH

Cells go through one cell cycle

early G2

early G2

100

200 300 Time (min)

400

fin1.3A

C G2

2

3

50 40

Fin1

30 20

1

10 0

0 0

100

200 300 Time (min)

400

500

70 60 50

2

40 30

1

20 10

0

0 0

Fin1 protein

Figure S3 Phosphorylation of S377, S526 and S698 of Fin1 by Sid2. The indicated strains were synchronised by size selection at 25°C. Each culture was split in two with one half shifted to 36°C while the other was maintained at 25°C. (a) For the sid2.as4 skp1.A4 mutant in panel a the shift was performed 180 minutes after size selection and the ATP analogue 3MB-PP1 was added to a final concentration of 20 mM to each culture at this time. For the fin1. KD skp1.A4 culture the shift was performed immediately after splitting the culture following size selection. (c-f) Mass spectrometric analysis of Fin1 phosphorylation sites. (c, d) Product ion spectra from two independent elastase generated peptides (sequences denoted). Product ions of particular analytical note are labelled with those fragments showing H3PO4 loss further denoted with *. Both spectrum (c) and (d) confirm the respective sequences and location of the site of phosphorylation unambiguously as S377. (e, f) Product ion spectra from two LysN generated peptides. Product ions of particular analytical

Septation index (%)

C

Septation index (%)

3

Fin1

asynchronous

Synchronization by size selection

collect cells for IP

b

g

i

Fin1 Septation index

100

200 300 Time (min)

400

500

Septation index

note are labelled. The spectrum in panel (e) confirms the denoted sequence and the location of the site of phosphorylation unambiguously as S526. The spectrum in (f) confirms the denoted sequence and the location of the site of phosphorylation unambiguously as S698. (g) A cartoon illustrating the procedure for sampling for the blots in Figure 2d. Small G2 cells were isolated by centrifugal elutriation. After one round of division the culture was split into two and the ATP analogue 3-MB-PP1 in MeOH or the solvent alone were added. 30 minutes later Fin1 was isolated by immunoprecipitation and probed with the phospho-specific and generic Fin1 antibodies to give the blots shown in Figure 2d. (h). The phenotypic characterisation of one of the three cultures used to create the samples for panel d of Figure 2d. (i-j) Fin1 protein levels as fin1.2A (i) or fin1.3A (j) cultures for which small G2 cells were isolated from asynchronous cultures at time 0. While modest changes in Fin1.2A protein levels were observed, no changes were seen in Fin1.3A levels.

4

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S U P P L E M E N TA R Y I N F O R M AT I O N Grallert et al. Supplementary Figure 4.

25

b

sid2.as4 fin1.KD sid2.as4

15

sid4.SA1sid2.as4 sid4.A1 sid2.as4

10

sid4.SA1 fin1.KD sid4.A1 fin1.KD sid2.as4 sid2.as4

30

5

cdc11.136 fin1.KD sid2.as4

15 10

100 Time (min)

0

150

wild type, shift to 36°C

C

C

40

4

30

G2

3

20

2

10

1 0 300

5

80

4

60

3

40

G2

2 1

20

0

0 150

350 Time (min)

40

1

20

0

0 300

400 Time (min)

500

sid4.SA1, shift to 36°C

6 5

C

G2

3

60 40

2

20

1 0

0 150

Binucleate cells

100 80

4

550

Fin1

g

f

60

G2

200

100

C

80

2

400

cdc11.136, shift to 36°C

6

100

G2

3

Fin1

200 Time (min)

150

C

4

No. of cells (%)

100

100

5

0 0

Time (min)

cdc7.A20 spg1.B8, shift to 36°C

Fin1

5

50

6

50 Septation index (%)

6

0

d

No. of cells (%)

50

c

Fin1

cdc11.136 sid2.as4

5 0

Fin1

fin1.KD sid2.as4

20

0

e

sid2.as4

25

No. of cells (%)

Mitotic index (%)

20

Mitotic index(%)

350 Time (min)

Septation index

Tetranucleate cells

cdc16.116 shift to 36°C

4

70 60

Fin1

3

50 40

2

30 20

1

10

0

Septation index (%)

add HU

Fin1 Septation index, 36°C, HU Septation index, 25°C, Septation index, 25°C, HU

0 0

h

100

200 300 Time (min)

400

500

i

sid1.c14, shift to 36°C

cdc14.332, shift to 36°C

4

80

4

80

3

60

3

60

2

40

2

40

1

20

1

20

0

0 150

250

350 Time (min)

Fin1

5 No. of cells (%)

100

Fin1

5

0

0 150

450

Fin1

100

Binucleate cells

Figure S4 SIN function is not required to generate the cycles of Fin1 accumulation/destruction. (a, b) “Analogue washout” experiments as executed for Figure 4b-f with the indicated strains. Restoration of Sid2 kinase activity was able to induce mitotic commitment in strains in which the function of the SIN anchor SPB protein Sid4 or the scaffold molecule Cdc11 had been compromised by conditional mutation. The lack of induction in these backgrounds when Fin1 is catalytically inactive indicates that loss of either protein does not lead to a re-routing of the controls from Sid2 to another primary target. (c-f, h,i) Plots of Fin1 levels in size selected cultures of the indicated strains. Culture were split into two after the first round of septation and one half was shifted to 36°C while the other was maintained at 25°C for the duration of the experiment (data not shown). Both phases of destruction were maintained in size selected wild type

