Telomere-Nuclear Envelope Dissociation Promoted by Rap1 Phosphorylation Ensures Faithful Chromosome Segregation

Current Biology 22, 1932–1937, October 23, 2012 ª2012 Elsevier Ltd All rights reserved

http://dx.doi.org/10.1016/j.cub.2012.08.019

Report Telomere-Nuclear Envelope Dissociation Promoted by Rap1 Phosphorylation Ensures Faithful Chromosome Segregation Ikumi Fujita,1,5 Yuki Nishihara,1,5 Makiko Tanaka,1,5 Hisayo Tsujii,1,5 Yuji Chikashige,3 Yuzo Watanabe,4 Motoki Saito,4 Fuyuki Ishikawa,4 Yasushi Hiraoka,2,3 and Junko Kanoh1,* 1Institute for Protein Research 2Graduate School of Frontier Biosciences Osaka University, Suita, Osaka 565-0871, Japan 3Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, Hyogo 651-2492, Japan 4Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto 606-8501, Japan

Summary Efficient chromosomal movements are important for the fidelity of chromosome segregation during mitosis; however, movements are constrained during interphase by tethering of multiple domains to the nuclear envelope (NE) [1]. Higher eukaryotes undergo open mitosis accompanied by NE breakdown, enabling chromosomes to be released from the NE, whereas lower eukaryotes undergo closed mitosis, in which NE breakdown does not occur [2]. Although the chromosomal movements in closed mitosis are thought to be restricted compared to open mitosis, the cells overcome this problem by an unknown mechanism that enables accurate chromosome segregation. Here, we report the spatiotemporal regulation of telomeres in Schizosaccharomyces pombe closed mitosis. We found that the telomeres, tethered to the NE during interphase [3], are transiently dissociated from the NE during mitosis. This dissociation from the NE is essential for accurate chromosome segregation because forced telomere tethering to the NE causes frequent chromosome loss. The phosphorylation of the telomere protein Rap1 during mitosis, primarily by Cdc2, impedes the interaction between Rap1 and Bqt4, a nuclear membrane protein, thereby inducing telomere dissociation from the NE. We propose that the telomere dissociation from the NE promoted by Rap1 phosphorylation is critical for the fidelity of chromosome segregation in closed mitosis. Results and Discussion S. pombe Telomeres Are Transiently Dissociated from the NE during M Phase To analyze the distance between telomeres and the NE by microscope, we visualized six telomeres of three chromosomes, the nuclear envelope (NE), and microtubules with Taz1-mCherry, Ish1-GFP [4], and GFP-Atb2 [5], respectively (Figure 1A). In S. pombe, newly born daughter cells are in early G2 phase, and they grow and increase in length during G2 (see Figure S1A available online) [6]. In G2 phase, telomeres moderately cluster themselves and continuously move inside the

5These authors contributed equally to this work *Correspondence: [email protected]

