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Rudolf Virchow, Die Cellularpathologie (1858)

All diseases involve changes of cells

All cells come from cells

Lecture 6 & 7:Cell biology 2014 (revised 4/2 -14)

Cells of all life forms have a common ancestor

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Controlled and co-ordinated divisions

somatic mutations &chromosomal instability

Cell cycle control in multi-cellular eukaryotes

Uncontrolled divisions Tumor (clonal origin!)

2

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 Uranus Pluto

SunMars

Minimal length of DNA replicated by (cells of) a human

“Simple” model systems for studies of eukaryotesBudding yeast, S. cerevisiae Polarized (bud & shmoo)

Fission yeast, S pombeSymmetric cell division

Yeast model systems: - Unicellular eukaryotic organism (autonomous cells)- Sexually active (mating & sporulation)- Short generation time - Haploid: phenotype of recessive (loss-of-function) mutations

vs.3

Primordial eukaryote(polarity & sex)

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)

Wild type protein

Functional(i.e. no phenotype)

26oC

36oC Non-functional(i.e. phenotype)

Principles of conditional mutants

4

Mutant protein

26o C

36o C

Missing!

Cell cycle Ts mutant or not?

26o C

House keepinggene mutated

Cell cycle controlgene mutated

36o C

Temperature sensitive yeast mutants 5

or(if lucky!)

= Ts clone

36o C+

Gene library(i.e., wild type genes cloned

into bacterial plasmids)

Yeast cellcycle Ts mutantMutated gene:(OK at 26oC but not 36oC)

Identification of cell division control (cdc) genes

ZZZ

ZZZZZZ

ZZZ

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OK

OK

Complementation by : OK

Cell cycle regulators are evolutionary conserved Budding yeast with a temperaturesensitive mutation in an essential cell division control (cdc) gene

cDNA library (copies of all human mRNA’s) identification of a human “ortholog”

The cell cycle control machinery is highly conserved in eukaryotsNobel prize in medicine 2001!

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36o C

36o C

Three distinct cell cycle regulated events

Increased size

Nuclear division followed by cytoplasmic division

DNA replication

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G1 (Gap 1)G2 (Gap 2)

S (DNA-Synthesis)

Cell cycle exit “G0”

Interphase (“between-phase(s)”)(90100% of a cell population)

Mitosis (Cell division phase)(010% of a cell population)

Chromosome segregationCell division

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Quiescent orpost-mitotic

Condensed chromatin”thread-like”

Interphase and mitosis

Chromatin“attract dyes”

G2 S

G1

GO

(Greek: mitos= thread, khrōma = color, soma = body)

Key events and checkpoints of the cell cycle

G2/M checkpoint

Spindle assembly checkpoint

G1/S checkpoint (“Start”)

G1

SG2

M1.

2.

3.

1.

2.

3.

Sense: Surroundings Cell size

Block: DNA replication

Sense: DNA replication status DNA damage Cell sizeBlock: Mitotic entry

Sense: Chromosome attachment to the mitotic spindleBlock: Chromosome separation and cytoplasmic division 10

Cell cycle regulation of proteinsAmount of protein

- Control of protein expression

Protein

DNA

mRNATranscription

Translation

- Control of protein turnover

Ub

UbUb

26S Proteosome

Activity of protein- Binding partners

+ =

+ =

- Phosphorylation

PP

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P

Cyclin dependent kinases (Cdk) – the controllers

O

Serine or threonine

Serine or threonine

O

O

O -P

Kinase

O

-

H

P

Cdk Cyclin

Kinase motifInhibitory domain (T-loop)

P

Cyclin substrate specificity

Both the cyclin and are requiredto activate the Cdk

P

Phosphatase

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G1/S(E)

S(A)

G1(D)

M(B)

G1Diffuse border

G2 MCyclin:

(expression)

Cdk 2

Cdk 4/6

Cdk 1

Cdk’s are stable while cyclin levels are “cyclic”

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Ras v-SNARE

NFAT

Hsp

70

Clathrin

COPII MHCACTH

cGM

P

Retinoic acid

Src R7

Notch

Wnt

Ci

RelA

cGMP

cGM

P

v-SNARE

v-SNARE

v-SNAREv-SNARE

The cell can not reside in two cell cycle phases simultaneously

The cell can not reside in two cell cycle phases simultaneously

G1M S

Interphase cyclins Mitotic cyclin

Cdk S

Mechanisms for interphase cyclin degradation

Cdk G1/S

Cdk S

Cdk G1

Cdk M

1.

