1903
Journal of Cell Science 113, 1903-1912 (2000)
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
cerevisiae, centromeres are clustered near one pole of the
nucleus as a rosette with the spindle pole body at its hub.
Opposite to the centromeric pole is the nucleolus.
Chromosome arms extend outwards from the centromeric
pole and are preferentially directed towards the opposite
pole. Centromere clustering is reduced by the ndc10
mutation, which affects a kinetochore protein, and by
the microtubule poison nocodazole. This suggests that
clustering is actively maintained or enforced by the
association of centromeres with microtubules throughout
interphase. Unlike the Rabl-orientation known from many
higher eukaryotes, centromere clustering in yeast is not
only a relic of anaphase chromosome polarization, because
it can be reconstituted without the passage of cells through
anaphase. Within the rosette, homologous centromeres are
not arranged in a particular order that would suggest
somatic pairing or genome separation.
INTRODUCTION
the remaining nuclear space or whether they occupy more
centromere-distant areas corresponding to increasing
chromosome arm lengths, as classical Rabl-orientation would
predict. It is also of interest, whether the centromere clustering
observed in budding yeast, which has an intranuclear mitotic
division, occurs by the same mechanism, i.e. anaphase
polarization, as Rabl-orientation in most higher eukaryotes.
Apart from being a mere mechanical consequence of
anaphase chromosome movement, the parallel centromeretelomere orientation of chromosomes could serve a functional
role. For example, it has been proposed that Rabl-orientation is
a major factor contributing to the vicinity (and possible
interaction) of homologous chromosome regions by assigning
them positions at the same latitude with respect to the
centromeric pole within the nucleus (Jin et al., 1998). It had also
been suggested that this specific arrangement of chromosomes
at interphase could persist into meiotic prophase and facilitate
meiotic homologous alignment (Fussell, 1987; Loidl, 1990;
Zickler and Kleckner, 1998). Here we have applied fluorescence
in situ hybridization (FISH) to centromeres and other
chromosome regions in combination with immunostaining of
the spindle pole body and microtubules to study the nonrandom
interphase arrangement of chromosome arms in the budding
yeast and its causes and consequences.
It is now becoming widely accepted that interphase nuclei
possess a high degree of spatial organization (see, e.g., Gilson
et al., 1993; Lamond and Earnshaw, 1998; Marshall et al.,
1997a). Order is dictated both by functional needs related to
interphase metabolism and by spatial constraints. Several
facets of nonrandom chromosome positioning have been
recognized. Among them are the association of transcriptionand replication-active chromosomal sites in foci, the existence
of distinct individual chromosome domains, the preferential
localization of chromosome ends at the periphery of the
nucleus and the sequestering of ribosomal DNA tracts to
separate compartments, namely the nucleoli. Also, nonrandom
relative positioning of the two parental sets of chromosomes in
diploids (like somatic pairing or genome separation) was
observed, but not all aspects of it are understood. In many
eukaryotes, the so-called Rabl-orientation of chromosomes
prevails at interphase (for reviews see, e.g., Fussell, 1987;
Dong and Jiang, 1998; Jin et al., 1998; Zickler and Kleckner,
1998). There, as a relic of anaphase movement, centromeres
cluster near one pole of the nucleus whereas chromosome arms
are arranged more or less in parallel and extend toward the
opposite pole. For the budding yeast it has been shown
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,
1993; Guacci et al., 1997a; Hayashi et al., 1998; Jin et al.,
1998). However, it remains to be demonstrated, whether the
outer chromosomal regions are distributed at random within
Key words: Yeast, Nuclear architecture, Chromosome arrangement,
Interphase, Centromere, SPB, Nucleolus, Saccharomyces cerevisiae
MATERIALS AND METHODS
Yeast strains and growth conditions
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
SK1
NKY857
DK4533-7-2
#872
MATa/α, HO/HO
MATa, ho::LYS2, lys2, leu2::hisG, his4X, ura3
MATα, cdc23-1(Ts−), leu2, ade2, ade3, can1, sap3, ura1, his7, gal1
MATa/α, cdc23-1(Ts−)/cdc23-1(Ts−)
4202-15-3a
MATa, ade2-1(ochre), his4-580 (amber), lys2-(ochre), trp1 (amber), tyr1 (ochre),
SUP4-3 (ts amber suppressor), bar1-1
MATα, ho, ura3, leu2, TRP1, ndc10-1(Ts−)
JK418
experiments, the diploid strain SK1 (Kane and Roth, 1974) was used.
Strain NK857, a haploid derivative of SK1, was kindly provided by
N. Kleckner (Harvard University, Boston, MA). Strains DK4533-7-2
(cdc23-1 Ts−), 4202-15-3a (bar1-1) and JK418 (ndc10-1 Ts−) were
gifts from D. Koshland (Carnegie Institution of Washington,
Baltimore, MD), L. Hartwell (Fred Hutchinson Cancer Res. Ctr.,
Seattle, WA), and J. V. Kilmartin (MRC, Cambridge, UK),
respectively. For mitotic and meiotic growth conditions see Jin et al.
