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C. elegans mitotic cyclins have distinct as well as overlapping
functions in chromosome segregation
Monique van der Voet1,†, Monique A. Lorson2,†,‡, Dayalan G. Srinivasan2,§, Karen L.
Bennett3, and Sander van den Heuvel1,2,*
1Developmental Biology, Utrecht University, Utrecht, The Netherlands 2The Massachusetts
General Hospital Cancer Research Center and Harvard Medical School, Charlestown, MA USA
3Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO
USA §Department of Ecology and Evolutionary Biology, Princeton, NJ USA
Abstract
Mitotic cyclins in association with the Cdk1 protein kinase regulate progression through mitosis in
all eukaryotes. Here, we address to what extent mitotic cyclins in the nematode
Caenorhabditis
elegans
provide overlapping functions or distinct biological activities.
C. elegans
expresses a
single A-type cyclin (CYA-1), three typical B-type cyclins (CYB-1, CYB-2.1 and CYB-2.2), and
one B3-subfamily member (CYB-3). While we observed clear redundancies between the
cyb
genes,
cyb-1
and
cyb-3
also contribute specific essential functions in meiosis and mitosis. CYB-1
and CYB-3 show similar temporal and spatial expression, both cyclins localize prominently to the
nucleus, and both associate with CDK-1 and display histone H1 kinase activity in vitro. We
demonstrate that inhibition of
cyb-1
by RNAi interferes with chromosome congression and causes
aneuploidy. In contrast,
cyb-3(RNAi)
embryos fail to initiate sister chromatid separation.
Inhibition of both cyclins simultaneously results in a much earlier and more dramatic arrest.
However, only the combination of
cyb-1, cyb-3
and
cyb-2.1/cyb-2.2
RNAi fully resembles
cdk-1
inhibition. This combination of redundant and specific phenotypes supports that in vivo
phosphorylation of certain Cdk targets can be achieved by multiple Cdk1/cyclin complexes, while
phosphorylation of other targets requires a unique Cdk1/cyclin combination.
Keywords
C. elegans
; cyclin B1; cyclin B3; mitosis; meiosis; cell cycle; chromosome congression;
chromosome segregation
Introduction
Cyclin-dependent kinases (CDKs) and their associated cyclin regulatory subunits control
progression through the eukaryotic cell-division cycle (reviewed in refs. 1 and 2). Activation
of the CDK1 kinase triggers entry into mitosis, while CDK1 inactivation is required for
mitotic exit. Expression, association and degradation of the cyclin subunits are critical in the
regulation of CDK activity.
© 2009 Landes Bioscience
*Correspondence to: Sander van den Heuvel; S.J.L.vandenHeuvel@uu.nl.
†These authors contributed equally to this work.
‡Current addresses: Bond Life Sciences Center; University of Missouri; Columbia, MO USA;
NIH Public Access
Author Manuscript
Cell Cycle
. Author manuscript; available in PMC 2013 April 02.
Published in final edited form as:
Cell Cycle
. 2009 December 15; 8(24): 4091–4102.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
All eukaryotes express a number of different cyclins. The members of this protein family
share similarity predominantly within the “cyclin box”, a conserved domain that mediates
binding and activation of CDKs. “G1 cyclins”, “S phase cyclins” and “mitotic cyclins” have
been defined by their pattern of expression, Cdk activation and cell cycle function. In
addition, cyclin subfamilies (e.g., A or B-type) have been distinguished based on sequence
similarity. These classifications are partly overlapping, as members of each subfamily share
cognate CDK partners and show similar patterns of expression and associated kinase
activity. Hence, the paradigm in the field has long been that specific CDK/cyclin
combinations phosphorylate specific target proteins and thereby promote distinct cell cycle
transitions. In metazoans, D-type cyclins together with Cdk4/Cdk6 promote progression
through G1 phase; E-type cyclins in association with Cdk2 support the onset of S phase; A-
type cyclins in complex with either Cdk2 or Cdk1 act in the S, G2 and M phases, and B-type
cyclins together with Cdk1 control progression through mitosis.3
Cyclins expressed in the same cell cycle phase are largely redundant in function. Four
different B-type cyclins (CLB1, CLB2, CLB3 and CLB4) regulate the progression through
mitosis in budding yeast. However, expression of CLB2 alone is sufficient and
GAL1
driven
expression of CLB1 complements the
clb1,2,3,4
deletion.4,5 Three different cyclins, a single
A-type and B-type cyclin and a member of the distinct B3 subfamily, cooperate during
mitosis in Drosophila. Mutation of
Dm
CycB or CycB3 does not interfere with viability.6
Furthermore, knockout of individual cyclins in mice result in limited defects, with the
exception of cyclins A2 and B1.7,8 The specific developmental defects associated with
cyclin D1, cyclin D2 and cyclin D3 knockout appear to result from distinct patterns of
expression of these cyclins, rather than specific functions.9–12 In support of this idea,
replacement of the cyclin D1 coding sequences with cyclin D2 rescues the cyclin D1
knockout phenotype.13,14 CDK knockout experiments have shown that substantial mouse
development is possible with Cdk1 alone, functioning in combination with a broad range of
cyclins.15 Such results are surprising, as specific functional properties would be expected to
underlie the conservation of distinct cyclin subfamilies throughout metazoan evolution.
Indeed, a recent analysis indicates that Drosophila mitotic cyclin A, B and B3 cyclins are
less redundant than previously concluded, as all three of these cyclins have specific critical
functions in the syncytial embryo.6,16 In fact, ample experimental evidence supports that
cyclins contribute to the substrate specificity of CDKs and that members of different
subfamilies cannot simply substitute for each other. Factors that contribute to cyclin
specificity are subcellular localization,17 and the use of a hydrophobic cyclin patch in
substrate contact.18,19 Thus far, the cyclin-substrate specificity is best characterized for
budding yeast CLB5 and mammalian cyclin A.18,19 For example, knock-in of
CLB2
into the
CLB5
locus in yeast prematurely initiates CLB2/CDC28 kinase activity and allows rescue of
clb1,2
lethality, but does not replace the role of
CLB5
in S phase initiation.20
Phosphorylation of pRb, p107 and E2F-1 by Cdk2/cyclin A involves binding of the cyclin A
hydrophobic patch to these substrates.18 This explains why Cdk2 in association with cyclin
A (but not cyclin B1) phosphorylates these substrates in vitro.21 Other cyclins may also
recruit specific targets through a distinct docking site or, alternatively, promote CDK
activity towards a broader range of targets. For most cyclins it remains poorly understood if
and how they confer target specificity to their CDK partners, which targets they recruit and
which functions they share with other cyclins.
