16
Xenopus
to contain a specifc sequence named the destruction- or
d-box required for their turnover (Glotzer et al., 1991).
Cdh1-dependent substrates were found to have a different
degradation-ensuring sequence motif, or “degron,” called
the KEN box, named for the Lysine (K)–Glutamic Acid
(E)–Asparagine (N) sequence required to signal for degradation ( Pfeger and Kirschner, 2000). The number of identifed APC substrates has continued to grow, suggesting
involvement of the APC in diverse intracellular pathways
of both actively dividing and post-mitotic cells (reviewed in
(Davey and Morgan, 2016)). Characterization of the APC/
Cyclosome is ongoing and continues to shape how we think
about the cell cycle and its regulation.
2.3.1. PAUSING THE CELL CYCLE
While the cell cycle is remarkably processive in egg extracts,
there are distinct pause points that have been documented
and used to better understand cell cycle control. In mature
Xenopus eggs, the cell cycle is arrested in metaphase of
meiosis II. This arrest is dependent on a factor called cytostatic factor (CSF), an activity that proved very challenging
to def ne at the molecular level but was ultimately identif ed
using Xenopus models. CSF, originally identifed in Rana
eggs, is an activity that results in cell cycle arrest following transfer of small amounts of egg cytoplasm into blastomeres of a cleavage stage embryo (Masui and Markert, 1971).
Cytological examination of the blastomeres of similarly
treated Xenopus embryos showed fully developed spindles
and condensed chromosomes in the arrested cells, consistent
with M phase arrest (Moses and Masui, 1989). The cytoplasmic transfer experiment proved that the activity of CSF is
dominant, causing cycling blastomeres to arrest with high
MPF levels at the next M phase following injection. The fact
that CSF could arrest post-fertilization embryos suggested
that CSF, while normally restricted to meiotic cells, was
able to arrest the mitotic cell cycle equally well. During CSF
arrest, the APC/C is inhibited, preventing degradation of
Cyclin and thus exit from M phase. Experiments over a number of years ultimately showed that CSF arrest requires both
the MOS-MAPK signaling that occurs during oocyte maturation (reviewed in Tunquist and Maller, 2003) and a protein
called Emi2/xErp1 (Tung et al., 2005). Emi2 binds to and
inhibits the APC/C and is degraded upon egg fertilization.
CSF transfer can both arrest cleavage-stage embryos and be
used in vitro to arrest egg extracts in M phase. Simply adding CSF arrested extract (prepared from unfertilized eggs) to
cycling extract results in CSF arrest of the recipient extract.
This approach has practical applications such as in the study
of mitotic chromosomes and spindles following DNA replication in vitro (Silva and Rankin, 2018; Song et al., 2012).
In somatic cells, the cell cycle can be paused or arrested
during mitotic divisions, and Xenopus models have helped
elucidate these mechanisms. The spindle checkpoint is a surveillance mechanism that prevents activation of the APC/C
(and therefore mitotic exit) in the presence of unattached
chromosomes. This mechanism was frst characterized in
budding yeast (Li and Murray, 1991), but critical experiments
in Xenopus showed that the mechanism was conserved in
vertebrates. Chen et al. showed that the checkpoint can be
triggered in egg extracts by the presence of high numbers
of chromosomes and the microtubule poison nocodazole,
which results in checkpoint signaling from unattached
kinetochores (Chen et al., 1996). They also conf rmed that
the frog ortholog of the yeast Mad2 protein was critical to
the arrest and that the Mad2 protein accumulated at the
unattached kinetochores. Mad2 activity was further characterized in egg extracts and proved to be an inhibitor of
the APC/C (Fang et al., 1998b; Li et al., 1997 ). The logic
of the pathway is clear: unattached kinetochores prevent
exit from mitosis by preventing Cyclin degradation. Once
chromosome attachments are made, the signaling pathway
is turned off, and mitotic exit can occur, ensuring accurate
chromosome segregation.
