Download The Cell Cycle

The Cell Cycle
The Mechanics of the cell cycle
G1
M
S
The cyclic process by which cells duplicate their
contents and partition into two daughter cells
G2
Emmy Verschuren
Institute for Molecular Medicine Finland (FIMM)
[email protected]
February 07 2013
Chapter 17 (but not mitosis)
Why understand The Mechanics of
the cell cycle?
The cell cycle simplified
> It only takes one rogue cell and its progeny to
kill a multicellular organism
G1
Tumor
Cells
M
Normal
Cells
Metastatic cancer invading liver
G0
Start
Restriction point
S
G2
The Cell Cycle has 2 major phases
Cell cycles in yeast: a genetically tractable unicellular eukaryote
G2
10-12 hours
G1
< 1 hour
M
S
G1
• Small, unicellular
• Cell size and shape reflects cell cycle phase
• Haploid genome makes genetics easy
Figure 17-2 Molecular Biology of the Cell (© Garland Science 2008)
Figure 17-5 Molecular Biology of the Cell (© Garland Science 2008)
Discovery of cell division cycle - cdc - genes
gain of function
cdc2 mutant
wild type cdc2
loss of function cdc2 mutant
• Excess function triggers premature cell cycle entry > smaller (shorter) cells
• Loss of function blocks cell cycle > larger (longer) cells
Figure 17-6 Molecular Biology of the Cell (© Garland Science 2008)
Xenopus egg biochemistry
Advantages:
• Large size (100.000 x more cytoplasm than average cell in the human body)
• Simple S-M cell cycles without intervening gap phases
Figure 17-9 Molecular Biology of the Cell (© Garland Science 2008)
Xenopus egg fertilisation mimicked in a test tube
Biochemical analysis of cycling sea urchin embryos
The identification of MPF: Mitosis Promoting Factor (later: cyclin B)
cyclins:
Cyclically expressed proteins whose appearance and
disappearance correlates with MPF (mitosis) activity
Figure 17-10 Molecular Biology of the Cell (© Garland Science 2008)
Mammalian cells proliferating in culture
note
Most cultured cell lines contain mutations so that they can be grown
(and studied) indefinitely = immortalisation
Figure 17-11 Molecular Biology of the Cell (© Garland Science 2008)
Analysis of cellular DNA reveals cell cycle phase
Thymidine analog bromo-deoxyuridine (BrdU)
labels S phase cells > BrdU pulse
Figure 17-12 Molecular Biology of the Cell (© Garland Science 2008)
Cell cycle control checkpoints
Two key components of the cell cycle control system
Cycle control system:
• Timer
• Correct order
• Each event only 1x per cycle
• On/off switches
• Robustness
• Adaptability
S. Pombe cdc2, Xenopus & Sea
Urchin & Mammalian MPF
are all the same
cyclin-dependent
kinase (cdk1) complex
Figure 17-14 Molecular Biology of the Cell (© Garland Science 2008)
Figure 17-15 Molecular Biology of the Cell (© Garland Science 2008)
Cyclins are the regulatory subunits of the Cdks
Core of the cell cycle control system
Yeast are unlike metazoans in having only one Cdk (Cdc2/
Cdk1) which interacts with different cyclins that regulate its
activity in different ways at various stages of the cell cycle
•G1 cyclins promote cell cycle entry
•G1/S cyclins promote passage through the late
G1 restriction point and into S phase
• S cyclins promote DNA replication
• M cyclins promote mitosis
Importantly: Cdk expression levels are
constant through the cell cycle
Oscillations of Cdk activities (through cyclical expression/destruction of cyclins)
leads to cyclical changes in the phosphorylation of proteins that regulate DNA
replication, mitosis and cytokinesis
Figure 17-16 Molecular Biology of the Cell (© Garland Science 2008)
Nomenclature of major cyclins and Cdks
While yeasts use only 1 Cdk, vertebrates use 4 different Cdks
Structural basis of Cdk activation
Important
The cyclin directs the Cdk activity to specific substrates
> each cyclin/Cdk complex has a unique set of substrates
Figure 17-17 Molecular Biology of the Cell (© Garland Science 2008)
Key negative controls of the cell cycle’s mechanics
Inhibitory phosphorylation of Cdks
