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First published online October 12, 2007
doi: 10.1242/10.1242/dev.010900
Research Report |
1 MRC Centre for Developmental Neurobiology, New Hunt's House, King's College
London, Guy's Campus, London SE1 1UL, UK.
2 Temasek Lifesciences Laboratory and Department of Biological Sciences, 1
Research Link, National University of Singapore, 117604, Singapore.
Authors for correspondence (e-mails:
c.slack{at}ucl.ac.uk;
wchia{at}tll.org.sg)
Accepted 7 August 2007
SUMMARY
Asymmetric cell divisions generate cell fate diversity during both invertebrate and vertebrate development. Drosophila neural progenitors or neuroblasts (NBs) each divide asymmetrically to produce a larger neuroblast and a smaller ganglion mother cell (GMC). The asymmetric localisation of neural cell fate determinants and their adapter proteins to the neuroblast cortex during mitosis facilitates their preferential segregation to the GMC upon cytokinesis. In this study we report a novel role for the anaphase-promoting complex/cyclosome (APC/C) during this process. Attenuation of APC/C activity disrupts the asymmetric localisation of the adapter protein Miranda and its associated cargo proteins Staufen, Prospero and Brat, but not other components of the asymmetric division machinery. We demonstrate that Miranda is ubiquitylated via its C-terminal domain; removal of this domain disrupts Miranda localisation and replacement of this domain with a ubiquitin moiety restores normal asymmetric Miranda localisation. Our results demonstrate that APC/C activity and ubiquitylation of Miranda are required for the asymmetric localisation of Miranda and its cargo proteins to the NB cortex.
Key words: Miranda, Anaphase promoting complex, Asymmetric division, Neuroblast, Drosophila
INTRODUCTION
Drosophila embryonic neuroblasts (NBs) divide asymmetrically along
the apicobasal (A/B) axis to generate a larger apical NB and a smaller basal
ganglion mother cell (GMC). A protein complex forms at the apical cortex of
the NB and directs both the basal localisation of neural cell fate
determinants and reorientation of the mitotic spindle to ensure that cell fate
determinants are segregated into the GMC upon cytokinesis
(Kaltschmidt et al., 2000
;
Jan and Jan, 2001;
Wang and Chia, 2005). An
apically localised protein cassette consisting of Par3 (Bazooka), Par6 and
atypical protein kinase C (aPKC) recruits Inscuteable (Insc), Partner of
Inscuteable (Pins, also known as Raps) and the G protein subunit G
i to
form an apical crescent at late interphase/early prophase
(Kraut and Campos-Ortega,
1996; Kraut et al.,
1996; Schober et al.,
1999; Parmentier et al.,
2000; Schaefer et al.,
2000; Wodarz et al.,
2000; Petronczki and Knoblich,
2001). During late prophase/early metaphase, cell fate
determinants and their respective adapter proteins Prospero-Miranda,
prospero mRNA-Staufen, Brat-Miranda and Numb-Partner of numb (Pon)
localise as crescents at the basal NB cortex
(Ikeshima-Kataoka et al., 1997;
Matsuzaki et al., 1998;
Schuldt et al., 1998;
Shen et al., 1998;
Betschinger et al., 2006;
Lee et al., 2006).
Basal but not apical crescent formation requires the cortically localised
tumour suppressor proteins Discs large (Dlg, also known as Dlg1) and Lethal
giant larvae [Lgl, also known as L(2)gl]
(Peng et al., 2000;
Albertson and Doe, 2003). The
precise mechanism whereby basally localised proteins are transported to the
basal NB cortex is not known, but requires the activity of both myosin II and
VI (Barros et al., 2003;
Petritsch et al., 2003). Also,
the mechanism by which the basal crescent is anchored to the NB cortex,
although shown to require an intact actin cytoskeleton, remains elusive
(Shen et al., 1998). Although
NBs divide in the larval central brain without a fixed A/B orientation, we
will continue to refer to Miranda, Pon, Numb, Pros as basal proteins and Insc,
Pins and the Par proteins as apical proteins.
The localisation of basal proteins is clearly tightly coordinated and
linked to changes in the cell cycle, but how this coordination is achieved is
not known. In this study we have identified a novel role for a key mitotic
regulator, the anaphase-promoting complex/cyclosome (APC/C), in regulating the
basal localisation of Miranda. The APC/C is a multi-subunit protein complex
with at least 11 core subunits that functions as an E3 ubiquitin ligase
(Vodermaier, 2004) normally
targeting proteins for degradation via the 26S proteasome
(Holloway et al., 1993).
