1 University of Siena, Department of Evolutionary Biology, Via Mattioli 4,
I-53100 Siena, Italy
2 University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2
3EH, UK
* Author for correspondence (e-mail: dmg25{at}mole.bio.cam.ac.uk )
Accepted 6 December 2001
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Summary |
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Key words: Asp, cytokinesis, meiosis, microtubules, contractile-ring, Drosophila
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Introduction |
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The central spindle is evident as a dense body of microtubules that forms
in the region between the two late anaphase-telophase nuclei. The mechanism
and dynamics of its formation are still poorly understood. The establishment
of the central spindle in Drosophila appears, in part, to depend on
at least two kinesin-like proteins: Klp3A
(Williams et al., 1995) and
Pavarotti (Pav-KLP) (Adams et al.,
1998
). The counterparts of Pav-KLP in other organisms, the
zen-4 gene product of C. elegans
(Raich et al., 1998
;
Severson et al., 2000
) or the
vertebrate Mklp1 (Vernos et al.,
1995
), have also been shown to be required for cytokinesis. In
addition to the contribution made by the spindle, several proteins, known as
chromosomal passengers, dissociate from the chromosomes at the
metaphase-anaphase transition and are deposited at the cell equator. The inner
centromere protein (INCENP) and the associated Aurora B kinase, for example,
transfer to the central spindle and the cell cortex and are necessary for
completion of cytokinesis (Adams et al.,
2000
; Adams et al.,
2001
; Kaitna et al.,
2000
; Giet and Glover,
2001
). The Polo-like kinases are also required for central spindle
formation, and Drosophila Polo-kinase and Pav-KLP are mutually
dependent for their localisation in the central spindle mid-zone (Adams et
al., 1988; Carmena et al.,
1998
). The analysis of the cytological phenotypes displayed by
mutants with disrupted meiotic cytokinesis in Drosophila males has
provided insight into an intimate relationship between the formation of the
central spindle and the contractile ring
(Giansanti et al., 1998
).
Mutations identified as disrupting the central spindle are found in genes that
encode a variety of actin-, microtubule- or septin-associated proteins
(reviewed by Field et al.,
1999
; Gatti et al.,
2000
; Glotzer,
1997
; Goldberg et al.,
1998
).
Mutations in abnormal spindle (asp) have not previously
been thought to affect cytokinesis. Male meiotic spindles in asp
mutants are bipolar, with particularly long and wavy microtubules
(Ripoll et al., 1985;
Casal et al., 1990
). Mitotic
cells accumulate at metaphase and show spindles with disorganized broad poles
at which
-tubulin has an abnormal distribution
(Avides and Glover, 1999
).
asp encodes a 220 kDa microtubule-associated protein found at the
spindle poles and centrosomes from prophase to early telophase. The protein
has consensus phosphorylation sites for CDK1 and MAP kinases, an actinin-type
actin-binding domain and multiple calmodulin-IQ-binding motifs
(Saunders et al., 1997
). Asp
and
-tubulin are present in partially purified centrosomes and are both
required for the organization of microtubules into asters
(Avides and Glover, 1999
). This
activity is dependent on the phosphorylation of Asp by the kinase Polo
(Avides et al., 2001
).
Here, we now show that asp function is also required for
cytokinesis in male meiosis and that the protein becomes localised at late
anaphase in a manner consistent with a function in organising the spindle
mid-zone. We have also examined the distribution of Asp protein in female
meiosis. These divisions are unusual because the first meiotic spindle is
acentriolar and appears not to contain any centrosomal proteins, such as
-tubulin and CP190 (Riparbelli and
Callaini, 1996
; Tavosanis et
al., 1997
). The female meiotic spindle microtubules are initially
nucleated from chromatin and require the minus-end motor Ncd to focus the
poles of the spindle for meiosis I (Endow
and Komma, 1996
). At telophase of meiosis I, a central pole body
is formed. Microtubules in the central part of the spindle dissociate and
reorganise with reverse polarity such that their minus ends are associated
with this central pole body. This results in the tandemly linked second
meiotic spindles (Endow and Komma,
1997
; Endow and Komma,
1998
; Riparbelli and Callaini,
1996
). Asp is a component of this central spindle pole body. We
suggest that Asp has a dual role not only organising microtubule-nucleating
centres at the poles at the onset of M-phase but also participating in
organising the central spindle region at telophase.
