Genetics: Published Articles Ahead of Print, published on October 26, 2010 as 10.1534/genetics.110.119628
1
Drosophila Mis12 complex acts as a single functional unit
essential for anaphase chromosome movement and a
robust spindle assembly checkpoint
Zsolt Venkei, Marcin R. Przewloka, David M. Glover*
Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom
Running title: mis12 and nsl1 kinetochore mutants
*Author for correspondence:
David M. Glover
Department of Genetics
University of Cambridge
Downing Street
Cambridge, UK
CB2 3EH
Tel: +44 (0)1223 333986
Fax: +44 (0)1223 333992
E-mail: [email protected]
Key words: BubR1, kinetochore, Mis12/MIND, Nsl1, spindle assembly checkpoint
2
The kinetochore is a dynamic multiprotein complex assembled at the centromere in
mitosis. Exactly how the structure of the kinetochore changes during mitosis and how its
individual components contribute to chromosome segregation is largely unknown. Here
we have focused on the contribution of the Mis12 complex to kinetochore assembly and
function throughout mitosis in Drosophila. We show that despite the sequential
kinetochore recruitment of Mis12 complex subunits Mis12 and Nsl1, the complex acts as
a single functional unit. mis12 and nsl1 mutants show strikingly similar developmental
and mitotic defects in which chromosomes are able to congress at metaphase, but their
anaphase movement is strongly affected. While kinetochore association of Ndc80
absolutely depends on both Mis12 and Nsl1, BubR1 localization shows only partial
dependency. In the presence of residual centromeric BubR1 the checkpoint still
responds to microtubule depolymerisation but is significantly weaker. These
observations point to a complexity of the checkpoint response that may reflect subpopulations of BubR1 associated with residual kinetochore components, the core
centromere, or elsewhere in the cell. Importantly our results indicate that core
structural elements of the inner plate of the kinetochore have a greater contribution to
faithful chromosome segregation in anaphase than in earlier stages of mitosis.
Introduction
Accurate segregation of sister chromatids in mitosis is crucial for viability and normal fate of
daughter cells. Missegregation leads to aneuploidy, a phenomenon associated with
developmental defects and cancer (KOPS et al. 2005). Accurate segregation of chromosomes
requires function of kinetochores, multi-protein complexes assembled on the outer sides of
centromeres on each sister chromatid (ALLSHIRE and KARPEN 2008; WELBURN and
CHEESEMAN 2008). The molecular composition and functions of the kinetochore change
during mitosis. Kinetochore assembly begins with the accumulation of the Mis12 complex on
centromeres in late G2 (GOSHIMA et al. 2003; KLINE et al. 2006). This is joined by
Spc105 and the Ndc80 complex to form the stable ‘core kinetochore’ on mitotic entry
(CHEESEMAN and DESAI 2008). The core kinetochore provides a platform for kinetochore
associated proteins recruited after nuclear envelope break down (NEBD; SANTAGUIDA and
MUSACCHIO 2009). After NEBD, microtubules emanating from the two opposite poles of
the spindle, form transient connections with kinetochores (VOROZHKO et al. 2008). Once
sister kinetochores have made attachments to opposite poles, the bioriented chromosomes are
3
able to congress to the equator of the mitotic spindle in prometaphase. Proper chromosome
biorientation and congression is monitored by the spindle assembly checkpoint (SAC) that
recognizes unattached kinetochores (WELBURN and CHEESEMAN 2008), reduced inter
and/or intrakinetochore tension (ELOWE et al. 2007; MARESCA and SALMON 2009;
UCHIDA et al. 2009) and responds by delaying Anaphase Promoting Complex/Cyclosome
(APC/C) activation. Erroneous MT kinetochore connections are recognized, disassembled and
corrected through an Aurora B mediated mechanism (RUCHAUD et al. 2007). Once
alignment has been achieved at metaphase, securely bound k-fiber microtubules (MTs) enable
the core kinetochores to sustain two anaphase functions: to maintain stable connections
between segregating chromosomes and the k-fiber MTs and to provide docking sites for
proteins that contribute to MT plus end dynamics and for motor proteins required for
chromosome movement (CHEESEMAN et al. 2006; DONG et al. 2007).
The Mis12 complex is an inner sub-complex of the core kinetochore that in the majority of
organisms comprises four sub-units, Mis12, Dsn1, Nnf1 and Nsl1. In cultured human cells,
localization of these subunits to the kinetochore was found to be inter-dependent (KLINE et al.
2006). Partial depletion of any of the components by RNAi led to reduced Ndc80 complex
and Blinkin/Spc105 at the kinetochore and similar defects in chromosome biorientation and
segregation (KLINE et al. 2006). Cenp-E and BubR1 kinetochore localization were also
reduced and SAC activity was compromised. In contrast, depletion of the Dsn1 homologue of
Caenorhabditis elegans was found to give a more severe, ‘kinetochore null’, phenotype than
depletion of the other subunits: chromosomes failed to align to the metaphase plate and failure
of chromosome segregation was associated with premature spindle elongation and mislocalization of all other kinetochore components (DESAI et al. 2003). Depletion of the Mis12,
Nnf1 and Nsl1 orthologs led to much less severe chromosome segregation and spindle
elongation defects and reduced but did not prevent recruitment of other core kinetochore
components (CHEESEMAN et al. 2004). The differences in phenotypes after depletion of
Mis12 complex subunits may reflect incomplete knock-down, but may be also interpreted as
real differences between function of individual proteins in different species. Recent highresolution light microscopy studies (JOGLEKAR et al. 2009; SCHITTENHELM et al. 2007; WAN
et al. 2009) and low resolution structural studies on yeast and human Mis12 complexes
(MASKELL et al. 2010; PETROVIC et al. 2010) suggest linear, sequential arrangement of
subunits in the complex. However, together these data do not currently provide a consistent
view about the position or the function of individual Mis12 complex subunits in the
architecture of the kinetochore.
4
Drosophila has one mis12, one nsl1 gene and two nnf1 genes that encode isoforms with
different developmental expression patterns (SCHITTENHELM et al. 2007). Despite extensive
studies, a Dsn1 ortholog has not been identified (MERALDI et al. 2006; PRZEWLOKA et al.