Regulation of APC/C activity by transient associations with the activating
subunits Cdc20 and Cdh1 promotes mitotic transitions via several key
processes, including the destruction of mitotic cyclins and inhibitors of
chromosome separation as well as the regulation of DNA replication, centrosome
duplication and mitotic spindle assembly
(Sigrist et al., 1995;
Zur and Brandeis, 2001;
Leismann and Lehner, 2003).
More recently, APC/C activity has been shown to play important roles in cell
cycle-independent processes, including the control of axon growth and
patterning in the developing mammalian brain
(Konishi et al., 2004) and the
regulation of synaptic size and transmission in both Caenorhabditis
elegans and Drosophila (Juo
and Kaplan, 2004; van Roessel
et al., 2004). A role for the APC/C has been described in
establishing the anteroposterior axis of the C. elegans embryo by
asymmetrically distributing PAR proteins and promoting the association of the
paternal pronucleus/centrosome with the actin-rich cortex
(Rappleye et al., 2002). We
describe a novel role for the APC/C in mediating NB asymmetric division in
Drosophila by regulating the asymmetric localisation and/or retention
of Miranda and its associated cargo proteins at the NB cortex.
MATERIALS AND METHODS
Flies
idaPL17 was isolated from a genetic screen
(Slack et al., 2006). Other
stocks used in this study include cdc27makos (D. Glover,
University of Cambridge, UK), mor3 and
mor5 (T. Orr-Weaver, MIT, Cambridge, MA),
lgl4 (C. Doe, University of Oregon, Eugene, OR),
cdh1rapie28 (C. Lehner, University of Zurich,
Switzerland), mirandaRR127 and
Df(3R)ora19 (F. Matsuzaki, RIKEN, Kobe, Japan).
cnnHK21, fzy1, idaD14,
Rpn6k00103, Tbp-104210b, Df(3L)Exel6098 and
polo9 were all obtained from the Bloomington stock centre.
NB mitotic clones were generated using the MARCM system as previously
described (Lee and Luo,
2001).
Cloning and constructs
The ida and miranda coding sequences were amplified by
PCR from 0 to 24 hour embryonic cDNA and subcloned into the pENTR/D/TOPO
vector (Invitrogen). Expression constructs were generated using the Gateway
Cloning System (Invitrogen) and vectors from the Terrence Murphy collection
distributed by the Drosophila Genomics Resource Center (DGRC). Full
details on the cloning of constructs are available on request. The
pCasPer-hsp70-HA-ubiquitin construct was a gift from A. Sehgal
(University of Pennsylvania Medical School, Philadelphia, PA).
Antibodies and immunofluorescence
For immunofluorescence, third instar larval brains were dissected in PBS
and fixed in 4% formaldehyde for 20 minutes. Depolymerisation of microtubules
was carried out by incubating dissected brains in 10 mM colchicine in PBS for
1 hour prior to fixing. Actin filaments were disrupted by incubation in 200
µM latrunculinB (Sigma) in PBS for 1 hour prior to fixing. Antibodies used
were mouse and rabbit anti-Miranda (F. Matsuzaki), rabbit anti-Insc, rabbit
anti-Pins, rat anti-Brat, mouse anti-Prospero (Developmental Studies Hybridoma
Bank, DSHB), rabbit anti-aPKC (Santa Cruz), rabbit anti-Numb and rabbit
anti-PON (Y. Jan, UCSF, San Fransisco, CA), rabbit anti-Staufen (Cai Yu,
Temasek Lifesciences Laboratory, University of Singapore), rabbit anti-CNN (T.
Kaufman, Indiana University, Bloomington, IN), rabbit anti-Nuf (W. Sullivan,
UCSC, Santa Cruz, CA), mouse anti-rab11 (Calbiochem), mouse and rabbit
anti-ßGal (Promega and Cappel), mouse and rabbit anti-GFP (Sigma and
Molecular Probes), mouse and rabbit anti-FLAG (Sigma), rabbit
anti-phosphohistone H3 (Upstate Biotechnology), mouse anti-ubiquitin FK2
(Biomol), mouse anti--tubulin (Sigma) and mouse anti-
-tubulin
(Sigma). Secondary antibodies conjugated to either Alexa-Fluor-488 or
Alexa-Fluor-546 were from Molecular Probes. DNA was visualised using ToPro-3
(Molecular Probes) and tissue was mounted in Vectashield (Vector Labs).