In the present study, we address to what extent mitotic cyclins have redundant versus
specific functions in early
Caenorhabditis elegans
development.
C. elegans
embryos are
particularly amenable for examination of mitosis and cytokinesis, as the early embryonic
cells are large and the spindle and chromosomes are cytologically observable. Moreover,
RNA-mediated interference (RNAi) provides an efficient reverse genetic technique to
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eliminate both maternal and zygotic gene functions.22 Consequently, single, double and
triple gene knockdown by RNAi may reveal specific as well as redundant gene functions in
early embryogenesis.
The
C. elegans
genome harbors orthologs of all major classes of metazoan cyclins: D, E, A,
B1, B2 and B3.23–28 We show that CYB-1 (Cyclin B1) and CYB-3 (Cyclin B3) follow
similar developmental expression patterns and largely overlapping subcellular localizations,
yet each of these cyclins is essential for specific processes in meiosis and mitosis.
Simultaneous inhibition of
cyb-1
and
cyb-3
results in an earlier and more severe M phase
arrest. However, only the combination of
cyb-1
,
cyb-2.1/2.2
and
cyb-3
RNAi resembles
cdk-1
inactivation. These data indicate that all B/B3-type cyclins act with CDK-1 and
provide overlapping as well as specific functions in meiosis and mitosis. Our results suggest
that phosphorylation of some mitotic Cdk targets can be accomplished by a variety of Cdk1/
mitotic cyclin complexes, while phosphorylation of other targets requires a specific Cdk1/
cyclin combination.
Results
Caenorhabditis elegans contains distinct subfamilies of mitotic cyclins
Previous studies by us and others identified the
Caenorhabditis elegans
cyclin genes
cyd-1
(Cyclin D),24,26
cye-1
(Cyclin E),25,28
cya-1
(Cyclin A),
cyb-1
(Cyclin B1) and
cyb-3
(Cyclin B3).23 The
C. elegans
Genome Sequencing project identified two additional B-type
cyclins,29 encoded by the Y43E12A.1 (
cyb-2.1
) and H31G24.4 (
cyb-2.2
) genes (Fig. 1). We
obtained cDNA clones from
cyb-2.1
and
cyb-2.2,
demonstrating that both genes are
expressed (Fig. 1A, Materials and Methods). These cDNAs are probably derived from the
full-length messages, as they contained SL1 trans-spliced leader sequences at their 5′-
ends.30 The predicted amino-terminal ends of all five
C. elegans
A, B and B3-type cyclins
include candidate destruction boxes (Fig. 1A and reviewed in ref. 23). This amino acid motif
is present in the N-termini of mitotic cyclins in other systems and is required for ubiquitin-
dependent proteolysis.31
The
cyb-2.1
and
cyb-2.2
coding sequences share 92% nucleotide identity, indicating a
relatively recent duplication. The CYB-2.1 and CYB-2.2 predicted proteins share 94%
aminoacid identity over their entire length. Each of these two cyclins is approximately 60%
identical to CYB-1. In contrast, CYB-3 is more closely related to cyclin B3 members in
other species than to the other mitotic cyclins in
C. elegans
(Fig. 1B). Similar to the B3
cyclins in other species, CYB-3 contains not only Cyclin B-type signature sequences but
also A-type sequences within the cyclin box.32
Knockdown of
cyb-1, cyb-2.1, cyb-2.2
or
cyb-3
by RNA interference (RNAi) resulted in
early embryonic cell division defects and embryonic lethality (see below). In contrast, upon
cyd-1
and
cya-1
RNAi all offspring developed into sterile larvae, and embryonic lethality
was not observed (data not shown). As previously reported,
cye-1(RNAi)
resulted in defects
in cell polarity establishment and arrest at the approximately 100-cell stage.25,26,33
Because of the close similarity at the DNA level, RNAi for either
cyb-2.1
or
cyb-2.2
is
expected to downregulate both genes. In addition,
cyb-2.1/2.2
and
cyb-1
share stretches of
approximately 80% nucleotide identity that could also lead to co-inhibition. To examine this
possibility, we injected a dsRNA fragment unique for
cyb-1
(nucleotide 11–270 of the open
reading frame). This specific
cyb-1
dsRNA fragment also resulted in a highly penetrant
embryonic lethality. However, injection of dsRNA corresponding to the most unique 107
nucleotides of the
cyb-2.2
coding region did not cause an apparent phenotype (data not
shown). We conclude that at least two different mitotic cyclins,
cyb-1
and
cyb-3,
have
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essential functions during embryonic development. For this reason, we focused our analysis
on
cyb-1
and
cyb-3
.
CYB-1 and CYB-3 specific antisera recognize active kinase complexes
We generated polyclonal antibodies against the full-length CYB-1 and CYB-3 proteins to
examine their expression, Cdk association and subcellular localization. Using total
C.
elegans
lysate in western blot experiments, the antisera reacted with proteins of the predicted
molecular weight for CYB-1 and CYB-3 (41 kD and 45 kD, respectively, Fig. 2A and B).
We examined the effects of
cyb-1
and
cyb-3
RNAi to test the specificity of the antibody
reactivity. Animals were soaked in dsRNA and their progeny collected and used in western
blotting experiments. RNAi treatment resulted in a significant and specific reduction of the
candidate CYB-1 and CYB-3 proteins (Fig. 2A). As expected, control RNAi treatment for
the essential mitotic gene
lin-5
did not reduce the reactivity with either cyclin antibody (Fig.
2A; reviewed in ref. 34).
RNAi treatment also eliminated specific antibody staining in immunohistochemical
experiments. Immunostaining of
C. elegans
embryos with CYB-1 and CYB-3 antisera
showed predominantly pronuclear staining at the time of migration before the first mitosis of
the fertilized egg (Fig. 2E).
cyb-1
RNAi strongly reduced reactivity with the anti-CYB-1
sera, while CYB-3 reactivity was unaffected. Likewise,
cyb-3
dsRNA completely eliminated
immunoreactivity with anti-CYB-3 antibodies, but not with CYB-1 (Fig. 2E). These results
demonstrate the specificity of the affinity-purified antibodies and the specificity of the
RNAi. However, co-inhibition of
cyb-2.1
/
2.2
and
cyb-1
was also observed in these
experiments. Injection of
cyb-2.1
or
cyb-2.2
dsRNA did not affect CYB-3 reactivity, but
partly reduced CYB-1 staining (data not shown).