(1998).
Cell preparation
Cells were prepared in three different ways. For good preservation of
their outer shapes and astral microtubules, cells were fixed and
permeabilized conventionally (procedure A). For some FISH
applications, cells were treated with a detergent prior to fixation
(procedure B) to exploit the enhanced cytological resolution offered
by spreading (Jin et al., 1998). Since this treatment tends to disrupt
cells, we applied another method where nuclei are moderately spread
by application of a detergent after the fixation treatment. This semispreading (procedure C) is a good compromise for obtaining a good
spatial resolution of nuclear contents and a reasonable maintenance
of cell integrity.
For procedures A and C, 5-ml samples were taken from cultures
with a density of ~1×107 cells/ml and formaldehyde was added to a
final concentration of 4%. Fixation took place at room temperature
for 30 to 60 minutes. Cells were washed with 2% KAc and collected
by centrifugation (2000 rpm for 4 minutes). The pellet was
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
mg/ml) were added. Digestion was performed for 20 minutes at 37°C.
After digestion, cells were washed with 2% KAc and recovered in 100
to 150 µl of the same medium. This suspension was stored on ice (for
up to 1 day) until used for the preparation of the slides.
For procedure A, slides were polylysine-coated (0.1% solution) to
reduce the loss of cells, whereas for procedures B and C after
spreading, cells readily stick to the slides due to their larger surface.
20 µl of the cell suspension were dropped onto a slide, spread out
evenly on its surface with the help of a glass rod and left for 2-3
minutes under humid conditions. For immunostaining, slides were
processed further without allowing the suspension to dry out
completely.
For procedure C, 20 µl of a cell suspension produced as above were
put on a slide and mixed with 4-fold amounts of both detergent (1%
aqueous solution of Lipsol; LIP Ltd, Shipley, UK) and fixative (4%
paraformaldehyde and 3.4% sucrose in distilled water). The mixture
was then spread out with a glass rod and left to solidify in a chemical
hood. The addition of sucrose has the advantage that the mixture is
hygroscopic and does not dry out completely. Therefore these
preparations can be used for immunostaining even after storage for
several days in the refrigerator (see Loidl et al., 1998a).
For procedure B, unfixed cells which had been spheroplasted with
Zymolyase, were mixed with detergent and fixative on a slide as
described above. For details of this spreading protocol see, e.g., Loidl
et al. (1991, 1998a).
Source/reference
Kane and Roth (1974)
N. Kleckner
D. Koshland
This study; derived from
DK4533-7-2 by 5×
backcross to NKY857
L. Hartwell
Goh and Kilmartin (1993)
Immunostaining
Specific labelling of the spindle pole body (SPB) was obtained with
polyclonal rabbit anti-Spc72p antibody (Knop and Schiebel, 1998;
kindly provided by E. Schiebel, Beatson Institute of Cancer Research,
Glasgow, UK). Microtubules and the SPB were immunolabelled with
YOL1/34 monoclonal rat anti-yeast tubulin antibody (Kilmartin et al.,
1982; purchased from Serotec, Kidlington, UK) according to a
standard protocol (see, e.g., Pringle et al., 1991). Slides were washed
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
were incubated with a drop of primary antibody (diluted 1:200 in 1×
PBS) under a coverslip at 4°C overnight. After two 5 minute washes
in 1× PBS, slides were incubated with FITC- or TRITC-conjugated
secondary antibody for 90 minutes at room temperature. The slides
were then washed 2 × 5 minutes in 1× PBS and mounted under a
coverslip in Vectashield anti-fading medium (Vector Laboratories
Inc., Burlingame, CA) supplemented with 0.5 µg/ml DAPI (4´6diamidino-2-phenylindole) as DNA-specific counterstain.
Images of immunostained cells were taken and their coordinates
were recorded. For subsequent FISH, the coverslip was rinsed off with
1× PBS, cells were postfixed for 5-10 minutes in paraformaldehyde
fixative (see above) and then subjected to the standard FISH procedure
(see below). In most cases, immunostaining was retained after FISH.
In some instances, however, it had faded and FISH images had to be
merged electronically with the corresponding images previously taken
from the same coordinates of immunostained nuclei (Loidl et al.,
1998b).
Fluorescence in situ hybridization (FISH)
The complete set of centromeres was highlighted by FISH with a pancentromeric probe (Jin et al., 1998). rDNA repeats were labelled with
a probe against fungal 25S rDNA (Scherthan et al., 1992). FISH
probes for painting the left arm of chromosome IV (IVL) and for a
region on XIIR were produced by PCR using the Expand Long
Template PCR System (Boehringer Mannheim GmbH, Mannheim,
Germany) according to the manufacturer’s instructions. Appropriate
primers were selected from the Saccharomyces Genome Database
(Cherry et al., 1998). Care was taken to amplify only regions without
major repeated DNA elements. For template sizes of around 8 kb we
applied the following conditions: 2 minutes at 94°C; 10 cycles with
10 seconds at 94°C, 30 seconds at 58°C, 6.5 minutes at 68°C; 20
cycles with 10 seconds at 94°C, 30 seconds at 58°C, 6.5 minutes at
68°C with a prolongation of 20 seconds per cycle and a final extension
of 7 minutes. The amplified PCR products were purified using the
EluQuick-System (Schleicher & Schuell, Dassel, Germany).