Immunostainings were analysed using laser scanning confocal microscopy (Zeiss
LSM 510).
S2 cell transfections and immunoprecipitations
Drosophila Schneider (S2) cells were cultured in
Drosophila serum-free media (Invitrogen) and transfections were
carried out using the Cellfectin transfection reagent (Invitrogen) according
to the manufacturer's instructions. For heat-shock treatments, cells were
incubated at 37°C for 2 hours and then at 25°C for 4 hours before
lysis. Cultured cells and larval brains were lysed in IP lysis buffer (150 mM
NaCl, 100 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100) containing
protease inhibitor cocktail (Calbiochem) and the proteasome inhibitor MG115
(Sigma). Cell extracts were immunoprecipitated using rabbit anti-FLAG (Sigma)
or rabbit anti-HA (Sigma) antibodies and immunocomplexes were bound to protein
G sepharose (Roche). Bound complexes were washed with IP lysis buffer and
subjected to immunoblotting using appropriate antibodies.
RESULTS AND DISCUSSION
Disruption to APC/C activity causes Miranda localisation defects
We have recently isolated a novel allele of imaginal discs
arrested (ida), homozygotes of which survive until early pupal
stages of development and fail to properly localise Miranda to the basal
cortex of mitotic neuroblasts (Slack et
al., 2006). Sequence analysis of this allele,
idaPL17, revealed a single nucleotide transversion within
the ida coding sequence, resulting in a premature stop codon at aa
334 (Q334
stop), the first residue of a putative tetratricopeptide (TPR)
motif. In mutant larvae, only 58% (n=72) of prophase NBs properly
localised Miranda to the cortex (Fig.
1B) compared with 100% (n=64) of wild-type prophase NBs
(Fig. 1A). A total of 41%
(n=209) of idaPL17 metaphase NBs showed Miranda
accumulation in a pericentrosomal compartment at the expense of cortically
localised protein, with 8% (n=209) of mutant NBs showing a complete
loss of cortically localised protein (Fig.
1D,N), whereas 91% (n=197) of wild-type metaphase NBs
showed cortically localised protein only
(Fig. 1C,N). We observed, at
low frequency, anaphase cells as defined by separated chromosome populations
displaced towards opposite poles of the cell, confirming previous observations
(Bentley et al., 2002) that
ida mutant neuroblasts are not arrested at metaphase. Miranda was
still mislocalised to pericentrosomal regions in these anaphase neuroblasts
(Fig. 1I). We did not observe
any phenotypic defects in idaPL17 homozygous mutant
embryos, presumably due to the perdurance of maternally provided protein, and
NB clones induced at early larval stages did not show any obvious mitotic or
Miranda-localisation defects, again indicative of protein stability (data not
shown). Attempts to induce maternal germline clones homozygous for
idaPL17 did not yield any surviving embryos, suggesting an
essential requirement for ida function during oogenesis (data not
shown). The idaPL17 mutant appears to be a genetic null,
because the penetrance of the Miranda localisation phenotype did not increase
in hemizygotes over a small deficiency that removes the entire ida
locus [Df(3L)Exel6098; 42% of NBs with pericentrosomal Miranda,
n=235] or in transheterozygotes over the mRNA null allele,
idaD14 (43% of NBs with pericentrosomal Miranda,
n=223) (Fig. 1N). We
were able to rescue the defects in Miranda localisation in ida mutant
NBs by expressing a GFP::Ida fusion protein, which, at all stages of the cell
cycle, was localised throughout the cytoplasm
(Fig. 1E and data not
shown).
ida encodes the Drosophila homologue of human APC5, a
subunit of the APC/C multiprotein complex
(Bentley et al., 2002). In
order to determine whether the defects in Miranda localisation are a specific
consequence of ida loss of function or are caused by a more general
disruption to APC/C activity, we analysed the effect of loss-of-function
mutations in genes encoding other APC/C subunits. We observed a significant
number of mitotic NBs with pericentrosomal Miranda accumulation in animals
homozygous for mutations in either cdc27 (13%, n=119;
Fig. 1F) or morula
(APC2; 39%, n=128; Fig.
