1 Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo
Alegre 823, 4150-180 Porto, Portugal
2 Instituto Superior de Ciências da Saúde-Norte, Grupo de Biologia
Molecular e Celular, Rua Central de Gandra 1317, 4580 Gandra PRD,
Portugal
3 ICBAS, Instituto de Ciências Biomédicas de Abel Salazar,
Universidade do Porto, 4000 Porto, Portugal
Author for correspondence (e-mail:
cesunkel{at}ibmc.up.pt)
Accepted 5 August 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: BubR1, Mitosis, Checkpoint, Embryogenesis, Polar body, Drosophila
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Current studies of the mitotic checkpoint proteins Mad2 and Bub3 indicate
that spindle checkpoint activity is required for proper chromosome segregation
within the epiblast of the mouse embryo at the time of rapid nuclear
proliferation (Dobles et al.,
2000; Kalitsis et al.,
2000
). In the Drosophila syncytial embryo, global
perturbations such as blocking DNA replication, inducing extensive DNA
damaged, or blocking spindle assembly are known to arrest or delay mitosis
throughout the syncytium (Callaini et al.,
1994
; Debec et al.,
1996
; Fogarty et al.,
1997
; Raff and Glover,
1988
). Interestingly, embryonic expression of a non-degradable
form of cyclin B or increase maternal cyclin B contribution can affect the
global behaviour of nuclear cycles (Ji et
al., 2002
; Su et al.,
1998
). In addition, syncytial mutant embryos for the mitotic
checkpoint protein Mps1 fail to arrest in mitosis in response to hypoxia
(Fischer et al., 2004
). It has
been recently proposed that embryogenesis between mammals and other metazoans
such as fly or frog should be aligned not from fertilisation but from the
rapid embryonic cell cycles (O'Farrell et
al., 2004
). Thus, mammalian peri-gastrulation cell cycles should
be homologous to the rapid cleavages cycles of other metazoans, such as
syncytial cycles in flies. Therefore, mitotic checkpoint proteins could have
an important and conserved role in monitoring rapid embryonic cell divisions
in different and evolutionary distant metazoans.
Mitotic checkpoint proteins detect microtubule attachment and/or tension
applied across kinetochore pairs to ensure that chromatids separate during
mitosis only when kinetochores form stable bipolar microtubule attachments
(Musacchio and Hardwick,
2002). Current studies indicate that the well-conserved Bub and
Mad proteins play an essential role in the spindle checkpoint function. These
proteins inhibit the activity of the anaphase promoting complex or cyclosome
(APC/C) until microtubule attachment/tension is achieved for all chromosomes
(Peters, 2002
;
Zachariae and Nasmyth, 1999
).
BubR1 is an essential spindle checkpoint protein conserved among higher
eukaryotic organisms (Chan et al.,
1998
; Chen, 2002
).
Recently, it has been shown that the previously identified Bub1 protein in
Drosophila (Basu et al.,
1999
) should be reclassified as BubR1
(Logarinho et al., 2004
). In
Drosophila somatic cells, BubR1 loss of function mutation causes an
accelerated transit through mitosis which leads to abnormal chromosome
segregation and apoptosis (Basu et al.,
1999
).
In order to analyse whether BubR1 is required during the nuclear
proliferation at the syncytial stages in Drosophila embryos, we
characterised a new semi-viable female sterile hypomorphic allele
(bubR1Rev1) that was produced by imprecise excision of a
previously characterised P element insertion
(Basu et al., 1999). In embryos
from homozygous bubR1Rev1 mutant females, syncytial nuclei
show defects similar to those observed in neuroblasts homozygous for the
bubR11 allele and do not arrest in mitosis in response to
microtubule depolymerisation. In addition, whereas in wild-type embryos the 10
first syncytial nuclear divisions are synchronous, embryos from homozygous
bubR1Rev1 mutant females show severe nuclear
de-synchronisation. Furthermore, BubR1 accumulates at the kinetochores of the
highly condensed polar body chromosomes. Analysis of fertilised embryos from
homozygous bubR1Rev1 mutant females indicates that BubR1
is required to maintain the condensed conformation of polar body chromosomes
and to exclude it from the mitotic oscillation.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Southern blotting
Genomic DNA was extracted according to Roberts
(Roberts, 1986) from adult
flies frozen in liquid nitrogen and digested with HindIII and
EcoRI (New England Biolabs). Southern blots were done according to
Sambrook et al. (Sambrook et al.,
1989
) and probed with a complete cDNA (see
Fig. 2A).
