© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 2952 doi:10.1242/jcs.176826
CORRECTION
A maternal effect rough deal mutation suggests that multiple
pathways regulate Drosophila RZZ kinetochore recruitment
Lé naé g Dé fachelles, Sarah G. Hainline, Alexandra Menant, Laura A. Lee and Roger E. Karess
There was an error published in J. Cell Sci. 128, 1204-1216.
A funding source was accidentally omitted. The complete funding section should have read as below.
Funding
This work was supported by the National Institutes of Health [grant numbers GM074044 to L.A.L., GM008554 to S.G.H.]. L.D. was
supported by fellowships from the French ‘Ministère Français de l’Enseignement Supérieur et de la Recherche’, and the Association pour la
Recherche sur le Cancer. The R.K. laboratory was supported by the Centre National de la Recherche Scientifique; the Agence Nationale de
la Recherche [grant number ANR-08-BLAN-0006]; and the Ligue National Contre le Cancer ‘Equipe Labellisée’ program 2012, and is a
member of the Laboratory of Excellence program No. ANR-11-LABX-0071/Investments for the Future program No. ANR-11-IDEX0005-01. Deposited in PMC for release after 12 months.
The authors apologise to the readers for any confusion that this error might have caused.
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2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1204–1216 doi:10.1242/jcs.165712
RESEARCH ARTICLE
A maternal effect rough deal mutation suggests that multiple
pathways regulate Drosophila RZZ kinetochore recruitment
ABSTRACT
Proper kinetochore recruitment and regulation of dynein and
the Mad1–Mad2 complex requires the Rod–Zw10–Zwilch (RZZ)
complex. Here, we describe rodZ3, a maternal-effect Drosophila
mutation changing a single residue in the Rough Deal (Rod) subunit
of RZZ. Although the RZZ complex containing this altered subunit
(denoted RZ3ZZ) is present in early syncytial stage embryos laid by
homozygous rodZ3 mothers, it is not recruited to kinetochores.
Consequently, the embryos have no spindle assembly checkpoint
(SAC), and syncytial mitoses are profoundly perturbed. The polar
body (residual meiotic products) cannot remain in its SACdependent metaphase-like state, and decondenses into
chromatin. In neuroblasts of homozygous rodZ3 larvae, RZ3ZZ
recruitment is only partially reduced, the SAC is functional and
mitosis is relatively normal. RZ3ZZ nevertheless behaves
abnormally: it does not further accumulate on kinetochores when
microtubules are depolymerized; it reduces the rate of Mad1
recruitment; and it dominantly interferes with the dynein-mediated
streaming of RZZ from attached kinetochores. These results
suggest that the mutated residue of rodZ3 is required for normal
RZZ kinetochore recruitment and function and, moreover, that the
RZZ recruitment pathway might differ in syncytial stage embryos
and post-embryonic somatic cells.
KEY WORDS: RZZ, Polar body, Spindle assembly checkpoint,
Mad1, Syncytial embryo, Kinetochore
INTRODUCTION
Cell division is driven by oscillations in the activity of cyclindependent kinases (Cdks) and their associations with regulatory
cyclin subunits. Mitotic entry is driven by high Cdk1–Cyclin-B
activity, and mitotic exit is dependent on mechanisms that
inactivate this complex and dephosphorylate its targets (reviewed
in O’Farrell, 2001; Lindqvist et al., 2009; Mochida and Hunt,
2012). The initiation of mitotic exit at the metaphase-anaphase
transition is under the control of the spindle assembly checkpoint
(SAC), which indirectly regulates the proteolytic degradation of
Cyclin B by controlling its ubquitinylation by the E3 ubiquitin
ligase known as the anaphase promoting complex (APC/C)
1
Equipe Labellisée Ligue Contre le Cancer, CNRS, Institut Jacques Monod,
UMR7592, Université Paris Diderot, Sorbonne Paris Cité, Paris Cedex 13 75205,
France. 2Department of Cell and Developmental Biology, Vanderbilt University
Medical Center, Nashville, TN 37232-8240, USA.
*These authors contributed equally to this work
`
Author for correspondence ([email protected])
Received 7 November 2014; Accepted 7 January 2015
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(reviewed in Musacchio, 2011; Lara-Gonzalez et al., 2012; Jia
et al., 2013). Among the key SAC proteins in this process are
Mad1 and Mad2, which form a complex at unattached
kinetochores and help generate an inhibitor of the APC/C.
Upon proper kinetochore attachment to the spindle, several SAC
components, including Mad1, Mad2 and the Rough Deal (Rod)–
Zw10–Zwilch (RZZ) complex are removed from the kinetochore
by a dynein-dependent process (Howell et al., 2000; Hoffman
et al., 2001; Howell et al., 2001; Wojcik et al., 2001; Basto et al.,
2004), thus dismantling the SAC apparatus and inactivating the
source of the APC/C inhibitor.
RZZ is an important component of the outer kinetochore
(Karess, 2005). It is a determinant of Mad1–Mad2 kinetochore
levels, as it promotes both Mad1–Mad2 recruitment (Buffin et al.,
2005; Kops et al., 2005) and recruitment of dynein and its
associated regulatory proteins (Starr et al., 1998; Griffis et al.,
2007; Chan et al., 2009). RZZ is also involved in regulating
kinetochore–microtubule attachments (Gassmann et al., 2008;
Gassmann et al., 2010; Cheerambathur et al., 2013), although
how it performs these functions, and how it is itself recruited to
kinetochores is poorly understood.
In the early division cycles of Drosophila embryogenesis,
nuclei divide synchronously in a syncytium, using a streamlined
cell cycle in which S- and M-phase oscillate, driven by stockpiles
of maternal mRNAs and proteins (reviewed in Foe et al., 1993;
Lee and Orr-Weaver, 2003). During these syncytial cycles, the
unfertilized nuclear products of female meiosis remain in Mphase at the periphery of the embryo. These nuclear products,
referred to as polar bodies, coalesce to form a ‘starburst’ of
condensed chromosomes and maintain this state until they are
culled from the embryo during cellularization.
Defective polar body M-phase maintenance has been reported
in mutants with either reduced Cdk1 activity or misregulated
levels of Cyclin B. For example, the Pan Gu (Png) kinase
complex promotes the translation of Cyclin B during female
meiosis and syncytial embryogenesis. Females mutant for the png
kinase or its regulatory subunits lay eggs with abnormally low
Cyclin B levels. As a result, polar bodies do not remain in Mphase but instead re-enter interphase and undergo unregulated
rounds of DNA replication, resulting in polyploid interphase-like
polar bodies (Fenger et al., 2000; Lee et al., 2001). The SAC,
which also regulates levels of Cyclin B, is another proposed
mechanism by which polar body M-phase is maintained. Indeed,
eggs laid by females homozygous for certain viable mutations of
the SAC genes Mps1 (also known as ald in Drosophila) and
BubR1 present similar polar body defects (Fischer et al., 2004;
Pérez-Mongiovi et al., 2005).