For various loci on the left arm of chromosome III, the right arm
of chromosome IV and the left arm of chromosome VII, cosmid or λ
clones #70884, #71013, #70884 and #70779 from the American Type
Culture Collection (ATCC; Gaithersburg, MD) were used as
hybridization probes. The chromosomal localization of these and the
other FISH probes used are shown in Fig. 1.
The probes were labelled by nick translation with Biotin-21-dUTP
(Clontech Laboratories Inc., Palo Alto, CA), Cy3-dUTP (red), Cy5dUTP (far-red; Amersham, Little Chalfont, England) or Fluorescein-
Interphase chromosome arrangement 1905
Fig. 1. Map of chromosome-specific FISH
probes used. White ovals denote
centromeres; dark boxes denote probes.
Probes and chromosomal loci are referred to
in the text by the chromosome number and
their location on the left (L) or right (R)
arm. RDN denotes the rDNA tandem repeat
(not drawn to scale). Not shown are the
probes constituting the pan-centromeric
probe-pool.
100 kb
I
IV
VII
XII
RDN
XVI
12-dUTP (green; Boehringer Mannheim) as described elsewhere
(Loidl et al., 1998a). The centromeric probes were pooled before
labelling as well as those for painting the left arm of chromosome IV.
Labelled probes were dissolved in hybridization solution (50%
formamide, 10% dextran sulfate, 2× SSC, 1 µg/µl salmon sperm
DNA) to a final concentration of approximately 30 ng/µl. After 5
minutes of denaturation at 95°C the probes were dropped onto slides,
denatured for 10 minutes at 90°C and hybridized for 12 to 48 hours
at 37°C. Posthybridization washes were performed in 50%
formamide/2× SSC (37°C), 2× SSC (37°C) and 1× SSC (room
temperature) for 5 minutes each. Biotinylated probes were detected
with FITC-conjugated avidin (green; Sigma, St Louis, MO). Some
probes were labelled with a mixture of Biotin-21-dUTP and Cy3dUTP and produced an orange signal.
Microscopy and evaluation
Preparations were examined with a Zeiss Axioskop or a Zeiss
Axioplan II epifluorescence microscope equipped with the
appropriate filter sets for the excitation of blue, green, red and far-red
fluorescence. Black and white images were captured separately for
each emission wavelength with a cooled CCD camera (Photometrics
Ltd, Tucson, AZ), pseudocolored and merged to produce multicolor
FISH and immunofluorescence images. FISH and immunostaining
were considered as successful if >80% of nuclei in a preparation
showed all of the expected colors. Slides which did not meet this
criterion were excluded from evaluation. Cells to be evaluated were
preselected on the basis of their undisrupted appearance in DAPI.
Evaluation was performed on stored electronic images. Centromeres
were classified as clustered if signals were fused in a single patch or
ring, or scattered over no more than about 30% of the nuclear area.
Observer bias was eliminated by blind evaluation of encoded slides
by two persons independently. Measurements of distances were
performed onscreen using tools of the IPLab image processing and
analysis software (Scanalytics, Fairfax, VA). Distances between FISH
signals were measured from center to center. The angular separation
between FISH signals within centromere rosettes was determined by
drawing an angle between the centers of the signals, with the reference
point at the center of the rosette.
cells was as high as 96%. 20% of cells contained bipolar
spindles of various lengths which were located entirely within
the mother cell. All but one of these cells (97%) had their
centromeres assembled in either one or two clusters. When the
mitotic spindle exceeded the length of ~3 µm (i.e. around the
onset of mitosis), sister centromeres split and two separate
centromere clusters could be discriminated probably due to the
splitting of sister centromeres. 8% of cells showed a long
mitotic spindle passing through the bud neck, which is
commonly referred to as anaphase. All of these cells showed
centromeres clustered near the two spindle poles. Therefore,
centromeres seem to travel at the leading edges of the growing
spindle during mitosis (see also Straight et al., 1997) and
remain clustered and closely attached to the spindle poles even
after degradation of the mitotic spindle, upon entry into
interphase.
RESULTS
Centromeres form a rosette with the spindle pole
body inside
A high proportion of interphase (i.e. monopolar spindle-stage)
nuclei with clustered centromeres (>50% in some series of
preparations) showed a circular arrangement of centromeres
both in spread (procedure B, see Materials and Methods, Fig.
2i) and semispread (procedure C) preparations (Fig. 2e; see
also Jin et al., 1998). The space inside the centromere rosette
showed reduced DAPI-staining, possibly due to lower
chromatin density (Fig. 2g). Immunostaining with an antibody
against the SPB revealed that the SPB was located in the center
of the rosette in 27 out of 36 nuclei (75%; Fig. 2h) and in 6
nuclei it was inside but not in the middle of the centromere
ring. Only in 3 nuclei was the SPB outside the rosette. From
this we conclude that in living nuclei the SPB is located at the
hub of the rosette. An area slightly larger than the SPB was
highlighted with YOL1/34 monoclonal antibody against the α
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
to the SPB (Kilmartin et al., 1982).
