Journal of Science 113, 1903-1912 (2000) 1903 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1165

Centromere clustering is a major determinant of yeast interphase nuclear organization

Quan-wen Jin, Jörg Fuchs and Josef Loidl* Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria *Author for correspondence (e-mail: [email protected])

Accepted 17 March; published on WWW 10 May 2000

SUMMARY

During interphase in the budding yeast, Saccharomyces interphase. Unlike the Rabl-orientation known from many cerevisiae, centromeres are clustered near one pole of the higher eukaryotes, centromere clustering in yeast is not nucleus as a rosette with the spindle pole body at its hub. only a relic of anaphase chromosome polarization, because Opposite to the centromeric pole is the . it can be reconstituted without the passage of cells through Chromosome arms extend outwards from the centromeric anaphase. Within the rosette, homologous centromeres are pole and are preferentially directed towards the opposite not arranged in a particular order that would suggest pole. Centromere clustering is reduced by the ndc10 somatic pairing or genome separation. mutation, which affects a kinetochore protein, and by the poison nocodazole. This suggests that clustering is actively maintained or enforced by the Key words: Yeast, Nuclear architecture, Chromosome arrangement, association of centromeres with throughout Interphase, Centromere, SPB, Nucleolus, Saccharomyces cerevisiae

INTRODUCTION the remaining nuclear space or whether they occupy more centromere-distant areas corresponding to increasing It is now becoming widely accepted that interphase nuclei chromosome arm lengths, as classical Rabl-orientation would possess a high degree of spatial organization (see, e.g., Gilson predict. It is also of interest, whether the centromere clustering et al., 1993; Lamond and Earnshaw, 1998; Marshall et al., observed in budding yeast, which has an intranuclear mitotic 1997a). Order is dictated both by functional needs related to division, occurs by the same mechanism, i.e. anaphase interphase metabolism and by spatial constraints. Several polarization, as Rabl-orientation in most higher eukaryotes. facets of nonrandom chromosome positioning have been Apart from being a mere mechanical consequence of recognized. Among them are the association of transcription- anaphase chromosome movement, the parallel centromere- and replication-active chromosomal sites in foci, the existence telomere orientation of chromosomes could serve a functional of distinct individual chromosome domains, the preferential role. For example, it has been proposed that Rabl-orientation is localization of chromosome ends at the periphery of the a major factor contributing to the vicinity (and possible nucleus and the sequestering of ribosomal DNA tracts to interaction) of homologous chromosome regions by assigning separate compartments, namely the nucleoli. Also, nonrandom them positions at the same latitude with respect to the relative positioning of the two parental sets of chromosomes in centromeric pole within the nucleus (Jin et al., 1998). It had also diploids (like somatic pairing or genome separation) was been suggested that this specific arrangement of chromosomes observed, but not all aspects of it are understood. In many at interphase could persist into meiotic prophase and facilitate eukaryotes, the so-called Rabl-orientation of chromosomes meiotic homologous alignment (Fussell, 1987; Loidl, 1990; prevails at interphase (for reviews see, e.g., Fussell, 1987; Zickler and Kleckner, 1998). Here we have applied fluorescence Dong and Jiang, 1998; Jin et al., 1998; Zickler and Kleckner, in situ hybridization (FISH) to centromeres and other 1998). There, as a relic of anaphase movement, centromeres chromosome regions in combination with immunostaining of cluster near one pole of the nucleus whereas chromosome arms the spindle pole body and microtubules to study the nonrandom are arranged more or less in parallel and extend toward the interphase arrangement of chromosome arms in the budding opposite pole. For the budding yeast it has been shown yeast and its causes and consequences. previously that centromeres are clustered in a region near the spindle-pole body throughout interphase and that telomeres reside outside the centromere cluster (Goh and Kilmartin, MATERIALS AND METHODS 1993; Guacci et al., 1997a; Hayashi et al., 1998; Jin et al., 1998). However, it remains to be demonstrated, whether the Yeast strains and growth conditions outer chromosomal regions are distributed at random within The yeast strains used in this paper are listed in Table 1. For most 1904 Q.-W. Jin, J. Fuchs and J. Loidl

Table 1. Yeast strains used in this study Name/number Relevant genotype Source/reference SK1 MATa/α, HO/HO Kane and Roth (1974) NKY857 MATa, ho::LYS2, lys2, leu2::hisG, his4X, ura3 N. Kleckner DK4533-7-2 MATα, cdc23-1(Ts−), leu2, ade2, ade3, can1, sap3, ura1, his7, gal1 D. Koshland #872 MATa/α, cdc23-1(Ts−)/cdc23-1(Ts−) This study; derived from DK4533-7-2 by 5× backcross to NKY857 4202-15-3a MATa, ade2-1(ochre), his4-580 (amber), lys2-(ochre), trp1 (amber), tyr1 (ochre), L. Hartwell SUP4-3 (ts amber suppressor), bar1-1 JK418 MATα, ho, ura3, leu2, TRP1, ndc10-1(Ts−) Goh and Kilmartin (1993)