2009; PRZEWLOKA et al. 2007; SCHITTENHELM et al. 2007). In contrast to the uniform
recruitment of Mis12 complex members in human cells, EGFP-tagged Mis12 and Nnf1a were
found to localize to centromeres of cultured cells in interphase and thus in advance of the
mitotic localization of Nsl1 and Nnf1b . In accord with this finding, kinetochore recruitment
of Drosophila Mis12 was independent of Nsl1 whereas Nsl1 recruitment required Mis12.
Knockdown of several centromeric (CID, CENP-C), Mis-12 complex (Mis12, Nsl1), or
Ndc80 complex (Nuf2, Ndc80, Spc25/Mitch) proteins led to similar phenotypes in cultured
cells: chromosome congression was impaired, anaphases showed chromosome missegregation and bridging, and spindles became markedly elongated (PRZEWLOKA et al.
2007).
Here we wished to clarify whether the apparent differences in organization and recruitment
of Mis12 complex components of between Drosophila tissue culture cells and other
metazoans had any significance in the whole organism. To this end we have investigated the
phenotypes of mutants for two representative genes of the Mis12 complex, mis12 and nsl1.
We confirm that their gene products are differentially localized to the kinetochore in the
embryonic cell cycles. Nevertheless, mis12 and nsl1 mutants show identical developmental
and mitotic defects in flies. Interestingly, chromosome congression can occur following
depletion of these molecules in the mutants. Residual Mis12 and Nsl1 molecules on
kinetochores are sufficient to perform the function required for the chromosome congression.
However, having a functional kinetochore and the correct stoichiometry of Mis12 complex
subunits appear to be essential for correct anaphase chromosome movement. Despite
kinetochore disruption, we found residual BubR1 still present in centromeric regions and the
spindle assembly checkpoint, although weakened, remained functional.
Results and Discussion
Drosophila Mis12 complex proteins, Mis12 and Nsl1, differ in the dynamics of their
recruitment to centromeres
We wished to determine whether the differences in timing of recruitment of EGFP-tagged
Mis12 or Nsl1 (PRZEWLOKA et al. 2007) or their endogenous counterparts (Fig. S1 and S2)
5
to the kinetochore in cultured Dmel-2 cells were also seen in cells in the intact organism. We
therefore constructed transgenic lines of flies expressing EGFP-tagged Mis12 or Nsl1 in order
to follow the dynamics of recruitment of these proteins to the kinetochore. We found that in
both the nuclear division cycles of the syncytium (Fig. 1A; Supp movies 1,3) or in the mitotic
domains of the cellularized embryo (Fig. 1B; Supp Movies 2,4), EGFP-Mis12, but not EGFPNsl1, was present at the centromere in interphase. The signal intensity of EGFP-Mis12 at the
centromere was however, weaker in interphase than in mitosis when it appeared to be
recruited to the kinetochore immediately before nuclear envelope break down. In contrast,
Nsl1 was first recruited to the kinetochore in prophase. In this aspect the timing of Nsl1
recruitment was more similar to the Ndc80 complex subunit Nuf2 than to Mis12 (Fig. 1; Supp
Movies 5, 6). Thus in Drosophila, association of a population of Mis12 with the centromere
in interphase appears to anticipate the assembly of the Mis12 complex on mitotic entry. This
is quite different from the weak localization of all members of the Mis12 complex to the
centromere during interphase and the wave of their recruitment just before mitosis in human
cells (GOSHIMA et al. 2003; HEMMERICH et al. 2008; KLINE et al. 2006; MCAINSH et
al. 2006). This could reflect either differences in organization of the Mis12 complex between
insects and mammals or the increase in centromere component complexity in mammals
(reviewed in PRZEWLOKA and GLOVER 2009).
mis12 and nsl1 mutants display embryonic lethality
To gain insight into the in vivo functions of these two Mis12 complex subunits in Drosophila
we have characterised mutant alleles of their genes. The mis12PBac{WH}f03756 allele has a
transposon insertion in the first intron of mis12 and was lethal when homozygous or when
placed over two different deficiency chromosomes, Df(3L)BSC27 or Df(3L)BSC224, that each
uncover only a narrow cytological region on the third chromosome. 95% of
mis12PBac{WH}f03756 homozygotes and 98% of mis12PBac{WH}f03756/Df(3L)BSC224 individuals
died during embryogenesis with the hatched mutant larvae dying uniformly at first larval
instar (L1). The nsl1P{lacW}G0237 allele has a P- transposon inserted into the second exon of nsl1
(after codon 87) and was lethal in homozygous and in nsl1P{lacW}G0237/Y hemizygous
combinations and when placed against a 50kb deficiency, Df(1)52, that removes the entire
nsl1 gene (MELLER and RATTNER 2002). Additionally Dp(1:Y)Bs v+ y+ [a translocation
including the nsl1 genomic region, (MELLER and RATTNER 2002)] completely rescued the
lethality of the nsl1P{lacW}G0237/Df(1)52 allele combination. We found that 65% of
nsl1P{lacW}G0237/– individuals died as embryos and 35% reached the L1 larval stage. The
6
hatched nsl1 mutant larvae were strongly retarded in movement and all of them died in the
first instar (n=400). Together, these data indicate that the mis12PBac{WH}f03756 and
nsl1P{lacW}G0237 alleles are strong hypomorphic or potential null alleles of mis12 and nsl1.
This was confirmed by our failure to detect Mis12 or Nsl1 proteins either by Western blotting
or immunostaining in the late mitotic cycles of embryonic development (Fig. S3).
Neither nsl1 nor mis12 mutant embryos displayed any major gross defects in their
morphological patterning at late stages (Bowne’s stages 15-17; data not shown). However
they did have body areas with lower cell densities in which cells displayed a high number of
micronuclei and/or presumptive polyploid nuclei, indicative of accumulated defects in
chromosome segregation from previous mitotic divisions (Figure 2A). In fixed mutant
embryos there were no detectable mitotic defects up to mitosis 14 indicating that their
maternal proteins can drive mitosis until this stage. Throughout cell division cycle 15 and 16
the size and shape of mitotic spindles in fixed mis12 and nsl1 mutant embryos did not differ
significantly from wild type (Supplementary Figure S4). We could detect no signs of any
chromosome condensation or congressional defects in the fixed (tubulin-, CID- and DNAstained) embryo preparations in mitoses 15 and 16 (data not shown). On the other hand there
were a high number of chromosome segregation defects in anaphase and telophase (Figure
2B) indicating a requirement of mis12 and nsl1 for the proper segregation of mitotic
chromosomes. At a stage where only a few CNS restricted cells of wild-type embryos perform
one last mitosis before hatching, both the mis12 and nsl1 mutants had an increased number of
cells in mitosis (see also below). Many of these cells were dispersed throughout the whole
body suggesting they represented mitotically delayed cells from earlier cycles.
