1 The Department of Chemistry and Biochemistry, Miami University, Oxford, OH
45056, USA
2 Department of Botany, Miami University, Oxford, OH 45056, USA
* Author for correspondence (e-mail: makaroca{at}muohio.edu)
Accepted 7 April 2003
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Summary |
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Key words: Meiosis, Arabidopsis, Cohesins, Synapsis
![]() |
Introduction |
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Sister chromatid cohesion also serves a critical role in meiosis; however, there are several important differences between its roles in meiosis and mitosis. In the first meiotic division, which is a reductional division, homologous chromosomes segregate. Attachment of sister kinetochores to spindles occurs with the same polarity, termed monopolar attachment, ensuring that homologs and not sister chromatids segregate in this first division. Therefore, whereas sister chromatids are attached to microtubules emanating from opposite poles during mitosis, they attach to microtubules from the same pole during meiosis I. Also, with few exceptions, recombination between homologous chromosomes occurs during meiotic prophase to form chiasmata, which hold maternal and paternal chromosomes together. In order for homologs to separate during anaphase I, chiasmata between homologous chromosomes must be resolved and sister chromatid cohesion released along the arms. Therefore, meiotic divisions require sister chromatid cohesion to be released in two steps. In the first step cohesion is released along the arms to facilitate resolution of chiasmata while it is maintained at the centromeres. Destruction of centromeric cohesion at anaphase II then allows the separation of sister chromatids in an equational division.
Sister chromatid cohesion during meiosis is mediated by cohesin complexes
that are similar to their mitotic counterparts and share many of the same
subunits (reviewed in Lee and Orr-Weaver,
2001; Orr-Weaver,
1999
). However, meiotic cohesin contains at least one, and in some
instances more, meiosis-specific forms of the core cohesin proteins, including
REC8 for SCC1 and in animal cells SMC1ß for SMC1 and STAG3 for SCC3
(Prieto et al., 2001
;
Revenkova et al., 2001
). All
meiotic cohesin complexes studied to date contain the meiosis-specific REC8
cohesin. Mutations in REC8 have been identified and characterized in a number
of organisms (Bai et al., 1999
;
Bhatt et al., 1999
;
Klein et al., 1999
;
Lin et al., 1992
;
Parisi et al., 1999
;
Pasierbek et al., 2001
;
StoopMyer and Amon, 1999
;
Watanabe and Nurse, 1999
). In
S. cerevisiae and S. pombe, rec8 mutations result in reduced
recombination, alterations in synaptonemal complex formation and premature
separation of sister chromatids (Molnar et
al., 1995
; Klein et al.,
1999
; Watanabe and Nurse,
1999
). In C. elegans, depletion of REC8 using RNAi
resulted in the formation of univalents and chromosome fragmentation at
diakinesis (Pasierbek et al.,
2001
). Finally, Arabidopsis plants containing mutations
in the REC8/SCC1 ortholog, referred to as SYN1/DIF1, exhibit alterations in
chromosome condensation and cohesion that lead to chromosome fragmentation at
metaphase I (Bai et al., 1999
;
Bhatt et al., 1999
).
REC8 localization studies on meiotic chromosomes have been conducted in
several organisms. S. pombe Rec8 is present from the time of
premeiotic DNA synthesis until after meiosis I
(Parisi et al., 1999). It is
localized as foci throughout chromosomes, with the highest concentration at
the centromeres (Watanabe and Nurse,
1999
). Likewise, S. cerevisiae Rec8 is found as punctate
foci along chromosomes in early prophase I. It subsequently localizes to
centromeric regions where it persists until approximately anaphase II
(Klein et al., 1999
). REC8
localization patterns in C. elegans were similar to those observed in
yeast. Specifically, REC8 was partially lost along chiasmata-distal portions
of the arms at anaphase I and at the centromeres at metaphase II
(Pasierbek et al., 2001
).