CDK-1/NCC-1 is the
C. elegans
cyclin-dependent kinase essential for progression through
mitosis.35 Based on results in other eukaryotes, this kinase is expected to act with a number
of different mitotic cyclins. CYB-1 and CYB-3 co-immunoprecipitated with the CDK-1
kinase, in contrast to control immunoprecipitations with non-specific antibodies (Fig. 2B).
In addition, both the CYB-1 and CYB-3 immunoprecipitates demonstrated kinase activity in
vitro towards the canonical CDK substrate histone H1 (Fig. 2C; reviewed in ref. 36). These
results are consistent with the hypothesis that CYB-1 and CYB-3 act to promote progression
through mitosis in
C. elegans
.
The CYB-1 and CYB-3 proteins show largely overlapping expression patterns
To examine whether CYB-1 and CYB-3 showed any marked differences in expression
during development, we prepared protein lysates from multiple developmental stages. As
expected for mitotic cyclins, the expression levels of CYB-1 and CYB-3 correlated with cell
division during each developmental stage (Fig. 2D). The most exponential cell proliferation
phase takes place from approximately two through seven hours of embryogenesis.
Coincident with expansive proliferation, the highest expression of CYB-1 and CYB-3
proteins was detected in embryos (Fig. 2D, left). CYB-1 and CYB-3 expression was
virtually absent in developmentally arrested first stage (Fig. 2D, L1 0 h) and dauer larvae
(not shown). Upon release from L1 arrest, sets of post-embryonic blast cells successively
initiate cell division,37 and CYB-1 and CYB-3 levels were found to increase during this
period (Fig. 2D, compare: L1 lanes 0 hr and 10 hr). CYB-3 protein levels remained low
during larval development, as compared to the levels in embryos, while CYB-1 levels were
fairly constant in developing animals.
To examine potential for distinct functions, we studied whether the subcellular localization
of CYB-1 and CYB-3 differs. Different subcellular localization has been reported for
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vertebrate cyclin B1 and B2,38 as well as for Drosophila and Chicken cyclin B1 and B3.32,39
In these systems, cyclin B2 and B3 are restricted to the nucleus, while cyclin B1
accumulates in the cytoplasm during G2 phase and is rapidly imported into the nucleus at the
onset of prophase. Immunostaining of
C. elegans
larvae showed expression of both cyclins
in dividing cells (data not shown). However, the strongest expression was detected in the
adult germline and early embryos, which we analyzed further.
Precursor germ-cell nuclei are formed in
C. elegans
by mitotic divisions in the distal ends of
the syncytial gonad. As these nuclei gradually move further from the distal tip cell, they exit
the mitotic cycle, initiate meiosis and go through pachytene of meiotic prophase I. These
germ nuclei initiate oocyte development when they reach the bend in the U-shaped gonad;
they exit from pachytene, cellularize, continue to enlarge and pause at diakinesis of meiosis
I. Meiosis I and II are completed following fertilization, which results in two polar bodies
that are expelled from the fertilized egg. Subsequently, a maternal pronucleus forms and
migrates towards the paternal pronucleus. The two pronuclei meet in the posterior of the
egg, migrate to the middle while rotating and initiate mitosis.
CYB-1 and CYB-3 were detected in the nuclei of the mitotic proliferating region of the
gonad (data not shown). During maturation of oocytes in the proximal gonad, CYB-1 and
CYB-3 levels were found to gradually increase in the nucleus (data not shown). CYB-1 and
CYB-3 remained diffusely distributed throughout the cytoplasm of the fertilized egg during
meiosis (Fig. 3A–D), and localized to the maternal and paternal pronuclei upon completion
of meiosis (Fig. 3E–H). Subsequently, during each mitotic division, CYB-1 and CYB-3
gradually disappeared from the nucleus as cells proceeded from prophase to metaphase (Fig.
3I–P, posterior cells, to the right). At approximately the time of anaphase initiation, both
cyclins also disappeared from the cytoplasm (Fig. 3I–P, anterior cells, to the left). Thus,
CYB-1 and CYB-3 show similar temporal and spatial localization. However, CYB-3
localization is mainly nuclear and perdures somewhat longer in metaphase, while a larger
fraction of CYB-1 is present in the cytoplasm. These differences are similar, though more
subtle, to those reported for cyclin B1 versus B3 localization in other metazoans.32,38,39
CYB-1 and CYB-3 have distinct meiotic functions
We used gene inactivation by RNAi to address whether
cyb-1
and
cyb-3
are required for
specific cell cycle events in
C. elegans
. Injection of either
cyb-1
or
cyb-3
dsRNA, or the
combination of the two, all caused highly penetrant embryonic lethal (Emb) phenotypes. We
closely followed early development in
cyb-1(RNAi)
and
cyb-3(RNAi)
embryos as well as
double
cyb-1(RNAi); cyb-3(RNAi)
embryos, using microscopic observations of live and
fixed specimens (Figs. 4–6). Interestingly, the three different RNAi experiments
reproducibly resulted in distinct cell cycle phenotypes, indicating specific cyclin functions.
Neither
cyb-1
nor
cyb-3
RNAi appeared to interfere with events in oogenesis, mature
oocytes were formed with chromosomes in typical arrangements of bivalents in diakinesis.
Oocytes initiated meiosis normally and became fertilized when entering the spermatheca
(not shown). However, meiosis was frequently defective, as was evident from aberrant
numbers of polar bodies and maternal pronuclei. In fixed and stained embryos,
approximately 56% (43/77) of the
cyb-1(RNAi)
embryos contained just one polar body.
Recording of embryogenesis with DIC optics revealed the formation of two maternal
pronuclei in 30% (14/46) of
cyb-1(RNAi)
embryos. Of the
cyb-3(RNAi)
embryos, 12%
(28/239) did not have any polar bodies and 59% (142/239) contained a single polar body.