Western blotting
Third larval instar brains, adult female ovaries and embryos were dissected
in 1xPBS, transferred to sample buffer [50 mM Tris (pH 6.8), 100 mM DTT,
2% SDS, 0,1% Bromophenol Blue and 10% glycerol] heated 3 minutes at 95°C
before SDS-page (7.5%) migration. Proteins were transferred onto Immobilon-P
membrane (Schleicher & Schuell) and probed with anti-DmBubR1 rabbit
polyclonal antibodies (Rb666 Ab)
(Logarinho et al., 2004),
diluted 1:2500 in PBST (1x PBS and 0.2% Triton-100) containing 1% BSA
(Sigma) and 2% dried non-fat milk. Anti-
-tubulin was detected using
mouse mAb B512 (Sigma-Aldrich), diluted 1:2000. Secondary antibodies
conjugated to HRP (Vector, UK) were used according to the manufacturer's
instructions. Detection of antibody signals was performed with the ECL system
(Amersham).
Immunofluorescence of embryos
Embryos were collected and aged at 25°C and processed as previously
(Sullivan et al., 2000).
Primary antibodies were: anti-BubR1 (Rb666) antibody pre-adsorbed on wild-type
0-2 hours embryos at a 1:10 dilution and used at 1:1000 dilution;
anti-
-tubulin (mouse mAb B512), used at 1:3000 (Sigma-Aldrich);
anti-phospho H3 rabbit polyclonal, used at 1:500 (Upstate Biotechnology);
anti-CID chicken polyclonal, used at 1:1000
(Blower and Karpen, 2001
);
anti-CNN rabbit polyclonal, used at 1:1000
(Heuer et al., 1995
);
anti-BrdU mouse monoclonal primary antibody, used at 1:50 (Sigma-Aldrich).
Secondary antibodies were: anti-rabbit Alexa Fluor 488 used at 1:2000
(Molecular Probes), anti-mouse Alexa Fluor 568 used at 1:2000 (Molecular
Probes) or anti-chicken Cy5 used at 1:200 (Jackson). Samples were mounted in
Vectashield (Vector, UK) containing 1 µg/ml of DAPI or 1.0 µg/ml
propidium iodide. Observation and images were obtained using Zeiss Axiovert
200M microscope (Zeiss, Germany) or a Leica TCS SP2 AOBS Confocal Microscope
(Leica Microsystems, Heidelberg). Images were deconvolved and processed with
Adobe Photoshop 7.0. For colchicine treatment, embryos were permeabilised in
n-heptane, containing 250 µM colchicine in 1x PBS for 30 minutes
before fixation. For BrdU incorporation, the embryos were permeabilised for 4
minutes in n-octane followed by 8-10 minutes BrdU pulse at 1 mg/ml in 1x
PBS before fixation.
Live imaging
Chromosomes in wild-type and bubR1Rev1 embryos were
visualised by time-lapse recording of the Histone-GFP transgene. Images were
taken every 10 or 15 seconds using a BioRad MRC600 Confocal Microscopy with
two scans per image. To determine the polar body Histone-GFP signal intensity
over time, we repeated three measurements of a restricted area of 16 pixels
taken from regions with higher intensity signal and use ImageJ software to
obtain an average and standard deviation for each image. Using the same
measurements, we calculated the average standard deviation pixel intensity of
the recorded period of time.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
BubR1 protein levels in wild-type and mutant alleles at various developmental times were determined by western blotting (Fig. 2C). High levels of BubR1 were detected in wild-type third instar larval brains and ovaries. In bubR11/bubR11 brains, no protein is detected, while in bubR1Rev1/bubR1Rev1 brains and ovaries there is a strong decrease in protein levels. BubR1 protein in ovaries results from somatic expression during the cell proliferation stages, the germline expression during cyst formation and the maternal contribution (data not shown). BubR1 could also be detected by western blot of embryo extracts at 30-90 minutes AEL using two wild-type embryos (Fig. 2C). However, in embryos derived from bubR1Rev1 homozygous females, the level of BubR1 was undetectable (Fig. 2C).
