Meiotic behaviours of chromosomes and microtubules
in budding yeast: relocalization of centromeres and
telomeres during meiotic prophase
Aki Hayashi1,2,a, Hideyuki Ogawa2,b, Kenji Kohno3, Susan M. Gasser4 and
Yasushi Hiraoka1,2,*
1
Kansai Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe, Japan
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan
3
Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara, Japan
4
Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland
2
Abstract
Background: Meiosis is a process of universal importance in eukaryotic organisms, generating variation
in the heritable haploid genome by recombination
and re-assortment of chromosomes. The intranuclear movement of chromosomes is expected to
achieve pairing and recombination of homologous
chromosomes during meiosis. Meiosis in the budding yeast Saccharomyces cerevisiae has been extensively studied, both genetically and by molecular
biology; here we report cytological observations of
meiotic chromosomal events in this organism.
Results: Using fluorescence microscopy, we have
examined the behaviour of chromosomes and
microtubules during meiosis in S. cerevisiae. We
first observed the dynamic behaviour of nuclei in
living cells using jellyfish green fluorescent protein
(GFP) fused with nucleoplasmin, a Xenopus oocyte
nuclear protein. The characterization of nuclear
movement in living cells was extended by an analysis
Introduction
Meiosis is a process of general importance in eukaryotic
organisms, generating variation in the heritable haploid
Communicated by: Masayuki Yamamoto
* Correspondence: Kansai Advanced Research Center,
Communications Research Laboratory, 588-2 Iwaoka,
Iwaoka-cho, Nishi-ku, Kobe, Japan; E-mail: yasushi
@crl.go.jp
Present addresses: aWhitehead Institute, Nine Cambridge,
MA02142, USA; bIwate College of Nursing, Ohgama,
Takizawa, Iwate 020-0151, Japan.
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of chromosomes and microtubules in fixed specimens. In addition, the nuclear localization of centromeres and telomeres was determined by indirect
immunofluorescence microscopy in synchronous
populations of meiotic cells. While telomeres
remain in clusters of 5–8 throughout meiosis,
centromeres change their nuclear localization
dramatically during the progression of meiosis:
centromeres are first clustered at a single site near
the spindle-pole body before the induction of
meiosis, and become scattered during the meiotic
prophase.
Conclusions: Our observations have demonstrated
that nuclear and cytoskeletal reorganization take
place with meiosis in S. cerevisiae. In particular,
the distinct relocalization of centromeres during
meiosis indicates a considerable movement of
chromosomes within the meiotic prophase nucleus.
genome by the recombination and re-assortment of
chromosomes. A large body of cytological observation
of meiotic chromosomal events has been accumulated,
primarily from studies in higher eukaryotes. During the
meiotic prophase, when pairing and recombination of
homologous chromosomes takes place, chromosomes
show a characteristic arrangement in which they are
bundled at the telomeres to form bouquet-like arrangements; this chromosome pattern has been observed in
a wide range of animals and plants, and is called
the ‘bouquet’ structure (reviewed in Fussel 1987; John
1990). Despite this study in a wide range of organisms,
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genetic control mechanisms for meiotic chromosomal
events have only been most extensively studied in the
budding yeast Saccharomyces cerevisiae, and a number of
mutants that are defective in meiosis have been isolated
(Hollingsworth et al. 1990; Rockmill & Roeder 1990;
Engebrecht et al. 1991; Malone et al. 1991; Menees et al.
1992; Ajimura et al. 1993). To understand meiosis, it is
important to combine cytological observations with
molecular biological analyses in a single organism. With
this goal in mind, cytological approaches have been
developed in the budding yeast S. cerevisiae (Hollingsworth et al. 1990; Klein et al. 1992; Scherthan et al.
1992; Weiner & Kleckner 1994; Sym & Roeder 1995)
as well as in the fission yeast Schizosaccharomyces pombe
(Uzawa & Yanagida 1992; Bähler et al. 1993; Funabiki
et al. 1993; Chikashige et al. 1994, 1997; Scherthan et al.
1994), for which, powerful molecular genetics tools are
available.
In particular, the continuous observation of chromosomal events in living meiotic cells would provide
information on meiotic chromosomal dynamics. In
most multicellular organisms, however, meiosis takes
place in specialized tissues, making it difficult to
examine the meiotic events in real time. In contrast,
yeast provides an opportunity to examine meiotic
events directly under a microscope, since meiosis is
easily induced and is completed within 8 h (Padmore
et al. 1991; Bishop et al. 1992). The fission yeast S.
pombe has provided a convenient experimental system in
which to study chromosome dynamics during meiosis.
In S. pombe, the continuous observation of chromosomes in living cells has demonstrated that the whole
nucleus exhibits an oscillatory movement between the
cell poles during the meiotic prophase; a further analysis
in fixed cells revealed that telomeres remain clustered at
the leading edge of the nuclear movement (Chikashige
et al. 1994). The universality of the clustering of telomeres during the meiotic prophase has been confirmed
in recent studies of nuclear organization in several
evolutionally distant organisms; mouse, human
(Scherthan et al. 1996) and maize (Bass et al. 1997).