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Materials and Methods |
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Reagents
A mouse monoclonal anti-ß-tubulin antibody (Boehringer, Mannheim UK)
was used at a 1:200 dilution; a rabbit polyclonal anti-Asp serum Rb3133
(Saunders et al., 1997) at
1:50 dilution; a rabbit anti-Pav-KLP polyclonal Rb3301
(Adams et al., 1998
) at 1:100;
a rabbit anti-
-tubulin polyclonal Rbcs1 at 1:100; a mouse monoclonal
anti-Peanut 4C9H4 at 1:4; a rabbit HsCen 1p polyclonal antibody
(Paoletti et al., 1996
) at
1:400 dilution; a rabbit polyclonal anti-centrosomin antibody
(Li and Kaufman, 1996
) at
1:400 dilution. Goat anti-mouse or anti-rabbit secondary antibodies coupled to
fluorescein or rhodamine (Cappel, West Chester, PA) were used at 1:600
dilution. DNA was stained with propidium iodide (Sigma, St. Louis, MO),
Hoechst 33258 (Sigma) or TOTO-3 (Molecular Probes, Europe, BV). The actin
cytoskeleton was stained with Rhodamin-phalloidin (Molecular Probes). Bovine
serum albumin (BSA) and Ribonuclease A (RNAse) were obtained from Sigma.
Indirect immunofluorescence and confocal images
Immunostaining was performed either by the methanol/acetone fixation method
described by Gonzalez and Glover (Gonzalez
and Glover, 1993) or by the ethanol/formaldehyde fixation method
as described by Hime et al. (Hime et al.,
1996
). Briefly, testes from pupae or newly eclosed adults were
dissected in phosphate-buffered saline (PBS) and placed in a small drop of 5%
glycerol in PBS on a glass slide. Testes were squashed under small cover
glasses and frozen on a copper bar precooled in liquid nitrogen.
To localise microtubules, Asp, Pav-KLP, -tubulin, Centrosomin,
Peanut and centrin, the frozen samples were fixed in methanol at -20°C,
washed for 15 minutes in PBS and incubated for 1 hour in PBS containing 0.1%
BSA (PBS-BSA) to block non-specific staining. For double labelling experiments
the samples were incubated overnight at 4°C with the specific antisera
against Asp, Pav-KLP,
-tubulin, Centrosomin, Peanut or centrin antigens
and then with anti-ß-tubulin antibody for 4-5 hours at room temperature.
After washing in PBS-BSA the samples were incubated for 1 hour at room
temperature with the appropriate secondary antibodies.
For simultaneous localisation of actin and tubulin, the frozen testes were immersed for 7 minutes in cold ethanol and 10 minutes in 4% paraformaldehyde. After washing in 0.2% Triton in PBS, the samples were incubated with the anti-ß-tubulin antibody for 1 hour at room temperature. Samples were then washed in PBS-BSA and incubated for 1 hour in the appropriate secondary antibody to which rhodamine-labeled phalloidin was added. In all cases DNA was stained with TOTO-3, Hoechst or propidium iodide. The samples were rinsed in PBS and mounted in 90% glycerol containing 2.5% n-propyl-gallate.
Metaphase I oocytes and oocytes at subsequent meiotic stages were obtained either by dissection of the ovaries or by collection from 5-7 day old females. Eggs were dechorionated in a 50% bleach solution for 2-3 minutes, rinsed in distilled water, dried on filter paper and transferred to a cold 1:1 mixture of heptane and methanol to remove the vitelline envelope. Eggs were then fixed for 10 minutes in cold methanol, washed in PBS and incubated for 1 hour in PBS containing 0.1% BSA. The eggs were incubated in the rabbit polyclonal anti-Asp serum Rb3133 overnight at 4°C and then with an anti-ß-tubulin antibody for 4-5 hours at room temperature. After washing in PBS-BSA the eggs were incubated for one hour with the appropriate secondary antibodies. For simultaneous DNA staining, the eggs were incubated in propidium iodide or TOTO-3 iodide. Eggs were mounted in small drops of 90% glycerol containing 2.5% n-propyl-gallate.