|
Analysis of nuclear divisions in bubR1Rev1 embryos
To determine whether BubR1 was required for nuclear divisions during early
embryogenesis, we analysed bubR1Rev1 embryos
(Fig. 3A-J). Quantitative
analysis of fixed bubR1Rev1 embryos (n=80) at 120
to 150 minutes AEL showed that 25% of the embryos stop development before
cycle 10 (when nuclei reach the cortex); 62% developed up to cycle 10-13,
although nuclei are not found evenly distributed throughout the cortex and
they did not cellularise; 10% initiated cellularisation; and only 3%
gastrulate but show pyknotic nuclei (Fig.
3H). bubR1Rev1 embryos accumulate defective
nuclei throughout the syncytial stages and after cycle 10, most had many
regions without cortical nuclei, extensive areas contained micronuclei,
abnormally larger sized nuclei or paired nuclei with extensive DNA bridges
(Fig. 3A-G).
bubR1Rev1 embryos also showed abnormal mitotic
progression, including precocious sister chromatid separation, lagging
chromosomes, DNA bridges, irregular chromosome segregation and aneuploidy
(Fig. 3E-G). A similar range of
abnormalities was also observed in nuclei within mitotic domains of
cellularised embryos (Fig.
3H).
To obtain a more accurate description of mitotic progression in
bubR1Rev1 embryos, we quantified the relative frequency of
mitotic figures in 30-90 minutes AEL embryos after fixation
(Fig. 3K). We also visualised
in vivo the behaviour of chromosomes using a GFP-Histone transgene
(Clarkson and Saint, 1999)
(Fig. 3I,J; see Movies 1 and 2
in the supplementary material). Quantification of the mitotic figures on fixed
material reveals that, whereas in wild-type embryos all nuclei undergo
synchronous division, almost half of the bubR1Rev1 embryos
show asynchronous nuclear behaviour following cycle 5. Among the synchronous
embryos, we observed a significant decrease in interphase nuclei and we never
observed proper metaphase alignment. Live recording shows that the divisions
in mutant embryos are highly asynchronous compared with the meta-synchronous
divisions in wild-type controls (see Movies 1 and 2 in the supplementary
material). In bubR1Rev1 embryos, nuclei in anaphase
(Fig. 3J at 9 minutes), in
prophase (Fig. 3J at 9 minutes)
and nuclei initiating sister chromatid separation
(Fig. 3J at 9 minutes) can be
found at the same time. At later times, some nuclei show unequal chromosome
segregation with lagging chromatids (Fig.
3J at 11 minutes) and chromatin bridges
(Fig. 3J at 14 minutes). In
addition, during mitotic progression, cytoplasmic movements are strongly
affected in mutant embryos (compare Movie 1 with 2 in the supplementary
material). Taken together these results indicate that BubR1 activity is
required during early embryonic development to maintain nuclear synchrony and
cytoplasmic flux, and to prevent abnormal chromosome segregation.
|
|
Owing to the abnormal mitotic progression of bubR1Rev1 embryos, we looked at the ability of centrosomes to nucleate microtubules and to organise proper mitotic spindles. Although all the centrosomes we observed were able to nucleate microtubule asters, a detailed analysis showed several abnormalities in spindle organisation and distribution with strong variation within each embryo (Fig. 4E-H). Spindles were abnormally distributed within the syncytium and some spindles were shorter in length (Fig. 4E arrowhead and arrow). In other cases, we observed fused spindles while neighbouring nuclei display an almost normal anaphase onset with traces of chromosome bridges (Fig. 4F, arrow and arrowhead). Free centrosomes were observed to replicate asynchronously from the cycle of DNA replication and were able to nucleate microtubules (arrowhead and arrow in Fig. 4G,H). In addition, chromosomes from neighbouring nuclei often shared the same spindle pole (asterisk in Fig. 4K,H). These results indicate that the abnormal mitotic progression observed in bubR1Rev1 embryos uncoupled DNA and centrosomes cycle.