Embryos examined before the first mitosis formed two maternal pronuclei in 45% (20/44) of
the
cyb-3(RNAi)
embryos. Extra pronuclei were always observed in embryos with less than
two polar bodies, indicating that failure to dispose of chromosomes within a polar body
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resulted in additional pronuclei. Similar observations were made following inactivation of
other genes involved in chromosome segregation and cytokinesis.34,40
These results indicate that
cyb-1
and
cyb-3
are each required during meiosis. Importantly,
time-lapse fluorescence microscopy of meiosis in utero showed that the meiotic defects are
distinct. Upon
cyb-1
RNAi, abnormal chromosome alignment and segregation was observed
(Fig. 6C, arrow), while
cyb-3
RNAi predominantly caused delayed progression through
meiosis II (Fig. 6D, arrow).
CYB-1 is required for chromosome congression, CYB-3 for sister chromatid separation
Regardless of whether two polar bodies were expelled, events immediately following
meiosis followed the normal pattern. As in the wild type, the maternal pronucleus migrated
towards the paternal pronucleus in the posterior of the egg (Fig. 4B, H and N), the two
nuclei migrated to the middle while rotating, the first mitotic spindle was formed and
nuclear envelopes disappeared (Fig. 4C, I and O). However, by the time of the metaphase/
anaphase transition, mitoses in the
cyb-1(RNAi)
and
cyb-3(RNAi)
embryos diverged from
the wild-type and from each other (Fig. 4D, J and P).
When examining wild-type embryos with Nomarski optics, the metaphase-aligned
chromosomes are invariably visible during the first mitotic division (Fig. 4C). Chromosome
alignment in metaphase is followed immediately by separation of the sister chromatids and
migration of sets of chromosomes to the poles during anaphase (Fig. 4D). Coincident with
chromosome segregation, the entire DNA/spindle complex migrates with rocking
movements to a more posterior position (reviewed in Strome and White).41 Subsequently,
cytokinesis occurs in a plane midway and perpendicular to the spindle, thereby generating
two cells of unequal size (Fig. 4E).
The mitotic metaphase plate appeared less well defined in
cyb-1(RNAi)
embryos (Fig. 4I).
Segregation of the DNA to opposite poles was initiated without detectable delay; however,
the metaphase and anaphase chromosomes continued to be less distinct. By late telophase,
multiple nuclei often appeared within a single cell (Fig. 4K and L). To further examine
mitotic defects in
cyb-1(RNAi)
embryos, we stained fixed embryos for tubulin and DNA
(Fig. 5). The mitotic spindles appeared normal in these embryos and DNA condensation
occurred at least partially (Fig. 5B). However, chromosomes often failed to align at the
metaphase plate, separated as diffuse masses during anaphase and formed more than two
nuclei during telophase. In cells containing multiple nuclei, the amount of DNA in each
nucleus varied considerably (data not shown). The mitotic defects observed are not
secondary consequences of an abnormal meiosis as similar chromosome congression defects
were detected in embryos that completed apparently normal meiosis I and II and extruded
two polar bodies (data not shown). These results suggest that
cyb-1
function is required for
full condensation and proper congression of the chromosomes at the metaphase plate.
The defects observed in
cyb-3(RNAi)
embryos were clearly distinct from those in
cyb-1(RNAi)
embryos. The formation of pronuclei, pronuclear migration, rotation and
bipolar spindle formation all proceeded with some delay (Fig. 4Y), mitosis was initiated and
a metaphase plate formed (Fig. 4M–O). However, sister chromatids failed to separate in
cyb-3(RNAi)
embryos (anaphase or telophase figures were not seen in any of the 88
embryos observed). The spindle movements were prolonged and more vigorous, apparently
attempting to separate the DNA. Exit from mitosis was significantly delayed in these
defective embryos (anaphase onset: 21.75 ± 1.40 vs. 13.22 ± 0.79 min in wild type).
Cytokinesis was initiated in the absence of chromosome segregation in all embryos, but the
cleavage furrow regressed upon unsuccessful cleavage in the majority of the embryos (Fig.
4P and Q). Duplication and separation of the centrosomes continued, giving rise to
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multipolar spindles (Figs. 4Q and 5C). Following abortive mitosis, DNA replication
eventually continued and
cyb-3(RNAi)
embryos usually arrested with a single polyploid
DNA mass and multiple centrosomes. Again, indistinguishable mitotic defects were
observed in embryos that did or did not complete meiosis, indicating that these defects are
not secondary consequences of an abnormal meiosis. The absence of sister chromatid
separation following
cyb-3
RNAi clearly indicates requirement of
cyb-3
in this process.
In summary, RNAi of
cyb-1
as well as
cyb-3
results in embryonic lethality due to failure of
a fundamental aspect of chromosome segregation. Because both cyclins are essential and
because their loss-of-function affects different aspects of mitosis, we conclude that CYB-1
and CYB-3 exert distinct mitotic functions.
CYB-1 and CYB-3 also have redundant functions
While RNA interference of
cyb-1
and
cyb-3
each produced a distinct mitotic defect, neither
of these defects was as severe or as early as those caused by loss of CDK-1. In
cdk-1(RNAi)
embryos, meiotic chromosome segregation was completely absent, paternal and maternal
pronuclei were formed but migrated slowly and the embryos entered a stable arrest upon
meeting of the pronuclei and prior to nuclear envelope breakdown.35 We inactivated both
cyb-1
and
cyb-3
simultaneously to examine whether these genes act redundantly in meiosis
and the initial mitotic events (Fig. 4S–X). Although meiosis was defective in the
cyb-1(RNAi); cyb-3(RNAi)
embryos nearly all of the embryos (131/134) expelled a single
polar body. Pronuclear migration was severely delayed, requiring up to three times the time
of migration in the wild-type embryo, and many embryos did not complete rotation of the
joined pronuclei. All embryos arrested prior to the first division upon meeting of the
pronuclei (Figs. 4X and Y; 5D). Most embryos contained multiple spindle asters but bipolar
spindles were not formed. In approximately half of the embryos (15/33) the paternal
pronucleus failed to decondense. In a substantial fraction of the embryos (17/103) the
paternal pronucleus migrated to meet with the maternal pronucleus in the anterior of the
embryo, opposite of wild-type migration. The fact that
cyb-1 cyb-3
double RNAi results in
more severe and earlier defects than either single RNAi indicates that these cyclins have
overlapping functions in addition to the specific roles described above.
