MOLECULAR AND CELLULAR BIOLOGY, Mar. 2004, p.

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0270-7306/04/$08.000 DOI: 10.1128/MCB.24.6.22152225.2004
Copyright 2004, American Society for Microbiology. All Rights Reserved.

Vol. 24, No. 6

MINIREVIEW
Anaphase-Promoting Complex in Caenorhabditis elegans
Foong May Yeong*
Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore 117597, Singapore

* Mailing address: Department of Biochemistry, Faculty of Medicine, National University of Singapore, MD 7, #05-09, 8 Medical Dr.,
Singapore 117597, Singapore. Phone: (65) 6874 8866. Fax: (65) 6779
1453. E-mail: [email protected].
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WD-40 repeat proteins known as Cdc20/Fizzy and its homologue, Cdh1/Fizzy-related (reviewed in references 25, 50, 54
and 78). These activators have been found to be highly conserved across species (reviewed in references 25 and 54).
While the functions of each individual APC subunit remain
unclear, a comprehensive picture of the roles of the APC in
mitosis has emerged through several important studies in budding yeast. The APCCdc20 and APCHct1 complexes have essentially been shown to be required for two main transitions in
mitosis, metaphase-to-anaphase transition and exit from mitosis. In metaphase, sister chromatids are held together by a
cohesin complex presently known to consist of at least four
subunits, Smc1, Smc3, Scc1/Mcd1/Rad21, and Scc3 (reviewed
in reference 48) (Fig. 1). In order that sister chromatids separate, Scc1 has to be cleaved by separase, a cysteine protease
belonging to the CD clan (68, 69). Separase, however, is normally held inactive during metaphase through its association
with the anaphase inhibitor securin. APCCdc20 functions to
promote sister chromatid separation by ubiquitination of securin and in so doing causes its destruction (reviewed in reference 48) (Fig. 1). The liberated separase is then free to
cleave Scc1, leading to loss of sister chromatid cohesion,
thereby allowing chromatids to be pulled apart by the spindle
microtubules (Fig. 1). Cdc20 is the target of the spindle assembly checkpoint, which is activated in the presence of spindle
defects. The spindle assembly checkpoint components such as
Mad1, Mad2, Mad3, Bub1, Bub3, and Mps1 are involved in a
pathway that serves to sequester Cdc20, thereby preventing the
onset of anaphase, when spindle microtubules are disrupted
(reviewed in reference 77).
Homologues of the APC subunits, securin, separase, and
cohesin subunits and even the spindle assembly checkpoint
components have been found in different organisms (reviewed
in reference 48), a strong indication that the pathway leading
to sister chromatid separation is perhaps rather well conserved.
APCCdc20 also triggers the destruction of S and M phase cyclins to initiate exit from mitosis so that cells can enter a new
round of cell division. The initial lowering of the mitotic kinase
activities by APCCdc20 (3, 30, 73, 76) allows APCHct1 to be
activated by the mitotic exit network (MEN), comprising components such as Tem1 (GTPase), Cdc15 (kinase), Dbf2 (kinase), and Cdc14 (phosphatase) (reviewed in reference 46).
The MEN pathway is the target for another type of spindle
checkpoint, the spindle position checkpoint. This checkpoint
depends upon Bub2 and monitors the orientation and position
of the spindle apparatus. In the event that the spindle apparatus is misoriented, Bub2 acts through the MEN pathway to

Progression through critical events in cell division relies to a

large extent on the destruction of key effectors of the cell cycle.
An example can be seen in the regulation of the major cell
cycle effector, the cyclin-dependent kinases (CDKs). CDKs
depend upon the binding of their activators, the cyclins, to
drive specific events at particular stages in the cell division
cycle. As different cyclins are synthesized at different phases of
the cell cycle, the CDK, when associated with cyclins, essentially exhibits distinct activities in each phase of the cell cycle.
The destruction of cell cycle stage-specific cyclins therefore
plays an important role in limiting the activities of the CDKs at
the end of each stage so that the CDKs can associate with yet
other cyclins to drive progression of the cell cycle (reviewed in
reference 45).
Cyclins were initially discovered as proteins whose levels
oscillated in a highly specific manner in sea urchin Arbacia
punctulata embryos undergoing successive rounds of cell division (12). Initial hints that the cyclical nature of the cyclins is
important for the regulation of the cell cycle came from the
observation that destruction of mitotic cyclins, leading to a
decrease in mitotic kinase activities, is required for the exit
from mitosis (47). Further analyses led to the understanding
that ubiquitination is key to cyclin destruction (19, 28). An E3
ubiquitin-ligase known as the anaphase-promoting complex
(APC) was subsequently purified from extracts of the clam
Spisula solidissima (29, 64) and African clawed frog Xenopus
laevis (33) and shown to be responsible for the ubiquitination
of mitotic cyclins.
In the process of ubiquitination, the E3 ligase adds a chain
of ubiquitin, a 76-residue polypeptide, to proteins destined for
destruction. The proteins marked with a ubiquitin chain are
recognized as substrates by the 26S proteosome and duly destroyed (reviewed in references 26 and 70). Studies in the
budding yeast Saccharomyces cerevisiae revealed that mutants
of the APC subunits such as cdc16 and cdc23 were defective for
anaphase progression (31), indicating that the APC is required
for mediating destruction of regulators for metaphase to anaphase transition in addition to the ubiquitination of mitotic
cyclins. To date, the evolutionarily conserved APC (reviewed
in references 25 and 54) has been shown to consist of at least
11 subunits (Table 1), and its activity is cell cycle regulated
such that it is active in mitosis (reviewed in reference 50).
Activation of the APC activity depends upon the association of

