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Cell biology 2014 (revised 4/2 -14)
Lecture 6 & 7:
All cells come
from cells
Cells of all life
forms have a
common ancestor
All diseases
involve
changes of
cells
1
Rudolf Virchow, Die Cellularpathologie (1858)
Cell cycle control in multi-cellular eukaryotes
somatic mutations
&
chromosomal instability
1013
Controlled and coordinated divisions
Uncontrolled divisions
 Tumor (clonal origin!)
Minimal length of DNA replicated by (cells of) a human
Human diploid genome: ~6 x109 bp (cost of MD education: ~6 x109 Skr/year)
(length of 1 bp) x (number of bp per cell)
(0.34 nm) x (6 × 109) = 2 m, 1013 cells  2 x 1013 m
0.5 x1013 m
Earth
Sun
Mars
Uranus
Pluto
2
“Simple” model systems for studies of eukaryotes
Primordial
eukaryote
Budding yeast, S. cerevisiae
Polarized (bud & shmoo)
(polarity & sex)
Fission yeast, S pombe
Symmetric cell division
Yeast model systems:
- Unicellular eukaryotic organism (autonomous cells)
- Sexually active (mating & sporulation)
- Short generation time
vs.
- Haploid:  phenotype of recessive
(loss-of-function) mutations
3
Principles of conditional mutants
Wild type protein
26oC
Mutant protein
Functional
(i.e. no phenotype)
36oC
Non-functional
(i.e. phenotype)
The mutant gene product is temperature sensitive (Ts)
OK at 26oC, but unfolds at 36oC
(i.e. the function of gene product can be switched off)
4
5
Temperature sensitive yeast mutants
= Ts clone
26o C
Missing!
36o C
Cell cycle Ts mutant or not?
26o C
36o C
or
(if lucky!)
House keeping
gene mutated
Cell cycle control
gene mutated
Identification of cell division control (cdc) genes
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ZZZ
Gene library
(i.e., wild type genes cloned
into bacterial plasmids)
ZZZ
ZZZ
+
36o C
OK
ZZZ
Yeast cell
cycle Ts mutant
OK
Mutated gene:
(OK at 26oC but not 36oC)
Complementation by
OK
:
Cell cycle regulators are evolutionary conserved
Budding yeast with a temperature
sensitive mutation in an essential
cell division control (cdc) gene
36o C
36o C
cDNA library (copies
of all human mRNA’s)
 identification of a
human “ortholog”
The cell cycle control machinery is highly conserved in eukaryots
Nobel prize in medicine 2001!
7
Three distinct cell cycle regulated events
DNA replication
Increased size
Nuclear division
followed by
cytoplasmic division
8
Interphase and mitosis
Quiescent or
post-mitotic
Mitosis (Cell division phase)
(010% of a cell population)
Chromosome segregation
Cell division
GO
G1
Condensed chromatin
”thread-like”
Interphase (“between-phase(s)”)
(90100% of a cell population)
S (DNA-Synthesis)
G1 (Gap 1)
G2 (Gap 2)
Cell cycle exit  “G0”
(Greek: mitos= thread, khrōma = color, soma = body)
G2
S
Chromatin
“attract dyes”
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Key events and checkpoints of the cell cycle
1. G1/S checkpoint (“Start”)
Sense: Surroundings
Cell size
Block: DNA replication
2. G2/M checkpoint
Sense: DNA replication status
DNA damage
Cell size
Block: Mitotic entry
3.
M
G1
1.
2.
