1 Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin,
Germany
2 Institute of Botany, University of Vienna, Vienna, Austria
* Author for correspondence (e-mail: schertha{at}molgen.mpg.de)
Accepted 4 March 2003
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Summary |
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Key words: Bouquet, Haploid, Kar3, Meiosis, Polyploid, Sir3, Telomeres, Yeast
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Introduction |
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In preparation for homologue pairing, dramatic changes in nuclear and
chromosome architecture occur: the premeiotic centromere and chromosome
distribution and organization are resolved, chromosomes elongate and
reposition within the nucleus, and the chromosome ends transiently form a
cluster at a limited region of the nuclear envelope (bouquet formation) (for
review, see Scherthan, 2001;
Zickler and Kleckner,
1998
).
Genetic and cytological analyses suggest that the formation of a
chromosomal bouquet in the synaptic meiosis of budding yeast occurs
independently of homologous recombination and synapsis
(Dresser and Giruox, 1988;
Rockmill and Roeder, 1998
;
Trelles-Sticken et al., 1999
).
In the vegetative (premeiotic) yeast nucleus, telomeres associate in a few
aggregates at the nuclear periphery (Gotta
et al., 1996
; Klein et al.,
1992
) and centromeres form a cluster near the spindle pole body
(SPB; the fungal microtubule-organizing centre)
(Goh and Kilmartin, 1993
;
Hayashi et al., 1999; Jin et al.,
2000
). Telomeres and centromeres depart from this vegetative
positioning when cells are starved for nitrogen and fermentable carbon
sources, and enter sporulation. Soon after the induction of meiosis,
centromeres disperse from the SPB (Hayashi et al., 1999;
Jin et al., 2000
) and
telomeres redistribute to cluster transiently at this location
(Trelles-Sticken et al.,
2000
). Thus, the bouquet stage of budding yeast resembles the
classical bouquet arrangement that occurs in a conserved manner during
prophase I of most, if not all, eukaryotes
(Zickler and Kleckner, 1998
;
Yamamoto and Hiraoka, 2001
;
Scherthan, 2001
).
The meiosis-specific telomer protein Ndj1p is required for ordered
cross-over distribution (interference), sister chromatid cohesion at meiotic
telomeres and segregation of small chromosomes in budding yeast meiosis
(Chua and Roeder, 1997;
Conrad et al., 1997
). It has
previously been shown that Ndj1p is essential for bouquet formation and
effective homologue pairing (Rockmill and
Roeder, 1998
; Trelles-Sticken
et al., 2000
).
In addition to its occurrence in synaptic and asynaptic organisms, meiosis
can also be induced in auto- and allopolyploids, and even in the absence of
homologues, as in haploid plants, in which heterosynapsis (synapsis between
non-homologous chromosomes) is observed
(Gillies, 1974;
Levan, 1942
;
Santos et al., 1994
).
Similarly, haploid yeast strains that express both mating-type loci can be
induced to undergo meiosis, and these meiocytes also display heterosynapsis
and foldback pairing (Loidl et al.,
1991
). The occurrence of heterosynapsis in haploid meiosis and the
association of non-homologous chromosomes in disomic meiosis
(Loidl et al., 1994
) suggests
the existence of a homology-independent `chromosome mover' mechanism a
task that has long been ascribed to meiosis-specific telomere clustering (for
reviews, see Loidl, 1990
;
Zickler and Kleckner, 1998
;
Scherthan, 2001
).
Chromosome movements and bouquet formation at meiosis might involve motor
proteins that move along microtubules, because microtubule-disrupting drugs
have been shown to interfere with chromosome movement, pairing and bouquet
formation (Cowan and Cande,
2002; Loidl, 1990
;
Svoboda et al., 1995
).
Observations of the asynaptic meiosis of Schizosaccharomyces pombe
have shown that bouquet formation and nuclear movements depend on the
integrity of the SPB, telomeres and the cytoplasmic dynein heavy chain motor
protein (reviewed in Yamamoto and Hiraoka,
2001
). In S. cerevisiae, the absence of the kinesin-like
microtubule motor Kar3p confers a defect in nuclear congression during mating
(Meluh and Rose, 1990
;
Rose, 1996
) and alters
microtubule dynamics, leading to longer, more abundant cytoplasmic
microtubules (Saunders et al.,
1997
). Because Kar3p has been found to interfere with synapsis and
recombination, it has been assumed to play a role in chromosome and telomere
movements during prophase I (Bascom-Slack
and Dawson, 1997
).
Finally, telomere clustering has also been observed to occur in polyploid
insects (e.g. Rasmussen and Holm,
1980) and plants
(Martinez-Perez et al., 1999
;
Schwarzacher, 1997
). In
plants, the bouquet forms during prophase I, whereas the sorting of homologous
centromeres takes place premeiotically
(Martinez-Perez et al., 2001
;
Martinez-Perez et al., 1999
).