Differences have also been reported concerning the release of sister
chromatid cohesion during meiosis. In yeast and C. elegans the
anaphase promoting complex (APC)-activated separase pathway is required for
the release of cohesin at the onset of anaphase I
(Buonomo et al., 2000;
Siomos et al., 2001
). In
contrast, experiments in Xenopus have suggested that chromosome
segregation at meiosis I takes place in the absence of APC activity and in the
presence of high levels of securin, the separase inhibitor
(Peter et al., 2001
;
Taieb et al., 2001
). This
suggests that removal of cohesin from the arms of Xenopus chromosomes
during meiosis I may occur by a mechanism similar to that observed for the
removal of cohesin from other vertebrate arms during mitotic prophase
(Darwiche et al., 1999
;
Losada et al., 1998
;
Sumara et al., 2000
;
Waizenegger et al., 2000
;
Warren et al., 2000
). Finally,
phenotypic differences have also been observed in cells containing mutations
in cohesin subunits. For example, in rec8 mutants of S.
cerevisiae, chromosomes segregate randomly at meiosis I
(Klein et al., 1999
), whereas
in S. pombe rec8 mutants, sister chromatids segregate equationally at
anaphase I (Watanabe and Nurse,
1999
). Therefore, a number of differences in the distribution and
release of REC8 as well as the effect of rec8 mutations have been
identified in the relatively few organisms studied to date. This suggests
that, while the general nature and properties of meiotic cohesin complexes are
similar, differences probably exist in the way cohesion is controlled in
different organisms.
As part of studies to better understand sister chromatid cohesion in
plants, we have further characterized the role of a putative
Arabidopsis REC8 ortholog, SYN1, which we previously identified in a
T-DNA-tagged, meiotic mutant (syn1) of Arabidopsis
(Bai et al., 1999;
Peirson et al., 1997
;
Peirson et al., 1996
).
Preliminary cytological studies indicated that syn1 plants exhibit
defects in chromosome cohesion and condensation that result in fragmentation
of the chromosomes and the formation of polyads
(Bai et al., 1999
;
Peirson et al., 1997
). In the
experiments described below, we show that SYN1 encodes a protein that
localizes to arms of meiotic chromosomes from approximately S phase to
anaphase I. The protein is not detected at the centromeres or after metaphase
I. Furthermore, fluorescence in situ hybridization (FISH) experiments on
microsporocytes from syn1 plants demonstrate that the mutation
eliminates arm cohesion as early as leptotene whereas centromere cohesion is
maintained until approximately anaphase I.
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Materials and Methods |
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SYN1 antibody production
A portion of the SYN1 cDNA containing amino acids 178 to 353 was cloned
into pET24b (Novagen), transformed into BL21(DE3)pLysS cells and overexpressed
as a histidine-tagged protein. Upon induction the overexpressed protein
accumulated in the insoluble fraction. Inclusion bodies were collected from
overexpressing cells, washed, solubilized in PBS containing 8 M urea and
purified using nickel chromatography. The isolated protein was further
purified by SDS polyacrylamide gel electrophoresis and used to inject New
Zealand White rabbits using standard procedures
(Harlow and Lane, 1988). The
antibody was affinity purified using the E. coli expressed protein
and found to be specific for SYN1; it did not cross-react with the three other
SYN1-like Arabidopsis proteins expressed in E. coli.
Immunolocalization
Inflorescences of 15-18 day old plants were fixed for 2 hours in Buffer A
(Dernburg et al., 1996)
containing 4% paraformaldehyde, washed twice and stored at 4°C in Buffer A
until needed. Buds were staged by squashing and staining an individual anther
in acetic orcein. The remaining anthers were squashed between two
perpendicular poly-L-lysine slides. Male meiocytes were covered with a thin
layer of agarose and treated with ß-glucuronidase
(Peirson et al., 1997
). After
washing in 1x PBS, the slides were blocked in 1x PBS containing 5%
BSA for 60 minutes and then incubated overnight at 4°C in a moist chamber
with anti-SYN1 antibody, diluted 1:250 in blocking buffer. After washing, the
slides were treated with Alexa-488-labeled goat anti-rabbit secondary antibody
for 2 hours at room temperature. After washing, the DNA was stained with 2
µg/ml propidium iodide (PI) and the slides mounted in DABCO antifade
mounting media. Samples were viewed with a Nikon PMC-2000 Confocal Microscope
System. Individual optical z-sections were captured, the three-dimensional
data were stacked (maximum intensity) using Image Pro Plus and were
represented as two-dimensional images.