There are also checkpoints that arrest the cell cycle in
interphase in response to DNA damage or incomplete DNA
replication. These mechanisms, which prevent mitotic entry
through signaling cascades that inhibit activation of MPF,
have been elucidated using Xenopus egg extracts and are
nicely reviewed elsewhere (Cupello et al., 2016; Garner
and Costanzo, 2009; Hoogenboom et al., 2017; Lin et al.,
2019; Lupardus et al., 2007; Peng et al., 2008; Smythe and
Newport, 1992). Interestingly, certain checkpoints appear
not to be fully active in Xenopus development until the early
cleavage cycles are over. The weakness of these checkpoints
in eggs and cleavage-stage embryos results in a strikingly
robust cell cycle prior to this time. The accumulation of
nuclei and the resulting change in nuclear-cytoplasmic ratio
that occur following cell cleavage divisions can be replicated in vitro through titration experiments. For example,
signaling from a single nucleus in a microliter of extract (or
an egg) is insuffcient to prevent mitotic entry when DNA
replication is blocked, but titration of nuclei into the same
extract can generate a signifcant signal and prevent M phase
entry. Similarly, the spindle checkpoint from a low concentration of unattached kinetochores does not cause M phase
arrest in egg extract, but addition of a large number of nuclei
with spindle attachment problems generates a robust mitotic
arrest (Chen et al., 1996 ). Thus, though the checkpoints are
not generally active until later in development, the components required are present in the egg and can be stimulated
to act under the certain conditions.
2.4. SYSTEMS BIOLOGY OF THE CELL CYCLE
Xenopus eggs and embryos have not only provided a system
to identify components that make up the cell cycle engine
but have also been instrumental in the development of mathematical models for how cell cycle transitions are controlled
and the feedback mechanisms that ensure switch-like transitions from interphase to M phase and back (Kim and Ferrell,
2007; Novak and Tyson, 1993; Pomerening et al., 2005;
Solomon et al., 1990). The ideas proposed by this modeling have been tested and validated using egg extract (Kim
Xenopus
to contain a specifc sequence named the destruction- or
d-box required for their turnover (Glotzer et al., 1991).
Cdh1-dependent substrates were found to have a different
degradation-ensuring sequence motif, or “degron,” called
the KEN box, named for the Lysine (K)–Glutamic Acid
(E)–Asparagine (N) sequence required to signal for degradation ( Pfeger and Kirschner, 2000). The number of identifed APC substrates has continued to grow, suggesting
involvement of the APC in diverse intracellular pathways
of both actively dividing and post-mitotic cells (reviewed in
(Davey and Morgan, 2016)). Characterization of the APC/
Cyclosome is ongoing and continues to shape how we think
about the cell cycle and its regulation.
2.3.1. PAUSING THE CELL CYCLE
While the cell cycle is remarkably processive in egg extracts,
there are distinct pause points that have been documented
and used to better understand cell cycle control. In mature
Xenopus eggs, the cell cycle is arrested in metaphase of
meiosis II. This arrest is dependent on a factor called cytostatic factor (CSF), an activity that proved very challenging
to def ne at the molecular level but was ultimately identif ed
using Xenopus models. CSF, originally identifed in Rana
eggs, is an activity that results in cell cycle arrest following transfer of small amounts of egg cytoplasm into blastomeres of a cleavage stage embryo (Masui and Markert, 1971).
Cytological examination of the blastomeres of similarly
treated Xenopus embryos showed fully developed spindles
and condensed chromosomes in the arrested cells, consistent
with M phase arrest (Moses and Masui, 1989). The cytoplasmic transfer experiment proved that the activity of CSF is
dominant, causing cycling blastomeres to arrest with high
MPF levels at the next M phase following injection. The fact
that CSF could arrest post-fertilization embryos suggested
that CSF, while normally restricted to meiotic cells, was
able to arrest the mitotic cell cycle equally well. During CSF
arrest, the APC/C is inhibited, preventing degradation of
Cyclin and thus exit from M phase. Experiments over a number of years ultimately showed that CSF arrest requires both
the MOS-MAPK signaling that occurs during oocyte maturation (reviewed in Tunquist and Maller, 2003) and a protein
called Emi2/xErp1 (Tung et al., 2005). Emi2 binds to and
inhibits the APC/C and is degraded upon egg fertilization.
