The eukaryotic cell division cycle
S. Ferrari, IMCR
Definition and structure of the cell cycle
Model systems used in cell cycle studies
Historical perspective on cell cycle studies
Example of control mechanism (checkpoint)
Cell division cycle (cdc)
“The series of events that leads from one mother cell
to two daughter cells”
Purpose:
propagating genetic information
G2
S
M
G1
G1= gap/growth-phase 1
I = interphase
S = synthesis (DNA)
G2= gap/growth-phase 2
M = mitosis
G0
Growth factors
M
G2
protein synthesis
mitosis
24 h
interphase
G1
S
DNA synthesis
preferential translation of mRNAs
(ribosome biogenesis)
protein synthesis
Interphase
G2
S
Antephase
Prophase
Prometaphase
Metaphase
M
Mitosis
Anaphase
Telophase
G1
G0
Quiescence
Mitosis
Antephase: sensitivity to stress, no visible changes
Prophase: chromosome condensation
Pro-metaphase: nuclear envelope breakdown,
chromosome attachment and congression
Metaphase: chromosome bi-orientation
Anaphase: spindle elongation
separation of sisters
Telophase: nuclear membrane formation
chromosome decondensation
Kyoto HeLa:
GFP-tubulin / cherry H2B
normal mitosis
abnormal mitosis
Model systems used in cell cycle studies
Model systems
S. cerevisiae
S. pombe
X. laevis
A. punctulata
(sea urchin)
H. sapiens
C. elegans
D. melanogaster
M. musculus
S-phase
mitosis
G1
human somatic cell
G2
cytokinesis
G1
0h
~20h
0h
1h
G2
~40h
Xenopus embryo
0h
1h
G1
G2
2h
4h
G1
G1
0h
2h
G1
G2
0h
budding yeast
G2
G2
sea urchin embryo
fission yeast
2h
1h
2h
3h
Drosophila embryo
0 min
24 min
H.P. Rusch / V.Gruter 1966 (Physarum polycephalum)
single cells
B
A
B
A
A
1
syncytium
B
2
3
4
5
time
AB
AB
AB
Conclusion: factors in A-cells can advance B-cells to mitosis
factor = protein kinase ?
Is transition through the cell cycle regulated?
1974, A. Pardee
1985, A. Zetterberg & O. Larsson
Interphase
G2
S
M
Restriction point
Start (yeast)
Mitosis
G1
mitogenic signals
GFs
G0
Quiescence
Cell fusion experiments: Johnson & Rao, 1970
~8-10 h
G1 cell
~3 h
G2 cell
S
G1 cell
S
+
S cell
M
G2 cell
M
+
M cell
?
G2 cell
+
S cell
?
M
S
Cell fusion experiments: Johnson & Rao, 1970
~8-10 h
G1 cell
~3 h
G2 cell
G2 cell
+
S cell
S
S
G1 cell
+
S cell
M
G2 cell
M
+
M cell
M
S
Conclusions:
- cell cycle phases are inter-dependent
- phase transitions depend on specific factors
Concept of promoting factors:
- SPF: S-phase promoting factor
- MPF: M-phase promoting factor
MPF
M
G2
mitosis
24 h
S
interphase
SPF
G1
Hartwell lab 1970:
- model system S. cerevisiae (budding yeast)
- screen for mutants presenting defects in DNA, RNA
or protein synthesis
- ~75 cell division cycle (CDC) mutants
[CDC28]
Lohka & Masui 1971, 1983:
- model system Xenopus laevis
- biochemical identification of M-phase promoting factor
Nurse lab: 1977
- model organism S. pombe (fission yeast)
- studying the mechanism of cell growth
- screening for “wee”-mutants (read-out = altered cytoplasm
to nucleus ratio)
wee-1: prevents the onset of mitosis before cells reach a critical size
wee1
cdc2
Hunt lab: 1983
- mechanism of mRNA translation in the cell cycle of sea urchin
cyclin
S
G2
M
cyclin
G1
S
G2
M
Lhoka & Maller: 1988, 1990
MPF is protein kinase composed of a cyclin and a CDK
catalytic
regulatory
CDK
cyclin
CDK1 = Sc CDC28 = Sp cdc2
Concept of promoting factors:
- SPF: S-phase promoting factor
- MPF: M-phase promoting factor
Cyclin/CDK
MPF
M
G2
mitosis
24 h
S
interphase
SPF
G1
CDK1
Cyclin B
CDK1
Cyclin A
M
G2
G1
CDK2
Cyclin A
S
CDK2
Cyclin E
CDK4
CDK6
Cyclin D
Cyclin D
Question:
- how do cyclins activate CDKs?
