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Lte1, Cdc14 and MEN-controlled Cdk inactivation in yeast coordinate rDNA decompaction with late telophase progression

2009; Springer Nature; Volume: 28; Issue: 11 Linguagem: Inglês

10.1038/emboj.2009.111

1460-2075

Elisa Varela, Kenji Shimada, Thierry Laroche, Didier Leroy, Susan M. Gasser,

Fungal and yeast genetics research

Article23 April 2009free access Lte1, Cdc14 and MEN-controlled Cdk inactivation in yeast coordinate rDNA decompaction with late telophase progression Elisa Varela Elisa Varela Friedrich Miescher Institute for Biomedical Research, Basel, SwitzerlandPresent address: Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, E-28029 Madrid Search for more papers by this author Kenji Shimada Kenji Shimada Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Thierry Laroche Thierry Laroche EPFL, BioImaging and Optics Platform, Lausanne, Switzerland Search for more papers by this author Didier Leroy Didier Leroy Medicines for Malaria Venture, International Center Cointrin, Geneva, Switzerland Search for more papers by this author Susan M Gasser Corresponding Author Susan M Gasser Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Elisa Varela Elisa Varela Friedrich Miescher Institute for Biomedical Research, Basel, SwitzerlandPresent address: Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, E-28029 Madrid Search for more papers by this author Kenji Shimada Kenji Shimada Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Thierry Laroche Thierry Laroche EPFL, BioImaging and Optics Platform, Lausanne, Switzerland Search for more papers by this author Didier Leroy Didier Leroy Medicines for Malaria Venture, International Center Cointrin, Geneva, Switzerland Search for more papers by this author Susan M Gasser Corresponding Author Susan M Gasser Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Author Information Elisa Varela1, Kenji Shimada1, Thierry Laroche2, Didier Leroy3 and Susan M Gasser 1 1Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 2EPFL, BioImaging and Optics Platform, Lausanne, Switzerland 3Medicines for Malaria Venture, International Center Cointrin, Geneva, Switzerland *Corresponding author. Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel 4058, Switzerland. Tel.: +41 61 697 7255; Fax: +41 61 697 3976; E-mail: [email protected] The EMBO Journal (2009)28:1562-1575https://doi.org/10.1038/emboj.2009.111 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The mechanism of chromatin compaction in mitosis has been well studied, while little is known about what controls chromatin decompaction in early G1 phase. We have localized the Condensin subunit Brn1 to a compact spiral of rDNA in mitotic budding yeast cells. Brn1 release and the resulting rDNA decompaction in late telophase coincided with mitotic spindle dissociation, and occurred asymmetrically (daughter cells first). We immunoprecipitated the GTP-exchange factor Lte1, which helps activate the mitotic exit network (MEN) in anaphase, with mitotic Brn1. In lteΔ cells Brn1 release was delayed, even at temperatures that do not impair mitotic exit. Mutations in MEN pathway components that act downstream of Lte1 similarly delayed rDNA decompaction. We found that Brn1 release in wild-type cells coincided with the release of Cdc14 phosphatase from the nucleolus and with mitotic CDK inactivation, yet it could be selectively delayed by perturbation of the MEN pathway. This may argue that different levels of Cdk inactivation control spindle disassembly and chromatin decompaction. Mutation of lte1 also impaired rotation of the nucleus in early G1. Introduction Passage through mitosis requires both temporally and spatially coordinated changes in chromatin compaction. The mechanisms that alter the level of chromatin compaction are not fully understood, although major players are the two large and related complexes of Condensin and Cohesin (Guacci et al, 1997; Ciosk et al, 1998; Lavoie et al, 2000). The Condensin complex contains a pair of related structural maintenance of chromosome (SMC) subunits that form a dimeric hinge, as well as three non-SMC subunits: Ycs4/XCAP-D2, Ycs5/XCAP-G and Brn1/Barren (Schleiffer et al, 2003). Brn1 is