nucleus (Figure S1B). Telomere clustering collapses around metaphase, and the replicated telomeres segregate in anaphase B (Figure S1C). Throughout G2 phase, most distances between telomere and the NE were <0.2 mm (overall G2-phase average, 0.21 mm; the S. pombe interphase nucleus is w2.2–2.5 mm in diameter; Figure 1B). Thus, the telomeres move within the vicinity of the NE during G2. In contrast, the telomere-NE distance during M phase was markedly increased (Figure 1B; see figure legend for the classification of M phase). At the MIV-S stage, the distance became shorter. In contrast to the telomere movements, the cut3 locus on chromosome II (w1,000 kb from the telomere and w600 kb from the centromere) was located apart from the NE during G2, and the distances between the cut3 locus and the NE became shorter at the M-II and M-III stages compared with those in G2, likely due to centromere relocation to the spindle pole body at the NE and chromosome condensation (Figure 1C). These data demonstrate that S. pombe telomeres are dissociated from the NE specifically in M phase. Dissociation of Telomeres from the NE during M Phase Is Required for Faithful Chromosome Segregation During interphase, telomeres are tethered to the NE through the interaction between the telomere binding protein complex Taz1-Rap1 and the nuclear membrane complex Bqt4-Bqt3 (Figure 2A) [3, 7–9]. To elucidate the importance of the mitotic telomere-NE dissociation, we fused the full length of Taz1 with GFP and the C-terminal region (amino acids 263–432) of Bqt4 (Bqt4DN), which possesses the NE localization ability and lacks the Rap1-binding domain [3]. Taz1-GFP-Bqt4DNDTM lacks the C-terminal 19 amino acids of Bqt4, which have been shown to be involved in the association with Bqt3 (Figure 2B) [3]. The Taz1-GFP-Bqt4DN and Taz1-GFPBqt4DNDTM proteins were expressed in a taz1D background at a level comparable to that of Taz1-GFP (data not shown). Strong punctate signals of Taz1-GFP-Bqt4DN were observed at the NE throughout the cell cycle, whereas Taz1-GFPBqt4DNDTM signals were moderately distributed in the nucleus, although some portion remained localized at the NE (Figure 2C). The telomere DNA length and telomere end protection in G1 phase were normal in the Taz1-GFP-Bqt4DNand Taz1-GFP-Bqt4DNDTM-expressing strains (Figures S2A and S2B), indicating that the C-terminal tagging of Taz1 with GFP-Bqt4DN or GFP-Bqt4DNDTM does not destroy Taz1 functions. In the GFP-Bqt4DN-expressing strain, the telomeres were localized at the NE during G2, whereas they were detached from the NE during mitosis (Figure 2D, left, M-II). In contrast, the telomeres were localized close to the NE throughout the cell cycle in the Taz1-GFP-Bqt4DN-expressing strain (Figure 2D, right). These data indicate that telomeres are anchored to the NE even during mitosis by the Taz1-GFPBqt4DN protein. The Taz1-GFP-Bqt4DN-expressing strain exhibited a considerably higher index of the entire M phase compared with the wild-type (WT) control (Figure S2C) and frequent lagging chromosomes, particularly the chromosomes stretched across two daughter nuclei after metaphase without the

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Figure 1. Telomeres Are Transiently Dissociated from the NE during M Phase (A) Visualization of telomeres, the NE, and microtubules with Taz1-mCherry, Ish1-GFP, and GFP-Atb2, respectively. Taz1 binds to the distal region of the subtelomere as well as to the telomere repeats [16]. Because chromosome III does not contain subtelomeres, the telomeres of chromosome III display considerably weaker Taz1-mCherry signals compared with other telomeres. Tel, telomere; cen, centromere. See Figures S1B and S1C for the live-cell images during G2 and M phases. (B) Scatterplots showing the distances between telomeres and the NE (right). Vertical bars in the graph indicate the average distances (p, Mann-Whitney U test). Inner gray-line circles indicate the NE, and black lines indicate mitotic spindles (left). G2 phase was divided into three stages based on cell length (early, <9.5 mm; middle, 9.5–11.5 mm; late, R11.5 mm). M phase was divided into four stages: M-I, when the mitotic spindles appear and extend to the opposite sides of the nucleus (zprophase-anaphase A); M-II, when the mitotic spindles and the nucleus extend to the opposite poles (zthe early period of anaphase B); M-III, when two daughter nuclei and mitotic spindles are observed (the latter period of anaphase B); and M-IV-S, when no mitotic spindle and no constriction at the septum position are observed. See Figure S1A for the S. pombe cell cycle. (C) Scatterplots showing the distances between the cut3 locus and the NE. The cut3 locus on chromosome II was visualized with GFP by the lac operator and repressor system [17]. The NE was visualized with mCherry-Bqt4. The cell-cycle stages were judged by the number and the position of Pcp1, a pericentrin protein [18]. Vertical bars in the graph indicate the average distances (p, Mann-Whitney U test versus WT in Figure 1B).

notable activation of DNA damage checkpoint (Figure 2E; Figure S2D). These phenotypes were alleviated in the Taz1GFP-Bqt4DNDTM-expressing strain (Figure 2E; Figure S2C). Furthermore, the Taz1-GFP-Bqt4DN-expressing strain lost minichromosome Ch16 [10] after cell division at a markedly high frequency (2.4 3 1022 per division), whereas the WT control and Taz1-GFP- and GFP-Bqt4DN-expressing strains rarely lost Ch16 (2.8 3 1024, 5.6 3 1024, and 3.9 3 1024 per division, respectively) (Figure 2F). The Taz1-GFPBqt4DNDTM-expressing strain lost Ch16 at a lower frequency (4.5 3 1023 per division) compared with that of the Taz1GFP-Bqt4DN-expressing strain. Moreover, the expression of the Taz1-GFP-Bqt4DN protein in the taz1+ strain resulted in a dominant effect on the Ch16 loss rate (1.0 3 1022 per division). These results indicated that the forced tethering of telomeres to the NE disturbs accurate chromosome movements and, therefore, strongly suggested that transient telomere dissociation from the NE during M phase is