In a screen for new cell-cycle regulators specifically required
during the syncytial stages of embryogenesis, we identified a
novel, maternal-effect allele of the rough deal (rod) gene, called
Journal of Cell Science
Lénaı̈g Défachelles1,*, Sarah G. Hainline2,*, Alexandra Menant1, Laura A. Lee2 and Roger E. Karess1,`
Journal of Cell Science (2015) 128, 1204–1216 doi:10.1242/jcs.165712
here rodZ3. This mutation only slightly perturbs RZZ activity in
homozygotes, which are viable and have relatively normal
mitoses. However, in eggs laid by rodZ3 mothers, RZZ is
incapable of kinetochore recruitment. As a result, such embryos
present decondensed polar bodies, profoundly perturbed syncytial
mitoses, and a non-functional SAC. The phenotype of this
mutation suggests that aspects of kinetochore assembly may
differ in maternal and zygotic Drosophila mitosis.
RESULTS
rodZ3 is a new maternal-effect lethal allele of rod with
abnormal syncytial mitoses and defective polar body Mphase maintenance
In a screen for new genes in Drosophila affecting polar body
maintenance, we identified a novel allele of rod in a large
maternal-effect lethal collection (Koundakjian et al., 2004). Flies
homozygous for this allele, called rodZ3, are viable and appear
normal. However, they are completely sterile; the females lay
eggs that fail to hatch (Table 1). Examination of 3–5-hour-old
eggs revealed a number of early developmental defects. None of
the embryos had reached gastrulation and most presented highly
abnormal distribution of blastoderm nuclei and unusually large
internal nuclei, suggesting problems with the syncytial mitoses
(Table 1; Fig. 1A; supplementary material Fig. S1). In contrast,
nearly all the wild-type (WT) embryos at this stage had either
begun gastrulating (73%) or were cellularizing (22%) (Table 1).
In younger (0–2 h) syncytial stage rodZ3-derived embryos, the
nuclei were frequently unevenly spaced and asynchronously
cycling (Fig. 1A,B; Table 1). The majority of the mitotic figures
in these embryos were abnormal, including multipolar spindles
with multiple centrosomes and poorly focused spindles lacking
one or both centrosomes (Fig. 1B). In fact, the overall phenotype
of these embryos is similar to that reported for germline clones of
zw10-null mutants (Williams and Goldberg, 1994).
In nearly all the eggs derived from rodZ3 females, the polar
body formed a large indistinct mass of interphase-like
decondensed chromatin instead of the starburst configuration of
condensed chromosomes found in WT embryos (Table 2;
Fig. 1A,C). The increased size of these polar bodies and the
intensity of their DNA stain compared to WT suggest that they
have an increase in DNA content, presumably owing to additional
rounds of DNA replication. Expression of the rod+tC3L9
transgene, which contains the WT rod genomic region, in the
rodZ3 background rescued this polar body defect, the embryonic
lethality (Table 1) and the mitotic phenotypes described below,
confirming that the mutation in rod was responsible for the
phenotypes.
Immunostaining rodZ3-derived embryos for phosphorylated
Histone H3 (PH3), a marker of M-phase chromosome condensation,
distinguished two classes of decondensed polar bodies. Roughly 40%
of the polar bodies were PH3-negative with rounded, interphase-like
chromatin (Fig. 1C; Table 2); the rest were PH3-positive with
irregularly-shaped and partially decondensed chromatin. WT polar
bodies were PH3-positive as has been reported by others (Fischer
et al., 2004; Pérez-Mongiovi et al., 2005). The irregularly-shaped,
semi-decondensed and PH3-positive polar bodies of rodZ3-derived
embryos also labeled with Spc25–RFP (a marker of the outer
kinetochore, present only during M-phase), indicating that
kinetochores were intact. Interestingly, these polar bodies
contained more than the expected 12 kinetochore signals
(Fig. 3A), suggesting that they had at least partially replicated
their DNA and returned to an M-phase-like state. The more
rounded interphase-like polar bodies lacked Spc25–RFP (Fig. 3A),
consistent with the interpretation that they were in interphase.
These results suggest that the polar bodies of rodZ3-derived
embryos might be cycling in and out of M-phase, as has been
proposed for other mutants (Fischer et al., 2004; Pérez-Mongiovi
et al., 2005).
The SAC is required for polar body maintenance
Decondensed polar bodies have been reported previously in
mutants of mps1 and BubR1 (Fischer et al., 2004; Pérez-Mongiovi
et al., 2005), two other genes encoding SAC components. rod is
the third SAC gene to be associated with this phenotype. To
determine whether rodZ3-derived syncytial embryos could
undergo SAC-dependent mitotic arrest in response to spindle
damage, syncytial-stage embryos were incubated with or without
colchicine for 30 min, and the fraction of mitotic embryos was
determined (see Materials and Methods for details). Whereas
nearly all WT embryos had become mitotic (Fig. 1D), neither
rodZ3 nor Mad2-null (Mad2P)-derived embryos (a negative
control) displayed any increase in the fraction of mitotic
embryos in response to colchicine treatment. These results
indicate that the SAC is defective in rodZ3-derived embryos and
suggests that the failure of polar bodies to remain in M-phase is
due to the defective SAC.
However, Mps1, BubR1 and the RZZ complex all participate
in other mitotic functions that do not involve the SAC (Starr
et al., 1998; Ditchfield et al., 2003; Jones et al., 2005; Lampson
and Kapoor, 2005; Maure et al., 2007; Jelluma et al., 2008;
Hewitt et al., 2010; Santaguida et al., 2010; Sliedrecht et al.,
2010; Althoff et al., 2012; Wainman et al., 2012). It is therefore
conceivable that the polar body decondensation is not specifically
connected to a defect in SAC activity. To confirm the role of the
SAC in the maintenance of polar body condensation, we
examined embryos laid by Mad2P females. Drosophila Mad2null mutants have a defective SAC, but do not exhibit the mitotic
defects associated with mutations in other SAC genes (Buffin
et al., 2007). We found that most of the polar bodies in Mad2Pderived embryos were large with semi- or fully decondensed
chromatin (Table 2), similar to those of rodZ3. This result
supports the hypothesis that SAC activity is required to
maintain polar body condensation, presumably by stabilizing
Cyclin B (Fischer et al., 2004; Pérez-Mongiovi et al., 2005). To
Table 1. Maternal-effect embryo phenotype of rodZ3
Percentage of Embryos
Maternal genotype
Hatched (n)
Normal gastrulation at 3–5 h (n)
Normal cellular blastoderm at
3–5 h (n)
Abnormal syncytial divisions
(n)
WT
rodZ3
rod+C3L9; rodZ3
94 (2355)
0 (945)
73 (1737)
73 (290)
0 (235)
–
22 (290)
0 (235)
–
2 (100)
60 (99)
8 (101)
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more rigorously test this latter point, we generated rodZ3 mutants
carrying one copy of a lethal allele of the shattered (shtd) gene
encoding APC/C subunit APC1 in Drosophila. We also directly
increased Cyclin B levels in rodZ3 flies by introducing two
additional copies of the WT Cyclin B (CycB) gene. Both
modifications partially suppressed the polar body phenotype of
rodZ3-derived embryos (Table 2). The frequency of abnormal
polar bodies fell to 69% in shtd3/+; rodZ3-derived embryos, and
to 53% in 26Cyclin B/+; rodZ3-derived embryos. These results
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argue that WT rod is required to maintain polar body M-phase
through its role in the SAC, which in turn inhibits APC/Cregulated degradation of Cyclin B.