a member of a conserved family of kleisins, which associate with and bridge between SMC head groups. The genes encoding Condensin subunits are essential for vegetative growth, yet conditional mutations were isolated and shown to impair mitotic chromatin compaction in budding yeast (Strunnikov et al, 1995; Freeman et al, 2000; Lavoie et al, 2000, 2004; Ouspenski et al, 2000; Bhalla et al, 2002). Conditional mutations in Cohesin subunits also have measurable effects on mitotic rDNA compaction (Lavoie et al, 2004). Mitotic kinases, such as the cyclin-dependent kinase Cdk, control the dynamics of these structural components of chromatin in early mitosis (Stegmeier et al, 2002; D'Amours et al, 2004; Lavoie et al, 2004; Sullivan et al, 2004; reviewed in Toth et al, 2007). For instance, activation of the anaphase promoting complex (APC) by Cdk leads to the destruction of Pds1 and cleavage of the kleisin subunit of Cohesin, Scc1 (Uhlmann et al, 1999). This coincides with a partial release of the Cdc14 phosphatase from its inhibitor Net1 (Queralt et al, 2006), which sequesters Cdc14 in the nucleolus (Shou et al, 1999; Visintin et al, 1999). This early anaphase release of Cdc14 (Stegmeier et al, 2002) correlates with both the accumulation of Condensin in the nucleolus and rDNA compaction, an event that facilitates rDNA segregation (D'Amours et al, 2004; Sullivan et al, 2004). The Ipl1/AuroraB kinase have also been implicated in chromatin compaction at the beginning of mitosis in yeast (Lavoie et al, 2004; Sullivan et al, 2004) and in mammalian cells (Lipp et al, 2007). The vertebrate AuroraB kinase was shown to control the localization of Condensin I to mitotic chromosomes, while the successive phosphorylation of vertebrate Condensin I and II by cyclin–Cdk complexes is thought to promote condensation (reviewed by Hirano, 2005). After sister chromatids are separated by extension of the mitotic spindle, cells exit mitosis. In budding yeast this is triggered by activation of the mitotic exit network (MEN), which controls the degradation of B-type cyclins and accumulation of the Cdk inhibitor Sic1 (D'Amours et al, 2004; Toth et al, 2007). Both are required to fully inhibit mitotic Cdk. Exit from mitosis requires the breakdown of the mitotic spindle and initiation of G1-phase-specific transcription events, although, unlike mammals, yeast has a closed mitosis that obviates the need to re-assemble the nuclear envelope around daughter nuclei. Nonetheless, in early G1-phase cells, the yeast nucleus rotates to position the nucleolus opposite the spindle pole body (SPB) (Bystricky et al, 2005). At the top of the MEN signalling cascade is a small GTPase called Tem1. Tem1 is negatively regulated by a GTPase-activating complex composed of Bub2 and Bfa1 (Geymonat et al, 2002) and is positively regulated by Lte1, which contains homology to guanine nucleotide exchange factors (GEF) of the Cdc25 family (Shirayama et al, 1994). Lte1 is not essential for mitosis at 30°C, yet cells that lack Lte1 are cold sensitive for progress through telophase (Shirayama et al, 1994). Indeed, GTP-bound Tem1 is required to activate Cdc15 kinase, which leads in turn to a second wave of Cdc14 phosphatase release. This final release of Cdc14 ensures inactivation of the mitotic Cdk by promoting the degradation of Clb2 by APCCdh1 and the accumulation of the Cdk1 inhibitor Sic1 (Visintin et al, 1998), which jointly signal entry into G1. In vertebrates, the reformation of the nuclear envelope and reassembly of nuclear lamina precede chromatin decompaction, yet to date no study has examined how chromatin decompaction is coordinated with exit from mitosis. Although chromatin decompaction correlates with breakdown of the long anaphase spindle in wild-type (wt) budding yeast, the temporal coincidence of two events in the cell cycle cannot be taken as evidence of coordinate control. For instance, bud emergence and the initiation of DNA replication coincide temporally in the yeast cell cycle, but are controlled by distinct pathways, which were identified and dissociated by mutagenesis (Hartwell et al, 1974). Here, we have examined the link between the well-characterized MEN pathway and chromosome decompaction. First, we show that