1G), suggesting that Miranda asymmetric cortical localisation is
disrupted when APC/C activity is attenuated. However, we did not see any
defects in Miranda localisation in homozygous mutants for strong
loss-of-function alleles for the two APC/C activators, cdc20 or
cdh1, indicating that Miranda targeting might occur independently of
these two proteins (data not shown). Loss of ida function causes
several mitotic defects, including an increased mitotic index, loss of cyclin
B degradation and hypercondensed chromosomes
(Bentley et al., 2002).
However, a strong hypomorphic allele of polo that shows similar
mitotic defects had normal cortical Miranda localisation (data not shown).
Furthermore, colchicine treatment of wild-type NBs to depolymerise
microtubules and induce metaphase arrest did not disrupt Miranda localisation,
therefore suggesting that the ida mutant phenotype is not a secondary
consequence of a delay or block in mitosis.
In C. elegans, APC/C function during embryonic anteroposterior
axis formation promotes the association of the paternal pronucleus/centrosome
with the embryonic cortex (Rappleye et
al., 2002). The pericentrosomal accumulation of Miranda in APC/C
mutant NBs led us to investigate a possible requirement for centrosomal
function during Miranda localisation. We examined loss-of-function mutants for
centrosomin (cnn), which encodes a core component of the
centrosome in Drosophila and is required for proper centrosome
assembly (Megraw et al.,
1999), and found that Miranda localisation to the NB cortex was
normal in these mutants (data not shown). Furthermore, cnn; ida
double mutants still accumulated pericentrosomal Miranda, suggesting that the
ida mutant phenotype is not dependent on intact centrosomal function
(Fig. 1L). We also note that we
did not see complete colocalisation of Miranda with Centrosomin in
ida mutant NBs, suggesting that Miranda is localised in a separate
compartment that itself localises to a region near to the centrosome.
Accumulation of Miranda in ida mutants was insensitive to colchicine
treatment to depolymerise microtubules, suggesting that the ida
mutant phenotype is not dependent on the mitotic spindle, although the
compartment in which Miranda localises separates from the region of the
centrosome upon colchicine treatment (Fig.
1M). We also observed pericentrosomal accumulation of Miranda in
ida mutants after latrunculin treatment, suggesting that the
ida mutant phenotype occurs independently of intact actin filaments
(data not shown).
|
;
Mollinari et al., 2002). Under
the conditions used for our experiments, we did not detect significant levels
of centrosomal Miranda localisation in wild-type NBs.
ida/APC5 is required for basal localisation of Miranda and its cargo proteins, but not for PON or Numb localisation or apical complex formation
The localisation of Miranda to the NB basal cortex requires the correct
localisation of the apical protein complex, which includes Inscuteable, Pins
and aPKC. In ida mutant NBs, Inscuteable localises normally to the
apical cortex during early prophase and is maintained as an apical cortical
crescent during metaphase (Fig.
2H,I). Similar results were obtained using anti-aPKC and anti-Pins
(data not shown), suggesting that the defects in Miranda localisation in
ida mutant NBs is not caused by a disruption to the apical complex
and that the APC/C functions downstream of or in parallel to the apical
components.
|
The ida mutant phenotype resembles that seen in mutants for the
tumour suppressor lgl, in which the targeting of Miranda and other
basally localised molecules to the NB cortex is disrupted so that Miranda no
longer forms a cortical crescent at metaphase but is instead mislocalised to
the centrosomes and mitotic spindle
(Ohshiro et al., 2000;
Peng et al., 2000). We
reasoned that, if the ida mutant phenotype resulted from a reduction
of Lgl function, then further reducing Lgl activity should enhance the Miranda
mislocalisation phenotype. However, removing one copy of lgl using a
null allele had no effect on the ida mutant phenotype and the number
of NBs with pericentrosomal Miranda was comparable to mutants for ida
alone (49% of NBs with pericentrosomal Miranda, n=103), suggesting
that Lgl is not a downstream effector of Ida activity. By contrast, removing
one copy of miranda using a deficiency that removes the entire
miranda locus [Df(3R)ora19] in the
idaPL17 mutant background strongly suppressed the Miranda
mislocalisation phenotype (20% of NBs with Miranda around the centrosomes,
n=98), suggesting that Miranda itself might be a target for APC/C
activity.