Important for checkpoint controls:
1. Inhibitory phosphorylation of Cdks by WeeI/Myt1
2. Cdk inhibition by Cdk Inhibitor (CKI) binding
3. Protein destruction catalyzed by E3 ubiquitin ligases
Car engine: it is much easier to detect a little
more stop than a little more start
Most important negative regulator of M-Cdk activity
Figure 17-18 Molecular Biology of the Cell (© Garland Science 2008)
Inhibition of Cdks by Cdk Inhibitor proteins (CKIs)
Control of protein proteolysis by SCF E3 ubiquitin ligase
• SCF directs protein destruction in G1/S (cyclins and certain CKIs)
• SCF ubiquitination requires phosphorylation of target proteins
Most important negative regulator of G1/S Cdk activity
Figure 17-19 Molecular Biology of the Cell (© Garland Science 2008)
Control of proteolysis by APC/C E3 ubiquitin ligase
Figure 17-20a Molecular Biology of the Cell (© Garland Science 2008)
The core rules of the cell cycle’s mechanics
•
•
•
•
•
•
Cyclin-dependent kinases control cell cycle transitions
Cyclins are expressed in a cyclic manner
G1, G1/S, S and M cyclins direct cell cycle-specific Cdk phosphorylation
Cyclin binding to the Cdk provides substrate specificity
Full Cdk activation requires phosphorylation by CAK
Cdk inhibition can be achieved by WeeI inhibitory phosphorylation, CKI
binding or cyclin proteolysis
• The SCF ubiquitin ligase mediates destruction in G1/S and requires
phosphorylation of substrates
• The APC/C ubiquitin ligase mediates destruction in M and requires
binding of activator proteins
• Cyclic transcription and gene expression also controls the cell cycle
• APC/C directs protein destruction in M phase
Figure 17-20b Molecular Biology of the Cell (© Garland Science 2008)
The Mechanics of the cell cycle
How do cyclin-Cdk switches initiate DNA replication in S phase
and chromosome segregation and cell division in M phase?
S- cdk low
The initiation of DNA replication only once per cell cycle
s-cdk high
What ensures that this only happens once per cell cycle?
Figure 17-23 Molecular Biology of the Cell (© Garland Science 2008)
DNA replication alternates with chromosome segregation
DNA
REplication
Inhibitory
circuits
Mitosis
REplication
origin
licencing
Figure 17-22 Molecular Biology of the Cell (© Garland Science 2008)
These mechanisms ensure that S phase
•! Starts in the right cell
•! Starts on the right DNA site
•! Starts at the right time
•! Occurs only once per cycle
MCM proteins as cancer biomarkers
Squamous intraepithelial lesions
The Mechanics of the cell cycle
How do cyclin-Cdk switches initiate chromosome segregation
and cell division in M phase?
What ensures that this only happens once per cell cycle?
Laskey and Coleman, Nature Reviews Cancer, 2005
G2/M cell cycle progression in less than a snapshot
the stages of mitosis
Activation of cyclin B/cdk1 and other mitotic kinases drives
mitosis through phosphorylation of key mitotic regulators
This is important for:
I. Nuclear envelope breakdown and setting up a bipolar mitotic spindle
> Mitosis entry
II. Protein destruction feedback
> Mitosis exit
Anti-Mitotics: TAXOL
32
However: in vivo cell proliferation rates are low
PREDECT public-private partnership to improve cancer models
Partner & EFPIA
clinical samples
MBoC Jan 2012 (17 years since discovery of Taxol)
2D/3D on Plastic
3D in Matrix
3D Co-culture
3D Bioreactor
Tissue Slices
Mouse models
Towards Systems Pathology
Tissues
Responses in the tumour microenvironment in vivo are complex,
and need to be understood to understand and model therapy responses
Cells
www.predect.eu
somatic cell proliferation
To start the cycle:
the mechanics of mitogenesis
Proliferation is not normally determined by nutrient availability
Strategic control of proliferation is through social signals
(mitogens)
Cells make their major decision about proliferation in late G1
Mitogens and
nutrients required
M
M
Mitogens
G1
G1
G0
What do mitogens do biologically ?