rodZ3 does not affect RZZ assembly
Sequencing the rodZ3 genomic DNA revealed a point mutation in
rod where glycine 1973 was replaced by a glutamic acid residue,
predicted to disrupt a short a-helix in the relatively conserved Cterminal region of the 2089-residue Rod protein. Drosophila Rod
Journal of Cell Science
Fig. 1. Embryos laid by rodZ3 mothers are defective in syncytial mitosis, polar body maintenance and the SAC. (A) Representative images of DNAstained early (left) and late (right) syncytial embryos from wild-type (WT) and rodZ3 females. rodZ3-derived embryos contain large decondensed polar bodies
(left). Also note the asynchrony of mitotic stages and the irregular distribution of nuclei in the rodZ3-derived embryos (right). Scale bars: 50 mm. Insets, larger
images of polar bodies (marked by arrows). (B) Representative images of prophase and metaphase figures from WT and rodZ3-derived syncytial embryos
stained for tubulin (green), centrosomin (red) and DNA (blue). rodZ3 mitotic figures have abnormal centrosome attachments. Broad acentrosomal and multipolar
spindles are common. Scale bar: 10 mm. (C) Details of polar bodies from WT and rodZ3-derived syncytial embryos stained for PH3 and DNA. rodZ3 polar bodies
are either PH3-positive (partially decondensed) or PH3-negative (fully decondensed). Scale bar: 10 mm. (D) The SAC is not functional in rodZ3-derived embryos.
Relative change in the fraction of mitotic syncytial embryos after 30 min incubation in colchicine (compared with untreated, set at 1) for the indicated genotypes.
rodZ3-derived embryos, like those derived from Mad2 null mothers (Mad2P, negative control), show no significant increase after treatment. *P,0.005;
**P,0.0001 (Fisher’s exact test; n580 embryos, except rescue-derived embryos where n5140).
RESEARCH ARTICLE
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Table 2. Polar body phenotype of rodZ3
PB phenotype
Maternal genotype (n)
Percentage normal
Condensed PBs
WT (145)
rodZ3 (176)
rod+C3L9; rodZ3 (157)
shtd3; rodZ3 (157)
2XCyclinB; rodZ3 (66)
Mad2P (147)
99
6
77
31
47
18
Decondensed PBs
Partially decondensed PBs
Fully decondensed PBs
1
94a,b
23
69a
53b
82
1
56
18
40
39
51
0
38
4
29
14
31
These differences are significant (*P,0.0001, Fisher’s exact test). PB, polar body.
has an overall predicted structure similar to that described for
human Rod (also known a KNTC1) (Çivril et al., 2010) with Nterminal b-folds and a long a-solenoid region extending to the Cterminus. Although not conserved in vertebrate Rod (where it is a
tyrosine or a serine residue), the glycine at position 1973 has been
conserved since the divergence of mosquitos and Drosophila,
over 250 million years (Wiegmann et al., 2011) (Fig. 2A).
The RZZ complex, with an apparent molecular mass of 600–
700 kDa, is believed to contain two copies of each of the three
subunits Rod, Zw10 and Zwilch, with Rod serving as the scaffold
(Scaërou et al., 2001; Williams et al., 2003; Çivril et al., 2010).
Immunoblotting revealed that levels of Rod, Zw10 and Zwilch in
total extracts of rodZ3-derived embryos were similar to those in
WT embryos (Fig. 2B). More importantly, Zw10 and Zwilch
co-immunoprecipitated with Rod equally well in extracts of WT
or rodZ3-derived embryos (Fig. 2B). Fractionating lysates from
WT and rodZ3-derived syncytial embryos by sucrose density
gradient centrifugation revealed that the rodZ3 mutation does not
affect the capacity of Rod to form high molecular mass
complexes (Fig. 2C). These data argue that the rodZ3 mutation
does not affect RZZ complex formation or steady-state levels.
RZZ is not recruited to kinetochores in rodZ3-derived
embryos but is recruited at reduced levels in
rodZ3 neuroblasts
With the knowledge that the RZZ complex was intact, we
next examined its behavior in rodZ3-derived syncytial embryos
by following GFP-tagged Zw10. WT embryos expressing
Fig. 2. The rodZ3 mutation changes a conserved glycine in the Rod_C domain, but does not affect RZZ assembly. (A) The mutation in rodZ3 results in a G
to E change at residue 1973 in a conserved C-terminal region of the protein. A Clustal alignment around the mutated region of Rod from mammals and dipterans
shows that G1973 (red box) has been conserved since the divergence of mosquitoes and flies. The corresponding position in mammalian Rod is always a polar
residue. (B) Levels of Rod, Zw10 and Zwilch are similar in extracts of WT and rodZ3-derived syncytial embryos, and immunoprecipitation (IP) of Rod brings down
Zw10 and Zwilch equally well in both WT and rodZ3 extracts. Immunoblots are for Rod, Zw10 and Zwilch in input lysates of WT or rodZ3 derived embryos (left)
(Tubulin is a loading control). Immunoprecipitates using anti-Rod, (middle), or control non-immune rabbit serum are shown on the right). The lower molecular
mass in the Zwilch blots is an unrelated contaminant. (C) RZZ from WT and rodZ3-derived syncytial embryos both form high-molecular-mass complexes.
Fractions of a sucrose density gradient centrifugation of embryo extracts immunoblotted for Rod. Arrows indicate migration of size markers.
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a,b
Percentage abnormal
Journal of Cell Science (2015) 128, 1204–1216 doi:10.1242/jcs.165712
Fig. 3. See next page for legend.
GFP–Zw10 and Spc25–RFP contained RZZ foci at the
kinetochores of the polar body and mitotic chromosomes
(Fig. 3A,B) as well as along metaphase spindle fibers (Fig. 3B),
reflecting the streaming of RZZ from attached kinetochores
(Wojcik et al., 2001; Basto et al., 2004). In contrast, no GFP
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signal was detected on any mitotic structure in rodZ3-derived
embryos (Fig. 3A,B). These data suggest that the rodZ3 mutation
interferes with kinetochore recruitment of the RZZ complex.
Although the absence of RZZ recruitment provided an
explanation for the severity of the phenotype in embryos laid
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Fig. 3. RZZ kinetochore recruitment is absent in rodZ3-derived embryos
but present, although reduced, in homozygous rodZ3 neuroblasts.
(A) RZZ does not localize to polar bodies in rodZ3-derived embryos. Polar
bodies from fixed WT and rodZ3-derived syncytial embryos expressing
Spc25–RFP (a kinetochore marker, red), GFP–Zw10 (an RZZ component,
green) and stained for DNA (blue). Scale bar: 10 mm. (n512 polar bodies
examined, all negative.) (B) RZZ does not localize to metaphase
kinetochores or spindle fibers in rodZ3-derived embryos. Scale bar: 10 mm.