Mitotic progression in bubR1Rev1 embryos after spindle damage
Previous findings in somatic cells of many organisms showed that treatment
with colchicine, a microtubule depolymerising agent, causes cells to arrest in
a prometaphase-like state dependent upon BubR1 accumulation at kinetochores
(Basu et al., 1999;
Chan et al., 1998
). However,
although colchicine treatment of Drosophila embryos during syncytial
stages causes a mitotic arrest (Zalokar
and Erk, 1976
), the role of spindle checkpoint proteins in this
arrest has never been analyzed. Thus, 0-2 hours AEL, wild-type and
bubR1Rev1 embryos were treated with colchicine at a
concentration of 250 µM for 30 minutes and the mitotic arrest was monitored
by immunodetection of Histone H3 phosphorylated at S10, which is diagnostic of
mitotic chromosome condensation (Fig.
5). For quantitative analysis, we divided embryos into three
categories, cycles 3 to 6, cycles 7 to 9 and cycles 10 to 13. The results show
that after colchicine incubation, all nuclei in wild-type embryos arrest in a
prometaphase-like state, with Phospho-Histone H3-positive chromatin
(Fig. 5A,A'',E) and
strong accumulation of BubR1 at kinetochores
(Fig. 5C). However, in
bubR1Rev1 embryos, we observed a decrease in the
proportion of nuclei showing mitotic arrest over time. Although 63% of the
embryos at cycle 3-6 arrest, only 40% at cycle 7-9 and 23% at cycle 10-13 are
Phospho-Histone H3 positive (Fig.
5E). The remaining embryos had mostly post-mitotic nuclei,
Phospho-Histone H3 negative (Fig.
5B) with no BubR1 accumulation at kinetochores
(Fig. 5D). Accordingly, our
results demonstrate that BubR1 checkpoint activity is required during
embryogenesis to allow mitotic arrest in response to spindle damage. In
addition, our results suggest that BubR1 maternal supply from
bubR1Rev1 allele although undetectable by western blotting
of individual embryos is sufficient to support partial checkpoint activity
during early nuclear cycles but as development proceeds it becomes
limiting.
|
|
Our observations of condensation-decondensation cycles, variation in Phospho-Histone H3 signal and increase number of CID-positive dots lead us to investigate whether in the absence of BubR1, the polar body became under the control of the mitotic oscillator that drives the nuclear cycles during the syncytial stages. Using a Histone-GFP transgene and time-lapse confocal microscopy, we recorded for a period of 40-80 minutes the polar body structure in wild type and bubR1Rev1 fertilised embryos collected at 0-30 min AEL (Fig. 7A,B, Movies 3 and 4 in the supplementary material). Then we quantified the relative Histone-GFP signal intensity over time (Fig. 7C) and calculated the pixel intensity±s.d. for the recorded period of time (Fig. 7D). As expected, in fertilised wild-type embryos the polar body always maintains a mitotic-arrest configuration during the recording period (Fig. 7A). The relative Histone-GFP signal over time remains constant with a low variation in pixel intensity (Fig. 7C,D). By contrast, bubR1Rev1 embryos display cycles of chromatin condensation and decondensation during the recorded period of time (Fig. 7B; Movie 4 in the supplementary material). Quantification of the relative Histone-GFP signal shows oscillation over time with a strong variation in pixel intensity for the recorded period of time, suggesting extra cycles of DNA replication (Fig. 7C,D). Indeed, although in wild type embryos, the polar body never incorporates BrdU, in bubR1Rev1 embryos, BrdU signal can be easily detected within the polar body chromosomes at 30-90 minutes AEL (Fig. 7D,E). Taken together, our results show that BubR1 is essential to maintain the mitotic arrest of the polar body and to prevent extra rounds of DNA replication.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In order to study BubR1 checkpoint activity during the syncytial nuclear
divisions, we characterised bubR1Rev1, a new hypomorphic
female sterile allele. Analysis of nuclear proliferation in
bubR1Rev1 embryos shows that starting at cycle 4-5 all
embryos present abnormal mitotic progression as described for
bubR11 mutant allele
(Basu et al., 1999). It has
been previously shown that BubR1 is involved in chromosome
alignment/congression (Ditchfield et al.,
2003
; Lampson and Kapoor,
2005
) and in the inhibition of APC/C activity by signalling lack
of tension at the kinetochore (Logarinho
et al., 2004
). The lagging chromatids, DNA bridges and aneuploidy
observed in bubR1Rev1 embryos indicate precocious anaphase
onset without stable microtubule-kinetochore attachment and an increase local
degradation of APC/C targets. Therefore, our observations of mitotic
progression on fix and live embryos, in addition to the failure to delay
mitosis exit in the presence of colchicine, support the notion that BubR1
spindle checkpoint activity is required during the rapid nuclear proliferation
of Drosophila embryo. In the absence of BubR1, nuclear mitotic exit
is initiated too rapidly, resulting in nuclear cycle asynchrony and
developmental failure.