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TABLE 1. Orthologues of APC subunits in C. elegans

Known APC subunitb

APC orthologue in C.
elegans (reference[s])

Function(s) in C. elegansa

emb-27/pod-6 (17, 56)

Progression through meiosis, a-p polarity

Cdc23 (TPR motif)

mat-3/pod-4 (9, 20, 56)

Progression through meiosis, mitosis, a-p polarity, affects

development (e.g., male tail)

Cdc27 (TPR motif)

mat-1/pod-5 (20, 56, 61)

Progression through meiosis, a-p polarity, affects

development (e.g., vulva and male tail)

Apc1 (Rpn1/2 homology)

mat-2/pod-3 (9, 20, 49)

Progression through meiosis, a-p polarity, affects

development of vulva and male tail

Apc2 (Cullin homology)

K06H7.6 (9)

Progression through meiosis

Apc4 (WD40 repeats)

emb-30 (17)

Progression through meiosis, mitosis, affects

development (e.g., vulva and male tail)

Apc5 (TPR motif)

M163.4 (9)

Mitosis

Apc-10

F15H10.3 (9)

Mitosis

Apc11

F35G12.9 (9)

Progression through meiosis

Cdc20 (WD40 repeats)

fzy-1 (36, 56)

Progression through meiosis, mitosis, a-p polarity

Cdh1 (WD40 repeats)

fzr-1 (14)

Mitosis, cyclin destruction, affects germ line proliferation

Cdc26
Doc-1
Ama-1

a-p, anterior-posterior.
b
TPR, tetratricopeptide repeat.

delay mitotic exit via Hct1 until the spindle apparatus is properly oriented (reviewed in reference 40).
It has also been shown in budding yeast that homologous
chromosome separation during meiosis takes place only in the
presence of a functional APC. As in sister chromatid segregation, the APC promotes the ubiquitination of securin in
meiosis I and II (59). Degradation of securin by the 26S proteosome frees separase (4, 69), which cleaves Rec8, a meiosisspecific cohesin subunit (37) that shows homology to Scc1 (see
above). During meiosis I, the Rec8 molecules localized to the
longitudinal arms are destroyed by separase, leading to homologous chromosome disjunction, while Rec8 in the proximity of
the centromeres is protected from proteolysis until meiosis II
(4, 37). The direct involvement of separase in meiosis II has yet
to be confirmed. The sequential destruction in meiosis of Rec8
homologues also occurs in other organisms, such as Schizosaccharomyces pombe (74), Mus musculus (39), and Arabidopsis
thaliana (5).
Insofar as our understanding of APC function in cell division
is concerned, the budding yeast has been instrumental in providing the initial insights. With the identification of homologues of the APC subunits and activators and the other components of chromosome separation and cohesion pathways
(reviewed in reference 48), it would be interesting to establish
the extent of conservation in the functions of the APC in
different organisms. Furthermore, the question now arises as
to whether the APC, which is essential in cell division, also

contributes to other processes in multicellular organisms. Indeed, recent reviews on the APC have alluded to the possibility
that the APC performs functions in cellular processes other
than mitosis and meiosis (25, 54). Support for additional roles
of the APC comes from studies showing that Hct1/Cdh1 and
APC subunits can be detected in postmitotic murine neuronal
cells (18) and that multiple copies of Hct1/Cdh1 genes exist in
chickens (72). Interestingly, however, work by various groups
on the APC in Caenorhabditis elegans in the past few years has
not only implicated APC subunits in meiosis and mitosis; more
significantly, the data indicate that the functions of the APC in
meiotic and mitotic divisions have important consequences in
the development of the worm (Table 1). This would suggest
that at least in C. elegans, additional roles of the APC, such as
in development, are a result of its contribution to proper cell
division.
Although C. elegans has not been the major organism of
choice for studying the APC, it has nonetheless lent itself as a
useful system for a better understanding of APC function for
several reasons. First, as the APC subunits, Cdc20/Fizzy and
Hct1/Fizzy-related have been found in C. elegans (Table 1),
comparisons can clearly be made to establish the similarities
and differences in APC function across different species. Second, an advantage of using C. elegans is the availability of
traditional genetic techniques which make the generation of
APC mutant worms for functional studies relatively easy. Also,
as a reverse genetics approach, the newly established double-

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Cdc16 (TPR motif)