SS
G2 M
M
G2
Mitogens engage (and maintain) expression
of multiple genes that promote or enable
cell proliferation
G1-CDK
E2F transcription
factors
Cell cycle entry
Restriction Point (R)
Three choices:
1. Proceed through cycle
2. Stay put for the time being
3. Enter long term quiescent state (e.g. G0 or terminal differentiation)
Mitogens stimulate G1-Cdk through transcriptional responses
Mitogens are found in blood
serum, e.g. PDGF, EGF
Inhibition of E2F G1/S transcription by Rb family pocket proteins
Rb/p107/p130
‘pocket’
Mitogens force
cells to (re)-enter
the cell cycle!!
cyclin D
Figure 17-62 Molecular Biology of the Cell (© Garland Science 2008)
e2f peptide
> Progressive phosphorylation of pRb proteins by G1-, G1/S-, S- and
M-Cdk inactivates pRb and liberates E2Fs to activate transcription
E2F TARGET GENES
Positive feedback loops drive S phase entry
CELL CYCLE
Rb ‘pocket’
cyclin D
e2f peptide
E2Fs
G1
S/G2
cyclin D1
Cycin D3
Jun
Myc
N-Myc
Aurora B kinase
cyclin A1
Cdk1
Cdc20
Cks1
Cks2
Hec
Ki-67
KIF4A
KNSL4
Polo kinase
Prc1
Smc2L1
Smc4L1
Stk12
G1/S
Cyclin E1
Cyclin E2
Cdc25A
Cdk2
E2F1
E2F2
E2F3
NPAT
Myb
Mybl2
TFDP1
DNA SYNTHESIS
CHECKPOINTS
DNA REPAIR
DEVELOPMENT
Ask
Cdc14B
Cdc45L
Cdc6
Cdc7L1
Cdt1
Dck
Dhfr
Dut
Lig1
Mcm2
Mcm3
Mcm4
Mcm5
Mcm6
Mcm7
Orc1L
PCNA
Pola
Pold1
Prim2A
RFC1
BRCA1
BRCA2
Bub1
Bub1B
Bub3
CENPE
Chk1
Mad2L1
p53
TTK
Bard1
Cstf1
Fen1
Mgmt
Mlh1
Msh2
Msh6
Pms2
Prkdc
Rad51
Rad54L
Ung1
Ung2
Bapx1
Eed
En2
Ezh2
Fos
Hey1
HoxA4
HoxA5
HoxA7
HoxA9
HoxA10
HoxA11
HoxB9
HoxD8
Pitx1
Six1
Suz12
RFC2
RFC3
RFC4
RPA1
RPA2
RPA3
Rrm1
Rrm2
TK1
Top2A
Tyms
ANTI-PROLIFERATIVE
APOPTOSIS
CDKN1
CDKN2
E2F7
Rb
RbL1
Apaf1
Bad
Bak1
Bcl-2
Bid
Bok
Caspase 3
Caspase 7
Caspase 8
MAP3K14
MAP3K5
p73
DIFFERENTIATION
Bmp2
Fts
TgfA
PparGC1
JunB
Tead4
Figure 17-62 Molecular Biology of the Cell (© Garland Science 2008)
DNA damage activates the p53 transcription factor
p53 activation halts the cell cycle so that
there is time to repair the damage
Figure 17-63 Molecular Biology of the Cell (© Garland Science 2008)
Active p53 arrests the cell cycle through the p21 CKI
> The cell cycle arrest allows for the DNA damage to be repaired
> If damage is severe or not repairable, cell death or senescence ensues
Figure 17-63 Molecular Biology of the Cell (© Garland Science 2008)
The cancer conundrum:
“cell-intrinsic tumour suppression”
Out of control mitogen stimulation
does NOT necessarily stimulate cell division