BubR1 is essential to maintain the mitotic arrest of the polar body
In wild-type Drosophila embryo, three out of the four haploid
meiotic products form the polar body which is formed by Phospho-H3-positive
condensed chromosomes containing radiating microtubules without centrosomes
(Foe et al., 1993). Although
it has been hypothesised that F-actin and myosin II are involved into pulling
and anchoring the polar body to the embryonic cortex with the plus ends of
microtubules attached to the kinetochores and the minus ends facing outwards
(Foe et al., 2000
), the
establishment and maintenance of the polar body mitotic arrest imply that
specific factors are required to exclude it from the mitotic oscillation as it
occurs in the same cytoplasm where neighbouring nuclei undergo synchronous
divisions. Surprisingly, our immunodetection study revealed that BubR1
accumulates at polar body kinetochores in unfertilised and fertilised
wild-type embryo. In bubR1Rev1 embryos; the polar body
fail to establish and maintain its structure. Instead, polar bodies undergo
cycle of DNA condensation-decondensation in phase with the mitotic cycle of
the neighbouring nuclei. Thus, our results strongly suggest that at least the
maintenance of the mitotic arrest and the exclusion of the polar body from the
mitotic oscillator activity in the cytoplasm are dependent on normal BubR1
levels. Accordingly, BubR1 localisation at the polar body kinetochores appears
to have a dominant effect by allowing maintenance of the arrest.
Similarly, it has been shown that the conserved mitotic checkpoint protein
Mps1 is require for polar body mitotic arrest
(Fischer et al., 2004) and
while BubR1 polar body localisation is affected by Mps1 mutation, its nuclear
syncytial localisation remains unaffected. Moreover, gnu, png and
plu gene products which form a multi-protein complex have been shown
to be required for entry and exit into mitosis during early syncytial cycles
in Drosophila embryos through Cyclin B stabilisation
(Fenger et al., 2000
;
Lee et al., 2003
;
Zhang et al., 2004
). Mutant
alleles of these genes induce nuclei to undergo DNA replication in the absence
of chromosome segregation and polar body de-condensation. Furthermore, it has
been shown that increase Cyclin B levels in a png mutant genetic
background can restore polar body chromosome condensation
(Lee et al., 2001
). These
observations lead us to speculate a potential genetic interaction between
BubR1, Mps1 and gnu-png-plu during the syncytial
stages. However, it remains to be determined whether Mps1 and BubR1 are
involved in the same pathway during early syncytial cycles and polar body
structure. Moreover, while polar body structure differs between bubR1
and gnu mutant embryos, it is possible that they interact genetically
to establish and maintain polar body structure through local cyclin B
stabilisation.
BubR1 checkpoint activity is required for synchrony of nuclear divisions
It has been recently proposed that syncytial nuclear proliferation should
be divided in three phases: a first phase of synchronous proliferation (cycle
1 to 6), a second phase being Cdk1/cyclinB dependent with local variation in
metaphase-anaphase duration (cycle 7 to 10), and a third phase of
meta-synchronous divisions that is DNA damage checkpoint dependent and shows
an increase in the duration of interphase and M phases
(Ji et al., 2004). Within each
embryo, the total nuclear cycle time between cycles 7 until 10 remains equal,
but those located at the centre undergo prolonged metaphase, which is
compensated by a shorter anaphase/telophase. These variations are regulated by
local Cdk1/cyclin B activity and the total cycle length can be modified and
influenced by variation in cyclin B maternal gene dose
(Ji et al., 2002
;
Ji et al., 2004
;
Stiffler et al., 1999
). It has
been proposed that the meta-synchronisation observed between cycle 7-10 is
solely induced by cytoplasmic flux and local oscillation in Cdk1/cyclin B
activity (Ji et al., 2004
) and
that early cycles are driven by the dynamics of the mitotic apparatus
(Edgar et al., 1994
). However,
our observations that in bubR1Rev1 embryos almost half of
the embryos show extensive loss of nuclear synchrony as early as cycle 4-5,
suggests that BubR1 checkpoint activity could be directly involved in the
process of nuclear synchrony.