The budding yeast S. cerevisiae provides another useful
experimental system for analysing the molecular
mechanisms for meiotic chromosomal events. Combined with the powerful molecular biological tools
available in S. cerevisiae, cytological studies of meiotic
chromosomal events in living cells should provide new
insights into the mechanisms of meiosis. In this study,
we have observed the dynamic behaviour of nuclei
throughout the progression of meiosis in living yeast
cells. Nuclear behaviour was followed in living meiotic
cells by the use of jellyfish green fluorescent protein
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(GFP), fused with nucleoplasmin, a nuclear protein
from Xenopus. The arrangement of microtubules during
the progression of meiosis was also examined in fixed
meiotic cells. A combined analysis of the results from
living and fixed cells revealed nuclear behaviours that are
associated with microtubule rearrangement. In addition, the nuclear location of centromeres and telomeres
were determined in fixed cells in synchronous populations of meiotic cells by indirect immunofluorescence
staining; analysis shows that centromeres are clustered at
a single location before the induction of meiosis, and
become scattered during the progression of meiotic
prophase, while telomeres remain in 5–8 focal clusters.
These results indicate that, in S. cerevisiae, chromosomes
re-organize their arrangement within a nucleus upon
entering to meiosis.
Results
GFP-nucleoplasmin as a fluorescent nuclear
marker in living yeast cells
We have used a GFP-nucleoplasmin fusion protein (Lim
et al. 1995) to follow nuclear dynamics in living yeast
cells. Nucleoplasmin is an abundant nuclear protein in
Xenopus oocytes (Earnshaw et al. 1980). When introduced
into mammalian cells, fluorescently tagged nucleoplasmin
is transported into a nucleus and stains the nucleus
when the nuclear membrane is intact (T. Haraguchi &
Y. Hiraoka, unpublished results). In the yeast, GFPnucleoplasmin was retained in the nucleus at all stages in
mitosis and meiosis, staining the nucleus in the living
state without affecting cell growth or spore viability.
Figure 1 demonstrates the exclusive nuclear staining of
GFP-nucleoplasmin, and shows that mitotic nuclear
division proceeds normally. The mitotic behaviour of
the yeast nucleus stained with the GFP-nucleoplasmin
was similar to that observed previously using a DNAspecific fluorescent dye or differential interference
contrast microscopy (Palmer et al. 1989; Yeh et al. 1991).
Mitotic nuclear division in S. cerevisiae is asymmetrical
between the mother cell and the daughter bud. During
mitosis, chromosome migration proceeds in two steps:
first, the daughter set of chromosomes migrates into the
daughter cell until the mass of chromosomes in the
daughter cell is roughly equal to that in the mother cell;
second, masses of chromosomes further segregate to
form two nuclei. In an example shown in Fig. 1, the
speed of the first migration step was about 1.4 mm/min,
and that of the second separation step was 0.4 mm/min.
These values are similar to those measured by following
a GFP marker of the spindle-pole body (SPB) (Kahana
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Nuclear dynamics in yeast meiosis
et al. 1995). We have also seen that meiotic chromosome segregations in this strain proceed with normal
timing (Fig. 2). These results indicate that the GFPnucleoplasmin acts as an innocuous fluorescent nuclear
marker in living yeast cells, both in mitosis and meiosis.
Live observation of nuclear behaviour during
the progression of meiosis
Figure 1 Mitotic nuclear division in a living yeast cell. Yeast
mitotic cells expressing the GFP-nucleoplasmin fusion protein
were observed in the SD medium at 30 8C on a microscope stage.
Time-lapse images at a single focal plane were obtained on a
cooled CCD at 1 min intervals. Numbers at the right side of
panels represent time in minutes. The arrow indicates the
position of the bud neck. Scale bar represents 2 mm.
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Using the GFP-nucleoplasmin fusion construct, the
behaviour of the nucleus in living cells was observed
during meiosis (Fig. 2). Meiosis can be induced synchronously in S. cerevisiae, as has been reported previously (Padmore et al. 1991; Bishop et al. 1992). The
progression of meiotic events after induction of meiosis
is summarized in Fig. 2A. DNA replication takes place
within the first 2 h of induction. The meiotic prophase
continues from 2 to 5 h, followed by the first and second
meiotic divisions, at about 5 h and 6 h, respectively.
In our experiments, yeast cells expressing the GFPnucleoplasmin fusion protein were transferred into the
sporulation medium to induce meiosis, and the process
of meiosis was followed under a fluorescence microscope over time (see Experimental procedures). An
example of our observations of nuclear behaviour in
living cells is shown in Fig. 2B, along with the reported
sequence of meiotic events (Fig. 2A). Nuclear divisions
took place at the expected timing in our live observations (Fig. 2B), indicating that our observation
conditions did not affect the progression of meiosis.
The progression of meiotic chromosomal events was
also examined in populations as an estimate for
synchrony (Fig. 2C); samples were obtained from the
same culture as was used in Fig. 2B. Similar temporal
sequences of chromosomal events were reproduced in
separate experiments using population analysis that are
described later in this paper.