Confocal images were obtained using a Leica TCS4D confocal microscope equipped with a Krypton/Argon laser (Leica Microsystems, Eidelberg). Images were collected using low laser emission to attenuate photobleaching and 8 frame-averaged scans made per image to improve the signal/noise ratio. Images collected at several focal planes were superimposed and merged into a single file and imported into Adobe Photoshop to adjust the size and contrast.
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Results |
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|
Whereas meiotic spindle poles appear well focused in asp
mutant meiosis, central spindle defects lead to a failure of cytokinesis
As mutations in asp are associated with disruptions in the compact
nature of the MTOCs in third instar larval neuroblasts, resulting in broad
spindle poles, we studied pole integrity during male meiosis in two
asp mutant alleles. We examined
asp1/aspdd4,
aspd44/aspdd4 and
aspdd4/Df(3R)H1
(White-Cooper et al., 1996
)
males by immunostaining with antibodies that revealed tubulin and the
centrosomal proteins Centrin,
-tubulin and Centrosomin. The
aspdd4 allele is a weak hypomorph that permits homozygotes
to survive to adulthood. Such flies show rough eyes and wing defects
characteristic of cell division cycle mutants, and both sexes show reduced
fertility. It has previously been placed in an allelic series on the basis of
the phenotypes seen in the sterile hemizygotes
(White-Cooper et al., 1996
).
Here we observed comparable phenotypes in male meiosis in each of the allelic
combinations studied. Our observations confirmed the meiotic spindle defects
that have previously been reported for asp, principally, the long,
wavy microtubules that are particularly evident at metaphase
(Table 1). Examination of the
spindle poles by immunostaining against Centrin (Figs
2A;
3AB),
-tubulin (Figs
2B;
3C) and Centrosomin
(Fig. 3D) revealed that they
were well organised in discrete structures but were often irregularly
positioned. Moreover, in contrast to the wild-type, the antibody against
Centrin failed to recognise pericentiolar material and only stained the
centrioles.
|
|
|
Whereas all cells in wild-type cysts are generally at similar stages of
meiotic progression, we found that meiocytes in asp cysts were at a
range of stages (Fig. 2).
Indeed we consistently observed a greater proportion of cells in all stages of
the first meiotic division in asp mutants and a reduction of the
proportion in the second division in comparison to wild type
(Table 2). This observation is
consistent with the abnormal spindles leading to a checkpoint response, which
in male meiosis is seen as a delay rather than an arrest in the progression
through prophase and metaphase (Savoian et
al., 2000; Rebollo and
Gonzalez, 2000
). It may also reflect difficulties in exit from the
first meiotic division cycle. In any event, excluding the unlikely possibility
that the phenotype is a direct consequence of earlier defects, the ability of
these cells to progress beyond metaphase reveals mutant defects later in the
meiotic cycle that suggest additional functions of the Asp protein other than
at the spindle poles. We found that in contrast to the well organised poles,
the central regions of the spindles of the late meiotic spindles were not
correctly organised at anaphase and telophase in asp testes (compare
the asp cells indicated with an arrowhead in
Fig. 3D with wild-type cells in
Fig. 1E,F). Depending upon the
allelic combination, we found that mid-zone microtubules were poorly organised
in 20-40% of cells within the cyst and absent in 5-15% of cells
(Table 1). The absence of the
mid-zone correlates with a failure of cytokinesis, which in turn leads to
tetranucleate cells at the end of meiosis II (e.g. the cell shown in
Fig. 3B). We were able to
quantify the extent of cytokinetic failure by counting the numbers of
multinucleate spermatids (Table
3). We found that in each allelic combination studied, up to
approximately half of the cells complete cytokinesis in both meiotic
divisions, but about 35% fail one and up to 10% fail both divisions. The
leakiness of this mutant phenotype probably reflects the hypomorphic nature of
the alleles under study.