The developmental consequences of Mis12 or Nsl1 depletions are not only very similar to
each other but also and to those in mutants of centromeric components cid (BLOWER et al.
2006), cenp-c (HEEGER et al. 2005) and cal1 (ERHARDT et al. 2008 and see below) or the
core kinetochore component Spc105 (SCHITTENHELM et al. 2009). In contrast,
hypomorphic and null mutants for Ndc80 complex subunit, mitch (WILLIAMS et al. 2007)
and nuf2 (our unpublished data) die only later in late larval stages. This could either reflect
prolonged maternal contributions of Ndc80 complex components or a greater ability to
tolerate the absence of the Ndc80 complex than the Mis12 complex from the kinetochore in
embryonic cycles. Further experimentation will be required to distinguish between these two
possibilities. The uniformity of spindle length between mis12 and nsl1 mutant and wild-type
cells contrasts with the dramatic increase in spindle length upon knock-down of these genes in
cultured cells. This may either represent different properties of the spindles per se between
7
these different cell types or physical constraints upon cell size within tissues of the organism
that are irrelevant for cultured cells. The depletion of other mitotic regulators, the Greatwall
kinase for example, also leads to spindle elongation in tissue culture cells (BETTENCOURTDIAS et al. 2004) but not in the larval CNS (ARCHAMBAULT et al. 2007; YU et al. 2004).
mis12 and nsl1 mutants show prometaphase delay and segregation defects
To understand more of the nature of the mitotic defects in mis12 and nsl1 hemizygous
embryos we carried out time-lapse imaging studies of mitoses 15 and 16 in which
chromosome segregation defects lead to formation of aneuploid daughter cells (Fig. 3, Supp.
Movies 9-11). We observed similar mitotic defects in both the mis12 and nsl1 mutants and
classified them into three broad categories. In the first category were cells that showed a
pronounced delay in anaphase onset (AO; Fig. 3B, C). Nevertheless, chromosomes did
congress to give a metaphase-like plate in this group and segregation then appeared to take
place normally at anaphase. The second, and major, category comprised cells that also
showed chromosome segregation defects (Fig. 3B, C). In the example of such a cell given in
Fig. 3B, the major chromosomes have remained at the equator of the cell where they have
eventually undergone decondensation upon mitotic exit. In contrast, the tiny fourth
chromosomes segregate to the poles. Indeed, we consistently observed cells in which smaller
chromosomes (4th and sex chromosomes) arriving at the spindle poles sooner than the larger
ones (2nd and 3rd). We found chromosome decondensation not accelerated or delayed in these
cells. The chromosomes seemed to decondense with the same timing as in wild type cells
(compare Figure 3A and panels 1’ and 2’ from B). Since many of sister chromatids in the
mutants were not fully detached as they started to decondense, the entangling of decondensing
chromosomes most probably strongly contributed to the formation of telophase bridges.
The third category comprised cells in which mitotic chromosomes congressed to the equator
of the cell and then remained there in a metaphase-like arrest (Fig. 3B, D).
The ability of chromosomes to congress in the great majority of mis12 and nsl1 mutant cells
indicates that full kinetochore function is not required and that redundant mechanisms are
being used for this process. Of these, chromosome movement utilizing chromokinesin
function seems likely to be most significant (AFSHAR et al. 1995). Although several outer
kinetochore components, supposedly missing from kinetochores of mis12 and nsl1 mutants
(e.g. Ndc80, see below) have been shown to be required for chromosome congression in
human cells, this process can occur in the absence of k-fibers (CAI et al. 2009). The fact that
chromosomes congress in the mis12 and nsl1 mutants may either reflect sufficiency of
8
alternative mechanisms for this process in Drosophila compared to vertebrates (see KOPS et
al. 2010 for review) or the incomplete depletion of Mis12 and Nsl12 in mutant embryos.
Perhaps even a low amount of residual Mis12 and Nsl1 on kinetochores is sufficient to
perform the function required for the chromosome congression. However, a functional
kinetochore and the correct stoichiometry of Mis12 complex subunits appear to be essential
for correct anaphase chromosome movement. The uniform rate of chromosome movement in
a normal anaphase may reflect the equivalent density of kinetochore fiber microtubules that
has been observed by electron microscopy for all chromosomes (MAIATO et al. 2006). It
may be that the ability of some chromosomes to move polewards at anaphase in the mis12 and
nsl1 mutants may reflect some residual ability of microtubules to connect to kinetochore
remnants that could be sufficient to move the smaller, but not the larger, chromosomes.
mis12 and nsl1 mutants fail to recruit Ndc80 complex but still have BubR1 at
centromeres
To uncover the extent to which kinetochore organization was disrupted in mis12 and nsl1
mutants we examined the localization of the Ndc80 protein, a key component of the Ndc80
complex that links the Mis12 complex to kinetochore fiber MTs. In addition to providing the
attachment site for kinetochore microtubules, the Ndc80 complex is also crucial for
recruitment of certain of the SAC proteins including the RZZ complex and Mad2 (DELUCA
et al. 2003; LIN et al. 2006; MARTIN-LLUESMA et al. 2002). We were unable to detect
Ndc80 at kinetochores in both mis12 and nsl1 mutant stage-14 embryos (Fig. 4). This accords
with the high levels of chromosome segregation defects observed. In contrast, the centromeric
localization of BubR1 was only partially diminished in mis12 or nsl1 mutant cells (Fig. 4 C,
D) and is similar to spc105 mutants, as previously reported (SCHITTENHELM et al. 2009).