It is assumed that this feature simplifies the homologue-alignment process and
leads to more rapid progress through prophase I
(Moore, 2002
). In agreement
with this assumption, polyploid plants (which often display a Rabl orientation
in somatic cells; Cowan et al.,
2001
; Dong and Jiang,
1998
) undergo meiosis faster than their diploid progenitors
(Bennett and Smith, 1972
;
Martinez-Perez et al.,
2000
).
Here, we have determined the timing of prophase progression and bouquet
formation in haploid and autotetraploid yeast meiosis, and in strains
deficient for the motor protein Kar3p and Sir3p, the latter being a component
of silent telomeric heterochromatin (for reviews, see Grunstein et al., 1998;
Shore, 2001). The experiments,
which all were done in the SK1 strain background, revealed that an increase in
ploidy delays prophase I progression, as assayed by occurrence of meiotic
divisions, centromere cluster resolution and bouquet formation. Delayed
prophase I progression was also noted in diploid and haploid
sir3
meiosis. Our data show that meiotic telomere clustering
occurs in the absence of Sir3p and Kar3p, with the latter possibly being
involved in control of telomere dynamics.
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Materials and Methods |
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To test haploid yeasts for the ability to form bouquets, we used a haploid
strain with SIR3 and SPO13 deleted
(Table 1)
(Loidl and Nairz, 1997) that
undergoes meiosis because of sir3
-induced mating-type
heterozygosity (Rine and Herskowitz,
1987
), whereas the spo13 mutation induces the cell to
skip the meiosis I division, leading to the formation of two viable spores
(Klapholz and Esposito, 1980
;
Wagstaff et al., 1982
). A
kar3
derivative of this strain
(Table 1) was constructed by
replacing the KAR3 gene with a KanMX cassette using a one-step
PCR-mediated technique (Wach et al.,
1994
). Furthermore, in both the KAR3 and the
kar3
haploid strain, the native NDJ1 gene was
substituted by a hemagglutinin (HA)-tagged version of the gene for improved
immunodetection of Ndj1p (Conrad et al.,
1997
; Trelles-Sticken et al.,
2000
).
As a control, we also used a diploid SK1 strain with a homozygous deletion
for SIR3. This strain was constructed by mating haploid strains that
had the chromosomal SIR3 loci disrupted and Sir3p expressed from
plasmid pJL276 (Loidl et al.,
1998). These plasmids, which carried the URA3 marker,
were eventually removed by selection on FOA.
Cell culture and preparation
For time-course analysis, cultures were grown in presporulation medium to a
density of 2x107 cells ml-1 followed by transfer
to sporulation medium (2% KAc) at a density of 4x107 cells
ml-1 (Roth and Halvorson,
1969). Briefly, aliquots from the sporulating cultures were
obtained during time-course experiments at induction of meiosis (transfer to
sporulation medium = 0 minutes) and from 180 minutes to 420 minutes at 10
minute or 20 minute intervals, or from 30 minutes up to 420 minutes at 30
minute intervals. In another set of experiments, aliquots were collected at 0
minutes and from 160 minutes to 300 minutes at 10 minute intervals. Aliquots
were immediately transferred to tubes on ice containing a tenth of a volume of
acid-free 37% formaldehyde (Merck). After 30 minutes, cells were removed from
fixative, washed with 1x SSC and spheroplasted with Zymolyase 100T (100
µg ml-1, Seikagaku) in 0.8 M sorbitol, 2% KAc, 10 mM
dithiothreitol. Adding 10 volumes of ice-cold 1 M sorbitol terminated
spheroplasting. Limited nuclear spreading was used to enhance cytological
resolution as described previously
(Scherthan and Trelles-Sticken,
2002
; Trelles-Sticken et al.,
2000
).
Meiotic time courses with diploid and tetraploid strains were only analysed when sporulation rates were >80%.
DNA probes and labelling
A composite pan-centromeric DNA probe was used to delineate all yeast
centromeres by FISH (cen-FISH) and a subtelomeric XY' plasmid probe that
delineates all telomeres in the SK1 strain (2n=32) investigated was
used to tag yeast telomeres (telo-FISH)
(Jin et al., 1998;
Trelles-Sticken et al., 2000
).
Cosmid h (pEKG011) (Thierry et
al., 1995
) was used to visualize a defined locus on the left arm
of chromosome XI. Probes were labelled either with dig-11-dUTP (Roche
Biochem) or with biotin-14-dCTP (Invitrogen) using a nick translation kit
according to the instructions of the supplier (Invitrogen).
