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HEMATOPOIESIS
From the Institut National de la Santé et de la
Recherche Médicale (INSERM) U362; Unité Mixte de Recherche
and Centre National de Recherche Scientifique (UMR 1599); Institut
Gustave Roussy, Villejuif, France; Station de Microscopie Confocale,
Institut Pasteur, Paris, France; and Amgen, Thousand Oaks, CA.
During differentiation, megakaryocytes increase ploidy through a
process called endomitosis, whose mechanisms remain unknown. As it
corresponds to abortive mitosis at anaphase and is associated with a
multipolar spindle, investigation of chromosome segregation may help to
better understand this cell-cycle abnormality. To examine this
variation, a new method was developed to combine primed in situ
labeling to label centromeres of one chromosome category and
immunostaining of tubulin. Human megakaryocytes were obtained from
normal bone marrow culture. By confocal microscopy, this study
demonstrates an asymmetrical distribution of chromosomes (1 or 7)
either between the spindle poles at anaphase stage of endomitosis and
between the different lobes of interphase megakaryocyte nuclei. The
metaphase/anaphase checkpoint appears normal on the evidence that under
nocodazole treatment megakaryocytes progressively accumulate in
pseudo-metaphase, without spontaneous escape from this blockage.
Immunostaining of p55CDC/hCDC20 with similar kinetochore localization
and dynamics as during normal mitosis confirms this result. HCdh1 was
also expressed in megakaryocytes, and its main target, cyclin B1, was
normally degraded at anaphase, suggesting that the
hCdh1-anaphase-promoting complex checkpoint was also functional. This
study found the explanation for these unexpected results of an
asymmetrical segregation coupled to normal checkpoints by careful
analysis of multipolar endomitotic spindles: whereas each aster is
connected to more than one other aster, one chromosome may segregate
symmetrically between 2 spindle poles and still show asymmetrical
segregation when the entire complex spindle is considered.
(Blood. 2001;97:2238-2247) Megakaryocytes are polyploid cells that increase
their DNA content through an original process called
endomitosis.1 Megakaryocyte progenitors proliferate
through normal 2N to 4N cycles under hematopoietic growth factor
control. They begin terminal differentiation with synthesis of specific
platelet proteins (promegakaryoblast stage), followed by a switch from
a mitotic to an endomitotic process, which is characterized by a
complete DNA replication without karyokinesis and cytokinesis. This
leads to cells that contain a single polylobulated typical nucleus with
a 2xN ploidy. Thereafter, terminal cytoplasmic maturation
occurs, leading to proplatelet formation and platelet production.
The main consequence of megakaryocyte polyploidization is to augment
cell size and, thus, to increase platelet production that originates
from its cytoplasmic fragmentation.2-4 Some researchers have also hypothesized that the level of megakaryocyte ploidy may
modify platelet size and functions by altering gene regulation (reviewed by Zimmet and Ravid5).
Two teams have shown that endomitosis in mouse and human megakaryocytes
correspond to an abortive mitotic process.6,7 Megakaryocytes undergo a normal cycle progression with G1, S, and G2
phases. It could be demonstrated by immunofluorescence that the first
stages of mitosis also normally occur with chromosome condensation and
a single multipolar spindle. More surprisingly, a nuclear membrane
breakdown was observed that is in contradiction with the theoretical
definition of an endomitosis. The first stage of anaphase (anaphase A)
characterized by sister chromatid separation and chromosome to pole
movement was also present. In both studies, however, absence of spindle
pole to pole elongation and chromosome segregation via microtubule
retraction was noticed and was interpreted by Nagata et al6
as a defect of anaphase B. Finally, telophase or cytokinesis was never
observed. Thus, megakaryocyte endomitosis is incomplete mitosis
characterized by a skip of late mitosis stages. However, the molecular
mechanisms responsible for this cell-cycle abnormality remain to be determined.
Late mitosis, from metaphase to cytokinesis, is characterized by timed
and coordinated events, which imply proper mechanisms of regulation
(reviewed in Morgan et al8). Most of these mechanisms have
been largely studied in genetic yeast models, leading to the
identification of key protein networks. Homologues of the main
components have been found in other species, including human, revealing
that this system is highly conserved through the evolution. The
fundamental principle of coordination of late mitotic events is based
on the sequential ubiquitin-dependent proteolysis of regulatory
proteins, catalyzed by the ubiquitin-ligase anaphase-promoting complex
(APC).8 The sequential association of the APC with 2 activators, namely CDC20 at the metaphase/anaphase transition, then
Cdh1 (p55CDC/hCDC209 and hCdh1 are the human
homologues10) at anaphase seems partially responsible for
the specific substrate targeting and timing.