|
|
To further correlate the central spindle defects described above with abnormal cytokinesis, we studied the distribution of three proteins that associate with the contractile furrow: Actin, Pav-Klp and Peanut (Fig. 4). In wild-type cells (inserts to Fig. 4), all three proteins are found in a ring-like structure at the central region of the spindle. A spectrum of localisation patterns of these proteins was seen in the asp mutant cells, which reflects the extent to which the central spindle had formed again, probably reflecting the leaky nature of the hypomorphic mutant combinations studied. We found the localisation of Actin to be most dramatically disrupted and scattered throughout the central part of the cell in fibrous aggregates (Fig. 4A). Nevertheless, some cells managed to produce a ring-like structure able to constrict the telophase cell (e.g. cell in bottom left of Fig. 4A). Similarly when stained to reveal Pavarotti, some meiotic cells had normal contractile rings (Fig. 4B, large arrow), whereas in others in the same filed Pav-KLP was more diffuse (Fig. 4B, small arrow). The presence of Pav-KLP in ring canals (Fig. 4B, arrowheads) indicated that cytokinesis had occurred correctly within the pre-meiotic rounds of mitosis. Staining to reveal the septin Peanut also showed that some cells are able to construct an apparently normal contractile ring (Fig. 4C, arrow) in association with an organised central spindle region (see the microtubule staining of the same cell, monochrome image). In other cells at a similar stage, however, both the distribution of Peanut and the central spindle microtubules were disorganised (Fig. 4C, arrowhead).
|
Asp associates with the spindle poles and the central microtubule
organising centre in female meiosis
In Drosophila female meiosis, an unusual microtubule organising
structure known as the central spindle pole body is formed between the
daughter nuclei at the end of the first division. Female meiosis occurs
without cytokinesis, and the shared pole of the second meiotic spindles is
formed at the end of meiosis I in a position at which cytokinesis would be
expected to occur in any other cell type. We therefore wished to determine the
localisation of Asp with respect to the spindle poles at this unusual central
microtubule organising center (MTOC). In female meiosis, the spindle is
organised primarily by the condensed chromosomes and becomes focused at the
poles by the concerted action of minus-end-directed motor proteins such as Ncd
(Endow and Komma, 1996).
Although these poles appear to lack
-tubulin, by the criterion of
immunostaining we found Asp protein at the poles at metaphase
(Fig. 5A) and anaphase
(Fig. 5B). At late anaphase or
early telophase of meiosis I, we saw Asp as a well defined ring in the array
of centrally nucleated microtubules (Fig.
5C). Prior to meiosis II, the region previously occupied by the
plus ends of microtubules in contact with chromosomes has to become the site
of microtubule minus ends at the shared central pole of the second meiotic
spindle. At anaphase II, we found that Asp also became concentrated at the
innermost spindle poles (Fig.
5D), and the ring-like staining at the central MTOC became more
dispersed as meiosis II proceeded.
|
The sperm aster fails to grow and pronuclear fusion does not take
place in asp mutant embryos
We then examined meiotic progression and behaviour of the pronuclei in eggs
derived from asp mothers. We were only able to do this in oocytes
derived from mothers homozygous for aspdd4 and
transheterozygous for aspdd4/asp1. Meosis I
progressed normally in such eggs, as can be seen by the spindle at early
telophase (Fig. 6A, arrowhead),
and the aster of microtubules that forms around the basal body, which was
contributed by the incoming sperm, had begun to form
(Fig. 6A, arrow). The second
meiotic division also appeared to proceed normally
(Fig. 6B, arrowhead). However,
the sperm aster did not increase in dimension
(Fig. 6B, arrow;
Table 4). This should be
contrasted with the sperm aster in wild-type eggs, where centrosomal proteins
such as -tubulin and CP190 are recruited from the egg cytoplasm around
the spindle basal body and the microtubules grow to reach the cortex of the
egg cytoplasm. Immunostaining of wild-type eggs revealed that Asp was also
recruited to this MTOC (Fig.