As all nuclei share a common cytoplasm, a key factor in the regulation of
mitotic progression is the transduction of a global state to the local nuclear
level so as to ensure proper synchrony. Although the abnormal mitotic
progression in bubR1Rev1 embryos can be easily explained
in terms of spindle checkpoint activity, the nuclear cycle asynchrony suggests
that BubR1 regulates the mitotic apparatus at a local level by timing the
proper progression of chromosome congression and anaphase onset. It has been
proposed that higher Cdk1/cyclin B activity decreases microtubule stability
and increases sister chromatids velocity at anaphase to maintain constant
nuclear cycle length within the embryo (Ji
et al., 2004; Stiffler et al.,
1999
). Our observations suggest that local variation in BubR1
checkpoint activity can provide a feedback mechanism to ensure proper
chromosome segregation and local variation in the timing of metaphase/anaphase
transition through regulation of microtubule attachment/tension at kinetochore
pairs and APC/C inhibition. However, our analysis of local cyclin B levels by
immunodetection did not provide conclusive results, probably owing to the
severe abnormalities we observed in bubR1Rev1 embryos
(data not shown). Furthermore, as an increase in cyclin B copy number induces
local changes in nuclear progression and only a global embryonic phenotype at
six extra copies (Ji et al.,
2002
; Ji et al.,
2004
), our observations lead us to speculate that variation in
BubR1/cyclin B copy number should induce local variation in timing mitotic
progression without affecting the total embryonic phenotype. Accordingly, a
decrease in BubR1 protein level should result in a decrease in metaphase delay
during cycle 7, as well as a reduced metaphase delay induced by higher cyclin
B levels.
In summary, our analysis of the requirement of BubR1 indicates that during syncytial development the spindle checkpoint appears to operate at various levels. It ensures that nuclei respond to global perturbations by imposing a mitotic arrest especially during later cycles. BubR1 also appears to work at a local level during early and late cycles to ensure proper synchrony of nuclear divisions. In parallel, BubR1 is required to sustain the mitotic arrest of the polar body so as to exclude it from undergoing further rounds of DNA replication during embryonic development.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/20/4509/DC1
* These authors contributed equally to this work
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Basto, R., Scaerou, F., Mische, S., Wojcik, E., Lefebvre, C., Gomes, R., Hays, T. and Karess, R. (2004). In vivo dynamics of the rough deal checkpoint protein during Drosophila mitosis. Curr. Biol. 14,56 -61.[CrossRef][Medline]
Basu, J., Bousbaa, H., Logarinho, E., Li, Z., Williams, B. C.,
Lopes, C., Sunkel, C. E. and Goldberg, M. L. (1999).
Mutations in the essential spindle checkpoint gene bub1 cause chromosome
missegregation and fail to block apoptosis in Drosophila. J. Cell
Biol. 146,13
-28.
Blower, M. D. and Karpen, G. H. (2001). The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions. Nat. Cell. Biol. 3, 730-739.[CrossRef][Medline]
Callaini, G., Riparbelli, M. G. and Dallai, R.
(1994). The distribution of cytoplasmic bacteria in the early
Drosophila embryo is mediated by astral microtubules. J. Cell
Sci. 107,673
-682.
Chan, G. K., Schaar, B. T. and Yen, T. J.
(1998). Characterization of the kinetochore binding domain of
CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1.
J. Cell Biol. 143,49
-63.
Chen, R. H. (2002). BubR1 is essential for
kinetochore localization of other spindle checkpoint proteins and its
phosphorylation requires Mad1. J. Cell Biol.
158,487
-496.