rDNA decompaction correlates with the release of Brn1 from the rDNA. We then found that several spindle-regulatory proteins, among them the Tem1-regulator Lte1, are associated with Brn1 in mitosis. We have used quantitative live microscopy to examine whether Lte1 and the MEN pathway control Brn1 localization. Indeed, lte1 deletion selectively delays Brn1 release and rDNA decompaction with respect to spindle disassembly and formation of the G1 nucleus. The deletion of bub2 rescued the lack of decondensation found in the lte1 mutant at low temperatures, implicating MEN pathway components downstream of Lte1. Decompaction of the rDNA furthermore correlated with the final release of Cdc14 phosphatase from the nucleolus. Moreover, in cells blocked at the cdc15 arrest point in late anaphase, premature inactivation of the cyclin-dependent kinase Cdc28 triggered both Brn1 delocalization and rDNA decompaction. We also found that the absence of Lte1 interferes with the rotation of the nucleus in early G1 phase, which positions the nucleolus opposite to the SPB. Our studies provide the first mechanistic analysis of the coordination of chromatin decompaction with entry into interphase. Results Decompaction of the rDNA starts in telophase To have a molecular marker for the process of chromatin compaction and decompaction in Saccharomyces cerevisiae, we tagged the Condensin subunit Brn1 and monitored its localization both by immunofluorescence (IF) and live microscopy of Brn1-GFP in dividing cells. Both Brn1-13myc and Brn1-GFP were integrated and expressed from the BRN1 promoter and fully complemented the growth arrest phenotype of brn1 deficient cells. High-resolution confocal microscopy of a fixed and immunostained exponential culture revealed a diffuse distribution of Brn1-13myc throughout the nucleus and nucleolus in interphase cells (Figure 1A; Supplementary Figure S1A). In anaphase, however, Brn1 assumed a compact but elongated spiral that extended the length of the nucleus, much like the rDNA (Figure 1D and E). Coincidence of mitotic Brn1 with the rDNA was confirmed by double staining and by colocalization of Brn1-GFP with the nucleolar marker Nop1-CFP (Figure 1D; Supplementary Figure S1A–D; Supplementary Movie 1). Time-lapse microscopy (Supplementary Movie 2) showed similar mitotic spiral structures in strains bearing Brn1-GFP. Finally, we note that in telophase cells Brn1 has a compact, punctate appearance that is lost in G1- and S-phase nuclei (Figure 1A). Figure 1.Brn1 localization in interphase and mitosis. (A) IF for Brn1-13myc labelled with anti-Myc (grey) and anti-tubulin (green) antibodies on strain GA-1656. The slightly punctate Brn1-staining in telophase panel can be contrasted to the diffuse staining seen in G1- and S-phase cells. d=daughter cell nucleus. Bar=5 μm. (B) Micrographs show IF for Brn1 and tubulin (as in A) coupled with DAPI for identification of DNA during late mitosis where spindles are either intact (bottom) or starting to disassemble (see arrowheads, upper panel). d=daughter cell nucleus. Bar=5 μm. (C) Selected frames from time-lapse imaging of Brn1-GFP (GA-2663; min in upper left) in which we observe Brn1-GFP segregation to the daughter cell and decompaction occurring initially in the daughter cell nucleus (d). Bar=5 μm. (D) Single confocal section showing Brn1-GFP (green) and Nop1-CFP (red) during chromosome segregation in two adjacent cells by live microscopy. Bar=5 μm. For live imaging of mitosis see Supplementary data. (E) Confocal sections of IF for Brn1-13myc with anti-Myc (green) and for phospho H3 (anti-H3PhosphoS10; red). Two mitotic figures are shown in the larger image, and an interphase cell is in the inset. Schematic figure depicts results from panels D and E. (F) Schematic representation of tetO and lacO array insertion on Chr 12. Underneath is the Perod-Kratky chain equation, where contour length Lc (nm) is the ratio of the genomic distance d (in bp) divided by the linear mass density of the chromatin chain c (in bp/nm) or Lc=d/c. Brn1 is restricted to the nucleolus in the net1-1 mutant as seen in this representative picture showing Brn1-GFP (green) and Nop1-CFP (red) in net1-1 cells (GA-3266; net1-1, Brn1-GFP and Nop1-CFP). Cells