required for the fidelity of chromosome segregation in closed mitosis. Rap1 Is Phosphorylated during M Phase To investigate the molecular mechanisms of the mitotic telomere-NE dissociation, we analyzed the nature of Rap1. Band shifts of Rap1 around M phase were observed, just before the mitotic cyclin-B (Cdc13) was completely degraded (20–50 min; Figure 3A). Consistently, the Rap1 band displayed a marked upshift at early M phase in the nda3-KM311 mutant, although slight band shifts were also observed in S and G2 phases (Figure S3A). Phosphatase treatment of Rap1 derived from cells in early M phase caused a marked downshift of the Rap1 band (Figure 3B). These data indicated that Rap1 is highly phosphorylated, particularly during M phase. To identify the phosphorylation sites in Rap1 that are responsible for its band shifts, we first examined whether the

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Figure 2. Telomere Dissociation from the NE Is Required for Faithful Chromosome Segregation (A) Schematic illustration of the protein complex that tethers telomeres to the NE during interphase. Taz1 directly binds to telomeric DNA and recruits Rap1 to telomeres. Bqt3 is a receptor for Bqt4 on the inner nuclear membrane. (B) Schematic illustration of the Taz1-GFP, GFP-Bqt4DN, Taz1-GFP-Bqt4DN, and Taz1-GFP-Bqt4DNDTM proteins. (C) Cellular localizations of each GFP protein. Strains JP1266 (Taz1-GFP), JP584 (GFP-Bqt4DN), JP558 (Taz1-GFP-Bqt4DN), and JP1323 (Taz1-GFPBqt4DNDTM) were grown in YES at 32
C. Cell images were taken using a DeltaVision microscope system and were processed with three-dimensional deconvolution. Broken lines indicate cell shapes. Arrowheads indicate the cells in anaphase. Scale bar represents 10 mm. See Figures S2A and S2B for the telomere DNA length and telomere end protection in each strain. (D) Telomeres are anchored to the NE during mitosis in the Taz1-GFP-Bqt4DN-expressing strain. Strains JP584 (GFP-Bqt4DN) and JP558 (Taz1-GFPBqt4DN) were grown in EMM at 32
C. Each GFP protein was visualized by indirect immunofluorescence staining (green). The subtelomere DNAs of chromosomes I and II were visualized by fluorescence in situ hybridization (red). Images on a single focal plane are shown. Scale bar represents 10 mm. (E) Abnormal chromosome segregation in the Taz1-GFP-Bqt4DN-expressing strain. Strains JP427 (WT control), JP1266 (Taz1-GFP), JP584 (GFP-Bqt4DN), JP558 (Taz1-GFP-Bqt4DN), JP1323 (Taz1-GFP-Bqt4DNDTM), and JP905 (rap1-5A) were grown to mid-log phase in YES at 32
C, fixed by 3% paraformaldehyde and 0.25% glutaraldehyde, and stained by DAPI (40 ,6-diamidino-2-phenylindole) for the observation of chromosomal DNA. Mitotic cells that had more than two masses of DNA signals were classified as follows: class I, daughter nuclei equally segregated; class II, a part of one or both daughter nuclei exhibited delayed segregation; class III, long stretches of chromosomes extended inward, forming a bridge between two nuclei; class IV, two daughter nuclei were segregated unequally; class V, one or several chromosomes were separated from two nuclei or fragmented. Typical images of chromosome segregation in class I (WT control) and class III (Taz1-GFP-Bqt4DN) are shown. Broken lines indicate cell shapes. Scale bar represents 5 mm. See Figures S2C and S2D for the mitotic index of each strain and the DNA damage signaling in the Taz1-GFP-Bqt4DN-expressing strain, respectively. (F) Ch16 loss rate of each strain. Strains JP427 (WT control), JP1266 (Taz1-GFP), JP584 (GFP-Bqt4DN), JP558 (Taz1-GFP-Bqt4DN), JP1323 (Taz1-GFPBqt4DNDTM), JP554 (control + Taz1-GFP-Bqt4DN), and JP905 (rap1-5A) were grown in YES at 32
C and incubated on YES plates containing 12 mg/ml adenine to assay loss of the ade6+ marker on Ch16. Error bars indicate SD (more than three experiments). P indicates p value of Student’s t test versus the WT control.