When we examined cal1 mutant embryos, in which the centromere does not form, as shown
by the absence of CID (Cal1 protein is involved in the CENP-A/CID loading onto
centromeric chromatin; ERHARDT et al, 2008), we were unable to detect BubR1 on
chromosomes (Fig. 4 C). In human cells the kinetochore localization of BubR1 seems to be
dependent on Spc105 (KIYOMITSU et al. 2007) and such an interaction is also possible in
Drosophila. Our studies of mis12, nsl1 and cal1 mutants as well as studies of spc105, and cid
mutants published by other groups (BLOWER et al. 2006,(SCHITTENHELM et al. 2009)
suggest that BubR1 may have at least two different binding sites: one on the centromere, the
other one on the kinetochore. Indeed two different localizations of BubR1, at inner and outer
kinetochore, have in fact been previously reported (JABLONSKI et al. 1998; reviewed in
9
HUANG and YEN 2009). We are at present unable to account for the mechanism of the
kinetochore independent centromeric localization of BubR1. Additionally, it is still necessary
to explain how BubR1 localization is restricted in space to the centromeric region of
chromosomes and in time to the prophase to metaphase interval. The fact that BubR1 is not
detectable at the centromeres of cid (BLOWER et al. 2006) and cal1 mutants suggests that
these two proteins might be essential for BubR1 localization at the centromere. BubR1 was
also recently described to be bound to DNA tethers that have been observed to form between
centric and acentric chromosome fragments of broken chromosome arms (ROYOU et al.
2010) and at unprotected telomeres (MUSARO et al. 2008). Understanding BubR1’s partners
on DNA tethers, uncapped telomeres, centromeres and kinetochores is an important challenge
requiring further investigation.
The extended time between NEBD and AO seen in mis12 and nsl1 mutants (Fig. 3C)
suggests that the SAC is able to induce mitotic delay in response to aberrant kinetochore
function. If indeed the checkpoint were functional, then the checkpoint response might be
enhanced following disruption of microtubules. To test this we treated both wild-type and
mutant embryos for 1h with colcemid at stage 14 (Fig. S5) and determined the effect upon the
mitotic index (Fig. 5). In both wild-type and mutant embryos the number of mitotic cells
increased significantly after MT disruption (Fig. 5), indicating the presence of an active SAC.
However the increase in mis12 and nsl1 mutant embryos was less than in wild-type
suggesting the SAC might be weakened as a result of reduced BubR1 levels at kinetochores.
Furthermore in cal1 and cid mutants, that fail to assemble the centromere and are unable to
recruit detectable amounts of BubR1 (Fig. 4C, BLOWER et al. 2006), the mitotic index was
less prominent, but still significantly increased after drug treatment (Fig. 5, BLOWER et al.
2006). Together, these data indicate that the SAC is able to work through pathways that do
not require BubR1 to be robustly located at the centromere. Irrespective of whether BuR1 is
present at the centromere in reduced amounts, following disruption of the Mis12 complex, or
absent, in cal1 and CID mutants, an increased number of cells arrest in mitosis following
destabilization of microtubules.
The facts that in mis12 and nsl1 mutants the SAC response to MT depolymerisation is
reduced and that this correlates with reduction of BubR1 at the centromere, suggest that both
centromeric and the kinetochore associated populations of BubR1 contribute to the robustness
of the SAC. It has previously been described that the SAC was unaffected in the absence of
centromere and kinetochore formation in cid null mutants (BLOWER et al. 2006). Our
finding of a SAC response to MT depolymerisation in cal1 mutants, where CID is also lost
10
from centromeres, is in accord with this earlier report. However, we also observe a weakening
of SAC activity in the cal1 mutants suggesting the possibility of a CID independent
involvement of Cal1 in SAC activity. This adds even greater complexity to the previous
conclusion that the SAC could function independently of the association of its component
molecules with the centromere (BLOWER et al. 2006). In addition to its role in the SAC,
BubR1 has been proposed to have additional functions to establish proper connections
between the kinetochore and microtubules and also as part of the basal timer of the interval
between prophase and anaphase onset (RAHMANI et al. 2009). Our data cannot address such
roles although we might speculate that a kinetochore associated sub-population of BubR1
might participate more in the k-fiber MT capturing function
In conclusion, we have demonstrated that despite the sequential recruitment of its subunits to
the kinetochore, the Drosophila Mis12 complex acts as a single functional unit in mitosis.
mis12 and nsl1 mutants show strikingly similar developmental and mitotic defects in flies and
confirm the necessity of the intact Mis12 complex in multiple kinetochore functions, of that
mediating anaphase chromosome movement is of principal importance for these particular
cell types. Finally, our findings reveal multiple levels of association of BubR1 with
chromosomes that are likely to affect the multiple functions of this checkpoint protein.
Materials and methods
cDNAs and constructs
Full length cDNA clones for Mis12 (RE19545), Nuf2 (SD05495), Ndc80 (LD33040) and
Nsl1 (RE03006) were ordered from the Drosophila Genomics Resource Centre (DGRC).
Constructs used in this study were generated by the Gateway Cloning System (Invitrogen).
Entry clones for C and N terminal protein fusions were generated for all of the cDNAs listed
above. Constructs for bacterial protein expression and for transgenic flies were made by LR
reactions between the entry clones and the following destination vectors: pDEST42 (for
expression in Escherichia coli with C-terminal 6xHis fusion, Invitrogen), pKM596 [for
expression in E. coli with N-terminal MBP fusion, (FOX et al. 2003)], pDEST12 and
pDEST13 (for UASp promoter driven expression of N-terminal and C-terminal EGFP fusions
in transgenic flies, constructed by Frederik Wirtz-Peitz in our laboratory).
Fly stocks
11
The Drosophila mutant stocks nsl1P{lacW}G0237 (PETER et al. 2002), mis12PBac{WH}f03756
(THIBAULT et al. 2004) and the Df(3L)BSC27, Df(3L)BSC224 deficiencies were obtained
from the Bloomington stock center. Stocks for Df(1)52 and Dp(1:Y)Bs- v+ y+ (MELLER and
RATTNER 2002) were kindly provided by Victoria Meller. We used GFP balancer
chromosomes to distinguish between homozygous or hemizygous mutants from sibling
progeny derived from heterozygous parents. For embryonic stages 9-12, we used the balancer
chromosomes FM7c, P{GAL4-Kr.C}DC1, P{UAS-GFP.S65T}DC5 or TM3, P{GAL4Kr.C}DC2, P{UAS-GFP.S65T}DC10, Sb1 (CASSO et al. 2000). In late embryos and in larval
stages we used the FM7i, P{Act-GFP}JMR3 or TM3, P{w[+mC]=Act-GFP}JMR2, Ser
balancer chromosomes (FERRANDON et al. 1998).
To investigate the function of mis12 and nsl1 in mitotic chromosome segregation in living
animals mutant alleles were combined with a second chromosome bearing the P{His2AvDEGFP.C} insertion (CREST et al. 2007). To investigate the cal1 phenotype in flies
cal1PBac{PB}cal1c03789 and Df(3R)Exel6176 were combined.
