Copyright ? 2010 by the Genetics Society of America
Functional Redundancy of Paralogs of an Anaphase Promoting
Complex/Cyclosome Subunit in Caenorhabditis elegans Meiosis
Kathryn K. Stein, Jessica E. Nesmith,1Benjamin D. Ross2and Andy Golden3
Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892
Manuscript received September 22, 2010
Accepted for publication October 11, 2010
The anaphase promoting complex/cyclosome (APC/C) mediates the metaphase-to-anaphase transition
by instructing the ubiquitination and turnover of key proteins at this stage of the cell cycle. We have
recovered a gain-of-function allele in an APC5 subunit of the anaphase promoting complex/cyclosome.
This finding led us to investigate further the role of APC5 in Caenorhabditis elegans, which contains two
APC5 paralogs. We have shown that these two paralogs, such-1 and gfi-3, are coexpressed in the germline
but have nonoverlapping expression patterns in other tissues. Depletion of such-1 or gfi-3 alone does not
have a notable effect on the meiotic divisions; however, codepletion of these two factors results in meiotic
arrest. In sum, the two C. elegans APC5 paralogs have a redundant function during the meiotic divisions.
.10 subunits, which is required for progression
through the cell cycle (Peters 2006). An activating
subunit associates transiently with the APC/C in a cell
cycle-dependent manner to positively regulate APC/C
activity: CDC20/fzy-1 at M phase and CDH1/fzr-1 during
the G1 phase of the cell cycle (Pesin and Orr-Weaver
2008). During meiosis and mitosis, the ubiquitination
of substrates by the APC/C results in protein turnover,
which drives these processes. One such APC/C target is
securin. Upon degradation of securin, separase, an en-
and promotes the metaphase-to-anaphase transition.
The timing of APC/C activation during M phase
must be carefully regulated because precocious or aber-
rant segregation of chromosomes can result in aneu-
ploidy of daughter cells, leading to embryonic death or
uncontrolled cell proliferation. At metaphase, the
APC/C is quiescent while the chromosomes are cap-
on the metaphase plate. At this time, the APC/C is held
in check by the components of the spindle assembly
checkpoint (SAC). The SAC regulates the activity of the
APC/C, depending on the state of chromosome attach-
ments (Musacchio and Salmon 2007). When the kinet-
HE anaphase promoting complex or cyclosome
(APC/C) is an E3 ubiquitin ligase composed of
ochores are unattached, the SAC holds the APC/C in the
inactive state by the inhibition of the positive APC/C
regulator, CDC20/FZY-1. Once the chromosomes are
properly aligned and oriented, the SAC releases CDC20
so that the APC/C is activated to ubiquitinate the appro-
priate substrates. The components of the SAC were first
discovered in Saccharomyces cerevisiae and the core pro-
teins are functionally conserved in multicellular organ-
isms (Musacchio and Salmon 2007).
In Caenorhabditis elegans, most of the APC/C subunits
are required for cell division. In homozygous null
mutants segregating from a heterozygous hermaphro-
postembryonic development; cell divisions in the late
larvae fail and the terminal phenotype of null mutants
is sterility due to the absence of germline proliferation
(Furuta et al. 2000). Temperature-sensitive mutants of
five APC/C subunits have been isolated (Furuta et al.
2000; Golden et al. 2000). When these hypomorphic
mutants are shifted to restrictive temperature following
the completion of the mitotic divisions required for
germline establishment, a brood of one-cell embryos
arrested at metaphase of meiosis I is produced. These
results indicate that there is a requirement for the
Golden et al. 2000; Shakes et al. 2003). RNAi-mediated
knockdown of individual APC/C subunits in late larval
hermaphrodites also leads to one-cell meiotic arrest
with the exception of two subunits, APC5 and APC10
(Davis et al. 2002), which exhibit arrest at a multicel-
lular stage when depleted. It recently has been estab-
lished that there are two genes encoding each of these
subunits in C. elegans (Tarailo et al. 2007). Therefore,
APC5 and APC10 may not exhibit an RNAi meiotic
1Present address: Biological and Biomedical Sciences Program, Univer-
sity of North Carolina, Chapel Hill, NC 27599.
2Present address: Department of Molecular and Cellular Biology,
University of Washington, Basic Sciences Division, Fred Hutchinson
Cancer Research Center, Seattle, WA 98109.
3Corresponding author: 8 Center Dr., Bldg. 8, Room 323, LBG, NIDDK,
National Institutes of Health, Bethesda, MD 20892.
Genetics 186: 1285–1293 (December 2010)
arrest phenotype because the two paralogs of each
subunit might function redundantly at the meiotic
divisions. Alternatively, itis possible that thesesubunits
are not required at the meiotic divisions.
specific temperature-sensitive loss-of-function allele of
mat-3/APC8/CDC23, mat-3(or180ts), to discover meiotic
regulators of the APC/C in C. elegans (Stein et al. 2007).
