RESEARCH ARTICLE 2627 In vivo localisation of fission -dependent kinase cdc2p and cdc13p during and

Anabelle Decottignies*, Patrick Zarzov and Paul Nurse Laboratory, Imperial Cancer Research Fund, London, WC2A 3PX, UK *Author for correspondence (e-mail: [email protected])

Accepted 23 April 2001 Journal of Cell Science 114, 2627-2640 (2001) © The Company of Biologists Ltd

SUMMARY

We investigated the in vivo localisation of fission yeast disappears. If cdc13p cannot be recognized by the cyclin-dependent kinase cdc2p during mitosis and meiosis. -promoting complex, cdc2-YFP and cdc13-YFP Fusion to yellow fluorescent protein (YFP) revealed that remain associated with the spindle. In mating cells, cdc2- cdc2-YFP is present in the cytoplasm at all stages of the cell YFP enters the nucleus as soon as the cells undergo fusion. cycle. Nuclear cdc2-YFP fluorescence oscillates with that of During karyogamy and meiotic , cdc2-YFP is cdc13-YFP cyclin. At G1/S, at least one of cdc13p, cig1p or highly enriched on the centromeres. In meiosis I, cig2p B-type is required for the accumulation of association of cdc2-YFP with the spindle and the spindle cdc2-YFP into the nucleus. Cdc2-YFP and cdc13-YFP are pole bodies shows differences to mitotic cells, suggesting highly enriched on the spindle pole body of cells in late G2 different mechanisms of spindle formation. This study or arrested at . Both accumulate on the spindle pole suggests that changes in cdc2p localisation are important bodies and the spindle in prophase and for both mitosis and meiosis regulation. independently of the microtubule-associated protein dis1p. In anaphase, the cdc2p/cdc13p complex leaves the spindle prior to sister chromatid separation, and cdc13-YFP is Key words: Schizosaccharomyces pombe, Mitosis, Meiosis, Cyclins, enriched at the nuclear periphery before fluorescence Cyclin-dependent kinase

INTRODUCTION kinase bound to cyclin), but not extracts, can convert fission yeast spindle pole bodies (SPBs, the equivalent of Cyclin-dependent kinases (CDKs)1 are required for the onset animal centrosomes) to a nucleation competent state (Masuda of S phase and M phase of the mitotic cell cycle (reviewed by et al., 1992). However, purified cdc2/ complex was Nurse, 2000). In higher eukaryotes, distinct CDKs associate unable to do so, suggesting that other factors besides cdc2 with several cyclins to control the G1/S transition, whereas kinase are also required for SPB activation (Masuda et cdk1/cyclin B is specifically required to control the G2/M al., 1992). In multicellular eukaryotes, duplication of the transition (reviewed by Nigg, 1995). In the fission yeast centrosome requires cdk2/ activity (reviewed by Schizosaccharomyces pombe, cdc2p/cdc13p complex controls Whitehead and Salisbury, 1999; Meraldi et al., 1999; Okuda the G2/M transition of the cell cycle, whereas cig2p B-type et al., 2000). Cdk1 localises to spindle MTs and metazoa cyclin associates with cdc2p to promote the G1/S transition, centrosomes via association with MT-associated proteins although this can be accomplished by the cig1p and cdc13p B- (MAPs), whose phosphorylation induces a change in MT type cyclins (Fisher and Nurse, 1996; Mondesert et al., 1996). dynamics at the onset of mitosis (reviewed by Andersen, 2000). Fission yeast possesses two other cyclins, puc1p and pas1p, In fission yeast, Alfa et al. showed by immunofluorescence that which are related to the Cln1-3p G1 cyclins of Saccharomyces cdc2p and cdc13p are localised to the SPBs during mitosis cerevisiae (Forsburg and Nurse, 1991; Tanaka and Okayama, (Alfa et al., 1990). In a more recent paper, cdc13-GFP fusion 2000). Puc1p may regulate the G1 phase progression in was shown to localise on the SPBs and the spindle from response to cell size (Martin-Castellanos et al., 2000), whereas prophase to metaphase (Yanagida et al., 1999). pas1p associates with a second non-essential CDK, pef1p, to S. pombe cdc2p is also required during the meiotic cell cycle activate the res2p-cdc10p transcriptional complex at the for premeiotic DNA synthesis, the second division and, very START point of the cell cycle (Tanaka and Okayama, 2000). likely, the first meiotic division (reviewed by Murakami CDKs are not only required for the onset of S phase and M and Nurse, 2000). Once karyogamy and premeiotic DNA phase of the mitotic cell cycle but also are involved in the replication have occurred, the meiotic prophase nucleus shows regulation of centrosomes and microtubule (MT) dynamics. In an elongated morphology, called a ‘horse-tail’, and oscillates Xenopus egg extracts, CDKs induce a change in MT dynamics back and forth between the cell poles (reviewed by Hiraoka, and steady-state length (Verde et al., 1990). Cytostatic factor- 1998). During this horse-tail movement, telomeres are arrested Xenopus egg extracts (which contain active cdc2 clustered at the SPB in a bouquet-like arrangement at the 2628 JOURNAL OF CELL SCIENCE 114 (14) leading end of the tail while centromeres from the three pairs nmt1 promoter. Mating and sporulation of cells was induced on of duplicated chromosomes are separated from the SPB EMM2 medium without NH4Cl. The S. pombe strains used in this (reviewed by Hiraoka, 1998). When this movement stops, the study are listed in Table 1. first meiotic division starts, leading to reductional segregation Gene tagging and deletion of homologous chromosomes, followed by separation of sister The cdc2-YFP, cdc2-CFP and cdc13-YFP genes were cloned into the chromatids in meiosis II (reviewed by Bickel and Orr-Weaver, SalI/SmaI-digested pREP5 plasmid, which contains the full-strength 1996). thiamine-repressible nmt1 promoter and the sup3-5 marker In this study, we used GFP- and YFP-tagged proteins to (Maundrell, 1993). The primers used for PCR amplifications are listed investigate the in vivo localisation of cdc2p/cyclin B in the in Table 2. The cdc2+ (primers 1 and 2) and cdc13+ (primers 3 and 4) fission yeast S. pombe and to establish its significance for both genes were amplified by PCR. The EYFP gene was isolated from the mitotic and meiotic cell cycle regulation. pEYFP plasmid (Clontech, cat. number 6004-1). The CFP gene was amplified by PCR on the pECFP plasmid (Clontech, cat. number 6075-1) (primers 5 and 6). The YFP and CFP fragments were digested with NotI, blunted with Klenow and digested with BamHI. The cdc2+ MATERIALS AND METHODS and cdc13+ ORFs were cloned as SalI/BamHI-digested fragments. Plasmids with Y(C)FP fusions were integrated at the own gene locus Yeast strains and media in S. pombe cells, generating strains AD143 and AD112 in which the Growth, transformation and genetic manipulation of S. pombe fusions are controlled by the endogenous gene promoters (see Fig. were performed using methods described previously 1B). In AD185 strain, the nmt1prom-cdc2 gene was replaced by the (http://www.bio.uva.nl/pombe/). Cells were routinely grown at 32°C kanR gene using a method described previously (Bähler et al., 1998). in either YE medium with supplements (YES) or minimal EMM2 For the cloning of cdc13∆81-YFP (lacking the first 81 amino acids) medium with or without addition of 15 µM thiamine to control the into pREP5 plasmid, the cdc13∆81 (primers 7 and 8) and the YFP

