1 Institute of Molecular and Cell Biology, National University of Singapore, 30
Medical Drive, Singapore 117609
2 MRC Centre for Developmental Neurobiology, King's College London, 4th Floor,
New Hunts House, Guy's Hospital Campus, London SE1 1UL, UK
* Author for correspondence (e-mail: mcbmw{at}imcb.nus.edu.sg)
Accepted 21 January 2003
![]() |
Summary |
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Key words: EAST, Meiosis, Mitosis, Chromosome congression, Nucleoskeleton
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Introduction |
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During the cell cycle, nuclear architecture undergoes dramatic changes as
the interphase nucleus is dismantled during entry into mitosis and reassembled
during exit from mitosis. During entry into mitosis, the nucleolus, nuclear
envelope (NE) and lamina dissolve and their components become vesiculated
(Moir et al., 2000;
Olson et al., 2000
). Proteins
of these structures are either degraded or are recycled for the reassembly of
the daughter nuclei. By contrast, other nonchromosomal proteins like CP60 and
Tpr continue to be associated with a `central nuclear remnant', even after NE
breakdown, suggesting that an interior `nuclear' structure derived from the
interphase nucleus persists into mitosis
(Oegema et al., 1997
;
Zimowska et al., 1997
). A
third group of proteins shuttle between the nucleus at interphase and
different parts of the mitotic apparatus such as centrosomes, spindle or
cleavage furrow, where they perform mitosis-specific functions. For instance,
Skeletor, a nuclear protein that is associated with chromosomes during
interphase, forms a spindle matrix at prophase and associates with the spindle
at metaphase (Walker et al.,
2000
). In male meiosis, the actin binding protein Anillin
localizes to the cleavage furrow and plays a role in cytokinesis
(Giansanti et al., 1999
).
We extended our studies on EAST by analyzing its potential role in the cell cycle. We show here that EAST is another member of a group of nonchromosomal nuclear proteins that remain associated with an internal remnant of the interphase nucleus during mitosis. Genetic studies show that east plays an important role in promoting the accuracy of both mitosis and meiosis. In syncytial blastoderm embryos, the removal of east leads to an increase in the frequency of mitotic errors and, as a result, to the elimination of surface nuclei. During mitosis in cellularized embryos, loss of east can delay the congression of chromosomes to the metaphase plate and consequently delay the onset of anaphase. Mutations of east also lead to nondisjunction of achiasmate chromosomes in female meiosis and to abnormal chromosome morphology in male meiosis. Our results indicate that after NE breakdown, a certain structure, which includes components derived from the interphase nucleus like EAST and CP60, acts to constrain chromosome mobility and facilitates the congression of chromosomes to the metaphase plate.
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Materials and Methods |
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Generation of germline clones
Germline clones for lethal east alleles were produced using the
FLP-recombinase dominant female sterility (FLP-DFS) technique
(Chou and Perrimon, 1992).
Lethal east-flippase recombination target (FRT) chromosomes were
generated by meiotic recombination of the east alleles hop-1, hop-5
and hop-7 with P[w+=wFRT]101 at 14A-B. The presence of the FRT site in lethal
recombinants was determined by PCR. The east-FRT recombinants were
crossed to ovoD1-P[w+=wFRT]101; P[ry+=hsFLP38]. Mitotic
recombination in their progeny was induced at the larval third instar stage by
heat shock at 37°C for 2 hours. For observations of live embryos,
easthop-1-FRT/FM7c was combined with the
His2AvDGFP marker. Germline clones were induced in
easthop-1 FRT/ovoD1;
HistoneGFP/+ animals. Control germline clones were induced in y,
w, FRT/ovoD1; HistoneGFP/+ larvae.