Most ofthesuppressors cloned from that studyfunction
in the SAC pathway. The SAC mutants probably sup-
press mat-3(or180ts) by increasing the level of APC/C
activity via a reduction of the strength of the spindle
checkpoint. The interdependence of the regulation of
the SAC and the APC/C is demonstrated by reciprocal
studies in which a loss-of-function in the SAC can be
suppressed by reduction-of-function APC/C mutants
(Furuta et al. 2000; Tarailo et al. 2007). Further,
Bezler and Go ¨nczy (2010, accompanying article in
this issue) assert that proper mitotic timing is achieved
through the negative regulation of the SAC by the
APC/C. In this study, we identify the mat-3 suppres-
sor av9gf as a gain-of-function allele of one of the
APC5 paralogs, such-1. In addition, our studies with
the such-1(h1960ts) loss-of-function allele and APC5
RNAi have led us to conclude that the APC5 paralogs
such-1 and gfi-3 function redundantly during the mei-
MATERIALS AND METHODS
Genetic mapping of av9: av9 was isolated in an EMS-based
genetic screen as a suppressor of mat-3(or180ts) embryonic
lethality at the nonpermissive temperature of 24?. Genome-
wide snip-SNP mapping was performed to localize this mutant
to chromosome III as described previously (Stein et al. 2007).
Classical three-factor genetic mapping was performed in the
with mat-3(or180ts) av9; him-8(e1489) males. In the dpy-18 unc-
25 interval 15/17 Unc non-Dpys were suppressed and 3/27
Dpy non-Uncs were suppressed; these data predict a map
position of ?10.01 6 1.45 on LG III. The such-1 gene is lo-
cated at 10.99 on LG III (www.wormbase.org).
RNAi: Two such-1 RNAi feeding constructs were made.
They contained genomic DNA from (1) a 275-bp fragment
spanning parts of exon and intron 10 and (2) exons 6–8 with
intervening introns. RNAi feeding constructs were created
using the Gateway system (Invitrogen, Carlsbad, CA) and a
customized L4440 vector (Timmons and Fire 1998), pCR88,
then transformed into HT115(DE3) competent cells for use.
2003) was used for gfi-3 RNAi experiments (Geneservice,
Cambridge, UK). This RNAi clone included 907 bp spanning
a genomic region from exons 14–16 and intervening introns.
At 24?, L4 hermaphrodites were fed bacteria containing the
RNAi construct for 24–28 hr and then moved to a new RNAi
plate for another 20–24 hr. Hermaphrodites were then
removed. After another 24 hr the second RNAi plate was
scored. Embryos were assessed microscopically for one-cell
arrest and larvae were counted. To control for RNAi
treatment, animals were grown on smd-1 bacteria, which does
not produce a phenotype. Animals of the following geno-
types were tested: wild type, mat-3(or180ts), mat-3(or180ts)
such-1(av9), and such-1(h1960ts). such-1(h1960ts) was isolated
from the such-1(h1960ts); mdf-1(gk2) line generated in the
Sequencing: Genomic DNA of such-1 was amplified by PCR
from the mat-3(or180ts) av9 strain and sequenced by Macro-
gen (Rockville, MD). A mutation was discovered in exon 10
(G to A 8260 nucleotides downstream of the start site), which
created a Snip-SNP not present in wild type. The mutation
was confirmed by digestion of a PCR fragment with AvaI.
Alignments were done with the CLUSTAL 2.0.11 multiple
sequence alignment program (Larkin et al. 2007).
Brood size: For each experiment, 5 L4 hermaphrodites of
each genotype were placed on OP50 plates at 16? or 24? and
moved each day to a new plate until embryos were no longer
observed. Embryos were counted 24 hr after hermaphrodites
were removed from each plate and larvae were counted 24 hr
later. Brood size was calculated by totaling all larvae and
unhatched embryos and percentage of hatching was equal
to the number of hatched embryos divided by the brood size
and multiplied by 100.