G2
S
3. Spindle assembly checkpoint
Sense: Chromosome attachment to the mitotic spindle
Block: Chromosome separation and cytoplasmic division
10
Cell cycle regulation of proteins
Amount of protein
- Control of protein expression
Activity of protein
- Binding partners
DNA
mRNA
Transcription
+
=
Translation
Protein
+
=
- Control of protein turnover
- Phosphorylation
P
26S Proteosome
P
P
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Cyclin dependent kinases (Cdk) – the controllers
Cdk
H
Cyclin
O
Serine or threonine
P
Phosphatase
Kinase
OO P
O-
O
Serine or threonine
P
Kinase motif
Inhibitory domain (T-loop)
Both the cyclin and P are required
to activate the Cdk
Cyclin  substrate specificity
12
Cdk’s are stable while cyclin levels are “cyclic”
Cdk 4/6
Cdk 2
Cdk 1
Diffuse border
M
Cyclin:
(expression)
G1
G1
(D)
S
G1/S
(E)
G2
S
(A)
M
G1
M
(B)
Interphase cyclins
Mitotic cyclin
MHC
13
Mechanisms for interphase cyclin degradation
1. High intrinsic
turnover
of G1 cyclin
2. P -dependent
ubiquitination of
G1/S cyclin
3. Cdk/M-cyclin
mediated P 
ubiquitination
of S cyclin
Cdk G1
Cdk +
Kinase X
P
Cdk G1/S
P
Cdk G1/S
Cdk +
Cdk M
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P
Cdk
S
P
Cdk
S
Cdk +
Cdk/cyclin control of progression and transitions
Active: Cdk X
Cdk
S
Cdk
S
Cdk G1
Transitions:
M
Cdk G1/S
G1
Cdk
S
G2
M
M
G1
G0
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External signals (mitogens)G1 cyclin expression
Hedgehog
XGF
Wnt
Receptor
Tyrosine
Kinase
GTP
Ras
16
= DNA
Myc
Myc
myc gene
G1
G1 cyclin gene
Rb phosphorylation by Cdk/G1 cyclin  E2F activation
Positive feedback loop
= DNA binding
protein
3.
Cdk
Cdk G1/S
G1/S
G1
1.
E2F
E2F
2.
Rb
S
repressor
P
P
activator
Rb
1. Phosphorylation by Cdk-G1 cyclin dissociates Rb from E2F
2. Transcription of E2F regulated genes
3. Enhancement by newly formed Cdk-G1/S cyclin
17
Transcriptional control of S phase components
E2F
E2F
E2F
Rb
E2F
S-phase components
Non-dividing cell:
Dominant repression by the E2F/Rb complex
P
Rb
P
E2F
G1/S
S-phase components
Proliferating cell:
E2F mediated transcription of “S phase genes”
S
Rb is mutated in ~40% of human tumors  constitutive production of S-phase components
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The stability of the genome: two levels of threat
S-phase: 46 x ~4 cm DNA has to
be replicated once (but only once!)
M-phase: sorting of 2 x 46
sister chromatids (no errors!)
G2
S-phase
(6 h)
~4 cm DNA= 130 x106 bp
~ 250 nt/s  144 h/ 4 cm
G1
G1
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Licensing of DNA for a single round of replication
Early G1
ORC
ORC
ORC
Cdc6 is recruited to ORCs
Cdc6
Cdc6
Cdc6
ORC
ORC
ORC
Formation of a pre-RC
(i.e., licensing of DNA):
Cdc6 dependent loading
of Mcm proteins onto DNA
Note - pre-replicative complexes
are formed after mitosis
independently of progression
into a new S-phase
Cdc6
Cdc6
Cdc6
ORC
ORC
ORC
20
Firing of pre-RC during S-phase
Cdc6
Late G1
ORC
Cdk
Firing of the first ORC
point of no return!
S
Phosphorylates
Cdc6 and Mcm
P
Cdc6
ORC
S
P
P
ORC
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DNA strand separation by helicase activity of Mcm proteins
A fool-proof system for prevention of re-replication
ORC
P
P
ORC
1. Phosphorylated Cdc6
dissociates from ORC
2.
2. Phosphorylated Cdc6 is
recognized by SCF
3. Ubiquitylation of Cdc6
by SCF (an E3-ligase)
4. Proteosomal degradation
of Cdc6
1.
P
Cdc6
S CF P
Cdc6
4.
3.
Proteosome
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Prevention of DNA licensing until the next G1-phase
Degradation of “free” Cdc6
Cdk
P
Cdc6
S
Cdk
P
Cdc6
S
Cdk
P
Cdc6
M
G1: “free” Cdc6 is
available
Cdc6
Cdc6
Cdc6
Cdc6
Cdc6
ORC
Cdc6 levels
S
G2
M
G1
23
Cell cycle entry and DNA replication
24
External signal  Activation of the Rb pathway!
Growth
G1
Cdk G1
factor
Myc
Positive
feedback loop
Rb
Rb/E2F = dominant
repressor of E2F
E2F
Cdk G1/S
Cdk
S
P
Cdc6
ORC
P
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p53: The guard against tumors
2.
Mdm2
p53
3.
3.
ATM
ARF
p53
p53
p53
p53
1.
p53 gene
4.