(n530 metaphases, all negative, from 12 embryos). (C) RZZ is recruited to
kinetochores in rodZ3 larval neuroblasts, but at reduced levels. In WT or rodZ3
heterozygous neuroblasts GFP–Zw10 localizes to kinetochores (marked by
Spc25–RFP) and spindle fibers. In homozygous rodZ3 cells (bottom), GFP–
Zw10 is detectable at kinetochores, but at lower intensity. Particles of RZZ
are visible between the kinetochores (white arrow) and the poles (yellow
arrows) in WT and heterozygous rodZ3, but not in homozygous rodZ3,
suggesting a problem with dynein-dependent streaming (see also Fig. 5).
Scale bars: 2 mm. (D) Quantification of RZZ levels on kinetochores. The
GFP–Zw10 signal, normalized to that of Spc25–RFP, was determined for WT
(blue, n57, ncolch5 26), rodZ3 homozygous (red, n57, ncolch5 8) or rodZ3/+
heterozygous (green) cells. Closed circles and open circles indicate cells
treated or untreated, respectively, with colchicine for 15 min. Results are
mean6s.e.m. *P,0.05, **P,0.01 (Student’s t-test). (E) The SAC is functional
in rodZ3 larval brains. Relative mitotic density in larval brains as a function of
time in colchicine. WT (black), rodZ3 (light gray), rod null (dark gray). Results
are mean6s.e.m. *P,0.05 (Student’s t-test, using at least five larval brains per
experiment). In rodZ3, the mitotic density increases as a function of time
in colchicine similar to WT, whereas in SAC-defective rod null brains, mitotic
cells do not accumulate, even after 1 h in colchicine. (F) Lagging chromatids
are common in rodZ3 neuroblast mitosis. Representative images of single
live neuroblasts of the indicated genotypes in metaphase and anaphase.
Kinetochores are visualized with Spc25–RFP (red) and the spindle is marked
with GFP–Jupiter (green). Indicated times (min:seconds) are relative to
anaphase onset (see also Table 3). Scale bars: 2 mm.
Z3
by homozygous rod mothers, it was also intriguing because the
homozygous rodZ3 offspring of heterozygous parents were
apparently normal. A mutation blocking RZZ kinetochore
recruitment would normally be a zygotic lethal. Indeed, null
mutations in any component of RZZ are homozygous lethal, dying
as a consequence of profound defects in chromosome segregation
during postembryonic development (Karess and Glover, 1989;
Williams et al., 1992; Williams and Goldberg, 1994; Williams
et al., 2003; Karess, 2005). We therefore examined RZZ behavior
in dividing tissues of homozygous rodZ3 third-instar larvae
(Fig. 3C). Unlike the rodZ3-derived embryos, rodZ3 larval
neuroblasts displayed substantial but limited recruitment of RZZ
to kinetochores during unperturbed mitosis. Average RZZ
kinetochore levels (normalized to the Spc25–RFP signal) were
approximately one-third that of WT (Fig. 3D). In addition to the
reduction of RZZ recruitment to kinetochores, there was little or no
RZZ on the spindle fibers and poles of rodZ3 neuroblasts,
suggesting a problem with streaming (see below and Fig. 5).
In WT cells, a brief colchicine or nocodazole treatment to
depolymerize kinetochore microtubules greatly increases the
levels of several outer kinetochore proteins, including RZZ, Mad1
and Mad2 (Hoffman et al., 2001; Williams and Goldberg, 1994;
Basto et al., 2004; Buffin et al., 2005). Strikingly, colchicine
treatment did not significantly increase the kinetochore signal of
GFP–Zw10 in rodZ3 neuroblasts; whereas in WT cells, the signal
more than doubled (Fig. 3D). This reduced recruitment of RZZ in
rodZ3 neuroblasts was confirmed by quantification of Rod levels by
immunostaining with anti-Rod antibody (data not shown).
To assess whether this limited RZZ recruitment is compatible
with a functional SAC, we assayed the ability of colchicine-treated
rodZ3 larval brains to accumulate mitotic cells (Fig. 3E). Both
rodZ3 and WT larval brains more than doubled their density of
mitotic cells after 1 h incubation in colchicine, whereas, rod-null
brains did not. These results indicate that the SAC is properly
functioning in rodZ3 larval neuroblasts.
Examination of karyotypes of mitotic cells in third-instar rodZ3
larval brains revealed relatively little aneuploidy (Table 3). Live
imaging of dividing rodZ3 neuroblasts (Fig. 3F), however, showed
that there was an elevated frequency of anaphases in which some
kinetochores migrated poleward more slowly than the others.
Indeed, the fraction of anaphases with at least one lagging
kinetochore was nearly as high in rodZ3 as in rod or Mad1-null
mutants (Emre et al., 2011), although only rarely was more than
one kinetochore involved (Table 3). As is the case for Mad1-null
mutants (Emre et al., 2011), these lagging chromatids were in most
cases correctly segregated, despite their delay in migration (given
that overall aneuploidy levels are low). In several cases, the lagging
chromatids appeared to be attached to spindle fibers emanating
from both poles (merotelic attachment), which is also a feature
associated with mitosis in Mad1-null mutants (Emre et al., 2011).
In summary, RZZ retains a limited capacity to associate with
the mitotic apparatus in rodZ3 larval neuroblasts, but this is
apparently adequate for mounting a SAC-mediated mitotic arrest
and providing sufficient functionality for maintaining reasonably
accurate mitosis. In contrast, RZZ appears to be incapable of
kinetochore recruitment in early embryos derived from rodZ3
mothers. These results largely explain the different mitotic
phenotypes of these two tissues.
Mad1 kinetochore recruitment rate and accumulation is
reduced in rodZ3 neuroblasts
We next asked whether rodZ3 affected the recruitment of Mad1
and Mad2 given that kinetochore levels of the Mad1–Mad2
complex depend in part on the presence of functional RZZ
(Buffin et al., 2005; Kops et al., 2005). In untreated rodZ3
neuroblasts, levels of Mad1 (Fig. 4A) and Mad2 (data not shown)
were often low, and sometimes undetectable on prometaphase
mitotic figures. On average, the Mad1–GFP kinetochore signal in
rodZ3 was not significantly different from that in rod-null cells
(Fig. 4B). The low levels of Mad1 might therefore help explain
why rodZ3 displays the elevated rate of lagging chromatids also
seen in Mad1 mutants. However, when treated with colchicine,
rodZ3 neuroblasts did accumulate Mad1 and Mad2 at kinetochores
(Fig. 4C). Both the rate of this accumulation and the final level
attained were reduced; reaching approximately one-third of the
levels found in WT after 8–10 min. This degree of Mad1 and Mad2
recruitment must be sufficient to elicit the robust SAC response of
colchicine-treated rodZ3 larval brains (Fig. 3E).
The relatively low levels of Mad1 recruitment after treatment
with colchicine might be a quantitative consequence of the
reduced RZZ at rodZ3 kinetochores or a qualitative consequence
of the structural change caused by the rodZ3 mutation (or both).
To gain insight into this question, we simultaneously measured
the fluorescent signals from GFP–Zw10 and Cherry–Mad1 on the
kinetochores of individual colchicine-treated neuroblasts and
displayed the values in a scatter plot (Fig. 4D). The plot shows
that the highest RZZ and Mad1 signals are (not surprisingly) only
found in the WT cells. However, considering only the range of
RZZ values overlapping between WT and rodZ3 (included within
the box of Fig. 4D), the distribution of Mad1 values was not
significantly different and the linear regression curves that fitted
the two data sets had similar slopes. This result indicates that the
recruitment levels of Mad1 are ‘normal’ in rodZ3 cells, given the
reduced amount of RZZ on unattached kinetochores.
