Department of Molecular Microbiology, Washington University School of Medicine, St. Louis MO 63110, USA
Author for correspondence (e-mail: naomi{at}borcim.wustl.edu )
Accepted 30 November 2001
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Summary |
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Key words: Apicomplexa, Centrin, Centriole, Centrosome, Colchicine, Dinitroaniline, Endodyogeny, Oryzalin, Spindle, Subpellicular Microtubule, Tubulin
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Introduction |
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The two populations of microtubules in Toxoplasma gondii
tachyzoites are spindle microtubules and subpellicular microtubules
(Fig. 1). The characteristic
crescent shape of Toxoplasma is maintained by an interaction between
the pellicle and the underlying twenty-two subpellicular microtubules
(Morrissette et al., 1997;
Nichols and Chiappino, 1987
).
The pellicle is composed of the plasma membrane and the closely apposed inner
membrane complex that comprises flattened vesicles. The subpellicular
microtubules (
5 µM long) have a characteristic organization and length
and are nucleated from the apical polar ring, a unique microtubule-organizing
center (MTOC) (Nichols and Chiappino,
1987
; Russell and Burns,
1984
). These microtubules are critically important for shape and
apical polarity (Fig. 1A1,B1).
When the subpellicular microtubules are disrupted, both cell shape and apical
polarity are lost (Morrissette and Roos,
1998
; Stokkermans et al.,
1996
). Because the subpellicular microtubules of extracellular
parasites are non-dynamic, the effects of microtubule-disrupting drugs are
only seen in intracellular (replicating) parasites
(Stokkermans et al., 1996
).
The spindle microtubules (
1-2 µM long) function to form an
intra-nuclear spindle (a closed mitosis) to coordinate chromosome segregation
(Fig. 1A2,B2). Spindle
microtubules originate in a dense plaque structure that is embedded in the
nuclear membrane adjacent to cytoplasmic centrioles
(Senaud, 1967
). A previous
study has demonstrated that disruption of microtubules with high
concentrations of oryzalin prevents parasite replication, although
intracellular tachyzoites continue to metabolize and grow in size
(Stokkermans et al.,
1996
).
|
Parasite replication occurs by means of internal budding termed endodyogeny
(Sheffield and Melton, 1968).
This process preserves the capacity of tachyzoites to invade the host
throughout the cell cycle by maintaining their critically important apical
specialization and crescent shape. Toxoplasma nuclear division is
followed by formation of two daughter cells within the mother parasite
(Fig. 1A3,B3). These daughter
cells are delimited by an inner membrane complex and associated subpellicular
microtubules, and each contains a complete set of apical organelles (conoid,
rhoptries and micronemes) and a nucleus, mitochondrion, Golgi and plastid.
Once daughter cells are mature, the maternal apical complex is disassembled
and the daughter parasites emerge from the maternal plasma membrane. Since
this method of replication is non-conventional, we have illustrated it in
Fig. 1 using diagrams and
immunofluorescence of tubulin.
In order to define the normal activity of the subpellicular microtubule and
spindle microtubule populations, we have analyzed their behavior during the
tachyzoite replication cycle. Because these individual microtubule populations
have independent MTOCs, we have probed the degree to which they are
coordinately regulated. The microtubule-disrupting agents colchicine and
oryzalin are capable of disrupting Toxoplasma microtubules
(Morrissette et al., 1997;
Morrissette and Roos, 1998
;
Shaw et al., 2000
;
Stokkermans et al., 1996
). We
have established that the subpellicular microtubules and the spindle
microtubules are differentially sensitive to disruption with these agents.
Moreover, these drugs can be used to uncouple the functions of the
subpellicular microtubules from those of the spindle microtubules, showing
that the checks that regulate cell division in other eukaryotes are not
present in Toxoplasma.
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Materials and Methods |
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Drug treatment
The dinitroaniline oryzalin was obtained from Riedel-deHaen (Germany), and
stock solutions were made up in DMSO. Colchicine and thymidine (Sigma) stock
solutions were made up in sterile DMSO and diluted into tissue culture media.