1. p53 is constitutively expressed but is normally…
2. degraded through ubiquitination by Mdm2 (E3-ligase)
3. DNA damage ( ATM ) or unbalanced/excessive proliferation
signaling ( ARF ) inhibits Mdm2  stabilization of p53
4. Transcriptional activation of p53 responsive genes
(ATM: ataxia telangiectasia-mutated, ARF: Alternative Reading Frame)
Target genes of p53 dependent transcription
p53
p21
Cell cycle block
DNA
repair
proteins
DNA repair
Bax
PUMA
Apoptosis
Loss of p53 function
Genetic instability
I´m gonna
live forever
26
Two distinct CdK Inhibitor (CKI) families
Specificity: G1, G1/S and S
G1 only
p21
Cdk
Cyclin
Inhibits: Catalytic activity
(Cip/Kip family)
Cdk
4/6
G1
Cyclin
Cyclin association
(Ink4 family)
In both cases, the Cdk activity is abolished
Molecular_models: 17.1-Cdk2
27
Growth
factor
PI3K
+++
Survival
signals
PKB/Akt
Cdk
Ras Myc
G1
p53 pathway detects:
1. Unbalanced/ excessive
proliferation signals (+++)
2. DNA damage
1.
ARF
2.
p16
Rb & p53 pathways cell cycle and apoptosis
Rb
ATM
 Cell cycle block
 Apoptosis
1.
E2F
p53
p21
Cdk G1/S
Cdk
S
P
Cdc6
ORC
Apoptosis
G1/S block
28
Three distinct cell cycle regulated events
DNA replication
Size growth
Nuclear division
followed by
cytoplasmic division
29
Cell division and cell growth
Oocytes grow without dividing
Fertilized eggs replicate and divide without growing
30
PI3-kinase signaling regulates protein synthesis
RTK
P.M.
P
P
P
P
P
PI-3 K
3
P
PDK1
P
3
P PKB/
Akt
PKB/Akt
P
PKB/Akt
P
+
Ribosomes
Translation
initiation
factor
+ mRNA =
Cellular
Proteins
size growth
31
32
Divergent (cooperating) RTK receptor signals
”Mitogen” signaling
(Rb-pathway)
Ras
G1
G1 G1
RTK
”Growth factor” signaling
P
P
P
P
P
PI-3 K
3
P
P
PTEN
3 P
P
PKB/Akt
P
Cell cycle
entry
Increased
cell size
Bad
Survival
Summary: G1-, S- and G2-phase (interphase)
DNA replication
”Mitogen” signaling:
G1
G1 G1
Ras
G1 G1 G1
G1 G1 G1 G1
Size growth
”Growth factor” signaling:
PI-3 K
+
Bad
Translation
Initiation
factor
=
33
The end of the cell cycle
Cell division = nuclear division + cytoplasmic division
M-phase
Mitosis
Sorting of 2 x 46
sister chromatids
Cytokinesis
Defines the division plane/symmetry
Protein sorting (if asymmetric division)
Topics of case 13: The cytoskeleton
34
Progression of cell division: two points of “No return”
Interphase (G2)
Prophase
Telophase/
cytokinesis
Prometaphase
Transition points
 Checkpoint
control
Anaphase
Video: 17.4
Animal_cell_division
Metaphase
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G2  M transition
Event
Cause
Mitotic spindle formation
Breakdown of
nuclear envelope
MAPs
Consequence
P
Change in
MT dynamics
P
Nuclear lamins
Disassembly of
nuclear lamina
P
Condensin
DNA packaging
Chromosome condensation
36
Checkpoint control of Cdk/M-cyclin activity
37
G2/M checkpoint control and regulation of Cdc25 activity:
DNA replication stall
DNA damage
Insufficient cell size
Dual feedback loops
 activity
burst!
Inhibitory sites: PP
Activating site: P
Cdk + M
Cdc25
wee1
CAK
Cdc25
Cdk M
PP
Cdk M
PP
Cdk PM
Cdk PM
Inactive
Inactive
Inactive
Active
G2
G2M
Chromosome segregation
Anaphase
initiation
+
Attached
during
S-phase
Sister chromatid
separation
(anaphase)
+
38
Cohesin
Separase
Securin
Checkpoint control of metaphase-anaphase transition
Cdc20
Inactive
APC/C
Cdc20
Active
 1 unattached kinetochore
(spindle assembly checkpoint)
Sorting of 2 x 46
chromosomes
Metaphase
+
APC/C
Anaphase
39
Mitotic exit and initiation of cytokinesis
Active Cdk P M
Checkpoint
Inactivation of Cdk
M
Mitotic exit and
cytokinesis
Cdk/M activity block initiation of cytokinesis!