5E), which eventually contributes to both poles of the gonomeric
spindles (Fig. 5F). In the
asp mutant cytoplasm, the microtubules of the sperm aster never
extended to the egg cortex. The sperm aster duplicated in the mutant eggs as
it does in wild type, but it was frequently found to detach from the spindle
that formed around the haploid male pronucleus
(Fig. 6C, arrow). We found that
these centrosome-associated asters continued to duplicate
(Fig. 6D, arrowheads) as do the
haploid male-derived nuclei that can undergo multiple rounds of haploid
mitosis (Fig. 6D, small arrows;
Table 4). However, the haploid
products of female meiosis frequently appeared to condense to form typical
polar body-like structures (Fig.
6D, large arrow).
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Discussion |
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The spindle poles in female meiosis differ further from those of either
mitosis or male meiosis. These spindles are initially nucleated by
chromosomes, and the spindle poles are focused by the action of
microtubule-associated motor proteins, particularly the ncd-encoded
kinesin-like protein (Endow and Komma,
1996; Endow and Komma,
1997
). Although the poles of the female meiotic spindle are not
stained by antibodies that recognize
-tubulin, progression through
female meiosis shows some dependence upon this protein
(Tavosanis et al., 1997
;
Wilson et al., 1997
). In
contrast to the low abundance of
-tubulin, we find Asp to be abundant
at the acentriolar poles of the female meiotic spindle. Nevertheless, meiosis
appears able to proceed, at least for the mutant asp alleles we have
selected for study. These are all hypomorphic and give leaky phenotypes that
generally allow the premeiotic mitoses to proceed in both sexes and so permit
the progression of both the primary spermatocytes and the oocyte to
meiosis.
Microtubules are also nucleated in the fertilised egg by a centrosome that
develops around the basal body of the sperm following its recruitment of
centrosomal proteins such as CP190 and -tubulin
(Callaini and Riparbelli, 1996
;
Riparbelli and Callaini,
1996
). This sperm aster normally comprises short microtubules
until metaphase II of female meiosis, at which time the microtubules begin to
grow and make contact with the cortex of the egg. We have found that growth of
the sperm aster does not take place in asp mutants. A similar
phenotype is seen in eggs derived from polo mutant mothers
(Riparbelli et al., 2000
). It
is possible that the dramatic effects of both polo and asp
mutants on growth of the sperm aster may reflect the ability of Polo kinase to
phosphorylate and activate the microtubule organising properties of Asp
(Avides et al., 2001
). In both
asp- and polo-derived eggs, as a consequence of the failure
of the sperm aster to grow, the female pronucleus cannot migrate to meet its
male counterpart, leading to the failure of the first gonomeric division of
the zygote. In both types of mutant cytoplasm, the centrioles associated with
the sperm aster can dissociate from the male pronucleus and undergo autonomous
replication cycles in the syncytium. The male pronuclei can also undergo
several rounds of haploid mitoses in both mutants. One noteworthy difference
between asp- and polo-derived eggs is that in asp,
the female pronuclei remain arrested as the polar body conglomerate whereas in
polo eggs they can escape from this arrest and also undergo several
rounds of haploid mitosis (Riparbelli et
al., 2000
).
An MTOC at cytokinesis?
In late anaphase-telophase of male meiosis, Asp localises to the spindle
mid-zone, but unlike other centrosomal antigens such as Pavarotti-KLP and Polo
kinase that become associated with the central region of the spindle mid-zone,
the majority of Asp decorates the very terminal regions of mid-zone
microtubules. At this stage the microtubules are positioned between the
telophase nuclei and the centre of the spindle. The terminal regions are
likely to be the minus ends of microtubules that have been released from the
centrosomes, which at this stage nucleate independent asters of microtubules.
This association of Asp with the spindle mid zone appears to be required for
the assembly of the correct structure of the late central spindle and in turn
for cytokinesis. The central spindle plays an essential role during
cytokinesis and there is a cooperative interaction between this structure and
the actinmyosin contractile ring: whenever one of the structures is disrupted
the other fails to assemble and function
(Gatti et al., 2000). In
keeping with this, we have found that many cells within asp mutant
cysts have abnormal central spindles lacking the characteristic
interdigitating microtubules. Moreover, molecules that participate in forming
parts of the contractile ring, Pavarotti-KLP, the septin Peanut, Polo kinase
and Actin, do not localise properly in asp mutant spermatocytes.
While our manuscript was in preparation, Wakefield et al.