Table 1. Yeast strains used in this study Strains Relevant genotype Origin AD112 h+ pREP5::cdc13-YFPint* This study AD117 h− pREP5::cdc2-CFPint pREP5::cdc13-YFPint This study AD143 h− pREP5::cdc2-YFPint This study AD152 h+ pREP5::cdc2-YFPint ∆nmtcdc2::kanR cut4-533 This study AD157 h− pREP5::cdc2-YFPint mts2 This study AD178 h− [pREP45::cdc13-YFP]‡ This study AD179 h− pREP45::YFP-cig2int This study AD185 h− pREP5::cdc2-YFPint ∆nmtcdc2::kanR This study AD189 h+ pREP5::cdc2-YFPint ∆nmtcdc2::kanR -22 This study AD192 h+ pREP5::cdc2-YFPint ∆nmtcdc2::kanR cdc10-V50 This study AD199 h− pREP5::cdc2-YFPint ∆nmtcdc2::kanR dis1-203 This study AD203 h− pREP45::cdc13∆81-YFPint This study AD205 h−∆cdc13::ura4+ pREP41::cdc13int pREP5::cdc2-YFPint cdc25-22 ∆nmtcdc2::kanR This study AD207 h+∆cdc13::ura4+ pREP41::cdc13int ∆cig1::ura4+ ∆cig2::ura4+ pREP5::cdc2-YFPint cdc25-22 ∆nmtcdc2::kanR This study AD210 h+∆cdc13::ura4+ pREP41::cdc13int ∆cig1::ura4+ pREP5::cdc2-YFPint cdc25-22 ∆nmtcdc2::kanR This study AD212 h− pREP5::cdc13-YFPint cdc10-V50 This study AD213 h− pREP5::cdc13-YFPint mts2 This study AD217 h− pREP5::cdc13∆81-YFPint This study AD219 h− dis1-GFP cdc25-22 This study AD226 h− pREP5::cdc13-YFPint cdc25-22 This study AD244 h+ cdc25-22 [pREP1::GFP-α2-tubulin]§ This study AD245 h− ∆cdc13::ura4+ pREP41::cdc13int ∆cig2::ura4+ pREP5::cdc2-YFPint cdc25-22 ∆nmtcdc2::kanR This study AD257 h− pREP5::cdc2-YFPint ∆nmtcdc2::kanR cyr1∆::LEU2 sxa2∆::ura4+ This study AD259 h− pREP5::cdc13∆81int This study AD264 h− pREP5::cdc13-YFPint dis1-203 This study AD265 h+ pREP5::cdc2-YFPint ∆nmtcdc2::kanR LacO repeat-lys1+ GFP-LacI-NLS-his7+ This study AD266 h+ pREP5::cdc13∆81int pREP5::cdc2-YFPint ∆nmtcdc2::kanR This study AD267 h− pREP5::cdc13-YFPint cut4-533 This study AD268 h− pREP5::cdc2-YFPint ∆nmtcdc2::kanR cdc25-22 [pREP1::GFP-α2-tubulin] This study MKY7A-4 h+ LacO repeat-lys1+ GFP-LacI-NLS-his7+ Nabeshima et al., 1995 PN671 h− dis1-203 Ohkura et al., 1988 PN745 h+ Lab collection PN1315 h−∆cdc13::ura4+ pREP41::cdc13int Fisher and Nurse, 1996 PN1351 h−∆cdc13::ura4+ pREP41::cdc13int ∆cig1::ura4+ ∆cig2::ura4+ Fisher and Nurse, 1996 PN1379 h+ cyr1∆::LEU2 sxa2∆::ura4+ Maeda et al., 1990 Kawamukai et al., 1991 PN1589 h− mts2 Gordon et al., 1993

*int, the plasmid is integrated into the yeast genome. ‡[ ], the plasmid is not integrated. §pREP1::GFP-α2-tubulin[LEU2] is a gift from Y. Hiraoka’s group. Fission yeast cdc2p localisation 2629

(primers 9 and 6) genes were amplified by PCR and processed as at 458 nm and YFP at 514 nm. Fluorescence emission was observed described above. The same strategy was used to clone cdc13∆81-YFP using a BP475-525 filter for CFP and a LP530 filter for YFP. and cdc13-YFP into pREP45 (medium-strength nmt1 promoter). In the pREP5:: cdc13∆81 plasmid, the cdc13∆81 gene was amplified by Cyclin B-dependent localisation of cdc2-YFP PCR (primers 7 and 10) and cloned into SalI/BamHI-digested pREP5 In Fig. 4, cdc25-22 cells were grown overnight at 25°C in EMM2 plasmid. For N-terminal YFP-tagging of cig2p, the YFP (primers 5 medium. Cultures were arrested at the G2/M transition by incubation and 11) and cig2+ (primers 12 and 13) genes were amplified by PCR. for 4 hours at 36°C. The cdc13 gene transcription was repressed by YFP was digested with SalI and KpnI and ligated to the SalI/BamHI- addition of 15 µM thiamine after either 3 hours (AD210) or 4 hours digested pREP45 plasmid together with the KpnI/BglII-digested (AD245 and AD207) at 36°C. Cells were pelleted and processed for fragment of cig2+. Plasmids pREP5::cdc13∆81-YFP, LFM at RT (23-25°C), as described above, or incubated at 25°C and pREP45::cdc13∆81-YFP, pREP5::cdc13∆81 and pREP45::YFP-cig2 processed for DNA content measurement by FACS as described were integrated into the genome of strains AD217, AD203, AD259 previously (http://www.bio.uva.nl/pombe/). and AD179, respectively. The transcription of all four genes is repressed by thiamine. C-terminal GFP-tagging of dis1p was Observation of cdc2-YFP and cdc13-YFP in cell cycle performed using a method described previously (Bähler et al., 1998), blocks where the endogenous gene is replaced by a fusion between the The following treatments were applied to the cells before LFM at RT: GFP(S65T) and the dis1+ genes and the transcription is controlled by thermosensitive (ts) strains were grown at 25°C in YES medium and the own gene promoter. Visualization of GFP-α2-tubulin was incubated at 36°C for either 4 hours (cdc10-V50 and cdc25-22) or 3 achieved by transformation of yeast with the pEG5 plasmid as hours (mts2 and cut4-533). Cold-sensitive dis1-203 cells (Ohkura et described (Ding et al., 1998). al., 1988) were grown at 32°C in YES and incubated for 8 hours at 20°C. Cell cycle block with hydroxyurea (HU) was performed by Western blotting and co-immunoprecipitation assays incubation of growing cells with 11 mM HU (Sigma) for 4 hours at The techniques used have been described previously 32°C in either YES (AD185 and AD112) or EMM2 medium (http://www.bio.uva.nl/pombe/). For co-immunoprecipitation assays, (AD179). For induction of cdc13∆81(−YFP) expression, cells were 2 mg of cell extract proteins were incubated with anti-cdc2p grown in YES medium, pelleted, washed and incubated for 16 hours polyclonal serum C2 (1:50, Simanis and Nurse, 1986). Proteins were at 32°C in EMM2 medium to allow switching ON of the nmt1 separated on a 12% SDS-polyacrylamide gel (Laemmli, 1970) and promoter. blotted onto Immobilon™-P membrane (Millipore). The antibodies used were anti-cdc13p mAb 6F (1:500; kind gift from Hayles and Observation of cdc2-YFP and cen1-GFP in mating and Steel) and anti-cdc2p mAb Y63 (1:500, Yamano et al., 1996). meiosis Immunoreactive bands were detected using ECL (Amersham). In Fig. 8, strains AD185 (cdc2-YFP) and PN745 were mixed on EMM2-NH4Cl plates for 8 hours at 25°C and observed by LFM. For Live fluorescence microscopy of GFP- and YFP-tagged observation of cdc2-YFP and cen1-GFP in the horse-tail nucleus (Fig. proteins 9), we crossed strains MKY7A-4 (Nabeshima et al., 1998) and AD185 Cells were grown exponentially, pelleted and spread onto a slide in the same conditions. GFP and YFP fluorescence were observed covered with a ~1 mm-thick layer of solid medium (2% low-melting separately using the procedure described above for CFP/YFP. Even agarose, 2% glucose and 225 µg/ml of adenine, histidine, uracil and though excitation of the GFP at 458 nm is not optimal, we were able leucine). In these conditions, cells were able to grow and divide for to detect the cen1-GFP signal. In Fig. 10, the cyr1∆sxa2∆ strain at least 3 hours at room temperature (RT, 23-25°C). Live fluorescence expressing cdc2-YFP was grown at 32°C in EMM2 medium before microscopy (LFM) was done at RT with a Zeiss Axioplan microscope, incubation for 8 hours at 30°C in the presence of 0.5 µg/ml P-factor, using a 100×, 1.3 oil immersion lens. GFP and YFP were excited with as described (Chikashige et al., 1997). a mercury lamp, using a HQ 480/40 filter. A HQ 535/50 filter was used for fluorescence emission and images were captured with a Hamamatsu CCD camera C5985 and processed with the Adobe RESULTS PhotoShop 5.5 software (Adobe Systems, San Jose, CA). The same procedure was used for the simultaneous observation of either cen1- Construction of cdc2-YFP and cdc13-YFP- GFP and cdc2-YFP (Fig. 3) or GFP-α2-tubulin and cdc2-YFP (Fig. 5D). For individual observation of cdc2-CFP and cdc13-YFP in the expressing strains same cell (Fig. 2D), we used a Zeiss LSM 510 laser-scanning confocal To follow the intracellular localisation of cdc2p and the cdc13p microscope and a 63×, NA 1.4 oil immersion lens. CFP was excited B-type cyclin in fission yeast, we constructed C-terminal