were grown at the permissive temperature (25°C), and microscopy was performed after 2 h at nonpermissive temperature (30°C). Bar=5 μm. Below is a schematic of the behavior of two distant points (blue and yellow) on a flexible polymer chain (no Condensin, green) as compared to a less flexible fibre that we propose results from Condensin association. (G) Distances between the centers of gravity of the two spots were measured on strains GA-3779 (wt) and GA-4114 (net1-1) which retains Condensin on the rDNA during interphase. >100 cells were scored for each stage except telophase (n=50). S-phase cells are budded, whereas G2/M have elongated nuclei. End-to-end distance measurements correspond to: 1⩽0.25 μm; 2⩽0.5 μm; 3⩽0.75 μm; 4⩽1 μm; 5⩽1.2 μm; 6⩽1.5 μm; 7⩽1.75 μm; 8⩽2.2 μm. Download figure Download PowerPoint Careful monitoring of both the spindle (tubulin) and Brn1 staining showed that Brn1 redistributed from a compact to a diffuse staining pattern in the final stages of mitosis (late telophase). This occurred first in daughter cells (73% daughter first, n=50; Figure 1B) when the extended mitotic spindle was still intact (56% intact spindle; Figure 1B). In the remaining 44% of the cells, spindle disassembly was just starting as Brn1 became diffuse (see arrowheads Figure 1B). Time-lapse microscopy further confirmed that Brn1 redistribution occurred first in daughter cell nuclei (see d, Figure 1C), suggesting that the unloading of Condensin in wt cells occurs before or coincident with spindle disassembly, and before establishment of the short G1-phase aster. The lack of coordination between mother and daughter nuclei suggests that decompaction may be controlled by local modifications, and not by a general cell-cycle 'timer'. To correlate the Brn1 binding with the compaction status of the rDNA chromatin, we tagged each end of the approximately 200 rDNA repeats with an array of lacO or tetO sites in a strain-expressing fusions of LacI-CFP and TetR-YFP (Figure 1F). Using 3D confocal microscopy (Schober et al, 2008), we monitored the end-to-end distances separating the two fluorescence tags in space (r in nm). Earlier work has shown that the chromatin fibre can be modelled as a flexible polymer chain using parameters described by the Perod–Kratky formula (Figure 1F; Kratky and Porod, 1994; Bystricky et al, 2004). In this equation, the spatial distance r that separates the two points on the polymer is a function of the persistence length (or stiffness, Lp) of the fibre and the linear mass density of the chromatin chain (in bp/nm). These parameters are not separable, as the more compact the chromatin fibre is, the stiffer it becomes, yielding a larger and less variable point-to-point separation for the two sites along the flexible fibre. In brief, the more condensed the local chromatin structure becomes, the less frequently the two distant points along the fibre will come into contact, leading to a larger mean separation in 3D (scheme Figure 1F, bottom). To correlate changes in compaction (r values) with Brn1 binding, we compared wt with the net1-1 mutant, in which the net1-1 protein fails to inhibit the Cdc14 phosphatase. In these cells, Brn1 staining remains condensed in G1 and is particularly compact in S-phase cells (Figure 1F, see arrowheads; Supplementary Figure S2). We measured the end-to-end distances for markers at the extremities of the rDNA In wt G1- and S-phase cells, which contain dispersed Brn1. Values ranged from 500 to 1500 nm, showing the broad variation typical for a flexible fibre. In net1-1 cells, particularly in S phase, end-to-end distances were concentrated between 1500 and 1750 nm, consistent with the compact Brn1 staining (bar 7, Figure 1G). Similarly, for wt cells in G2/M and early anaphase, the end-to-end distances spanning the rDNA were larger and less variable, suggesting that mitotic rDNA fibre increases its persistence length, that is, becomes stiffer. Thus, coincident with the binding of Brn1, the mitotic rDNA chromatin fibre becomes compact and stiff, reflecting an increase in mass density (more nucleosomes per μm). This resembles mitotic condensation events in mammalian chromosomes (Hirano, 2005). We can clearly distinguish the staining of