No. of cells (%)

a

250

350 Time (min)

450

Tetranucleate cells

cultures (c). Perturbation of SIN function by mutation of the G protein/Pak kinase cassette Spg1/Cdc7 (d), the scaffold protein Cdc11 (e) or the SPB anchor protein Sid4 (f), had no impact upon the cycles in Fin1 levels. (g) SIN induction according to an approach detailed previously17. Hydroxyurea was added to small G2 wild type or cdc16.116 cells 60 minutes after they had been isolated from an asynchronous population by centrifugal elutriation. 230 minutes later each culture was split into two and one half shifted to 36°C. This temperature shift inactivated the SIN inhibitor Cdc16 driving SIN activation and consequently induced septation (grey triangles) in the S phase arrested cdc16.116 culture, but did not impact upon SIN function in the wild type control. Fin1 levels were not altered by this form of ectopic induction of SIN activity. Sid1 (h) and Cdc14 (i) are required for the G2 phase accumulation and destruction of Fin1.

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S U P P L E M E N TA R Y I N F O R M AT I O N Grallert et al. Supplementary Figure 5.

a

b

wild type ring fix

wild type

sid2.c13myc

wild type 10µM

10µM

Sid2

Sid2

Sid2

Sid2

DAPI

DAPI

DAPI

DAPI

Sad1

DAPI+DIC

DAPI+DIC

DAPI+DIC

DAPI+DIC

DAPI

c

Sid2

40

Sid2

30 1

20 10

DAPI+DIC

Sid2 Cdc2

0 0

100 Sid2

cdc10.V50

200

300

400

500

0 20 60 80 100 120 140 160 168 176 184 192 200 208 216 224 232 240 248 256 264 272 280 288 296 304 312 320 340 360 380 400 420 440 460 480

0

Septation index (%)

50 2

d

sid2.cGFP

Time (min) Septation index

cdc25.22

e

nda3.KM311

cdc11.136

sid4.A1 10µM

10µM

Sid2

Sid2

Sid2

Sid2

Sid2

DAPI

DAPI

DAPI

DAPI

DAPI

DAPI+DIC

DAPI+DIC

DAPI+DIC

DAPI+DIC

DAPI+DIC

Figure S5 Sid2 recruitment to mitotic but not interphase SPBs. a, b, d, e) Sid2 immunofluorescence/DAPI/combined DAPI and differential interference contrast (DIC) images of the indicated strains. For all panels in the figure the “SPB fix” (10 mins 3% formaldehyde) was used with the exception of the indicated panel in (a) where “ring fix” (30 mins 3% formaldehyde) was employed. For (b) the immunofluorescence imaging combined Sid2 and Sad1 imaging as indicated to show that the Sid2 signal was absent from short, uninucleate, G2 cells in which a clear Sad1 signal could be detected. (a) In addition to staining of the cytokinetic ring, the Sid2 signal was seen on the mitotic SPBs of sid2+ and sid2.c13myc cells. In contrast, the ring staining was accompanied by association with the SPBs irrespective of cell cycle status in the sid2.GFP background. We conclude that the finding that Sid2 is recruited to SPBs throughout the cell cycle16 is misleading as it is based upon a behavioural shift in the Sid2.GFP fusion protein that is generated by the addition of the GFP sequences. Rather we suggest that Sid2 binds to SPBs of mitotic cells from just after the separation of the two

SPBs following mitotic commitment until the degradation of the septum (staining persists on the SPBs while the background fluorescence in cells indicates that the primary septum is full formed). (c) Sid2 levels in western blots of whole cell extracts (right panel) remain constant as cells transit the cell division cycle (left). (d) Sid2 immunofluorescence of cdc10.v50, cdc25.22 or nda3.KM311 cells after, 5, 4.5 or 9 hours at their restrictive temperatures, respectively, consolidates the view that Sid2 only associates with the SPBs during mitosis (nda3.KM311), but neither G1 (cdc10. v50) nor G2 (cdc25.22) phase of the cell cycle. We conclude that Sid2 associates with mitotic but not interphase SPBs and that this association with the SPB is regulated at the level of recruitment and not synthesis/ stability. (e) Given that the fusion of GFP to Sid2 kinase influences it’s recruitment to the SPB we assessed the dependency of the wild type (Sid2+) protein upon the function of the SPB anchor and SIN scaffold molecules Sid4 and Cdc11. Like Sid2.GFP, wild type Sid2+ relies upon these molecules to dock to the SPB.

6

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S U P P L E M E N TA R Y I N F O R M AT I O N

References 1. 2. 3.

Hanks, S.K., Quinn, A.M. & Hunter, T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42-52 (1988). Bishop, A.C. et al. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395-401 (2000). Blethrow, J., Zhang, C., Shokat, K.M. & Weiss, E.L. Design and use of analog-sensitive protein kinases. Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] Chapter 18, Unit 18 11 (John Wiley & Sons, 2004).

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