All mitotic defects observed in the
cyb-1 cyb-3
double RNAi embryos were also seen in
cdk-1(RNAi)
embryos.35 However, upon
cyb-1 cyb-3
double RNAi meiosis I was still
completed (Fig. 6E), in contrast to
cdk-1(RNAi)
embryos (Fig. 6B). This might indicate that
CDK-1 functions with yet other cyclins during the first meiotic division. The defects
observed in
cyb-2.1/cyb-2.2(RNAi)
embryos resembled those in
cyb-1(RNAi)
embryos
(data not shown). As described above, we cannot exclude that the
cyb-2.1/cyb-2.2
phenotype results from co-inhibition of
cyb-1
. However, RNAi for
cyb-2.1/cyb-2.2
further
increased the severity of the
cyb-1; cyb-3
RNAi phenotype. Knockdown of all four mitotic
cyclins fully phenocopied the
cdk-1(RNAi)
phenotype: the condensed chromosomes
remained in typical diakinesis arrangement after fertilization and a meiotic spindle failed to
form (Fig. 6F). Exit from meiosis occurred with normal timing (formation of a maternal
pronucleus 28 min. post fertilization). The maternal pronucleus migrated slowly towards the
posterior and met with the paternal pronucleus, after which the fertilized egg remained
stably arrested. We conclude that the different B and B3 cyclins act in part redundantly in
meiotic and mitotic M phase, in addition to unique cyclin B versus cyclin B3 functions.
Discussion
All eukaryotes express a series of cyclins that are positive regulators of cell division and
likely are derived from a single ancestor.1,2 Specific cyclins accumulate during different
phases of the cell cycle, and thus they can activate CDK partners at different times. Some
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cyclins show specificity for distinct CDKs, which allows them to act in specific processes.
However, it is less clear if and how different cyclins target the same CDK to different
substrates. Although several in vitro studies have indicated substrate specificity for distinct
cyclin-CDK complexes, data from in vivo studies are limited, in particular for mitotic
cyclins.
Here we show that the
C. elegans
mitotic cyclins CYB-1 and CYB-3 are present in dividing
cells at overlapping subcellular locations and with similar cell cycle profiles, and that both
cyclins form active complexes with the CDK-1 catalytic subunit. Despite these similarities,
each single cyclin is essential and their inactivation results in specific mitotic defects. Loss
of
cyb-1
function leads to defects in alignment of the chromosomes at the metaphase plate,
while inactivation of
cyb-3
prevents segregation of sister chromatids. Mitosis is prevented
altogether when both cyclins are inactivated simultaneously, resembling the mitotic, but not
meiotic,
cdk-1(RNAi)
phenotype. Together, these results demonstrate that
C. elegans
cyclins
CYB-1 and CYB-3 act in the regulation of mitosis, with overlapping functions in promoting
initiation of mitosis and distinct roles in the execution of the chromosome segregation
process.
Whether cyclins CYB-2.1 and CYB-2.2 have distinct contributions in mitosis is currently
uncertain. Injection of dsRNA corresponding to a small unique fragment of
cyb-2.1
/
2.2
did
not cause any apparent abnormalities. Injection of a large dsRNA fragment caused defects
similar to
cyb-1
RNAi, but this also reduced CYB-1 levels. While uncertain for mitosis,
cyb-2.1
/
2.2
clearly contribute to meiosis. Meiotic chromosome segregation was entirely
blocked only when
cyb-2.1
/
2.2
RNAi was combined with
cyb-1
and
cyb-3
RNAi. Thus,
CYB-2.1/2.2 can drive partial progression through meiosis in the absence of CYB-1 and
CYB-3, but it cannot do so during the subsequent mitotic division.
A specific function in cell cycle progression may have been expected, at the least for cyclin
B3. Cyclin B3 contains sequence motifs of both A- and B-type cyclins and is considered a
distinct subfamily of mitotic cyclins, although slightly more closely related to the B
subfamily.32 B3 cyclins are conserved from
C. elegans
to mammals and, in contrast to other
cyclins, are encoded by single genes.27 In any given species, B3 cyclins are more closely
related to B3 cyclins in other metazoans than to A- and B-type cyclins within the same
species (Fig. 1B). The selective pressure for conservation of a distinct B3 cyclin throughout
metazoan evolution may be expected to indicate some functional specificity.
On the other hand, the initial characterization of cyclin B3 in Drosophila revealed
predominantly overlapping functions with cyclin B. Paradoxically, the Drosophila cyclins
appear to behave more dissimilarly than their
C. elegans
counterparts. Accumulation and
destruction of Cyclin B3 in Drosophila succeeds that of cyclin A and cyclin B.39 In contrast,
CYB-1 and CYB-3 seemed present during the same part of the cell cycle and both
disappeared very close to the onset of chromosome segregation. Drosophila cyclin B3 is
exclusively nuclear, in contrast to cyclin A and B, which accumulate in the cytoplasm and
translocate to the nucleus.6
C. elegans
CYB-3 is also mainly or exclusively nuclear, while
substantial levels of CYB-1 are present in the cytoplasm. However, we have not observed
cell cycle dependence in the nuclear translocation of CYB-1. Expression of truncated
Drosophila cyclins lacking the destruction box motif also affected mitotic progression in
specific ways: Δcyclin A caused metaphase delay, Δcyclin B early anaphase arrest and
Δcyclin B3 late anaphase arrest.39 We have been unable to arrest mitosis in
C. elegans
by
expression of truncated cyclins, possibly because the required expression levels were not
reached.
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A recent study addressed the cyclin contribution to rapid syncytial divisions of Drosophila
embryos,16,42 based on injection of dsRNA and time-lapse fluorescence microscopy. At this
time of development, the functional similarities between Drosophila CycB and
C. elegans
CYB-1, as well as Drosophila CycB3 and
C. elegans
CYB-3 are striking. Knockdown of
only CycB interfered with chromosome congression in metaphase, while CycB3 depletion
disrupted the transition to anaphase. A critical difference is the contribution of cyclin A:
while CycA is a major mitotic cyclin in Drosophila, we did not detect a role for its closest
C.
elegans
homolog, CYA-1, in embryonic divisions. The Drosophila studies by Jacobs et al.6
and McCleland et al.42,43 demonstrate that the maternal contribution of CycB and CycB3 is
more critical than their zygotic functions. We focused our analysis on the contribution of B
and B3-type cyclins in meiosis and the first embryonic divisions, which require maternal
functions. Although beyond the scope of this study, cyclin knockdown through soaking
RNAi of first stage larvae and examination of deletion mutants showed that
cyb-3
and, to a
lesser extent,
cyb-1
are also essential for progression through mitosis during larval
development (data not shown).