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Journal of Cell Science (2015) 128, 1204–1216 doi:10.1242/jcs.165712
Table 3. Mitotic phenotype of rodZ3 larval neuroblasts.
Zygotic genotype
Percentage aneuploid (s.d.)a
Percentage abnormal anaphase (one or
more lagging kinetochore)b (n)
Mean number of laggards per abnormal
anaphase
WT
rodZ3
rodnull
Mad1null
0.2 (0.22)c
2.9 (1.2)
37 (4.5)
Less than 1d
12.5 (16)
64 (22)
70 (23)
63 (28)
1
1.8
2.3
2.3
a
The percentage of aneuploidy (.2n only) was determined on aceto-orcein-stained fixed tissue. bAnaphases were determined on live cells. cFrom Buffin
et al. (Buffin et al., 2007). dFrom Emre et al. (Emre et al., 2011).
To determine whether the rodZ3 mutation is affecting steadystate turnover of Mad1 at unattached kinetochores, we subjected
Mad1–GFP to fluorescence recovery after photobleaching (FRAP)
analysis (Fig. 4E). The Mad1–GFP that had accumulated on rodZ3
kinetochores after 15 min in colchicine displayed the same
turnover dynamics as that in WT. A fast turnover pool, ,40% of
the total, recovered with a half-life of 10–12 seconds, whereas the
remaining 60% was essentially stable, showing little or no
recovery. Thus, the steady-state turnover properties of Mad1 are
not disrupted in rodZ3 neuroblasts.
In summary, the primary cause of the reduced kinetochore
Mad1 signal in colchicine-treated rodZ3 neuroblasts is the low
levels of RZZ recruitment itself, and not a specific effect of the
rodZ3 mutation on the ability of RZZ to recruit Mad1.
Nevertheless, the systematic reduction in the kinetochore Mad1
signal seen in unperturbed mitosis (Fig. 4A) to levels found in
rod-null cells, and the fact that Mad1 is slower to accumulate on
unattached kinetochores (Fig. 4C), suggests the rodZ3 mutation
might specifically alter the rate of Mad1 accumulation.
considered the possibility that the RodZ3 protein was directly
interfering with the ability of RZZ to stream. Consistent with this
idea, rodZ3/+ heterozygous neuroblasts also displayed reduced
streaming relative to WT (Fig. 5A), suggesting that rodZ3 has a
dominant effect in disrupting RZZ function. To test this idea, we
co-expressed two Rod proteins with different fluorescent tags in a
rod-null background: Cherry–Rod+ and either GFP–Rod+ or
GFP–RodZ3, expressed from a transgene containing the identical
mutation as the rodZ3 allele (Fig. 5D). When two WT transgenes
were expressed, both green and red particles of RZZ streamed on
kinetochore-attached microtubules, whereas when GFP–RodZ3
was co-expressed with Cherry–Rod+ the streaming of both the
red (WT) and green (mutant) RZZ was greatly reduced (Fig. 5D).
These results are consistent with a partially functional GFP–
RodZ3 subunit assembling into the multi-subunit RZZ complex
and inhibiting its ability to be transported from kinetochores.
RZZ streaming is disrupted in rodZ3 neuroblasts
In a search for maternal effect mutants affecting the regulation of
early embryonic divisions, we found a unique allele of rod, called
rodZ3, which profoundly affects the ability of RZZ to be recruited
to kinetochores in the early embryo.
Although previous studies have shown that Cdk1 and Cyclin B
activity are required for polar body condensation, we have
established that polar bodies maintain Cdk1–Cyclin-B activity
through activation of the SAC. We show here that the RZZ
complex, like Mps1 and BubR1, localizes to kinetochores in polar
bodies and is required to maintain polar body condensation in
Drosophila. rodZ3-derived embryos, like those of Mad2, Mps1
and BubR1 mutants, contain large interphase-like polar bodies.
Pérez-Mongiovi et al. (Pérez-Mongiovi et al., 2005) found
evidence that in BubR1Rev1-derived embryos, polar bodies cycle
between periods of condensation and decondensation with
accompanying DNA replication. They suggested that these
polar body cycles are under the control of the embryonic
mitotic oscillator. Because polar bodies in rodZ3 embryos are not
always fully decondensed, it is possible that they undergo similar
cycles of DNA replication and condensation. The fact that the
semi-decondensed polar bodies have more than the expected 12
kinetochores (labeled with the outer kinetochore protein Spc25;
Fig. 3A), is consistent with this idea.
Directly or indirectly increasing Cyclin B levels in rodZ3derived embryos partially suppresses the polar body condensation
defects. This result argues that Rod and the other SAC
components maintain polar body condensation by inhibiting the
APC/C-mediated degradation of Cyclin B. The Png kinase
complex is also required to maintain Cyclin B and thus polar
body condensation (Lee et al., 2001). However, rod and png
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Several transient outer kinetochore components, including RZZ
and Mad1–Mad2, display a characteristic movement from
kinetochores to poles along kinetochore fibers called shedding,
stripping or streaming (Howell et al., 2000; Howell et al., 2001;
Wojcik et al., 2001; Karess, 2005; Famulski et al., 2011). This
dynein-dependent process is believed to contribute to the
extinction of the SAC signal following proper kinetochore–
microtubule attachment. In live images of GFP–Zw10 expressed
in rodZ3 neuroblasts (Fig. 3C), this streaming appeared to be
substantially reduced. Because streaming is highly variable, even
in WT, we quantified this apparent difference by scoring mitotic
cells into three classes according to their degree of streaming:
none (all GFP–Zw10 signal restricted to kinetochores), low
(GFP–Zw10 signal mostly on kinetochores) and high (signal on
kinetochores, spindle and poles). By these criteria, rodZ3
neuroblasts consistently failed to show any streaming (Fig. 5A).
Given that streaming is a dynein-dependent process and RZZ is
required for the recruitment of the dynein–dynactin complex to
kinetochores (Starr et al., 1998; Bader and Vaughan, 2010;
Raaijmakers et al., 2013), we asked whether rodZ3 specifically
reduced kinetochore dynein levels. We measured the accumulation
of GFP–DLIC2, a dynein subunit, relative to Spc25–RFP, on
mutant or WT cells incubated 15 min in colchicine (Fig. 5B,C). In
90% of rodZ3 cells, GFP–DLIC2 was detectable at kinetochores,
averaging about half the signal found in WT. As was the case for
kinetochore Mad1–GFP, the range of GFP–DLIC2 levels was very
broad and probably reflects the variation in RZZ recruitment levels.