FISH
All preparations were subjected to two colour FISH as described previously
(Scherthan and Trelles-Sticken,
2002). The hybridization solution contained two differently
labelled probes: the pan-telomere XY' probe and either the
pan-centromeric DNA probe or the cosmid probe mapping near the centromere on
the left arm of chromosome XI (h;
Trelles-Sticken et al., 2000
).
Immunofluorescent detection of hybrid molecules was carried out with
Avidin-FITC (Sigma) and rhodamine-conjugated sheep anti-dig Fab fragments
(Roche Biochemicals) (for details, see
Scherthan and Trelles-Sticken,
2002
). Prior to microscopic inspection, preparations were embedded
in antifade medium (Vector Labs, Burlingame) containing 0.5 µg
ml-1 DAPI (4'-6-diamidino-2-phenylindole) as DNA-specific
counterstain.
Immunostaining
A polyclonal antiserum against S. cerevisiae SPB component (Tub4;
Marschall et al., 1996) was
used to stain the SPB in conjunction with telomeres (for details, see
Trelles-Sticken et al., 1999
).
A rabbit antiserum against Zip1p transverse filament protein
(Sym et al., 1993
) of the
yeast synaptonemal complex was applied to identify nuclei at all stages of
synapsis. Ndj1p was stained in freshly prepared, mildly spread nuclei obtained
from the strains that express HA-tagged Ndj1p using a monoclonal anti-HA-tag
antibody (Biotec Santa Cruz) and secondary anti-mouse Cy3-conjugated
antibodies (Dianova).
Microscopic evaluation
Signal patterns in spread nuclei were investigated using a Zeiss Axioskop I
epifluorescence microscope equipped with a double-band-pass filter for
simultaneous excitation of red and green fluorescence, and single-band-pass
filters for excitation of red, green and blue (Chroma Technologies). Digital
images were obtained using a cooled greyscale CCD camera (Hamamatsu)
controlled by the ISIS fluorescence image analysis system (MetaSystems). More
than 110 nuclei were scored for each time point and probe combination directly
in the microscope in randomized preparations. Fluorescence signal patterns
were analysed in nuclei with an undisrupted homogeneous appearance in the DAPI
image.
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Results |
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To determine the timing of meiotic divisions in the different SK1 strains,
DAPI-stained aliquots of cells that were collected at consecutive time points
in sporulation and scored for bi- and tetranucleated cells as divisions. It
was found that the tetraploid strain passed through the meiotic divisions with
a significant delay (2 hours) with respect to the diploid wild type. This
was observed in three independent experiments
(Fig. 1) and argues against an
acceleratory effect of increased ploidy on meiosis I progression in yeast.
|
Centromere cluster resolution is delayed in tetraploid meiosis
To determine whether changes in nuclear architecture are affected by the
prolonged prophase of the tetraploid yeast strain, we next measured the
dissolution of the centromere cluster upon entry into meiosis. In the
premeiotic (vegetative) yeast nucleus centromeres are tightly clustered at the
spindle pole body (SPB; the fungal microtubule-organizing centre), whereas
induction of meiosis leads to centromere dispersion throughout the nucleus
(Hayashi et al., 1999; Jin et al.,
1998; Jin et al.,
2000
). In tetraploid meiosis, we noticed that the frequency of
nuclei with one centromere cluster (premeiotic nuclear architecture) dropped
only gradually, which contrasted with rapid dissolution of centromere
clustering in diploid wild-type SK1 meiosis
(Fig. 3). The former aligns
with the delayed meiotic divisions in tetraploid meiosis
(Fig. 1).
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Altogether, the significantly delayed timing of nuclear divisions and chromosomal events in tetraploid yeast meiosis, like centromere cen-cluster dissolution and bouquet formation (Figs 3, 4), suggests that an increase in ploidy retards prophase I progression in yeast meiosis, which contrasts with the situation in plants.
Chromosomal events in haploid meiosis
In yeast, meiosis can also be induced in haploid and disomic strains, and
genetic analysis suggests that these strains might undergo bouquet formation
(Loidl et al., 1991;
Rockmill and Roeder, 1998
). To
see whether a bouquet forms in the absence of homologous chromosomes, we
investigated meiosis in a haploid spo13
sir3
SK1 strain that expresses both mating types and skips the reductional division
(Klapholz and Esposito, 1980
;
Wagstaff et al., 1982
).
Two independent experiments revealed that meiotic divisions in the haploid
spo13 sir3
strain occurred with a delay
compared with the diploid wild type (Fig.
1). However, centromere cluster dissolution progressed with
wild-type kinetics after induction of meiosis in the haploid SK1 strain, which
suggests that earliest chromosomal events of haploid prophase I are not or
only mildly affected by SPO13 or SIR3 mutation (see below).