CDK2 with bound ATP
De Bondt HL, 1993, Nature 363, 595-602
Molecular mechanism of CDK activation
Cyclin
ATP binding
Cyclin
Substrate binding
Cyclin
Cyclin binding
Cyclin
Cyclin
Cyclin
Thr160 phosphorylation
K
CDK
Cyclin
Substrate
SP
xK
T-loop
RX
L
hydrophobic
patch
+
CKS1
P
cationic pocket
Question:
- what is the role of cyclin/CDKs
in cell cycle transitions?
=
centrosome duplication, DNA replication
chromosome condensation, spindle formation
Sequential and interdependent
triggering
Central controller
Experimental evidence obtained with inhibitors (HU, Caffeine)
CycD/CDK4
CycE/CDK2
restriction
point
centrosome
duplication
CycA/CDK2
CycB/CDK1
DNA
synthesis
NMB
spindle
assembly
Cyclin/Cdk = controller
Question:
- who controls the controllers ?
(how are cyclin/CDKs regulated) ?
4. Mitotic machinery:
Are chromosomes aligned?
Are kinetochores properly attached?
3. Cell growth:
Is size appropriate?
Is DNA decatenated?
spindle checkpoint
G2/M checkpoint
M
G2
1. Environment:
Are reserves adequate?
What signals come from
outside?
controller
Intra S-phase checkpoint
G1
S
G1 checkpoint
2. DNA replication machinery:
Is all DNA replicated?
Is DNA intact?
What is a checkpoint?
Checkpoint:
“Control mechanism enforcing dependency in the cell cycle”
Hartwell & Weinert, Science 1989
Central controller
Checkpoint
Activating
Silencing
Example of input cascade:
1. mitotic entry
The MPF subsystem
S
G2
M
CDK1
CycB
S
G2
M
The MPF subsystem
DNA replication
CHK1
CAK
WEE1
CDK1
Y15
T14
T161
T161
Cdc25C
CDK1
CDK1
CycB
CycB
Wee1
CycB
G2
M
yc
C
B
Example of input cascade:
1. mitotic entry
2. DNA damage
The MPF subsystem
DNA replication
DNA damage
CHK1
CHK2
T161
p53
p21
p38MAPK
p21
WEE1
CDK1
CycB
MK2
CAK
Y15
T14
Cdk1
T161
T161
Cdc25C
CDK1
CDK1
CycB
CycB
Wee1
CycB
G2
M
yc
C
B
Cell cycle and cancer
G2
S
G2/M CHKPT
M
G1/S CHKPT
Restriction point
(Start)
G1
G0
Mitogenic signals
SAC CHKPT
Literature
Malumbres M and Barbacid M
Cell cycle, CDKs and cancer: a changing paradigm
Nature Rev. Cancer 9, 2009, p.153
Ganem NJ, Godinho SA and Pellman D
A mechanism linking extra centrosomes to chromosomal instability
Nature 460, 2009, p.278
Vogelstein B. and Kinzler KW
Cancer genes and the pathways they control
Nature Medicine 10, 2004, p.789
Luo J, Solimini NL, and Elledge SJ
Principles of Cancer Therapy: Oncogene and Non-oncogene Addiction
Cell 136, 2009, p.823
chromosome aberrations
/Securin
Draviam VD et al., 2004, Curr.Op. Gen & Dev. 14, 120-125