CSF transfer can both arrest cleavage-stage embryos and be
used in vitro to arrest egg extracts in M phase. Simply adding CSF arrested extract (prepared from unfertilized eggs) to
cycling extract results in CSF arrest of the recipient extract.
This approach has practical applications such as in the study
of mitotic chromosomes and spindles following DNA replication in vitro (Silva and Rankin, 2018; Song et al., 2012).
In somatic cells, the cell cycle can be paused or arrested
during mitotic divisions, and Xenopus models have helped
elucidate these mechanisms. The spindle checkpoint is a surveillance mechanism that prevents activation of the APC/C
(and therefore mitotic exit) in the presence of unattached
chromosomes. This mechanism was frst characterized in
budding yeast (Li and Murray, 1991), but critical experiments
in Xenopus showed that the mechanism was conserved in
vertebrates. Chen et al. showed that the checkpoint can be
triggered in egg extracts by the presence of high numbers
of chromosomes and the microtubule poison nocodazole,
which results in checkpoint signaling from unattached
kinetochores (Chen et al., 1996). They also conf rmed that
the frog ortholog of the yeast Mad2 protein was critical to
the arrest and that the Mad2 protein accumulated at the
unattached kinetochores. Mad2 activity was further characterized in egg extracts and proved to be an inhibitor of
the APC/C (Fang et al., 1998b; Li et al., 1997 ). The logic
of the pathway is clear: unattached kinetochores prevent
exit from mitosis by preventing Cyclin degradation. Once
chromosome attachments are made, the signaling pathway
is turned off, and mitotic exit can occur, ensuring accurate
chromosome segregation.
There are also checkpoints that arrest the cell cycle in
interphase in response to DNA damage or incomplete DNA
replication. These mechanisms, which prevent mitotic entry
through signaling cascades that inhibit activation of MPF,
have been elucidated using Xenopus egg extracts and are
nicely reviewed elsewhere (Cupello et al., 2016; Garner
and Costanzo, 2009; Hoogenboom et al., 2017; Lin et al.,
2019; Lupardus et al., 2007; Peng et al., 2008; Smythe and
Newport, 1992). Interestingly, certain checkpoints appear
not to be fully active in Xenopus development until the early
cleavage cycles are over. The weakness of these checkpoints
in eggs and cleavage-stage embryos results in a strikingly
robust cell cycle prior to this time. The accumulation of
nuclei and the resulting change in nuclear-cytoplasmic ratio
that occur following cell cleavage divisions can be replicated in vitro through titration experiments. For example,
signaling from a single nucleus in a microliter of extract (or
an egg) is insuffcient to prevent mitotic entry when DNA
replication is blocked, but titration of nuclei into the same
extract can generate a signifcant signal and prevent M phase
entry. Similarly, the spindle checkpoint from a low concentration of unattached kinetochores does not cause M phase
arrest in egg extract, but addition of a large number of nuclei
with spindle attachment problems generates a robust mitotic
arrest (Chen et al., 1996 ). Thus, though the checkpoints are
not generally active until later in development, the components required are present in the egg and can be stimulated
to act under the certain conditions.
2.4. SYSTEMS BIOLOGY OF THE CELL CYCLE
Xenopus eggs and embryos have not only provided a system
to identify components that make up the cell cycle engine
but have also been instrumental in the development of mathematical models for how cell cycle transitions are controlled
and the feedback mechanisms that ensure switch-like transitions from interphase to M phase and back (Kim and Ferrell,
2007; Novak and Tyson, 1993; Pomerening et al., 2005;
Solomon et al., 1990). The ideas proposed by this modeling have been tested and validated using egg extract (Kim