Transgenic strains expressing kinetochore proteins fused to EGFP were generated by
standard P-element-mediated germ line transformation and by selection based on the use of
mini-white dominant marker gene. Functionality of Mis12::EGFP and Nsl1::EGFP transgenes
were confirmed by rescue experiments. Features of the transgenes are summarized in
supplementary Table S1. To express fusion proteins in the early embryo for the purpose of
live imaging we used the α-tubulin4-GAL4-VP16 driver (DUFFY 2002). Single copies of the
driver and the UASp transgenes were combined in female flies by standard crosses. The
animals were raised on standard food and kept on at 25°C.
Cell culture and RNAi experiments
The Dmel-2 cell line (Invitrogen) was cultured in Drosophila Express Five SFM medium
(Invitrogen) supplemented with Pen/Strep and L-Glutamine at 25 °C according to standard
protocols. dsRNAs for mis12, nsl1 and ndc80 were synthesized and RNAi treatments were
carried out as described previously (PRZEWLOKA et al. 2007).
Antibodies and immunofluorescence microscopy
Polyclonal antibodies were generated in rabbits against bacterially expressed Ndc80-6xHis,
Nsl1-6xHis, MBP-Mis12 and against the mixture of two Mis12 peptides:
DFNSLAYDQKFFNF and CKLKQFWNQVPLQIKKETD; IgGs were purified from the sera.
12
An anti-MBP-Mis12 antibody was generated in rabbits and serum was preabsorbed to MBP
before using it in Western blot experiments.
For immunofluorescence imaging cultured cells were fixed and stained as described before
(BETTENCOURT-DIAS et al. 2004). Blocking and staining with primary and secondary
antibodies was carried out as described previously (SULLIVAN et al. 2000). Briefly,
embryos of the requested age were collected, dechorionated by incubation in 50% bleach for 2
min, rinsed in water and immobilized on coverslips. Immobilized embryos were covered by a
drop of fixation buffer (as used for cultured cells). The vitelline membranes were
permeabilized mechanically by a glass needle at the ends of the embryos. The fixative was
allowed to diffuse into the embryos for 15 min. Fixed embryos were manually removed from
the vitelline membrane, rinsed twice in PBS and once in PBT.. Primary rabbit antibodies were
used in 1:500 dilutions and were against: Mis12 peptide, Nsl1, Ndc80, and Ser10-Phosphohistone H3 (Abcam) and BubR1 (generated in our laboratory). A mouse monoclonal αtubulin DM1A (SIGMA) was used at a 1:1000 dilution. Chicken anti-CID antibody
(generated in our laboratory) was used at a 1:3000 dilution. For Westerns anti-MBP-Mis12
and anti-Ndc80 antibodies were used at 1:2000 dilutions.
Drug treatment of embryos
mis12, nsl1 mutant and wild type stage 14 embryos dechorionated using 50% domestic
bleach. The vitelline membrane of the embryos was permeabilized by brief octane treatment
(SU et al. 1999). Subsequently embryos were incubated for one hour in in Drosophila
Express Five SFM medium (Invitrogen) supplied with 25µM Colcemid or with equal volume
of DMSO. After drug treatment embryos were fixed and processed for immunofluorescence
as described above.
Live imaging
To characterize mitotic defects in mis12 and nsl1 mutants, eggs were collected from fertilized
Drosophila females having the P{His2AvD-EGFP.C} insertion on the second chromosome
and nsl1P{lacW}G0237/FM7i, ftz-lacZ or mis12PBac{WH}f03756/ TM3, ftz-lacZ genotype and aged for
180-210 min. Partner males had the P{His2AvD-EGFP.C} insertion on the second
chromosome and Y/FM7i, ftz-lacZ or Df(3L)BSC224/TM3, ftz-lacZ chromosome
combinations respectively. Staining for lacZ activity enabled fixed mutant and wild type
embryos to be distinguished after live imaging. Embryos were dechorionated, aligned and
immobilized on coverslips. After covering embryos with halocarbon oil, spinning disc
13
confocal laser microscopy was carried out on an inverted Zeiss Axiovert 200 microscope
equipped with Perkin Elmer UltraVIEW RS system . A Zeiss 63x/1.4 oil immersion objective
was used for time-lapse imaging of GFP fluorescence signals. GFP signals in the embryonic
cortex were filmed through mitoses 14-16. Z-stacks of 25 images (stack step 0.5 µm) were
acquired every 20 sec. Experiments were carried out at 24°C in a temperature-controlled
room.
Acknowledgements
We would like to thank members of our laboratory for helpful discussions. This work was
supported by a project grant from the BBSRC.
References
AFSHAR, K., N. R. BARTON, R. S. HAWLEY and L. S. GOLDSTEIN, 1995 DNA binding
and meiotic chromosomal localization of the Drosophila nod kinesin-like protein. Cell
81: 129-138.
ALLSHIRE, R. C., and G. H. KARPEN, 2008 Epigenetic regulation of centromeric
chromatin: old dogs, new tricks? Nat Rev Genet 9: 923-937.
ARCHAMBAULT, V., X. ZHAO, H. WHITE-COOPER, A. T. CARPENTER and D. M.
GLOVER, 2007 Mutations in Drosophila Greatwall/Scant reveal its roles in mitosis
and meiosis and interdependence with Polo kinase. PLoS Genet 3: e200.
BETTENCOURT-DIAS, M., R. GIET, R. SINKA, A. MAZUMDAR, W. G. LOCK et al.,
2004 Genome-wide survey of protein kinases required for cell cycle progression.
Nature 432: 980-987.
BLOWER, M. D., T. DAIGLE, T. KAUFMAN and G. H. KARPEN, 2006 Drosophila
CENP-A mutations cause a BubR1-dependent early mitotic delay without normal
localization of kinetochore components. PLoS Genet 2: e110.
CAI, S., C. B. O'CONNELL, A. KHODJAKOV and C. E. WALCZAK, 2009 Chromosome
congression in the absence of kinetochore fibres. Nat Cell Biol 11: 832-838.
CASSO, D., F. RAMIREZ-WEBER and T. B. KORNBERG, 2000 GFP-tagged balancer
chromosomes for Drosophila melanogaster. Mech Dev 91: 451-454.
CHEESEMAN, I. M., J. S. CHAPPIE, E. M. WILSON-KUBALEK and A. DESAI, 2006 The
conserved KMN network constitutes the core microtubule-binding site of the
kinetochore. Cell 127: 983-997.