Given that dynein recruitment is only partially reduced,
whereas dynein-dependent streaming is entirely absent, we
DISCUSSION
The SAC is required for polar body M-phase maintenance
in Drosophila
Journal of Cell Science (2015) 128, 1204–1216 doi:10.1242/jcs.165712
Fig. 4. See next page for legend.
regulate Cyclin B levels through different mechanisms. During
embryogenesis, Png kinase promotes the translation of Cyclin B
by regulating the polyadenylation of its mRNA and antagonizing
the translational repressor Pumilio (Vardy and Orr-Weaver,
2007). The SAC components, by contrast, recognize the polar
body kinetochores as improperly attached to the spindle, and
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Fig. 4. Mad1 kinetochore recruitment is slow, but its steady state
dynamics are normal in rodZ3 neuroblasts. (A) Images of live WT (top) or
rodZ3 (bottom) prometaphase neuroblasts expressing Mad1–GFP and
Spc25–RFP. Scale bars: 2 mm. (B) Quantification of Mad1–GFP kinetochore
recruitment, normalized to Spc25–RFP in untreated WT (blue, n543), rodZ3
(red, n544), or rod-null (gray, n529) cells. Horizontal bars represent the
mean. In most rodZ3 cells, little or no Mad1 is detectable at kinetochores,
similar to rod null. **P,0.01; Student’s t-test. (C) Mad1–GFP (top) and GFP–
Mad2 (bottom) accumulate slowly on kinetochores, but reach substantial
levels in colchicine-treated rodZ3 neuroblasts. GFP signals of kinetochore
Mad1 or Mad2 were monitored in individual, colchicine-treated WT (blue) or
rodZ3 (red) neuroblasts, normalized to Spc25–RFP, and plotted as a function
of time after nuclear envelope breakdown (NEB). The maximum
accumulation is about 35% that of WT. P,0.05 (Student’s t-test). (D) The
relationship between Mad1 and RZZ levels is unchanged in rodZ3 cells.
Larval brains expressing Cherry (Ch)–Mad1 and GFP–Zw10 were imaged
after a 30-min colchicine treatment. The intensities of kinetochore Cherry and
GFP were quantified in individual WT (blue) or rodZ3 (red) cells. Levels of
GFP–-Zw10, representing RZZ, were plotted on the x-axis and those of Ch–
Mad1 were plotted on the y-axis. The two data sets show a similar correlation
between Mad1 and RZZ. The regression curves shown for WT and rodZ3 fit
the data points within the black box, which correspond to kinetochores
displaying the range of GFP–Zw10 signals common to the two genotypes.
The difference in slope is not significant (ANOVA test). (E) rodZ3 does not
alter turnover dynamics of Mad1–GFP on unattached kinetochores. Left,
FRAP analysis of WT (blue) or rodZ3 (red) neuroblasts expressing Mad1–
GFP (top) or GFP–Spc25 (bottom) treated with colchicine for 15 min.
Fluorescence recovery was monitored over 200 s and normalized to the prebleach signal. Neither the size of the fast-turnover pool of Mad1 (,40%) nor
its half-life (,10 s) are altered in rodZ3 cells. The stable outer kinetochore
component Spc25 was used as a control. Right, representative images of
Mad1–GFP in (a) WT and (b) rodZ3 cells, and (c) GFP–Spc25 in WT cells,
before and after photobleaching kinetochores. The bleached area is circled
in yellow. Scale bars: 5 mm.
generate the inhibitor of APC/C-mediated proteolytic degradation
of Cyclin B.
rodZ3 sterility is due to defects in RZZ function beyond its role
in the SAC
rodZ3-derived embryos undergo aberrant syncytial mitosis and a
developmental arrest prior to gastrulation. These embryos
accumulate asynchronously dividing nuclei with centrosome
attachment defects throughout the syncytial divisions. Defects
in mitotic spindle function during syncytial embryogenesis can
uncouple the nuclear and centrosomes cycles (Archambault and
Pinson, 2010). The resulting replication of detached centrosomes
and fusion of free centrosomes to neighboring mitoses leads to
additional aberrant mitoses. Furthermore, centrosome detachment
during syncytial embryogenesis inhibits the nuclear positioning
and migration required for morphogenesis, all of which might
contribute to the developmental arrest of rodZ3-derived embryos.
In Drosophila, inactivation of the SAC has only minor
consequences on mitosis and on embryonic viability (Buffin
et al., 2007). Comparing the embryonic phenotype of rodZ3 with
that of Mad2 nulls is instructive. Both lack a functional SAC in
embryos and consequently the polar body fails to arrest in Mphase. However, although 60–70% of Mad2-null embryos hatch
and reach adulthood (Buffin et al., 2007), no rodZ3-derived
embryos reach the gastrulation stage. The different phenotypes of
Mad2-null and rodZ3-derived embryos argue strongly that it is not
the absence of the SAC per se that is responsible for the lethality
of rodZ3-derived embryos, but rather the combination of
defects caused by simultaneously removing the SAC and nonSAC functions of RZZ, the latter regulating dynein and
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kinetochore–microtubule attachments (Starr et al., 1998; Yang
et al., 2007; Gassmann et al., 2008; Gassmann et al., 2010).
rodZ3 affects the recruitment and behavior of RZZ and Mad1
The primary defect we have identified in RZ3ZZ is its failure to
associate with syncytial and polar body kinetochores in the early
embryo. Most of the other phenotypes can be explained as
secondary consequences of this recruitment failure. The striking
difference between the maternal and zygotic phenotypes of rodZ3
can be ascribed to the fact that RZ3ZZ partially retains its ability
to be recruited to kinetochores in dividing larval tissues.
Why is RZ3ZZ capable of kinetochore recruitment in larval
neuroblasts and not in maternally-derived early embryos? One
possibility is that the unusual rapidity of syncytial mitosis might
render it more sensitive to the structural change in RZ3ZZ.
However, the fact that RZ3ZZ is also absent from polar bodies
argues that this is unlikely to be a complete explanation because
the polar body does not rapidly cycle but remains in M-phase. It
would, in this scenario, still have enough time to recruit RZ3ZZ.
We suggest that there are factors involved in recruiting RZZ to
kinetochores that subtly differ between the syncytial stage and
postembryonic mitoses. For example, there might be two, partially
redundant, recruitment pathways for RZZ. One would be employed
in postembryonic mitoses, such as in larval neuroblasts, but not in
the early embryo. The other recruitment pathway would normally
be used in both tissues, but is damaged in rodZ3. These two
‘pathways’ could be as simple as two different kinetochore-binding
domains of RZZ and two corresponding docking sites on
kinetochores. This idea might also provide an explanation for the
second striking feature of RZ3ZZ behavior: in larval neuroblasts, it
shows no further kinetochore accumulation beyond the basal level
when microtubules are depolymerized with colchicine. This
supplemental RZZ binding might also depend on the second
recruitment pathway, explaining why it is defective in rodZ3. The
mutation in rodZ3 might thus affect a residue important to RZZ
kinetochore binding, about which very little is known, particularly
in Drosophila. Kinetochore proteins implicated in RZZ recruitment
in vertebrates such as ZWINT (Starr et al., 2000; Famulski et al.,
2008) and CENP-I (Matson and Stukenberg, 2014), have not been
found in the fly genome (Przewloka et al., 2007).