Centromere clustering and the mitotic spindle
To establish the relationship of centromeres to the spindle poles
throughout all stages of the cell cycle, we performed
simultaneous immunostaining of microtubules and FISH to
centromeres (pancentromeric FISH) in a logarithmically
growing culture of SK1. 281 cells from a conventional
preparation (procedure A) were analyzed (Fig. 2a-d). 72% of
cells showed a single microtubule aster and no sign of a bipolar
spindle, which identified them as being in G1 to early S phase
(Byers and Goetsch, 1975). Centromere clustering in these
Centromere clustering is enforced by the attachment
of centromeres to microtubules
Centromere clustering is only slightly reduced when nuclei are
arrested at interphase for an extended period. A bar1 strain
(which is highly sensitive to yeast mating pheromone; see
Barkai et al., 1998) was arrested in G1 (95% unbudded cells,
n=200) by exposure to α-factor (15 µg/ml; for experimental
details see also Jin et al., 1998) and showed only a weak
reduction of clustering from 82% to 72% after 3 hours (Fig.
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 spindle
apparatus 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
interval between mitoses under normal growth conditions,
diffusion should not produce a notable loss of centromere
clustering. Only in cultures which had been kept under
stationary conditions for 24 hours or longer, was centromere
clustering found to be reduced to between 30 and 40% (Jin et
al., 1998).
Interphase clustering of the centromeres could be due to
their passive persistence at the positions they occupied at
telophase, even after degradation of the mitotic spindle in the
absence of a disruptive force. Alternatively, it could be actively
maintained by the permanent association of centromeres with
components of the spindle apparatus or the nuclear scaffold
(see Marshall and Sedat, 1999). We performed experiments to
Interphase chromosome arrangement 1907
a
100
90
% of CEN clustering
80
70
60
50
wildtype (37°C)
40
ndc10 (37°C)
30
20
10
0
0
30
60
90
120
150
180
min
b
100
% of CEN clustering
90
80
70
60
50
40
30
Nocodazole
20
alpha factor
10
0
0
30
60
90
120
min
Fig. 3. Loss of centromere clustering induced by the temperaturesensitive ndc10-1 mutation and by the micrutubule poison
nocodazole. (a) exponentially growing cultures were transferred from
permissive temperature (23°C) to restrictive temperature (37°C) and
aliquots were taken at the indicated time-points. A rapid reduction of
centromere clustering was found both in spread (procedure B) and
semispread (procedure C) ndc10-1 nuclei. Clustering remained high
in wild-type nuclei which indicates that the effect is not due to
increased temperature per se. (b) Also nocodazole-treated (15 µg/ml)
nuclei showed a clear reduction in centromere clustering. Cells of a
bar1 mutant MATa strain were arrested at interphase (G1) by
exposure to α-factor (see Results) and showed only minimal
reduction of centromere clustering over the same period. Thus,
centromere clusters do not simply disperse in the absence of the
mitotic spindle, but the dispersal is due to the ndc10-1 mutation and
the nocodazole treatment. Each experiment was performed three
times and 100 nuclei were scored for each time-point in each run.
see if the loss of microtubules or of the microtubulekinetochore association has an influence on centromere
clustering. In one experiment, we shifted the temperaturesensitive mutant ndc10-1 (Goh and Kilmartin, 1993), which is
defective in a component of the kinetochore, to restrictive
temperature (37°C). This resulted in a very strong disruption
of centromere clustering. Whereas ~70% of nuclei had
clustered centromeres at 23°C, centromere clustering was
reduced to ~20% after 1 hour at 37°C and to ~10% after 3 hours
(Fig. 3). Shift of a wild-type strain to 37°C did not notably
reduce centromere clustering. This confirms that the effect
observed in the ndc10-1 mutant was not caused by the elevated
temperature per se (Fig. 3a).
We also treated logarithmic cultures of the strain SK1 with
nocodazole, an inhibitor of microtubule polymerization, at a
concentration of 15 µg/ml. The effect of nocodazole treatment
was less pronounced than that of the ndc10 mutation. On
average, we found a reduction in the number of nuclei with
clustered centromeres from over 80% to 40-50% after 60 to 90
minutes exposure (Fig. 3b). Similarly, Guacci et al. (1997a)
and Marshall et al. (1997b) had observed an increase of
distances between centromeres III in nocodazole-treated cells.
Thus, from the partial disruption of clusters by ndc10 and
nocodazole we conclude that interphase centromere clustering
is not due to the passive persistence of the anaphase
configuration (which would only be possible in the absence of
diffusional motion – see Discussion), but that it is enforced by
the active maintenance of a SPB-kinetochore connection via
microtubules.