Expression of GFP-tagged EAST protein
Two versions of EAST (EASTFL: amino acids 1-2336, EASTC: aa 1-1573)
were expressed with a C-terminally fused green fluorescent protein (GFP) tag
using the GAL4 system (Brand and Perrimon,
1993
). The corresponding coding regions of the east cDNA
were joined in frame with the coding region of GFP derived from the vector
pEGFP-N1 (Clontech, Palo Alto, CA). The joined fragments were cloned into
pUAST (germline transformation vector containing the upstream activating
sequence for the GAL4 transcription factor). Transgenic flies were generated
using standard P-element-mediated transformation techniques
(Spradling, 1986
). Expression
of EASTGFP in mitotic cells of embryos was achieved using the
ubiquitous drivers matalpha4-GAL4-VP16. Expression of EASTGFP
in salivary glands was accomplished using the ftz-GAL4 driver.
Immunocytochemistry
Embryos were fixed in 37% formaldehyde as previously described
(Tio et al., 1999) and double
labeled with mouse anti-EAST (1:1000, ED3) and rat anti-
-tubulin (1:50,
Harlan Sera-Lab, Loughborough, UK). Cy3-conjugated goat anti-mouse and
Cy5-conjugated donkey anti-rat secondary antibodies were used for detection.
Stained embryos were mounted in sonicated phenylenediamine-derived intense
fluorochrome (SPIF) (Lundell and Hirsh,
1994
) for the detection of DNA and analyzed by laser-scanning
confocal microscopy. Testes were dissected out of late male pupae before
eclosion. Sex bristles and red eye pigmentation identified mutant
easthop-7 males. Dissection and fixation of whole-mount
testes was based on a published protocol for squashed testes, which preserves
chromosome morphology very well (Bonaccorsi
et al., 2000
). This protocol also achieved a good preservation of
microtubule morphology in prophase and metaphase, but not in prometaphase. In
brief, testes were dissected in 0.7% NaCl, fixed in 4% paraformaldehyde in PBS
and stained with mouse phospho-histone H3 (Ser10) 6G3 monoclonal antibody
(1:200, Cell Signalling Technology, Beverley, MA). Tissues were counterstained
with rat anti-
-tubulin and TO-PRO-3 (1:1000, Molecular Probes, Eugene,
OR). Images of fluorescently labeled samples were acquired using a Biorad MRC
1024 or a Zeiss LSM 500 laser-scanning confocal microscope.
Live investigation of GFP-tagged proteins
Embryos were collected on yeast-agar plates, dechorionated by hand and
transferred to a drop of halocarbon oil (Voltalef S10) on a 32x22 mm
coverslip. The coverslip was inverted and, using double-sided tape, attached
on top of two columns, which were each composed of two layers of 18x18
mm coverslips, thus allowing oxygen exchange. Embryos in the hanging drop were
observed under an upright Zeiss Axioplan microscope attached to a Bio-Rad MRC
1024 confocal laser scanhead. Live images were acquired using a 40x lens
and 2x or 3x zoom at 15 second intervals. Third instar larval
salivary glands were prepared as previously described
(Wasser and Chia, 2000). The
cell-permeable nucleic acid dye Syto-17 (Molecular Probes) was used at
concentration of 5 µM in Ringer's solution to visualize polytene
chromosomes. Image processing was carried out using Confocal Assistant 4.0 and
Adobe Photoshop 5.5. Time-lapse movies were assembled using Adobe ImageReady
2.0.
Determination of nondisjunction
Crosses of east
(y+w+)/FM7c females against
yw males were carried out at 25°C. The rates of nondisjunction
are based on estimates of the total number of progeny. The numbers of
east/yw regular daughters (lethal alleles only) were doubled to
account for the dead east/Y hemizygotes. The numbers of FM7c males
were substituted by those of yw/FM7c females as FM7c males showed a
reduced viability. The numbers of exceptional offspring (yw males and
east/FM7c females) were doubled as their nullo-X (0/0) and triplo-X
(east/yw/FM7c) siblings are not viable. Hence the rates of
nondisjunction (ND) were determined as follows: ND=(2x
yw+2x east/FM7c)/(2x east/yw+2x
yw/FM7c+2x yw+2x east/FM7c). The
segregation defect is specific to achiasmate chromosomes in meiosis I as
east/+ mothers did not give rise to exceptional progeny. Moreover,
the progeny of homozygous east germline clones was exclusively
female, indicating that they did not produce nullo-X gametes.