experiments, the diploid strain SK1 (Kane and Roth, 1974) was used. Immunostaining Strain NK857, a haploid derivative of SK1, was kindly provided by Specific labelling of the spindle pole body (SPB) was obtained with N. Kleckner (Harvard University, Boston, MA). Strains DK4533-7-2 − − polyclonal rabbit anti-Spc72p antibody (Knop and Schiebel, 1998; (cdc23-1 Ts ), 4202-15-3a (bar1-1) and JK418 (ndc10-1 Ts ) were kindly provided by E. Schiebel, Beatson Institute of Cancer Research, gifts from D. Koshland (Carnegie Institution of Washington, Glasgow, UK). Microtubules and the SPB were immunolabelled with Baltimore, MD), L. Hartwell (Fred Hutchinson Cancer Res. Ctr., YOL1/34 monoclonal rat anti-yeast antibody (Kilmartin et al., Seattle, WA), and J. V. Kilmartin (MRC, Cambridge, UK), 1982; purchased from Serotec, Kidlington, UK) according to a respectively. For mitotic and meiotic growth conditions see Jin et al. standard protocol (see, e.g., Pringle et al., 1991). Slides were washed (1998). twice for 5 minutes in 1× PBS (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4, pH 7.5), excessive liquid was drained and the slides Cell preparation were incubated with a drop of primary antibody (diluted 1:200 in 1× Cells were prepared in three different ways. For good preservation of PBS) under a coverslip at 4°C overnight. After two 5 minute washes their outer shapes and astral microtubules, cells were fixed and in 1× PBS, slides were incubated with FITC- or TRITC-conjugated permeabilized conventionally (procedure A). For some FISH secondary antibody for 90 minutes at room temperature. The slides applications, cells were treated with a detergent prior to fixation were then washed 2 × 5 minutes in 1× PBS and mounted under a (procedure B) to exploit the enhanced cytological resolution offered coverslip in Vectashield anti-fading medium (Vector Laboratories by spreading (Jin et al., 1998). Since this treatment tends to disrupt Inc., Burlingame, CA) supplemented with 0.5 µg/ml DAPI (4´6- cells, we applied another method where nuclei are moderately spread diamidino-2-phenylindole) as DNA-specific counterstain. by application of a detergent after the fixation treatment. This semi- Images of immunostained cells were taken and their coordinates spreading (procedure C) is a good compromise for obtaining a good were recorded. For subsequent FISH, the coverslip was rinsed off with spatial resolution of nuclear contents and a reasonable maintenance 1× PBS, cells were postfixed for 5-10 minutes in paraformaldehyde of cell integrity. fixative (see above) and then subjected to the standard FISH procedure For procedures A and C, 5-ml samples were taken from cultures (see below). In most cases, immunostaining was retained after FISH. with a density of ~1×107 cells/ml and formaldehyde was added to a In some instances, however, it had faded and FISH images had to be final concentration of 4%. Fixation took place at room temperature merged electronically with the corresponding images previously taken for 30 to 60 minutes. Cells were washed with 2% KAc and collected from the same coordinates of immunostained nuclei (Loidl et al., by centrifugation (2000 rpm for 4 minutes). The pellet was 1998b). resuspended in 500 µl 2% KAc, and 10 µl 0.5 M dithiotreitol and 14 µl of a Zymolyase 100T (Seikagaku Co., Tokyo) stock solution (10 Fluorescence in situ hybridization (FISH) mg/ml) were added. Digestion was performed for 20 minutes at 37°C. The complete set of centromeres was highlighted by FISH with a pan- After digestion, cells were washed with 2% KAc and recovered in 100 centromeric probe (Jin et al., 1998). rDNA repeats were labelled with to 150 µl of the same medium. This suspension was stored on ice (for a probe against fungal 25S rDNA (Scherthan et al., 1992). FISH up to 1 day) until used for the preparation of the slides. probes for painting the left arm of chromosome IV (IVL) and for a For procedure A, slides were polylysine-coated (0.1% solution) to region on XIIR were produced by PCR using the Expand Long reduce the loss of cells, whereas for procedures B and C after Template PCR System (Boehringer Mannheim GmbH, Mannheim, spreading, cells readily stick to the slides due to their larger surface. Germany) according to the manufacturer’s instructions. Appropriate 20 µl of the cell suspension were dropped onto a slide, spread out primers were selected from the Saccharomyces Genome Database evenly on its surface with the help of a glass rod and left for 2-3 (Cherry et al., 1998). Care was taken to amplify only regions without minutes under humid conditions. For immunostaining, slides were major repeated DNA elements. For template sizes of around 8 kb we processed further without allowing the suspension to dry out applied the following conditions: 2 minutes at 94°C; 10 cycles with completely. 10 seconds at 94°C, 30 seconds at 58°C, 6.5 minutes at 68°C; 20 For procedure C, 20 µl of a cell suspension produced as above were cycles with 10 seconds at 94°C, 30 seconds at 58°C, 6.5 minutes at put on a slide and mixed with 4-fold amounts of both detergent (1% 68°C with a prolongation of 20 seconds per cycle and a final extension aqueous solution of Lipsol; LIP Ltd, Shipley, UK) and fixative (4% of 7 minutes. The amplified PCR products were purified using the paraformaldehyde and 3.4% sucrose in distilled water). The mixture EluQuick-System (Schleicher & Schuell, Dassel, Germany). was then spread out with a glass rod and left to solidify in a chemical For various loci on the left arm of chromosome III, the right arm hood. The addition of sucrose has the advantage that the mixture is of chromosome IV and the left arm of chromosome VII, cosmid or λ hygroscopic and does not dry out completely. Therefore these clones #70884, #71013, #70884 and #70779 from the American Type preparations can be used for immunostaining even after storage for Culture Collection (ATCC; Gaithersburg, MD) were used as several days in the refrigerator (see Loidl et al., 1998a). hybridization probes. The chromosomal localization of these and the For procedure B, unfixed cells which had been spheroplasted with other FISH probes used are shown in Fig. 1. Zymolyase, were mixed with detergent and fixative on a slide as The probes were labelled by nick translation with Biotin-21-dUTP described above. For details of this spreading protocol see, e.g., Loidl (Clontech Laboratories Inc., Palo Alto, CA), Cy3-dUTP (red), Cy5- et al. (1991, 1998a). dUTP (far-red; Amersham, Little Chalfont, England) or Fluorescein- Interphase chromosome arrangement 1905