The first critical step in late mitosis is an accurate chromosome
segregation, which ensures an equivalent DNA repartition between the 2 daughter cells and confers genomic stability. It is initiated as early
as prometaphase, which allows the bipolar attachment of chromosomes,
followed by their alignment on the equatorial plate at metaphase.
Although all the chromosomes are attached, the p55CDC-APC complex
triggers the anaphase inhibitor Pds1 for degradation, leading to the
sister chromatid separation with cohesin release (reviewed in
Nasmyth et al11). This process is monitored by a conserved
spindle checkpoint that is able to delay the anaphase onset and to
inhibit the mitotic exit, in case of spindle assembly abnormality or a
failure in chromosome attachment (reviewed by Rudner and
Murray12 and Hardwick13). Several proteins
including Mad1, 214,15 and Bub1, 316,17 are
implicated that form complexes at the kinetochores of unattached
chromosomes.18 If a single chromosome is misaligned, the
main component Mad2 blocks the activation of the p55CDC-APC and
maintains the metaphase.19,20
The second crucial event during the mitosis is the cyclin B1
degradation, leading to Cdk1 inactivation. In somatic vertebrate cells,
cyclin B1 destruction has been shown to be initiated at metaphase and
complete at the beginning of anaphase,21 consistent with
the notion that p55CDC-APC might target cyclin B1 (as well as anaphase
inhibitors) for proteolysis.8 Late mitotic abnormalities such as anaphase delay, aberrant anaphase, and a failure of mitotic exit have also been described after microinjection of an anti-p55CDC antibody in a human cell line, arguing for a similar function of P55CDC
in humans.22 In yeast, cyclin destruction by the CDC20-APC initiates a drop in Cdk1 activity, leading to the subsequent activation of the Cdh1-APC.23,24 Therefore, high Cdh1-APC activity at the end of mitosis induces a complete cyclin B1 degradation that allows
cytokinesis, and the maintenance of a mitotic cyclin low level allows
G1 phase transition followed by DNA replication.25 Furthermore, a separate pathway of the spindle checkpoint has been
defined, implying Bub2 protein located at the spindle
poles,26 which monitors the completion of anaphase and
prevents activation of Cdh1-APC, degradation of the B type cyclin, and
the final mitosis exit (reviewed by Burke27). The precise
role of Cdh1 during mitosis has not been clearly defined in
eukaryotes,28 although homologues of this regulation
pathway, such as hCdc14 phosphatase and GAPCenA, have been identified
in humans.29 Different timing of p55CDC and hCdh1 has been
explained through distinct mechanisms of regulation. p55CDC expression
has been demonstrated to fluctuate during cell cycle, in parallel to
change in cellular localization. Its level peaks at the onset of the
mitosis, when the protein is localized in the nucleus at prophase and
concentrates at the kinetochores at prometaphase. Finally, p55CDC is
abruptly degraded at the end of mitosis. HCdh1 also displays
fluctuations through the cell cycle, whereas its main activation is
regulated by the nonphosphorylated status of the
protein.28
In this report, we investigated how chromosome segregation occurred on
a multipolar spindle in endomitotic megakaryocytes. Indeed, previous
immunofluorescence studies with kinetochores staining were limited to
properly analyze separation and segregation of chromosomes. Because of
the high number of chromosomes in polyploid cells, they only gave a
rough appreciation of this phase. Whether this process was complete for
all the chromosomes remained an important point to define, because an
abnormal anaphase A might prevent cells from undergoing the following
anaphase B and cytokinesis. To perform this study, we elaborated a new
approach, associating both primed in situ labeling (PRINS) to stain
chromosome centromeres and immunofluorescence to visualize
megakaryocyte spindle. This procedure allowed us to demonstrate that
the mitotic checkpoint is functional in megakaryocytes. However, an
asymmetrical repartition of chromosomes in polyploid megakaryocytes
occurs that is the consequence of the spindle organization but not to a
defect in sister chromatid separation. Moreover, in contrast with
previous published results,30 mechanisms of endomitosis
seem independent of cyclin B1 regulation.