The C-terminal domain of Miranda is required for its ubiquitylation and efficient cortical localisation
Our data demonstrates that the correct localisation of Miranda to the basal
NB cortex requires APC/C. Because the APC/C normally functions as an E3
ubiquitin ligase, we tested whether this role of the APC/C could be mediating
the effects on Miranda localisation. In order to determine whether Miranda can
be ubiquitylated, we performed immunoprecipitations on protein extracts of
Drosophila S2 cells in which we constitutively expressed FLAG-tagged
Miranda and expressed HA-tagged ubiquitin under the control of a heat-shock
promoter. After immunoprecipitation using anti-HA antibody, we were able to
detect FLAG-Miranda in the immune complex in extracts only from cells in which
HA-ubiquitin expression had been induced by heat shock
(Fig. 3A). We performed further
immunoprecipitations on protein extracts both from S2 cells and larval brains
expressing only FLAG-Miranda without expressing exogenous HA-tagged ubiquitin.
In both cases, we were able to detect ubiquitylated Miranda in the immune
complex after immunoprecipitation using anti-FLAG antibodies
(Fig. 3B,C). These results
using brain and S2 extracts clearly demonstrate that Miranda can be
ubiquitylated both in vivo and in S2 cells. Although the antibody used
recognises both mono- and poly-ubiquitin conjugates, we only observed a single
band on the western blots probed for ubiquitylated Miranda. The absence of
higher molecular weight Miranda species, even in the presence of proteasome
inhibitors, suggests that Miranda might be mono- rather than
poly-ubiquitylated.
The C-terminal region of Miranda contains a putative APC/C-recognition
motif (amino acids 811 to 814: GKEN) that shows homology to the KEN box, a
motif required for Cdh1-dependent APC/C-mediated ubiquitylation of substrate
proteins (Castro et al., 2003).
To examine the effects of the removal of this motif on Miranda localisation,
we used the mirandaRR127 allele, in which the C-terminal
103 amino acids of the encoded protein, including the GKEN motif, are replaced
by an unrelated stretch of 112 amino acids
(Ikeshima-Kataoka et al.,
1997). We examined the localisation of this truncated form of
Miranda in mitotic larval NBs by generating somatic clones using the MARCM
system, which allows the generation of homozygous-mutant NB clones that
express membrane-bound CD8::GFP in an otherwise heterozygous background
(Lee and Luo, 2001). In
mirandaRR127 mutant NBs, Miranda localisation was similar
to that seen in ida mutant NBs: the truncated protein was exclusively
cytoplasmic during early prophase (5/5 NBs;
Fig. 4B,B') and
accumulated in a pericentrosomal compartment at the expense of cortical
protein during metaphase (20/20 NBs; Fig.
4D,D'). As in ida mutant NBs, accumulation of
Miranda in mirandaRR127 mutant NBs was insensitive to
colchicine treatment to depolymerise microtubules
(Fig. 4E,E') and we also
observed accumulation of pericentrosomal Prospero in these mutant NBs
(Fig. 4F,F'). Removal of
this C-terminal domain prevented ubiquitylation of Miranda in S2 cells (see
Fig. 3A,B). Furthermore, NBs
that overexpress this truncated protein (20%, n=30) showed a similar
mislocalisation of the expressed protein (data not shown) and replacement of
this C-terminal domain with ubiquitin restored normal localisation
(Fig. 4G,G').
Interestingly, mutation of the GKEN motif itself did not prevent Miranda
ubiquitylation and had no effect on the localisation of the protein (data not
shown), suggesting that mutation of this site alone is insufficient to disrupt
ubiquitylation of Miranda.
|
Ubiquitylation by the APC/C normally targets proteins for degradation via
the 26S proteasome. Although it is possible that a proportion of ubiquitylated
Miranda is targeted for degradation, disruption to proteasome function caused
markedly different phenotypes than those observed when APC/C activity was
attenuated. Although we observed Miranda accumulating in the region of the
centrosomes in NBs mutant for the proteasome regulatory subunits Rpn6
or Tbp-1 (Fig.