I 100 kb Fig. 1. Map of chromosome-specific FISH probes used. White ovals denote centromeres; dark boxes denote probes. IV Probes and chromosomal loci are referred to in the text by the chromosome number and VII their location on the left (L) or right (R) arm. RDN denotes the rDNA tandem repeat XII (not drawn to scale). Not shown are the RDN probes constituting the pan-centromeric probe-pool. XVI

12-dUTP (green; Boehringer Mannheim) as described elsewhere cells was as high as 96%. 20% of cells contained bipolar (Loidl et al., 1998a). The centromeric probes were pooled before spindles of various lengths which were located entirely within labelling as well as those for painting the left arm of chromosome IV. the mother cell. All but one of these cells (97%) had their Labelled probes were dissolved in hybridization solution (50% × µ µ centromeres assembled in either one or two clusters. When the formamide, 10% dextran sulfate, 2 SSC, 1 g/ l salmon sperm mitotic spindle exceeded the length of ~3 µm (i.e. around the DNA) to a final concentration of approximately 30 ng/µl. After 5 minutes of denaturation at 95°C the probes were dropped onto slides, onset of ), sister centromeres split and two separate denatured for 10 minutes at 90°C and hybridized for 12 to 48 hours centromere clusters could be discriminated probably due to the at 37°C. Posthybridization washes were performed in 50% splitting of sister centromeres. 8% of cells showed a long formamide/2× SSC (37°C), 2× SSC (37°C) and 1× SSC (room mitotic spindle passing through the bud neck, which is temperature) for 5 minutes each. Biotinylated probes were detected commonly referred to as anaphase. All of these cells showed with FITC-conjugated avidin (green; Sigma, St Louis, MO). Some centromeres clustered near the two spindle poles. Therefore, probes were labelled with a mixture of Biotin-21-dUTP and Cy3- centromeres seem to travel at the leading edges of the growing dUTP and produced an orange signal. spindle during mitosis (see also Straight et al., 1997) and Microscopy and evaluation remain clustered and closely attached to the spindle poles even after degradation of the mitotic spindle, upon entry into Preparations were examined with a Zeiss Axioskop or a Zeiss Axioplan II epifluorescence microscope equipped with the interphase. appropriate filter sets for the excitation of blue, green, red and far-red Centromeres form a rosette with the spindle pole fluorescence. Black and white images were captured separately for body inside each emission wavelength with a cooled CCD camera (Photometrics Ltd, Tucson, AZ), pseudocolored and merged to produce multicolor A high proportion of interphase (i.e. monopolar spindle-stage) FISH and immunofluorescence images. FISH and immunostaining nuclei with clustered centromeres (>50% in some series of were considered as successful if >80% of nuclei in a preparation preparations) showed a circular arrangement of centromeres showed all of the expected colors. Slides which did not meet this both in spread (procedure B, see Materials and Methods, Fig. criterion were excluded from evaluation. Cells to be evaluated were 2i) and semispread (procedure C) preparations (Fig. 2e; see preselected on the basis of their undisrupted appearance in DAPI. also Jin et al., 1998). The space inside the centromere rosette Evaluation was performed on stored electronic images. Centromeres showed reduced DAPI-staining, possibly due to lower were classified as clustered if signals were fused in a single patch or chromatin density (Fig. 2g). Immunostaining with an antibody ring, or scattered over no more than about 30% of the nuclear area. against the SPB revealed that the SPB was located in the center Observer bias was eliminated by blind evaluation of encoded slides of the rosette in 27 out of 36 nuclei (75%; Fig. 2h) and in 6 by two persons independently. Measurements of distances were performed onscreen using tools of the IPLab image processing and nuclei it was inside but not in the middle of the centromere analysis software (Scanalytics, Fairfax, VA). Distances between FISH ring. Only in 3 nuclei was the SPB outside the rosette. From signals were measured from center to center. The angular separation this we conclude that in living nuclei the SPB is located at the between FISH signals within centromere rosettes was determined by hub of the rosette. An area slightly larger than the SPB was drawing an angle between the centers of the signals, with the reference highlighted with YOL1/34 monoclonal antibody against the α point at the center of the rosette. subunit of tubulin under conditions (procedure B) which disrupt astral microtubules (Fig. 2i). This is in accordance with the reported binding of this antibody to microtubules attached RESULTS to the SPB (Kilmartin et al., 1982).