five Ser or Thr-Pro motifs in Rap1 (Figure S3B) could be Cdc2 substrates in vitro using affinity-purified p13Suc1-associated kinases (the major kinase is Cdc2) [11]. We found that Ser213, Thr378, Ser422, and Ser513, but not Ser549, are substrates for p13Suc1-associated kinases (Figures S3C–S3E); however, a clear band shift remained in Rap1213/378/422/513A at

M phase (Figure S3F), suggesting the existence of other M phase-specific phosphorylation sites in vivo. The mass spectrometry identified 20 amino acid residues as putative phosphorylation sites (Table S1). We therefore screened for an alanine mutation that abolishes the M phase-specific band shift of Rap1213/378/422/513A and found that the band shifts of

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Figure 3. Rap1 Is Phosphorylated during M Phase

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(A) Strain JK905 (cdc25-22) was grown in YES to early log phase and arrested in late G2 phase by Cdc13 a temperature shift to 35.5
C for 4 hr and then Rap1 (kDa) (Cyclin-B) released from arrest by a temperature shift to 90 25
C. The percentage of cells with septa was Cdc2 determined by counting >200 cells for each time point. The whole-cell extracts were analyzed by 100 nda3-KM311 C immunoblotting using anti-Rap1, anti-Cdc13 (cy80 clin-B), and anti-PSTAIR for Cdc2 (loading S 60 control). See Figure S3A for the Rap1 band shift As M As M 40 in various cell-cycle mutants. 117 (B) Strain JK317 (WT) was grown in YES at 32
C to 20 (kDa) Rap1 M G2 89 mid-log phase (asynchronous). Strain JK2326 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 (nda3-KM311) was grown in YPD at 32
C and Time after the release from cdc25-22 block (min) Cdc13 then arrested in early M phase by a temperature shift to 20
C for 12 hr. Rap1 proteins were immunoprecipitated from the cell extracts using anti0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 (min) Cdc2 D Rap1 and treated with calf intestinal alkaline pS213 phosphatase (CIAP) for 1 hr at 32
C with or without phosphatase inhibitors. The samples pT378 were analyzed by immunoblotting using antiRap1. pS422 (C) Strains JK3347 (nda3-KM311, WT) and JK3379 (nda3-KM311 rap1213/378/422/456/513A; pS456 rap1-5A) were grown in YPD to mid-log phase (asynchronous, As) at 32
C and then pS513 arrested in early M phase (M) by a temperature shift to 20
C for 12 hr. The whole-cell extracts Rap1-HA were analyzed by immunoblotting using antiCdc13 Rap1, anti-Cdc13, and anti-PSTAIR for Cdc2 (loading control). See Figures S3B–S3E and Cdc2 S3F and Table S1 for in vitro kinase assays using p13Suc1-associated kinases, the band100 shift assay of Rap1213/378/422/513A, and the 80 S mass-spectrometry for Rap1 phosphorylation, S 60 respectively. 40 (D) Cells of the strain JK2515 (cdc25-22 rap120 M G2 M 2HA6His:ura4+) were synchronized as described 0 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 in Figure 3A. Affinity-purified Rap1-2HA6His Time after the release from cdc25-22 block (min) proteins were analyzed by immunoblotting using each phosphospecific antibody and anti-HA (for Rap1). The whole-cell extracts were also analyzed by immunoblotting using anti-Cdc13 and anti-PSTAIR for Cdc2 (loading control). See Figures S3G and S3H for the specificity of each phosphospecific antibody. See Figure S3I for the summary of the Rap1 phosphorylation sites. 89

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Rap1 in M phase mostly disappeared when Ser456 was substituted with alanine in Rap1213/378/422/513A (rap1-5A; Figure 3C). We next analyzed the timing of the phosphorylations at the five residues (Ser213, Thr378, Ser422, Ser456, and Ser513) using phosphospecific Rap1 antibodies (Figure 3D; Figures S3G and S3H). Phosphorylation at Ser213 was nearly constant throughout the cell cycle. In contrast, phosphorylations at the other residues peaked during M phase, although they were not completely synchronized. These results demonstrate that Rap1 is phosphorylated at Ser213, Thr378, Ser422, Ser456, and Ser513 during M phase in vivo (Figure S3I). The conservation of Rap1 phosphorylation in other species is currently unknown. Rap1 Phosphorylation Promotes Telomere-NE Dissociation by Inhibiting Rap1-Bqt4 Interaction To test whether Rap1 phosphorylation is involved in the mitotic telomere-NE dissociation, we substituted the five phosphorylation sites with alanine (the nonphosphorylated form rap1-5A) and with glutamic acid or aspartic acid (the phosphomimic forms rap1-5E and rap1-5D, respectively). Strikingly, the rap1-5A mutation increased the efficiency of the Rap1-Bqt4 interaction, whereas the rap1-5E and rap1-5D