Brn1-labelled rDNA from that of genomic DNA in mitosis (see histone H3Ser10 phosphorylation; Figure 1E) and from that of DNA topoisomerase II (TopoII; Supplementary Figure S1E). This is reminiscent of observations made for Condensin and TopoII in mammalian chromosomes (Maeshima and Laemmli, 2003). We note that in yeast H3Ser10-P is not required for chromatin compaction (Lavoie et al, 2002), whereas the role of TopoII in yeast is still unclear (D'Amours et al, 2004; Sullivan et al, 2004; D'Ambrosio et al, 2008). Given that TopoII does not bind the string-like spiral of mitotic rDNA, whereas Brn1 does, we conclude that this Condensin subunit is the better marker for rDNA compaction. Brn1 is stable throughout the cell cycle To understand what triggers Brn1 release and rDNA decompaction, we asked whether Brn1 would undergo cleavage like Scc1, the 'kleisin' counterpart in Cohesin (Schleiffer et al, 2003). Alternatively cell-cycle-dependent modifications of Brn1 might coincide with its relocalization from the rDNA fibre, although Brn1, unlike Barren in higher eukaryotes, does not contain SP/TP consenses for Cdk modification. A western blot for Brn1-13myc on samples taken as cells traverse the cell cycle showed that Brn1 protein levels do not vary significantly through the cell cycle, making it unlikely that its release is mediated by degradation (Figure 2A and B). When low percentage gels were run, we note a ladder of larger bands in mitosis, each one representing a shift of Brn1 by 10–20 kDa (see 60 min, Figure 2B). This modification coincided with the presence of metaphase spindles (1.5–3 μm in length) and disappeared as cells progressed through mitosis to anaphase and telophase (spindle length, 3–10 μm). In a comprehensive analysis of SUMO-conjugated proteins in yeast (Denison et al, 2005), it was shown that Brn1 is sumoylated. We assume, therefore, that the mitosis-specific retardation observed here reflects Brn1 sumoylation, a modification reported for other Condensin subunits as well (D'Amours et al, 2004). However, given that only a fraction of Brn1 is modified during metaphase and that desumoylation does not correlate with rDNA decompaction, SUMO seems unlikely to regulate Brn1 binding and/or release. Figure 2.Brn1 is stable during mitosis and precipitates Lte1. (A) Western blot for Brn1-13myc in protein extracts from GA-1656. Samples were taken at the indicated time points after release from α-factor arrest. Blotting for a cytoplasmic p42 RNase serves as a loading control and the graph below shows the budding index and the level of Brn1 relative to the control. (B) As A, except that a 6% polyacrylamide gel was used to resolve larger forms of Brn1. Loading control was Mcm2. The graphs below show cell-cycle phases based on cell morphology at the given time points. Synchrony is lost by 135 min. (C) Silver-stained gel of anti-Myc IP from mitotic extracts of an untagged control strain (GA-180, labelled) or the same strain carrying endogenous Brn1-13myc (GA-1656). Dots indicate specific Brn1-precipitated proteins. This was repeated four times with similar results. The proteins (indicated to right) identified by MALDI ToF mass spectrometry were confirmed by at least five peptides. The specificity of the anti-Myc blot for Brn1-13myc was confirmed (see western blot, right panel). (D) Western blot analysis after chromatin fractionation of nuclear extracts from strain GA-2975. WCE=whole cell extract; Sup=soluble fraction; Chr=chromatin fraction. Download figure Download PowerPoint Brn1 associates with Lte1 To get a handle on other proteins that might control Condensin's association with chromatin, we next looked for Brn1-interacting partners in mitotic cell extracts. Reciprocal immunoprecipitation (IP) with Smc1, a subunit of Cohesin, and Smc2, a subunit of Condensin, confirmed that Brn1-13myc is indeed part of the Condensin and not the Cohesin complex, although we find that Smc2 and Smc4 are generally less soluble than Brn1 in detergent-lysed cell extracts (data not shown). By IP we recovered Brn1-13myc from the soluble fraction of lysed mitotic spheroplasts and we identified the co-precipitating factors by mass spectroscopy. The cells used for this experiment were blocked in mitosis