For cell cycle experiments, CTK11 tachyzoites were treated with 10 µM
thymidine for 4.0 hours. Although the normal doubling time of RH tachyzoites
is approximately 6.5 hours, treatment with thymidine is relatively toxic to
tachyzoites and the bulk of parasites are synchronous and viable at 4.0 hours.
Oryzalin washout experiments were carried out by infecting and growing
Toxoplasma tachyzoites in 0.5 or 2.5 µM oryzalin for 24 or 48
hours, after which the drug was removed, the monolayer rinsed and fresh medium
without drug was added. Samples were collected at the time of drug washout, 3
hours after drug washout, and at 24 hour intervals for the next four days.
To quantify microtubule behavior, 30 random microscope fields were scored from two coverslips within an experiment. Independent washout experiments were quantified. Values representing the sum of the 30 fields from a coverslip were normalized to fractions of a total of 100 parasites. The average value of the samples within an experiment was averaged between experiments and the standard error of the mean was calculated. The resulting values were plotted in Excel. To document nuclear division in 0.5 µM oryzalin, parasites were scored as (1) carrying out nuclear division and segregation correctly, (2) carrying out nuclear division correctly but showing a defect in nuclear segregation to daughter buds or (3) showing unequal nuclear division or arrested without nuclear division. To quantify recovery from treatment with 0.5 or 2.5 µM oryzalin, parasites were scored as (1) first generation replicating parasites (equal or greater than 8 parasites per parasitophorous vacuole), (2) first generation aberrant vacuoles, (3) second generation non-replicating parasites (single crescent-shaped parasites) or (4) second generation replicating parasites (2 or 4 parasites per parasitophorous vacuole).
Immunofluorescence
Intracellular parasites on 12 mm circular glass coverslips were fixed,
permeabilized and stained as previously described
(Morrissette et al., 1994).
They were mounted in Vectashield Mounting Media with DAPI (Vector). Phase
contrast and immunofluorescence images were collected on a Zeiss Axioskop
using the Axiovision camera and software. Images were exported as tif files
and manipulated in Photoshop 5.5. Confocal images were collected on a Leica
TCS SP2 Confocal microscope. Half-micron step optical sections were converted
into parallel projections using the Leica software, and these images were
overlaid and modified using Adobe Photoshop 5.5.
Antibodies
A Toxoplasma-specific rabbit anti-tubulin polyclonal antiserum was
raised against the peptide KGEMGAEEGA conjugated to hen egg albumen
(Cocalico). Mouse antiserum generated against this peptide was kindly provided
by John Boothroyd (Stanford University). Anti-centrin monoclonal (20H5) and
polyclonal antibodies were kindly provided by Jeffrey Salisbury (Mayo Clinic).
The monoclonal antibody 45.15 against the subpellicular network component
IMC-1/net-1 was provided by Gary Ward (University of Vermont). Secondary
antibodies conjugated to Alexa 568 and Oregon Green were obtained from
Molecular Probes as was the ToPro3 DNA stain used in the confocal samples.
Electron microscopy
Cells in 60 mm dishes were infected with tachyzoites and treated with drugs
as above. These samples were fixed at 4° for 30 minutes in `double fix'
containing 1% glutaraldehyde and 1% osmium tetroxide in 25 mM phosphate
buffer, pH 6.2. After three washes in cold deionized water, the samples were
postfixed in 1% aqueous uranyl acetate for 3 hours at 4°. These samples
were embedded in Epon and processed for electron microscopy.
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Results |
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When replicating Toxoplasma were treated with serial dilutions of the microtubule disrupting drug oryzalin, nascent subpellicular microtubules were more sensitive to disruption than spindle microtubules were. Phase contrast microscopy of Toxoplasma tachyzoites grown in HFF fibroblasts showed the continued budding of tachyzoites in 0.5 µM oryzalin, although daughter parasites were round rather than crescent-shaped (Fig. 3A). At 2.5 µM oryzalin, both spindle and subpellicular microtubules were disrupted and tachyzoites grew as enormous intracellular inclusions, incapable of cell division (Fig. 3B). Electron microscopy of Toxoplasma grown in 0.5 µM oryzalin showed the segregation of daughter nuclei and organelles into daughter buds (Fig. 3C). Fig. 3D illustrates the non-dividing, non-polarized nature of the intracellular tachyzoite in 2.5 µM oryzalin. Both the round dividing and the round, non-dividing oryzalin-treated tachyzoites are incapable of reinvasion after lysing out of host cells (N.M., unpublished).