Double mutant analysis: Genetic enhancement was evalu-
ated between the loss-of-function mutant, such-1(h1960ts),
and temperature-sensitive alleles of APC/C subunit mutants:
mat-3(or180ts) dpy-1(e1), emb-27(g48ts) unc-4(e120), and unc-
74(x19) mat-1(ax144ts), by mating such-1(h1960ts) males with
hermaphrodites of the marked lines. In the F2generation,
marked hermaphrodites were picked to single plates at 16?
and scored for sterility, maternal-effect lethality (Mel), or
production of a normal brood. In the case of emb-27(g48ts)
and mat-1(ax144ts), it was anticipated that 25% of the F2
would also be homozygous for the unmarked such-1(h1960ts)
mutation because the two genes reside on different chromo-
somes. Hermaphrodites that were sterile or Mel were
genotyped for the presence of such-1(h1960ts), utilizing the
Snip-SNP created by the such-1(h1960ts) mutation and the
SgrA1 enzyme. Because mat-3 and such-1 are both on LG III,
were picked from each plate to detect an F2recombinant
animal by the presence of F3sterile animals or F4embryonic
lethality at the permissive temperature. Animals were geno-
typed to confirm the presence of such-1(h1960ts).
Most of the double mutants were generated with marked or
unmarked alleles of APC/C subunits and dpy-18(e364) such-
1(av9). The following strains marked with a morphological
marker werecombinedwithanunmarkedsuch-1(av9gf) allele:
unc-74(x19) mat-1(ax144ts), mat-1(ax212ts) unc-13(e51), and
mat-3(ax148ts) dpy-1(e1). In all cases, control strains contain
the morphological marker but not the test allele. In each
experiment, at least 400 embryos were evaluated from the
experimental and control double mutants. Double mutants
were shifted to the restrictive temperature at the L4 stage for
24 h, and then were removed. Embryos and larvae were
counted the next day and embryos were closely examined
with a Nikon SMZ-2B dissecting microscope to assess the stage
of arrest, one-cell or multicellular. Percentage of hatching was
calculated as described above (n ¼ 3). Double mutants be-
tween unc-17(e245) mdf-2(av14) or fzy-1(av15gf) unc-4(e120)
by molecular genotyping. Five animals were shifted to 24?
and brood size and hatching was determined for each
experiment. The Dpy Unc phenotype of these animals
sometimes led to a small brood and premature death of the
hermaphrodites, as a result of egg-laying defects. To account
for the early death of hermaphrodites in the brood size
calculations, total progeny for each day was calculated by
adding the number of embryos and larvae on the plate and
1286K. K. Stein et al.
dividing by the number of adults that were alive on that plate
at the beginning of the day to obtain a value of progeny per
animal for that day. The progeny per animal per day were
then summed for the course of the experiment to produce
the value of total brood per animal.
Transgenic animals: For both such-1 and gfi-3, the 59
promoter region (750 bp upstream of the translational start
site for such-1, 885 bp for gfi-3) and 39-UTR (739 bp
downstream of stop codon for such-1, 758 bp for gfi-3) were
amplified by PCR and entry clones were generated using
Gateway technology (Invitrogen). All entry clones were
sequence confirmed. These regulatory regions were then
recombined with mCherryTH2B coding sequence in a
destination vector containing a wild-type copy of unc-119.
unc-119(ed3) animals were bombarded by biolistic transfor-
mation (Wilm et al. 1999) using a Model PDS-1000/He
biolistic particle delivery system (BioRad, Hercules, CA) and
plated on fifteen 100-mm plates. Postbombardment, animals
were allowed to grow for 7 days at 24? until the plate was
starved. A pedestal of rescue (a small piece of agar seeded
with bacteria) was then introduced to each plate and after 48
hr pedestals were examined for wild-type (non-unc-119)
animals. Transformed animals contain the unc-119 rescuing
construct and are normally motile. Therefore, transformed
animals, but not Unc animals, are able to crawl up the piece
of agar and access the food, which allows them to reproduce
and also facilitates detection. Wild-type animals were picked
to single plates to determine which were stable trangenics
and to distinguish between integrated and extrachromo-
somal lines. Finally, animals were examined microscopically
to assess if any were expressing mCherryTH2B. Positive lines
were maintained and used for further studies. For such-1,
four lines were generated with an identical expression
pattern. One was integrated and the remainder expressed
extrachromosomal arrays. For gfi-3, three lines were gener-
ated with an identical expression pattern. Two were in-
tegrated and the other expressed an extrachromosomal
array. Live animals and embryos were examined using a
Nikon Eclipse E800 microscope equipped with a PerkinElmer
Ultraview LCI CSU10 scanning unit (PerkinElmer, Fremont,
CA) and an ORCA ER cooled CCD camera (Hamamatsu,
Japan). The objective was a 340 Nikon Plan Apo oil with a
numerical aperature of 1.0.
such-1(h1960ts)/Df RNAi experiments: To generate such-
1(h1960ts)/ctDf3 animals, such-1(h1960ts) males were mated
into ctDf3/qC1 dpy-19(e1259) glp-1(q339) hermaphrodites and
cross progeny were individually moved to RNAi or control
OP50 plates at 25?. Hermaphrodites were moved to new
RNAi plates after 24 hr and left on these plates for an
additional 24 hr. Unhatched embryos and larvae were
counted forthe 24-to 48-hrplateand percentage of hatching
was calculated. Plates segregating sterile Dpy animals were
judged to contain qC1 dpy-19(e1259) glp-1(q339) and were
used as a control. All other plates were such-1(h1960ts)/ctDf3.