mutations decreased the efficiency in the yeast two-hybrid system (Figure 4A). Additionally, Rap1-5E or 5D associated with GST-Bqt4 with lower efficiencies compared with the WT Rap1 in the S. pombe cell lysates (Figure S4A). These data suggest that Rap1 phosphorylation has an inhibitory effect on the Rap1-Bqt4 association. In marked contrast, the mutations at the Rap1 phosphorylation sites did not have a significant effect on the interactions between Rap1 and the other Rap1 interactors, Taz1 and Poz1, which are involved in telomere length regulation [7, 12], and Bqt1 and Bqt2, which are involved in meiotic telomere clustering [13] (Figure 4A). Consistently, the mutations caused no notable effects on Rap1 protein level, telomere DNA length, or spore formation after meiosis (Figures S4B–S4D). These observations suggest that the phosphorylation of Rap1 has a specific inhibitory effect on the Rap1-Bqt4 interaction. We further found that Rap1 phosphorylation influences the position of the telomeres in the nucleus. The deletion of bqt4+ or rap1+ resulted in substantial increases in the telomere-NE distances in G2, whereas the distances in M phase of the rap1D mutant were similar to those in the WT (Figure 4B). The shorter distances in G2 compared with those in M phase in the rap1D mutant imply that chromosomal domains other than telomeres remain tethered to the NE in G2. Intriguingly, the

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Figure 4. Rap1 Phosphorylation Primarily by Cdc2 Promotes Telomere Dissociation from the NE (A) Yeast two- and three-hybrid assays. pACT2rap1+ was mutated at the five phosphorylation sites as indicated. Transformants were assayed for b-galactosidase activity. See Figures S4A and S4B–S4D for the GST pull-down assays and the phenotypes of each rap1 mutant, respectively. (B) Scatterplots showing the distances between the telomeres and the NE in strains JP842 (bqt4D) and JP836 (rap1D). A plot of the data for WT (JK81) during the entire G2 phase (taken from Figure 1B) is shown as the control. Vertical bars in the graph indicate the average distances (p, Mann-Whitney U test). (C) Scatterplots showing the distances between the telomeres and the NE in strains JP446 (rap1-5A), JP448 (rap1-5E), and JP450 (rap1-5D). Vertical bars in the graph indicate the average distances (p, Mann-Whitney U test). (D) Yeast two-hybrid assays for the interactions between Rap1 and Bqt4. For the rap1-4A or rap1-1A series, four or one of the five phosphorylated residues were mutated to alanine residues, respectively. For the rap1-4A series, the remaining WT residue is indicated. Transformants were assayed for b-galactosidase activity. See Figure S4E for identification of the Bqt4-binding regions in Rap1. (E) Scatterplots showing the distances between the telomeres and the NE in strains JP1699 (rap1213A), JP1701 (rap1378A), JP1703 (rap1422A), JP1705 (rap1456A), JP1707 (rap1513A), and JP1119 (rap1513E) in G2 phase. A plot of the data for WT (JK81) during the entire G2 phase (taken from Figure 1B) is shown as the control. Vertical bars in the graph indicate the average distances (p, Mann-Whitney U test). (F) Strains JK3113 (WT) and JP1945 (cdc2-33) were grown in YES at 32
C overnight (left) or at 25
C overnight and shifted to at 35.5
C for 4 hr (right). Affinity-purified Rap1-2HA6His proteins were analyzed by immunoblotting using each phosphospecific antibody and anti-HA (for Rap1). See Figures S4F and S4G for the phenotypes of the rap1-3PA mutant.