Our findings agree with the SPO13 mutation causing a delay in the
meiosis I division (Klapholz and Esposito,
1980
; McCarroll et al., 1994) through the meiosis-I spindle
checkpoint (Lee et al., 2002
;
Shonn et al., 2001). The low frequency of divisions seen in the haploid time
courses might be due to the low frequency of sporulation, which, in agreement
with earlier reports (Loidl et al.,
1991
), never exceeded 40% and might indicate the presence of a
synaptic checkpoint that arrests the cells before meiosis I (see
Discussion).
Combinatorial telo-FISH and SPB immunostaining on mild nuclear spreads
obtained 270 minutes after induction of sporulation revealed that telomeres
aggregate at the haploid SPB (Fig.
2C), which demonstrates the occurrence of a bouquet in haploid
yeast meiosis. Meiotic time courses in the haploid spo13
sir3
SK1 strain reproducibly revealed peak bouquet frequencies
at 300 minutes (12.9±2.6% SD, three time courses). This timing matches
with a bouquet peak at 300 minutes in diploid sir3
meiosis but
contrasts with bouquet formation in our diploid wild-type SK1 time courses,
where peak bouquet frequencies (20.5±4% SD, three time courses) formed
reproducibly around 210 minutes in sporulation medium
(Fig. 4). Hence, nuclei with
bouquet topology accumulate late in haploid spo13
sir3
prophase I, which is probably due to heterosynapsis and
delayed repair of double-stranded breaks (DSBs) in the haploid condition
(De Massy et al., 1994
;
Loidl and Nairz, 1997
) and to
pleiotropic effects elicited by the deletion of SIR3 (see below).
SIR3 deletion disturbs centromere cluster resolution in diploid
meiosis
Because Sir3p is a structural component of telomeric heterochromatin
(Hecht et al., 1996;
Moretti et al., 1994
), whose
absence could potentially influence telomere positioning at meiosis, we
determined whether a SIR3 deletion in the SK1 strain background has
an impact on meiotic chromosome behavior. Like the haploid SIR3
mutant, diploid sir3
meiosis exhibited delayed meiotic
divisions (Fig. 1). Cen FISH to
meiotic time courses revealed that induction of sporulation in SK1 strain
leads to a drop of cells with one centromere cluster over the first 180
minutes, which matches the course of centromere (cen) cluster resolution in
haploid meiosis. Subsequently, the decrease in nuclei with one cen cluster
slowed to follow the more gradual reduction seen in the tetraploid time course
(Fig. 3). Repeated time courses
confirmed a reproducible delay of cen-cluster resolution in the diploid
sir3
time courses compared with the diploid wild type
(Fig. 5). The differences
between the two data sets were highly significant (the Wilcoxon rank sum test
and the one-sided t test gave values of 0.000398 and 0.0001 at
P=0.05, respectively). These observations suggest an over-all delayed
generation of meiotic nuclear architecture in a significant proportion of
sir3
diploid cells, which might in turn retard events
downstream of cen clustering, such as bouquet formation and homologue pairing
(see below). We consider it unlikely that failed initiation of meiosis in a
significant subset of cells could be a cause of the changes observed, because
sporulation was >80% in all diploid sir3
and wild-type time
courses investigated. Rather, it seems that a proportion of diploid
sir3
cells enters meiosis and initiates changes in nuclear
architecture with a delay.
Meiotic telomeres cluster in the absence of Sir3p
Combinatorial telo-FISH and SPB IF to diploid Sir3p-deficient meiocytes
obtained at 300 minutes after induction of meiosis revealed a single telomere
FISH signal cluster at the diploid sir3 SPB
(Fig. 2C). Frequencies of
nuclei with one telo-FISH signal cluster increased similar to the wild type
over the first 180 minutes after meiosis induction, then dropped and formed a
second peak at 270 and 300 minutes (Fig.
4). The biphasic nature of bouquet frequency probably relates to
the tardy dissolution of centromere clustering in a substantial proportion of
sir3
cells (Figs
3,
5) and is consistent with a
delayed occurrence of meiotic divisions in diploid sir3
meiosis (Fig. 1). In line with
the haploid data (Figs 2,
4), it is evident that bouquet
formation bypasses the requirement for Sir3p.
SIR3 mutation delays homologue pairing
Given the promotion of homologue pairing by bouquet formation
(Rockmill and Roeder, 1998;
Trelles-Sticken et al., 2000
),
delayed telomere clustering would predict retarded homologue pairing. Thus, we
determined the pairing of a cosmid-tagged centromere-close region on the left
arm of chromosome XI (Trelles-Sticken et
al., 2000
). In the wild type, we noticed that significant levels
of premeiotic homologue pairing were reduced between 60 and 120 minutes after
induction of meiosis, which probably reflects the resolution of vegetative
homologue contacts during the premeiotic S phase
(Weiner and Kleckner, 1994
).