Centromere clustering and the mitotic spindle Centromere clustering is enforced by the attachment To establish the relationship of centromeres to the spindle poles of centromeres to microtubules throughout all stages of the , we performed Centromere clustering is only slightly reduced when nuclei are simultaneous immunostaining of microtubules and FISH to arrested at interphase for an extended period. A bar1 strain centromeres (pancentromeric FISH) in a logarithmically (which is highly sensitive to yeast mating pheromone; see growing culture of SK1. 281 cells from a conventional Barkai et al., 1998) was arrested in G1 (95% unbudded cells, preparation (procedure A) were analyzed (Fig. 2a-d). 72% of n=200) by exposure to α-factor (15 µg/ml; for experimental cells showed a single microtubule aster and no sign of a bipolar details see also Jin et al., 1998) and showed only a weak spindle, which identified them as being in G1 to early S phase reduction of clustering from 82% to 72% after 3 hours (Fig. (Byers and Goetsch, 1975). Centromere clustering in these 3), possibly by diffusion of the chromosomes (see, e.g., 1906 Q.-W. Jin, J. Fuchs and J. Loidl

Fig. 2. FISH and immunostaining of nuclei of the strain SK1 to show the arrangement of chromosomes and their relationship to elements of the during mitosis and at interphase (all cells except in (k) are diploid, (k) shows the haploid derivative NKY857). (a-d) Nuclei at different stages of mitosis (preparation according to procedure A), showing that centromeres (red) are clustered throughout the whole cell cycle and in permanently close contact with the spindle poles (green, anti-tubulin staining). (a) Early to mid interphase with monopolar spindle. (b) Beginning of the separation of the spindle poles. (c) Metaphase. (d) Late mitosis (anaphase); centromeres have reached opposite poles of the daughter nuclei. (e) Ring shaped centromere cluster in mitotic interphase; (f) dispersed centromeres in early meiosis (e and f by procedure C, FISH with pan-centromeric probe). (g) A DAPI stained interphase nucleus with a small dark spot on one side and a larger dark area on the opposite side. The dark spot (arrow) corresponds to the region inside the centromere ring. (This was apparent from pan-centromeric FISH on this particular nucleus, which is not shown here for better discrimination of the dull region from the surrounding chromatin.) The larger area is the nucleolus which is largely devoid of DNA. The nucleolus organizing regions on chromosome XII can be seen as two threads (arrowheads) inside the nucleolus (procedure B). (h) Nucleus with SPB inside the centromere ring (procedure C, simultaneous immunostaining with anti-tubulin antibody (orange due to the use of a mixture of FITC- and TRITC-conjugated secondary antibodies), and FISH with pan-centromeric probe (red)). (i) Nucleus with centromere cluster and SPB near one pole and RDN tracts inside the nucleolus at the opposite pole. Chromosome arms XIIR can be seen to loop back at the nucleolus organizing region (RDN) (same preparation and staining as in h but FISH with additional rDNA probe (green)). (j) Ring-shaped centromere cluster (red) and rDNA tracts (arrowheads) inside the nucleolus occupy opposite poles of the nucleus. A region distal to RDN on chromosomes XIIR (arrows) is located in the zone between the nucleolus and the centromere cluster due to the U-turn of the rDNA tracts (procedure B). (k) ‘Painting’ of chromosome arm IVL (procedure C). The intercalary probes (green) are mostly flanked by the centromere- and telomere-near probes (red and orange, respectively), which suggests a linear arrangement of the chromosome arm. Rarely, it appears sharply bent. (l) Polarized arrangement of chromosomes at interphase. A distal probe on IVR (green) shows a larger intranuclear distance from the centromeres (red) than a more proximal probe (orange) on the same arm (procedure B). White dots denote the likely courses of chromosome arms. (m) Examples for highlighting specific centromeres (centromeres XVI – green) within centromere rosettes. The angular separation of homologous centromeres was measured (Fig. 6). The large green area in (j) and (l) is the nucleolus, unspecifically stained by secondary antibody. Bars: in l and m, 1 µm in (a-l) and (m), respectively.

Marshall et al., 1997b). Thus, during the short ~90 minutes Interphase clustering of the centromeres could be due to interval between mitoses under normal growth conditions, their passive persistence at the positions they occupied at diffusion should not produce a notable loss of centromere telophase, even after degradation of the mitotic spindle in the clustering. Only in cultures which had been kept under absence of a disruptive force. Alternatively, it could be actively stationary conditions for 24 hours or longer, was centromere maintained by the permanent association of centromeres with clustering found to be reduced to between 30 and 40% (Jin et components of the spindle apparatus or the nuclear scaffold al., 1998). (see Marshall and Sedat, 1999). We performed experiments to Interphase chromosome arrangement 1907

a We also treated logarithmic cultures of the strain SK1 with 100 nocodazole, an inhibitor of microtubule polymerization, at a 90 concentration of 15 µg/ml. The effect of nocodazole treatment 80 was less pronounced than that of the ndc10 mutation. On 70 average, we found a reduction in the number of nuclei with 60 clustered centromeres from over 80% to 40-50% after 60 to 90 50 wildtype (37°C) minutes exposure (Fig. 3b). Similarly, Guacci et al. (1997a) 40 ndc10 (37°C) and Marshall et al. (1997b) had observed an increase of 30 distances between centromeres III in nocodazole-treated cells.