Table 2. Primer sequences Primer number Sequence 15′ACGCGTCGACGGTTTGCAATGGAGAATT 25′CGGGATCCCCATGAAAATCACGAAGATA 35′ACGCGTCGACTTTCCTCATGACTACCCG 45′CGGGATCCCATTCTTCATCTTTCAT 55′CGGGATCCCGTGAGCAAGGGCGAG 65′CGCTCAGTTGGAATTCTAG 75′CGCGTCGACTTCCTCATGAATGTCAGACACACTACTGCTTCTGTCAGT 85′CGGGATCCCATTCTTCATCTTTCAT 95′CGGGATCCCGGAGGCGGTGCGGGCGCTGGCGTGAGCAAGGGCGAGGAGCTG 10 5′CGGGATCCACTAAATTACCATTCTTC 11 5′CGAGATCTCGTTCGTTAGTGACCATCAT 12 5′GGGGTACCGCTCTCTATTCAATTTCAAAG 13 5′CGAGATCTCGTTCGTTAGTGACCATCAT 2630 JOURNAL OF CELL SCIENCE 114 (14) fusions between the corresponding genes and the yellow fluorescent protein-encoding gene (YFP, reviewed by Tsien, 1998). The fusions were expressed using the inducible nmt1 promoter in the pREP5 plasmid (see Materials and Methods). Although the cdc2-YFP fusion fully complemented the cdc2- 33 mutation, the cdc13-YFP construct failed to rescue the cdc13-117 mutation. However, overexpression of the cdc13- YFP fusion resulted in the appearance of short septated cells indicating premature entry into mitosis, suggesting that the fusion had some biological activity (not shown). Further support for this suggestion were obtained by expressing a truncated version of cdc13-YFP (lacking the first 81 amino acids), which resulted in an anaphase block, as described before (reviewed by Yanagida, 1998), and a high level of kinase/cyclin complex (Fig. 1A, lanes 2,4). In addition, co- immunoprecipitation experiments with anti-cdc2p antibody showed that cdc13-YFP and cdc13p interacted with cdc2p with similar efficiency (Fig. 1A, lanes 1,3). Fusion at the N-terminus of both cdc2p and cdc13p proteins gave similar results to the C-terminal fusions (not shown). The C-terminal fusions in the pREP5 plasmid were integrated at their own locus by homologous recombination, generating strains with the YFP-fusions controlled by the cdc2 and cdc13 gene promoters and with the untagged genes controlled by the nmt1 promoter (Fig. 1B, top, and lanes 1-2,5- 6). The nmt1-cdc2 gene was replaced by the kanamycin resistance gene, leaving the cdc2-YFP fusion as the only source Fig. 1. Analysis of cdc2- and cdc13-YFP proteins. (A) Co- ∆ of cdc2p in the cell (Fig. 1B, lane 3). The cdc13-YFP- immunoprecipitation assay of either cdc13-YFP or cdc13 81-YFP expressing strain grew normally in the presence of thiamine and cdc2p. Cells expressing cdc13-YFP (AD178, lanes 1,3) or cdc13∆81-YFP (AD203, lanes 2,4) were grown for 20 hours at 32°C (YES), even though expression of the untagged cdc13p was in EMM2 without thiamine. Immunoprecipitation from cell extract low compared with wild-type cells grown as control (Fig. 1B, proteins was performed with polyclonal anti-cdc2p serum. Cdc13p lane 6 vs WT in lane 4), giving further support for partial (tagged or untagged) was detected in total cell extracts (lanes 1,2) or functionality of cdc13-YFP. Cdc13-YFP was present at WT immunoprecipitated complexes (lanes 3,4) by western blot using levels (Fig. 1B, lane 6 vs lane 4). This strain enabled us to anti-cdc13p mAb. Lanes 1,2, 50 µg of cell extract proteins. Lanes monitor cdc13-YFP behaviour in cells behaving like WT, even 3,4, immunoprecipitation. (B) pREP5 plasmids with the cdc2- (lanes though they are expressing only very low amounts of untagged 1-3) and cdc13-YFP (lanes 5-6) fusions were integrated at the gene cdc13p. locus as shown on the cartoon and strains were grown for 20 hours in either EMM2 without thiamine (−T) or YES (+T) medium. 50 µg of In vivo localisation of cdc2-YFP and cdc13-YFP in cell extract proteins were loaded and detected using mAbs against the mitotic cell cycle cdc2p (1-3) and cdc13p (4-6). Lane 1, pREP5::cdc2-YFPint (AD143) −T; lane 2, pREP5::cdc2-YFPint (AD143) +T; lane 3, pREP5::cdc2- We followed cdc2-YFP and cdc13-YFP localisation in living YFPint ∆nmtcdc2::kanR (AD185) –T; lane 4, control WT strain cells using the cdc2-YFP (AD185) and cdc13-YFP (AD112)- (PN745); lane 5, pREP5::cdc13-YFPint (AD112) −T; and lane 6, expressing strains grown in YES (see Materials and Methods). pREP5::cdc13-YFPint (AD112) +T. In the conditions of our experiments, the nmt1::cdc13 gene expression remained OFF during microscopic observations. Quantification of cdc2-YFP fluorescence revealed that at least was lowest in late mitosis when cdc13-YFP fluorescence was 60-70% of the total cdc2-YFP was present in the cytoplasm of undetectable (Fig. 2Ak,B,C). Nuclear signals gradually G2 cells (Fig. 2A, top; B), whereas all the cdc13-YFP increased once mitosis had been completed (Fig. 2A1-m,B). fluorescence was detected in the nucleus (Fig. 2A, bottom). We Nuclear fluorescence of both cdc2-YFP and cdc13-YFP was believe that the fact that we can detect the cell outlines in clearly detectable in septated cells known to undergo S phase cdc13-YFP-expressing cells does not reflect a cytoplasmic (Nasmyth et al., 1979) but was not as high as in G2 cells (Fig. localisation of the cyclin since autofluorescence was also 2A, m vs a and b, B). In late G2, a bright spot of both cdc2- observed in control cells that do not express any YFP-fusion YFP and cdc13-YFP appeared at the nuclear periphery and (Fig. 2An). The fluorescence of mitochondrial flavoproteins split into two dots during early mitosis (Fig. 2Ab-c). These dots (Kunz et al., 1997) is likely to contribute to the green appeared to correspond to the SPBs because they separated autofluorescence of yeast cells. The cable-like structures that apart with a spindle between them during mitosis as confirmed can be seen in the cytoplasm of some cells in Fig. 2A (bottom) by simultaneous observation of cdc2-YFP and and GFP-α2- may therefore correspond to the fluorescence of mitochondria tubulin (cf. Fig. 5Dp-t). From prophase to metaphase, cdc2- aligned with cytoplasmic MTs (interphase MTs and post- YFP and cdc13-YFP were enriched on the forming spindle and anaphase array), as described (Yaffe et al., 1996). SPBs (Fig. 2Ac-f). At mitotic exit, the fluorescence of both For both cdc2-YFP and cdc13-YFP, nuclear fluorescence proteins disappeared, first from the middle of the nucleus, then Fission yeast cdc2p localisation 2631