In vitro cultures of megakaryocytes from CD34+
cells
CD34+ cells were grown for 5 days in Iscoves modified
Dulbecco medium (Gibco BRL, Cergy Pontoise, France) that contained
penicillin (100 U/mL), streptomycin (100 µg/mL), glutamine (2 mM)
(Sigma Chemical, St Louis, MO), PRINS and immunofluorescence
Fluorescence in situ hybridization and immunofluorescence Similar megakaryocyte preparations as described above were used. Immunodetection of the von Willebrand factor (vWF) was first performed by using a rabbit polyclonal antibody (1/200; Dako, Trappes, France). Then slides were fixed 5 minutes again in methanol 75/acetic acid 25 and dehydrated in ethanol 70%, 85%, and 100% before the following fluorescence in situ hybridization (FISH) procedure on DNA. A labeled 11q probe was used according to the distributor recommendations (LSI 11q, Vysis, Voinsins-le Bretonneux, France). Codenaturation of probe and specimens was made in a single step (2 minutes at 85°C) followed by 8 hours of hybridization at 37°C by placing the slides on a hot plate. Slides were immediately immersed in 0.4 × SSC/0.3% NP-40 at 72°C for 2 minutes, also in 2 × SSC/0.1% NP-40 for 30 seconds, and were finally mounted, using 4',6-diamidino-2phenilindole (DAPI) and Vectashield (Vector Laboratories).Indirect immunofluorescence Megakaryocytes were cytocentrifuged onto slides, fixed in 2% paraformaldehyde (Serva, Heidelberg, Germany) for 10 minutes, and rinsed in 1 × PBS. Cells were permeabilized with 0.1% Triton X-100 before incubation with primary antibodies for 30 minutes. Three washes in 1 × PBS preceded incubation with secondary antibodies for 30 minutes. DNA was stained with TOTO3-iodide diluted at 1/1500 for 1 hour, or DAPI finally mounted with antifading. The primary antibodies included mouse anti- , anti- -tubulin (1/50, Sigma) and
anti-cyclin B1 (1/100, GNS-1 Pharmingen) antibodies, rabbit anti-p55CDC (1/200, Amgen), anti-hCdh1 (1/200, kindly provided by
J. M. Peters, Research Institute of Molecular Pathology, Vienna, Austria) antibodies, a goat anti-MKLP1 (Santa Cruz Biotechnology, Santa
Cruz, CA) antibody, and a human antikinetochore antibody (1/200,
obtained from patients with calcinosis cutis, Raynaud phenomenon,
esophageal motility disorder, sclerodactyly, and telangiectasis [CREST] syndrome and kindly provided by J. C. Brouet, Hopital St
Louis, Paris, France). Secondary antibodies were donkey fluorescein isothiocyanate (FITC)- or TRITC-labeled antimouse, FITC- or
TRITC-conjugated antirabbit, TRITC-labeled antigoat, and Cy5-labeled
antihuman F(ab')2 fragments (1/50, Jackson Immunoresearch Laboratories).
Image acquisition and analysis Confocal images were captured using a laser scanning confocal microscope LMS 510 (Zeiss, Oberkochen, Germany) either with Plan-apochromat × 63/NA1.4 or × 100/NA1.4 objectives. Three laser excitations were used (ie, 488 nm, 543 nm, 633 nm for FITC, TRITC, and Cy5 or TOTO 3-iodide, respectively). Serial optical sections of 0.7 µm (images collected at 0.5-µm intervals) in the z-axis of the cell were collected sequentially for each marker and overlaid to obtain a 3-dimensional reconstruction.Other images were captured with the use of an epifluorescence microscope, using either × 60 or × 40 objectives (Nikon, Tokyo, Japan) and processed using Adobe Photoshop 5.0.
To observe chromosome segregation during megakaryocyte polyploidization, we elaborated a new method, which couples PRINS for labeling centromeres of one chromosome and tubulin immunostaining to visualize the endomitotic spindle. In that case, PRINS provided an advantageous approach to label centromeres of chromosomes in comparison to classical FISH for 2 reasons. First, it is a quicker method, giving best quality cell samples for the subsequent immunostaining procedure; second, in situ primer elongation gave a stronger and more specific centromeric signal, compared to what can be obtained by probe hybridization. Chromosomes 1 or 7 centromeres were arbitrarily chosen for labeling because specific probes were available for these 2 chromosomes.33 Simultaneous painting of more than one chromosome would have been confusing for the image analysis because of the high number of generated signals in polyploid cells. Interphase nuclei contained green (FITC) dots, corresponding to
centromeres of chromosomes 1 or 7, depending on the specific primers
used for the experiments. Their number could be correlated to cell
ploidy, because most interphase nuclei contained 2 copies when they
were diploid, and 2xN copies when their DNA content was
polyploid. During mitosis, these dots were localized on the spindle
labeled in red (TRITC) by immunofluorescence (Figure
1).