Clarkson, M. and Saint, R. (1999). A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior. DNA Cell Biol. 18,457 -462.[CrossRef][Medline]
Cooley, L., Kelley, R. and Spradling, A. (1988). Insertional mutagenesis of the Drosophila genome with single P elements. Science 239,1121 -1128.[Medline]
Debec, A., Kalpin, R. F., Daily, D. R., McCallum, P. D., Rothwell, W. F. and Sullivan, W. (1996). Live analysis of free centrosomes in normal and aphidicolin-treated Drosophila embryos. J. Cell Biol. 134,103 -115.[Abstract]
Ditchfield, C., Johnson, V. L., Tighe, A., Ellston, R., Haworth,
C., Johnson, T., Mortlock, A., Keen, N. and Taylor, S. S.
(2003). Aurora B couples chromosome alignment with anaphase by
targeting BubR1, Mad2, and Cenp-E to kinetochores. J. Cell
Biol. 161,267
-280.
Dobles, M., Liberal, V., Scott, M. L., Benezra, R. and Sorger, P. K. (2000). Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell 101,635 -645.[CrossRef][Medline]
Edgar, B. A., Sprenger, F., Duronio, R. J., Leopold, P. and O'Farrell, P. H. (1994). Distinct molecular mechanism regulate cell cycle timing at successive stages of Drosophila embryogenesis. Genes Dev. 8,440 -452.[Abstract]
Fenger, D. D., Carminati, J. L., Burney-Sigman, D. L.,
Kashevsky, H., Dines, J. L., Elfring, L. K. and Orr-Weaver, T. L.
(2000). PAN GU: a protein kinase that inhibits S phase and
promotes mitosis in early Drosophila development.
Development 127,4763
-4774.
Fischer, M. G., Heeger, S., Hacker, U. and Lehner, C. F. (2004). The mitotic arrest in response to hypoxia and of polar bodies during early embryogenesis requires Drosophila Mps1. Curr. Biol. 14,2019 -2024.[CrossRef][Medline]
Foe, V. E., Odell, G. M. and Edgar, B. A. (1993). Mitosis and morphogenesis in the Drosophila embryo: point and counterpoint. In The Development of Drosophila (ed. M. Bate and A. M. Arias), pp. 149-300. New York: Cold Spring Harbor Laboratory Press.
Foe, V. E., Field, C. M. and Odell, G. M.
(2000). Microtubules and mitotic cycle phase modulate
spatiotemporal distributions of F-actin and myosin II in Drosophila syncytial
blastoderm embryos. Development
127,1767
-1787.
Fogarty, P., Campbell, S. D., Abu-Shumays, R., Phalle, B. S., Yu, K. R., Uy, G. L., Goldberg, M. L. and Sullivan, W. (1997). The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity. Curr. Biol. 7,418 -426.[CrossRef][Medline]
Heuer, J. G., Li, K. and Kaufman, T. C. (1995).
The Drosophila homeotic target gene centrosomin (cnn) encodes a novel
centrosomal protein with leucine zippers and maps to a genomic region required
for midgut morphogenesis. Development
121,3861
-3876.
Howell, B. J., McEwen, B. F., Canman, J. C., Hoffman, D. B.,
Farrar, E. M., Rieder, C. L. and Salmon, E. D. (2001).
Cytoplasmic dynein/dynactin drives kinetochore protein transport to the
spindle poles and has a role in mitotic spindle checkpoint inactivation.
J. Cell Biol. 155,1159
-1172.
Ji, J. Y., Haghnia, M., Trusty, C., Goldstein, L. S. and
Schubiger, G. (2002). A genetic screen for suppressors and
enhancers of the Drosophila cdk1-cyclin B identifies maternal factors that
regulate microtubule and microfilament stability.
Genetics 162,1179
-1195.
Ji, J. Y., Squirrell, J. M. and Schubiger, G.
(2004). Both cyclin B levels and DNA-replication checkpoint
control the early embryonic mitoses in Drosophila.
Development 131,401
-411.
Kalitsis, P., Earle, E., Fowler, K. J. and Choo, K. H.
(2000). Bub3 gene disruption in mice reveals essential mitotic
spindle checkpoint function during early embryogenesis. Genes
Dev. 14,2277
-2282.