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stranded RNA interference (dsRNAi) method provides a powerful and convenient tool for targeting genes in knockdown
experiments in C. elegans (15). Furthermore, the well-characterized behavior of the homologous chromosomes (five pairs of
autosomes and one pair of sex chromosomes in the hermaphrodite) in worm oocytes has been useful for examining meiotic
progression, while the single-celled embryo has characteristics
which allow cytological observations of mitotic division processes such as spindles, nucleus, and centrosomes by Nomarski
optics. Finally, with the complete documentation of the entire
cell lineage (65) and an anatomy that has been well studied
(reviewed in reference 38), C. elegans can potentially serve as
an important model for investigating the role of the APC in
development. This review takes a look at what is currently
known about the contributions of APC in cell division as well
as how the functions of the APC in cell division impinge upon
the development of C. elegans.
APC IN MEIOSIS
The evidence from budding yeast that the APC is essential
for progression through meiosis is clear. Nonetheless, despite
the conservation of APC subunits and cohesin components in
different organisms (reviewed in reference 48), whether there
is indeed a widespread requirement for the APC in chromosome segregation during meiosis is far from certain. In fact,
two studies have shown that the APC is dispensable for meiosis
in Xenopus laevis (53, 66), casting some doubt on the relevance
of extrapolating the data from budding yeast to higher organisms. In this regard, C. elegans has provided evidence showing

support for the data obtained with budding yeast that the APC
is indeed essential for meiotic progression.
The hermaphrodite reproductive system in C. elegans has
been a useful tool for the examination of both mitotic and
meiotic cell divisions. The hermaphrodite reproductive system
is a U-shaped structure, with one arm of the U extending
towards the anterior of the worm and the other towards the
posterior (Fig. 2A). Each arm serves as an ovary which ends off
with a spermatheca joined to a uterus (Fig. 2A). A vulval
opening in the middle of the uterus allows fertilized oocytes to
be laid. At the distal end of each arm, germ cells divide mitotically but transit from mitotic to meiotic divisions as they move
towards the proximal end near the spermatheca (Fig. 2B). The
mitotic proliferation of the germ cells is under the influence of
the distal tip cells (Fig. 2B and C), which provide signals via
molecules such as GLP-1 (for germ-line proliferation-defective; a transmembrane protein homologous to Notch), LAG-1
(a highly conserved transcription factor acting downstream of
GLP-1), and LAG-2 (a transmembrane Delta-like protein
which acts as the signaling ligand for GLP-1). Once the cells
move to a distance greater than 20 cell diameters away from
the distal tip cells, cells exit mitosis and enter meiosis in the
transition zone (Fig. 2B, trans zone) as they develop into oocytes. However, the oocytes do not complete meiosis but remain arrested in diakinesis of meiosis I as they move into the
proximal arm.
Meiosis in the oocyte proceeds to completion only upon
fertilization by a sperm (reviewed in reference 60). Once fertilized, the oocyte chromosomes first undergo meiosis I, during
which homologous chromosomes or bivalents separate into

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FIG. 1. Diagrammatic representation of sister chromatid separation. Sister chromatids are held together by cohesin complexes (short black
bars) during metaphase. APCCdc20 ubiquitination of securin (red) leads to liberation of separase (blue). Separase cleaves Scc1, one of the subunits
of the cohesin complex, and sister chromatids are pulled apart by spindle microtubules (long black bars).

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of APC activity. Nonetheless, the data from the sperm provide

additional indications that the APC is responsible only for
promoting proper chromosome segregation and not for spermatid differentiation or for early events in embryogenesis.
The requirement for APC activity in meiosis was further
established through studies of another class of mutants isolated
recently, known as metaphase-to-anaphase (mat) transitiondefective mutants (20) (Table 1). Inactivation of mat genes
such as mat-1 (CDC27), mat-2 (APC1), and mat-3 (CDC23) in
temperature-sensitive mutants or by dsRNAi led to an arrest in
meiosis I in the embryos (9, 20) (Table 1). Other APC subunits,
identified through sequence homologies to APC2 and APC11
(K06H7.6 and F35G12.9, respectively) (Table 1), are also essential in meiosis I (9). However, growth of several temperature-sensitive mutant alleles of mat-1 at semirestrictive temperatures led to the observation of different phenotypes; a
subset of the mat-1 mutants arrested in the one-celled, meiosis
I stage, a second subset of mutants underwent the first meiotic
division to give rise to one polar body, and a third group of
mutants completed meiosis I and II, resulting in the extrusion
of two polar bodies, though the meiosis was by no means
normal (61). These observations show that mat-1 contributes
to both meiosis I and II, though it appears that different levels
of APC activities are required for meiosis I and II. The meiotic
defects due to the partial inactivation of the mat-1 mutants also
contribute to developmental abnormalities, for instance, failure to establish a proper anterior-posterior axis and laying
down of an impermeable, three-layer eggshell (see below).
Defects in homologous chromosome separation in C. elegans
can also be seen in worms where the APC activator Cdc20/
Fizzy was disrupted. C. elegans fzy-1 was isolated in a suppressor screen by Kitagawa and coworkers (36) by using the gk2
mutant allele of mdf-1, a homologue of the MAD1 spindle
assembly checkpoint component (35). Localization of FZY-1
to chromosomes in antibody staining analysis of worm embryos
lends support to the idea that FZY-1 functions in metaphaseto-anaphase transition during mitosis and meiosis (36). In addition, Kitagawa and coworkers (36) proposed that a securinseparase pair exists in C. elegans which plays an important role
in chromosome separation similar to that in the budding yeast.
The authors isolated the securin orthologue, or ify-1 (for interactor of fizzy-1) in a yeast two-hybrid screen with FZY-1 as
the bait. Hermaphrodites in which ify-1 function was abolished
by dsRNAi produced single-celled embryos with abnormal
chromosome morphology due to defective chromosome segregation in meiosis I (36). dsRNAi experiments revealed that
IFY-1 may also be required in processes such as centrosome
duplication, spindle organization, and cytokinesis (36). Nonetheless, the identification of a destruction box motif in IFY-1
and the stabilization of IFY-1 protein level in an temperature-