To precisely analyze chromosome segregation, the whole nucleus was analyzed by confocal microscopy. In diploid cells from interphase to metaphase, 2 green dots (2 copies of the same chromosome) were observed; 4 dots were present when sister chromatids separated from the beginning of anaphase to the onset of cytokinesis, 2 on each side of the equator, as would be expected from a regulated chromosome segregation (data not shown). In case of polyploid cells, which had complex spherical multipolar spindles, a simultaneous nuclear DNA painting was necessary to properly determine the precise stage of endomitosis. Asymmetrical chromosomes repartition in polyploid megakaryocytes CD34+ cell-derived cultures did not contain only megakaryocytes (about 50% purity). A double labeling with anti-vWF and antitubulin antibodies was first performed, which confirmed that polyploid cells were exclusively megakaryocytes, and allowed us to use the protocol described above without need of a megakaryocytic marker. In the subsequent set of experiments, we analyzed endomitotic megakaryocytes and observed that the centromeres of chromosome 1 were not symmetrically distributed along the multipolar spindle of all endomitotic cells examined. Indeed, some spindle poles did not bear any chromosome 1s as illustrated in Figure 2 in either 4N (Figure 2A), 8N (Figure 2B), or 16N (Figure 2C) megakaryocytes. Identical experiments were performed by labeling the centromere of chromosome 7s, leading to the same results (data not shown). These observations demonstrate that the chromosome repartition is performed asymmetrically on the multipolar spindle, regardless of the chromosome category (either chromosome 1 or 7) or the degree of cell ploidy.
Megakaryocytes have a normal metaphase/anaphase checkpoint but an asymmetrical segregation of chromosomes at anaphase The adequate bipolar attachment of chromosome to microtubules from different centrosomes occurs at prometaphase and metaphase and is essential for the ensuing accurate segregation of chromosomes at anaphase onset. This stage is normally monitored by the mitotic checkpoint. The observation of an asymmetrical repartition of chromosomes along the megakaryocytic spindle suggested an abnormality of chromosome attachment to microtubules, which had been missed by the mitotic checkpoint. Therefore, we investigated if the control of this checkpoint was normal in megakaryocytes as had been previously suggested.6 To explore the metaphase/anaphase checkpoint in megakaryocytes, nocodazole was added to the cultures, and cells were studied at various times up to 24 hours (0, 3, 5, 7, and 24 hours). Mitotic index was determined using nuclear staining, and cells were considered to be in pseudo-metaphase arrest when chromosomes were condensed. A total of 400 cells were examined at each time period, and the average of a triplicate determination was calculated. Typical pseudo-metaphase arrest of megakaryocytes was observed as early as 3 hours following exposure to nocodazole, whereas mitotic index increased from 1% up to 25% after 24 hours. The pseudo-metaphase arrest was maintained as long as the drug was present. In contrast, after nocodazole withdrawal, megakaryocytes exit from mitosis (Figure 3A) as judged by decondensed chromosomes. As megakaryocyte culture contains a portion of diploid cells with normal mitotic cell cycle, an additional score on 500 cells was similarly determined, taking into account only polyploid cells. Similar results were observed (Figure 3B)
As the mitotic checkpoint is normal in megakaryocytes, it suggests that
chromatid separation should occur normally. By careful analysis of
endomitotic megakaryocytes we had previously analyzed, we observed that
the number of hybridization dots was similar to that of spindle poles:
it meant that the sister chromatids had not yet separated (Figure
2A-C). The endomitosis of these previous experiments corresponded to
prometaphase or metaphase, but not to anaphase, which is consistent
with the respective duration of both phases. Thereafter, improved
culture conditions and microscopic examination of up to 500 endomitotic
megakaryocytes allowed us to visualize anaphases and chromatid
separation. At this stage, regardless of ploidy, the nucleus was
characterized by a unique shape with chromosomes forming rings around
each aster (Figure 4). This organization
was the consequence of chromosome segregation and migration to each
spindle pole. In that case, the dot number, which is equivalent to the