0.21

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distances in the rap1-5A mutant during G2 were markedly shorter than in the WT (Figure 4C). In G2 phase, the Rap1 protein is maintained as a hypophosphorylated form, rather than as a nonphosphorylated form (Figure S3A). Therefore, the nonphosphorylated form (Rap1-5A) likely led to the stronger telomere-NE association compared with the WT. In contrast, the distances in G2 were clearly greater in the rap15E and rap1-5D mutants than in the WT. These data suggest

that Rap1 phosphorylation promotes telomere-NE dissociation by inhibiting the Rap1-Bqt4 interaction. In the rap1-5A mutant, the telomereNE distances during M phase were shorter than in the WT (M-I, M-II, and M-IV-S; Figure 4C), indicating that Rap1 phosphorylation is one of the factors for the mitotic telomere-NE dissociation. The rap1-5A mutant lost minichromosome Ch16 at a 3-fold higher frequency than did the WT, although it did not display a notable defect in chromosomal movements (Figures 2E and 2F), suggesting that Rap1 phosphorylation contributes to the fidelity of chromosome segregation. It should be noted that, even in the rap1-5A mutant, the telomere-NE distances during M phase were increased compared with those in G2. This result implies the presence of other mechanisms regulating the telomere-NE dissociation in addition to Rap1

Role of the Telomere in Closed Mitosis 1937

phosphorylation and/or the presence of other Rap1 phosphorylation sites critical for the mitotic telomere-NE dissociation. Rap1 Phosphorylation by Cdc2 Is Important for Telomere Dissociation from the NE Yeast two-hybrid analyses suggested that the mutation at Ser513 critically affects the Rap1-Bqt4 interaction (Figure 4D). Moreover, rap1513A, but not any of the other single-alanine mutants, showed markedly shorter telomere-NE distances in G2 (Figure 4E). In contrast, the rap1513E mutant exhibited a moderate elongation of the distance. Furthermore, the regions of Rap1 containing Ser513 are required for the Rap1Bqt4 interaction in the yeast two-hybrid system (Figure S4E). These results suggest that phosphorylation at Ser513 is the key for Rap1-Bqt4 dissociation and thereby for the mitotic telomere-NE dissociation. We next investigated the contribution of Cdc2 in the Rap1 phosphorylation and the telomere-NE dissociation. Rap1 phosphorylations at Thr378, Ser422, and Ser513 were markedly reduced by the inactivation of Cdc2 in the cdc2-33 mutant (Figure 4F) [14]. Consistently, these phosphorylations were observed during the early to middle stages of mitosis, when Cdc2 is highly active (Figure 3D). Moreover, these residues were efficiently phosphorylated by p13Suc1-associated kinases (the major kinase is Cdc2) in vitro (Figures S3D and S3E). Furthermore, the phosphorylations at Thr378, Ser422, and Ser513 were completely abolished when the proline residues following the three phosphorylation sites were mutated to alanine (rap1-3PA) to destroy the consensus target sequence (Ser or Thr-Pro) of Cdc2 (Figure S4F). These data strongly suggest that Thr378, Ser422, and Ser513 are the targets of Cdc2 in vivo. The telomere-NE distances in the rap1-3PA mutant were clearly shorter than in the WT in G2 (Figure S4G), and those in M phase were shorter than in the WT (M-II and -IV-S). These results suggest that Cdc2 participates in the telomere-NE dissociation by phosphorylating Thr378, Ser422, and Ser513 of Rap1. Other phosphorylation events may also contribute to the telomere-NE dissociation because the rap3PA mutation exhibited less of an effect on the telomere-NE distance than the rap1-5A mutation (Figure 4C; Figure S4G). From this study, we propose a model in which the telomere plays an important role in the dynamism of chromosomes during M phase. Upon entry into mitosis, the phosphorylation of Rap1, primarily by Cdc2, induces its detachment from Bqt4. This reaction, possibly in collaboration with other unknown mechanisms such as chromosome condensation, promotes telomere dissociation from the NE. Consistently, telomeres are transiently released from the NE in S. cerevisiae closed mitosis [15]. Therefore, the mitotic telomere-NE dissociation is not specific for S. pombe but, rather, is conserved in closed mitosis. In a sense, the release of chromosomes from the NE may be the common mechanism that is required for the proper movement of chromosomes in both closed and open mitoses. Supplemental Information Supplemental Information includes four figures, two tables, and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2012.08.019. Acknowledgments We thank Y. Watanabe, A. Sugimoto, K. Tanaka, and S. Saitoh for critical reading of the manuscript; M. Sato and Y. Kakui for communicating unpublished data; the Yeast Genetic Resource Center for strains; Y. Hirano,