FIG. 2. (A) Adult hermaphrodite showing mouth, anus, and tail with U-shaped gonad. The spermatheca, uterus containing embryos, and vulva
are also highlighted (more details in text). (B) Magnified view of one-half of the U-shaped gonad from the adult hermaphrodite with oocyte
chromosomes in newly fertilized embryos undergoing meiosis in the uterus (adapted from reference 57). Embryo 1, fertilization of oocyte (grey
circle, 4n) by the sperm; embryo 2, completion of meiosis I by oocyte chromosomes (grey circle, 2n) and extrusion of the first polar body; embryo
3, completion of meiosis II and extrusion of the second polar body; embryo 4, fusion of oocyte and sperm nuclei (see the text for more description).
(C) Chromosome segregation during meiosis in the C. elegans oocyte (57). Six pairs of homologous chromosomes undergo meiosis I upon
fertilization, followed by sister chromatid separation during meiosis II (see the text for details).

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two sets of univalents, with the one closer to the surface of the
embryos extruded as a polar body (Fig. 2B and C). During
meiosis II, sister chromatid segregation occurs, with the extrusion of a second polar body (1). The chromosomes in C. elegans
during meiosis can be easily observed under standard 4,6diamidino-2-phenylindole (DAPI) staining and fluorescent microscopic examination of the oocytes. Also, the generation of a
histone H2B-green fluorescent protein transgenic worm strain
by Judith Austins laboratory has enabled time-lapse imaging
in live oocytes of chromosome segregation that can help reveal
chromosome dynamics during meiosis and mitosis.
The role of APC in C. elegans meiosis was recently revealed
in studies of mutations of two loci, emb-30 and emb-27, previously identified to be essential for embryogenesis (6). The
emb-30(g53ts) mutant was initially found to produce one-celled
embryos carrying multiple nuclei, while the emb-27(g48) mutant produced one-cell embryos defective in polar body formation when the mutants were grown at the restrictive temperature (6). Detailed analyses showed that homologous
chromosomes of emb-30(tn337ts) (17) and emb-27(g48) (20)
mutants failed to separate, an indication that the embryonic
lethality was due to defects in meiosis. Sequence similarities
found between EMB-30 and the human Apc4 and fission yeast
Lid1 APC subunit (17), and EMB-27 with Cdc16 (20) therefore implicate the APC in progression through meiosis.
The APC subunits appear to be involved in a general function of homologous chromosome separation, as early events
prior to anaphase I such as eggshell deposition, chromosome
condensation, and localization of sperm and oocyte chromosomes near the embryo cortex were unaffected by the mutations in both emb-30 and emb-27 mutants (17, 20). Furthermore, the APC is also required in meiosis during
spermatogenesis. In general, the main features of spermatogenesis are relatively similar in hermaphrodites and males.
Basically, germ cells first enter meiosis to form spermatocytes,
which then undergo meiosis to generate spermatids (reviewed
in reference 27). Loss of emb-30 or emb-27 function led to a
failure to properly segregate chromosomes in the mutant
sperm during spermatogenesis (17, 20) though interestingly,
cell division continued, resulting in the production of spermatids devoid of chromosomal material (58). These anucleate
spermatids surprisingly retained their ability to crawl towards
and fertilize the oocytes. The oocytes, once fertilized, became
activated and proceeded to complete meiosis. Other events,
such as proper eggshell formation, migration of the oocyte
pronucleus, and establishment of the resulting embryo, appeared unaffected by mutations in the APC subunits. Therefore, unlike oocytes, which remained arrested in meiosis I, the
spermatids were able to continue with the differentiation program to form somewhat functional spermatozoa in the absence