During unperturbed mitosis, kinetochore Mad1 levels are as low
in rodZ3 as they are in rod-null larval neuroblasts (Fig. 4A,B). We
have previously reported that Mad1-null neuroblasts display a
relatively mild mitotic phenotype. Despite having a defective SAC,
they display little aneuploidy, but have a high incidence of lagging
chromatids and merotely (Emre et al., 2011). It is therefore not
surprising that we find a similar phenotype in rodZ3 cells, which
have greatly reduced kinetochore Mad1 levels.
The mutation in rodZ3 does not seem to alter the ratio of Mad1
recruited per unit of RZZ. On unattached kinetochores, given
enough time, Mad1 accumulates to levels approximately
proportional to the amount of RZZ present (Fig. 4C,D). The
recruited Mad1 also displays the same steady-state turnover
dynamics as in WT cells (Fig. 4E). However, the time required
to reach that maximal recruitment is longer than in WT
(Fig. 4C). We speculate that the slower recruitment rate of
Mad1 might explain its low levels on kinetochores during
unperturbed mitosis. There is little known about the molecular
mechanism by which RZZ helps to recruit Mad1. It is not even
known if they interact directly, although a fraction of RZZ and
Mad1 can be co-immunoprecipitated from mitotic cells (our
unpublished work).
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Fig. 5. The mutation in rodZ3 affects RZZ streaming in neuroblasts.
(A) Quantification of RZZ streaming in WT, rodZ3, and rodZ3/+ neuroblasts.
Cells were classed by the intensity of GFP–Zw10 streaming: high, low, and
no streaming. RZZ streaming is not observed in homozygous rodZ3 cells
and is substantially reduced in rodZ3/+ cells. (B) Dynein recruitment is
reduced but not eliminated in rodZ3 neuroblasts. Images of GFP–DLIC2 at
kinetochores in live WT, rodZ3 and rod-null cells treated with colchicine.
Dynein is recruited to unattached kinetochores in WT and rodZ3 cells, but not
in rod-null cells. Scale bars: 2 mm. (C) Quantification of GFP–DLIC2 at
kinetochores, normalized to Spc25–RFP, in colchicine-treated WT (blue,
n536), rodZ3 (red, n534) or rod-null (gray, n518) cells. In 90% of rodZ3 cells,
GFP–DLIC2 is detectable at kinetochores but its signal is reduced compared
to that in WT cells. Horizontal bars represent the mean. **P,0.01 (Student’s
t-test). (D) RodZ3 dominantly disrupts RZZ streaming. Top, metaphase
spindles of neuroblasts expressing two different colors of WT Rod [Cherry
(Ch)–Rod+ and GFP–Rod+, first row, or one WT copy and one mutant copy
(Ch–Rod+ and GFP–RodZ3, second row] in a rod-null background. In cells
expressing only WT Rod, streaming is normal, and particles containing both
colors are found on the spindle fibers. When GFP–RodZ3 is co-expressed
with Ch–Rod+, the streaming of both is reduced. Scale bars: 2 mm. Bottom,
quantification of Rod streaming, using criteria described in A. The fraction of
cells with robust streaming is substantially reduced when GFP–RodZ3 is
present.
Finally, rodZ3 has a perceptible dominant effect on the dyneindependent streaming of RZZ. This can best be seen in Fig. 5D,
where RFP–RodZ3 co-expressed with GFP-Rod+ interferes with
the streaming of both tagged proteins from kinetochores. The
RZZ complex is believed to contain two copies of each of its
three subunits, with Zw10 and Zwilch binding to the N-terminal
half of the Rod protein (Starr et al., 1998; Scaërou et al., 2001;
Williams et al., 2003; Çivril et al., 2010; Cheerambathur et al.,
2013). This stoichiometry means that a given RZZ particle could
contain one WT and one mutant copy of RZZ, if both versions are
present. Given that the effect of rodZ3 on streaming is more
severe than its effect on dynein recruitment, we suggest that the
mutation in rodZ3 might also perturb an interaction with the
dynein motor complex.
In summary, the mutation in rodZ3 affects three activities of
RZZ: its own kinetochore recruitment, its capacity to rapidly
recruit Mad1 and its capacity to be removed from attached
kinetochores by dynein-dependent streaming. The G to E
mutation of rodZ3 at residue 1973 resides in the most highly
conserved portion of the Rod protein, known as Rod_C domain
(pfam10493), which is predicted to fold into several short ahelices. This domain is only found in known or suspected
homologs of Rod, although no specific function or interacting
proteins have been are associated with it. Thus, characterization
of rodZ3 provides a unique opportunity to examine how the Rod
C-terminus contributes to RZZ function.
MATERIALS AND METHODS
Drosophila stocks
Flies were maintained at 25 ˚C using standard techniques. A y w stock was
used as wild type for embryo experiments. The stock bw; rodZ3 st/TM6B
was a gift from Charles Zuker (Columbia University, New York, NY).
The rodZ3 allele had designation Z3-0733 in the Zuker stock collection.
We shortened and superscripted it to indicate that it is an allele of rod.
The following mutations and transgenes have been previously described:
rod-null alleles rodAG1 and rodX2 and the transgene rod+tC3L9 (Scaërou
et al., 1999), GFP-Rod (Basto et al., 2004), RFP-Rod (Buffin et al.,
2005), the null allele Mad11, the transgene Mad1-GFP and Cherry-Mad1
(Emre et al., 2011), the null allele Mad2P (Buffin et al., 2007), the
2XCyclin B stocks, and transgenes Spc25-GFP and Spc25-RFP
(Schittenhelm et al., 2007) (gifts from Christian Lehner, University of
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RESEARCH ARTICLE
Zurich, Zurich, Switzerland), the shtd3 stock (from the Bloomington stock
center), transgene GFP-DLIC2 (gift from Jordan Raff, Oxford, UK).The
GFP-Zw10 construct will be described elsewhere. Transgenic lines
Cherry-Rod, and GFP-RodZ3 were obtained by P-element transposition.
The transgenes correspond to genomic sequence (including the promoter
region) as originally described previously (Basto et al., 2004). Fly
transformation was performed by BestGene (Chino Hills, CA).
Embryo immunostaining and colchicine treatment
0–2-h embryos (unless otherwise noted) were collected as described
previously (Rothwell and Sullivan, 2000). For Tubulin, Centrosomin and
DNA staining, embryos were dechorionated in 50% bleach, and fixed and
devitellinized by shaking in a 1:1 mixture of methanol and heptane. For
visualizing RFP or GFP fluorescence during cortical divisions, 0–30-min
embryos were collected, aged for 1 h 15 min, dechorionated and fixed
and devitellinized as above. For PH3 immunostaining, embryos were
dechorionated, fixed in a 1:1 mixture of 4% formaldehyde (in PBS) and
heptane, and devitellinized as described above. For colchicine treatment,
0–1-h embryos were collected, dechorionated and incubated in 1:1
mixture of 250 mM colchicine (in PBS) and heptane for 30 min. Treated
embryos were then fixed with formaldehyde and devitellinized as above.