Lampson, M. A. and Kapoor, T. M. (2005). The human mitotic checkpoint protein BubR1 regulates chromosome-spindle attachments. Nat. Cell. Biol. 7, 93-98.[CrossRef][Medline]
Lee, L. A., Elfring, L. K., Bosco, G. and Orr-Weaver, T. L.
(2001). A genetic screen for suppressors and enhancers of the
Drosophila PAN GU cell cycle kinase identifies cyclin B as a target.
Genetics 158,1545
-1556.
Lee, L. A., Van Hoewyk, D. and Orr-Weaver, T. L.
(2003). The Drosophila cell cycle kinase PAN GU forms an active
complex with PLUTONIUM and GNU to regulate embryonic divisions.
Genes Dev. 17,2979
-2991.
Logarinho, E., Bousbaa, H., Dias, J. M., Lopes, C., Amorim, I.,
Antunes-Martins, A. and Sunkel, C. E. (2004). Different
spindle checkpoint proteins monitor microtubule attachment and tension at
kinetochores in Drosophila cells. J. Cell Sci.
117,1757
-1771.
Musacchio, A. and Hardwick, K. G. (2002). The spindle checkpoint: structural insights into dynamic signalling. Nat. Rev. Mol. Cell. Biol. 3, 731-741.[CrossRef][Medline]
O'Farrell, P. H., Stumpff, J. and Su, T. T. (2004). Embryonic cleavage cycles: how is a mouse like a fly? Curr. Biol. 14,R35 -R45.[Medline]
Peters, J. M. (2002). The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. Cell 9, 931-943.[CrossRef][Medline]
Raff, J. W. and Glover, D. M. (1988). Nuclear and cytoplasmic mitotic cycles continue in Drosophila embryos in which DNA synthesis is inhibited with aphidicolin. J. Cell Biol. 107,2009 -2019.[Abstract]
Roberts, D. B. (1986). Basic Drosophila care and techniques. In Drosophila: A Practical Approach (ed. D. B. Roberts), pp. 1-38. Oxford, England: IRL Press.
Sambrook, J., Fritsch, F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Sibon, O. C., Kelkar, A., Lemstra, W. and Theurkauf, W. E. (2000). DNA-replication/DNA-damage-dependent centrosome inactivation in Drosophila embryos. Nat. Cell. Biol. 2, 90-95.[CrossRef][Medline]
Stiffler, L. A., Ji, J. Y., Trautmann, S., Trusty, C. and
Schubiger, G. (1999). Cyclin A and B functions in the early
Drosophila embryo. Development
126,5505
-5513.
Su, T. T., Sprenger, F., DiGregorio, P. J., Campbell, S. D. and
O'Farrell, P. H. (1998). Exit from mitosis in Drosophila
syncytial embryos requires proteolysis and cyclin degradation, and is
associated with localized dephosphorylation. Genes
Dev. 12,1495
-1503.
Sullivan, W., Ashburner, M. and Hawley, R. S. (2000). Drosophila Protocols. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Takada, S., Kelkar, A. and Theurkauf, W. E. (2003). Drosophila checkpoint kinase 2 couples centrosome function and spindle assembly to genomic integrity. Cell 113,87 -99.[CrossRef][Medline]
Wojcik, E., Basto, R., Serr, M., Scaerou, F., Karess, R. and Hays, T. (2001). Kinetochore dynein: its dynamics and role in the transport of the Rough deal checkpoint protein. Nat. Cell. Biol. 3,1001 -1007.[CrossRef][Medline]
Yu, K. R., Saint, R. B. and Sullivan, W. (2000). The Grapes checkpoint coordinates nuclear envelope breakdown and chromosome condensation. Nat. Cell. Biol. 2,609 -615.[CrossRef][Medline]
Zachariae, W. and Nasmyth, K. (1999). Whose end
is destruction: cell division and the anaphase-promoting complex.
Genes Dev. 13,2039
-2058.
Zalokar, M. and Erk, I. (1976). Division and migration of nuclei during early embryogenesis. J. Microbiol. Cell 25,97 -106.
Zhang, X. H., Axton, J. M., Drinjakovic, J., Lorenz, L.,
White-Cooper, H. and Renault, A. D. (2004). Spatial and
temporal control of mitotic cyclins by the Gnu regulator of embryonic mitosis
in Drosophila. J. Cell Sci.
117,3571
-3578.
|