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essential for both meiosis and mitosis through its role in localizing cohesin subunits adds to the number of important players
required for chromosome segregation in the worm.
APC IN MITOSIS
The embryonically lethal phenotype of APC mutations due
to defects in meiosis could potentially obscure observations as
to whether the APC functions during mitosis in C. elegans.
Indeed, of the APC subunits examined, only the inactivation of
apc-5 and apc-10 by dsRNA did not result in embryonic lethality at the meiotic one-cell stage; rather, the embryos died at the
multicellular stage (9). These data indicate that apc-5 and
apc-10 contribute to postmeiotic events, perhaps in mitosis.
The observation that mothers in which either apc-5 or apc-10
was disrupted by dsRNAi eventually became sterile due to a
failure of germ line maintenance (9) points to the established
role of the APC in mitosis (reviewed in references 25, 54, and
78), more specifically, in germ line mitosis. One possible explanation for the absence of a meiotic defect in the apc-5 and
apc-10 dsRNAi experiments could be that not all APC subunits
are required strictly for meiosis (see above) and that some of
the subunits may be needed for proper mitotic divisions and/or
specific processes during embryogenesis. Alternatively, it could
well be that the dsRNAi failed to completely knock down the
gene functions of apc-5 and apc-10. The partial APC activity
allowed the embryos to progress through meiosis even though
the reduced activity was insufficient for subsequent processes.
Other lines of evidence supporting the contribution of APC
to postmeiotic events can be seen in the emb-30 class I and
mat-1(ax144) and mat-1(ax212) mutant larvae (61). By allowing these emb-30 class I mutants to grow at the permissive
temperature to bypass the meiotic defect due to emb-30 mutation, the embryos were able to develop into adult worms,
albeit with developmental defects such as an everted vulva
(17). Double mutants carrying either the mat-1(ax212) or mat1(ax227) allele in combination with other APC mutant alleles
were able to develop to the adult stage, but these gave birth to
dead embryos at the permissive temperature, although the
single mutants were fully fertile (61). These data demonstrate
that by compromising APC activity in worms carrying a combination of two mutant alleles of the APC, defects can occur
even at the permissive temperature, perhaps due to the APCs
contribution to somatic cell division during embryonic and
postembryonic development (see below).
The indication that the APC is indeed required for mitosis in
somatic tissues came from detailed analysis of mat-1(ax144)
and mat-1(ax212) mutant embryos allowed to develop at the
permissive temperature into male larvae. Phospho-histone
staining revealed an increase in the posterior ends of the male
larvae when they were shifted to the restrictive temperature
(61), a sign that the cells failed to progress through mitosis.
The mitotic progression phenotype in the APC mutants correlates with the well-accepted role of the APC for the ubiquitination of the anaphase inhibitor securin (reviewed in reference 48). However, the early function in meiosis of the C.
elegans securin, ify-1, and separase, sep-1 (see above), could
have precluded the identification of their role in mitosis,
though it is not unreasonable to expect that they should also
contribute to sister chromatid segregation, given that the con-

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sensitive emb-30 mutant is consistent with the idea that the

APC, together with FZY-1, is responsible for ubiquitination of
IFY-1, perhaps for setting free SEP-1 (36), the orthologue of
separase.
The notion that IFY-1 and SEP-1 act as securin and separase, respectively, is supported by the physical interaction between IFY-1 and SEP-1 in yeast two-hybrid and glutathione
S-transferase assays (36) as well as the meiosis I chromosome
segregation phenotype observed in a sep-1 mutant grown at the
restrictive temperature (63). The meiosis-specific cohesin
REC-8 has also recently been identified in C. elegans and
found to localize to chromosomal axes in oocytes at diakinesis
(51). Like that of S. cerevisiae, C. elegans REC-8 is partially
destroyed in meiosis I and rec-8 depletion in the oocytes caused
aberrant separation of chromosomes, resulting in 24 separate
chromatids and chromosome fragments (51). This is a strong
indication that REC-8 normally serves as a cohesin subunit in
keeping chromosomes together.
Other cohesin subunits in C. elegans have also been found
through sequence homologies to the families of Smc1, Smc3,
and Scc3 subunits from the genome database (44). Furthermore, work by Chan and colleagues established that REC-8
colocalizes with SMC-1, SMC-3, and SCC-3 to meiotic chromosomes (7). Intriguingly, TIM-1, believed to be the paralogue
of the Drosophila clock protein TIMELESS, has been coimmunoprecipitated in a cohesin complex comprising SMC-1,
SMC-3, SCC-1, and SCC-3 (7). dsRNAi data show that tim-1 is
essential for chromosome segregation, as temperature-sensitive tim-1 worms were defective in chromosome separation in
meiosis and mitosis. Immunofluorescence studies revealed that
non-SMC subunits such as REC-8 and SCC-1 failed to localize
to chromosomes in worms where tim-1 was knocked down by
dsRNAi, a strong hint that TIM-1 is important for targeting of
non-SMC cohesin subunits to the chromosomes.
Taken together, these findings show that, in contrast to meiosis in Xenopus laevis, chromosome separation in C. elegans
meiosis is mediated by an APC-dependent pathway similar to
that found in S. cerevisiae. Despite this evidence supporting the
idea that APC is required for meiosis, it could still be argued
that the data from S. cerevisiae and C. elegans are not as useful
for understanding meiosis in vertebrates and that the finding in
Xenopus laevis (53, 66) may better reflect the situation in
higher organisms. However, work from mouse oocytes reveals
that the progression through meiosis also depends upon the
APC and securin (67), further demonstrating the functional
conservation of the APC in meiosis. Terret and coworkers (67)
suggested that the apparent lack of requirement of the APC in
Xenopus meiosis could be due either to technical difficulties in
inactivating the APC in Xenopus oocytes or to the possibility
that Xenopus laevis has evolved a unique mechanism of chromosome separation in meiosis.
It should be noted that the complete picture of meiosis in C.
elegans is not as straightforward as in budding yeast. Reports
showing that the inactivation of ify-1 failed to rescue the mat
phenotype in an emb-30 mutant background (36) and that
depletion of rec-8 was unable to rescue the apc-11 phenotype
(9) point to the possibility that factors other than securin/
IFY-1 have to be degraded by the APC for proper meiosis I
progression and that REC-8 is probably not the sole substrate
of separase in C. elegans. The finding that TIM-1 (7) is also

