Biology Reference
In-Depth Information
FIGURE 14.9
Bistability at the G2/M
transition in frog egg extracts. From Sha et al.
[85]
; used by permission. Cytoplasmic extracts
of frog eggs are used to measure the amount of
cyclin B necessary to induce or sustain the
kinase activity of CycB:Cdk1 heterodimers
(referred to in this paper as MPF, mitosis
promoting factor). Sperm nuclei in the extract
(stained blue for chromatin) are photographed
to stage the extracts. Interphase: round nuclei,
dispersed chromatin, intact nuclear membrane,
low activity of MPF (confirmed in separate
experiments, not shown). Mitosis: highly
condensed chromatin, no nuclear membrane,
high activity of MPF. (A) Cyclin threshold for
activation of MPF. Extracts are prepared in
interphase (t
¼
0) in the presence of cyclohex-
imide, to block all protein synthesis, including
the synthesis of endogenous cyclin B. Samples
of the extract are injected with increasing
amounts of non-degradable cyclin B
(CycB
D
90) at t
¼
0 and photographed at
intervals thereafter (t
¼
90 min time point
shown here). Extracts containing 0
e
32 nM CycB
D
90 have insufficient MPF activity to enter mitosis, but 40 nM CycB
D
90 or larger is enough to activate
MPF and drive the nuclei into mitosis. (B) Cyclin threshold for inactivation of MPF. Extracts are prepared in interphase (t
¼
0) in the absence of
cycloheximide and injected with increasing amounts of CycB
D
90. Whether the injected amount is small or large, synthesis of endogenous cyclin B drives
the extract into mitosis by t
¼
60 min. At t
¼
60 min the extracts are treated with cycloheximide to prevent any further synthesis of endogenous cyclin B.
As the extracts try to exit from mitosis, they activate Cdc20 and degrade the endogenous cyclin B proteins but leave behind the non-degradable CycB
D
90
molecules. Extracts containing 24 nM CycB
D
90 or higher retain MPF in the active form and block the nuclei in mitosis. Extracts containing 16 nM
CycB
D
90 or lower have inactive MPF (confirmed in separate experiments, not shown) and return to interphase. For CycB
D
90 concentrations in the range
24
e
32 nM, the MPF control system is bistable: it can persist in an MPF-inactive state (panel A) or in an MPF-active state (panel B) under identical
conditions on cyclin B concentration in the extract. Which state the extract adopts depends on whether it was initially in the MPF-inactive state (above) or
in the MPF-active state (below).
FIGURE 14.10
Additional checkpoints in late G1 and telo-
phase. Left: a cell that has passed the restriction point (aka, Start)
can still be blocked in late G1, with high levels of starter kinase, if
a new checkpoint is created by a saddle-node bifurcation at the far
left. Right: similarly, a cell that has completed the M/A transition
can still be blocked in telophase (T), if a new checkpoint is created
by a saddle-node bifurcation at the far right.
These checkpoints are imposed and lifted by saddle-node
bifurcations; hence, just like the restriction point and exit
from mitosis, they are dynamically irreversible transitions.
On the other hand, unlike RP and exit, they are not universal.
For instance, in contrast to budding yeast, mammalian cells
do not have a telophase checkpoint. In mammalian cells, as
in most cells, the division plane is determined late in the cell
cycle, by the location of the mid-zone of the late anaphase
spindle (which is the position where the metaphase plate
assembled). Hence, as long as the M/A transition has been
successfully completed, then cell division will automatically
separate the two new daughter nuclei. There is no need for
a telophase checkpoint, as in budding yeast, where exit from
mitosis occurs in two stages.
Furthermore, the DNA damage checkpoint in budding
yeast works completely differently than in mammalian
cells. Damage in G1 phase causes a specific phosphoryla-
tion of Swi6 that blocks transcription of Cln2, the starter