40
Molecular events during mitotic exit
41
Active APC/C  cyclin degradation  inactive Cdk  mitotic exit
APC/C
Cdc20
+
Cdk
P
M
Cdk
P
M
Cdk +
Constitutively active phosphatases drives the cell out of mitosis
P
MAPs
MAPs
Phosphatase
P
Nuclear lamins
CondensinP
Nuclear lamins
Mitotic exit
Condensin
Mitosis versus meiosis:
Animation: 21.2-meiosis
Checkpoints function: block of premature transitions
Spindle assembly checkpoint
Spindle attachment to chromosomes
APC/C
Cdc20
G2/M checkpoint
DNA status and
cell size
M G1
G2
S
G1/S checkpoint
The surroundings
Mitogen
E2F
Cdc25
PP
Cdk PM
Cdk PM
Checkpoints verify that
all processes at each
cell cycle phase have
been completed before
transition into the next
P
42
Cell death in multi-cellular eukaryots
Cell death
The word apoptosis is
greek for fallen leaves
Necrosis
- ”Murder”
- Associated with
inflammation
(and vice versa)
Apoptosis
- ”Suicide”
- Death for the benefit
of the organism
- No inflammatory response
43
Importance of apoptosis
Development
Homeostasis
Immune
survelliance
x
Elimination of
superfluous cells
between the
developing fingers
Apoptosis and cell
proliferation must
balance each other
x
Killing of virus
infected cells
44
Cellular changes during apoptosis
Normal cell
Different stages of apoptosis
Cell shrinkage
Membrane ruffling
DNA condensation
DNA fragmentation
Phagocytic cells recognizes phosphatidyl serine ( )
which becomes exposed on the surface during apoptosis
Burp!
45
Video: 18.1 Apoptosis
Apoptosis – a protease cascade
Initiator
Apoptotic signal
caspase
multimers
monomers
Cytoskeleton
DNA
Other things
Active effector
caspase
A
t
=
46
Active initiator
caspase
Inactive effector
caspase
47
Control of apoptosis
• Two pathways for initiation of apoptosis:
Extrinsic (receptor-mediated) and Intrinsic (mitochondrial)
• Apoptosis is controlled by the balance of
Pro- and Anti-apoptotic regulatory proteins
External and internal signals
Pro-apoptotic
Anti-apoptotic
Cell survival
Apoptosis
The intrinsic apoptotic pathway
Proapoptotic
Antiapoptotic
Cyt. C
BH123
Pore former
48
Cyt. C
Bcl-2
Inhibitor of
pore formation
Cyt. C
Cyt. C
Cyt. C
Note - Bcl2 has all four BH domains
(Albert et al: Fig. 18-9)
Pore formation in the outer
mitochondrion membrane
 Cyt. C release
Cytochrome C in the cytosol triggers apoptosis
Apaf1 : APoptosis Activating Factor
Cyt. C Apaf1
Apoptosome
Caspase 9
Cyt. C Apaf1
Caspase 9
Cyt. C
Caspase 3
Caspase 9
Target proteins
Caspase 3
T a rg e t pr o t e i ns
49
Regulation of the intrinsic apoptotic pathway
50
Antiapoptotic
Proapoptotic
p53 pathway: Pro-apoptotic
downstream mediators
BH123 (family)
BH3-only
Bcl-2
(family)
Note - Bcl2 has all four BH domains
 per definition! (Albert et al: Fig. 18-9)
Bax
p53
PUMA
ATM
ARF
Summary on apoptosis
Extrinsic apoptosis
Intrinsic apoptosis
Survival
signals
Ligand
BH123
Bcl-2
Cyt. C
Death
signals
BH3-only
Initiator caspase
Death
receptor
Initiator caspase
Effector caspases
- DNA
- Cytoskeleton
- Other things
A
t
51
Determinants of survival and proliferation
Proapoptotic
Antiapoptotic
G1 cyclin
Regulation of cell survival
CKI
Regulation of cell proliferation
x
x
Apoptosis
or
+
+
Cell proliferation
+
or
52
Quiescent cell (G0)
A single external signal may serve multiple functions
Mitogen
G1 cyclin
Growth factor
Ribosome
+
Survival factor
Translation
initiation
factor
One specific ligand/receptor complex may transduce several
distinct signals
G1
+
Translation
Initiation
factor
53
Recommended reading
Chapter 17
Chapter 18
1053-1080
1092-1094
1101-1112
1115-1128
Alberts et al
5th edition
54