If their temporal and spatial expression is similar, what is the critical difference between
CYB-1 and CYB-3? Cyclins positively regulate CDKs by changing their conformation to an
active state and by contributing to the docking of substrates. The crystal structures of several
CDK complexes have provided insights into these cyclin roles.44 Studies of the cyclin A/
Cdk2 structure have shown that cyclin binding changes the position of the so-called T loop
in the CDK, so that it no longer blocks access to the catalytic cleft. In addition, upon binding
to cyclin, the CDK α-helix that includes the conserved PSTAIRE domain changes position,
so that a critical glutamic acid in this domain can contribute to coordinating the phosphate
atoms of Mg2+ATP.44,45
These roles in CDK activation are thought to be universal and are most likely accomplished
by CYB-1 as well as CYB-3. However, specificity may be provided at the level of substrate
interaction. The crystal structure of a cyclin A/Cdk2/p27Kip1 complex has revealed contact
sites between cyclin A and p27Kip1.46 A hydrophobic patch on the cyclin A surface contacts
residues of the RXL motif present in these substrates, which is essential for phosphorylation
of RXL-containing substrates but not for histone H1 phosphorylation.18 The hydrophobic
pocket contains the MRAIL sequence and is conserved in CYB-1: including the critical
residues M143, L147, W150, Q188 and L189 (Marked in Fig. 1A by asterisks).
Surprisingly, two of the most critical hydrophobic residues, M143 and L189, are changed to
hydrophilic residues T and K, respectively, in CYB-3 (Fig. 1A). Although additional
domains may be involved, these differences between CYB-1 and CYB-3 probably affect
substrate specificity, as they concern the only substrate-docking site in cyclins known to
date.47
A compelling question remains as to what specific substrates are phosphorylated by the
CYB-1 and CYB-3/CDK-1 kinases. The loss-of-function phenotypes of these cyclins evoke
some interesting candidates. The
cyb-1(RNAi)
phenotype is consistent with partially
penetrant defects in chromosome condensation and/or microtubule-kinetochore attachments.
A number of critical proteins have been identified in these processes (e.g., Desai et al.).48
However, the fact that most cell divisions complete successfully in
cyb-1(RNAi)
embryos
indicates that the actual defects could be subtle. In contrast, lack of sister-chromosome
separation in
cyb-3(RNAi)
embryos is a specific and fully penetrant defect. Most mitotic
processes in these embryos either occur normally or fail subsequent to chromosome-
separation failure. For chromosome segregation to occur, cohesin molecules that hold sister
chromatids together need to be degraded. This process involves several sequential steps:
activation of the anaphase-promoting complex (APC) leads to degradation of Securin, which
allows the protease Separase to become active and to cleave the Scc1 Cohesin subunits.
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Phosphorylation by a CYB-3/CDK-1 kinase could regulate any step in this process.
Interestingly, in yeast, Cdc28-dependent phosphorylation of APC components is required
specifically to promote the Cdc20-dependent (anaphase promoting) activity of the APC, and
not for its G1 activity.49 Future studies may reveal whether this function is exerted
specifically by CYB-3/CDK-1 in
C. elegans
.
Materials and Methods
Culture conditions and strains
C. elegans
strains were cultured using standard techniques as described by Brenner50 and
were derived from the wild-type Bristol N2 strain. Strain SV1010 was used for live-imaging
(
ruIs57[unc-119
(+)
pie-1
-GFP-α-tubulin
]; itIs37[unc-119
(+)
pie-1
-mCherry-H2B] (may
contain unc-119 (eds) III)).
Isolation of cyb-2.1 and cyb-2.2 cDNAs
The genomic and cDNA sequences of
cyb-1
and
cyb-3
were previously reported.23 The
C.
elegans
genome project revealed two additional B-type cyclin genes: Y43E12A.1/
cyb-2.1
located 0.1 map units to the right of
cyb-1
(+4.78, chromosome IV), and H31G24.4/
cyb-2.2
located at −1.65 mu on chromosome I. A 1.5 kb
cyb-2.1
cDNA was kindly provided by Yuji
Kohara (National Institute of Genetics, Mishima, Japan). DNA sequence analysis confirmed
the open reading frame as predicted by GENEFINDER. A partial cDNA from
cyb-2.2/
H31G24.4 was obtained by reverse transcriptase/PCR amplification. Briefly, 1 μg of
poly(A)+ RNA from mixed stage animals was primed using 1 nmol/μl random hexamer
primers and copy DNA synthesized with reverse transcriptase. The cDNA was used in
standard PCR amplification reactions, cloned into pBSK and examined by DNA sequence
analysis.
Antibody production
Full-length
cyb-3
and
cyb-1
cDNAs were cloned into the pET19b expression vector
(Novagen) and expressed in
E. coli
. The purified His-tagged CYB-1 and CYB-3 proteins
were injected into mice and rabbits according to standard procedures.51 The specificity of
the polyclonal antisera obtained was further improved by adsorption to bacterial proteins
and affinity purification. The anti-CYB-1 serum initially contained antibodies recognizing a
centrosomal protein, which were removed during the purification.
Western blot analysis and immunohistochemistry
C. elegans
protein extract was obtained from developmentally staged wild-type animals.
Protein samples were separated on a 10% polyacrylamide gel using SDS-PAGE and the
protein was immunoblotted using standard procedures.51 Mouse monoclonal anti-α-Tubulin
antibodies N356 and DM1A (1:3,000, Sigma) were used as loading controls.
Immunostaining of
C. elegans
embryos was as described.35 Antibodies used for these
studies are listed with their respective dilutions: mouse monoclonal anti-α-Tubulin DM1A
(1:100; Sigma), mouse polyclonal anti-CYB-3 (1:20), rabbit polyclonal anti-CYB-3 (1:200),
rabbit polyclonal anti-CYB-1 (1:20), rabbit polyclonal anti-CDK-1/NCC-1 1755 (1:100).