|
In colchicine-sensitive host cells (such as HFF cells), parasites arrested
after host cell invasion in the presence of relatively low concentrations of
colchicine. In colchicine-resistant CV2-8 cells, parasites in colchicine
entered aberrant replication cycles that parallel behavior in oryzalin. This
suggests that parasites do not proceed with intracellular replication in HFF
cells because host microtubules are required to set up a functional
parasitophorous vacuole following invasion (data not shown). This notion is
supported by observations of the `evacuoles' that arise when
Toxoplasma entry is arrested but rhoptry secretion continues; these
evacuoles move along host microtubules
(Hakansson et al., 2001;
Sinai et al., 1997
).
Tachyzoites grown in colchicine-resistant CV2-8 CHO cells displayed
differential microtubule stability. Tachyzoites in 1.0 mM colchicine continue
budding but were round rather than crescent-shaped; 5.0 mM colchicine
disrupted both spindle and subpellicular microtubules and tachyzoites grew but
were incapable of cell division (phase contrast data not shown). Electron
microscopy of tachyzoites grown in CV2-8 CHO cells in 1.0 mM colchicine showed
the round, budding phenotype observed with tachyzoites in 0.5 µM oryzalin
(Fig. 3E) and in 5.0 mM
colchicine the round, non-budding appearance akin to the shape of tachyzoites
in 2.5 µM oryzalin (Fig.
3F).
Replicating tachyzoites discard excess organelles and cytoplasm in a
posteriorly located structure termed the residual body. This structure can
resemble the rounded daughter buds observed in oryzalin-treated parasites. In
order to distinguish daughter parasites from the residual body, we used an
antibody (45.15, anti-IMC-1) that recognizes an intermediate-filament-like
component of a network that is associated with the inner membrane complex
(IMC). In untreated tachyzoites, IMC-1 labeling was localized directly below
the plasma membrane and ran from a region directly under the extreme apex of
the parasite to a region close to the posterior of the tachyzoite
(Fig. 4A, top row). It began in
a region coincident with the subpellicular microtubules but extended
significantly beyond them. The residual body was not labeled with the IMC-1
antibody. Immunofluorescence with the Toxoplasma-specific tubulin
antibody demonstrated that at 0.5 µM oryzalin, the subpellicular
microtubules were shortened or absent (Fig.
4A middle row), but nuclear division proceeded with correct
segregation of the centrioles (Fig.
4B middle row). In contrast, in 2.5 µM oryzalin, both spindle
and subpellicular microtubules were disrupted
(Fig. 4A bottom row), nuclear
division and budding ceased and centrioles continued to duplicate unchecked
(Fig. 4B bottom row).
Equivalent results are obtained with colchicine-treated Toxoplasma in
CV2-8 cells (not shown). Fig.
4C shows two daughter parasites (demarcated by the IMC-1 antibody)
that have completed nuclear division (DAPI) and budded (tubulin and IMC-1
labeling). One nucleus was correctly segregated to a daughter bud (DB), but
the other bud failed to capture a nucleus, which was retained within the
residual body (RB). Quantification of nuclear division and budding behavior in
0.5 µM oryzalin demonstrated that the majority of replicating parasites
(60%) correctly divided and segregated their nuclei
(Fig. 4D). A smaller set of
parasites underwent nuclear division but retained one or both nuclei in the
residual body (
20%). A similar number of parasites (
20%) either
underwent aberrant nuclear division (producing unequally sized nuclei) or
arrested prior to nuclear division although daughter buds were formed. The
innate failure rate of division in untreated cells is >1% as judged by DAPI
staining. The important conclusion of this quantification is that nuclear
division was unaffected in 80% of replicating parasites, and the bulk of these
parasites correctly segregated their nuclei to daughter cells under conditions
that disrupted the subpellicular microtubules.