Centromere clustering is independent of anaphase
chromosome polarization
We wanted to test whether the formation of centromere clusters
requires the mitotic spindle or if it can be brought about by the
action of intranuclear microtubules which are present during
interphase. To this end we destroyed centromere clusters and
observed under which conditions they reform. We took
advantage of the rapid and highly synchronous resolution of
the centromere clusters, which occurs due to a major
reorganization of the nucleus at meiotic prophase (Fig. 2f; Jin
et al., 1998; Trelles-Sticken et al., 1999).
The strain SK1 was transferred to sporulation medium and
samples were taken at regular intervals to determine the time
when centromere clustering was lowest. Cultures which had
reached that point were transferred to rich medium which
causes them to leave the meiotic pathway and to return to
mitotic growth (RTG; see, e.g., Zenvirth et al., 1997).
Centromere clustering was found to reappear around 60
minutes after RTG (Fig. 4a). Phase contrast microscopy and
immunostaining of microtubules from the same samples
revealed that large buds and long bipolar anaphase spindles
reappeared about 30 minutes later (Fig. 4a), suggesting that
centromere clustering occurs prior to anaphase. (A few meiotic
nuclei were also present due to meioses occurring in
presporulation medium and to cells which failed to return to
the mitotic cycle. However, their frequency was too low (<5%)
to explain the observed centromere clustering by meiotic
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
cycle is arrested at metaphase because the sister chromatids
cannot be separated due to a defect in the anaphase promoting
complex (Irniger et al., 1995). Although cdc23 mutant cells do
not do well in meiosis even at the permissive temperature
(23°C), between 40 and 55% of the nuclei lost centromere
clustering upon transfer to sporulation medium. Centromere
clusters reappeared after transfer to rich medium at the
restrictive temperature (37°C; Fig. 4b). After only 1 hour upon
RTG, centromere clustering reached almost premeiotic levels.
Although the meiosis-induced reduction in centromere
clustering was low (due to the poor performance of cdc23 cells
in sporulation medium), this reduction and reappearance of
centromere clustering was consistent in 3 independent
1908 Q.-W. Jin, J. Fuchs and J. Loidl
a
100
centromere clustering
90
80
RTG
70
60
50
% of cells
40
30
20
10
SPM
YPD
50
long bipolar spindles
40
30
20
10
15
0
-3,5h
45
30
90
60
120
150
min
b
80
% of CEN clustering
RTG
70
60
50
SPM
23°C
40
-7
-6
-5
-4
-3
YPD
37°C
-2
-1
0
1
2
3
4
5
hrs
experiments. To dismiss the possibility that mutant cells had
escaped from arrest, we examined aliquots of the cultures after
RTG for the presence of anaphase spindles and elongated
anaphase nuclei in anti-tubulin and DAPI stained nuclei. No
anaphase spindles were found in 500 nuclei, and only 18% of
the cells had elongated nuclei. But strikingly, even those had
their centromeres organized in a single cluster without any
sign of beginning anaphase separation. This indicates that
metaphase arrest had occurred in the mutant. Thus, we
conclude that the poleward movement or the assembly of
centromeres at the poles of dividing nuclei during
anaphase/telophase is not required for centromere clustering.
Rather, it occurs prior to or concomitantly with the formation
of the bipolar spindle.
A relaxed centromere-telomere polarization prevails
in interphase nuclei
We asked if chromosome arrangement in yeast interphase
nuclei bears the signs of Rabl-orientation, i.e. the ± straight
orientation of chromosome arms from the centromeric to the
opposite pole. In DAPI stained nuclei, the nucleolus appeared
as a weakly stained area opposite the dark spot that marks the
region surrounding the SPB, confirming that centromeres and
nucleolus mark two opposite poles in the yeast nucleus (see
Fig. 4. Centromere clustering upon return to growth (RTG). Cell
samples at various timepoints before and after RTG were drawn and
examined for frequency of centromere clustering. Centromere
clustering was high during premeiotic growth and decreased due to
cells entering meiotic prophase when grown on sporulation medium
(SPM). When centromere clustering reached a minimum level (at a
stage corresponding to zygotene/pachytene of meiotic prophase; see
Jin et al., 1998) cells were transferred from sporulation medium to
full growth medium (YPD). The point of transfer is labelled as RTG.
Soon after RTG, centromere clustering increased again. In the upper
panel of (a) three experiments with the strain SK1 are shown.
Centromere clustering (according to our definition in Materials and
Methods) occurred after a ~60 minute lag following RTG. However,
even earlier a tendency for centromeres to assemble was apparent.
(a) lower panel: Aliquots from the culture were checked in parallel
for the presence of long bipolar (anaphase) spindles as a measure for
the resumption of mitoses upon RTG. Anaphase spindles occurred in
20-30% of cells growing in presporulation medium, dropped to
below 5% after RTG and reached frequencies of up to 40% ~150
minutes after RTG (no centromere clustering values shown for the
late time point). Thus, centromere clusters seem to reappear ~30-60
minutes prior to anaphases. (b) 3 experiments with the temperaturesensitive cell cycle mutant cdc23 are shown. The meiotic decrease of
centromere clustering was slower and less prominent in the mutant.