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FIG. 3. First mitotic division in the embryo establishes the anterior-posterior axis. The first cell division is asymmetric, resulting in a larger
anterior AB cell and a smaller posterior P1 cell.

APC IN DEVELOPMENT
Mitotic cell divisions are important during the development
of multicellular organisms for the generation of the correct cell
numbers or cell types important for the proper formation of
the organisms. Aberrant cell divisions occurring during embryonic or postembryonic development of the worm can result in
developmental defects. In the first mitotic division of the onecelled embryo, P0, two unequal daughter cells, a larger anterior
AB and a smaller posterior P1 (Fig. 3), are generated, and each
subsequently contributes to cells forming different tissue types
(65). This asymmetric division depends on the anterior-posterior axis, established through the interactions among the sperm
pronucleus/centrosome complex, the associated microtubules
and the actin-rich cortex upon fertilization of the oocyte. The
sperm pronucleus/centrosome complex triggers cytoplasmic
flow and cortical rearrangements that cause the polarized localization of specific cellular determinants such as the PAR
proteins. These PAR proteins play an important role in the

displacement of the mitotic spindle to ensure an asymmetric

division (reviewed in references 41 and 52).
A screen for polarity- and osmosis-defective (pod) mutants
identified a role for the APC in anterior-posterior axis formation during division of the P0 (56) (Table 1). Complementation
analyses of pod and mat mutants showed that the pod mutations constitute different alleles of the mat locus (Table 1). The
finding that the mat alleles led to meiosis I arrest (see above)
while pod alleles bypassed the meiotic defect suggests that the
mat alleles correspond to more severe mutations leading to a
greater reduction of APC function (20). Detailed analysis of
the pod-3(or319) mutant by Rappleye and coworkers (56) indicated that a partial disruption of the APC-1 subunit orthologue led to the inability of the sperm pronuclei to associate
with the cortex, which is key to the establishment of the anterior-posterior axis (71). As a consequence, PAR-3 granules,
normally localized to the anterior of the embryo (11), were
observed throughout the cortex of the embryo and PAR-2
proteins were displaced to the cytoplasm in the pod embryos
instead of their usual location in the posterior cortex. In addition, other cytoplasmic determinants such as the germ cell
granules, which normally localize to the posterior of the P0
cells in wild-type worms, accumulated in the middle of the
one-celled embryos in the different pod mutants. Shakes and
coworkers (61) suggested that the contribution of the APC in
establishing proper polarity as seen in the pod mutants could
be indirect, as they observed polarity defects due to mat-1
inactivation only in the presence of gross meiotic defects in
embryos where only one polar body was formed. This would
mean that the progression through an abnormal meiosis could
indirectly precipitate the polarity defect.
The proper establishment of the anterior-posterior axis also
depends on ubiquitin and the APC activator CDC20/FZY-1, as
disruption of either ubiquitin gene ubq-2 or cdc20/fzy-1 by
dsRNAi abolished asymmetric division of the P0 embryo (56).
Rappleye and coworkers (56) proposed that APCCDC20/FZY-1
contributes to anterior-posterior polarity through the activation of separase in the P0 cell, as the disruption of sep-1 also led
to a defect in anterior-posterior polarity establishment. APCactivated separase triggers the association of the sperm centrosome with the posterior cortex, causing the displacement of
the PAR-3-containing complex to the anterior cortex. PAR-2
on the other hand is able to localize to the posterior cortex,
thereby setting up an asymmetric distribution of cellular determinants in the one-cell P0. A polarized localization of these

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servation in the components in the APC and the cohesin complex appears to extend across different organisms (reviewed in
reference 48).
That an emb-30 mutation can partially rescue the abnormal
chromosome separation in an mdf-1 background where chromosome segregation occurs in the presence of a defective
spindle assembly checkpoint (17) further indicates that the
APC is needed for proper chromosome separation. Analysis of
smc-1, smc-3, and scc-3 in C. elegans by dsRNAi revealed that
these cohesin subunits are required for proper chromosome
segregation during mitosis in embryos (46). More recent work
in Chans group with immunoprecipitation assays found
COH-2 in the SMC-1, SMC-3, and SCC3 cohesin complex,
providing strong evidence that COH-2 is the functional orthologue of Scc1 (7). Also, tim-1 was immunoprecipitated in the
cohesin complex and appears to be essential for mitotic chromosome segregation in both germ line and somatic cells. Furthermore, Kitagawa and Rose found that mdf2, the C. elegans
orthologue of the MAD2 spindle assembly checkpoint component in budding yeast, was able to rescue the benomyl-sensitive
phenotype in a mad2 deletion strain of budding yeast (35). This
work points to the conservation of the sister chromatid segregation pathway, as Mad2 has been shown to act through sequestration of Cdc20 from the APC, inhibiting chromosome
segregation upon spindle damage.