Online supplemental information
Quicktime time-lapse videos of post-syncytial embryos expressing GFP-tagged
proteins were produced as described in the Materials and Methods section on
live investigation of GFP-tagged proteins. The dynamics of EASTGFP and
histoneGFP in mitotic cells were recorded at 15 second intervals.
Quicktime Movie 1 corresponds to Fig.
2; Movies 2 and 3 correspond to
Fig. 5
(http://jcs.biologists.org/supplemental).
|
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![]() |
Results |
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EASTGFP fusion protein in dividing cells of live embryos
To study the temporal and spatial dynamics of EAST localization in dividing
cells in vivo, full-length EAST was tagged with a C-terminally fused GFP.
Transgenic expression in embryos was achieved using the GAL4-system
(Brand and Perrimon, 1993). To
test whether this fusion protein (EASTFLGFP) showed the same
localization as the endogenous wild-type (wt) protein, ectopic expression was
first performed in salivary glands. EASTFLGFP, like wt EAST, is
targeted to the extrachromosomal and extranucleolar domains of the nucleoplasm
(Fig. 2A). As expected,
EASTFLGFP also localizes to the nuclei of diploid embryonic cells. In
addition, this GFP-tagged fusion protein appears to show the same functional
properties as the untagged counterpart. Ectopic expression of both transgenic
proteins in larval salivary gland cells lead to the expansion of END regions
between chromosomes and between chromosomes and nuclear lamina. Moreover,
persistent overexpression of both proteins in embryos causes lethality (see
below).
To analyze the dynamics of EAST localization during mitosis, time-lapse
recordings of EASTFLGFP (Fig.
2C) were compared with those of histone-H2AGFP
(Fig. 2E)
(Clarkson and Saint, 1999).
Because the ectopic expression of EASTFLGFP did not discernibly alter
the time difference between nuclear envelope breakdown (marked by the
diffusion of EASTFLGFP and histoneGFP from the nucleus into the
cytoplasm) (Fig. 2C,E; timepoint +00:00 minutes) and onset of anaphase (stretching of the rounded
cell in the case of EASTFLGFP, +04:45), compared with that of the
histoneGFP marker, time-lapse movies of both GFP fusion proteins in
different embryos could be aligned. At prometaphase (+01:00), a strongly
labeled center surrounded by a dimly labeled peripheral zone indicates that
the bulk of EASTGFP still remains associated with the nuclear remnant.
The central fluorescence decays towards late metaphase (+03:00), giving rise
to a more evenly distributed fluorescence. At early anaphase (+04:45),
EASTFLGFP clusters along a central line, which corresponds well to the
clump of EAST between the segregating sister chromatids seen in fixed tissues
(see Fig. 1E). EASTFLGFP
shows a diffuse distribution at late anaphase (+06:15) and telophase. After
completion of cytokinesis, EASTFLGFP is rapidly recruited to the
daughter nuclei (+7:45). In summary, the distribution of EAST revealed by
real-time analysis is in good agreement with its distribution derived from
fixed tissues. EAST remains associated with the remnant of the interphase
nucleus until early anaphase and becomes incorporated into the newly assembled
interphase on exit from mitosis.
The C-terminus of EAST is required for association with the mitotic
remnant of the nucleus
A truncated version of EAST comprising the first 1573 residues coupled to
GFP (EASTCGFP) was also analyzed in vivo. This fusion protein,
like its full-length counterpart, which contains the N-terminal 2332 residues,
is imported into interphase nuclei of all developmental stages and
preferentially accumulates in extrachromosomal regions of giant larval nuclei
(Fig. 2B), showing that the
N-terminal two thirds of EAST are sufficient for nuclear import and correct
intranuclear distribution. However, the C-terminally deleted fusion protein
differs from the full-length version in its behavior during mitosis
(Fig. 2D). During prometaphase
(+01:00), EAST
CGFP quickly disperses into the cytoplasm and does
not associate with a central nuclear remnant. Its distribution remains diffuse
until it accumulates again in the daughter nuclei at early interphase
(+07:45). The failure of this fusion protein to be retained in the center of
the cell at prometaphase suggests that the C-terminus of EAST is required for
its anchorage to the remainder of the nucleus that has been stripped of its
outer shell (nuclear envelope and lamina). Moreover, EASTGFP and
EAST
CGFP show differences in toxicity, if expressed from the
ubiquitous daughterlessGAL4 driver. Persistent overexpression
of EASTFLGFP or wt EAST causes embryonic lethality, whereas
EAST
CGFP overexpression does not affect development to viable
adulthood.