Y. Tange, H. Maekawa, M. Ohsugi, H. Kosako, K. Furuya, A. Matsuura, F. Esashi, K. Ishii, T. Kobayashi, and T. Toda for advice; and all laboratory members and the Shinohara laboratory (IPR) for general support. This work was supported by the Osaka University Life Science Young Independent Researcher Support Program of JST, Grants-in-Aid for Scientific Research (KAKENHI), the Astellas Foundation for Research on Metabolic Disorders, the Takeda Science Foundation, the Sumitomo Foundation, and the Novartis Foundation for the Promotion of Science to J.K. Received: March 28, 2012 Revised: June 28, 2012 Accepted: August 9, 2012 Published online: September 6, 2012 References 1. Gu¨ttinger, S., Laurell, E., and Kutay, U. (2009). Orchestrating nuclear envelope disassembly and reassembly during mitosis. Nat. Rev. Mol. Cell Biol. 10, 178–191. 2. Sazer, S. (2010). Nuclear membrane: nuclear envelope PORosity in fission yeast meiosis. Curr. Biol. 20, R923–R925. 3. Chikashige, Y., Yamane, M., Okamasa, K., Tsutsumi, C., Kojidani, T., Sato, M., Haraguchi, T., and Hiraoka, Y. (2009). Membrane proteins Bqt3 and -4 anchor telomeres to the nuclear envelope to ensure chromosomal bouquet formation. J. Cell Biol. 187, 413–427. 4. Taricani, L., Tejada, M.L., and Young, P.G. (2002). The fission yeast ES2 homologue, Bis1, interacts with the Ish1 stress-responsive nuclear envelope protein. J. Biol. Chem. 277, 10562–10572. 5. Tatebe, H., Goshima, G., Takeda, K., Nakagawa, T., Kinoshita, K., and Yanagida, M. (2001). Fission yeast living mitosis visualized by GFPtagged gene products. Micron 32, 67–74. 6. Knutsen, J.H., Rein, I.D., Rothe, C., Stokke, T., Grallert, B., and Boye, E. (2011). Cell-cycle analysis of fission yeast cells by flow cytometry. PLoS ONE 6, e17175. 7. Cooper, J.P., Nimmo, E.R., Allshire, R.C., and Cech, T.R. (1997). Regulation of telomere length and function by a Myb-domain protein in fission yeast. Nature 385, 744–747. 8. Chikashige, Y., and Hiraoka, Y. (2001). Telomere binding of the Rap1 protein is required for meiosis in fission yeast. Curr. Biol. 11, 1618–1623. 9. Kanoh, J., and Ishikawa, F. (2001). spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast. Curr. Biol. 11, 1624–1630. 10. Niwa, O., Matsumoto, T., and Yanagida, M. (1986). Construction of a mini-chromosome by deletion and its mitotic and meiotic behaviour in fission yeast. Mol. Gen. Genet. 203, 397–405. 11. Brizuela, L., Draetta, G., and Beach, D. (1987). p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. EMBO J. 6, 3507–3514. 12. Miyoshi, T., Kanoh, J., Saito, M., and Ishikawa, F. (2008). Fission yeast Pot1-Tpp1 protects telomeres and regulates telomere length. Science 320, 1341–1344. 13. Chikashige, Y., Tsutsumi, C., Yamane, M., Okamasa, K., Haraguchi, T., and Hiraoka, Y. (2006). Meiotic proteins Bqt1 and Bqt2 tether telomeres to form the bouquet arrangement of chromosomes. Cell 125, 59–69. 14. Dickinson, J.R. (1983). Nucleoside diphosphokinase and cell cycle control in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 60, 355–365. 15. Hediger, F., Neumann, F.R., Van Houwe, G., Dubrana, K., and Gasser, S.M. (2002). Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast. Curr. Biol. 12, 2076–2089. 16. Kanoh, J., Sadaie, M., Urano, T., and Ishikawa, F. (2005). Telomere binding protein Taz1 establishes Swi6 heterochromatin independently of RNAi at telomeres. Curr. Biol. 15, 1808–1819. 17. Nabeshima, K., Nakagawa, T., Straight, A.F., Murray, A., Chikashige, Y., Yamashita, Y.M., Hiraoka, Y., and Yanagida, M. (1998). Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle. Mol. Biol. Cell 9, 3211–3225. 18. Flory, M.R., Morphew, M., Joseph, J.D., Means, A.R., and Davis, T.N. (2002). Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe. Cell Growth Differ. 13, 47–58.