% of CEN clustering 20 Thus, from the partial disruption of clusters by ndc10 and nocodazole we conclude that interphase centromere clustering 10 is not due to the passive persistence of the anaphase 0 configuration (which would only be possible in the absence of 03060 90 120 150 180 min diffusional motion – see Discussion), but that it is enforced by the active maintenance of a SPB-kinetochore connection via b microtubules. 100 90 Centromere clustering is independent of anaphase 80 chromosome polarization 70 We wanted to test whether the formation of centromere clusters 60 requires the mitotic spindle or if it can be brought about by the 50 action of intranuclear microtubules which are present during 40 interphase. To this end we destroyed centromere clusters and 30 observed under which conditions they reform. We took

% of CEN clustering Nocodazole 20 advantage of the rapid and highly synchronous resolution of alpha factor the centromere clusters, which occurs due to a major 10 reorganization of the nucleus at meiotic prophase (Fig. 2f; Jin 0 0 30 60 90 120 et al., 1998; Trelles-Sticken et al., 1999). min The strain SK1 was transferred to sporulation medium and samples were taken at regular intervals to determine the time Fig. 3. Loss of centromere clustering induced by the temperature- when centromere clustering was lowest. Cultures which had sensitive ndc10-1 mutation and by the micrutubule poison nocodazole. (a) exponentially growing cultures were transferred from reached that point were transferred to rich medium which permissive temperature (23°C) to restrictive temperature (37°C) and causes them to leave the meiotic pathway and to return to aliquots were taken at the indicated time-points. A rapid reduction of mitotic growth (RTG; see, e.g., Zenvirth et al., 1997). centromere clustering was found both in spread (procedure B) and Centromere clustering was found to reappear around 60 semispread (procedure C) ndc10-1 nuclei. Clustering remained high minutes after RTG (Fig. 4a). Phase contrast microscopy and in wild-type nuclei which indicates that the effect is not due to immunostaining of microtubules from the same samples increased temperature per se. (b) Also nocodazole-treated (15 µg/ml) revealed that large buds and long bipolar anaphase spindles nuclei showed a clear reduction in centromere clustering. Cells of a reappeared about 30 minutes later (Fig. 4a), suggesting that bar1 mutant MATa strain were arrested at interphase (G1) by centromere clustering occurs prior to anaphase. (A few meiotic α exposure to -factor (see Results) and showed only minimal nuclei were also present due to meioses occurring in reduction of centromere clustering over the same period. Thus, centromere clusters do not simply disperse in the absence of the presporulation medium and to cells which failed to return to mitotic spindle, but the dispersal is due to the ndc10-1 mutation and the mitotic cycle. However, their frequency was too low (<5%) the nocodazole treatment. Each experiment was performed three to explain the observed centromere clustering by meiotic times and 100 nuclei were scored for each time-point in each run. divisions.) To define the temporal relationship between centromere clustering and spindle formation more precisely, we used a temperature-sensitive cdc23 mutant. In this mutant the cell see if the loss of microtubules or of the microtubule- cycle is arrested at metaphase because the sister chromatids kinetochore association has an influence on centromere cannot be separated due to a defect in the anaphase promoting clustering. In one experiment, we shifted the temperature- complex (Irniger et al., 1995). Although cdc23 mutant cells do sensitive mutant ndc10-1 (Goh and Kilmartin, 1993), which is not do well in meiosis even at the permissive temperature defective in a component of the kinetochore, to restrictive (23°C), between 40 and 55% of the nuclei lost centromere temperature (37°C). This resulted in a very strong disruption clustering upon transfer to sporulation medium. Centromere of centromere clustering. Whereas ~70% of nuclei had clusters reappeared after transfer to rich medium at the clustered centromeres at 23°C, centromere clustering was restrictive temperature (37°C; Fig. 4b). After only 1 hour upon reduced to ~20% after 1 hour at 37°C and to ~10% after 3 hours RTG, centromere clustering reached almost premeiotic levels. (Fig. 3). Shift of a wild-type strain to 37°C did not notably Although the meiosis-induced reduction in centromere reduce centromere clustering. This confirms that the effect clustering was low (due to the poor performance of cdc23 cells observed in the ndc10-1 mutant was not caused by the elevated in sporulation medium), this reduction and reappearance of temperature per se (Fig. 3a). centromere clustering was consistent in 3 independent 1908 Q.-W. Jin, J. Fuchs and J. Loidl

a Fig. 4. Centromere clustering upon return to growth (RTG). Cell 100 samples at various timepoints before and after RTG were drawn and centromere clustering 90 examined for frequency of centromere clustering. Centromere 80 clustering was high during premeiotic growth and decreased due to RTG 70 cells entering meiotic prophase when grown on sporulation medium (SPM). When centromere clustering reached a minimum level (at a 60 stage corresponding to zygotene/pachytene of meiotic prophase; see 50 Jin et al., 1998) cells were transferred from sporulation medium to 40 full growth medium (YPD). The point of transfer is labelled as RTG. 30 Soon after RTG, centromere clustering increased again. In the upper 20 panel of (a) three experiments with the strain SK1 are shown. Centromere clustering (according to our definition in Materials and