Pairing gained meiosis-specific levels over the next 180 minutes, with the
maximum values in this wild-type time course (52-56%) being reached after 300
minutes in sporulation medium (Fig.
6). In diploid sir3
meiosis, wild-type pairing
values were reached only after 480 minutes in sporulation
(Fig. 6). A second meiotic time
course experiment revealed 62% pairing at 300 minutes in the wild type and 63%
in the sir3
diploid strain at 420 minutes After induction of
meiosis, which confirms a delay in homologue pairing in the mutant and
suggests that the slow prophase I progression retards homologue pairing in the
sir3
diploid strain. Eventually, however, wild-type levels of
homologue pairing were reached in the absence of Sir3p.
|
Chromosomal events in Kar3p-deficient haploid meiosis
Motor proteins are expected to move meiotic telomeres, and so we
investigated the role of Kar3p kinesin in bouquet formation. For this
analysis, we used the bouquet-proficient haploid SK1 strain, because we
repeatedly failed to obtain sporulating kar3 diploids in the
SK1 background. Kar3p is a kinesin-like motor protein
(Meluh and Rose, 1990
) that is
required for proper positioning of the metaphase spindle (for review, see
Hildebrandt and Hoyt, 2000
)
and karyogamy (the congression stage when parental nuclei migrate and fuse
with each other during syngamy) (Rose,
1996
). In addition to its role in mitosis, it has been shown that
Kar3p deficiency leads to reduced meiotic recombination rates and defects in
chromosome synapsis (Bascom-Slack and
Dawson, 1997
), which has led to the assumption that Kar3p could
drive meiotic telomere clustering
(Bascom-Slack and Dawson, 1997
;
Zickler and Kleckner, 1998
).
To assess the requirement of bouquet formation for Kar3p, we deleted
KAR3 in the bouquet-proficient spo13
sir3
haploid SK1 strain. The kar3
spo13
sir3
triple mutant and a KAR3
spo13
sir3
haploid control strain were induced to
undergo meiosis. Although mitotic growth was not dramatically affected,
repeated meiotic experiments revealed sporulation rates of 10% in the
kar3
haploid, whereas the haploid control strain showed 40%
sporulation. The reduced sporulation in the kar3
haploid
agrees with the disruptive effect of the KAR3 deletion on diploid
meiosis (Bascom-Slack and Dawson,
1997
).
KAR3-deficient meiosis shows wild-type centromere cluster
resolution
To investigate the order of chromosomal events in kar3
meiosis, we first determined resolution of vegetative nuclear architecture
after induction of meiosis (i.e. the drop of frequency of nuclei with one
centromere cluster; see above). Time-course experiments at 30-minute intervals
up to 7 hours after transfer to sporulation medium revealed that, at 0
minutes, 58% and 56% of nuclei displayed one centromere FISH cluster in the
haploid control and kar3
strain, respectively. The cen cluster
frequencies decreased similarly in both strains but with a slight delay
between 180 and 420 minutes in kar3
sporulation
(Fig. 7). Eventually, both time
courses reached similar values of cen clustering in the control (17%) and
kar3
(16%) at 420 minutes. The more gradual cen-cluster
resolution between 180 and 420 minutes in a proportion of kar3
spo13
sir3
triple mutant cells compared with
the spo13
sir3
double mutant meiocytes
(Fig. 7) could result from
altered microtubule dynamics in the absence of Kar3p
(Cottingham et al., 1999
).
Altogether, it appears that Kar3p is dispensable for centromere cluster
resolution after induction of meiosis.
|
|
Expression of Ndj1p and Zip1p is altered in the absence of Kar3p
Time-course analysis revealed a rapid increase in Ndj1p-HA-positive nuclei
after 150 minutes in sporulation in the haploid control, whereas the increase
in kar3 meiosis was more gradual over the entire meiotic time
course, with Ndj1-HA-positive kar3
meiocytes being more
abundant at early time points (Fig.
8). The frequencies of meiotic cells peaked at 49% at 420 minutes
in the kar3
haploid, whereas 74% of cells in the haploid
control strain were Ndj1-HA positive after 420 minutes in sporulation
(Fig. 8). The latter figure
indicates that far more cells enter haploid meiotic prophase (74% in control,
49% in kar3
) than sporulate 40% in the control and 10%
in kar3
, respectively. To validate these results, we also
monitored the expression of Zip1p, which is an synaptonemal complex (SC)
protein that connects homologous chromosomes partially during zygotene and
entirely during pachytene (Sym et al.,
1993
). As for Ndj1p, a more gradual increase in meiocytes
expressing Zip1p was noted in the kar3
haploid
(Fig. 8). The observed
expression profiles for Ndj1p and Zip1p agree with the report that
kar3-mutant cells display derepression of premeiotic IME1
transcription and thus ectopic sporulation, whereas induction of meiosis does
not further increase IME1 expression in prophase I
(Meluh, 1992
;
Keeney, 2001
). The
recombination defects in diploid kar3
meiosis
(Bascom-Slack and Dawson, 1997
)
might thus relate to a more gradual entry of fewer cells into meiosis (see
above). Furthermore, it is possible that a checkpoint responds to the absence
of Kar3p function (Hardwick et al.,
1999
; Shanks et al.,
2001
).