For several hours at 37°C after RTG, mitoses did not occur at a
notable frequency (not shown) but centromere clustering reached
premeiotic levels after 1 hour in all three experiments. In all
experiments, 100 nuclei were scored for each time point.
Yang et al., 1989; Guacci et al., 1997a; Oakes et al., 1998). The
reduced DAPI staining of the nucleolus suggests that it
contains little DNA; only thin DAPI-positive threads were
sometimes visible within this area (Fig. 2g). FISH with a probe
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
Guacci et al., 1994). The ca. 1000 kb long RDN array on
chromosome XIIR, which comprises the rDNA repeats, begins
~300 kb away from the centromere (see Cherry et al., 1998).
This physical distance of 300 kb between centromere XII and
RDN is sufficient to span the intranuclear distance between the
centromeric pole and the nucleolus. In diploid nuclei, the two
RDN arrays sometimes appear separate (Fig. 2g,i) and at other
times associated (Fig. 2j). The occasional association is either
due to their joint formation of a nucleolus or to somatic pairing
of the rDNA sequences (see below). Quite often, the RDN tract
within the nucleolus appears loop-shaped (Fig. 2i) suggesting
that the chromosome arm XIIR is folded back toward the
centromeric pole (see also Guacci et al., 1994). Simultaneous
hybridization to centromeres, rDNA and a locus distal to RDN
confirmed that the distal portion of XIIR occupies the region
between the centromeres and the nucleolus (Fig. 2j).
Since the U-turn of XIIR may be an exception because of
the presence of the nucleolus, we checked the orientation of
the more typical 450 kb chromosome arm IVL by
‘chromosome painting’. For that purpose, a mixture of ten
PCR-amplified DNA fragments with a length between 8 kb and
10 kb was used as a hybridization probe (see Fig. 1). The
probes closest to the centromere and the telomere were labelled
with Cy3-dUTP and Cy5-dUTP, respectively, whereas all
interstitial loci were labelled with fluorescein-dUTP. The
specificity of the probes was verified in spread and semispread
pachytene nuclei (not shown) where individual bivalents
Interphase chromosome arrangement 1909
appear as rods with compact painting signals (Loidl et al.,
1995). In interphase nuclei, a continuous hybridization signal
or the maximum number of eight interstitial signals was rarely
observed, which may be due to differential condensation
along the chromosomes or the occasional failure of the small
individual probes to generate a detectable signal. Nevertheless,
the array of signals allowed the course of the chromosome arm
to be determined. In 25 out of 36 (69%) semispread nuclei
(procedure C) from logarithmically growing cultures, the
interstitial FISH signals were between the centromeric and
the telomeric signals, although not normally in a straight line.
This suggests that, while a general centromere-telomere
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
among the interstitial ones. Since this arrangement of signals
may also result if a nucleus is viewed from the top (from the
centromeric or opposite pole), it is estimated that less than 30%
of nuclei fail to exhibit centromere-telomere polarization of
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
distances from the centromere. In the case of a random
course of chromosome arms, no significant differences in
the intranuclear centromere-telomere distances should be
observed, whereas a more linear orientation (with or without
meandering of the interstitial DNA) would result in a positive
correlation of physical and intranuclear distances (Fig. 5a).
Multicolor FISH with probes specific for a region including the
centromere of chromosome IV and for regions 273 kb and 1041
kb (Fig. 1) away on its long arm was carried out (Fig. 2l). In
75% of nuclei, intranuclear distances from the centromere were
larger for the distal than for the proximal locus (Fig. 5b).
However, in only 10% of nuclei the distal locus had a more
than twice as large intranuclear distance from the centromere
than the proximal locus, although its physical distance from the
centromere is about four times as large. This suggests that the
proximal part of the chromosome arm points away from the
centromere more directly, whereas the distal part tends to loop
back or to meander.
The arrangement of homologous centromeres
within the rosette
To detect a possible nonrandom arrangement of homologous
chromosomes within the centromere rosettes of diploid
interphase nuclei (see above), centromeres of chromosomes I,
IV, VIII and XVI were labelled differentially from the rest of
centromeres (Fig. 2m), and the angular separation between
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
each of the four chromosomes. As can be seen from Fig. 6,
angles between 0° and 180° were present at approximately
equal frequencies. 108 of the 200 homologous pairs of
centromeres were separated by an angle of less than 90°, which
is close to the 50% that would be expected if there is no
preferential location of two homologous centromeres either in
the same or the opposite halves of the ring (Fig. 6). This result
refutes both, somatic pairing of centromeric or centromerenear chromosome regions and separation of the parental
a
C
C
IN
ID
D
IN
ID
N
N
D
b
3
2
ID
IN
1
0
0
20
40
60
80
nuclei ranked according to increasing
100
ID
IN
Fig. 5. Test for the polarized orientation of chromosome arms by the
demonstration that intranuclear distances between FISH labelled loci
(in µm) increase with their physical distances (in kb) on chromosome
arms. (a) Principle of the experiment. Intranuclear distances (I)
between the centromere (C) and a centromere-near locus (N) were
compared to the distance between the centromere and a distant locus
(D). Measured distances ID > IN indicate that a chromosome arm
shows a ± polarized orientation, whereas ID < IN indicate that it folds
back. (b) Ratios of intranuclear distances from the centromere of
physically distant (1041 kb) and physically close (273 kb) loci on
IVR. For y-values >1 the locus with the larger physical distance from
the centromere was also found to have a larger intranuclear distance.