% of cells of % 10 SPM YPD Methods) occurred after a ~60 minute lag following RTG. However, even earlier a tendency for centromeres to assemble was apparent. 50 long bipolar spindles (a) lower panel: Aliquots from the culture were checked in parallel 40 for the presence of long bipolar (anaphase) spindles as a measure for 30 the resumption of mitoses upon RTG. Anaphase spindles occurred in 20 20-30% of cells growing in presporulation medium, dropped to 10 below 5% after RTG and reached frequencies of up to 40% ~150 minutes after RTG (no centromere clustering values shown for the

-3,5h 0 15 30 45 60 90 120 150 late time point). Thus, centromere clusters seem to reappear ~30-60 minutes prior to anaphases. (b) 3 experiments with the temperature- min sensitive cell cycle mutant cdc23 are shown. The meiotic decrease of b centromere clustering was slower and less prominent in the mutant. 80 For several hours at 37°C after RTG, mitoses did not occur at a RTG notable frequency (not shown) but centromere clustering reached premeiotic levels after 1 hour in all three experiments. In all 70 experiments, 100 nuclei were scored for each time point.

60 Yang et al., 1989; Guacci et al., 1997a; Oakes et al., 1998). The reduced DAPI staining of the nucleolus suggests that it 50 contains little DNA; only thin DAPI-positive threads were SPM YPD sometimes visible within this area (Fig. 2g). FISH with a probe % of CEN clustering 40 23°C 37°C against rDNA highlighted relatively strongly condensed tracts of RDN repeats inside the nucleolus (Fig. 2i,j). Other chromosomal regions seem to be excluded from it (see also -7 -6 -5 -4 -3 -2 -1 0 1 234 5 Guacci et al., 1994). The ca. 1000 kb long RDN array on hrs chromosome XIIR, which comprises the rDNA repeats, begins ~300 kb away from the centromere (see Cherry et al., 1998). experiments. To dismiss the possibility that mutant cells had This physical distance of 300 kb between centromere XII and escaped from arrest, we examined aliquots of the cultures after RDN is sufficient to span the intranuclear distance between the RTG for the presence of anaphase spindles and elongated centromeric pole and the nucleolus. In diploid nuclei, the two anaphase nuclei in anti-tubulin and DAPI stained nuclei. No RDN arrays sometimes appear separate (Fig. 2g,i) and at other anaphase spindles were found in 500 nuclei, and only 18% of times associated (Fig. 2j). The occasional association is either the cells had elongated nuclei. But strikingly, even those had due to their joint formation of a nucleolus or to somatic pairing their centromeres organized in a single cluster without any of the rDNA sequences (see below). Quite often, the RDN tract sign of beginning anaphase separation. This indicates that within the nucleolus appears loop-shaped (Fig. 2i) suggesting metaphase arrest had occurred in the mutant. Thus, we that the chromosome arm XIIR is folded back toward the conclude that the poleward movement or the assembly of centromeric pole (see also Guacci et al., 1994). Simultaneous centromeres at the poles of dividing nuclei during hybridization to centromeres, rDNA and a locus distal to RDN anaphase/telophase is not required for centromere clustering. confirmed that the distal portion of XIIR occupies the region Rather, it occurs prior to or concomitantly with the formation between the centromeres and the nucleolus (Fig. 2j). of the bipolar spindle. Since the U-turn of XIIR may be an exception because of the presence of the nucleolus, we checked the orientation of A relaxed centromere-telomere polarization prevails the more typical 450 kb chromosome arm IVL by in interphase nuclei ‘chromosome painting’. For that purpose, a mixture of ten We asked if chromosome arrangement in yeast interphase PCR-amplified DNA fragments with a length between 8 kb and nuclei bears the signs of Rabl-orientation, i.e. the ± straight 10 kb was used as a hybridization probe (see Fig. 1). The orientation of chromosome arms from the centromeric to the probes closest to the centromere and the telomere were labelled opposite pole. In DAPI stained nuclei, the nucleolus appeared with Cy3-dUTP and Cy5-dUTP, respectively, whereas all as a weakly stained area opposite the dark spot that marks the interstitial loci were labelled with fluorescein-dUTP. The region surrounding the SPB, confirming that centromeres and specificity of the probes was verified in spread and semispread nucleolus mark two opposite poles in the yeast nucleus (see pachytene nuclei (not shown) where individual bivalents Interphase chromosome arrangement 1909 appear as rods with compact painting signals (Loidl et al., a C C 1995). In interphase nuclei, a continuous hybridization signal I or the maximum number of eight interstitial signals was rarely D I observed, which may be due to differential condensation N I D along the chromosomes or the occasional failure of the small N I individual probes to generate a detectable signal. Nevertheless, N D the array of signals allowed the course of the chromosome arm N to be determined. In 25 out of 36 (69%) semispread nuclei (procedure C) from logarithmically growing cultures, the D interstitial FISH signals were between the centromeric and the telomeric signals, although not normally in a straight line. b This suggests that, while a general centromere-telomere 3 polarization prevails, the chromosome arms follow a more or less meandering path (Fig. 2k). In the remaining 31% of nuclei, the centromeric and telomeric FISH signals were interspersed 2 among the