Bouquet formation in kar3 meiosis
To test whether a Kar3p-deficient strain can cluster meiotic telomeres, we
performed HA-Ndj1p IF together with SPB IF on meiotic time courses of the
haploid control and the corresponding KAR3-deleted SK1 strain. The
formation of a single telomere signal cluster at the SPB disclosed the
formation of a true bouquet in haploid kar3 meiosis
(Fig. 2C). When the proportion
of bouquet cells with a single Ndj1p signal cluster was determined among
NDJ1-HA-expressing haploid kar3
and control meiocytes, it was
found that chromosomal bouquets formed in the two strains, with more bouquet
nuclei being present in the kar3
mutant from early time points
onwards. This was also seen by telo-FISH in preparations from the same time
courses (not shown). Because more cells enter meiosis in the control strain
than in the kar3
strain (see above), it appears that the
Kar3p-deficient time courses contain more bouquet nuclei
(Fig. 7). Telo-FISH time-course
experiments on strains that lacked the HA-tagged version of Ndj1p (not shown)
revealed a similar excess of bouquet nuclei in haploid kar3
meiosis (peak value 27.5%) compared with controls (peak value 22.5%).
Altogether, it appears that telomere cluster formation in budding yeast
meiosis tolerates the absence of Kar3p. Higher frequencies and earlier
appearance of bouquet nuclei in kar3
haploid meiosis indicate
altered bouquet duration. Thus, Kar3p might be involved in the control of
tubulin-dependent telomere dynamics and release of telomere clustering.
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Discussion |
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Besides other possibilities, the prophase I delay in the polyploid yeast is
in agreement with the assumption that a genome with increased chromosome
number will require a more elaborate and time-consuming homologue search
during prophase I (Dorninger et al.,
1995; Pfeifer et al.,
2001
). How, then, according to such a scenario, is prophase I
progression accelerated in polyploid plants? This is probably related to the
fact that the homologue search and alignment process and the correction of
non-homologous premeiotic centromere associations in polyploid plants takes
place prior to the onset of meiotic prophase
(Martinez-Perez et al., 2001
).
By contrast, the redistribution of pericentromeric heterochromatin
(centromeres) in mammals and yeast occurs during the onset of first meiotic
prophase (Hayashi et al., 1999; Pfeifer et
al., 2001
; Scherthan et al.,
1996
; Trelles-Sticken et al.,
1999
). Therefore, the elaborate architectural changes in the
diploid meiocyte nucleus that occur after the initiation of meiosis probably
extend the time required for the transit though prophase I, whereas the rapid
prophase I progression in polyploid plants obviously benefits from the
premeiotic alignment of homologous pericentromeres
(Martinez-Perez et al.,
2001
).
In our polyploid yeast, an extended meiotic cell cycle might also be
induced by altered amounts of the microtubule/kinetochore-associated proteins
like Bik1p, which have been shown to be limiting in cells of higher ploidy
(see below) (Lin et al.,
2001), and the repression of G1 cyclin genes (Galitski et al.,
1988). The latter requires the cell to be larger before it passes through
START and replication (for a recent review, see
Rupes, 2002
). Accordingly,
cells of higher ploidy will require more time before they enter premeiotic
S-phase and initiate first meiotic prophase. This could explain the generally
retarded entry into prophase I, signified by the delay of cen-cluster
resolution and bouquet formation in the tetraploid strain. Thus, the transient
increase of the frequency of cells with a single cen-cluster between 180 and
240 minutes after induction of meiosis
(Fig. 3) probably relates to a
delayed entry of a subpopulation of polyploid cells, possibly smaller daughter
cells that will require longer to pass through the premeiotic divisions
(Rupes, 2002
). This feature
will probably reduce synchrony in the tetraploid and other time courses, which
becomes particularly evident when cultures are investigated by cytology (see
below) (Trelles-Sticken et al.,
1999
).