Secondary FITC- or Texas Red-conjugated antibodies were used at 1:100 dilutions (Jackson
ImmunoResearch Laboratories). DNA was stained with 1 μg/ml 4,6-diamidino-2-
phenylindole (DAPI; Sigma). Samples were mounted on slides in Prolong Antifade Gold.
van der Voet et al. Page 10
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Immunoprecipitation and H1 kinase assays
Lysates for immunoprecipitations were obtained from
C. elegans
embryos, lysed and
sonicated in lysis buffer (10 mM HEPES pH 7.4, 0.5 mM EDTA, 2.5 mM EGTA, 2.5 mM
MgSO4, 15% glycerol, 250 mM NaCl, 0.5% Triton X-100, 1 μg/ml aprotinin, 1 μg/ml
leupeptin, 1 μg/ml trypsin inhibitor, 1 mM NaF and 2 mM PMSF). CYB-1, CYB-3 and
CDK-1 were immunoprecipitated from embryonic lysates containing 500 μg total protein,
using 20 μl Protein A beads cross-linked to antibody. The beads were washed 4X in lysis
buffer. Half of the beads were boiled in 2X sample buffer, followed by SDS-PAGE, western
blotting and probing with the anti-PSTAIRE monoclonal antibody (1:2,000 dilution). The
remaining beads were washed 2X in kinase buffer (50 mM HEPES pH 7.4, 25 mM MgCl2,
5 mM DTT) and incubated in a 50 μl reaction mix containing kinase buffer, 1 μg histone H1
(Gibco) and 5 μCi [γ-32 P]ATP for 30 minutes at 30°C. The beads were boiled in 2X
sample buffer and the supernatant was run on a 12% SDS polyacrylamide gel. Preimmune
rabbit serum was used as a negative control.
Isolation of synchronized developmental stages
To obtain protein samples representing the developmental stages of the worm, synchronized
C. elegans
wild-type animals were isolated from eggs, larval stages 1–4, adults, gravid
adults and developmentally-arrested dauer larvae. Eggs were obtained by hypochlorite
treatment of gravid adults. For L1 arrested animals, eggs were hatched in M9 in the absence
of food for 24 hours and then harvested. For L1, L2, L3, L4, adult and gravid adult animals,
embryos were hatched in the presence of food and incubated at 20°C for 10, 20, 29, 39, 52
and 70 hrs., respectively, and then harvested. All stages were analyzed by microscopy before
harvesting to ensure proper development. For protein lysate, animals staged above were
collected, an equal volume of 2X Laemmli buffer was added and proteins were released by
boiling for 5 minutes.
RNA interference
Plasmids used for in vitro transcription contained full-length
cyb-1
,
cyb-2.1, cyb-2.2, cyb-3
or
lin-5
cDNA, a 260 bp
cyd-1
fragment, corresponding to nucleotides 11–270 of the open
reading frame (ORF), or a 107 bp fragment of
cyb-2.2
, corresponding to nucleotides 53–160
of the ORF. Templates were linearized and transcribed using T3 or T7 polymerase
(Ambion). RNA was phenol extracted, ethanol precipitated and dissolved in 1X injection
buffer (20% polyethylene glycol 8000, 200 mM KPO4 pH 7.5, 30 mM KCitrate pH 7.5) to a
final concentration of ~1 mg/ml. The antisense and sense transcripts were annealed and
injected into early adult N2 hermaphrodites as described by Fire et al.22 Injected wild-type
animals were singled onto plates and transferred every 12 hours to score the entire brood.
For western blot analysis, synchronized L4 larvae were soaked in a
cyb-1
or
cyb-3
dsRNA
solution for 24 hours at 20°C, transferred to a plate with food to accumulate eggs, followed
by hypochlorite treatment to harvest embryos. For larval RNAi, L1 animals synchronized in
the absence of food were incubated in
cyb-1
or
cyb-3
dsRNA solution for 24 hours before
placing them on plates with food.
Nomarski observations and recordings
Meiotic divisions were followed in utero after anesthetization of adult worms with 0.1%
tricaine and 0.01% tetramisole on 2% agarose pads. Early embryonic events were recorded
using time-lapse video microscopy typically from pronuclear formation to the four-cell stage
(one image every 10 seconds). Embryos from either wild-type or dsRNA-injected
hermaphrodites were dissected and mounted onto agarose pads as described.52
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Microscopy and image acquisition
A Zeiss Axioplan II microscope was used for Nomarski and immunofluorescence
microscopy. Cell divisions were recorded with a 100x 1.4 numerical aperture
PlanApochromat objective lens on a motorized microscope (Zeiss Axioplan). Meiotic
divisions were recorded with a 30-sec interval with 400 ms fluorescent exposure.
Fluorescent excitation light was filtered to 10% transmission with neutral density filters, and
binning was set to 2 × 2 with automatic gain adjustments. Microinjections were performed
using a Nikon inverted microscope and Narishige micromanipulator.
Acknowledgments
We thank Andy Golden for CDK-1/NCC-1 specific antibodies, Masakane Yamashita for anti-PSTAIRE antibodies,
and Yuji Kohara for
cyb-2.1
cDNA. We acknowledge Mike Boxem for critically reading the manuscript. This work
was supported by the NIH, RO1 GM057990 to S.v.d.H., by a postdoctoral fellowship from the Medical Foundation
to M.A.L., and by a William Starr Fellowship to D.G.S.
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Figure 1.
Distinct mitotic cyclin A, B and B3 genes are conserved in
Caenorhabditis elegans
. (A)
Alignment of
C. elegans
CYA-1, CYB-1, CYB-2.1, CYB-2.2 and CYB-3 coding sequences
with ClustalW2 and BoxShade. Asterisks (*) indicate predicted residues of the hydrophobic
patch involved in substrate docking. Note that Met144 and Leu191 are not conserved in
CYB-3 (B) Phylogenetic tree of the predicted A-, B- and B3-type cyclins from
C. elegans
(Ce)
,
D. melanogaster (Dm), X. laevis (Xl)
and B-type cyclins of
S. cerevisiae (Sc)
visualized with Phylip DrawTree. Note that the B3-type cyclins are more closely related to
cyclin B3 in other species than to other A- and B-type cyclins within the same species.