CHEESEMAN, I. M., and A. DESAI, 2008 Molecular architecture of the kinetochoremicrotubule interface. Nat Rev Mol Cell Biol 9: 33-46.
CHEESEMAN, I. M., S. NIESSEN, S. ANDERSON, F. HYNDMAN, J. R. YATES, 3RD et
al., 2004 A conserved protein network controls assembly of the outer kinetochore and
its ability to sustain tension. Genes Dev 18: 2255-2268.
CREST, J., N. OXNARD, J. Y. JI and G. SCHUBIGER, 2007 Onset of the DNA replication
checkpoint in the early Drosophila embryo. Genetics 175: 567-584.
14
DELUCA, J. G., B. J. HOWELL, J. C. CANMAN, J. M. HICKEY, G. FANG et al., 2003
Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2
to kinetochores. Curr Biol 13: 2103-2109.
DESAI, A., S. RYBINA, T. MULLER-REICHERT, A. SHEVCHENKO, A. HYMAN et al.,
2003 KNL-1 directs assembly of the microtubule-binding interface of the kinetochore
in C. elegans. Genes Dev 17: 2421-2435.
DONG, Y., K. J. VANDEN BELDT, X. MENG, A. KHODJAKOV and B. F. MCEWEN,
2007 The outer plate in vertebrate kinetochores is a flexible network with multiple
microtubule interactions. Nat Cell Biol 9: 516-522.
DUFFY, J. B., 2002 GAL4 system in Drosophila: a fly geneticist's Swiss army knife. Genesis
34: 1-15.
ELOWE, S., S. HUMMER, A. ULDSCHMID, X. LI and E. A. NIGG, 2007 Tension-sensitive
Plk1 phosphorylation on BubR1 regulates the stability of kinetochore microtubule
interactions. Genes Dev 21: 2205-2219.
ERHARDT, S., B. G. MELLONE, C. M. BETTS, W. ZHANG, G. H. KARPEN et al., 2008
Genome-wide analysis reveals a cell cycle-dependent mechanism controlling
centromere propagation. J Cell Biol 183: 805-818.
FERRANDON, D., A. C. JUNG, M. CRIQUI, B. LEMAITRE, S. UTTENWEILER-JOSEPH
et al., 1998 A drosomycin-GFP reporter transgene reveals a local immune response in
Drosophila that is not dependent on the Toll pathway. EMBO J 17: 1217-1227.
FOX, J. D., K. M. ROUTZAHN, M. H. BUCHER and D. S. WAUGH, 2003 Maltodextrinbinding proteins from diverse bacteria and archaea are potent solubility enhancers.
FEBS Lett 537: 53-57.
GOSHIMA, G., T. KIYOMITSU, K. YODA and M. YANAGIDA, 2003 Human centromere
chromatin protein hMis12, essential for equal segregation, is independent of CENP-A
loading pathway. J Cell Biol 160: 25-39.
HEEGER, S., O. LEISMANN, R. SCHITTENHELM, O. SCHRAIDT, S. HEIDMANN et al.,
2005 Genetic interactions of separase regulatory subunits reveal the diverged
Drosophila Cenp-C homolog. Genes Dev 19: 2041-2053.
HEMMERICH, P., S. WEIDTKAMP-PETERS, C. HOISCHEN, L. SCHMIEDEBERG, I.
ERLIANDRI et al., 2008 Dynamics of inner kinetochore assembly and maintenance
in living cells. J Cell Biol 180: 1101-1114.
JOGLEKAR, A. P., K. BLOOM and E. D. SALMON, 2009 In vivo protein architecture of the
eukaryotic kinetochore with nanometer scale accuracy. Curr Biol 19: 694-699.
KLINE, S. L., I. M. CHEESEMAN, T. HORI, T. FUKAGAWA and A. DESAI, 2006 The
human Mis12 complex is required for kinetochore assembly and proper chromosome
segregation. J Cell Biol 173: 9-17.
KOPS, G. J., A. T. SAURIN and P. MERALDI, 2010 Finding the middle ground: how
kinetochores power chromosome congression. Cell Mol Life Sci. DOI:
10.1007/s00018-010-0321-y
KOPS, G. J., B. A. WEAVER and D. W. CLEVELAND, 2005 On the road to cancer:
aneuploidy and the mitotic checkpoint. Nat Rev Cancer 5: 773-785.
LIN, Y. T., Y. CHEN, G. WU and W. H. LEE, 2006 Hec1 sequentially recruits Zwint-1 and
ZW10 to kinetochores for faithful chromosome segregation and spindle checkpoint
control. Oncogene 25: 6901-6914.
MAIATO, H., P. J. HERGERT, S. MOUTINHO-PEREIRA, Y. DONG, K. J.
VANDENBELDT et al., 2006 The ultrastructure of the kinetochore and kinetochore
fiber in Drosophila somatic cells. Chromosoma 115: 469-480.
15
MARESCA, T. J., and E. D. SALMON, 2009 Intrakinetochore stretch is associated with
changes in kinetochore phosphorylation and spindle assembly checkpoint activity. J
Cell Biol 184: 373-381.
MARTIN-LLUESMA, S., V. M. STUCKE and E. A. NIGG, 2002 Role of Hec1 in spindle
checkpoint signaling and kinetochore recruitment of Mad1/Mad2. Science 297: 22672270.
MASKELL, D. P., X. W. HU and M. R. SINGLETON, 2010 Molecular architecture and assembly
of the yeast kinetochore MIND complex. J Cell Biol 190: 823-834.
MCAINSH, A. D., P. MERALDI, V. M. DRAVIAM, A. TOSO and P. K. SORGER, 2006
The human kinetochore proteins Nnf1R and Mcm21R are required for accurate
chromosome segregation. EMBO J 25: 4033-4049.
MELLER, V. H., and B. P. RATTNER, 2002 The roX genes encode redundant male-specific
lethal transcripts required for targeting of the MSL complex. EMBO J 21: 1084-1091.
MERALDI, P., A. D. MCAINSH, E. RHEINBAY and P. K. SORGER, 2006 Phylogenetic
and structural analysis of centromeric DNA and kinetochore proteins. Genome Biol 7:
R23.
JOGLEKAR, A. P., K. BLOOM and E. D. SALMON, 2009 In vivo protein architecture of the eukaryotic kinetochore
with nanometer scale accuracy. Curr Biol 19: 694-699.