The SAC response in embryos was determined by comparing the
percentage of mitotic embryos following a 30 min incubation with or
without colchicine. ‘Mitotic’ embryos were defined as those having at
least 50% PH3-positive nuclei. For immunostaining, fixed embryos were
rehydrated in PBS and incubated in 1 mg/ml RNaseA for 1 h, then
incubated in primary antibody overnight at 4 ˚C, washed, then incubated
in Cy2- and/or Cy5-conjugated secondary antibodies (Jackson
ImmunoResearch) for 3 h at room temperature. Embryos were stained
with propidium iodide and mounted in clearing solution (Fenger et al.,
2000) or in Prolong-Gold with DAPI (Life Technologies). Embryos were
visualized with a Nikon Eclipse 80i microscope equipped with a
CoolSNAP ES camera (Photometrics). Statistical analysis of imaging
quantifications was performed using the Fisher Exact test. Primary
antibodies used were anti-a-Tubulin YL1/2 (AbD Serotec) or antiTubulin DM1a (Sigma-Aldrich), anti-Centrosomin (gift from William
Theurkauf, University of Massachusetts Medical School, Worcester,
MA), anti-PH3 (06-570, Millipore), anti-Rod (Scaërou et al., 1999), antiZw10 and anti-Zwilch (gifts from Michael Goldberg, Cornell University,
Ithaca, NY).
Embryo immunoblotting
0–1-h embryos were homogenized in non-denaturing lysis buffer
(NDLB) (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5 mM EDTA, 1%
Triton X-100). For immunoprecipitations, 500 mg of embryo lysate was
incubated with 5 ml anti-Rod serum (Scaërou et al., 1999) or normal
rabbit serum overnight at 4 ˚C. Lysates were then incubated with 25 ml of
Protein-G–Sepharose beads (GE Healthcare) for 3 h at 4 ˚C. Beads were
pelleted, washed three times in NDLB, and boiled in 30 ml 66 sample
buffer. The resulting supernatant and 20 mg of input lysate were analyzed
by SDS-PAGE and by immunoblotting using standard techniques.
Horseradish peroxidase (HRP)-conjugated secondary antibodies and
chemiluminescence were used to detect primary antibodies.
For sucrose gradient analysis, 2 mg of embryo lysate was layered on
top of a sucrose gradient column of 5%, 10%, 20% and 30% sucrose, then
centrifuged in a L8-70M ultracentrifuge (Beckman) with a SW55Ti rotor
for 4 h at 250,000 g and 4 ˚C. A total of 19 fractions were collected and
8% of each fraction was analyzed by SDS-PAGE and immunoblotting.
Journal of Cell Science (2015) 128, 1204–1216 doi:10.1242/jcs.165712
acquired with a Zeiss Axio Imager.Z1 equipped with a Plan-Apo 1006
NA 1.4 oil objective and an apotome module. Acquisition times were
400 ms for Alexa Fluor 594 and 300 ms for Alexa Fluor 488 and images
were stacks of five planes at 1-mm intervals. Fluorescence was quantified
using ImageJ.
For determination of aneuploidy rates and to assay the SAC response,
third-instar larval brains were fixed and stained in aceto-orcein following
incubation in colchicine (1024 M) for 5, 30 or 60 min, and the mitotic
density (the average number of mitotic figures per microscope field with
at least one mitosis) was determined as described previously (Buffin
et al., 2007).
In vivo observation of larval neuroblasts
Larval brains were prepared as described previously (Emre et al., 2011).
Fluorescence time-lapse videos were acquired with an Olympus IX-70
inverted microscope, a focused xenon lamp and an OrcaER camera
(Hammamatsu), piloted by the Cell-R hardware and software system
(Olympus). Acquisition times per frame were 100 ms for GFP–Zw10 and
150 ms for RFP–Rod, and images were obtained as stacks of five planes
at 1-mm intervals taken every 15 s with a 606 NA 1.4 oil objective.
FRAP analysis
Neuroblasts from third instar larval brains were treated 15 min with
colchicine at 1024 M. FRAP experiments were performed using a
confocal microscopy system (Zeiss Axiovert with LSM780) equipped
with a Plan-Apo 636 NA 1.4 oil objective (during experiments with
Mad1–GFP, Spc25–GFP and GFP–Rod) or a Plan-Apo 406 NA 1.3 oil
objective (during experiments with GFP–Zw10). Imaging was controlled
by Zeiss confocal software (Zen 2012). A circular area of 0.9 mm (GFP–
Zw10) or 4 mm (Mad1–GFP, Spc25–GFP) diameter was bleached with
the 488 nm and 514 nm lines of an argon laser at 25 mW. Images were
acquired with a temporal resolution of 1 s (for Mad1–GFP) or 5 seconds
(for Spc25–GFP and GFP–Zw10) with a GaAsP detector (490–650 nm).
Fluorescence intensity in a region of interest was quantified using
ImageJ. The exponential kinetics of FRAP was analyzed by nonlinear
regression fitting using Origin software.
Assay for perdurance of maternally supplied Rod
To test the hypothesis that perdurance of a small amount of WT Rod
(protein or mRNA), initially loaded into the cytoplasm of the egg laid by
a rodZ3/+ heterozygous mother, might be responsible for the relatively
mild phenotype of homozygous rodZ3 larvae, we assayed for GFP–Rod+
of strictly maternal origin in third-instar larval brains. From the cross of y
w; GFP-Rod/y+Cy; +/+ females to y w/Y males, phenotypically y+ larvae
should not inherit the GFP–Rod transgene. Third-instar larval brains from
these y+ larvae were examined both by immunoblotting, probing with
anti-GFP antibody, and by fluorescence microscopy following colchicine
treatment, for evidence of GFP signal on kinetochores. Neither approach
detected a positive signal of GFP–Rod.
Acknowledgements
We thank the ImagoSeine facility, a member of France BioImaging infrastructure,
supported by the French National Research Agency [grant number ANR-10INSB-04, ‘Investments of the Future’].
Competing interests
The authors declare no competing or financial interests.
Author contributions
Neuroblast cytology
Immunostaining used the method described previously (Williams et al.,
2003). Drosophila third-instar larval brains expressing Spc25–RFP were
dissected in 0.7% NaCl and treated for 15 min in 1024 M colchicine.
After formaldehyde fixation and methanol dehydration, brains were
immunostained with a mouse monoclonal anti-RFP (Abcam, ab658556;
1:500) and rabbit anti-Rod (1:1000) antibodies. Secondary antibodies
were Alexa-Fluor-594-conjugated anti-mouse-IgG and Alexa-Fluor-488conjugated anti-rabbit-IgG (1:500; Life Technologies). Images were
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All authors contributed to the conception and design of experiments, analysis of
data, and manuscript preparation. Experiments were performed principally by L.D.
and S.G.H. A.M. contributed to the initial analysis of the rodZ3 zygotic phenotype.
Funding
This work was supported by the National Institutes of Health [grant numbers
GM074044 to L.A.L., GM008554 to S.G.H.]. L.D. was supported by fellowships
from the French ‘Ministère Français de l’Enseignement Supérieur et de la
Recherche’, and the Association pour la Recherche sur le Cancer. The R.K.
laboratory was supported by the Centre National de la Recherche Scientifique,
Journal of Cell Science
RESEARCH ARTICLE
and La Ligue National Contre le Cancer ‘Equipe Labellisée’ program 2012, and is
a member of the Laboratory of Excellence program No. ANR-11-LABX-0071/
Investments for the Future program No. ANR-11-IDEX-0005-01. Deposited in
PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.165712/-/DC1
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