labeled sister chromatid number (chromosome 1), was twice the aster
number, as expected. It was clearly visualized during endomitosis of 4N
megakaryocytes, which have 8 labeled chromosome 1s on their
tetrapolar-spindle at anaphase (Figure 4A). These data support the
contention that sister chromatid separation was complete and normal in
megakaryocytes as previously suggested.6 However, the
numbers of chromosome 1s at each aster were different (from 0 to 4 during 4N to 8N endomitosis), suggesting that chromosome migration
toward the spindle poles is asymmetrical. Then, at the end of anaphase,
the number of each category of chromosome is not identical for each
aster in polyploid megakaryocytes (Figure 4A-C). Identical results were
obtained with centromere labeling of chromosome 7 (not shown). However,
2 potential biases could limit the proper interpretation of these
results: first, PRINS as well as other in situ fluorescence labeling
techniques can give false signals. These false signals were clearly
distinguished from the real signals by confocal microscopy, considering
the colocalization of the last one with the labeled DNA. Second, 2 signals could superimpose on a cell view and appear as one dot: it was
resolved by analyzing different angles of the same cell using the
3-dimensional cell reconstruction (not shown). Thus, we were able to
clearly demonstrate that chromosome segregation was asymmetrical; this
asymmetry was independent of the chromosome category (either chromosome
1 or 7) and the degree of cell ploidy.
P55CDC/hCDC20 protein is expressed and normally regulated in polyploid megakaryocytes P55CDC/hCDC20 is involved in metaphase/anaphase checkpoint, which appears to be normal in megakaryocytes but also may regulate late phases of the mitosis.22 We, therefore, studied p55CDC/hCDC20 expression in megakaryocytes and examined its cell-cycle changes during endomitosis. By immunostaining experiments, p55CDC was detected in megakaryocyte nuclei (Figure 5A). Double labeling with a human CREST antibody colocalized p55CDC to each kinetochore at prometaphase and metaphase. Finally, when using a third staining with an antitubulin antibody, the p55CDC localization and the kinetics of its disappearance could be studied during an endomitotic cell cycle (Figure 5B,C). The results were consistent with those previously obtained by Kallio et al22 during a normal mitosis. P55CDC accumulated in the nucleus at prophase; it concentrated at kinetochores at prometaphase with a heterogeneous staining intensity between chromosomes: Those closest to the spindle poles, which have not yet been correctly attached to the microtubules, had a brighter staining than those close to the metaphase plate. At metaphase, all chromosomes accumulated in the center of the spindle, forming a complex metaphase plate, had a quite uniform and less intense p55CDC signal. At anaphase p55CDC could not be detected. Thus, p55CDC has a normal expression in megakaryocytes.
Spindle organization explains the paradox between an asymmetrical segregation of chromosomes and a normal mitotic checkpoint Endomitotic megakaryocytes are characterized by a multipolar spindle. According to immunofluorescence section analysis of these spindles, several asters are bound together and form a complex array of interconnected microtubules (Figure 6). At metaphase, chromosomes migrated to the center of the spindle and formed a multibranched structure. At anaphase, sister chromatid separation occurred asynchronously, depending on the spindle localization (Figure 2D). The separated chromatids migrated toward each spindle pole, resulting in an asymmetrical distribution between the different poles. This asymmetry was related to the complex structure of the spindle, provided one aster is connected to more than one other aster. Thus, at the level of a "bipolar spindle" (ie, connection between 2 asters), chromosome segregation was symmetrical, but it became asymmetrical when the entire spindle was considered. In a manner similar to mitotic diploid cells, chromosomes clustered into a ring around each aster. In the absence of spindle elongation and disassociation movements of the chromosomes surrounding the aster, the different rings remained connected to each other. Consequently, the nuclear membrane may reform around a single polyploid nucleus. At this stage the spindle was degraded, and the chromosomes decondensed. Each chromosome ring appears to correspond to one lobe of the megakaryocytic nucleus (Figure 5C).