Fig. 2. Cellular localisation of cdc2- YFP and cdc13-YFP in the mitotic cell cycle. Cells were grown exponentially in YES medium, layered onto solid medium-coated slides and observed by LFM at RT. (A) Fluorescence of cdc2-YFP (AD185) and cdc13-YFP (AD112) was observed in different cells at various stages of the cell cycle: G2 interphase (a); G2/M boundary (b); mitosis from prophase to anaphase B (c-k); G1 and S phase (l- m). Bar, 5 µm. (B) Quantification of nuclear and cytoplasmic cdc2-YFP. Pictures from cells (strain AD185) at different stages of the cell cycle were taken and analysed using PhotoShop 5.5 to estimate the total cdc2-YFP fluorescence in the nucleus and the cytoplasm. Image fields were focused with non- fluorescent optics and fluorescence was observed only by the camera, using the same exposure time in each case. 15-25 cells were analysed at each stage and the bars give the s.d. Fluorescence measurements are in a single focal plane with the diameter of the nucleus at its maximum. Therefore, the graph gives the maximum proportion of nuclear cdc2-YFP at each stage of the cell cycle. The line shows the average cell length. Bar, 5 µm. (C) Cdc13-YFP fluorescence (strain AD112) was enriched at the nuclear periphery of cells exiting from mitosis in (a) to (i). Bar, 5 µm. (D) Cdc2-CFP and cdc13-YFP fluorescence were observed separately in strain AD117 using laser scanning confocal microscopy: anaphase B (a); S phase (b); G2 interphase (c); and mitosis (d). Bar, 5 µm. In addition, the black and white cdc2-CFP and cdc13-YFP pictures are shown below the colour pictures. from the spindle, and finally from the SPBs (Fig. 2Ag-i). In CFP, using the same strategy as that described for the cdc2- anaphase, cdc13-YFP was mainly detected at the nuclear YFP strain. Cells at different stages of the cell cycle are periphery (Fig. 2Ai-j,Cb-h) and completely disappeared by presented in Fig. 2D with cdc13-YFP shown in the left panel, nuclear division (Fig. 2Ak,Ci). Cdc2-YFP nuclear fluorescence cdc2-CFP in the middle panel and merged images in the right was reduced by 75% in anaphase but did not accumulate at the panel. In anaphase, cdc2-CFP and cdc13-YFP did not co- nuclear periphery, and some residual cdc2-YFP fluorescence localise at the nuclear periphery (Fig. 2Da), whereas in S followed the sister chromatids towards the cell ends (Fig. 2Ai- phase, cdc2-CFP and cdc13-YFP fluorescence was lower than k, top, B). Observation of the same cell revealed that transition that observed in G2 cells or in mitosis (Fig. 2D, b vs c and d). from stage (c) to (h) and (h) to (m) required 4 and 45 minutes, Finally, we investigated the dynamics of cdc2p and cdc13p respectively (not shown). The above data indicate that the level at mitotic exit. By monitoring cells in real time, we found that of cdc13p and cdc2p in the nucleus change together during the both cdc13-YFP and cdc2-YFP leave the mitotic spindle cell cycle. To confirm this, the levels of cdc2p and cdc13p were rapidly, in less than 1 minute (Fig. 3A, 3′ vs 2′, B, 2′ vs 1′). compared within the same cell by fusing the cdc2+ gene to Expression of cdc2-YFP in a strain with the centromeric region 2632 JOURNAL OF CELL SCIENCE 114 (14) of chromosome I marked by GFP fluorescence (cen1-GFP; Nabeshima et al., 1998) revealed that cdc2-YFP leaves the mitotic spindle immediately prior to sister chromatid separation (Fig. 3B, 3′ vs 2′). In this experiment, YFP and GFP fluorescence were observed simultaneously and the separation of sister chromatids was detected when the two dots of cen1- GFP moved apart. Cdc2p requires the B-type cyclins to accumulate in the nucleus after completion of mitosis Because cdc2-YFP nuclear fluorescence decreases dramatically upon cdc13p degradation at mitotic exit, we next tested whether B-type cyclins are required for the Fig. 3. Cdc2-YFP leaves the mitotic accumulation of cdc2p in the nucleus. Cdc13p is the essential spindle in early anaphase A, before cyclin at the G2/M transition and is required for entry into S sister chromatid separation.Time-lapse phase in the absence of cig1p and cig2p (Fisher and Nurse, series showing cdc13-YFP (A, AD112) 1996). Using cig1 and cig2 deletions (Bueno et al., 1991; and cdc2-YFP/cen1-GFP (B, AD265) Obara-Ishihara and Okayama, 1994; Fisher and Nurse, 1996) localisation at the metaphase/anaphase in combination with a cdc13 thiamine-repressible allele transition. Pictures from cells were (Hayles et al., 1994; Fisher and Nurse, 1996) and the cdc25- taken every 1 or 2 minutes as indicated. 22 ts mutation, we constructed four strains to test our Cdc2-YFP fluorescence disappeared from the spindle (B, 2′) immediately prior to separation of cen1-GFP hypothesis (see Materials and Methods). Strains were grown dots (arrowheads) on sister chromatids (B, 3′). Bar, 5 µm. at 25°C without thiamine before shifting the temperature to 36°C for 4 hours to block cells at the G2/M transition. Thiamine was added either after 3 hours at 36°C for cig2+ strains (Fig. 4Ad,Bb) or at the time of release for the cig2-deleted strains (Fig. 4Ae-f,Bc-d) to allow cell progression through mitosis. These conditions produced cells containing solely cdc13p (Fig. 4Ac),