Fluorescence in situ hybridization (FISH)
Inflorescences were fixed in acetic alcohol (ethanol:glacial acetic acid,
3:1) for 2 hours at room temperature and stored at -20°C after
replenishing the fixative. Staged buds were subjected to FISH using previously
published procedures (Caryl et al.,
2000; Fransz et al.,
1996
). The following probes were used in this study: (1) pAL1
containing a pericentromeric 180 bp repeat
(Martinez-Zapater et al.,
1986
); and (2) BAC probes F15E21 and MFG13 corresponding to the
lower arms of chromosomes one and five, respectively. Southern blotting
demonstrated that the BAC clones hybridized to a single copy region of the
genome. The pAL1 probe was generated by primary PCR amplification using the
M13 forward and reverse primers followed by random primer labeling in the
presence of biotin-labeled dUTP (Roche). The BAC probes were digested with
EcoR1 to fragment the DNA followed by random primer labeling in the
presence of biotin-labeled dUTP. Biotin-labeled probes were used in
hybridization solution at 10 µg/ml and detected with 10 µg/ml
fluorescein-labeled streptavidin. Slides were counterstained with PI, mounted
and viewed as above.
Dual FISH and immunolocalization
Buds were fixed and spread as described above for immunolocalization
experiments. In situ hybridization using DNA probes was conducted essentially
as described previously (Dernburg et al.,
1996). Specifically, agarose-covered, poly-L-lysine slides
containing spread PMCs were washed twice in Buffer A, twice in 20% deionized
formamide/2x SSC, twice in 40% deionized formamide/2x SSC/0.1%
Tween-20 and twice in 50% deionized formamide/2x SSC/0.1% Tween-20.
Hybridization solution (50% deionized formamide/2x SSC/0.1% Tween-20/10%
Dextran Sulfate) containing a PCR fragment (15 µg/ml) corresponding to the
pericentromeric 180 bp repeat labeled with digoxygenin-labeled dUTP (Roche)
was added and the specimen covered with a coverslip and sealed with rubber
cement. The slides were incubated at 40°C for 30 minutes, denatured at
96°C for 6 minutes and then hybridized overnight at 37°C. After
hybridization the slides were washed in 50% deionized formamide/2x SSC
at 37°C and 20% deionized formamide/2x SSC/0.1% Tween-20, 2x
SSC/0.1% Tween-20 and 2x SSC/5% BSA, all at room temperature. They were
then incubated in binding solution (2x SSC/0.1% Tween-20/5% BSA)
containing 20 µg/ml mouse anti-digoxygenin (Roche) for 1 hour at 37°C.
After washing, the slides were incubated in binding solution containing 20
µg/ml Texas Red goat anti-mouse IgG (Jackson Labs) for 1 hour at room
temperature, washed in 2x SSC/0.1% Tween-20 and incubated overnight at
4°C in binding solution containing anti-SYN1 antibody (1:250 dilution).
After washing the slides were incubated in 20 µg/ml Alexa-488-labeled goat
anti-rabbit IgG (Molecular Probes) in binding solution at 37°C for 1 hour,
washed, mounted and viewed as above.
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Results |
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Syn1 plants are defective in arm cohesion but maintain centromere
attachment until anaphase I
Sister chromatid cohesion and homologous chromosome pairing was
investigated using Alexa-488-labeled BAC clones, which correspond to the arms
of chromosomes one and five. Similar results were obtained with both probes.
In wild-type plants two FISH signals were typically observed in cells during
meiotic interphase and early leptonema when probes corresponding to chromosome
arms were used (Fig. 1A).
During zygonema in wild-type meiocytes, the number of arm signals was reduced
from two to one as homologous chromosomes paired
(Fig. 1B). One signal was
typically observed through diakinesis (Fig.
1C-D). In contrast, when probes corresponding to chromosome arms
were used in FISH against syn1 microsporocytes, four distinct signals
were normally observed from meiotic interphase to metaphase
(Fig. 1E-H). The presence of
four arm signals was consistent with a lack of sister chromatid cohesion.