This hypothesis was verified by further analysis of interphase megakaryocytes. We observed a heterogeneous distribution of chromosome 11 labeled by FISH in the different lobes of the polyploid nuclei (Figure 2E). The significance of the megakaryocyte nucleus polylobulation was not well understood, but these results suggest that each lobe corresponds to the incompletely segregated chromosomes surrounding one aster as a consequence of the anaphase B defect. Timing of cyclin B1 degradation is conserved in polyploid megakaryocytes As we have first demonstrated that p55CDC/hCDC20 and the "first branch" of the spindle checkpoint were functional in polyploid megakaryocytes, we investigated the second main regulation pathway leading to mitotic exit.27We first investigated expression of hCdh1, the second APC
coactivator, by immunofluorescence experiments. We could observe that
this protein was normally expressed in polyploid megakaryocytes (Figure
7A). The nuclear punctuate staining was
homogeneous in interphase (Figure 7Ai), consistent with previous
experiments in human neuronal cells.34 During endomitosis,
hCdh1 colocalized with the spindle poles (a common localization with
cyclin B1) (Figure 7Aii,B), and these results were confirmed in the
myeloid KG1a cell line. Nevertheless, as hCdh1 is activated through
dephosphorylation more than through its expression level, these results
do not demonstrate that this molecule is functional during endomitosis.
Thus, we decided to study indirectly Cdh1 function through the analysis of the main hCdh1-APC complex substrate: cyclin B1. With the use of
cyclin B1 immunostaining, we confirmed expression of cyclin B1 in
polyploid megakaryocytes. Moreover, by analyzing many endomitosis situations, we showed that timing of the mitotic cyclin expression was
analogous to that previously described in normal mitosis21 (Figure 7B). During endomitosis, cyclin B1 displayed a strong nuclear
labeling in prophase, then colocalized with spindle poles in
prometaphase, whereas staining completely disappears at anaphase. Altogether, these results demonstrate that cyclin B1 is expressed in
endomitotic megakaryocytes with a normal timing. Normal degradation of
cyclin B1 concomitant with expression of hCdh1 provides a
molecular environment25 in megakaryocytes favorable
for G1-phase transition accomplishment and DNA re-replication
despite an abortive mitosis.
Expression of MKLP1 in megakaryocytes Finally, we investigated whether one of the main kinesin MKLP1 implicated in anaphase and cytokinesis35,36 was expressed in megakaryocytes. Immunofluorescence experiments showed that MKLP1 was normally present in the midzone of the multipolar spindle of megakaryocytes (Figure 8) like in other diploid mitotic cells, revealing that the first stages of the cleavage furrow organization occurs. Subsequent bundles of the microtubules were, however, not observed, as expected.
Previous studies have provided some evidence that endomitosis could be a consequence of both a multipolar spindle that limits chromatid separation and the absence of cytokinesis.6,7 Our purpose was to investigate whether chromosomes segregate normally during endomitosis. We addressed this question by localizing centromeres of either chromosome 1 or 7 in endomitotic human megakaryocytes. Our main difficulty was to elaborate a reliable strategy to visualize one category of chromosomes and to follow their localization along the spindle during the different stages of mitosis. PRINS labeling is an alternative to FISH that has already proved its ability to label repeated sequences of DNA-like centromeres.37 For our purpose, it turned out to be an advantageous approach because of its simplicity, quickness, specificity, and the possibility to couple it with an immunostaining procedure. With this technique we observed an asymmetrical repartition of labeled chromosome 1s along the spindle at prometaphase and metaphase of each endomitosis from 4N to 32N cells. Indeed, megakaryocytes usually had the expected number of labeled chromosome 1s, but they were not symmetrically coupled to a spindle pole. A defined subnuclear localization of chromosomes, depending on their category, has been described.38 To demonstrate that the asymmetrical repartition was not due to a particular localization of chromosome 1s, we performed the same experiments with labeling of chromosome 7 centromeres and observed a similar asymmetrical repartition of this chromosome along the spindle of polyploid megakaryocytes. The metaphase/anaphase checkpoint explored by spindle disruption with nocodazole treatment was normal because the endomitotic index increased with time in the presence of the drug, suggesting that the entry in anaphase is normally controlled. Thus, our goal was to demonstrate how chromosome segregation per se occurs during the endomitotic anaphase. A major obstacle was the scarceness of anaphase on slides, inasmuch as endomitotic megakaryocytes corresponded to less than 1% of the cell samples. Therefore, there was an obvious need to analyze a very large number of asynchronous endomitosis to observe a few anaphases. By this approach we could prove that chromosome disjunction was always a complete process for chromosome 1, 7, or 11. This result is in agreement with previous experiments, which have suggested by immunostaining that chromatid separation occurs in megakaryocytes.6,7 However, despite this normal centromere dissociation, the final repartition of sister chromatids was always asymmetrical between all the spindle poles leading to different numbers of chromosomes at each pole. It was possible to extrapolate these conclusions whatever the ploidy of human megakaryocytes (from 4N to 64N). Furthermore, by studying late stages of endomitosis, we found that each lobe of the megakaryocyte nucleus corresponded to chromosomes that have migrated to one aster and thus are the equivalent of one nucleus. However, this asymmetrical repartition of chromosomes explains why previously it has been shown that each individual lobe does not have a 2N ploidy.39 To our knowledge, there is no precedent that an "atypical"