This was the case in 75% of the nuclei.
genomes. (The separation of parental chromosomes into two
haploid sets on opposite sides of prometaphase rosettes has
been claimed to occur in human fibroblasts and HeLa cells
(Nagele et al., 1995) but was disputed recently (Allison and
Nestor, 1999).) However, our observation of a random
distribution of homologous centromeres within the rosette does
not rule out the possibility that regions at more distal positions
of chromosome arms show preferential homologous
associations (Keeney and Kleckner, 1996; Kleckner, 1998;
Burgess et al., 1999).
DISCUSSION
Chromosome arrangement at interphase
Here we describe the highly organized spatial distribution of
chromosomes in interphase nuclei of the budding yeast. Its
most prominent feature is the clustering of centromeres. Goh
and Kilmartin (1993), who immunostained Ndc10p, a putative
kinetochore protein, found a label mostly in the region of the
SPB, and proposed that centromeres may be attached to
intranuclear microtubules and cluster near the SPB even in
1910 Q.-W. Jin, J. Fuchs and J. Loidl
180
probe Cen16
probe Cen1-near
probe Cen4
probe Cen8
160
angle (degrees)
140
120
100
80
60
40
20
0
0
10
20
30
40
50
number of centromere rosettes
Fig. 6. Arrangement of homologous centromeres within centromere
rosettes. Angular separation of homologous centromeres was
measured in 50 nuclei with well-expressed, circular rosettes (see
examples in Fig. 2m) for each of the chromosomes I, IV, VII and XVI.
All angles between 0° and 180° are represented at roughly the same
frequency. 108 of 200 homologous centromere-pairs (i.e. 54%) are
separated by angles smaller than 90° and therefore occupy the same
half of the rosette. Thus, there is no recognizable tendency towards a
preferential vicinity of homologs within the rosette. Inserts show
schematized examples of rosettes with angles drawn between
homologous centromeres.
interphase. This centromere clustering was confirmed by
Guacchi et al. (1997a) and Jin et al. (1998) using FISH
labelling of centromeres.
The circular arrangement of centromeres which we report
here, can also be observed in intact nuclei (see also Goh and
Kilmartin, 1993; Jin et al., 1998). It may be due to the
presence of a core bundle of ‘continuous’ microtubules which
is surrounded by a shell of kinetochore microtubules (Winey
et al., 1995). Therefore, it may be imagined that centromeres
form a rosette around this microtubule bundle, and that
chromosome arms are displaced from the interior of the
nucleus and confined to the peripheral domain during mitosis.
Since intranuclear microtubules are present throughout the
whole cell cycle (Byers and Goetsch, 1975), this arrangement
is possibly maintained in interphase nuclei (Murray and
Szostak, 1985; Goh and Kilmartin, 1993; Guacci et al.,
1997a).
Whilst the classical Rabl-orientation is believed to be a
consequence of the anaphase movement of the centromeres
toward the spindle poles (Fussell, 1987; Dong and Jiang, 1998;
Zickler and Kleckner, 1998), it does not seem to be the only
means by which centromeres cluster. In several animals some
degree of centromere clustering has been observed in the
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
mutant Ndc10 centromeric protein and to the spindle poison
nocodazole supports the existence of a microtubule-dependent
process which stabilizes centromere clusters throughout
interphase. In the absence of a stabilizing force, centromere
clusters would be probably disrupted by Brownian motion
inside the nucleus. However, Marshall et al. (1997b) observed
only limited diffusion of centromere-near loci, and they
suggested that it was constrained by elements of the
cytoskeleton. This active maintenance of centromere clustering
would suggest that it serves a function perhaps in the context
of a general higher organization within the yeast nucleus. It is
possible that centromere clustering is functionally equivalent
to the prometaphase congression of centromeres at the cellular
equator, which occurs in higher eukaryotes.
In the classical Rabl-orientation, centromere clustering is
accompanied by the largely parallel orientation of chromosome
arms which is the consequence of the trailing of the arms at
anaphase. Since in yeast centromere clustering is brought about
(at least in part) by a different mechanism, it was of interest to
see how arms are oriented relative to the centromeres.
Although we found a general centromere-telomere polarization
(Fig. 2k), chromosome arms seem to meander and to loop back
occasionally. This is particularly true for chromosome arm
XIIR which carries the rDNA repeats. The RDN array occupies
a position ~300 kb away from the centromere. Distal to it there
is another 610 kb of chromosome XII DNA. Since the
nucleolus occupies a region opposite to the centromere cluster,
the RDN array forms a U-turn within the nucleolus, and the
distal parts of chromosome XIIR are located in the zone
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
centromere-telomere polarization, we prefer to designate the
observed interphase chromosome arrangement in yeast as
Rabl-like arrangement.