Our live observations allowed an identification of
interesting aspects of meiotic behaviour that had not
been recognized in fixed specimens. During the meiotic
prophase, we observed the deformation of the nucleus;
nuclei became elliptic and continuously changed their
elliptic shape during this period (Fig. 2B, t ¼ 2:30–
5:00) whereas nuclei were spherical during the first
2–2.5 h of meiosis (Fig. 2B, t ¼ 1:25–2:15) as well as
during mitotic interphase (data not shown). While
nuclear deformation was prominent, the translational
movement was rather subtle, unlike the striking nuclear
movement observed in fission yeast meiotic prophase
(Chikashige et al. 1994). During meiotic nuclear
division, unlike mitotic nuclear division, a symmetrical
separation of the nuclear masses was observed (Fig. 2B,
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t ¼ 5:00–5:10; compare with Fig. 1); chromosomal
DNA was segregated into two equal masses during both
meiotic divisions I and II. More examples are shown
in Fig. 3. The speed of chromosome separation was
0.4 mm/min at meiosis I and 0.2 mm/min at meiosis II
(average from eight and six cases, respectively). An
Figure 2 Progression of chromosomal events during yeast
meiosis. (A) The temporal sequence of meiotic events in yeast
SK1 strain after the synchronous induction of meiosis (Padmore
et al. 1991; Bishop et al. 1992). When committed to meiosis,
yeast cells proceed with meiotic events with relatively high
synchrony: a single round of DNA replication, homologous
chromosome pairing, meiotic recombination, and two consecutive divisions of chromosomes producing four haploid
gametes. (B) Yeast cells producing GFP-nucleoplasmin fusion
protein were observed in the SPM medium at 30 8C on a
microscope stage. Time-lapse images at a single focal plane were
obtained on a cooled CCD at 5 min intervals. Numbers on the
images indicate time in hours and minutes after induction to
meiosis. Scale bar represents 2 mm. (C) Percentage of cells
containing 1 (filled circle), 2 (triangle) or 4 (open circle) nuclei is
plotted as a function of time after meiotic induction in the SPM
medium. Cells were sampled every hour, fixed with ethanol, and
stained with DAPI; 100 cells were counted for each time point.
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Figure 3 Meiotic divisions. Examples of meiotic chromosome
segregations are shown for three individual cells. Two segregated
nuclear masses re-approach each other after meiotic division I,
and then segregate into four masses at meiotic division II. The
asterisk shows the time point when the nuclear masses approach
closest to each other in each example. Scale bar represents 2 mm.
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Nuclear dynamics in yeast meiosis
additional intriguing observation is the backlash motion
of the divided nuclear masses during interkinesis, the
stage between the first and second meiotic divisions
(Figs 2 and 3). The two nuclear masses that had divided
in meiosis I approached each other once again (Fig. 2B,
t ¼ 5:20–5:40; Fig. 3A, t ¼ 30–75 min; Fig. 3B, t ¼ 30–
60 min; Fig. 3C, t ¼ 35–50 min; the asterisk in Fig. 3
indicates the period when the two nuclear masses
approached closest to each other), and then segregate
into four haploid masses in meiosis I (Fig. 2B, t ¼ 6:00;
Fig. 3, frames after the asterisk). This backlash motion
of the nucleus probably reflects a unique behaviour of
the nuclear membrane during S. cerevisiae meiosis (see
Fig. 6, and also Discussion section).
Meiotic behaviour of chromosomes and
microtubules examined in fixed specimens
The nuclear behaviours observed in living cells were
supplemented by imaging chromosomes in fixed specimens. Yeast cells were induced to enter meiosis and
were fixed with formaldehyde at appropriate times after
the induction of meiosis. Chromosomes were stained
with a DNA-specific fluorescent dye, 40 ,6-diamidino2-phenylindole (DAPI); microtubules were stained
with an antitubulin antibody in the same specimens to
provide a benchmark for the specific stages of meiosis.
At time 0 after the transfer of cells to the sporulation
medium for the induction of meiosis, microtubules
were observed only as a single dot located at the SPB
(Fig. 4A). At 2 h after induction to meiosis, astral microtubules radiating at the SPB were observed (Fig. 4B). At
4 h after meiotic induction, the astral microtubules
disappeared, and were replaced by a short bundle of
microtubules (Fig. 4C). These stages, from 2 to 4 h,
coincident with the period of meiotic prophase, occur
when nuclei showed their transformation, although we
can not demonstrate microtubule involvement in such
nuclear behaviour.
We next followed the arrangement of microtubules
during meiotic chromosome segregations at 7 h after
meiotic induction (Fig. 5). During meiotic division I,
spindle microtubules elongate to segregate chromosomes (Fig. 5A–D). After spindle microtubules reach
their maximal length at meiotic division I, microtubules
gradually disassemble, leaving microtubules at the SPBs
Figure 4 Arrangement of microtubules in
meiotic prophase. Microtubules stained with
antitubulin antibody (left) and chromosomes
stained with DAPI (middle) are shown
together with a merged image (right).
Cells were fixed at 0 h (A), 2 h (B) and 4 h
(C) after induction to meiosis. Images were
obtained at a single focal plane. An aster of
microtubules (B) and a bundle of microtubules (C) are shown by an arrow. Scale bar
represents 5 mm.
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only (Fig. 5D-F). Upon microtubule disassembly, divided
chromosome masses gradually approach each other, as
was seen in living cells, almost to the point where they
form a single nuclear mass (Fig. 5F–H). Within a single
nucleus, two sets of spindle microtubules began their
elongation for meiosis II (Fig. 5G–H). The spindle
microtubules further elongate and four equal masses of
the chromosomes migrate in directions defined by two
sets of spindle microtubules, parallel or crossing each
other (Fig. 5I–K). Combining the results obtained from
living and fixed cells, we summarize in Fig. 6 the various
behaviours of the nuclei and microtubules during
meiosis.