4H,H' and data not shown), this process was microtubule
dependent, whereas in both ida mutant NBs and NBs mutant for the
mirandaRR127 allele, accumulation of Miranda was observed
even after microtubule depolymerisation with colchicine
(Fig. 4I,I', compare with
Fig. 1M). In addition, we did
not see pericentrosomal accumulation of either Prospero or Staufen in
proteasome-mutant NBs (Fig.
4J,J' and data not shown). Furthermore, we did not observe
any significant differences by western blot in Miranda protein levels between
wild-type and ida mutant brain extracts (see Fig. S1 in the
supplementary material), suggesting that the pericentrosomal localisation of
Miranda in ida mutants is not caused by excessive Miranda
accumulation and therefore reflects the disruption of a process other than
proteasomal degradation. Recently, several proteasome-independent processes
regulated by ubiquitylation have been identified, including protein kinase
activation, vesicle trafficking, DNA repair and gene silencing
(Sun and Chen, 2004).
The asymmetric localisation of cell fate determinants during NB division is
tightly coordinated with changes in the cell cycle. The formation of an apical
complex of proteins during early prophase not only directs the correct
orientation of the mitotic spindle during metaphase but is also required for
the formation of a basal crescent of cell fate determinants and their adapter
molecules during late prophase/metaphase
(Kaltschmidt et al., 2000;
Jan and Jan, 2001;
Wang and Chia, 2005). It thus
appears likely that multiple components of the cell cycle machinery that
coordinate cell cycle transitions might also be involved in the regulation of
basal protein localisation, as has been previously shown for cdc2
(Tio et al., 2001) and Aurora
A (Wang et al., 2006). We have
shown that the efficient localisation of the adapter protein Miranda to the NB
basal cortex requires the activity of the APC/C mitotic regulator. Mutations
in several APC/C core subunits showed reduced cortically localised Miranda,
with cytosolic accumulation of Miranda in an as yet unidentified
pericentrosomal compartment. By contrast, apical complex formation was
unaffected in these mutant NBs, showing that the APC/C acts downstream of or
in parallel to the apical complex to ensure proper basal protein localisation.
Furthermore, the basal localisation of PON/Numb were also unaffected by loss
of APC/C activity, suggesting that Miranda itself might be a specific target
for APC/C activity in mitotic NBs. This is further supported by the
observation that the ida mutant phenotype can be partially rescued by
specifically reducing Miranda protein levels.
|
;
Sun and Chen, 2004).
Ubiquitylation of Miranda could function as a signal to regulate its transport
to the basal cell cortex perhaps by influencing its association with motor
proteins that mediate basal protein targeting. Alternatively, ubiquitylation
of Miranda could regulate the retention of Miranda at the basal cell cortex by
influencing its association with anchoring or scaffolding molecules. Efficient
localisation and/or retention of Miranda to the basal cortex clearly requires
APC/C activity, but the presence of Miranda protein at the basal cortex in
APC/C mutants, albeit at a much reduced level compared with wild-type,
indicates that other processes and molecules might also be involved.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/21/3781/DC1
ACKNOWLEDGMENTS
We thank B. Bello, C. Yu, F. Yu, A. Gould, D. Glover, Y.N. Jan, T. Kaufman, C. Lehner, F. Matsuzaki, J. Raff, A. Sehgal, W. Sullivan, T. Orr-Weaver, DSHB (University of Iowa), the Bloomington and Szeged Stock Centres, and Flybase for generously providing antibodies, fly stocks and information. We thank members of the Tear and Chia labs for help and suggestions. The authors were supported by a Wellcome Trust Programme Grant, a Wellcome Trust Advanced Training Fellowship (R.I.T.) and Wellcome Trust Principal Research Fellowship (W.C.).
Footnotes
* Present address: Biology Department, University College London, Darwin
Building, Gower Street, London WC1E 6BT, UK 
REFERENCES
Albertson, R. and Doe, C. Q. (2003). Dlg, Scrib and Lgl regulate neuroblast cell size and mitotic spindle asymmetry. Nat. Cell Biol. 5,166 -170.[CrossRef][Medline]
Barros, C. S., Phelps, C. B. and Brand, A. H. (2003). Drosophila nonmuscle myosin II promotes the asymmetric segregation of cell fate determinants by cortical exclusion rather than active transport. Dev. Cell 5,829 -840.[CrossRef][Medline]
Bentley, A. M., Williams, B. C., Goldberg, M. L. and Andres, A.