A minimum of 25 single plates were assessed for such-1(exon
10) RNAi, gfi-3 RNAi, and smd-1 RNAi. Embryos were visually
examined to ascertain whether they were arrested at a
one-cell or multicellular stage.
av9 is a gain-of-function allele of the APC5 homolog,
such-1: av9 was isolated in a large-scale genetic screen
as a semidominant suppressor of the embryonic le-
thality of the temperature-sensitive APC8 subunit
mutant, mat-3(or180ts) (Stein et al. 2007). mat-3(or180ts)
is a maternal-effect lethal mutation that produces a
brood of one-cell embryos arrested at metaphase of
meiosis I when shifted to restrictive temperature. In the
and become fertile adults (Stein et al. 2007). av9 was
unc-25 using snip-SNP and classical genetics methods.
This region of LG III was examined for candidate genes
involved in cell cycle regulation or the spindle assembly
checkpoint. One candidate, Y66D12A.17, encodes such-
1 (suppressor of spindle checkpoint defect), a homolog
of the APC5 subunit. RNAi was used to test whether av9
was likely to be an allele of such-1. If av9 was a loss-of-
function allele of such-1, RNAi of such-1 would be
expected to restore viability to mat-3(or180ts) embryos
at the restrictive temperature. If av9 was a gain-of-
function allele of such-1, RNAi of such-1 would eliminate
the viability of mat-3(or180ts) av9 embryos at the re-
strictive temperature. Wild-type animals are viable on
such-1 RNAi (Figure 1). We found that knockdown
of such-1 by RNAi did not rescue the lethality of mat-
3(or180ts) at 24? but depletion of such-1 in a mat-
3(or180ts) av9 background eliminated the suppression
semidominant suppressor, av9, may contain a gain-of-
function mutation in the such-1 gene and will hereafter
be referred to as such-1(av9gf).
The such-1 exons were sequenced from the av9gf
strain and found to contain a point mutation that
results in a glutamic acid (E)-to-lysine (K)-amino-acid
change at residue 693. The APC5 homologs have few
regions of extended conservation between species and
only a single putative TPR domain has been reported
(Bentley et al. 2002). Thus, there does not appear to
be a conserved functional role for this amino acid
across species. C. elegans contains an additional ortho-
logof APC5, GFI-3 [GEI-4 (Four)-Interacting protein],
Figure 1.—such-1 RNAi eliminates the suppression of mat-
3(or180ts) by such-1(av9). Wild-type, mat-3(or180ts), and mat-
3(or180ts) such-1(av9)animalswere grownat 24?onsmd-1 control
or such-1(exons 6-8) RNAi and embryonic hatching was assessed.
1C indicates the presence of arrested one-cellembryos. Statistics,
Student’s T-test; asterisk indicates a P-value ,0.001. Error bars
indicate standard deviation.
APC5 in C. elegans Meiosis1287
which is 38% identical and 71% similar to SUCH-1 at
the amino acid level, including identity at the site that
is mutant in av9gf (for an amino acid alignment, see
Tarailo et al. 2007). All other model organisms
contain only a single copy of APC5. Identity between
each C. elegans paralog and the ortholog in human,
Drosophila, and S. cerevisiae is comparable, indicating
(Tarailo et al. 2007).
To investigate whether the such-1(av9gf) allele has a
phenotype on its own, we tested the viability of this
mutant in an otherwise wild-type background. When
compared to the wild-type strain, there were no signif-
icant differences between such-1(av9gf) alone and wild-
type animals with regard to brood size or embryonic
hatching (Figure 2,AandB).Therefore,whiletheav9gf
mutation is capable of significant suppression of mat-
3(or180ts),ithas nodeleterious effects onthe viability or
fecundity of the animal.