|
To assess the recovery of microtubule function in parasites treated with oryzalin, we treated tachyzoites with drug for 24-48 hours, removed the oryzalin and followed recovery (Fig. 5). We observed that parasites treated with 0.5 µM oryzalin for 48 hours (Fig. 5A) recovered the ability to form subpellicular microtubules (Fig. 5B). Invasiveness was restored in these parasites, suggesting that the subpellicular microtubules played a role in tachyzoite invasion. These parasites continued to invade and replicate correctly, indicating that their chromosomes were correctly segregated during the 48 hour exposure to oryzalin (six to seven doublings). When oryzalin was washed out from Toxoplasma tachyzoites treated with 2.5 µM oryzalin for 48 hours (Fig. 5C), the polyploid nuclear mass was not correctly segregated (Fig. 5D). Daughter parasites were made that contained large aggregates of DNA, contained only an apicoplast genome or lacked DNA altogether. Astoundingly, parasites without nuclei completed budding and escaped from the parasitophorous vacuole (Fig. 5E1-4).
|
Washout experiments consisting of a 48 hour oryzalin treatment followed by a 48 hour recovery were quantified to assess the subsequent recovery of tachyzoites. Parasitophorous vacuoles containing equal to or greater than eight parasites per parasitophorous vacuole were assumed to be vacuoles that were existent during the oryzalin treatment and washout/recovery (primary parasitophorous vacuoles). Vacuoles containing large aberrant masses were considered to be irretrievably altered parasites in primary parasitophorous vacuoles. Parasitophorous vacuoles containing 1-4 parasites were considered to be parasites that lysed from the original parasitophorous vacuoles and invaded new host cells during the washout/recovery phase, creating `secondary' parasitophorous vacuoles. Parasites were scored as (1) primary vacuoles with replicating parasites, (2) primary vacuoles with aberrant parasites, (3) secondary vacuoles with non-replicating parasites (single crescent-shaped parasites) or (4) secondary vacuoles with replicating parasites (2 or 4 parasites per parasitophorous vacuole). Parasites treated with 0.5 µM oryzalin retained the capacity to undergo correct nuclear division and scission despite lacking the bulk of their subpellicular microtubules. After oryzalin was removed, the tachyzoites recovered their subpellicular microtubules and lysed out of host cells, reinvaded and continued to replicate as second generation parasites (Fig. 6, left panel). Slightly more abnormal versus normal primary vacuoles were observed in these samples because many of the normal first generation vacuoles have lysed and the parasites have gone on to make secondary vacuoles. When oryzalin was removed from parasites treated with 2.5 µM of the drug, an increased number of aberrant masses in primary vacuoles was observed. Second-generation parasites bud off of these masses and escape from the parasitophorous vacuole but they contained irregular nuclei or lacked nuclei altogether. The majority of these second generation parasites could not initiate growth and replication because they lacked adequate genetic material (Fig. 6, right panel).
|
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Discussion |
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Nuclear division in the Apicomplexa proceeds without nuclear membrane
breakdown. Spindle pole plaques (variously termed centrocones, centriolar
equivalents or centriolar plaques) organize the spindle microtubules of
apicomplexans (Chobotar and Scholtyseck,
1982; Senaud,
1967
). The Apicomplexan spindles terminate in poorly defined
regions of electron density located within invaginations of the nuclear
membrane; these regions (the spindle pole plaques) are found in close
proximity to extranuclear centrioles (Fig.
2). In turn, the centrioles are intimately associated with the
apicoplast (Striepen et al.,
2000
) and are also close to the forming daughter buds. The
processes of nuclear division and budding in Toxoplasma are somewhat
akin to these behaviors in Chlamydomonas. The biflagellated alga
Chlamydomonas also has a closed nuclear division
(Preble et al., 2000
).
Membrane-associated rootlet microtubules originate from the basal bodies, and
these microtubules enfold the daughter nuclei during replication, similar to
the behavior of the daughter subpellicular microtubules in Toxoplasma.