Mutations of east affect chromosome segregation during
female meiosis
Mutations of east dominantly interfere with the segregation of
achiasmate chromosomes in female meiosis. Females carrying east
mutations over the X-chromosome balancer FM7c produced exceptional gametes,
which contained either none or two X-chromosomes
(Fig. 3). Defects in meiosis
were only associated with lethal east alleles. Five out of ten
recessive lethal alleles displayed nondisjunction (ND) frequencies ranging
from 2.6% to 23.7%, whereas all four viable east alleles tested were
not affected. The wt X-chromosome over FM7c displayed a ND rate of 0.4%. A ND
rate of up to 1% has been reported for control X-chromosomes over the FM7c
balancer (Moore et al., 1994).
Because the original P-element insertion ETX3 (the parental strain from which
the various east alleles were derived) balanced over FM7c was not
associated with a significant ND rate, this phenotype probably resulted from
imprecise excisions of the P-element out of the east locus. The
segregation of east X-chromosomes from wt X-chromosomes was not
perturbed, suggesting the phenotype is specific to achiasmate chromosomes.
Even homozygous east germline clones did not give rise to exceptional
progeny (see below). Genetic tests also showed that ND occurs during the
segregation of homologs in meiosis I and not during the segregation of sister
chromatids in meiosis II (see Materials and Methods). The highest frequencies
of ND were associated with the 5' deletion of the east
transcription unit (easthop-7) and a deletion of the
upstream region (easthop-5)
(Wasser and Chia, 2000
). ND
was limited to achiasmate X-chromosomes. Similar to previously identified
mutants, east does not affect the segregation of the achiasmate
chromosome 4 (Sekelsky et al.,
1999
). Apart from the ND phenotype, heterozygous east
females did not show any other discernible abnormalities.
|
Loss of east function increases the frequency of mitotic
defects
The presence of east mRNA in unfertilized eggs
(VijayRaghavan et al., 1992)
and the detection of EAST protein in nuclei of the syncytial blastoderm stage
indicate maternal expression. To study a possible role of east in the
mitosis of early embryos, the maternal component of east expression
east(mat) was removed by generating germline clones for the three
lethal alleles easthop-1, easthop-5
and easthop-7 using the DFS-FRT technique
(Chou and Perrimon, 1992
).
Maternal east expression is not essential for viability because, for
all three alleles tested, some of the embryos lacking the maternal component
and carrying only one paternal wt copy of the east gene can still
develop into fertile females. However, the presence of maternally provided
east gene products improves the rate of survival
(Table 1). Although some
heterozygous embryos without maternal east developed into normal
adults (Fig. 4A,B), others
showed severe developmental defects that resulted in embryonic lethality
(Fig. 4C,D). The X-chromosomal
FM7-Act-GFP balancer was used to determine the lethality of
easthop-1 (maternal -/-, zygotic +/-) embryos; the lack of
maternal east was lethal for approximately 40% of zygotically
heterozygous east embryos. Such heterozygous adults often (around 25%
of escapers for allele easthop-1) lacked body structures
such as tergites on the abdominal cuticle or one of the legs
(Fig. 4B). This phenotype was
never observed in females born as control germline clones (maternal +/+,
zygotic +/-) or in heterozygous east flies (maternal +/-, zygotic
+/-) that inherited maternal gene products from heterozygous mothers.
|
|
To examine the consequences of loss of east on the synchronous
nuclear cycles of the syncytial blastoderm, embryos lacking germline
east were stained with anti-histone and antitubulin antibodies. In a
small proportion of syncytial embryos (approximately 10% in
easthop-1 or easthop-5), vast areas of
the embryonic surface were depleted of nuclei
(Fig. 4F). This phenotype was
not observed in control embryos (Fig.