Fig. 4. Cyclin B-dependent accumulation of cdc2-YFP into the nucleus after mitotic exit. (A) Cells from strains AD205 (a-b), AD207 (c, f), AD210 (d) and AD245 (e) were grown overnight at 25°C in the absence of thiamine. Cultures were incubated at 36°C for 4 hours to block the cells at the G2/M transition (time 0). In d-f, the cdc13 gene expression was switched off by addition of 15 µM of thiamine after either 3 hours (d) or 4 hours (e-f) at 36°C. Cells were observed by LFM for 2 hours 30 minutes at RT. Cdc2-YFP nuclear fluorescence was similar during the first 30 minutes of release (from G2/M to metaphase) in all strains (a). From anaphase B to post-, cdc2-YFP nuclear fluorescence (b-f, arrowheads) was dependent on the cyclin B present in the strain (cdc13p, cig1p and cig2p (b), cdc13p (c), cig2p (d), cig1p (e) or none of them (f)). Bar, 5 µm. (B) In the same experiments, DNA content was measured by FACS on fixed cells: asynchronous cultures at 25°C (AS), after 4 hours at 36°C (0) and at different time points during the release at 25°C as indicated. AD207 (a and d), AD210 (b) and AD245 (c). (C) Cdc2- and cdc13-YFP fluorescence were not observed in the nucleus of cdc10-V50 ts cells incubated for 4 hours at 36°C (AD192 and AD212). Note the cdc2-YFP aggregation in the cytoplasm of cells incubated at 36°C. This aggregation at high temperatures has also been reported recently for the GFP alone (Fukuda et al., 2000). Bar, 5 µm. Fission yeast cdc2p localisation 2633 cig2p (Fig. 4Ad) or cig1p (Fig. 4Ae), or lacking all of these B- 1996; Blanco et al., 2000). After incubation for 4 hours at 36°C type cyclins (Fig. 4Af). After 30 minutes of release into in a cdc10-V50 ts mutant, nuclear cdc2-YFP fluorescence mitosis, the cdc2-YFP fluorescence was found to be enriched levels were similar to those in the cytoplasm (Fig. 4C, left). on the mitotic spindle in all strains (Fig. 4Aa) and faded by the Cytoplasmic cdc2-YFP often appeared as bright blobs, onset of anaphase. In the presence of all three B-cyclins, cdc2- possibly due to aggregation of cdc2-YFP at 36°C as recently YFP fluorescence never completely disappeared from the reported for GFP (Fukuda et al., 2000) (Fig. 4C, left). This daughter nuclei at mitotic exit and started to increase again at phenomenon has also been observed in WT cells expressing G1/S (Fig. 4Ab). The cdc2-YFP nuclear fluorescence profile cdc2-YFP after incubation at 36°C and is therefore not related was similar when cdc13p was the only B-type cyclin present to the ts mutation (not shown). In cdc10-V50-arrested cells, (Fig. 4Ac,Ba). However, when only cig2p was present, the cdc13-YFP was completely absent from the nucleus (Fig. 4C, nuclear fluorescence was lower at G1/S and remained right). This observation is in contrast with previous work in unchanged as cells proceeded through S phase (Fig. 4Ad,Bb). which cdc13p was detected by immunofluorescence in the When cig1p was the only cyclin present, the cdc2-YFP nucleus of cdc10-129 cells after 6 hours at 36°C (Booher et al., fluorescence disappeared immediately after mitosis, and only 1989). This is probably because of leak-through of this allele, became detectable again in septated cells after 2 hours of and we made similar observations with our cdc13-YFP fusion release (Fig. 4Ae). The FACS profile showed a major 1C peak after prolonged incubation of cdc10-V50 cells at 36°C (not after 1 hour and 40 minutes of release and the following S shown). phase was very slow (Fig. 4Bc). In the absence of all three The above results show that B-type cyclins, cdc13p, cig1p cyclins, nuclear staining of cdc2p never appeared again (Fig. or cig2p, are required to allow cdc2-YFP accumulation in the 4Af). The FACS profile showed a major 1C peak at the time nucleus after completion of mitosis. In the absence of all three of daughter cell separation and some of the post-mitotic cells B-type cyclins, the concentration of cdc2-YFP is similar in the took a long time or failed to separate, resulting in a broad FACS nucleus and in the cytoplasm. profile (Fig. 4Bd). To confirm the above data, we looked at cdc2-YFP nuclear Cdc2p/cyclin complex accumulates on the SPB of fluorescence in cdc10-arrested cells, when cdc13p, cig1p and cells arrested at S phase or at the G2/M transition cig2p cyclins are absent (Hayles et al., 1994; Mondesert et al., We next examined the association between cdc2p/cdc13p and

Fig. 5. Cdc2-YFP accumulates on the SPB of cells arrested at the G2/M transition or treated with HU. (A) Cdc2-YFP (AD185, a-e) fluorescence is very low on the SPB of septated cycling cells growing at 32°C in YES medium (c-e). SPB fluorescence was not observed in binucleate cells before septation (a- b). Bar, 5 µm. (B) Cdc2-YFP (AD185, a) and cdc13-YFP (AD112, b) accumulate in the nucleus and on the SPB of cells treated with 11 mM HU for 4 hours at 32°C. YFP-cig2 fluorescence was also enriched on the SPB of cells grown for 16 hours in the absence of thiamine at 32°C and incubated with 11 mM HU for 4 hours (AD179, c). Bar, 5 µm. (C) SPB accumulation of cdc2-YFP (AD189, a) and cdc13-YFP (AD226, b) is stronger in cdc25-22 ts cells after incubation for 4 hours at 36°C. Aggregation of cytoplasmic cdc2- YFP is observed at 36°C (see Fig. 4 legend). Bar, 5 µm. (D) cdc25-22 cells were arrested at the G2/M transition by incubation for 4 hours at 36°C and released into mitosis at RT: cdc13-YFP (AD226, a-e), cdc2- YFP (AD189, f-j), GFP-α2-tubulin (AD244, k-o) or both cdc2-YFP and GFP-α2-tubulin (AD268, p-t). G2/M boundary (a f,k,p) and mitosis (b-e,g- j,l-o,q-t). Bar, 5 µm. 2634 JOURNAL OF CELL SCIENCE 114 (14) the SPBs. In an asynchronous population, cdc2-YFP and stf1.1 is a semi-dominant mutation that bypasses the cdc13-YFP were strongly associated with the SPB in 20% of requirement for cdc25p-mediated activation of cdc2p at the the cells, consistent with an enrichment in late G2 or early G2/M transition (Hudson et al., 1990). Isolation of the cut12 mitosis. However, low cdc2-YFP fluorescence could be gene then revealed that cut12+ is allelic to stf1+ (Bridge et al., observed at the SPB of some binucleate cells after septation 1998). As cut12p was found to be localised to the SPB has occurred (Fig. 5Aa-e). Therefore, we tested whether we throughout the cell cycle, the authors suggested that could detect SPB-associated fluorescence in cells arrested in stf1.1/cut12.G17V allows the formation of cdc2p/cdc13p early S phase with HU. In HU-treated cells, cdc13-YFP and complexes at the SPB that would not be Tyr15-phosphorylated cdc2-YFP accumulated strongly in the nucleus and a bright dot by wee1p/mik1p kinases, thereby promoting mitosis in the was often seen at the nuclear periphery, indicating that the absence of cdc25p function. We tested whether cdc2-YFP complex is most probably localised to the SPB (Fig. 5Ba,b). would go prematurely to the SPB of stf1.1 cells but we did not Observation of the same cell releasing from an HU block find any significant difference with the WT situation (not confirmed that the bright dot observed at the nuclear periphery shown). co-localises with the SPB since the dot further split into two dots that separated apart with a spindle between them during At mitotic exit, cdc13p recognition by the APC is mitosis (data not shown). The B-type cyclin cig2p level is also required for cdc2p to leave the spindle increased in HU-arrested cells (Mondesert et al., 1996), and We next addressed what would happen to cdc2p localisation cig2p appears to be localised to the SPB as revealed by YFP- during mitosis if cdc13p was not degraded. The ‘destruction- cig2 LFM (Fig. 5Bc). To address the question of whether the box’ motif on the cyclin and a functional anaphase-promoting strong accumulation of the cdc2p/cdc13p complex observed complex (APC) are required for cdc13p degradation and APC at later stages of the cell cycle required the Tyr15- mutant cells become blocked in anaphase (reviewed by dephosphorylation of cdc2p, we looked at the SPB Yanagida, 1998). In the cut4-533 ts mutant, which lacks a fluorescence of cells arrested at the G2/M transition in a cdc25- functional APC, cdc2-YFP and cdc13-YFP fluorescence 22 ts mutant (Fig. 5C). After 4 hours incubation at 36°C, cells remained associated with the spindle, the SPBs, and had a high level of nuclear cdc2-YFP and cdc13-YFP and both throughout the nucleus (Fig. 6c-d). Overexpression of a proteins became strongly accumulated at the SPB (Fig. truncated cdc13p, lacking the 81 first amino acids, including 5Ca,b,Da,f), showing that cdc2p/cdc13p complexes with low the ‘destruction box’ required for recognition by the APC, kinase activity can locate to the SPB. In the G2/M block, resulted in an anaphase block (Yamano et al., 1996) and cytoplasmic MTs are still present (Fig. 5Dk, GFP-α2-tubulin), strong cdc2-YFP and cdc13∆81-YFP fluorescence remained establishing that the enrichment of cdc2p/cyclin B on the SPB associated with the spindle, SPBs and nucleus (Fig. 6e-f). In occurs prior to the reorganization of the cytoplasmic array of most cells, the mitotic spindle failed to elongate. Deletion of MTs. Upon release into mitosis at 25°C, cdc2-YFP and cdc13- the first 106 amino acids of cdc13p, including a region of YFP were still enriched on the separating SPBs and on the cdc13p not present in cig2p, did not impair its ability to bind forming spindle (Fig. 5Db-e,g-j,l-o,q-t). to mitotic MTs (not shown). Ts mutants of the mts2 subunit Our data suggest that a fraction of S. pombe cdc2p is present of the proteasome complex that become arrested at the at the SPB in G1/S or S phase. More kinase then accumulates metaphase/anaphase transition (Gordon et al., 1993) still on the SPB of G2 cycling cells. SPB-association was also accumulated the cdc2p/cdc13p complex in the nucleus and on detected in cells arrested in early S phase and at the G2/M the mitotic spindle and SPBs (Fig. 6a-b). transition prior to the reorganization of the cytoplasmic array These data indicate that interaction of cdc13p with the APC of MTs and mitotic spindle formation when the bright dots of occurs on the spindle and the SPBs during anaphase, and that cdc2-YFP and cdc13-YFP separate and spindle MTs form recognition of cdc13p by the APC is required for cdc2p to between them. leave the spindle at mitotic exit. If cdc13p cannot be recognized Next, we looked at the localisation of cdc2-YFP in a stf1.1 by the APC, or if a functional APC is absent, then cdc2p and mutant. Indeed, Hudson et al. have shown that fission yeast cdc13p remain associated with mitotic MTs.