segregation of chromosomes is allowed by a normal mitotic checkpoint, 2 statements which are apparently contradictory. One explanation could be
found in the structure of megakaryocytic spindles. A 2-pole spindle has
only 2 microtubule arrays, and there is theoretically no problem of
"choice" for chromosome attachment. In contrast, immunofluorescence
studies have shown that the megakaryocytic spindle contained several
asters that turned out to be interconnected. In that case, the
chromosomes can be effectively bound in a stable manner to microtubules
emanating from 2 different asters, which seem to be randomly chosen.
Then sister chromatids separate normally and migrate symmetrically
between these 2 poles. However, chromosome distribution is ultimately
asymmetrical when considering the entire multipolar spindle. Thus, the
definitive asymmetrical localization of chromosomes appears to be due
to the existence of multiple interconnections between the different
asters. These results are summarized in a model in Figure
9.
Previously, the phosphoepitope 3F3/2 was shown to disappear during endomitosis,7 a result also in favor of a normal regulation of the metaphase/anaphase transition.40 To further demonstrate this hypothesis, we investigated the expression and localization of P55CDC/hCDC20 in megakaryocytes. P55CDC plays a major role in mitosis by conferring the specificity of the proteolysis that occurs at the metaphase/anaphase transition and may also be involved in the regulation of mitotic exit. The level of P55CDC is regulated during the cell cycle with a peak at mitotic entry and a fall at mitotic exit. Immunofluorescence demonstrates that p55CDC is maximally expressed at prometaphase and is localized on kinetochores. The staining is lost in anaphase. The same dynamics and localization of P55CDC were observed in megakaryocytes at all levels of ploidy. Previous studies have involved cyclin B1 in the endomitotic process with controversial results. Two found a high level of cyclin B1 in polyploid endomitotic megakaryocytes.6,7 In contrast, other studies in megakaryocytic cell line and primary megakaryocytes showed a reduced level of cyclin B1 correlated with a modified dynamics during endomitosis, related to an enhanced ability to degrade cyclin B1.30 Our immunofluorescence experiments definitively show that cyclin B1 has strictly the same timing in endomitotic megakaryocytes as in other human cells with a strong nuclear staining in G2 prophase, a localization at the spindle poles in prometaphase, followed by an abrupt disappearance at anaphase.21 By the same method, hCdh1 was detected in megakaryocytes, but its functionality and role in endomitosis remain to be confirmed. Nonetheless, as described in other vertebrate somatic cells, the Cdh1-APC is supposed to be functional in G1 phase, allowing the re-replication of the genome since megakaryocytes have degraded cyclin B1 and exit of endomitosis.25 Taken together, our observations lead to the conclusion that neither chromosome segregation nor cyclin B1 regulation abnormalities are implicated in the endomitotic mechanism. Thus, endomitosis might be the consequence of microtubule motor proteins, as previously suggested by Nagata et al,6 so that the nuclear membrane finally reforms around a unique polyploid nucleus without telophase and cytokinesis. In diploid cells, the metaphase spindle steady-state structure is the result of antagonistic forces generated on one hand by the C terminal kinesins and on another hand by bipolar kinesins and the MKLP1 kinesins located at the midzone (reviewed in Sharp et al41). Anaphase B spindle elongation is usually correlated with the inactivation of "inward force." Then, during endomitosis, the absence of anaphase B might correspond to 3 mechanisms. The first mechanism is the lack of microtubule motor proteins that participate in the separation of the 2 polar microtubule arrays. This seems unlikely as the first states of spindle assembly and metaphase plate normally occur and microtubule motors likely operate in a group with extensive overlapping function.42,43 Immunofluorescence studies also confirmed that the MKLP1 protein was present in megakaryocytes with an expected localization at the midzone of the spindle at anaphase. More than a structural defect, the endomitotic mechanism could effectively reside in an abnormal regulation of such "outward proteins" that may depend on the aurora kinase and polo kinase pathways.36,44 In favor of this hypothesis, the aurora kinase AIM1 has been shown to be down-regulated in the megakaryocyte.45 Finally, a defect of C kinesin degradation might also compromise the anaphase B process in megakaryocytes. All these hypotheses have to be confirmed in the future with the help of other eukaryote cell models.