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Figure 2.
CYB-1 and CYB-3 bind CDK-1, show associated H1 kinase activity in vitro, and are
expressed coincident with cell division. (A) Western blot showing that CYB-1 and CYB-3
antisera are specific. Lanes contain total embryonic protein lysate, obtained after soaking
adults in
cyb-1
(left),
cyb-3
(middle) or
lin-5
(right, control) dsRNA. CYB-1 (top), CYB-3
(middle) or anti-α-tubulin antibodies (bottom; loading control) were used for detection. (B)
CYB-1 and CYB-3 associate with CDK-1 and (C) form active kinase complexes. Total
lysate or immunoprecipitates with the indicated antibodies were used. (D) CYB-1 and
CYB-3 protein expression correlate with cell division during development. Protein lysates of
synchronized wild-type animals were immunoblotted and probed with the indicated anti-
cyclin antibodies. Larval stages are shown. L1 0 hr: developmentally arrested first stage (L1)
larvae; L1 10 hr: L1 larvae 10 hr after stimulation of development by food addition. α-
Tubulin protein levels serve as a loading control. (E)
cyb-1
and
cyb-3
RNAi specifically
reduce expression of the corresponding proteins. Wild-type or RNAi-treated embryos were
triple-stained for DNA (DAPI, left), CYB-1 (middle) and CYB-3 (right). Anterior is to the
left, scale bar approx. 10 μm.
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Figure 3.
CYB-1 and CYB-3 show largely overlapping protein localizations. (A and B) CYB-1 and (C
and D) CYB-3 localization are cytoplasmic in meiosis. (E–H) Following the completion of
meiosis, CYB-1 and CYB-3 localize to the maternal pronucleus in the anterior (left arrow)
and paternal pronucleus (right arrow). CYB-1 in particular is also present in the cytoplasm.
(I–L) CYB-1 and CYB-3 remain present in the cytoplasm and nucleus during prophase, but
are undetectable in anaphase. Two-cell embryos in which the anterior AB cell is in anaphase
(arrowhead) and the posterior P1 cell in prophase (arrow). CYB-1 and CYB-3 are present in
prophase but not in anaphase. (M–P) Similar but somewhat later embryos, in which P1 is in
metaphase. Embryos were stained for DNA (DAPI) and anti-CYB-1 or CYB-3 antibodies.
Anterior is to the left, scale bar approx. 10 μm.
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Figure 4.
Mitotic defects associated with CYB-1 and CYB-3 knockdown. (A–F) Development of a
wild-type embryo from pronuclear formation until the four-cell stage. The metaphase plate
is indicated by a single arrow; segregating chromosomes in anaphase by the double arrows.
(G–L)
cyb-1(RNAi)
embryo. Alignment of the chromosomes at the metaphase plate is
incomplete; however, chromosome segregation continues, frequently followed by the
formation of multiple nuclei within a single cell. Arrows in (K and L) show the formation of
multiple nuclei in the posterior P1 cell following the first mitosis and in the EMS cell after
division of P1. (C)
cyb-3(RNAi)
embryo. Black arrows point to metaphase-aligned
chromosomes, white arrow point to furrow ingression (in P) and spindle poles (in Q). Sister
chromatids fail to separate (note the expanded time in the right image). In most embryos, a
cleavage furrow forms, fails to complete abscission and subsequently regresses (D)
cyb-1;cyb-3
double RNAi embryo arrests prior to initiation of the first mitosis. A (single)
polar body is expelled during meiosis and the pronuclei move together slowly. No further
development is observed. Selected images are from time-lapse DIC recordings of living
embryos. Scale bar approx. 10 μm. (Y) Timing of events following meiosis, appearance of a
maternal pronucleus was taken as
t =
0. Wild-type (n = 3),
cyb-1
RNAi (n = 3),
cyb-3
RNAi
(n = 4),
cyb-1;cyb-3
RNAi (n = 6). (1) 5/6
cyb-1;cyb-3
RNAi embryos failed pronuclear
centration, (2) 5/6
cyb-1;cyb-3
RNAi embryos failed pronuclear rotation, (3) 1/4
cyb-3
RNAi embryos failed cytokinesis.
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Figure 5.
Distinct mitotic defects in
cyb-1(RNAi), cyb-3(RNAi)
and
cyb-1-;cyb-3
double RNAi
embryos. Images show similar stage embryos, double-stained for DNA (left) and α-tubulin
(middle). Merged images are to the right. (A) wild-type two-cell embryo, with bipolar
spindles and AB cell in anaphase (left) and P1 in metaphase (right). (B) two-cell stage
cyb-1(RNAi)
embryo, arrows indicate unequal DNA segregation in anaphase of AB (see
arrow), and a presumed lagging chromosome in metaphase of P1 (right cell, arrow). (C)
cyb-3(RNAi)
embryo demonstrating lack of chromosome segregation, with continued
spindle poles duplication. (D)
cyb-1;cyb-3
double RNAi embryo arrested before fusion of
the maternal and paternal pronuclei. Anterior is to the left, scale bar 10 μm.
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Figure 6.
Time-lapse fluorescent microscopy demonstrates meiotic defects. Images show still-shots of
meiotic time-lapse movies in utero. (A) wild-type meiosis I and II; chromosomes align at a
metaphase spindle (
t
= 5:30; 19:30), the spindle rotates by 90° (
t
= 7:30; 23:00), and
segregates the chromosomes during anaphase (
t
= 11.30; 28.00). (B) RNAi of
cdk-1
.
Chromosomes remain in a diakinesis arrangement with limited microtubule organization, the
spindle fails to form, but exit from meiosis happens with normal timing. (C) RNAi of
cyb-1
results in meiotic defects in chromosome alignment and segregation (white arrow). (D)
RNAi of
cyb-3
results in a substantial delay in metaphase of meiosis II. (E) Double RNAi of
cyb-1
and
cyb-3
results in a dramatic meiosis II defect, while meiosis I is delayed but still
completes. (F) Triple RNAi of
cyb-1, cyb-2.1/2.2, cyb-3
results in diakinesis arrest, similar
to
cdk-1
RNAi. Red: H2B::Cherry, green: α-tubulin::GFP. Time after entry into the uterus is
indicated in each panel. Scale bar is 2 μm, the future anterior cortex is to the left.
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