Nuclear positioning of telomeres and
centromeres during the meiotic prophase
The diploid nucleus of S. cerevisiae has 32 centromeres
and 64 telomeres. Because the relocation of centromeres and telomeres within a meiotic prophase nucleus
has been reported in several organisms (Chikashige et al.
1994; Scherthan et al. 1996; Bass et al. 1997), we wished
to examine the dynamics of these chromosomal domains
during the meiotic prophase. To achieve this goal, we
have determined the nuclear locations of telomeres and
centromeres in synchronous populations of meiotic
cells. Telomeres were detected using the chromosomal
integration of GFP-Rap1 fusion construct (see Experimental procedures) or an antibody against Rap1 protein
(Klein et al. 1992), which has been shown to co-localize
with telomeric repeats by combined fluorescence in situ
hybridization and immunofluorescence studies (Gotta
et al. 1996). The localization of Rap1 protein at the
chromosome ends can be seen in spread preparations of
pachytene chromosomes (Fig. 9F). Centromeres were
detected using an antibody against a yeast centromere
Figure 5 Microtubules and chromosomes during meiotic
chromosome segregations. Microtubules stained with antitubulin
antibody (left) and chromosomes stained with DAPI (middle) are
shown together with a merged image (right). Cells were fixed at
7 h after induction to meiosis and fluorescently stained; images
were obtained at a single focal plane. Formation and elongation
of spindle microtubules during meiotic division I (A–D);
disappearance of spindle microtubules (E–F); reformation and
elongation of microtubules during meiotic division II (G–K).
Two masses of chromosomes approach each other coincident
with the disappearance of spindle microtubules after meiotic
division I, and continue approaching during the formation of
short spindles (E–H). A further elongation of spindle microtubules segregate the chromosomes into four masses (I–K). Scale
bar represents 2 mm.
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Nuclear dynamics in yeast meiosis
Figure 6 (A) schematic diagram of the behaviour of nuclei and
microtubules during the progression of meiosis. The arrangement of microtubules is summarized along with the temporal
sequence of meiotic events. The behaviour of nuclear membrane
is based on the previous report (Moens & Rapport 1971).
binding protein Ndc10 (Goh & Kilmartin 1993).
Localization of Ndc10 protein at the centromeres
during meisosis was confirmed in spread preparations
of pachytene chromosomes (Fig. 10G).
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Three-dimensional data of telomere localization
were obtained from living cells stained with GFPRap1 (Fig. 7) and fixed cells stained with anti-Rap1
antibody (data not shown); basically the same results
were obtained with GFP-Rap1 and anti-Rap1 antibody. Each panel of Fig. 7A–E represents a throughfocus series of a nucleus stained with GFP-Rap1.
Unlike the situation in S. pombe, a single cluster of
telomeres was not observed at any of the stages during
meiosis in S. cerevisiae, but instead, telomeres were
clustered at several sites around the nuclear periphery.
Distinct signals which were seen at early stages of meiosis
(thick arrow in Fig. 7A,C) become diffused at 2–3 h
after the induction of meiosis (arrowhead in Fig. 7C,D;
counted as a single signal), and smaller separate signals
became prominent at later stages (thin arrow in Fig. 7E).
The number of telomere clusters was counted in a threedimensional data stack; the average number of telomere
clusters ranged from 5 to 8 during the 0–4 h period after
induction of meiosis, and rather slightly increased (Fig.
8). The tendency of an increase in the number of
telomere signals was more clearly seen when nuclei were
spread by a gentle detergent treatment. In the spread
preparations, the number of small signals of the telomeres
increased towards the pachytene stage (Fig. 9). In early
stages of meiosis (0–1 h), the telomeres seemed to be
tightly clustered (thick arrow in Fig. 9A); the clusters of
telomeres appeared to loosen at 2–3 h after the induction
of meiosis (arrowhead in Fig. 9C,D). Individual signals
can be seen at the ends of the pachytene chromosomes
(thin arrow in Fig. 9F). These results suggest that the
interaction between telomeres loosens during the
progression of the meiotic prophase.
On the other hand, centromeres exhibited clear
changes in their nuclear localization during progression
through meiosis (Fig. 10). Centromeres were clustered
at a single location near the SPB before the induction of
meiosis (Fig. 10A,B). After induction, the cluster of
centromeres first becomes scattered along a line, and
then extends within the nucleus (Fig. 10C–E). At 5 h
after the induction of meiosis, which corresponds to the
pachytene stage when synaptomenal complexes form
between homologous chromosomes, a scattered pattern
of centromere localization is frequently observed (Fig.
10E). In a mutant strain, ndt80-1, in which cells are
arrested in pachytene (Xu et al. 1995), a majority of
the cells exhibit a scattered distribution of centromeres
(Fig. 10F). We have classified the patterns of centromere
location into three groups: a single cluster, a linear
alignment and a scattered distribution (indicated by a
thick arrow, an arrowhead and a thin arrow respectively,
in Fig. 10A–E). The frequency of each group was
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Figure 7 Nuclear position of telomeres
during meiosis. Telomeres were detected by
GFP-Rap1 in living meiotic cells. Threedimensional image data were obtained at
0.2 mm focus intervals, and computationally
processed to remove out-of-focus image
information using an iterative deconvolusion
method (Agard et al. 1989). Five images from
left to right in each panel (A–E) are a throughfocus series of a single nucleus at 0.2 mm focus
intervals. The images were obtained from
different cells at each time point: 0 h (A), 1 h
(B), 2 h (C), 3 h (D) and 4 h (E) after meiotic
induction. Scale bar represents 2 mm.
measured in synchronous populations after induction of
meiosis. Results indicate a clear tendency for the
relocalization of centromeres during meiosis (Fig. 11).