The authors thank J. M. Peters (Research Institute of Molecular Pathology, A-1030 Vienna, Austria) and J. C. Brouet (Hôpital St Louis, Paris, France) for the human antibodies, anti-hCdh1 and antikinetochores, respectively.
Submitted May 30, 2000; accepted November 28, 2000.
Supported by Fondation de la Recherche Médicale (FRM) (L.R.) and Association de la Recherche contre le Cancer (ARC 9728) (N.D.) from La Ligue Nationale contre le Cancer.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: William Vainchenker, INSERM U362, PR1, Institut Gustave Roussy, 39 rue Camille Desmoulins, Villejuif, 94805, France; e-mail: verpre{at}igr.fr.
1. Odell TT, Jackson CWJ, Reiter RS. Generation cycle of rat megakaryocytes. Exp Cell Res 1968;53:321-327[CrossRef]. 2. Behnke O. An electron microscope study of the megacaryocyte of the rat bone marrow, I: the development of the demarcation membrane system and the platelet surface coat. J Ultrastruct Res 1968;24:412-433[CrossRef][Medline] [Order article via Infotrieve].
3.
Cramer E, Norol F, Guichard J, Breton-Gorius J, Vainchenker W, Debili N.
Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.
Blood
1997;89:2336-2346
4.
Italiano JE Jr, Lecine P, Shivdasani RA, Hartwig JH.
Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes.
J Cell Biol
1999;147:1299-1312 5. Zimmet J, Ravid K. Polyploidy: occurrence in nature, mechanisms, and significance for the megakaryocyte-platelet system. Exp Hematol 2000;28:3-16[CrossRef][Medline] [Order article via Infotrieve].
6.
Nagata Y, Muro Y, Todokoro K.
Thrombopoietin-induced polyploidization of bone marrow megakaryocytes is due to a unique regulatory mechanism in late mitosis.
J Cell Biol
1997;139:449-457
7.
Vitrat N, Cohen-Solal K, Pique C, et al.
Endomitosis of human megakaryocytes are due to abortive mitosis.
Blood
1998;91:3711-3723 8. Morgan DO. Regulation of the APC and the exit from mitosis. Nat Cell Biol 1999;1:E47-E53[CrossRef][Medline] [Order article via Infotrieve].
9.
Weinstein J.
Cell cycle-regulated expression, phosphorylation, and degradation of p55Cdc.
J Biol Chem
1997;272:28501-28511 10. Fang G, Yu H, Kirschner MW. Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1. Mol Cell 1998;2:163-171[CrossRef][Medline] [Order article via Infotrieve].
11.
Nasmyth K, Peters JM, Uhlmann F.
Splitting the chromosome: cutting the ties that bind sister chromatids.
Science
2000;288:1379-1385 12. Rudner AD, Murray AW. The spindle assembly checkpoint. Curr Opin Cell Biol 1996;8:773-780[CrossRef][Medline] [Order article via Infotrieve]. 13. Hardwick KG. The spindle chekpoint. Trends Genet 1998;14:1-4 |
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Abstract
Introduction
, 2 µg/mL) was added to the medium of culture of
megakaryocytes obtained from CD34+ cells with PEG-rhuMGDF
and SCF. Megakaryocytes were regularly sampled (at 0, 3, 5, 7, and 24 hours) centrifuged onto slides and fixed with paraformaldehyde, then
nuclei were counterstained with DAPI. Mitotic index was calculated for
400 cells at each time and determined on the average of triplicate
experiments. In parallel, nocodazole was withdrawn (
) at 7 hours and
mitotic indexes were similarly determined at 24 and 48 hours (A). Panel
B shows a similar experiment in which only polyploid megakaryocytes
were taken into account to determine the mitotic index (based on a
500-cell count for each sample).
