BC4 – The Cell Cycle Boris Pfander Max Planck Ins9tute of Biochemistry [email protected] +49-­‐89-­‐8578-­‐3050 www.biochem.mpg.de/pfander 1 Topics Overview on the Cell Cycle The discovery of CDK – the master regulator of the cell cycle S-­‐phase and DNA replica9on Checkpoints that sense DNA damage M-­‐phase – mitosis and cytokinesis Chromosome condensa9on & cohesion 2 Cell cycle stages and transitions
G1>S
START
G2>M
metaphase > anaphase
& mitotic exit
3 Mitosis 4 5 5 Phases of Mitosis
6 6 Changes in chromosome morphology during mitosis
Condensin
Cohesin
7 7 Condensin shapes mitotic chromosomes
Condensin
Condensin
DNA
Condensin
Cohesin
P
Condensin
Houlard et al., NCB, 2015
8 Condensin might trap loops of DNA.
Condensin
Cdk1-Cyclin B target
9 Cohesin mediates sister chromatid cohesion.
Cohesin
Cohesin
Cohesin
The cohesin ring model
10 Metaphase > anaphase transition
11 Cyclin levels drop at the metaphase to anaphase
transition
Cdk1 activity drops!
12 Cyclin B levels and (kinase) activity of MPF
change in parallel in cycling Xenopus egg extracts
13 Metaphase to anaphase transition
14 -> exit from mitosis requires degradation of cyclin B
by what mechanism?
Glotzer, Murray & Kirschner, 1991:
1) radio-label cyclin B
↓
incubation in Xenopus egg extracts,
which are in anaphase or interphase
↓
SDS-PAGE and auto-radiography
anaphase
cyclin B
T [min.]:
interphase
↓
extract in:
0
30
0
30
15 2) radio-label cyclin B or ubiquitin
↓
incubation in Xenopus egg extracts,
which are in anaphase or interphase
↓
after 10 min.: SDS-PAGE and auto-radiography
(overexposure!!!)
I125-labeled:
cyclin B
ubiquitin
A
A
x4
x3
ubiquitin x 1
cyclin B
cyclin B
↓ ↓
x2
extract in:
I
I
(A = anaphase; I = interphase)
at the time of its degradation
cyclin B is covalently modified by addition of ubiquitin
è
16 Proteolysis controls late mitotic events
Exit from mitosis requires inactivation of Cdk1 by
degradation of mitotic cyclin
The degradation is mediated by the anaphase-promoting-complex
(APC), an E3 ubiquitin ligase
APC activation requires
MPF activity
(ensures correct order of events)
Cdk1 facilitates its own
inactivation
(mitotic checkpoint and unknown
mechanisms ensure delay
to give Cdk1 enough time to act)
17 Anaphase-promoting complex/cyclosome
An atomic model of APC/C determined by cryo-EM.
By David Barford
APC – a gigantic E3 ubiquitin ligase
18 Metaphase to anaphase transition
and mitotic exit
19 VOTE: How can we explain these results?
A. Chromosome segregation and exit from mitosis are
regulated independently.
B. Chromosome de-condensation occurs only in G1.
20 APCCdc20
???
Are really both dependent on APCCdc20?
M cyclin
stable cyclin B
anaphase
mitotic exit
21 amount of DB-peptide
è
experimental evidence for existence of another APC substrate,
which inhibits anaphase (Holloway & Murray, 1993)
APCCdc20
anaphase
inhibitor
anaphase
destruction-box (DB) peptide
M cyclin
= APC recognition sequence (RxxL);
competitive inhibitor of APC
mitotic exit
22 What inhibits anaphase?
What keeps the replicated chromosomes together?
Metaphase spread
Cohesin blocks chromosome
segregation.
23 Cohesin cleavage promotes sister separation
Anaphase
Cohesin Scc1 subunit
Scc1
Protease
Scc1 Ct fragment
Uhlmann and Nasmyth, 1999
APC
Protease inhibitor
24 Sister chromatid cohesion & separation
Nasmyth lab
25 Sister chromatid cohesion
Emergence of sister chromatid cohesion
ê
memory of which chromatids
belong to each other
ê
"division of labor" made possible:
timely separation of reduplication and
segregation of chromosomes
ê
evolution of large genomes with many chromosomes
26 APC controls (all) late mitotic events
Spindle assembly checkpoint (SAC)
27 Summary metaphase-to-anaphase onset,
mitotic exit
1.  Maximal activity of Cdk-mitotic cyclin (cyclin B)
2.  Chromosomes align on metaphase plate, attached
and ready to segregate into the daughter cells.