Haploid meiocytes undergo wild-type telomere redistribution
We also investigated prophase I progression in haploid yeast meiosis. Our
haploid SK1 strain can undergo meiosis because of the deletion of
SIR3, which simulates heterozygosity of the mating type locus
(Rine and Herskowitz, 1987),
and SPO13 disruption, which induces the cell to skip the meiosis I
division, leading to the formation of two viable spores
(Klapholz and Esposito, 1980
;
Wagstaff et al., 1982
). We
consider the spo13 mutation not to be relevant to the analysis of
chromosomal events during the onset of prophase I in the haploid SK1 strain,
because the spo13-dependent delay is mediated through the meiosis I
spindle checkpoint (Lee et al.,
2002
; Shonn et al.,
2002
) and because centromere cluster resolution occurred at a
similar pace in diploid wild-type and haploid meiosis
(Fig. 3). Further analysis is
required to tease apart the spo13
- and
sir3
-induced defects in haploid prophase I.
Little, however, is known about the effects of the SIR3 deletion
on chromosomal events in meiosis. SIR (silent information regulator) proteins
mediate transcriptional silencing at silent mating type loci and in the
vicinity of telomeres, the latter being known as telomere position effect
(TPE) (Gottschling et al.,
1990; Stone and Pillus,
1998
). Telomeric heterochromatin is built on the interactions of
Sir2p, Sir3p and Sir4p with Rap1p and histones H3 and H4
(Hecht et al., 1996
;
Kyrion et al., 1993
; reviewed
by Grunstein et al., 1998).
Although it has been shown that sir3 mutations lead to moderately
shortened telomeres and delocalize Rap1p in diploid cells
(Palladino et al., 1993),
Sir3p and Sir4p have been found to be dispensable for the localization and
clustering of vegetative telomeres (Gotta
et al., 1996
). We also observed that the haploid
sir3
SK1 meiosis displayed centromere dispersion comparable to
that in the diploid wild type, whereas telomeres congregated at the haploid
sir3
SPB. This indicates that the programme for bouquet
formation is triggered by nitrogen starvation and the expression of both
mating type loci in the same cell. These observations agree with genetic data
indicating that bouquets form in haploid yeast
(Rockmill and Roeder, 1998
).
Because a chromosomal bouquet has been observed in haploid rye
(Santos et al., 1994
), it
appears that the signal for telomere clustering in synaptic meiosis does not
depend on the presence of homologous chromosomes.
During diploid wild-type SK1 sporulation, telomere clustering peaked
reproducibly around 210 minutes (see Results)
(Trelles-Sticken et al.,
1999), whereas peak values were seen at 300 minutes after
induction of haploid meiosis. Increased bouquet frequencies also persisted in
prophase I of diploid spo11
and rad50S recombination
mutants, in which DSB formation or repair, respectively, is blocked
(Trelles-Sticken et al.,
1999
). In haploid meiosis, DSB formation is reduced and repair is
delayed compared with diploid meiosis (De
Massy et al., 1994
; Loidl and
Nairz, 1997
; Loidl,
1995
). Given the similar pace of centromere cluster resolution in
the control and kar3
haploid compared with diploid wild-type
SK1 meiosis, the prevalence of haploid bouquet cells at later time points
might relate to the absence of appropriate conditions or signals from the
machinery that monitors the initiation and progression of meiosis-specific
events like DSB formation and/or synapsis
(Roeder and Bailis, 2000
;
Keeney, 2001
).
In the absence of homologous chromosomes, one escape route from a potential
checkpoint that may monitor chromosomal events (synapsis)
(Odorisio et al., 1998;
Roeder and Bailis, 2000
) could
be heterosynapsis between non-homologous chromosomes or chromosome arms
(foldback pairing) and repair of DSBs among sister chromatids. It is
intriguing that foldback pairing is observed in haploid meiosis of plants and
yeast (Levan, 1942
;
Loidl et al., 1991
;
Santos et al., 1994
).
Heterosynapsis is thought to occur late in first meiotic prophase, when
homology is no longer required (Moses et
al., 1984
; von Wettstein et
al., 1984
). However, some sort of checkpoint seems to operate
during haploid yeast meiosis, because only 54% of the cells that entered
meiosis passed the MI division.
Sir3p is dispensable for telomere clustering in diploid meiosis
Because haploid meiosis represents an experimental system, we also
investigated the impact of SIR3 disruption on diploid meiosis.