Chlamydomonas has prominent fibers formed from centrin that link the
axoneme, basal bodies/centrioles and nucleus
(Baron et al., 1995
;
Salisbury et al., 1988
;
Taillon et al., 1992
;
Wright et al., 1989
). The
Chlamydomonas vfl2 mutant has a point mutation in centrin and a
defect in organelle segregation, indicating that centrin fibers play a role in
correct segregation during replication
(Taillon et al., 1992
). Since
the Toxoplasma centrioles do not appear to be nucleating microtubules
directly, we suggest that they may function to organize centrin fibers that in
turn link the subpellicular microtubules to the apicoplast and nucleus.
Cytokinesis in higher eukaryotes is regulated by the position of the
spindle poles (Rappaport,
1961; Wheatley,
1999
). The cleavage furrow forms perpendicular to the plane of the
spindle, thus ensuring that each daughter cell will inherit a nucleus. The
apical polar ring MTOC drives Toxoplasma budding, whereas nuclear
division is controlled by the spindle pole plaque structure. This arrangement
permits Toxoplasma to replicate while maintaining a fully
differentiated phenotype. In the related parasite Plasmodium, this
arrangement permits schizogeny, the accumulation of multiple nuclei prior to a
synchronous budding of
64 daughter parasites. One consequence of having
multiple MTOC is that it is possible to disconnect nuclear division and
budding to create anucleate daughter parasites. We have generated anucleate
parasites by disrupting the synchrony of nuclear division and parasite budding
(Fig. 5). Anucleate `zoids'
were previously identified in the unrelated protozoan parasite Trypanosoma
brucei after treatment with drugs (such as rhizoxin) that disrupt
microtubules (Ploubidou et al.,
1999
; Robinson et al.,
1995
). In this case, the spindle microtubules appear to be more
susceptible to disruption than the subpellicular microtubules. When T.
brucei is treated with rhizoxin, the nuclear genome is replicated but
spindle function and nuclear segregation is inhibited. These parasites divide,
creating a diploid daughter cell and an anucleate zoid, both bounded by
subpellicular microtubules and containing a functional flagellum. Trypanosome
zoids contain kinetoplast DNA, reminiscent of the Toxoplasma zoids
described here, which generally contain the apicoplast genome
(Fig. 5E).
Shaw and Tilney have previously studied the effect of 1-5 µM oryzalin on cell division in Toxoplasma. These authors used morphological observations to conclude that oryzalin prevented parasite budding (cytokinesis) but did not block centriole replication or mitotic spindle formation. In the present study, we have used functional assays to quantify the effects of low (0.5 µM) and high (2.5 µM) levels of oryzalin on nuclear and cell division in Toxoplasma. Our studies reveal that the spindle and subpellicular microtubules have different sensitivity to oryzalin. Thus, while we would agree with Shaw and Tilney that spindle microtubules are resistant to low levels of oryzalin (which disrupt subpellicular microtubule function), we observed disruption of both subpellicular and spindle microtubule populations at high levels of drug. This was most dramatically shown by the reversibility experiments. Removal of 0.5 µM oryzalin permitted daughter cells to establish functional subpellicular microtubules during cytokinesis. In turn, these restored subpellicular microtubules re-establish infectivity in daughter parasites. A dramatically different outcome was observed when parasites were removed from 2.5 µM oryzalin. These parasites failed to correctly segregate nuclei or centrioles into daughter cells, although cytokinesis generated anucleate progeny. We interpret this to mean that in 2.5 µM oryzalin, nuclear spindles were disrupted. Since DNA replication continued unchecked, upon oryzalin removal parasites could not segregate chromosomes correctly and were unable to resolve this defect in nuclear division.
Observations of nuclear division in 0.5 µM oryzalin also suggest that the subpellicular microtubules are not required for scission of the horseshoe-shaped nucleus during mitosis. Both the washout studies and staining with DAPI indicate that nuclear division was accomplished in 0.5 µM oryzalin, where the subpellicular microtubules are greatly shortened or absent. It is, however, clear that the subpellicular microtubules play an important role in segregation of organelles to daughter buds. Although daughter buds (with correctly assembled inner membrane complex) form in 0.5 µM oryzalin, in approximately 20% of the population the divided nuclei do not segregate into daughter cells but are retained in the residual body. In 0.5 µM oryzalin, unequal segregation can also be observed for the apical complex organelles such as the rhoptries and micronemes (N.M., unpublished).