The such-1 gain-of-function allele may not reflect the
true function of this gene. Therefore, we examined the
such-1 loss-of-function phenotype. Two loss-of-function
alleles exist for such-1, a penetrant paternal-effect lethal
Go ¨nczy 2010) and such-1(h1960ts), a point mutant that
exhibits near-normal hatching at 20? but reduced
hatching and brood size at 25? (Tarailo et al. 2007).
such-1(h1960ts) embryos that fail to hatch arrest at a
range of embryonic stages, from rare one-cell embryos
to multicellular embryos. The absence of a complete
meiotic one-cell arrest in such-1(h1960ts) embryos at
the restrictive temperature raised the possibility that
APC5 does not serve as a canonical APC/C subunit in
C. elegans. Characteristically, the temperature-sensitive
APC/C mutants isolated to date enhance each other
when combined at the permissive temperature and
double mutants are sterile or produce broods of un-
hatched embryos (Shakes et al. 2003; our unpublished
observations). To address the possibility that such-
1(h1960ts) encodes a functional APC/C subunit, the
loss-of-function allele was combined with three repre-
sentative temperature-sensitive APC/C loss-of-function
mutants at permissive temperature and assayed for
viability. Double mutants between such-1(h1960ts) and
1(ax144ts) exhibited a sterile or maternal-effect lethal
phenotype (Table 1). This finding is consistent with a
role for such-1 in APC/C function. such-1(h1960ts) may
not exhibit a one-cell arrest because it is a weak
hypomorphic allele or alternatively, it has overlapping
function with the APC5 paralog, gfi-3.
such-1(av9gf) suppresses APC/C subunit mutations
but does not interact genetically with SAC component
mutations: We have shown previously that many of the
suppressors isolated in the mat-3(or180ts) suppressor
screen are capable of suppressing mutations in other
APC/C subunits (Stein et al. 2007). The majority of
these suppressors were spindle assembly checkpoint
genes, so it is thought that they suppress through a
of the APC/C itself, we wondered whether this sup-
pressor would be as promiscuous or whether suppres-
sion might be limited to mat-3(or180ts). To address
this hypothesis, we made double mutants of such-
1(av9gf) with a strong and weak allele of each of the
five APC/C subunits for which temperature-sensitive
alleles are available (Table 2) (Golden et al. 2000). In
addition, we generated a double mutant with emb-
1(hc62ts), an allele that also exhibits a one-cell–arrest
our unpublished observations). We found that such-
1(av9gf) was a suppressor of three weak APC/C alleles in
and emb-1(hc62ts) (Table 2). Notably, suppression of the
Figure 2.—such-1(av9gf) animals are fertile and healthy.
Wild-type and such-1(av9gf) animals were grown at 16? and
24?. Brood size (A) and percentage of embryonic hatching
(B) were evaluated and found to be not significantly diffe-
rent. Statistics, Student’s T-test; error bars indicate standard
The loss-of-function mutant, such-1(h1960ts), enhances
temperature-sensitive APC/C mutants
APC/C geneAPC/C subunit % P0sterility % Mel
Embryonic hatching of such-1(h1960ts) was evaluated alone
(wild type) or in genetic combination with other APC/C sub-
unit mutants at the permissive temperature, 16?. The animals
that were scored as maternal-effect lethal (Mel) laid broods
exclusively composed of dead embryos. NA, not applicable.
1288 K. K. Stein et al.
a small percentage of embryos were capable of hatching,
probably due to mitotic defects arising later in develop-
ment that were not suppressed by such-1(av9gf). These
results indicate that such-1(av9gf) suppression is not
limited to mat-3(or180ts) and, in addition, is not specific
for the MAT-3 subunit. Thus, such-1(av9gf) appears to
increase the activity of various mutant forms of the
We have shown that such-1(av9gf) does not confer an
apparent mutant phenotype in an otherwise wild-type
background (Figure 2). This observation indicates that
a gain-of-function mutation, which likely increases the
activity of the APC/C, on the basis of its ability to
suppress mat-3(or180ts), does not have detrimental
effects on the normal development and function of
the organism. However, if APC/C activity was elevated
even further, what would be the consequences for the
cell and organism? We tested this question in two ways.
First, we reduced the spindle checkpoint activity by
using a loss-of-function mutation in mdf-2. mdf-2 is
1999), which inhibits the APC/C by sequestering
of FZY-1, the positive regulator of the APC/C and
ortholog of CDC20, should be available to drive APC/C
activity. Previous studies have shown that mdf-2(av14) is
a strong suppressor of APC/C loss-of-function mutants
and also that its embryonic lethality is enhanced by
another SAC loss-of-function mutation in san-1/mdf-3,
the MAD3 ortholog (Nystulet al. 2003). We combined
mdf-2(av14) with such-1(av9gf) and examined these
double mutants for the presence of any enhanced
phenotype. We found that such-1 and mdf-2 do not
appear to have any genetic interaction (Figure 3, A and
B). We then tested whether increasing fzy-1 activity
directly through a fzy-1 gain-of-function mutation
would lead to overactivated APC/C when combined
with such-1(av9gf). However, the fzy-1(av15gf) allele,
which is healthy on its own (like av9gf), does not
exhibit a synthetic phenotype when in combination
with av9gf (Figure 3, C and D). Thus, the change in
APC/C activity provided by these two mutants either is
not detrimental to the cell or is insufficient to alter the
bulk of APC/C activity in an appreciable way.