We found that if there is no or only a short bipolar spindle
(<3 µm) present, centromeres form a single cluster. It splits in
two when the spindle lengthens (Fig. 2a-d). This is in
accordance with Straight et al. (1997) who observed in living
cells a sudden separation of sister centromeres when the
spindle was between 2.5 and 3 µm long. Centromeres then
performed anaphase A movement towards the poles in less than
26 seconds. The shortness of this stage and probably a highly
synchronous behaviour of all centromeres could explain why
we did not observe a notable number of nuclei with
centromeres scattered between the two spindle poles. On the
other hand, Guacci et al. (1997a) found that in cdc20 and cdc23
mutant cells arrested in metaphase, centromeres were dispersed
and detached from the spindle poles. It is therefore possible
that also in unarrested metaphase centromeres transiently
oscillate along the short bipolar spindle in the course of initial
orientation. Whereas a limited number of individual
centromeres may be seen to be widely separated from each
other (Guacci et al., 1997a), the entirety of centromeres may
still form a cluster of about 2 µm in diameter, as in our case.
Interphase chromosome arrangement 1911
The question of somatic pairing*
The Rabl-like orientation has an effect on the colocalization of
homologous chromosome regions in diploids since, with
reference to the centromeric pole, homologous regions will be
found at similar latitudes of the nucleus. In addition to this
effect, Keeney and Kleckner (1996), Kleckner (1998) and
Burgess et al. (1999) have reported a small preference for
somatic pairing. However, in our analysis of the relative
distribution of homologous centromeres within the centromere
rosette we found no tendency of a preferential arrangement. It
is highly improbable that the order of centromeres within the
circle is upset by the spreading exerted during preparation,
therefore we can safely assume that the observed lack of an
ordered arrangement reflects the natural state. In a previous
paper we have reported an experiment in which the distances
between the centromeres of chromosome IV were on average
slightly shorter than the distances between the centromere of
chromosome IV and a centromere-near region on chromosome
III (Fig. 3 in Jin et al., 1998). However, this slight difference
may have been caused by the fact that the probe on
chromosome III maps to a region ~30 kb from the centromere
and thus resides slightly outside the rosette, at a different
nuclear latitude.
The random arrangement of homologous centromeres which
we report here, does not preclude local homologous
interactions further down the chromosome arms. However, in
view of the relatively weak preference for homologous
colocalization (own unpublished results; Guacci et al., 1994;
Keeney and Kleckner, 1996; Kleckner, 1998; Burgess et al.,
1999), we are inclined to believe that it is not necessary to
invoke specific mechanisms for homologous interaction in
vegetative yeast cells. Chromosome sorting due to spatial
constraints, in addition to the Rabl-like organization, could
occur within the nucleus. For instance, not only distances from
the centromeres, but also the position of loci on long vs. short
arms could be crucial. Since telomeres are located at the
nuclear periphery (Klein et al., 1992; Gotta et al., 1996), loci
on short arms would be located near the nuclear surface,
whereas interstitial regions on long arms at a similar latitude
would tend to occupy the interior of the nucleus. This would
create a tendency of arms of similar lengths (and thus of
homologs) to colocalize.
The meiotic clustering switch: a discussion
We have shown previously that centromere clustering is
resolved in meiotic prophase (Jin et al., 1998). At around the
same time, telomeres cluster in a region near the SPB, causing
chromosomes to loop back on themselves and to form the socalled bouquet (Trelles-Sticken et al., 1999). This centromeretelomere clustering switch seems to be conserved between
organisms as diverge as higher plants (Schwarzacher, 1997;
Moore, 1998), Schizosaccharomyces pombe (Chikashige et al.,
1997), and the budding yeast. The Rabl-orientation or Rabllike orientation and the bouquet are similar in that both are
characterized by a roughly parallel arrangement of
chromosome arms between a centromeric and a telomeric
domain. However, as was pointed out by Zickler and Kleckner
(1988), the meiotic bouquet configuration is not a simple
*We follow the common use of ‘somatic pairing’ to designate pairing or association of
homologous chromosomes in vegetative (=non-meiotic) cells, although, strictly speaking,
yeast does not have somatic cells.
reinforcement of the Rabl-orientation because centromeres and
telomeres switch positions with respect to the microtubule
organizing center. Thus, the clustering switch is a major
nuclear restructuring event during which chromosomes turn
around by 180 degrees. Even so, Rabl-orientation could
provide a nonrandom chromosome disposition which
facilitates bouquet formation (Fussell, 1987) or some other
aspect of meiotic nuclear reorganization (Loidl, 1990).
We are indebted to John Kilmartin, Nancy Kleckner, Doug
Koshland, Lee Hartwell and Elmar Schiebel for providing strains and
antibodies. We thank Dieter Schweizer and Franz Klein for valuable
suggestions and Andrew Murray for critical reading of the manuscript.
This work was supported by grant no. S8202-GEN from the Austrian
Science Fund (F.W.F.).
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