4E). We observed a variety of mitotic abnormalities in a small
subset of nuclei, including incomplete separation of chromosomes at anaphase,
resulting in polyploid nuclei (Fig.
4G) and nuclei dropping from the surface into the interior of the
egg, leaving behind orphan centrosomes at the cortex
(Fig. 4H). Interestingly, other
maternal-effect mutations were also reported to cause both an elimination of
nuclei in embryos of the syncytial blastoderm stage and missing appendages in
surviving adults (Sullivan et al.,
1990; Zalokar et al.,
1975
).
Nuclear cycles in live embryos lacking maternal east
To test whether loss of maternal east caused any subtle phenotypes
that might not be discernible in fixed embryos, we studied the synchronous
nuclear cycles in vivo using the histoneGFP marker. Germline clones of
the genotype easthop-1 FRT were compared to yw
FRT control germline clones. Live recordings starting from either nuclear
cycle 11 or 12 and ending in early interphase of cycle 14 were acquired for 14
mutant east and 11 control embryos. The subset of nuclei observed
represented approximately 4% of the nuclei on the surface of each embryo (see
Materials and Methods). To quantify mitotic errors, the numbers of nuclei that
showed delayed condensation in prophase, failed to separate completely in
anaphase, remained condensed in the following interphase and subsequently
dropped into the interior of the egg were scored
(Table 2). The error
frequencies were up to four times higher in mutant than wild-type embryos. The
absolute average numbers of nuclei affected were less than 7%. More faulty
divisions were observed in the faster nuclear cycle (NC) 12 than in the slower
NC 13. The density of nuclei was slightly reduced in mutants compared to wt in
NC 13 and 14. Only in one case (out of 14) did we see a large patch of nuclei
in a mutant embryo sinking into the interior after an apparently normal NC 12.
The resulting hole was found to cover around one third of the surface. In
summary, the studies on syncytial blastoderm embryos did not provide any
evidence that east plays an essential or crucial role in the
progression of nuclear cycles. However, the increase in the frequency of
mitotic abnormalities that are observed when maternal east is removed
indicates that it plays an important role in promoting the efficiency and
accuracy of the process.
|
Removal of east affects the congression of chromosomes at
prometaphase
Live observations of easthop-1 (maternal -/-) embryos
carrying the histoneGFP marker were also extended to mitotic domains,
clusters of synchronously dividing cells of post-syncytial embryos
(Foe, 1989). A subset of
dividing cells in mutant embryos displayed abnormal chromosome movements
during prometaphase. During prometaphase of wt cells, chromosomes usually
remain clustered in the center of the cell and become aligned in a plate-like
configuration that is oriented perpendicular to the future axis of division
(Fig. 5A, +2:00 minutes). In
mutant cells, two types of unusual maneuvers were observed. On nuclear
envelope breakdown, chromosomes moved away from each another, giving rise to
spatially separated groups of chromosomes instead of one contiguous cluster
(Fig. 5B, +1:00 to +4:30).
Distinct clusters of chromosomes in prometaphase were observed in 44% of
mutant cells (30 out of 68 in 15 embryos). By comparison, 14% of control cells
(6 out of 43 in 8 embryos) showed a disjoint configuration at this stage that
never appeared as dramatic as in mutants. At a lower frequency (5-10%),
chromosomes in east cells strayed away from the center of the cell
towards the plasma membrane (Fig.
5C). This phenotype was never seen in control embryos. Abnormal
movements of chromosomes during congression to the metaphase plate were
associated with a delayed onset of anaphase. In controls, the onset of
anaphase occurred 3.5-6.0 minutes (average 4.5±0.5) after the beginning
of prometaphase (see Fig. 6 for
distribution diagram). The same period in east (maternal -/-) embryos
showed a higher degree of variability, ranging from 4.0 to 9.75 minutes
(average 5.4±1.1). Discrepancies in duration and choreography of
chromosome congression could be seen within the same mitotic domain; e.g.