Fig. 6. Cdc2-YFP does not leave the mitotic spindle if cdc13p is not recognized by the APC. Fluorescence of cdc2- and cdc13-YFP was observed in the nucleus and on the SPBs and spindle of ts mutants defective in either proteasome (mts2) or APC (cut4-533) function after incubation for 3 hours at 36°C. AD157 (a), AD213 (b), AD152 (c) and AD267 (d). Aggregation of cytoplasmic cdc2-YFP is observed at 36°C (see Fig. 4 legend). Upon overexpression of indestructible cdc13p (lacking the first 81 aa) after 16 hours incubation at 32°C in thiamine-free medium, cdc2-YFP was present in the nucleus and on the SPBs and spindle of anaphase-arrested cells (AD266, e). Overexpressed cdc13∆81-YFP showed similar localisation (AD217, f). Bar, 5 µm. Fission yeast cdc2p localisation 2635 Dis1p is not required for the localisation of cdc2- increased, and the nucleus of the cdc2+ strain became YFP and cdc13-YFP onto the mitotic spindle fluorescent, showing that cdc2-YFP could enter the nucleus at The above data suggest that cdc2p contributes to the regulation this stage of conjugation (Fig. 8Bb). When nuclei adopted a of MT dynamics in mitosis. In Xenopus, XMAP215 is known drop-shape, their SPB-associated extremities became very to target cdk1 to the mitotic MTs and to be a major MT- bright (Fig. 8Bc). This was followed by karyogamy when the stabilizing factor (reviewed by Andersen, 2000). Therefore, we bright dots migrated from the site of nuclear fusion towards the tested whether S. pombe dis1p, a homologue of XMAP215, opposite end of the nuclei, probably due to cdc2-YFP becoming plays a similar role in fission yeast. associated with the clustered centromeres as they detached from We replaced the dis1+ gene by a dis1-GFP fusion in a cdc25- the SPBs (Fig. 8Bd-f). After completion of karyogamy, ‘horse- 22 background (Materials and Methods) and investigated the tail’ movements started and cdc2-YFP was concentrated into 1- localisation of dis1-GFP in mitosis. In agreement with previous 3 bright dots in the elongated zygotic nucleus (Fig. 8Bg-k). The studies (Nabeshima et al., 1995), dis1-GFP was present on the same diploid cell was followed for 70 minutes (Fig. 8Bi-s). metaphase spindle (Fig. 7Ab-e) but, in contrast to the earlier When horse-tail movements stopped, cdc2-YFP was found to study, did not appear to be enriched on the SPBs during be located in several fuzzy dots throughout the nucleus that metaphase. By following the same cells in a cdc25-22 block- disappeared when cdc2-YFP relocalised to the spindle (Fig. and-release experiment, dis1-GFP was found to be associated 8Bl-u). After the first meiotic division had been completed, with dots likely to correspond to the centromeres, which cdc2-YFP fluorescence reappeared on the SPBs and the spindle become located near the SPBs during anaphase (Fig. 7Ab-f). of the two nuclei in the second meiotic division (Fig. 8Bv-x). The extremities of the mitotic spindle often showed more than The appearance of cdc2-YFP on the meiosis I spindle was one dot of dis1-GFP consistent with a localisation on the different in appearance to either mitosis or meiosis II. Instead centromeres (Fig. 7Ag,h). Moreover, we found that dis1-GFP of being first located on the SPBs and then on the spindle, cdc2- and bub1-GFP, a kinetochore-binding protein (Bernard et al., YFP appeared all along the spindle as it formed during meiosis 1998), had similar localisation patterns in metaphase (not I (Fig. 8Bp-s), suggesting distinct pathways of spindle shown). Because of its association with the metaphase spindle, formation, reminiscent of the formation of ‘acentrosomal’ we tested whether dis1p was required for the targeting of fission meiotic spindles in many oocytes and in some spermatocytes yeast cdc2p/cyclin complex to mitotic MTs (Fig. 7B) using a (reviewed by Merdes and Cleveland, 1997). cold-sensitive (cs) dis1-203 mutant. The dis1-203 mutation is a nonsense mutation resulting in the formation of a truncated dis1p protein containing only the first 265 amino acids (wild-type dis1p is an 882-amino-acid protein) and therefore lacking the central domain of dis1p, which comprises the cdc2p phosphorylation sites and is thought to be responsible for the binding of dis1p to MTs (Nabeshima et al., 1995). The dis1-203 cs mutant arrests in mitosis with condensed chromosomes scattered along the elongated and disrupted spindle (Ohkura et al., 1988). Cdc2-YFP and cdc13-YFP were found to be present on the SPBs and on the disrupted spindle at 20°C (Fig. 7Ba,b). These results show that dis1p MAP function is not required for the targeting of cdc2p/cdc13p complex to the mitotic MTs. Dis1-GFP may associate with centromeres in mitosis, in agreement with a role of this protein in the dynamic movement of centromeres during metaphase (Nabeshima et al., 1998).

Localisation of cdc2-YFP in karyogamy and Fig. 7. Association of cdc2-YFP and meiosis cdc13-YFP with mitotic microtubules In S. pombe meiotic prophase, telomeres cluster near does not require dis1p function. (A) The the SPB to form a bouquet-like arrangement endogenous dis1+ gene was replaced by (reviewed by Hiraoka, 1998) followed by separation the dis1-GFP fusion in a cdc25-22 of the centromeres from the SPB as cells proceed background (AD219). Cells were towards the first meiotic division (Chikashige et al., arrested at the G2/M transition after 4 1994; Chikashige et al., 1997) (Fig. 8A). A cdc2- hours incubation at 36°C and dis1-GFP + fluorescence was observed in vivo at RT. Dis1-GFP was found on cytoplasmic MTs YFP-expressing strain was crossed with a cdc2 in the block (a) and then relocalised to discrete dots along the forming spindle (b-e). strain and the cross monitored by LFM (Fig. 8B). At Fluorescence was enriched at the extremities of the elongating spindle (f-j). c-f show onset of conjugation, cdc2-YFP fluorescence was a time-lapse series of the same mitotic cell as it progresses through mitosis. Bar, 5 detected in only one of the two cells (Fig. 8Ba). As µm. (B) In cold-sensitive dis1-203 cells incubated for 8 hours at 20°C, cdc2-YFP conjugation proceeded, the two cells fused, the (AD199, a) and cdc13-YFP (AD264, b) were still associated with the unsegregated nuclear fluorescence of the cdc2-YFP-expressing cell chromosomes, the SPBs and the spindle of mitosis-arrested cells. 2636 JOURNAL OF CELL SCIENCE 114 (14)

Fig. 8. Localisation of cdc2-YFP during mating, karyogamy and meiosis. (A) Cartoon summarizing the localisation of chromosomes, centromeres (᭹), telomeres (᭺) and SPB (᭿) during mating, karyogamy and meiotic prophase in S. pombe as described (Chikashige et al., 1994; Chikashige et al., 1997). (B) A cdc2-YFP-expressing strain (AD185) and a cdc2+ strain (PN745) were crossed on nitrogen-depleted medium and observed by LFM. Conjugating cells (a-b). Both nuclei become fluorescent (b). During karyogamy, cdc2-YFP is enriched in bright dots (c-f, arrowheads). In the horse-tail nucleus, cdc2-YFP is concentrated into 1-3 bright dots (g-k). Nucleus movement stops and meiosis I starts (l-s). We followed cdc2-YFP fluorescence in the same diploid cell for 70 minutes from meiotic prophase (i) to metaphase of the first meiotic division (s). Meiosis I (s- u). Meiosis II (v-x).