Occasionally cells with two or three signals were observed; however, most
cells contained four (average=3.9, Table
1). This indicated that in addition to a lack of sister chromatid
arm cohesion, the arms of homologous chromosomes failed to pair in
syn1 plants.
|
|
Cohesion and pairing of centromeric regions was also examined by FISH using
PAL1, a 180 bp tandemly repeated sequence that localizes to the central domain
of the pericentromeric heterochromatin of all 10 Arabidopsis
chromosomes (Fransz et al.,
1998). In wild-type plants approximately 10 centromere signals
(eight to 10) were observed during meiotic interphase and leptonema
(Table 1). As expected, during
zygotene and pachytene between two and five centromere signals were observed,
with averages of 4.2 and 4.9 respectively. During diplotene/diakinesis in
wild-type meiocytes, five to six signals were typically observed. These
results are consistent with those previously observed in wild-type
Arabidopsis (Armstrong et al.,
2001
). Syn1 meiocytes resembled wild-type plants during
meiotic interphase and leptotene, exhibiting approximately 10 (six to 10)
centromere signals (Fig. 2A-B).
This result contrasts with results obtained with arm-specific probes and
suggests that sister chromatid cohesion at the centromeres of syn1
meiocytes was maintained during prophase. Consistent with a general lack of
pairing, syn1 meiocytes continued to exhibit approximately 10
centromere signals throughout prophase
(Fig. 2C-D;
Table 1). In a number of cells,
fewer than 10 centromere signals (six to nine) were observed
(Table 1), suggesting that some
pairing of centromeric regions may have occurred. However, we believe that it
is more likely that the reduced number of signals in these cells is not due to
chromosome pairing, but rather because of the generally intertwined and sticky
nature of chromosomes in syn1 meiocytes, and possibly general
centromere clustering.
|
Approximately eight (five to 10) centromere signals were detected in optical sections of syn1 meiocytes at metaphase I (Fig. 2E). In contrast, 12 to 18 signals were present in the 43 cells observed at telophase I (average=14.8; Fig. 2F). Because of the highly condensed nature of the chromosomes at telophase I, we believe that this number may actually be an under-representation of the true number of centromere signals. Nonetheless, the presence of more than 10 centromere signals indicates that although sister chromatids remain associated at their centromeres up to metaphase I, centromere cohesion is lost by telophase I. During metaphase I, centromere signals were always found in the condensed chromosome mass at the center of the cell (Fig. 2F), suggesting that the presence of a centromere was sufficient for movement of the chromosome to the spindle assembly. Consistent with this is the observation that centromere signals were always found at the spindle poles during anaphase I. In contrast, acentric chromosome fragments, present in syn1 meiocytes, failed to attach to the spindles, although they too were often found in the center of the cell (Fig. 2F). The absence of centromere sequences confirmed that they represented chromosome fragments rather than univalents. Taken together these results confirm that syn1 meiocytes are defective in sister chromatid cohesion and homologous chromosome pairing; however, sister chromatids remain attached at their centromeres up to metaphase I.
SYN1 localizes to the arms of meiotic chromosomes from approximately
interphase to metaphase I
To examine the distribution of SYN1 on chromosomes during meiosis,
antibodies were raised to the central portion (amino acids 176-353) of SYN1.
This region was chosen because it is the least conserved portion of the
protein. Arabidopsis contains four SCC1/REC8 paralogues. Like all
SCC1/REC8 proteins, the greatest similarity is found at the N- and C-terminal
regions of the proteins. In contrast the central portions of the proteins show
very little (less than 15% identity) sequence conservation
(Dong et al., 2001).
Consistent with this observation, the SYN1 antibodies did not crossreact with
E. coli expressed protein for the three other Arabidopsis
cohesin proteins, SYN2, 3 and 4 (data not shown).