seven cells of one east mutant mitotic domain entered mitosis and
spent the following different times in prometaphase and metaphase: 5.75, 4.75,
8.0, 9.0, 4.75, 4.5, 9.75 minutes (cells entered mitosis in the same order as
their respective time periods are listed). Apart from one case, all cells
eventually entered anaphase and completed mitosis. In summary, these
observations suggest that east might contribute to the process of
chromosome alignment by constraining the mobility of chromosomes at
prometaphase. Loss of east can therefore lead to a delay of
chromosome congression, but not to mitotic arrest.
|
Abnormal chromosome behavior in spermatocytes
To test whether the loss of east might affect meiosis, the testes
of easthop-7 hemizygotes in the late pupal stage were
stained with an antibody against histone H3 phosphorylated at serine10 (PH3).
PH3 labeling is correlated with chromosome condensation and allows the
identification of dividing cells between prophase and anaphase
(Hendzel et al., 1997;
Wei et al., 1998
). We observed
abnormalities in different stages of meiosis I. In early prophase of wt
primary spermatocytes, characterized by aster formation and lack of PH3
labeling, the replicated genome is organized into three or four spatially
separated clusters of DNA (Fig.
7A) (Cenci et al.,
1994
). In east mutants, DNA appears to be more diffuse
and does not aggregate into distinct clumps
(Fig. 7B). A lower intensity of
TO-PRO-3 staining indicates a lower degree of DNA condensation. The clustering
of chromosomes in wt is preserved through the onset of H3 phosphorylation
later in prophase until prometaphase when the nuclear envelope breaks down
(Fig. 7C). During the beginning
of H3 phosphorylation in east spermatocytes, chromosome clusters
continue to show differences in shape and quantity from wt controls. The
quantity of clumps of DNA can exceed four
(Fig. 7D) and chromosomes can
show a more scattered distribution (Fig.
7E). In contrast to the tightly packed organization of condensed
chromosomes in wt cells, loose chromosome arms can be observed in mutants.
During metaphase in wt cells, the bivalents are captured by the spindle and
congregate to form one cluster (Fig.
7F). Approximately 50% of east primary spermatocytes in
metaphase exhibited an abnormal arrangement of chromosomes. Chromosomes were
observed to stray away from the major cluster and adopt a more scattered
configuration (Fig. 7G).
|
Furthermore, mutant (Fig. 7J,K), but never wt (Fig. 7H,I) testes, contained cells with round and condensed PH3-positive chromosomes. These cells, which were observed throughout the testes individually or in groups of two, showed cortical tubulin staining that is characteristic of interphase cells (Fig. 7K, bottom panels), although PH3 only marks mitotic cells in wt. Because the size of these cells was much smaller than that of primary spermatocytes, we infer that they are probably spermatogonia arrested in mitosis. By contrast, PH3-positive cells of similar size in wt were located near the tip of the testis and showed chromosome and microtubule morphologies characteristic of mitotic cells (Fig. 7H,I).
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Discussion |
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Possible role of a nuclear remnant in mitosis
Not even a complete deletion of the east locus causes a block or
any other catastrophic defect in cell-cycle progression, therefore we can rule
out whether east has any obligatory function in cell division.
However, the loss-of-function phenotypes suggest that its activity contributes
to the accuracy and efficiency of mitosis and meiosis. EAST, as a component of
an internal nucleoskeleton, appears to facilitate the congression of
chromosomes at prometaphase by constraining their mobility
(Fig. 8). During prometaphase
in embryonic east mutant cells, chromosomes were observed to adopt a
more relaxed configuration than in wt, drift apart from one another and tumble
from central to cortical regions of the cell. The abnormal movements can delay
the onset of anaphase. These observations suggest that, although the
disassembly of the nuclear exoskeleton (NE, lamina) has to take place to allow
interactions between chromosomes and microtubule spindle, an intact nuclear
endoskeleton seems to be required to keep chromosomes clustered together in
the central region of the cell. The abnormal movements of chromosomes,
resulting in a delay of anaphase, were limited to cell divisions and not
detected during the syncytial nuclear cycles. An explanation might be that
mitosis in syncytial embryos is semiclosed, which means that the lamina is
only punctured and not dissolved until late in metaphase when chromosome
alignment is completed (Paddy et al.,
1996). Therefore, during the nuclear cycles, the lamina limits the
mobility of chromosomes.
|
Functional studies on other proteins of a putative nucleoskeleton are rare.