To eliminate the possibility of stage-specific separation
of the antigen from the centromere, localization of the
NDC10 antigen at the centromere was confirmed by
immunofluorescence staining in spread preparations of
pachytene chromosomes, as the signals of NDC10
staining were located at the primary constriction of
those chromosomes (Fig. 10G).
Discussion
were extremely sensitive to UV illumination, making it
difficult to observe meiotic nuclear behaviour for
extended periods of time. It is not known whether
the UV sensitivity of Hoechst 33342-stained cells results
from the SK1 genetic background that we use in our
studies. In this study, to avoid the use of the DNAbinding fluorescent dye under UV illumination, we
used GFP fused to a Xenopus oocyte nuclear protein,
nucleoplasmin, as a fluorescent nuclear marker (Lim
et al. 1995). By fusing GFP to a nonessential, foreign
nuclear protein and illuminating with blue light, it has
become possible to follow meiotic nuclear behaviour
for several hours in a single cell.
Live observations of yeast nuclei
The dynamic behaviour of nuclei has been successfully
visualized by the use of a DNA-specific fluorescent dye,
DAPI or Hoechst 33342, in living yeast cells (Palmer
et al. 1989; Chikashige et al. 1994) as well as in mammalian cells (Haraguchi et al. 1997). It was found,
however, that Hoechst 33342-stained yeast cultures
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Genes to Cells (1998) 3, 587–601
Nuclear division in meiosis
Our results have shown that the mode of chromosome
segregation in meiosis was apparently different from
that in mitosis. In mitosis, nuclear division takes place
asymmetrically, that is, a portion of the nucleus gradually
enters into the bud, as demonstrated previously (Palmer
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Nuclear dynamics in yeast meiosis
Figure 8 Number of telomere clusters during meiosis. The
number of telomere spots was counted in three dimensions. Each
panel of plots is labelled by time in hours after the induction of
meiosis. Total number of nuclei examined (N) and average
number of telomere spots (av) are given in each panel.
et al. 1989; also see Fig. 1 in this paper). In contrast,
meiotic chromosome segregations take place symmetrically; the nuclear mass is divided into two equal
masses both in meiosis I and II. This apparent difference
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Figure 9 Localization of telomeres in spread preparations.
Location of telomeres in spread preparations of detergent-treated
nuclei. Telomeres are detected by anti-Rap1 (left panel). In the
right panels, signal of telomeres (green) are superimposed on the
DAPI counterstain of chromosomes (red). Time after induction
of meiosis is 0 h (A), 1 h (B), 2 h (C), 3 h (D) 4 h (E) and 5 h (F).
Scale bar represents 2 mm.
is probably due to the budding mode of cell division in
mitosis. In addition to the asymmetry of the mother–
bud geometry itself, the mitotic chromosomes must pass
through the narrow channel of the bud neck, making it
difficult for the entire mass of chromosomes to migrate
together. On the other hand, meiotic chromosomes
segregate within a single cell with no geometrical
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Figure 10 Dynamics of centromeres during meiosis. (A) Centromeres before induction of meiosis are shown. In the right panel, signals
of centromeres (green) are superimposed on the counter stain of nuclei (red). (B) Centromeres (top) and SPB (bottom) in the same cells;
in the middle panel, centromeres (green) are superimposed on the nuclear counter stain (red). (C–E) Centromeres at 2 h (C), 4 h (D)
and 5 h (E) after induction of meiosis. (F) Centromeres in a mutant strain ndt80-1 after a 6-h arrest. In the bottom panel of each set in
C–F, signals of centromeres (green) are superimposed on the counter stain of nuclei (red). (G) Signals of centromeres (green) are
superimposed on spread pachytene chromosomes (red) at 5 h after induction of meiosis. Scale bar represents 5 mm. The scale bar under
the panel B is for the panels A–F.
constraints, enabling the symmetrical segregation of the
chromosome masses. In fact, in a mutant of cytoplasmic
dynein, in which a nucleus fails to enter the bud, mitotic
nuclear division takes place symmetrically within a
mother cell (Yeh et al. 1995), similar to the behaviour
we have observed in meiosis.
An interesting observation is the backlash motion of
the nucleus during interkinesis. As spindle microtubules
disappeared after meiosis I, the divided masses of the
chromosomes approached each other once again to
form a single mass of chromosomes. The backlash
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motion probably reflects a unique feature of the nuclear
membrane during meiosis in S. cerevisiae, in which two
rounds of meiotic chromosome segregation take place
without a separation of the nuclear membrane, as has
been shown in electron microscopy (Moens & Rapport
1971). Such a backlash motion is not observed in S.
pombe (Chikashige et al. 1994), in which the nuclear
membrane divides to form an individual nucleus for
each set of the segregated chromosomes each time at
meiosis I and II (Tanaka & Hirata 1982). Thus, in S.
cerevisiae, even after homologous sets of chromosomes
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Nuclear dynamics in yeast meiosis
Figure 11 Nuclear location pattern of centromeres in meiotic prophase. The nuclear
localization of centromeres is classified into
three groups as defined in Fig. 9. The frequency of occurrence of each group is plotted
as a function of time after induction of meiosis
in SK1 cells as well as in a mutant strain
ndt80-1 arrested at the pachytene stage of
meiosis for 6 h. Numbers on top of the plot
are the total number of nuclei counted for
each time point.
are spatially separated at meiosis I, they are still confined
within a single undivided nucleus. After the tension
generated by microtubules is lost, divided masses of
chromosomes are drawn back, possibly by the surface
tension of the nuclear membrane (Fig. 5E–G; Fig. 6). It
should be noted that the nuclear mass at this stage often
appears as almost a single mass, and thus these nuclei can
be mistaken for a undivided nucleus with nuclear
staining alone in a population of fixed cells. Our results
demonstrate that live observations provide a unique
opportunity in which to detect transient events such as
the backlash motion in interkinesis, which cannot be
detected in fixed specimens.