In a time-lapse series during horse-tail movements (Fig. 9A), cells occurred prior to or after the detachment of centromeres cdc2-YFP fluorescence was concentrated into 2 or 3 dots. To from the SPB. Chikashige et al. have shown that, in a see whether these bright dots were associated with the sxa2∆cyr1∆ mutant responding to P-factor, telomeres cluster centromeres, we crossed the cen1-GFP and the cdc2-YFP- together with the SPB and the centromeres to form a bouquet- expressing strains, distinguishing GFP (Fig. 9Bb) from YFP like arrangement (Chikashige et al., 1997) (Fig. 10, left). Cdc2- fluorescence (Fig. 9Ba). Cells in meiotic prophase showed that YFP was enriched close to a darker portion of the nucleus, the cen1-GFP signal overlapped with one of the cdc2-YFP dots which is likely to be the nucleolus (corresponding to in the horse-tail (Fig. 9Bc). The fact that the GFP and YFP chromosome III telomeres; Fig. 10, arrowheads) in signals were not fully superimposed is probably due to the 30 sxa2∆cyr1∆ cells incubated for 8 hours at 30°C in the presence kb distance between the cen1-GFP signal and the centromere of P-factor. Cdc2-YFP was enriched in either a bright single of chromosome I. The co-localisation of cen1 with one cdc2- dot or multiple dots very close to each other (Fig. 10, arrows). YFP dot suggests that the remaining bright dots of cdc2-YFP The fact that these bright dots of cdc2-YFP were not observed correspond to the two other pairs of centromeres. at the nuclear periphery but in the nucleoplasm is consistent The above data show that cdc2-YFP enters the nucleus very with a localisation on the centromeres and not on the SPB. early in conjugating cells, with cdc2-YFP becoming enriched Therefore, in the absence of a mating partner, cdc2-YFP is at the SPB-associated end of the fusing nuclei. Cdc2-YFP then likely to become enriched on the SPB-associated centromeres migrates towards the opposite end of the nucleus, becoming prior to their detachment from the SPB during mating. associated with the clustered centromeres. In the horse-tail nucleus, cdc2-YFP is enriched in 1-3 bright dots, at least one of which co-localises with the centromeric region of DISCUSSION chromosome I. We have followed the in vivo localisation of YFP-tagged cdc2p Cdc2-YFP is enriched on the telomeres-SPB- and cdc13p during the fission yeast cell cycle. Our study is centromeres cluster in the absence of a mating consistent with previous immunofluorescence data obtained partner during S. pombe mitotic cell cycle (Booher et al., 1989; Alfa Finally, we addressed the question of whether the accumulation et al., 1990) but our in vivo analysis gives more information of cdc2p on the centromeric regions of chromosomes in mating and reveals new dynamics previously unsuspected during both Fission yeast cdc2p localisation 2637