J. (2002). Phenotypic characterization of Drosophila ida
mutants: defining the role of APC5 in cell cycle progression. J.
Cell Sci. 115,949
-961.
Betschinger, J., Mechtler, K. and Knoblich, J. A. (2006). Asymmetric segregation of the tumour suppressor Brat regulates self-renewal in Drosophila neural stem cells. Cell 124,1241 -1253.[CrossRef][Medline]
Castro, A., Vigneron, S., Bernis, C., Labbe, J. C. and Lorca,
T. (2003). Xkid is degraded in a D-box, KEN-box, and
A-box-independent pathway. Mol. Cell. Biol.
23,4126
-4138.
Holloway, S. L., Glotzer, M., King, R. W. and Murray, A. W. (1993). Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor. Cell 73,1393 -1402.[CrossRef][Medline]
Ikeshima-Kataoka, H., Skeath, J. B., Nabeshima, Y., Doe, C. Q. and Matsuzaki, F. (1997). Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions. Nature 390,625 -629.[CrossRef][Medline]
Jan, Y. N. and Jan, L. Y. (2001). Asymmetric cell division in the Drosophila nervous system. Nat. Rev. Neurosci. 2,772 -779.[CrossRef][Medline]
Juo, P. and Kaplan, J. M. (2004). The anaphase-promoting complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of C. elegans. Curr. Biol. 14,2057 -2062.[CrossRef][Medline]
Kaltschmidt, J. A., Davidson, C. M., Brown, N. H. and Brand, A. H. (2000). Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system. Nat. Cell Biol. 2,7 -12.[CrossRef][Medline]
Konishi, Y., Stegmuller, J., Matsuda, T., Bonni, S. and Bonni,
A. (2004). Cdh1-APC controls axonal growth and patterning in
the mammalian brain. Science
303,1026
-1030.
Kraut, R. and Campos-Ortega, J. A. (1996). inscuteable, a neural precursor gene of Drosophila, encodes a candidate for a cytoskeleton adaptor protein. Dev. Biol. 174, 65-81.[CrossRef][Medline]
Kraut, R., Chia, W., Jan, L. Y., Jan, Y. N. and Knoblich, J. A. (1996). Role of inscuteable in orienting asymmetric cell divisions in Drosophila. Nature 383, 50-55.[CrossRef][Medline]
Lee, C. Y., Wilkinson, B. D., Siegrist, S. E., Wharton, R. P. and Doe, C. Q. (2006). Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal. Dev. Cell 10,441 -449.[CrossRef][Medline]
Lee, T. and Luo, L. (2001). Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci. 24,251 -254.[CrossRef][Medline]
Leismann, O. and Lehner, C. F. (2003).
Drosophila securin destruction involves a D-box and a KEN-box and promotes
anaphase in parallel with Cyclin A degradation. J. Cell
Sci. 116,2453
-2460.
Matsuzaki, F., Ohshiro, T., Ikeshima-Kataoka, H. and Izumi, H. (1998). miranda localizes staufen and prospero asymmetrically in mitotic neuroblasts and epithelial cells in early Drosophila embryogenesis. Development 125,4089 -4098.[Abstract]
Megraw, T. L., Li, K., Kao, L. R. and Kaufman, T. C. (1999). The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila. Development 126,2829 -2839.[Abstract]
Meyer, H. H., Wang, Y. and Warren, G. (2002). Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4. EMBO J. 21,5645 -5652.[CrossRef][Medline]
Mollinari, C., Lange, B. and Gonzalez, C. (2002). Miranda, a protein involved in neuroblast asymmetric division, is associated with embryonic centrosomes of Drosophila melanogaster. Biol. Cell 94,1 -13.[CrossRef][Medline]
Ohshiro, T., Yagami,T., Zhang, C. and Matsuzaki, F. (2000). Role of cortical tumour-suppressor proteins in asymmetric division of Drosophila neuroblast. Nature 408,593 -596.[CrossRef][Medline]
Parmentier, M. L., Woods, D., Greig, S., Phan, P. G., Radovic,
A., Bryant, P. and O'Kane, C. J. (2000). Rapsynoid/partner of
inscuteable controls asymmetric division of larval neuroblasts in Drosophila.
J. Neurosci. 20,RC84
.