Nuclear reorganization during meiotic
prophase
In the interphase nuclei of vegetatively growing cells,
several clusters of telomeres have been observed (Gotta
et al. 1996), and centromeres are clustered at a single
location within a nucleus (Jin et al. 1998). In our
experiments, the telomeres were found in 5–8 clusters
throughout meiosis, and the distinctive bouquet
structure with a single cluster of telomeres was not
detected at any stage of meiosis in S. cerevisiae. This is in
contrast to S. pombe, in which the telomeres of all of the
chromosomes remain clustered at the SPB during the
entire period of meiotic prophase (Chikashige et al. 1994,
1997). However, we cannot rule out the possibility that
a subset of telomeres contact the SPB at one time after
another, or that the telomeres transiently form a single
cluster in a very short period of the meiotic prophase. In
fact, the bouquet structure of the chromosomes is
transient, and is only observable during a limited period
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in the meiotic prophase in mouse, human (Scherthan
et al. 1996) and maize (Bass et al. 1997) whereas it is
stable in S. pombe. It is also thought that a single transient
cluster of telomeres can be detected at a low frequency
under certain situations during S. cerevisiae meiosis
(H. Scherthan, personal communication). On the other
hand, we have observed a clear relocalization of centromeres after the induction of meiosis. In S. cerevisiae,
centromeres are clustered at a single location near the
SPB before the induction of meiosis and are redistributed during meiotic prophase. The clustered centromeres first become scattered along a line, and are
subsequently scattered within the nucleus in the
pachytene stage of meiosis. Similar observations of
centromere relocalization during S. cerevisiae meiosis
have been reported in Jin et al. (1998).
Apparent differences in chromosome dynamics
between S. pombe and S. cerevisiae may reflect the
mechanistic features of chromosomes in these organisms. It should be pointed out that in S. cerevisiae, the
arrangement of chromosomes within a nucleus is
spatially restricted because of the small size of chromosomes. The size of chromosome arms (the portion of
the chromosome from the centromere to either end
of the chromosome) ranges from 90 kbp to 1.1 Mbp in
S. cerevisiae (Goffeau et al. 1997) whereas it ranges from
1.5 Mbp to 3.7 Mbp in S. pombe (Fan et al. 1989). When
centromeres are clustered at a single site, the localization
of the telomeres of small chromosomes is limited by the
length of chromosome arms. The scattering of centromeres in the meiotic prophase may increase the freedom
of chromosomal movement. In addition, results obtained
from spreads of meiotic nuclei indicate that telomere
interactions become looser during the meiotic prophase.
Genes to Cells (1998) 3, 587–601
597
A Hayashi et al.
This alteration in telomere–telomere contacts may
reflect a general enhancement in the dynamics of
meiotic chromosomes, which is necessary for the spatial
rearrangement of homologous chromosomes.
Recent studies have demonstrated the role of S.
pombe telomeres in aligning homologous chromosomes
(Shimanuki et al. 1997; Cooper et al. 1998; Nimmo et al.
1998). It remains to be examined whether the dynamics
of telomeres and centromeres are directly associated
with meiotic chromosomal events in S. cerevisiae. In S.
cerevisiae, studies of the progression of meiosis in haploid
cells disomic for a circular or a linear extra chromosome
has concluded that telomeres play a role in homologous
chromosome recognition (Rockmill & Roeder 1998).
One candidate for mediating recognition is the Tam1/
Ndj1, a meiosis-specific telomere binding protein
(Chua & Roeder 1997; Conrad et al. 1997). Since the
genetic controls of meiosis also differ greatly between
budding and fission yeasts (Yamamoto et al. 1997), it
will be useful to compare and integrate the knowledge
obtained from both organisms, as well as from animals
and plants, in order to understand the underlying
molecular mechanisms for meiotic chromosome
dynamics.
Experimental procedures
Yeast strains
The Saccharomyces cerevisiae strains used in this study are diploid
derivatives of the SK1 strain (Alani et al. 1990) which is isogenic for
ho::LYS2/ho::LYS2, lys2/lys2, ura3/ura3, leu2::hisG/leu2::hisG,
arg4-nsp/arg4-bgl. AHY110 (ho::LYS2/ho::LYS2, lys2/lys2,
ura3/ura3, leu2::hisG/leu2::hisG, arg4-nsp/arg4-bgl) was used as
a wild-type diploid strain. A diploid ndt80-1 strain is AHY115
(ho::LYS2/ho::LYS2, ura3/ura3, lys2/lys2, his4::LEU2/HIS4,
arg4/arg4, rap1::RAP1-GFP-LEU2/rap1::RAP1-GFP-LEU2,
ndt80-1/ndt80-1). A diploid strain carrying the RAP1-GFP
fusion construct, AHY111, is described in the next section.