C. elegans contains two APC5 paralogs: To date, C.
elegans is unique among model organisms, as it con-
tains two genes, such-1 and gfi-3, which are equally
APC5 genes in C. elegans? It is possible that these two
paralogs of APC5 have a redundant function in the
same tissue or cell type, serve different functions in the
same cell, or have the same function in different
tissues, such as the soma and the germline. To un-
derstand the role of APC5 in C. elegans, we first
determined the expression pattern of such-1 and gfi-3
and then further investigated the loss-of-function
phenotype of each gene to establish the processes for
which each gene is required.
We examined the expression pattern of such-1 and
generating transgenic animals. The fusion protein
mCherryThistone H2B was driven by the promoter
and 39-UTR of either such-1 or gfi-3. such-1pTmChT
H2BTsuch-1 39-UTR is expressed throughout the de-
veloping germline (Figure 4A) as well as in the meiotic
embryo (Figure 4B) and throughout embryogenesis
of the adult, including some head neurons and vulval
precursor cells (data not shown). gfi-3pTmCh:H2BTgfi-3
39-UTR was similarly expressed throughout the germ-
line, meiotically, and in all embryonic stages (Figure 4,
E–G). Only the gfi-3 transgene is observed in the soma
of the L1–L4 larval stages and is also expressed in the
gut cells of the adult animal (Figure 4E, data not
shown). In hermaphrodites, both such-1– and gfi-3–
driven transgene expression is prominent in mature
sperm stored in the spermatheca. We generated male
transgenics and found that these transgenes are ex-
pressed throughout the male germline (Figure 4, D and
H). such-1 and gfi-3 exhibit overlapping expression
patterns in the germline, but are expressed specifically
1 and gfi-3 have the opportunity to act redundantly
during germline development and meiosis.
such-1 and gfi-3 function redundantly during meiosis:
The expression data suggest that such-1 and gfi-3 could
be redundant during meiosis, explaining the lack of
meiotic arrest phenotypes observed with the such-
1(h1960ts) loss-of-function mutant. To test this, two
such-1 RNAi constructs were used, derived from geno-
mic DNA containing exons 6–8 or exon 10, to knock
such-1(av9gf) suppression is not specific to mat-3(or180ts)
Gene APC subunit % hatching
Five–10 hermaphrodites were shifted to the restrictive tem-
perature at the L4 stage of development. At least 400 embryos
were assayed for each double mutant. For each gene, the
strongest allele is listed first.
APC5 in C. elegans Meiosis1289
down the message levels in wild-type animals (Figure
5A). Wild-type animals fed bacteria expressing either
such-1 RNAi construct exhibited no embryonic lethality
(Figure 5B and data not shown). While the such-1 RNAi
was sufficiently effective to block the suppression of the
sensitized mat-3(or180ts) such-1(av9gf) strain (see Figure
1), such-1 RNAi of wild-type animals does not recapitu-
late the phenotype of the such-1(h1960ts) loss-of-function
Figure 3.—such-1(av9gf) does not genetically interact with the spindle checkpoint mutants mdf-2(av14) or fzy-1(av15gf). Double
mutants were made with dpy-18(e364) such-1(av9gf) and two spindle checkpoint mutants, unc-17(e245) mdf-2(av14) (A and B) and
fzy-1(av15gf) unc-4(e120) (C and D). Brood size per animal (A and C) and embryonic hatching (B and D) are not significantly
different between the control and experimental strain. Statistics, Student’s T-test; error bars indicate standard deviation.
Figure 4.—such-1 and gfi-3 transcriptional fusions are expressed predominantly in the adult germline and embryo. such-1T
mCherryTH2BTsuch-1 and gfi-3TmCherryTH2BTgfi-3 stable transgenes were imaged by fluorescence microscopy (A–H) and
DIC (B, C, F, and G). such-1 (A) and gfi-3 (E) adult hermaphrodites exhibit expression in the germline extending from the distal
tip (inset) to the ?1 oocyte (arrowhead, A and E). Sperm expression is observed in the spermathecae (arrow, A and E). Somatic
gut nuclei (asterisk, E) also express gfi-3. such-1 is expressed in meiotic (arrow, B) and mitotic embryos (C). gfi-3 is expressed
at all embryonic stages, including meiosis (arrow, F) and mitosis (G). Male germlines also express the such-1 (D) and gfi-3
(H) transgenes; mature sperm are indicated with an arrow. Bars, 10 mm.
1290 K. K. Stein et al.
allele (Figure 5B). To further reduce SUCH-1 levels,
such-1(h1960ts) loss-of-function animals were grown on
such-1 RNAi at the restrictive temperature of 25?.