with nocodazole, which generally yielded approximately 70% G2/M (G2/metaphase) and 25% anaphase cells. A similarly arrested mitotic extract from cells lacking the 13-Myc tag were used as a control. Four high-molecular-weight bands, ranging from 116 to 205 kDa, were selectively recovered from the tagged-Brn1 extract (Figure 2C). Peptide analysis by MALDI-TOF mass spectrometry showed that bands 3 matched the known Condensin subunit Ycs4, and band 4 was Brn1 itself. As expected from fractionation studies, Smc2 and Smc4 were not recovered in the soluble fraction. Novel Brn1-interacting factors included Spa2, a protein involved in polarized growth, Stu1, which stabilizes the mitotic spindle (Higuchi and Uhlmann, 2005), and Chd1, a chromodomain ATPase that is part of SAGA and helps regulate rDNA transcription (http://db.yeastgenome.org). Finally, the fourth protein recovered in significant levels was the MEN-regulatory factor, Lte1 (Low temperature essential 1). Lte1 localizes to the bud cortex for most of the cell cycle, yet in late mitosis and G1 it redistributes throughout the cell (Supplementary Figure S3E; Bardin et al, 2000; Jensen et al, 2002; Seshan et al, 2002). Given that Lte1 was recovered with Brn1-13myc in mitotic extracts, we asked whether Lte1 could also be recovered with the chromatin fraction after spheroplast lysis. Indeed, after chromatin fractionation, we recovered a nuclear subpool of Lte1 that, like Brn1, was chromatin bound (Figure 2D). We nonetheless are unable to tell whether the interaction between Brn1 and Lte1 is direct, as reciprocal IP was unsuccessful. This failure may stem from the instability of Lte1 in cell extracts. Loss of Lte1 delays decompaction of the rDNA The possibility of crosstalk between MEN and Condensin prompted us to examine Brn1 localization and rDNA decompaction in a lte1 deletion strain. LTE1 is not an essential gene, but its deletion renders cells cold sensitive for growth and leads to an anaphase arrest at 14°C (Shirayama et al, 1994). Wild-type (wt) and lte1 deletion cells expressing Brn1-GFP and CFP-Tub1 were synchronized in G1 at 30°C and released at either 30°C or at the semipermissive temperature, 16°C. At 16°C, progression through anaphase and telophase is slower in both wt and mutant cells (Figure 3A and B), allowing us to carefully monitor the timing of Brn1 release. We scored cell-cycle stage by the presence of a bud and the length of the spindle, which is extended in telophase. In cells containing only short microtubule staining (the G1 aster), we scored whether Brn1 was compact (compact, Figure 3C) or dispersed in the nucleoplasm (diffuse, Figure 3C). Figure 3.rDNA decompaction is delayed in the lte1 mutant. (A) Cells were arrested in G1 with α-factor and released for strains GA-3263 (BRN1-GFP, CFP-TUB1; wt ▪) and GA-3042 (lte1Δ, BRN1-GFP, CFP-TUB1; lte1Δ □) at the indicated temperatures. Cells were fixed and analysed by confocal microscopy for the indicated phenotypes. 100 cells were scored for each genotype and condition. Panels are labelled 1–8 to facilitate textual reference. (B) Protein extracts were prepared from the same experiment as in A at 16°C. Western blot analysis was performed on all samples for Clb2, and for Mcm2 as a loading control. (C) Brn1-GFP is scored by live microscopy as compact or diffuse in 100 G1-aster containing cells of strains GA-3263 and GA-3042 at the indicated temperatures. Micrographs show representative images of Brn1 fluorescence. (D) Strains GA-3263, GA-3042, and GA-4864 (BRN1-GFP, CFP-TUB1, lte1Δ bub2Δ) were arrested in G1 with α-factor at 30°C and released at 14°C. Samples were taken at indicated times. Mitotic arrest is scored by the number of dumbbell-shaped cells. In cells with G1-phase asters (no matter what the cellular morphology), Brn1 staining was scored as compact or diffuse (c or dif). This frequency is plotted relative to the entire cell population, so that the sum of Brn1-c and Brn1-dif does not equal 100%. Download figure Download PowerPoint As shown above, in wt cells Brn1 becomes diffuse in daughter nuclei in late telophase just as the spindle disassembles (Figure 1B). At 30°C, the lte1 defect for MEN activation is fully compensated by other pathways or by