Fig. 10. Cdc2-YFP is enriched in the cluster of SPB-centromeres- telomeres in response to P-factor, in the absence of a mating partner. In the nucleus of h− cyr1∆sxa2∆ cells responding to P-factor, telomeres (᭺), centromeres (᭹) and SPB (᭿) cluster together at one end of the nucleus (Chikashige et al., 1997) (cartoon). The h− cyr1∆sxa2∆ cells expressing cdc2-YFP (AD257) were grown exponentially in EMM2 medium at 32°C before incubation for 8 hours at 30°C in the presence of 0.5 µg/ml P-factor. Arrows indicate cdc2-YFP-enriched dots. Arrowheads show the dark portion of the nucleus that corresponds most probably to the rDNA and, therefore, Fig. 9. Cdc2-YFP co-localises with cen1- to the telomeres of chromosome III. GFP during meiotic prophase. (A) A cdc2- YFP-expressing strain (AD185) and a cdc2+ strain (PN745) were crossed on nitrogen- depleted medium. A time-lapse series et al., 1984). This model suggests that cdc13p associates with showing cdc2-YFP fluorescence in the cdc2p in the cytoplasm and that the complex is then rapidly horse-tail nucleus of meiotic prophase is transported into the nucleus, explaining why cdc13p could not presented (time is indicated in minutes). be detected in the cytoplasm. An alternative hypothesis would Cdc2-YFP was found in the nucleoplasm be that the actual function of the B-type cyclins is to retain and was concentrated into 2 or 3 bright dots cdc2p in the nucleus. In this model, the uncomplexed cdc2p during nuclear movement. (B) A cdc2-YFP- may be able to enter the nucleus freely and would only expressing strain (AD185) and a cen1-GFP associate with the cyclin in the nucleoplasm. Once complexed strain (MKY7A-4) were crossed and a cell in meiotic prophase was observed in vivo to the cyclin, the kinase may not be able to get out of the using a laser scanning confocal microscope nucleus anymore, unless the cyclin is degraded. In both these to distinguish cdc2-YFP (a) from cen1-GFP models, it appears that the level of cyclin in the cell is the (b) fluorescence. In all cases, one cdc2-YFP limiting step for nuclear accumulation of cdc2p. dot (arrows) co-localised with the GFP In cycling cells, low amounts of cdc2-YFP were detected on fluorescence associated with the centromere the SPB of septated cells (undergoing S phase), whereas cdc2- of chromosome I (arrowheads) (c). YFP, cdc13-YFP and YFP-cig2 accumulate more clearly on the SPB of cells arrested in early S phase with HU. This may be related to the role of cdk2/ or E complex in higher mitosis and meiosis. During the mitotic cell cycle, cdc13-YFP eukaryote centrosome duplication during G1-S (reviewed by was detected only in the nucleus, whereas cdc2-YFP Whitehead and Salisbury, 1999; Meraldi et al., 1999; Okuda et fluorescence was found in both the cytoplasm and the nucleus. al., 2000). In late G2 cycling cells, we found that both cdc2p The level of cdc2-YFP and cdc13-YFP fluorescence in the and cdc13p are much more strongly associated with the SPB, nucleus changed together during the cell cycle, in agreement suggesting that the kinase activity required on the SPB of cells with previous immunofluorescence data (Booher et al., 1989). entering mitosis is higher. Cdc25p tyrosine-phosphatase The nuclear level of both was highest from late G2 to activity is not required for the enrichment of cdc2- and cdc13- metaphase, dropped in anaphase, and reappeared around the YFP on the SPB. Upon release of a cdc25-22 ts mutant at 25°C, completion of S phase. In early G1 cells, cdc13-YFP was not SPB separation occurred quickly, suggesting that the detected and cdc2-YFP levels were similar in the nucleus and cdc2p/cdc13p complex is activated on the SPB upon entry into cytoplasm. As cyclin levels increased at G1/S, nuclear cdc2- mitosis. This is consistent with the work on activation of the YFP fluorescence also increased. Using cyclin B-depleted human centrosomal cdk1/cyclin B1 complex (De Souza et al., strains, we established that the accumulation of cdc2-YFP 2000) and with work in budding yeast showing that separation fluorescence in the nucleus of post-mitotic cells required at of duplicated SPBs requires Tyr-19 dephosphorylation of least one of the three B-type cyclins, cig1p, cig2p or cdc13p. Cdc28 (Lim et al., 1996). We propose that active fission yeast In a HU block, the cyclin B level is highly increased, resulting cdc2p regulates in vivo SPB MT-nucleating activity and in the accumulation of more than 80% of the cdc2-YFP cellular spindle formation. However, the nature of the signal that content in the nucleus. The above data show that cdc2p requires triggers accumulation of the cdc2p/cdc13p complex on the the B-type cyclins to accumulate in the nucleus after mitotic SPB of G2 cells is unknown. We do not think that it is only exit. One hypothesis is that cdc2p requires the cyclins to enter dependent on cell size since the cdc2-YFP/cdc13-YFP the nucleus, in agreement with the fact that the cdc2p amino fluorescence observed on the SPB of elongated HU-arrested acid sequence does not contain any nuclear localisation signal cells is lower than the one observed in a cdc25-22 block. One (NLS), whereas all three S. pombe B-type cyclins contain hypothesis might be that SPB accumulation of cdc2p/cdc13p regions of homology to the NLSs of SV40T antigen (Kalderon requires maturation of the duplicated SPB, which occurs 2638 JOURNAL OF CELL SCIENCE 114 (14) during the G2 interphase of cycling cells (reviewed by Adams centrosomes and the mitotic spindle (reviewed by Peters, and Kilmartin, 2000). 1999; Tugendreich et al., 1995). In mitosis, the cdc2p/cdc13p complex accumulates on the In the second part of our work we investigated the SPBs and the spindle from prophase to metaphase, in localisation of cdc2p during mating, karyogamy and meiosis. agreement with recent data obtained with a cdc13-GFP fusion In conjugating cells, we found that cdc2-YFP enters the (Yanagida et al., 1999). In anaphase, we observed that cdc2- nucleus before karyogamy starts, possibly because of the YFP and cdc13-YFP disappeared rapidly from the mitotic requirement of cdc2p kinase activity for premeiotic DNA spindle, immediately prior to sister chromatid separation. This synthesis (reviewed by Murakami and Nurse, 2000). When is shortly before the switch in MT dynamics (Mallavarapu et nuclei adopted the drop-shaped profile, cdc2-YFP was al., 1999) at the onset of anaphase B. Because CDKs from a enriched on the cluster of centromeres-SPB-telomeres. As number of different species regulate MT dynamics in mitosis karyogamy proceeded, nuclei fused together at their cdc2- via the association with MAPs (reviewed by Andersen, 2000), YFP enriched ends, followed by the appearance of one bright we searched for S. pombe MAPs homologues that could dot in the middle of each fusing nucleus, suggesting that associate with cdc2p/cdc13p on the MT spindle in metaphase. cdc2-YFP was associated with the clustered centromeres We found homology between S. pombe ORF SPAC23A1.17 known to leave the SPB at that stage (Chikashige et al., 1997). and Xenopus XMAP4 (Shiina and Tsukita, 1999) (BLAST In the horse-tail nucleus, cdc2-YFP was in the nucleus and score of 10−11), but the GFP-tagged protein was localised to was enriched in 1-3 bright dots. Because one of the dots co- the septum and the cell ends and not on the mitotic spindle (not localised with cen1-GFP in the centromeric region of shown). Also, dis1p (Nabeshima et al., 1995), which shows chromosome I, we conclude that the other cdc2-YFP dots co- high identity with Xenopus XMAP215, a cdk1-associated MT- localise with the centromeres of chromosomes II and III. The stabilizing factor (reviewed by Andersen, 2000; Tournebize et enrichment of cdc2-YFP with the cluster of centromeres- al., 2000), was not required for the location of cdc2p/cdc13p SPB-telomeres in shmooing cells does not require a mating to the mitotic MTs. Dis1-GFP was associated with centromeres partner since a bright cdc2-YFP dot(s) was observed in in metaphase, and because dis1p is phosphorylated on cdc2p cyr1∆sxa2∆ cells responding to P-factor. These data suggest consensus sites and is required for centromere movement in that cdc2p plays a role at the centromeres early during the metaphase (Nabeshima et al., 1995; Nabeshima et al., 1998), mating process. One possibility would be that cdc2p activity we suggest that cdc2p has an effect on centromere dynamics. regulates the centromere-SPB detachment that occurs at early It has been suggested that, in metaphase, kinetochore proteins stages of karyogamy (reviewed by Hiraoka, 1998). Cdc2p bind loosely to MTs whereas, during anaphase, they become target proteins at the centromeres might need to be strongly attached (Zhai et al., 1995). Cdc2p might phosphorylated throughout meiotic prophase to avoid early phosphorylate centromeric dis1p during metaphase resulting in reassociation of centromeres with the SPB. Given cdc2p low stability of kinetochore MT attachment to the centromere localisation we propose that cdc2p might influence the proper and, on displacement of cdc2p/cdc13p from the spindle, segregation of chromosomes in meiosis I, and we have dephosphorylation of dis1p might result in strong binding to preliminary evidence that inactivation of cdc2p during the kinetochore MTs followed by sister chromatid separation meiotic prophase increases the frequency of equational in anaphase A. division of sister chromatids (not shown). Surprisingly, We also showed that, in anaphase A and early anaphase B, cdc13-YFP nuclear fluorescence was much lower in cdc13-YFP fluorescence was mainly detected at the nuclear karyogamy and meiosis compared to mitosis (not shown), periphery, possibly corresponding to its degradation by the suggesting a possible association of cdc2p with another proteasome, since subunits of the 19S regulatory cap have cyclin(s) in the nucleus of meiotic cells. Although the been shown to localise to the inner face of the nuclear fluorescence intensity was low, we detected the association of membrane in S. pombe (Wilkinson et al., 1998). As this was cdc13-YFP with the centromeres during horse-tail movement not observed for the cdc2-YFP protein, the dissociation of the of the nucleus in meiotic prophase. cdc2p/cdc13p complex may need to occur prior to cdc13p Association of cdc2-YFP with centromeres was no longer degradation. This is consistent with studies showing that, in detected in the first meiotic division. After the horse-tail Xenopus egg extracts, dissociation of the cyclin B-cdk1 movements had stopped, cdc2-YFP relocalised to the spindle complex occurs prior to cyclin B degradation (Nishiyama et at the first meiotic division. Observation of MT-associated al., 2000). When a stable form of cdc13p was overexpressed, cdc2-YFP fluorescence in meiosis I revealed that cdc2-YFP both cdc2-YFP and cdc13∆81-YFP remained associated with fluorescence appeared all along the spindle while, in mitosis or the spindle. A similar localisation was observed in a cut4 ts meiosis II, fluorescence is first enriched on the duplicated SPBs mutant, defective in APC function, suggesting that cdc13p before extending to the elongating spindle. This suggests that needs to be recognized by the APC to leave the spindle. regulation of spindle formation in meiosis I might be different Interaction between the APC and cdc13p appears to occur on from mitotic division and influence the distribution of sister the spindle itself and recognition of cdc13p by the APC chromatids in meiosis I. This observation suggests the appears to be required for cdc2p to leave the spindle. The fact existence of a different pathway of spindle formation in that cdc2p dissociation from the spindle first requires cdc13p fission yeast meiosis I, and possibly the formation of an dissociation is in agreement with the observation that the ‘acentrosomal’ meiotic spindle as observed previously in many association of mammalian cdk1/cyclin B complex with oocytes and in some spermatocytes (reviewed by Merdes and spindle MTs occurs through the interaction of cyclin B with Cleveland, 1997). Thus it appears that modifications in cdc2p MAPs (Ookata et al., 1995; Charasse et al., 2000). Our data localisation play a role in changing chromosomal behaviour in are also consistent with the co-localisation of APC with the meiosis from that observed in mitosis. Fission yeast cdc2p localisation 2639

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