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MINIREVIEW

FUTURE PERSPECTIVES
The coordination of cell division, cell death, and differentiation is critical for the development of a multicellular organism. The development of a multicellular organism can be affected by the cell division cycle in one of several ways. For
example, the number of division cycles, which correlates with
the number of progeny cells resulting from a progenitor cell, is

an important factor in organogenesis for proper shape or size

and hence functioning of tissues or organs. For instance, in C.
elegans a defect in lin-23, which functions as part of the stem
cell factor ubiquitin-ligase complex involved in the degradation
of G1 cyclins, led to unrestrained cell proliferation and hyperplasia of all cell types (34). In another C. elegans mutant carrying a truncated cyclin E homolog, cye-1, underproliferation
of postembryonic blast lineages was observed (14). The cell
cycle stage apparently also affects the differentiation program
of the developing organisms. In the vulval development of C.
elegans, the vulval progenitor cells at G1 or pre-S phase in the
cell cycle can adopt the primary vulval precursor cell fate,
whereas only cells in post-S phase can be induced to the secondary vulval precursor cell fate (2). It is therefore noteworthy
that the studies on the C. elegans APC described above have
provided further links between the machinery involved in cell
divisions and proper development of the worm.
A better understanding of how the APC is regulated during
cell division could help shed some light on how cell division is
coordinated with developmental processes. To this end, C.
elegans continues to serve as a useful tool, as genetic screens
can be performed relatively easily for isolating regulators
and/or substrates of the APC. Screens for mutations affecting
cell division, such as the one carried out by Gonczy et al. (21),
are useful, as additional genes involved in cell division can be
identified. In that particular screen, mutations in 34 loci on
chromosome III which affected cell division in the single-cellstage embryo were obtained. The fact that the spindles, centrosomes, and nuclei of the mutant embryos can be easily
visualized and documented by time-lapse differential interference contrast microscopy demonstrates the strength of using
the C. elegans one-celled embryo for studying cell division.
Using this technique, the authors were able to categorize the
mutants into distinct mutant phenotypes, such as defects in
spindle assembly, anaphase spindle positioning, chromosome
segregation, and cytokinesis, among others, for further studies.
Genetic interactions between genes identified from such
screens and genes encoding APC can be tested by standard
genetic crosses and relevant candidates can be further characterized in biochemical assays. It would be interesting if distinct
pathways leading to APC activation were found for meiotic
and mitotic cell division due to differential regulation of the
APC. Mutant analyses could possibly lead to the discovery of
as yet unidentified subunits of the APC which may assemble
different APCs for meiosis and mitosis. A subset of cell division
mutants could also allow the isolation of substrates of the APC.
Conversely, a more directed approach to screening for genes
involved in cell division is provided by reverse genetics with
dsRNAi. Large-scale screens by various groups have been used
to target specific clusters of genes, such as those expressed in
the worm ovary (55) and genes on chromosome I (16, 79) and
chromosome III (22), as well as a genome-wide dsRNAi endeavor by Maeda and colleagues (42) and Kamath and coworkers (32). Consistent with the data showing that the APC is
essential for meiosis in C. elegans, dsRNAi of the genes on
chromosome III revealed that of the 11 genes required for
progression through meiosis, 10 encode components of the
proteosome, APC subunits, or ubiquitin (22). The availability
of the dsRNAi data on the Wormbase (http://www.wormbase
.org) will no doubt speed up the hunt for genes involved in cell

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known cytoplasmic determinants in the wild-type P0 is required