Centromere cluster dissolution was biphasic during diploid
sir3 sporulation. Delayed resolution of the centromere cluster
suggests that the switch from vegetative to meiotic nuclear architecture be
perturbed by the SIR3 mutation in the diploid condition. It is
possible that this is a consequence of the distorted gene expression that
occurs in the absence of functional Sir3p deletion of SIR3
has been reported to lead to transcriptional repression of, for example, the
RME1 repressor of meiosis (Wyrick
et al., 1999
; Mitchell and
Herskowitz, 1986
). SIR3 deletion has also been shown to
alter the expression of several genes required for S-M phase progression, like
BRN1 (Ouspenski et al.,
2000
; Lavoie et al.,
2000
), BIK1 (Berlin et
al., 1990
; Lin et al.,
2001
) and Cdc15 (Schweitzer and Philippsen, 1997;
Tinker-Kulberg and Morgan,
1999
). Interestingly, Bik1p, a yeast mitogen-associated protein
(MAP) that is required for microtubule attachment at the kinetochore
(Berlin et al., 1990
;
Lin et al., 2001
), interacts
with Ndc10p (Cbf2p), a component of the yeast kinetochore complex
(Lin et al., 2001
), which is
necessary for vegetative centromere clustering
(Jin et al., 2000
). This makes
it likely that kinetochore detachment (centromere cluster resolution) at
meiosis could be actively controlled. Semi-quantitative RT-PCR data indicate
derepression of BRN1 and BIK1 expression that persists into
diploid sir3
meiosis (E.T-S. and H.S., unpublished). An
altered pool of the yeast condensin Brn1p and the kinteochore MAP Bik1p could
influence the progression of sir3
diploids through the last
premeiotic division and/or distort centromere dispersion during prophase I.
Interestingly, the administration of microtubule-disrupting drugs 120 minutes
after induction of meiosis (when premeiotic mitoses have ceased) delayed
centromere-cluster resolution (E.T-S. and H.S., unpublished). Thus, it seems
likely that centromere cluster resolution during prophase I is an active
process that in some way requires intact microtubules.
Probably as a consequence of the retarded resolution of centromere
clustering and/or altered gene expression in the absence of Sir3p, peak
frequencies of bouquet nuclei and homologue pairing were also delayed in
diploid sir3 meiosis. The observation that the telomeres
cluster at the haploid and diploid sir3
SPBs suggests that the
telomeric heterochromatin protein Sir3p is not directly required for meiotic
telomere movements.
Role of Kar3p in meiotic telomere clustering
Bouquet formation in S. cerevisiae has been shown to depend on the
presence of the meiosis-specific telomere protein Ndj1p
(Chua and Roeder, 1997;
Conrad et al., 1997
;
Trelles-Sticken et al., 2000
).
Furthermore, it has long been assumed that the repositioning of telomeres
during meiotic prophase requires the action of motor proteins
(Loidl, 1990
;
Zickler and Kleckner, 1998
).
In the asynaptic prophase of the fission yeast S. pombe, it has been
found that microtubule-disrupting drugs and mutations in the myosin heavy
chain motor protein disrupt nuclear motility during prophase I and reduce the
rate of recombination and homologue pairing
(Svoboda et al., 1995
;
Yamamoto et al., 1999
).
In budding yeast, the kinesin-like motor protein Kar3p
(Meluh and Rose, 1990) is
required for the congression step of haploid nuclei, during which these
approach each other to form a zygote nucleus
(Rose, 1996
). Based on the
observation that a KAR3 deletion can lead to reduced levels of
recombination and deterioration of synapsis, it has been suggested that Kar3p
could be a telomere motor (Bascom-Slack et al., 1997). Thus, we directly
tested whether a KAR3 deletion does impair meiotic telomere
clustering. We had to make use of haploid meiosis because several attempts to
generate sporulating diploid kar3
strains failed in the SK1
strain background. Analysis of the expression of the meiosis-specific telomere
protein Ndj1p in haploid control and kar3
cultures allowed to
investigate exclusively meiotic cells. It was found that meiotic telomere
clustering at the kar3
SPB, which provides physical evidence
that the meiotic defect mediated by KAR3 deletion does not impair
bouquet formation in the haploid and likely in other conditions too.
Surprisingly, the frequency of haploid bouquet-stage nuclei was elevated in
the kar3
condition, which indicates that the bouquet stage
might be extended in absence of Kar3p. Thus, Kar3p might be involved in the
control of meiotic telomere clustering and especially in its release, which is
compatible with the microtubule-destabilizing properties of Kar3p
(Cottingham et al., 1999
;
Saunders et al., 1997
).
Given that the induction of haploid meiosis is less effective in the
absence of Kar3, it appears that the reduced levels of recombination
observed by molecular analysis in diploid kar3 meiosis
(Bascom-Slack et al., 1997) might relate to the reduced levels of
meiosis-specific events at any time point in meiosis compared with the
control, and/or to the response of a meiotic checkpoint
(Shanks et al., 2001
). This
reasoning is supported by the observation that the ndj1
bouquet mutant, unlike kar3
(Bascom-Slack et al., 1997), only
displays minor defects in recombination
(Chua and Roeder, 1997
).
Although meiotic telomere clustering occurs in the absence of Kar3p, it
remains to be seen whether other yeast microtubule motor proteins
(Hildebrandt and Hoyt, 2000
)
could play a more dominant role in telomere movements at meiosis.
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References |
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