KLINE, S. L., I. M. CHEESEMAN, T. HORI, T. FUKAGAWA and A. DESAI, 2006 The human Mis12 complex is
required for kinetochore assembly and proper chromosome segregation. J Cell Biol 173: 9-17.
MASKELL, D. P., X. W. HU and M. R. SINGLETON, 2010 Molecular architecture and assembly of the yeast
kinetochore MIND complex. J Cell Biol 190: 823-834.
MUSARO, M., L. CIAPPONI, B. FASULO, M. GATTI and G. CENCI, 2008 Unprotected Drosophila melanogaster
telomeres activate the spindle assembly checkpoint. Nat Genet 40: 362-366.
PETROVIC, A., S. PASQUALATO, P. DUBE, V. KRENN, S. SANTAGUIDA et al., 2010 The MIS12 complex is a
protein interaction hub for outer kinetochore assembly. J Cell Biol 190: 835-852.
SCHITTENHELM, R. B., R. CHALECKIS and C. F. LEHNER, 2009 Intrakinetochore localization and essential
functional domains of Drosophila Spc105. EMBO J.
SCHITTENHELM, R. B., S. HEEGER, F. ALTHOFF, A. WALTER, S. HEIDMANN et al., 2007 Spatial organization of a
ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes. Chromosoma 116:
385-402.
WAN, X., R. P. O'QUINN, H. L. PIERCE, A. P. JOGLEKAR, W. E. GALL et al., 2009 Protein architecture of the
human kinetochore microtubule attachment site. Cell 137: 672-684.
PETER, A., P. SCHOTTLER, M. WERNER, N. BEINERT, G. DOWE et al., 2002 Mapping
and identification of essential gene functions on the X chromosome of Drosophila.
EMBO Rep 3: 34-38.
PETROVIC, A., S. PASQUALATO, P. DUBE, V. KRENN, S. SANTAGUIDA et al., 2010 The MIS12
complex is a protein interaction hub for outer kinetochore assembly. J Cell Biol 190:
835-852.
PRZEWLOKA, M. R., and D. M. GLOVER, 2009 The kinetochore and the centromere: a
working long distance relationship. Annu Rev Genet 43: 439-465.
PRZEWLOKA, M. R., Z. VENKEI and D. M. GLOVER, 2009 Searching for Drosophila
Dsn1 kinetochore protein. Cell Cycle 8: 1292-1293.
PRZEWLOKA, M. R., W. ZHANG, P. COSTA, V. ARCHAMBAULT, P. P. D'AVINO et
al., 2007 Molecular analysis of core kinetochore composition and assembly in
Drosophila melanogaster. PLoS ONE 2: e478.
RAHMANI, Z., M. E. GAGOU, C. LEFEBVRE, D. EMRE and R. E. KARESS, 2009
Separating the spindle, checkpoint, and timer functions of BubR1. J Cell Biol 187:
597-605.
ROYOU, A., M. E. GAGOU, R. KARESS and W. SULLIVAN, 2010 BubR1- and Polocoated DNA tethers facilitate poleward segregation of acentric chromatids. Cell 140:
235-245.
16
RUCHAUD, S., M. CARMENA and W. C. EARNSHAW, 2007 Chromosomal passengers:
conducting cell division. Nat Rev Mol Cell Biol 8: 798-812.
SANTAGUIDA, S., and A. MUSACCHIO, 2009 The life and miracles of kinetochores.
EMBO J 28: 2511-2531.
SCHITTENHELM, R. B., R. CHALECKIS and C. F. LEHNER, 2009 Intrakinetochore
localization and essential functional domains of Drosophila Spc105. EMBO J 28:
2374-2386.
SCHITTENHELM, R. B., S. HEEGER, F. ALTHOFF, A. WALTER, S. HEIDMANN et al.,
2007 Spatial organization of a ubiquitous eukaryotic kinetochore protein network in
Drosophila chromosomes. Chromosoma 116: 385-402.
SU, T. T., S. D. CAMPBELL and P. H. O'FARRELL, 1999 Drosophila grapes/CHK1 mutants
are defective in cyclin proteolysis and coordination of mitotic events. Curr Biol 9:
919-922.
SULLIVAN, W., ASHBURNER, M. and R. S. HAWLEY, 2000 Drosophila Protocols
Cold Spring Harbor Laboratory Press, New York
THIBAULT, S. T., M. A. SINGER, W. Y. MIYAZAKI, B. MILASH, N. A. DOMPE et al.,
2004 A complementary transposon tool kit for Drosophila melanogaster using P and
piggyBac. Nat Genet 36: 283-287.
UCHIDA, K. S., K. TAKAGAKI, K. KUMADA, Y. HIRAYAMA, T. NODA et al., 2009
Kinetochore stretching inactivates the spindle assembly checkpoint. J Cell Biol 184:
383-390.
VOROZHKO, V. V., M. J. EMANUELE, M. J. KALLIO, P. T. STUKENBERG and G. J.
GORBSKY, 2008 Multiple mechanisms of chromosome movement in vertebrate cells
mediated through the Ndc80 complex and dynein/dynactin. Chromosoma 117: 169179.
WAN, X., R. P. O'QUINN, H. L. PIERCE, A. P. JOGLEKAR, W. E. GALL et al., 2009 Protein
architecture of the human kinetochore microtubule attachment site. Cell 137: 672-684.
WELBURN, J. P., and I. M. CHEESEMAN, 2008 Toward a molecular structure of the
eukaryotic kinetochore. Dev Cell 15: 645-655.
WILLIAMS, B., G. LEUNG, H. MAIATO, A. WONG, Z. LI et al., 2007 Mitch a rapidly
evolving component of the Ndc80 kinetochore complex required for correct
chromosome segregation in Drosophila. J Cell Sci 120: 3522-3533.
YU, J., S. L. FLEMING, B. WILLIAMS, E. V. WILLIAMS, Z. LI et al., 2004 Greatwall
kinase: a nuclear protein required for proper chromosome condensation and mitotic
progression in Drosophila. J Cell Biol 164: 487-492.