Previous work has shown that the subpellicular microtubules are
extraordinarily stable during isolation and are heavily decorated with
associated proteins (Morrissette et al.,
1997; Nichols and Chiappino,
1987
; Russell and Burns,
1984
; Russell and Sinden,
1982
). Drugs such as colchicines or the dinitroanilines that
disrupt dynamic microtubules are completely ineffective against the
subpellicular microtubules of extracellular apicomplexan parasites, indicating
that these microtubules are not dynamic
(Russell, 1983
;
Stokkermans et al., 1996
).
Once parasites are intracellular and replicating, colchicine or the
dinitroanilines disrupt the nascent (dynamic) microtubules of daughter
parasites. Consistent with this extraordinary stability, at the completion of
endodyogeny, the maternal subpellicular microtubules, apical polar ring and
conoid are removed to the residual body at the posterior of budding cells
(Fig. 2E). This relocation may
be a prelude to microtubule disassembly and recycling or to degradation of the
complex. In either case, maternal microtubule disassembly is not coincident
with daughter cell budding.
It is intriguing that the subpellicular microtubules that are highly stable in vitro should be more susceptible to disruption by drugs such as oryzalin or colchicine. The differential stability of the microtubule populations may be explained by the influence of an associated protein or proteins that specifically interact with only one of the microtubule populations. Alternatively, the intranuclear nature of the Toxoplasma spindle may afford some protection from the destabilizing drugs. However, we favor the hypothesis that the tachyzoites can still build short microtubules in lower concentrations of oryzalin or colchicine. These shorter microtubules may be adequate to make a functional spindle but are incapable of providing sufficient scaffolding to generate crescent-shaped rather than spherical tachyzoites. As would be predicted, centriole segregation (as assessed by centrin staining) occurs correctly as long as the spindle microtubules are intact. In the presence of lower concentrations of the microtubule-disrupting drugs colchicine or oryzalin, the centrioles segregate as long as spindle formation is unimpaired. In contrast, centriole segregation fails, although duplication remains unchecked, in concentrations of the drugs that disrupt both spindle and subpellicular microtubule populations (Fig. 4B).
Although Toxoplasma microtubules are disrupted by colchicine,
compared with oryzalin, they are relatively insensitive to it (0.5-2.5 µM
oryzalin versus 1.0 to 10.0 mM colchicine is required). This is consistent
with the `plant-like' nature of apicomplexan tubulin revealed by phylogenetic
analysis (Stokkermans et al.,
1996). Plant tubulins are exquisitely sensitive to oryzalin (a
commercial herbicide) and relatively insensitive to colchicine (a plant
product). The related apicomplexan Plasmodium is also susceptible to
similarly high concentrations of colchicine that inhibit nuclear division and
re-invasion. A previous study of the behavior of the Plasmodium
erythrocytic stage in colchicine established that there were concentrations
(10 µM-1mM) where schizont nuclear division and budding continued but
invasion was inhibited, and higher concentrations (>10 mM) where nuclear
division and schizogeny were affected
(Bejon et al., 1997
). Owing to
the much smaller size of Plasmodium merozoites, the appearance of
microtubules was not directly examined; however, they produced remarkably
similar results to the data presented here, which suggests that
Plasmodium also has differential stability for spindle and
subpellicular microtubules.
In this paper we have investigated the dynamics of spindle microtubules and subpellicular microtubules. These two populations of microtubules are each organized by a different MTOC and are differentially sensitive to disruption by drugs. These observations have permitted us to isolate the essential functions of these microtubules. The presence of subpellicular microtubules is necessary for host cell invasion. Parasites lacking intact subpellicular microtubules are incapable of invading host cells. Parasites containing subpellicular microtubules but lacking a nucleus are capable of completing scission from the maternal cell and are capable of invasion. Conversely, spindle microtubules are necessary and sufficient for chromosome segregation and nuclear scission. Apicomplexans use multiple MTOCs to independently control nuclear division and cell polarity/cytokinesis. Having multiple MTOCs permit greater flexibility but eliminates the opportunity for checks for accurate nuclear division and correct cytokinesis found in other eukaryotes.
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Acknowledgments |
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