Immunolocalization experiments on wild-type microsporocytes with SYN1 antibody revealed a strong signal in the nucleus beginning at approximately meiotic interphase. Meiocytes at interphase displayed diffuse chromatin and SYN1 labeling (Fig. 3A). Although some labeling was observed in the centrally located nucleolus, the SYN1 signal was clearly stronger in the surrounding nucleoplasm. During early leptotene, the SYN1 signal associated with the condensing chromatin (Fig. 3B). As meiocytes proceeded through leptotene, SYN1 labeling went from a relatively diffuse pattern (Fig. 3C) at early stages to approximately 100 large foci at the leptotene/zygotene transition (Fig. 3D). During zygotene and pachytene, the SYN1 signal was distributed over most of the chromosomes (Fig. 3E-G). As the chromosomes began to condense during diplotene, SYN1 labeling was reduced and began to shift from the chromosomes into the nucleoplasm (Fig. 3H). As the cells proceeded through diplotene and diakinesis, labeling in the nucleoplasm became progressively stronger until it completely filled the nucleus (Fig. 3I,J). No labeling was detected in the nucleolus. By the time the nuclear envelope had broken down SYN1 was only detected on prometaphase chromosomes, suggesting that after release the protein is degraded (Fig. 2K). As cells proceeded through metaphase I the SYN1 signal became progressively weaker until it was no longer detectable by the beginning of anaphase I (Fig. 3L-N). SYN1 was never observed on chromosomes after the onset of anaphase I. However, we were able to detect very weak SYN1 staining in the nucleus of interphase II cells (Fig. 3O). Staining of cells at this stage was always weak and short-lived.
|
SYN1 labeling was never observed in somatic cells of the anther or in
Arabidopsis cell cultures (data not shown). This was consistent with
results from in situ hybridization experiments, in which SYN1 transcripts were
only detectable in the locules of stage 8 and 9 anthers (data not shown).
Likewise, SYN1 labeling was not detected in meiocytes of syn1 plants
(data not shown), which confirms that the antibody is specific for SYN1. Taken
together these results support phylogenetic evidence
(Bai et al., 1999), indicating
that SYN1 is the Arabidopsis REC8 ortholog.
Results shown in Fig. 3H-J indicated that most SYN1 was lost from the chromosomes during diplotene/diakinesis. Fig. 4A-D clearly shows that during diplotene SYN1 labeling associated with the condensing chromosomes is dramatically reduced. By diakinesis most of the SYN1 labeling is not associated with the chromosomes, rather it appears to be free in the nucleoplasm. At these stages signal was never associated with the nucleolus. During metaphase I, SYN1 was detectable on the chromosomes, but not in the nucleoplasm (Fig. 4E,F). In contrast to its localization during zygotene and pachytene, SYN1 labeling was more narrowly focused on metaphase chromosomes. By early anaphase I SYN1 was clearly no longer detectable (Fig. 4H). SYN1 signal was never observed in the approximately 50 anaphase I cells that were examined. However, during meiotic interphase II, SYN1 was briefly detected in the nucleus of meiocytes. This signal was very transient and disappeared before metaphase II (data not shown). Therefore, SYN1 was detectable in meiocytes from approximately interphase I to interphase II. Most of the protein appeared to disassociate from the chromosomes at diplotene/diakinesis, and labeling of the chromosomes was not detected after metaphase I.
|
Results from our immunolocalization studies suggested that SYN1 was localized preferentially along the arms of meiotic chromosomes and not at the centromeres. In order to investigate this possibility further we conducted dual SYN1 immunolocalization/centromere FISH experiments. Centromere labeling patterns and the distribution of SYN1 in the dual immunolocalization/centromere FISH experiment resembled the results obtained for the individual experiments. During leptotene, zygotene and early pachytene (Fig. 5A-C), SYN1 labeling was clearly evident on chromosome arms. However, the dispersed nature of the chromosomes made it difficult to determine if the centromeres were also labeled with the SYN1 antibody. Beginning at late pachytene (Fig. 5D), as the centromeres started to become distinguishable from the chromosome arms, SYN1 labeling was not detected at centromeric regions (verified by 3D analysis; however only 2D projections are shown). Likewise, as overall SYN1 levels decreased during diplotene, diakinesis and metaphase, labeling was found primarily along the chromosome arms and not at the centromeres (Fig. 5E-H). In the approximately 350 centromeres examined in >70 cells observed at these stages, overlap between the SYN1 and centromere signals was observed 38 times (11%). In cells that were oriented such that the centromeres were clearly distinguishable from the arms, no overlap in labeling was detected between SYN1 and the centromere repeat clone. Therefore, SYN1 is generally not detectable at the centromeres.