3.  Cdk-cyclin B activates APC by phosphorylation;
4.  APC is a large E3 ubiquitin ligase complex, for its
activity in mitosis it requires a co-factor Cdc20
5.  Phosphorylated APC ubiquitylates cyclin B –
degradation and decrease of Cdk activity.
6.  APC ubiquitylates SECURIN, which frees SEPARASE
– sister chromatid cohesion is removed from sister
chromatids – onset of anaphase
7.  Multiple different proteins are degraded through APC
activity.
8.  Cdk activity decreases at the end of mitosis, proteins
are dephosphorylated by phosphatases.
28 Transition into the next G1
29 Two antagonistic oscillators control the cell cycle.
CDK off
CDK on
CDK off
APC on
APC off
APC on
30 The G1 phase of somatic cells cycles
is a state of stable Cdk inactivity
How do you establish a G1 (a prolonged, stable state of Cdk inactivity)?
Problem?
Cdk-cyclin B activate APCCdc20 – only phosphorylated APCCdc20 is functional
APCCdc20 degrades cyclin B – decrease in Cdk activity
Less Cdk activity – less APCCdc20 phosphorylated – cyclin concentrations start
to increase again – cell cannot move out of mitosis to reach G1
31 The G1 phase of somatic cells cycles
is a state of stable Cdk inactivity
How do you establish a G1 (a prolonged, stable state of Cdk inactivity)?
Problem?
Cdk-cyclin B activate APCCdc20 – only phosphorylated APCCdc20 is functional
APCCdc20 degrades cyclin B – decrease in Cdk activity
32 The G1 phase of somatic cells cycles
is a state of stable Cdk inactivity
How do you establish a G1 (a prolonged, stable state of Cdk inactivity)?
Cdh1
1) keep APC active after mitosis
How? > 2nd APC-activator: Cdh1;
APCCdh1 is inhibited by MPF > becomes
active only upon decrease of MPF activity
followed by dephosphorylation of Cdh1
2) activate a CKI (e.g. Sic1 in S. cerevisiae)
33 Two flavors of APC/cyclosome
APCCdc20
Becomes active at mitotic entry upon activation by
M-cyclin/CDK –
Cdc20 binds only phosphorylated APC
APCCdh1
Active from the anaphase onset to the end of G1 phase
- Ensured by at least 2 different mechanisms
1. Inactivating phosphorylation of Cdh1 by M-cyclin/CDK
2. Inhibitors of Cdh1 activated during interphase
ONLY unphosphorylated Cdh1 binds unphosphorylated APC
The two versions of APC are active at different times during cell
cycle, they are differently regulated and target different substrates
Finishing mitosis, one step at a time
Matt Sullivan & David O. Morgan
Nature Reviews Molecular Cell Biology 8, 894-903 (November 2007)
34 The G1 phase of somatic cells cycles is a state of stable
Cdk inactivity
35 Test yourself! 1. 
2. 
3. 
4. 
5. 
6. 
7. 
8. 
Which a2ributes of the cell cycle are conserved throughout eukaryotes? Order of phases? Length of phases? Presence of alternaGng S-­‐ and M-­‐
phases? Presence of alternaGng G1 and G2 phases? Mutants of Cdc28 in budding yeast arrest in G1; mutants of Cdc2 in fission yeast arrest in M-­‐phase? Both genes encode for CDK, how is this possible? Why are fission yeast wee mutants small? Explain! Describe two mechanisms that contribute to the G1/S cell cycle switch at start! The introducGon of many replicaGon origins brings about a specific challenge for eukaryotes. Name it and describe how cells regulate replicaGon iniGaGon in order to avoid this problem! Can the DNA damage checkpoint be arGficially acGvated in the absence of DNA damage? How? How are sister chromaGds held together? By which mechanism is this linkage removed at the metaphase-­‐to-­‐anaphase transiGon? Two different forms of the APC are acGve during the cell cycle. Describe similariGes and differences and why cells rely on two forms of the APC. 36 Thank you and good luck with the cell cycle!
37