Tem1 activation (Shirayama et al, 1994), and, therefore, we scored no significant accumulation of telophase spindle structures (Figure 3A, panel 2). Nonetheless, we detected compact Brn1 labelling in approximately 15% of the cells bearing G1-phase asters, indicating inefficient Brn1 unloading, a state that persisted for 2 h (Figure 3A, panel 3). In contrast, the coincidence of compact Brn1 with G1 asters was highly transient in wt cells at 30°C, being lost by 100 min (Figure 3A, panel 3, no spindle). This suggested that there might be a loss of coordination between Brn1 release and spindle disassembly in lte1Δ cells. At semipermissive temperature (16°C), the loss of coordination between Brn1 unloading and spindle disassembly is sharply aggravated in lte1Δ cells. Although disassembly of the telophase spindle was complete by 250 min, disappearing with almost wt kinetics, compact Brn1 staining was seen to coincide with G1 asters in over 30% of the lte1 cells (Figure 3C, panel 7). This value was 8% in wt cells at this temperature. Given that 16°C is semipermissive for the lte1 strain, Brn1 does eventually become dispersed, although even at 250 min, 10% of the lte1 cells retained compact Brn1 staining (Figure 3A, panel 7). We conclude that the release of Brn1 is inefficient in the lte1 mutant, being delayed by 30–45 min relative to spindle disassembly. A delay can also be detected for the complete degradation of Clb2, which occurs in wt cells by 205 min, but at 250 min in lte1 cells (Figure 3B). To eliminate possible artefacts due to low temperature, we monitored Brn1 release in wt and lte1 mutant cells at a higher semipermissive temperature. At 25°C, we also found that Brn1-GFP was present in a compact structure together with G1 asters in 38% of the mutant cells, which compares with 3.6% in a wt culture under identical conditions (Figure 3C). Again the absence of Lte1 delayed Brn1 release from the rDNA, even though the switch from an anaphase spindle to G1 aster was affected only slightly (16°C) or not at all (30°C). We conclude that Lte1-controlled events either help coordinate rDNA decompaction with spindle disassembly or directly trigger decompaction by facilitating Brn1 release. MEN pathway is upstream of Brn1 release At the top of the MEN cascade, the GTPase Tem1 is negatively regulated by an inhibitory GAP, Bub2, whereas it is positively regulated by Lte1. To see whether the lte1 defect illustrated in Figure 3 correlates with impaired activation of the MEN pathway by Tem1, we tested whether we can suppress the delay in Brn1 release by restoring Tem1 activation indirectly through bub2 deletion (Bardin et al, 2000; Pereira et al, 2000; Wang et al, 2000; Adames et al, 2001; Lee et al, 2001; Geymonat et al, 2002). The effects were monitored by comparing the frequency with which compact Brn1 and small G1 asters coincide in wt, lte1 and double lte1 bub2 mutants, at the restrictive temperature for lte1Δ (14°C). Cultures were grown at 30°C, blocked in G1 with α-factor and released into precooled media at 14°C. Samples were collected hourly from 4.5 to 6.5 h after release, as within this window full Brn1 release is observed in wt cells (Figure 3D). We scored the abundance of dumbbell (large budded) cells, the disappearance of which signals progression into G1 phase (Figure 3D). As expected, at fully restrictive temperature nearly 80% of the lte1 mutant cells remain dumbbell shaped at 6.5 h, whereas both wt and lte1 bub2 double mutant strains progress into the next cell cycle and show unbudded or budded single cells. The coincidence of compact Brn1 with G1 asters is seen rarely in both wt and lte1 bub2 cells, confirming that Brn1 is usually released by the time that cells bear G1 asters (right hand columns, Figure 3D). However, as seen at semipermissive temperatures, some lte1 cells broke through the late telophase block, and these retained compact Brn1 staining despite the disassembly of the anaphase spindle morphology (Figure 3D). This argues that bub2 deletion largely suppresses the lte1 defect for Brn1 relocalization, as for other events of mitotic exit. We conclude that the MEN pathway has a role in controlling rDNA decompaction. Mutants of the MEN pathway delay chromatin