GFP fusion constructs
The plasmid construction of pAGN2 bearing the Xenopus GFPnucleoplasmin fusion gene is described in Lim et al. (1995): the
plasmid contains the LEU2 gene as a selection marker and the
fusion gene which produces the GFP fused to N-terminus of
nucleoplasmin under regulation of ADH1 promoter.
Chromosomal integration of the GFP-Rap1 fusion construct
was obtained as follows: Plasmid pAH50 was constructed by
smaI-XhoI self-ligation at the multicloning site of pRS405 which
contains the LEU2 gene as a selection marker (Stratagene). The
3.0 kb PvuII-NheI fragment of RAP1 gene, which encodes
amino acid residues 19-827 of the RAP1 protein, was cloned into
pAH50 between Ecl136I and XbaI sites to construct pAH51.
598
Genes to Cells (1998) 3, 587–601
GFP-S65T was PCR-amplified using primers, 50 -GGAGGCCT
TATGAGTAAAGGAGAAGAACT-30 and 50 -GAAGGCCTT
GTTTGTATAGTTCATCCATGC-30 , and inserted into the
pAH51 at its NruI site within coding region of the RAP1 gene,
which corresponds to the position at amino acid residue 235 of
the Rap1 protein; the resulting GFP-RAP1 fusion plasmid is
designated pAH52. To obtain strains carrying chromosomal
integration of the GFP-Rap1 fusion construct, haploid strains,
TNY026 (ho::LYS2, lys2, ura3, leu2::hisG, arg4-nsp) and
TNY111 (ho::LYS2, lys2, ura3, leu2::hisG, arg4-bgl), were
transformed with pAH52 after digestion with PstI which resides
within a portion of the RAP1 coding region at the 50 side to the
GFP gene. Transformants were selected for LEU2þ. The
resulting GFP-RAP1 fusion construct contains the chromosomal
RAP1 gene intervened by the GFP gene under regulation of the
authentic RAP1 promoter. The transformed haploid strains were
mated with each other to generate a diploid strain AHY111
(ho::LYS2/ho::LYS2, lys2/lys2, ura3/ura3, leu2::hisG/leu2::hisG,
arg4-nsp/arg4-bgl, rap1::RAP1-GFP-LEU2/rap1::RAP1-GFPLEU2).
Media and sporulation conditions
Media and conditions for the routine culture of the yeast strains
are as described previously (Sherman et al. 1983; Padmore et al.
1991; Shinohara et al. 1992). The MYPL medium contains 0.3%
malt extract, 0.3% yeast extract, 0.5% polypeptone and 2%
sodium lactate pH 4.5 (Adzuma et al. 1984). Complete medium,
YPD, contains 1% yeast extract, 2% bactopeptone and 2%
glucose. Synthetic minimum medium, SD, contains 0.67% yeast
nitrogen base and 2% glucose supplemented with the appropriate
amino acids (Sherman et al. 1983).
The synchronous meiotic culture was prepared as described
previously (Padmore et al. 1991; Bishop et al. 1992). Cells from a
frozen stock were grown on an MYPL plate at 30 8C for 12 h.
The cells were then streaked on a YPD plate and incubated at
30 8C for 2 days. A single colony was incubated in the YPD liquid
medium at 30 8C for 24 h. Then the cells were diluted into the
presporulation medium YPA (1% yeast extract, 2% bactopeptone
and 1% potassium acetate) to a final concentration of
OD600 ¼ 0.2, and incubated with vigorous aeration at 30 8C
for 12 h until the cell density reached to OD600 ¼ 1.5. The cells
were then harvested and transferred into the sporulation medium
SPM (1% potassium acetate supplemented with appropriate
amino acids) to induce meiosis.
In case for the cells bearing the GFP-nucleoplasmin fusion
gene, a single colony was selected on an SD plate lacking leucine
after growth at 30 8C for 2 days, and was grown in SD liquid
medium lacking leucine at 30 8C for 36 h. The cells were induced
to undergo meiosis by passage through the presporulation medium
YPA and the sporulation medium SPM, as described above.
Microscope system
Fluorescence images were obtained on a cooled, charge-coupled
device (CCD) as an image detector. A Peltier-cooled CCD
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Nuclear dynamics in yeast meiosis
camera (Photometrics Ltd, Tucson, Arizona), with a 1340 × 1037
pixel CCD chip (KAF1400) coated to improve short-wavelength
sensitivity (Metachrome II coating, Photometrics Ltd), was
attached to an Olympus inverted microscope IMT-2. The microscope lamp shutter, focus movement, CCD data collection and
filter combinations were controlled by a Silicon Graphics Personal
Iris 4D35/TG workstation. For wavelength switching during
data collection, the excitation and barrier filters were mounted
on revolving wheels controlled by the Silicon Graphics workstation. Details of the microscope system set-up were described
in Hiraoka et al. (1991) except that the MicroVAX was replaced
by a Silicon Graphic workstation. For the observations of living
cells, a microscope system built in a custom-made, temperaturecontrol room was used; the room temperature could be controlled in a range from 10 8C to 50 8C with a precision of 0.1 8C
(Haraguchi et al. 1997; Ding et al. 1998). A computer and other
control units were placed outside the room and the microscope
controlled remotely.