interstitial ones. Since this arrangement of signals I may also result if a nucleus is viewed from the top (from the D I centromeric or opposite pole), it is estimated that less than 30% N of nuclei fail to exhibit centromere-telomere polarization of 1 chromosome arm IVL. To quantify centromere-telomere polarization of chromosome arms, we measured the intranuclear distances between loci at the centromeres and at different physical 0 020406080100 distances from the centromere. In the case of a random I nuclei ranked according to increasing D course of chromosome arms, no significant differences in IN the intranuclear centromere-telomere distances should be observed, whereas a more linear orientation (with or without Fig. 5. Test for the polarized orientation of chromosome arms by the meandering of the interstitial DNA) would result in a positive demonstration that intranuclear distances between FISH labelled loci µ correlation of physical and intranuclear distances (Fig. 5a). (in m) increase with their physical distances (in kb) on chromosome Multicolor FISH with probes specific for a region including the arms. (a) Principle of the experiment. Intranuclear distances (I) between the centromere (C) and a centromere-near locus (N) were centromere of chromosome IV and for regions 273 kb and 1041 compared to the distance between the centromere and a distant locus kb (Fig. 1) away on its long arm was carried out (Fig. 2l). In (D). Measured distances ID > IN indicate that a chromosome arm 75% of nuclei, intranuclear distances from the centromere were shows a ± polarized orientation, whereas ID < IN indicate that it folds larger for the distal than for the proximal locus (Fig. 5b). back. (b) Ratios of intranuclear distances from the centromere of However, in only 10% of nuclei the distal locus had a more physically distant (1041 kb) and physically close (273 kb) loci on than twice as large intranuclear distance from the centromere IVR. For y-values >1 the locus with the larger physical distance from than the proximal locus, although its physical distance from the the centromere was also found to have a larger intranuclear distance. centromere is about four times as large. This suggests that the This was the case in 75% of the nuclei. proximal part of the chromosome arm points away from the centromere more directly, whereas the distal part tends to loop genomes. (The separation of parental chromosomes into two back or to meander. haploid sets on opposite sides of prometaphase rosettes has been claimed to occur in human fibroblasts and HeLa cells The arrangement of homologous centromeres (Nagele et al., 1995) but was disputed recently (Allison and within the rosette Nestor, 1999).) However, our observation of a random To detect a possible nonrandom arrangement of homologous distribution of homologous centromeres within the rosette does chromosomes within the centromere rosettes of diploid not rule out the possibility that regions at more distal positions interphase nuclei (see above), centromeres of chromosomes I, of chromosome arms show preferential homologous IV, VIII and XVI were labelled differentially from the rest of associations (Keeney and Kleckner, 1996; Kleckner, 1998; centromeres (Fig. 2m), and the angular separation between Burgess et al., 1999). homologs was determined. Angles between 0° (signals fused) and 180° (signals positioned on opposite sides of the rosette) were measured (see Materials and Methods) in 50 nuclei for DISCUSSION each of the four chromosomes. As can be seen from Fig. 6, angles between 0° and 180° were present at approximately Chromosome arrangement at interphase equal frequencies. 108 of the 200 homologous pairs of Here we describe the highly organized spatial distribution of centromeres were separated by an angle of less than 90°, which chromosomes in interphase nuclei of the budding yeast. Its is close to the 50% that would be expected if there is no most prominent feature is the clustering of centromeres. Goh preferential location of two homologous centromeres either in and Kilmartin (1993), who immunostained Ndc10p, a putative the same or the opposite halves of the ring (Fig. 6). This result kinetochore protein, found a label mostly in the region of the refutes both, somatic pairing of centromeric or centromere- SPB, and proposed that centromeres may be attached to near chromosome regions and separation of the parental intranuclear microtubules and cluster near the SPB even in 1910 Q.-W. Jin, J. Fuchs and J. Loidl

absence of Rabl-orientation (for references see Jin et al., 1998). Here we have shown for yeast that if centromere clustering is disrupted experimentally, it is reconstituted even in the absence of anaphase. This reformation of clusters seems to be executed by intranuclear microtubules which are present throughout interphase (see above). Also the sensitivity of clustering to a 180 mutant Ndc10 centromeric protein and to the spindle poison probe Cen16 nocodazole supports the existence of a microtubule-dependent 160 probe Cen1-near process which stabilizes centromere clusters throughout 140 probe Cen4 interphase. In the absence of a stabilizing force, centromere probe Cen8 clusters would be probably disrupted by Brownian motion 120 inside the nucleus. However, Marshall et al. (1997b) observed only limited diffusion of centromere-near loci, and they 100 suggested that it was constrained by elements of the . This active maintenance of centromere clustering 80 would suggest that it serves a function perhaps in the context