(Wakefield et al., 2001) have
also analysed the role of the Asp protein at the spindle poles and in
cytokinesis. We see no conflicts between their conclusion that Asp plays a
role in microtubule bundling at the spindle poles and an earlier report that
Asp contributes to the integrity of mitotic MTOCs
(Avides and Glover, 1999
).
However, Asp may not be sufficient to bundle microtubules, as when centrosomal
antigens are dispersed following the disruption of the
-tubulin ring
complex, spindle poles can be severely splayed even though Asp is present at
their minus ends (Barbosa et al.,
2000
). Our present results confirm and extend the findings of
Wakefield et al. (2001
) that
Asp is required to organise microtubules at the central region of the late
mitotic spindle to enable cytokinesis
(Wakefield et al., 2001
). We
speculate that the minus ends of these microtubules have dissociated from the
spindle poles and now rely on the Asp protein for their stability in order to
form a mid-zone structure for the central spindle. Moreover, the idea that the
central spindle results from de novo nucleation of microtubules by transient
organising centres located in the region between the two daughter nuclei is
substantiated by a requirement for
-tubulin in the equatorial region at
the time of central spindle formation
(Julian et al., 1993
;
Shu et al., 1995
).
Asp localisation in female meiosis a unifying model?
Female meiosis differs from male meiosis and mitosis in Drosophila
not only in that the spindle poles lack centrioles but also in that the first
division is not followed by cytokinesis. Instead the central spindle has been
postulated to undergo reorganisation to reverse the polarity of microtubules
around an unusual central spindle pole body and so form two tandemly oriented
spindles for meiosis II. A model for how this occurs has been presented by
Endow and Komma (Endow and Komma,
1998) and is modified in Fig.
7B. This central spindle pole body recruits centrosomal antigens,
including CP190 and
-tubulin. It is also striking that it additionally
recruits molecules that are known to function in cytokinesis. These include
Pav-KLP (Riparbelli et al.,
2000
) and Asp (this paper).
|
We note that this specialised central pole body of female meiosis in Drosophila has several features in common with the central spindle of conventional divisions, and we speculate that Asp may be one of several centrosomal/central-spindle-associated proteins that play common roles in setting up these structures. In late anaphase, during both the conventional divisions and female meiosis I, a subpopulation of microtubules have to become detached from the initial poles. In effect the two half spindles of female meiosis I separate but remain focused at their original poles. The parallel step in male meiosis or in a conventional mitosis is seen by the ability of the original centrosomes to continue to nucleate asters of microtubules. In contrast, microtubules in the central part of the female meiosis I spindle must be reorganised such that their polarity is reversed to form the linked central poles of the tandemly arranged meiosis II spindles. The Asp-containing central pole body that develops in this region appears first as a ring like structure that nucleates a broad mid-zone region of microtubules that only later becomes independently focused as the meiosis II spindles migrate apart. In mitosis or male meiosis, Asp appears to be at the ends of the reorganised microtubules that form the central spindle. As telophase develops, this mid-zone becomes compacted as it coordinates the formation of the contractile ring. Indeed in male meiosis, it will ultimately be incorporated into the ring canal.
Microtubule-nucleating centres that reorganise the late central region of
the spindle may be a highly conserved feature of cell division. In fission
yeast, a transient MTOC nucleates a post-anaphase array of microtubules in the
central part of the cell before the initiation of septation
(Hagan and Hyams, 1988). This
is probably the fission yeast counterpart of the central spindle region, as
its formation is promoted by overexpression of the fission yeast homologue of
Polo kinase encoded by plo1, and it drives septation at inappropriate
stages of cell cycle progression (M.G.R., G.C., D.M.G. and M.d.C.A.,
unpublished).
Progression through M-phase appears to require the coordination of the activities of several MTOCs. The chromosomes themselves can provide nucleating centres for the plus ends of microtubules, and centrosomes, if present, can contribute a minus-end organising activity. Our results suggest, however, that components of such a minus-end organising activity may not be restricted to the poles but may later in M-phase be regrouped to participate in organising the spindle mid-zone in order to successfully execute cytokinesis.
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Acknowledgments |
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References |
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