Reduction of such-1 by RNAi reduces the viability of
such-1(h1960ts) mutants (Figure 5B), indicating that
such-1(h1960ts) is not null, consistent with its weak
phenotype. such-1(h1960ts) hermaphrodites grown on
either such-1 RNAi produce embryos with a significantly
reduced hatching rate, and one-cell–arrested embryos
are only rarely observed (Figures 5, B andC). To further
1(h1960ts) [such-1(h1960ts)/ctDf3] and grew them on
such-1 RNAi. At 25?, the hatching rate of untreated
such-1(h1960ts)/ctDf3 is 33%. When animals of this
genotype are grown on such-1(exon 10) RNAi, a 2.4%
hatching rate is observed; however, all of the such-1
RNAi-treated embryos are multicellular (data not
shown). These results indicate that a severe depletion
of the levels of such-1 is not sufficient to yield a meiotic
one-cellarrest typicallyobservedwith anAPC/C mutant
or RNAi of an APC/C subunit (Davis et al. 2002).
Because such-1 depletion does not produce a one-
cell arrest, we sought to deplete gfi-3 and such-1 in
combination to directly address the possible redun-
dancy of these two genes. There are no mutant alleles
available for gfi-3 and wild-type animals fed bacteria
expressing gfi-3 RNAi do not exhibit lethality (Figure
5B). When such-1 and gfi-3 RNAi is introduced as a mix,
wild-type animals show a slight increase in embryonic
lethality (Figure 5B). such-1(h1960ts) hermaphrodites
grown on gfi-3 RNAi produce embryos with a signifi-
cantly reduced hatching rate and a significant popula-
tion of one-cell embryos (Figure 5, B and C). Under
conditions where such-1 and gfi-3 are simultaneously
depleted by RNAi in combination with such-1(h1960ts)
loss-of-function, a brood composed almost exclusively of
that hatch before RNAi becomes fully effective become
and gfi-3 are redundant at the metaphase-to-anaphase
animals hemizygous for such-1(h1960ts) [such-1(h1960ts)/
ctDf3] on gfi-3 RNAi at 25? and found that hatching
is reduced to 0.7% (n ¼ 1659). Importantly, the embryos
laid on gfi-3 RNAi are almost all meiotic one-cell embryos,
in contrast to the such-1 RNAi-treated embryos, which
are multicellular. In sum, gfi-3 and such-1 are likely
redundantly required for the first meiotic division.
APC5 is required for viability in all organisms where
it has been studied to date. In the yeasts Schizosacchar-
omyces pombe and S. cerevisiae, null mutations in APC5
Figure 5.—such-1 and gfi-3 act redundantly in meiosis. (A) Schematic of such-1 and gfi-3 gene structure and position of genomic
fragments used in RNAi constructs (solid bars below gene structure). The scale bar to the bottom right of each gene structure
indicates 100 base pairs (bp). Total genomic length is to the right of the gene structure. (B) Percentage of embryonic hatching of
wild-type and such-1(h1960ts) animals grown on several different RNAi conditions at 25?. N . 750 for each sample. (C) Percentage
of dead embryos exhibiting a one-cell–arrest phenotype at 25?. N . 435 for each sample. Statistics, Student’s T-test; asterisk in-
dicates a significant difference between sample and smd-1 control, with a P-value ,0.05. Error bars indicate standard deviation.
APC5 in C. elegans Meiosis1291
exhibit the ‘‘cut’’ phenotype or arrest at metaphase,
typical of an APC/C subunit mutant in each species
(Yu et al. 1998; Zachariae et al. 1998; Ors et al. 2009).
In Drosophila, null alleles of the APC5 ortholog, ida,
result in larval lethality following the depletion of wild-
type maternal stores during embryogenesis. When ida
is manipulated so that expression is eliminated in the
germline, flies produce an extremely small brood,
which is 100% embryonic lethal (Bentley et al.
2002). However, ida mutant cells are not arrested in
metaphase, but rather seem to enter anaphase pre-
cociously, which generates aneuploid cells. The dispa-
rate phenotypes observed between organisms suggest
that APC5 may have divergent functions within the
APC/C complex. In this study, we demonstrate that C.
elegans has two APC5 subunits that are redundantly
required for meiosis. Although we have not been able
to generate a null for both APC5 subunits, a severe loss
of the function of both of these genes together leads to
meiotic metaphase arrest, suggesting that APC5 in C.
elegans operates as a classical APC/C subunit. Our
findings and the previous studies in fungi suggest that
thefunction of Drosophila idais divergent and that the
canonical role for APC5 is in the metaphase-to-anaphase
The two C. elegans APC5 paralogs, such-1 and gfi-3,
both function during the meiotic divisions. Depletion
of such-1 alone does not result in a penetrant one-cell–
arrest phenotype, although we occasionally observe a
few one-cell embryos from such-1(h1960ts) mothers at
the restrictive temperature. gfi-3 RNAi in combination
with a reduction of such-1 yields a brood almost ex-
clusively composed of meiotic one-cell embryos, which
further supports redundancy of thesegenes during the
meiotic divisions. Itisformallypossiblethat such-1 is, in
fact, the sole APC5 subunit required for the meiotic
divisions and that such-1 is not completely depleted
by the combination of the such-1(h1960ts) allele, the
deficiency chromosome and such-1 RNAi; however, we
consider this very unlikely.