angle (degrees) of a general higher organization within the yeast nucleus. It is 60 possible that centromere clustering is functionally equivalent to the prometaphase congression of centromeres at the cellular 40 equator, which occurs in higher eukaryotes. In the classical Rabl-orientation, centromere clustering is 20 accompanied by the largely parallel orientation of chromosome arms which is the consequence of the trailing of the arms at 0 01020304050anaphase. Since in yeast centromere clustering is brought about (at least in part) by a different mechanism, it was of interest to number of centromere rosettes see how arms are oriented relative to the centromeres. Fig. 6. Arrangement of homologous centromeres within centromere Although we found a general centromere-telomere polarization rosettes. Angular separation of homologous centromeres was (Fig. 2k), chromosome arms seem to meander and to loop back measured in 50 nuclei with well-expressed, circular rosettes (see occasionally. This is particularly true for chromosome arm examples in Fig. 2m) for each of the chromosomes I, IV, VII and XVI. XIIR which carries the rDNA repeats. The RDN array occupies All angles between 0° and 180° are represented at roughly the same a position ~300 kb away from the centromere. Distal to it there frequency. 108 of 200 homologous centromere-pairs (i.e. 54%) are is another 610 kb of chromosome XII DNA. Since the separated by angles smaller than 90° and therefore occupy the same nucleolus occupies a region opposite to the centromere cluster, half of the rosette. Thus, there is no recognizable tendency towards a preferential vicinity of homologs within the rosette. Inserts show the RDN array forms a U-turn within the nucleolus, and the schematized examples of rosettes with angles drawn between distal parts of chromosome XIIR are located in the zone homologous centromeres. between the centromeric and the nucleolar pole of the nucleus (Fig. 2j; see also Guacci et al., 1994, 1997a,b). Thus, because of its different mode of origin and a relatively relaxed interphase. This centromere clustering was confirmed by centromere-telomere polarization, we prefer to designate the Guacchi et al. (1997a) and Jin et al. (1998) using FISH observed interphase chromosome arrangement in yeast as labelling of centromeres. Rabl-like arrangement. The circular arrangement of centromeres which we report We found that if there is no or only a short bipolar spindle here, can also be observed in intact nuclei (see also Goh and (<3 µm) present, centromeres form a single cluster. It splits in Kilmartin, 1993; Jin et al., 1998). It may be due to the two when the spindle lengthens (Fig. 2a-d). This is in presence of a core bundle of ‘continuous’ microtubules which accordance with Straight et al. (1997) who observed in living is surrounded by a shell of kinetochore microtubules (Winey cells a sudden separation of sister centromeres when the et al., 1995). Therefore, it may be imagined that centromeres spindle was between 2.5 and 3 µm long. Centromeres then form a rosette around this microtubule bundle, and that performed anaphase A movement towards the poles in less than chromosome arms are displaced from the interior of the 26 seconds. The shortness of this stage and probably a highly nucleus and confined to the peripheral domain during mitosis. synchronous behaviour of all centromeres could explain why Since intranuclear microtubules are present throughout the we did not observe a notable number of nuclei with whole cell cycle (Byers and Goetsch, 1975), this arrangement centromeres scattered between the two spindle poles. On the is possibly maintained in interphase nuclei (Murray and other hand, Guacci et al. (1997a) found that in cdc20 and cdc23 Szostak, 1985; Goh and Kilmartin, 1993; Guacci et al., mutant cells arrested in metaphase, centromeres were dispersed 1997a). and detached from the spindle poles. It is therefore possible Whilst the classical Rabl-orientation is believed to be a that also in unarrested metaphase centromeres transiently consequence of the anaphase movement of the centromeres oscillate along the short bipolar spindle in the course of initial toward the spindle poles (Fussell, 1987; Dong and Jiang, 1998; orientation. Whereas a limited number of individual Zickler and Kleckner, 1998), it does not seem to be the only centromeres may be seen to be widely separated from each means by which centromeres cluster. In several animals some other (Guacci et al., 1997a), the entirety of centromeres may degree of centromere clustering has been observed in the still form a cluster of about 2 µm in diameter, as in our case. Interphase chromosome arrangement 1911

The question of somatic pairing* reinforcement of the Rabl-orientation because centromeres and The Rabl-like orientation has an effect on the colocalization of telomeres switch positions with respect to the microtubule homologous chromosome regions in diploids since, with organizing center. Thus, the clustering switch is a major reference to the centromeric pole, homologous regions will be nuclear restructuring event during which chromosomes turn found at similar latitudes of the nucleus. In addition to this around by 180 degrees. Even so, Rabl-orientation could effect, Keeney and Kleckner (1996), Kleckner (1998) and provide a nonrandom chromosome disposition which Burgess et al. (1999) have reported a small preference for facilitates bouquet formation (Fussell, 1987) or some other somatic pairing. However, in our analysis of the relative aspect of meiotic nuclear reorganization (Loidl, 1990). distribution of homologous centromeres within the centromere rosette we found no tendency of a preferential arrangement. It We are indebted to John Kilmartin, Nancy Kleckner, Doug is highly improbable that the order of centromeres within the Koshland, Lee Hartwell and Elmar Schiebel for providing strains and antibodies. We thank Dieter Schweizer and Franz Klein for valuable circle is upset by the spreading exerted during preparation, suggestions and Andrew Murray for critical reading of the manuscript. therefore we can safely assume that the observed lack of an This work was supported by grant no. S8202-GEN from the Austrian ordered arrangement reflects the natural state. 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