for generating a differential pulling force exerted by the mitotic
spindles such that asymmetric division occurs (23). Interestingly, the absence of a proper anterior-posterior axis resulted
also in the altered timing and misorientation of spindles during
the second cell division (56).
The homologue of FZY-1, FZR-1 (for Fizzy-related, a homologue of Cdh1), apparently has no involvement in polarity
determination (14). fzr-1 was isolated in a synthetic lethal
screen in a lin-35/Rb background (14). lin-35 fzr-1 double mutants were mostly nonviable, though several survived and exhibited hyperproliferation of the seam cells, vulval tissue, gut
cells, and tumorous phenotype in the gonadal arm. The fzr-1
single mutant displayed milder defects in hyperproliferation of
tissues, although complete inactivation of fzr-1 in RNAi led to
a drastic reduction in fertility. The overexpression of cyclins A
and E by Fay and Han in the fzr-1 mutant background resulted
in an increase in mitotic divisions in the germ cells at the distal
arm (14). This led to multiple-armed gonads and has been
described as the Shiva (after the Hindu god Shiva, who is
usually depicted with four arms) phenotype. This is most likely
due to the reduced ability to degrade G1 cyclins such as cyclin
E and A. Hence, FZR-1 is required for the proper regulation
of cell division during the development of the worm, most
probably due to its role in controlling cyclin levels.
The function of the APC subunits in mitotic cell divisions
has consequences in the development of the worm during
postembryonic development for both germ and somatic cells.
For example, emb-30(tn337ts) mutant embryos laid at the permissive temperature but allowed to develop at the restrictive
temperature were defective in germ line proliferation, with
many of the mitotic germ cells arrested in metaphase (17).
Additional emb-30 mutant alleles isolated, such as those in the
emb-30(class I) mutants, further point to the requirement of
the APC in vulva development, somatic gonad development,
and male tail morphogenesis (17). These defects were not due
to differentiation defects, as analysis of vulval precursor cells
showed that they either delayed or blocked mitosis. Everted
vulva and male tail morphogenesis defects were also observed
in mat-2 mutants (9) and mat-1 mutants (61), suggesting that
certain mutant alleles led to reduced levels of APC activity,
which allowed meiosis I progression but not postembryonic
development. Male tail abnormalities such as missing rays and
complete absence of rays were also found in mat-1(ax144) and
mat-1(ax212) mutant male larvae (61). These sensory rays,
used during mating for sensing of hermaphrodites, are derived
from blast cells through division and differentiation (reviewed
in reference 10). As the APC is implicated in mitotic divisions
in the worm, the absence of such rays in the mat-1(ax144) and
mat-1(ax212) mutant male larvae could be attributed to the
lack of or improper cell division.

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ensure the proper execution of cell division and development

are will be important, as the decoupling of these processes can
have dire consequences, such as oncogenesis (reviewed in reference 8). Given the conservation in the APC function in C.
elegans and the range of laboratory manipulations afforded by
the worm, studies in the worm can yet provide meaningful
insights into the coordination of cell division and development
in higher organisms.
ACKNOWLEDGMENTS
I thank Goh Phuay Yee and Tham Foong Yee for critical comments
on the manuscript and the two anonymous reviewers for their very
constructive and insightful comments.
Y.F.M. is supported by the Faculty of Medicine start-up fund (grant
number R-183-000-104-112) and Provost start-up fund (grant number
R-183-000-605-101), National University of Singapore.
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division and development. One could essentially perform

searches at Wormbase for all possible worm genes implicated
in a specific cellular or developmental process based on phenotypes. Genes identified through such searches can then be
used to determine functional interactions with the APC.
Studying the developmental context in which the APC functions could pose a bigger challenge for C. elegans, given that
the abolishment of APC activity more often than not leads to
an early embryonic defect. However, as in the studies by Furuta and coworkers (17) and Shakes et al. (61), when conditional mutants such as temperature-sensitive APC mutants are
available, it could still be possible to investigate the regulation
of the APC during the development of the worm. By culturing
the temperature-sensitive mutants at the permissive temperature to circumvent the embryonically lethal phenotypes, one
can shift the mutants to the restrictive temperature and examine developmental defects once the embryos have passed embryonic development. Such temperature shift regimens or perhaps the use of conditional knockouts based on dsRNAi (e.g.,
heat-inducible promoters) will permit the dissection of various
cell division processes involving the APC during postembryonic development.
A screen performed by OConnell and coworkers (49) for
either a temperature-sensitive embryonically lethal (Emb) or a
postembryonic phenotype known as sterile and uncoordinated
(Stu) led to the identification of novel genes which are involved
in cell division. Detailed examination revealed that some of the
Stu mutants had defects in meiosis or mitosis, demonstrating
that a developmental defect such as Stu is a good predictor for
postembryonic cell division defects. Conversely, the mutations
leading to the Stu phenotype also caused problems during
embryonic cell divisions, yet again indicating the close link
between the processes of cell division and development. While
these mutants could be used for testing for genetic interactions
with mutants of the APC subunits, the general strategy employed by OConnell and colleagues (49) to obtain conditional
mutants can be employed for isolating more players involved in
cell division and development.
An established function of the APC which has not been well
explored in C. elegans is the destruction of mitotic cyclins for
the regulation of exit from mitosis (reviewed in reference 46).
Although fzr-1 has been shown to be required for cyclin destruction (13) and components required for mitotic exit such as
the MEN have been identified through sequence homologies
(24), not much is known about how conserved the pathway
leading to mitotic exit is in C. elegans. In addition, while the
MEN components have been implicated in cytokinesis (reviewed in reference 43), the APC has only recently been shown
to be required for cytokinesis in the sea urchin and sand dollar
(62), adding yet another contribution to its regulation of the
cell division cycle. C. elegans will no doubt provide a useful
system for further work on the APC and the control of exit
from mitosis and cytokinesis. How the processes of exit from
mitosis and cytokinesis are regulated during the development
of the worm also awaits further investigation.
As it stands, studies with C. elegans remain relevant for
understanding APC functions. It is known that there are tight
controls that maintain the balance between cell division and
developmental processes in a multicellular organism (reviewed
in reference 8). Understanding what the mechanisms which

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MINIREVIEW

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