The APC5 paralogs are likely not redundant at all
cell divisions. such-1(h1960ts) exhibits some embryonic
lethality, suggesting a nonredundant role for embryonic
viability. Additionally, the such-1(h1960ts) loss-of-function
allele was originally recovered as a suppressor of the
deletion allele of the spindle checkpoint gene, MAD1/
mdf-1 (Tarailo et al. 2007). If such-1 and gfi-3 were
completely redundant, this allele would not be antici-
pated to suppress the deletion mutant phenotype on
its own. It is likely that the two APC5 genes are partially
redundant and that such-1(h1960ts) reduces the activity
of the APC/C in a subset of somatic cells to allow
survival of the mdf-1 deletion strain. The recovery of
such-1(h1960ts) and the novel gain-of-function allele,
such-1(av9gf), underscores the utility of genetic screens
to isolate unique genetic alleles.
Why might there be two redundant APC5 genes in
Caenorhabditis? Although there is an overlap in
function at meiosis, our studies with transgenic ani-
mals indicate that larval and adult somatic expression
is not equivalent. For example, GFI-3 may play a more
prominent role during larval development, where it is
the primary APC5 paralog expressed. Alternatively, it is
possible that the activity of the APC/C containing
either subunit is appreciably different. For this reason,
one form of the APC/C may be preferable in certain cell
However, it remains to be determined whether there
elegans also has two paralogs of APC10. No comprehen-
these subunits is not known. It is possible that the two
APC10 subunits are also redundant at meiosis because
RNAi of one of the subunits does not produce a one-cell
meiotic-arrest phenotype (Davis et al. 2002). RNAi of
the second paralog has not yet been reported.
What is the possible mechanism of suppression in
mat-3(or180ts) such-1(av9gf) animals? Structural studies
have recently produced models of how the APC/C is
assembled, and these studies cast some light on the
mechanism of suppression. APC5 sits at the junction of
the two arms of the APC/C, one containing the target-
binding TPR domain-containing proteins and the
other the catalytic subunits (Thornton et al. 2006).
In that study, APC5 and CDC23/APC8/MAT-3 were
strongly suppress mat-3(or180ts) to viability may be
attributable to a compensatory modification of this
interaction in the two mutants. Mutations in two other
APC/C subunits, CDC27/APC3/mat-1 and APC4/emb-
30, and emb-1, are suppressed at the meiotic stage by
such-1(av9gf), but most embryosdo not hatch. Thismay
be due to subtle differences in strength and require-
ments of these alleles. mat-1(ax212ts) is defective in
postembryonic divisions; these animals are sterile if
shifted to the restrictive temperature at the L1 stage.
Perhaps such-1(av9gf) is not sufficient to suppress the
APC/C in this context. Additionally, while such-
1(av9gf) maysuppressthemeiotic divisions, the fidelity
of these divisions may be compromised and result in
multicellular embryonic lethality due to aneuploidy. It
is likely that the overall APC/C structure or assembly is
disrupted in the mat-3(or180ts) mutants and the av9gf
allele is able to compensate for the mat-3 mutant and
hold the complex together more effectively, which
might influence the ubiquitin ligase activity of APC/C.
Exploration of the mechanism of suppression awaits
future biochemical analysis of APC/C complexes bear-
ing this APC5 mutation.
We thank Paula Fearon for critical reading of the manuscript and
Risa Kitagawa for the such-1(h1960) allele. We also thank Kevin
O’Connell for the ‘‘pedestal of rescue’’ technique for recovering
transgenes from ballistic transformations as well as his useful sugges-
1292K. K. Stein et al.
tions for the manuscript. This research was supported by the Intra- Download full-text
mural Research Program of the National Institutes of Health (NIH),
National Institute of Diabetes and Digestive and Kidney Diseases.
Some nematode strains used in this work were provided by the
Caenorhabditis Genetics Center, which is funded by the NIH National
Center for Research Resources.
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The spindle-assembly check-
Regulation of APC/C
Suppressors of spin-
Specific interference by ingested
Communicating editor: D. I. Greenstein
APC5 in C. elegans Meiosis1293