|
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Discussion |
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Defects in sister chromatid cohesion interfere with homologous
chromosome pairing in Arabidopsis
Varying phenotypes have been observed for rec8 mutants in other
organisms. For example, when REC8 is depleted using RNAi in C.
elegans, meiotic chromosomes undergo presynaptic alignment, but not
synapsis (Pasierbek et al.,
2001). Separation of sister chromatids is observed as early as
leptotene but is typically not wide spread until diakinesis. In contrast,
approximately 70% of S. cerevisiae cells carrying a rec8
mutation exhibit FISH labeling patterns consistent with defects in both
cohesion and chromosome pairing/alignment
(Klein et al., 1999
).
Interestingly, in S. pombe cells containing the rec8-110
mutation, pairing of interstitial and centromeric chromosome regions was
strongly impaired, whereas pairing at chromosome ends was less impaired
(Molnar et al., 1995
).
Our results indicate that the phenotype of syn1 meiocytes is similar to that observed in S. cerevisiae; specifically, SYN1 is required for sister chromatid arm cohesion and homologous chromosome pairing. Results from FISH experiments on syn1 plants revealed, in general, four arm-specific signals throughout prophase consistent with both a lack of sister chromatid arm cohesion and homologous chromosome pairing. Approximately 10 (six to 10) centromere signals were observed from meiotic interphase to anaphase I, when on average 15 (12 to 18) signals were observed (Table 1). These results are also consistent with a lack of homologous chromosome pairing but suggest that sister chromatids remain attached at their centromeres until anaphase I. At this point it is not clear why we see differences in cohesion between chromosome arms and the centromeres. One possibility is that the centromeres remain topologically intertwined during prophase. It is also possible that another protein helps link the centromeres prior to anaphase I in Arabidopsis.
SYN1 is not detectable at the centromeres of prophase chromosomes or
in association with chromosomes after anaphase I
SYN1 is clearly detectable in the nuclei of meiocytes at approximately
interphase. It is likely that SYN1 is present as early as S phase, as appears
to be the case in S. pombe
(Parisi et al., 1999).
However, because it is difficult to accurately assess the stage of cells
within this period, we can not conclude with certainty that SYN1 is present
during S phase. As meiocytes proceed through leptotene, SYN1 labeling
progresses from a relatively diffuse pattern to approximately 100 large foci
at the leptotene/zygotene transition (Fig.
2D). During zygotene and pachytene, chromosome arms are completely
covered with SYN1 (Fig. 2E-G).
During diplotene and diakinesis, much of the SYN1 signal is released and moves
into the nucleoplasm as the chromosomes condense
(Fig. 2H). By prometaphase I,
unbound SYN1 is no longer detected in the cell. It is, however, still
localized to discreet regions of the chromosomes where it persists until the
metaphase/anaphase transition. By the beginning of anaphase I, SYN1 was no
longer detectable (Fig. 2M). These results for SYN1 were generally consistent with those observed in other
organisms.
S. cerevisiae Rec8 was initially found as punctate foci during
leptotene and zygotene (Klein et al.,
1999). By pachytene it formed a continuous line along the
longitudinal axes of the chromosomes. By anaphase I, Rec8 labeling was
dramatically reduced but still detectable as a number of small foci that
remained visible in the vicinity of the centromeres until shortly after the
onset of anaphase II. In S. pombe, Rec8 was highly enriched in
centromeric regions from pre-meiotic S phase through metaphase II
(Parisi et al., 1999
).
Finally, C. elegans REC8 was found as dots along unsynapsed
chromosome axes during leptotene/zygotene
(Pasierbek et al., 2001
).