Fluorescence microscopy in living cells
Yeast cells producing the GFP-nucleoplasmin fusion protein
were transferred to a glass-bottomed culture dish (MatTek Corp.,
Ashland, MA) coated with 1 mg/mL concanavalin A. Living
fluorescently stained cells were observed on the temperaturecontrolled CCD microscope system using an Olympus oil
immersion objective lens (Dplan Apo 100/NA ¼ 1.3). Mitotic
cells were observed in the synthetic SD medium at 30 8C; meiotic
cells were observed in the SPM medium at 30 8C. Images of
GFP-nucleoplasmin were obtained on the cooled CCD with an
exposure of 0.1–0.2 s under the illumination of a mercury arc
lamp using a high-selectivity filter combination for fluorescein
(Chroma Technology, Brattleboro, VT).
Immunofluorescence microscopy
The immunofluorescence staining of yeast cells was performed as
described in Kilmartin & Adams (1984). Meiotic cells were fixed
with 3% formaldehyde (freshly prepared from paraformaldehyde)
for 10 min at room temperature. Fixed cells placed on a glass slide
were digested with 3 mg/mL of Zymolyase (Seikagaku Kogyo,
Tokyo) for 30 min at 30 8C, and then permeabilized by treatment
with methanol and acetone at ¹20 8C. After treating with PBS
containing 3% BSA at room temperature for 10 min, the
specimens were incubated with an appropriate antibody.
For staining microtubules, the specimens were incubated with
1:500-diluted antitubulin antibody YOL1/34 (Sera-Lab, Sussex,
UK) in PBS containing 1% BSA and 0.1% Tween-20 (PBT) at
4 8C for 12–16 h. The specimens were washed four times with
PBT, followed by treatment with PBS containing 3% BSA.
Immunofluorescence signals were detected by incubating the
specimens with 1:200-diluted anti-rat IgG antibody conjugated
with fluorescein (Cappel, Durham, NC) in PBT at room
temperature for 1 h. Centromeres were stained with 1:125diluted affinity-purified antibody against Ndc10 protein (a gift
of Dr J. Kilmartin, Cambridge, UK). Telomeres were stained
q Blackwell Science Limited
with 1:50-diluted affinity-purified antibody against Rap1 protein.
The immunofluorescence signals of centromeres or telomeres
were detected with anti-rabbit IgG antibody conjugated with
fluorescein (Cappel). SPB was stained with 1:25-diluted monoclonal antibody against Nuf2 protein (a gift of Dr P. Silver,
Cambridge, MA) and anti-mouse IgG antibody conjugated with
rhodamine (Cappel). The anti-Nuf2 antibody detected the S.
cerevisiae SPB and the mouse centrosome (Osborne et al. 1994).
After four washes using PBT, the nuclei were stained with
1 mg/mL DAPI, and were mounted with 90% glycerol containing 0.1% p-phenylenediamine. Microscope images were obtained
on the computerized CCD microscope system using an Olympus
oil immersion objective lens (DPlan Apo 100/NA ¼ 1.3). Highselectivity excitation and barrier filter combinations (Chroma
Technology, Brattleboro, Vermont) for DAPI, fluorescein and
rhodamine were used on revolving wheels under computer
control. For double- or triple-staining fluorescence images, a
single dichroic mirror with triple-band pass properties designed
for wavelengths of DAPI, fluorescein, and Texas Red (Chroma
Technology, Brattleboro, Vermont) was used to eliminate the
significant displacement of images during wavelength switching,
and thus no further alignment was necessary (Hiraoka et al.
1991).
Spread preparation of chromosomes
Spheroplasts were prepared by digesting meiotic cells with
3 mg/mL of Zymolyase (Seikagaku, Tokyo) for 30 min at 30 8C.
Spread preparations were prepared from spheroplasts as described
in Loidl et al. (1991). A suspension of spheroplasts (15 mL) was
placed on to a glass slide. To this suspension, the following
solutions were added sequentially: 40 mL fixative (4% formaldehyde, freshly prepared from paraformaldehyde, in 3.4% sucrose),
80 mL 1% Lipsol (alkaline laboratory cleaning detergent), and
80 mL fixative, as above. The glass slides were laid flat, allowed to
dry overnight, and the next day they were gently rinsed with
0.2% Photo-Flo400 (Kodak) and left to dry in a vertical position.
The specimens were used for immunofluorescence staining as
described above.
Acknowledgements
We would like to thank John Kilmartin for the anti-Ndc10
antibody, Pamela Silver for the anti-Nuf2 antibody and Masahiro
Ajimura for the ndt80-1 strains; Beth Rockmill, Schirleen
Roeder, and Harry Scherthan for communicating their results
prior to publication. We also thank Ayumu Yamamoto, Da-Qiao
Ding, Yuji Chikashige, Akira Nabetani and Tokuko Haraguchi
for a critical reading of the manuscript. The use of GFPnucleoplasmin as a nuclear marker in living yeast cells was advised
by Tokuko Haraguchi, Ayumu Yamamoto and Da-Qiao Ding.
This work was supported by grants from the Japanese Ministry of
Posts and Telecommunications, the Japanese Ministry of Culture,
Science and Education, and the Science and Technology Agency
of Japan. Aki Hayashi was supported by a predoctoral fellowship
from the Japanese Society for Promotion of Science.
Genes to Cells (1998) 3, 587–601
599
A Hayashi et al.
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Received: 13 July 1998
Accepted: 19 August 1998
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