Figure legends
FIGURE 1.— Localization of EGFP tagged Mis12 and Ndc80 complex proteins in living
transgenic embryos. (A) In syncytial blastoderm a subpopulation of Mis12::EGFP is
constantly present on centromeres. In contrast, Nsl1 does not localize to interphase
centromeres and EGFP::Nuf2 is excluded from interphase nuclei. In mitosis all the three
17
fusion proteins localize identically to kinetochores. (B) Following cellularization these three
proteins show similar subcellular localization to syncytial blastoderm: Mis12 is present
throughout the cell cycle but increases in levels at the kinetochore during mitosis; Nsl1 and
Nuf2 only associate with chromosomes at kinetochores during mitosis. Microtubules were
detected by injected rhodamine conjugated tubulin. Examples of single mitotic cells are
surrounded by intact lines, single interphase cells are surrounded by dotted lines. Scale bars
represent 10 µm.
FIGURE 2.— Cellularized embryos of mis12 and nsl1 mutants show similar mitotic
defects (A) Distribution and morphology of nuclei (stained with DAPI to reveal DNA) in
mis12 and nsl1 mutant embryos. Areas showing a lower nuclear density (*), high numbers of
micronuclei (arrowheads), or large, presumably polyploid nuclei (arrows) are characteristic
for both mutants. (B) Wild-type, mis12 and nsl1 mutant embryos stained to reveal β-tubulin
(red), CID (green) or DNA (blue). Lagging chromosomes and bridges were seen in anaphase
cells of the mis12 and nsl1 mutants in mitosis 15 and 16. Scale bars represent 10 µm.
FIGURE 3.— Progression of mitosis 15 in living mis12 and nsl1 mutant embryos. (A)
Congression and segregation of His2AvD::EGFP-marked chromosomes in wild-type embryos
at stage 9. (B) Three examples of congression and segregation of His2AvD::EGFP-marked
chromosomes in mis12 mutant embryos. In the majority of mutant cells the breadth of the
congressed metaphase plate did not differ from wild-type cells. Segregation defects were
more severe for the larger chromosomes ( 2nd and 3rd). The smaller chromosomes (4th and sex
chromosomes) were less effected (series 1’ time point 2:00). (C) Prolonged time between
NEBD and AO is a characteristic of both mutants. This time interval was measured in 8 nuclei
from 2 wild-type embryos and for 12 or 15 nuclei from 3 embryos of mis12 and nsl1 mutants.
Error bars represent ±1 SD . (D) Categories of chromosome segregation phenotypes in wildtype, mis12,and nsl1 mutant embryos. n=80 nuclei from 3 embryos for wild type; 150 nuclei
from 6 embryos for mis12 mutants; and 150 nuclei from 5 embryos for nsl1 mutants.
FIGURE 4.— Recruitment dependencies of Nsl1 (A), Ndc80 (B), and BubR1(C and D) in
mis12 and nsl1 mutant stage 14 embryos. Green staining indicates the Nsl1, Ndc80 and
BubR1 proteins relative to CID (red) and DNA (blue). Localization of BubR1 in cal1 mutant
is also shown for comparison in. (D) Error bars represent ±1 SD .
18
FIGURE 5.— SAC response to microtubule depolymerisation in stage 14 embryos. (A)
Distribution of mitotic cells in fixed embryos stained to reveal DNA (blue) and Ser 10phospho-histone H3 (red; PH3) after treatment for 1h with DMSO or Colcemid. (B) Numbers
of mitoses per embryo in wild-type, mis12, nsl1 and cal1 mutant stage 14 embryos following
treatment of embryos of the indicated genotypes with DMSO or Colcemid. 10 embryos scored
for each measurement. Error bars represent ±1 SD. Number of mitotic cells significantly
increased in the presence of Colcemid for all genotype categories (two-tailed independent ttest, P<0.001).
1
FIGURE 1.
Mitosis 12
A
Metaphase
Interphase
+Tubulin
EGFP::Nuf2
Nsl1::EGFP
Mis12::EGFP
+Tubulin
B
EGFP::Nuf2
Nsl1::EGFP
Mis12::EGFP
Mitosis 14
Tubulin
Merged
2
FIGURE 2.
A
PBac{WH}f03756
Wild type
mis12
H
P{lacW}G0237
nsl1
/–
*
*
*
α-tubulin
mis12
PBac{WH}f03756
/– nsl1
P{lacW}G0237
/–
Wild type
B
CID
DNA
Merge
d
/–
3
FIGURE 3.
0:20
0:40
1:00
1:20
2:00
2:40
3:00
3:40
6:00
10
8
6
4
2
0
0:40
1:00
1:20
2’
3:00
3:40
6:00
0:00
0:20
0:40
1:00
1:20
100
Wild type
mis12PBac{WH}f03756/–
80
H
nsl1P{lacW}G0237/–
60
40
20
2:40
3:00
3:40
25:00
Metaphase
arrest
2:00
Missegregation
3’
0
Wild type like
anaphase
PBac{WH}f03756
2:40
mis12
2:00
Rations of phenotype categories (%)
0:20
/–
0:00
mis12
1’
D
/–
0:00
12
P{lacW}G0237
6:00
C
nsl1
3:40
1:20
/–
3:00
1:00
WT
B
2:40
0:40
PBac{WH}f03756
Wild type
2:00
0:20
Time from NEBD to AO (min)
0:00
A
cal1
/–
/–
P{lacW}G0237
nsl1
0
/–
C
/–
P{lacW}G0237
nsl1
/–
P{lacW}G0237
nsl1
/–
/–
PBac{WH}f03756
mis12
PBac{WH}f03756
mis12
Wild type
Wild type
Ndc80
mis12
Wild type
A
BubR1 signal intensity at the kinetochore
/–
BubR1
Nsl1 CID DNA
Wild type
PBac{WH}f03756
/– mis12
P{lacW}G0237
nsl1
Nsl1
PBac{WH}f03756
PBac{PB}cal1c03789
4
FIGURE 4.
Ndc80 CID DNA
B
BubR1 CID DNA
D
4000
3000
2000
1000
5
FIGURE 5.
A
P{lacW}G0237
Wild type
nsl1
PBac{WH}f03756
/–
mis12
/–
cal1
PBac{PB}cal1c03789
/–
PH3 DNA
PH3
+ DMSO
PH3 DNA
PH3
+ 25µM Colcemid
Number of mitotic cells per embryo
B
+ DMSO
+ 25µM Colcemid
600
Δ=145 (+29%)
Δ=111
(+22%)
Δ=166 (+48%)
400
Δ=188 (+97%)
200
0
Wild type
P{lacW}G0237
nsl1
/–
PBac{WH}f03756
mis12
/–
cal1
PBac{PB}cal1c03789
/–