Peng, C. Y., Manning, L., Albertson, R. and Doe, C. Q. (2000). The tumour-suppressor genes lgl and dlg regulate basal protein targeting in Drosophila neuroblasts. Nature 408,596 -600.[CrossRef][Medline]
Petritsch, C., Tavosanis, G., Turck, C. W., Jan, L. Y. and Jan, Y. N. (2003). The Drosophila myosin VI Jaguar is required for basal protein targeting and correct spindle orientation in mitotic neuroblasts. Dev. Cell 4, 273-281.[CrossRef][Medline]
Petronczki, M. and Knoblich, J. A. (2001). DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3, 43-49.[CrossRef][Medline]
Rappleye, C. A., Tagawa, A., Lyczak, R., Bowerman, B. and Aroian, R. V. (2002). The anaphase-promoting complex and separin are required for embryonic anterior-posterior axis formation. Dev. Cell 2,195 -206.[CrossRef][Medline]
Schaefer, M., Shevchenko, A., Shevchenko, A. and Knoblich, J. A. (2000). A protein complex containing Inscuteable and the Galpha-binding protein Pins orients asymmetric cell divisions in Drosophila. Curr. Biol. 10,353 -362.[CrossRef][Medline]
Schober, M., Schaefer, M. and Knoblich, J. A. (1999). Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts. Nature 402,548 -551.[CrossRef][Medline]
Schuldt, A. J., Adams, J. H., Davidson, C. M., Micklem, D. R.,
Haseloff, J., St Johnston, D. and Brand, A. H. (1998).
Miranda mediates asymmetric protein and RNA localization in the developing
nervous system. Genes Dev.
12,1847
-1857.
Shen, C. P., Knoblich, J. A., Chan, Y. M., Jiang, M. M., Jan, L.
Y. and Jan, Y. N. (1998). Miranda as a multidomain adapter
linking apically localized Inscuteable and basally localized Staufen and
Prospero during asymmetric cell division in Drosophila.
Genes Dev. 12,1837
-1846.
Sigrist, S., Jacobs, H., Stratmann, R. and Lehner, C. F. (1995). Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3. EMBO J. 14,4827 -4838.[Medline]
Slack, C., Somers, W., Sousa-Nunes, R., Chia, W. and Overton, P. (2006). A mosaic genetic screen for novel mutations affecting Drosophila neuroblast divisions. BMC Genet. 7, 33.[CrossRef][Medline]
Sun, L. and Chen, Z. J. (2004). The novel functions of ubiquitination in signaling. Curr. Opin. Cell Biol. 16,119 -126.[CrossRef][Medline]
Tio, M., Udolph, G., Yang, X. and Chia, W. (2001). cdc2 links the Drosophila cell cycle and asymmetric division machineries. Nature 409,1063 -1067.[CrossRef][Medline]
van Roessel, P., Elliott, D. A., Robinson, I. M., Prokop, A. and Brand, A. H. (2004). Independent regulation of synaptic size and activity by the anaphase-promoting complex. Cell 119,707 -718.[CrossRef][Medline]
Vodermaier, H. C. (2004). APC/C and SCF: controlling each other and the cell cycle. Curr. Biol. 14,R787 -R796.[CrossRef][Medline]
Wang, H. and Chia, W. (2005). Drosophila neural progenitor polarity and asymmetric division. Biol. Cell 97,63 -74.[CrossRef][Medline]
Wang, H., Somers, G. W., Bashirullah, A., Heberlein, U., Yu, F.
and Chia, W. (2006). Aurora A acts as a tumour suppressor and
regulates self-renewal of Drosophila neuroblasts. Genes
Dev. 20,3453
-3463.
Wodarz, A., Ramrath, A., Grimm, A. and Knust, E.
(2000). Drosophila atypical protein kinase C associates with
Bazooka and controls polarity of epithelia and neuroblasts. J. Cell
Biol. 150,1361
-1374.
Zur, A. and Brandeis, M. (2001). Securin degradation is mediated by fzy and fzr, and is required for complete chromatid separation but not for cytokinesis. EMBO J. 20,792 -801.[CrossRef][Medline]
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Cterm)
under the control of an actin promoter were cotransfected into
Drosophila S2 cells with DNA encoding HA-ubiquitin under the control
of a heat-shock promoter. Extracts were prepared from cells both without (-)
and with (+) heat shock. FLAG-Miranda but not
FLAG-Miranda