Antibody perturbation studies of the nuclear Skeletor protein showed a role in
spindle assembly during mitosis (Walker et
al., 2000). Similarly, the abnormal chromosome movements in
east embryos during metaphase might result from the disruption of a
spindle matrix that is composed of nuclear proteins and guides the outgrowth
of the spindle. However, two lines of evidence argue against this possibility.
First, EAST, unlike Skeletor, does not show colocalization with the mitotic
spindle at metaphase. Second, abnormal DNA staining patterns can be detected
in prophase of male meiosis before the NE breaks down and chromosomes
establish interactions with spindle microtubules.
In contrast to higher mobility resulting from a loss of function, the gain
of function of east can impose additional constraints on the mobility
of chromosomes. Within the spatial limits of the nuclear envelope, the
expansion of the END caused by the overexpression of EAST can lead to an
exclusion of chromosomes from a ring zone underneath the lamina in larval
salivary cells (see Fig. 2A,B)
and spermatogonia (Wasser and Chia,
2000). A study on chromosome motion in live Drosophila
spermatocytes showed evidence of constraints on the mobility of chromosomes
inside the interphase nucleus (Vazquez et
al., 2001
). The constraints in mobility were proposed to be
mediated by the interactions of chromosomes with the nuclear envelope and
other internal nuclear structures.
Interphase chromosome organization and chromosome inheritance
A nucleoskeleton could also assist in the segregation of nonexchange
chromosomes in meiosis. Chiasmata formation assures the alignment of
homologous chromosomes and their co-orientation during metaphase of meiosis I.
Genetic studies in Drosophila have established that a backup system
termed `distributive disjunction' helps to partition even those chromosomes
(such as balancers) that do not undergo homologous recombination. Achiasmate
chromosomes are paired via heterochromatic regions throughout prophase of
meiosis I, disjoin at metaphase and are positioned towards opposite spindle
poles, whereas chiasmate chromosomes remain in a central chromatin mass
(Dernburg et al., 1996;
Karpen et al., 1996
;
Theurkauf and Hawley, 1992
). A
reasonable explanation for the high frequency of nondisjunction observed for
multiple east alleles is that, following nuclear envelope breakdown
in east mutant oocytes, paired achiasmate homologues might, due to a
defective nucleoskeleton, drift away from the zone previously occupied by the
nucleus. In this scenario, two homologues, after splitting up, could end up
near the same spindle pole and, at anaphase, be partitioned to the same
daughter nucleus. A link between chromosome organization and chromosome
transmission has been shown for genes involved in position effect variegation
(PEV). Su(var)2-10 regulates chromosome structure and organization and is
required for the faithful inheritance of chromosomes
(Hari et al., 2001
).
Our data on primary spermatocytes are consistent with an involvement of
east in the spatial arrangement of chromosomes. During prophase and
prometaphase of male meiosis I, chromosomes are normally clustered into three
major spatially separated clumps of DNA
(Cenci et al., 1994). In
east mutant spermatocytes, both the shape and number of DNA clusters
differ from wt, indicating a defective spatial organization of chromosomes
within the nucleus. A recent study on live cultures of spermatocytes showed
that homologous chromosomes and sister chromatids separate in late G2
(Vazquez et al., 2002
). It was
proposed that, besides heterochromatic association and chromatin entanglement,
pairing of homologous chromosomes is maintained by restricting the homologs to
discrete chromosome territories. Our data are consistent with the notion that
the EAST protein might be involved in stabilizing these chromosome
territories.
In summary, our data support the model that chromosomes in the interphase nucleus and in mitosis are embedded into a structure that can restrict their mobility. The putative nuclear endoskeleton has to be elastic and permeable, thus permitting movements of chromosomes and penetration of the microtubule spindle. The challenge ahead lies in identifying additional components of this nuclear structure, and further understanding of how EAST, as well as these components, act.
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Acknowledgments |
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Footnotes |
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References |
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