During pachytene, REC8 antibodies associated with the SC. From anaphase I
through metaphase II, REC8 signals were less intense and restricted to the
region between the centromere and the chiasmata. Therefore, in both S.
cerevisiae and C. elegans REC8 appeared to localize to the SC
during pachytene, and REC8 was localized to the centromeres of all three
organisms from anaphase I to metaphase II. Although the resolution of our
experiments does not allow us to say with certainty that SYN1 is associated
with the SC, on the basis of its distribution and the results from other
systems, this is highly likely.
The greatest difference between our results and those obtained in S.
cerevisiae, S. pombe and C. elegans is our observation that SYN1
antibody does not localize to centromeric regions or to meiotic chromosomes
after metaphase I. This raises the interesting possibility that SYN1 is not
involved in maintaining centromeric cohesion and that one of the other three
Arabidopsis cohesin proteins (SYN2, SYN3 or SYN4) may be responsible
for centromeric cohesion. If true, then this would suggest that meiotic
chromosome cohesion is controlled differently in plants than in other
organisms. However, several observations suggest that this is not the case.
First, SYN1 staining was detected in the nucleus of interphase II cells
(Fig. 3O). Although this
staining was always weak and short-lived, it was reproducible. This suggests
that low levels of the protein may be present on the chromosomes of anaphase I
cells but that it is inaccessible to the antibody. This is similar to results
obtained with antibodies to vertebrate SCC1, which failed to detect the
protein on metaphase chromosomes (Darwiche
et al., 1999; Losada et al.,
1998
; Sumara et al.,
2000
). Through the use of myc-tagged SCC1 Waizenegger et al. were,
however, able to demonstrate that SCC1 is present on metaphase I chromosomes
and that it localizes to the centromeres
(Waizenegger et al., 2000
).
Our observation that the SYN1 signal is relatively weak during diplotene is
consistent with the theory that chromosome conformation and possibly the
location of the protein on the chromosomes has a major effect on the observed
signal. During pachytene the SYN1 signal is very strong
(Fig. 3F), whereas during
diplotene the total level of SYN1 signal is dramatically reduced as the
chromosomes condense (Fig. 3H).
SYN1 signal is again relatively high at late diakinesis when most of the
signal is no longer associated with the chromosomes
(Fig. 3J). Finally, similar to
other rec8 mutants, meiocytes in syn1 plants exhibit defects
in centromere cohesion beginning at approximately anaphase I. This indicates
that SYN1 plays an important role in centromere cohesion. Therefore, we
believe that SYN1 is present at the centromeres of meiotic chromosomes but
that it is not detectable with our antibody. We are currently investigating
this question further through the use of GFP- and epitope-tagged versions of
SYN1 and detailed localization studies for SYN2, SYN3 and SYN4.
Most SYN1 crossreactive material is released from Arabidopsis
chromosomes beginning at approximately diplotene. From diplotene to
prometaphase I SYN1 labeling is very strong in the nucleoplasm. This is in
contrast to results obtained in C. elegans where REC8 was observed
along the axes of diplotene/diakinesis chromosomes. It was not until the cells
entered metaphase I that REC8 labeling appeared to become progressively less
intense. The release of SYN1 from the chromosomes coincident with chromosome
condensation during diplotene/diakniesis resembles the situation in mitotic
cells of animals where the bulk of cohesin is removed from chromosomes during
prophase (Losada et al.,
1998). This early release of cohesin from chromosome arms is
referred to as the prophase pathway and is separase independent
(Waizenegger et al., 2000
).
The segregation of chromosomes at meiosis I in Xenopus also appears
to take place in the absence of APC activity and in the presence of high
levels of securin, the separase inhibitor
(Peter et al., 2001
;
Taieb et al., 2001
). In
contrast, in S. cerevisae and C. elegans, the resolution of
chiasmata along meiotic chromosome arms depends on the cleavage of REC8 by
separase at the onset of anaphase I
(Buonomo et al., 2000
;
Siomos et al., 2001
). At this
time it is not known if the removal of SYN1 from meiotic chromosome arms is
separase dependent. The Arabidopsis genome does, however, contain a
putative separase homologue, suggesting that the APC-mediated pathway is
utilized for the release of centromeric and/or possibly arm cohesion.
Experiments are underway to investigate this question.
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Acknowledgments |
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References |
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