2.

3.

High intrinsicturnover of G1 cyclin

Cdk +

-dependent ubiquitination of G1/S cyclin

PCdk G1/S

PUb

UbUb

Cdk +

PUb

UbUb

P PUb

UbUb

+

PUb

UbUb

Cdk

Cdk/M-cyclinmediated ubiquitinationof S cyclin

Kinase X

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P

P

G1 S G2 M

Cdk G1/S

Cdk G1

Cdk M

G0

S Cdk

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Transitions:

Cdk/cyclin control of progression and transitions

G1M

Cdk S Active: Cdk X

External signals (mitogens)G1 cyclin expression

Wnt

Receptor Tyrosine Kinase

Hedgehog

G1

G1 cyclin geneMyc

Myc

myc gene

XGF

GTPRas

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= DNA

E2FRb

E2F

repressor activator

Cdk G1/S

DNAreplisome

Cdk G1 G1/S

S

1. 2.

2.

Phosphorylation by Cdk-G1 cyclin dissociates Rb from E2F 1.

Positive feedback loop

RbP P

Rb phosphorylation by Cdk/G1 cyclin E2F activation

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3.

Enhancement by newly formed Cdk-G1/S cyclin3.Transcription of E2F regulated genes

= DNA binding protein

DNAreplisome

E2F

Proliferating cell:E2F mediated transcription of “S phase genes”

E2FRb

Non-dividing cell:Dominant repression by the E2F/Rb complex

Transcriptional control of S phase components

Rb is mutated in ~40% of human tumors constitutive production of S-phase components

S-phase componentsS

G1/S RbP P

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E2F

S-phase components

E2FE2F

The stability of the genome: two levels of threat

G2

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S-phase: 46 x ~4 cm DNA has to be replicated once (but only once!)

S-phase(6 h) ~4 cm DNA= 130 x106 bp

~ 250 nt/s 144 h/ 4 cm

G1

M-phase: sorting of 2 x 46 sister chromatids (no errors!)

G1

Licensing of DNA for a single round of replication

Early G1 ORC

ORCCdc6

Formation of a pre-RC (i.e., licensing of DNA): Cdc6 dependent loading of Mcm proteins onto DNA

Cdc6 is recruited to ORCs

Note - pre-replicative complexes are formed after mitosis independently of progression into a new S-phase

ORC ORC

ORCCdc6

ORCCdc6

ORCMcm

Mcm

Cdc6ORCM

cm

Mcm

Cdc6

ORCMcm

Mcm

Cdc6

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DNAreplisome

Cdk S

Firing of pre-RC during S-phase

DNAreplisome

DNAreplisome

Firing of the first ORC point of no return!

Late G1 ORCMcm

Mcm

Cdc6

S Mcm

Mcm

ORCCdc6

ORC

P

DNA strand separation by helicase activity of Mcm proteins

Phosphorylates Cdc6 and Mcm

P P

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A fool-proof system for prevention of re-replication

2.

3.

Cdc6P

Cdc6P

S CFUb

UbUb 4.

2.

3.

1.

4.

Phosphorylated Cdc6dissociates from ORC

Phosphorylated Cdc6 isrecognized by SCF

Ubiquitylation of Cdc6by SCF (an E3-ligase)

ProteosomeProteosomal degradationof Cdc6

DNAreplisome

DNAreplisomeM

cm

Mcm

ORC

ORC1.

P P

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Cdc6

Prevention of DNA licensing until the next G1-phase

G1S G2 M

Cdc

6 le

vels

PCdc6

Ub

UbUb

Cdk S

PCdc6

Ub

UbUb

PCdc6

Ub

UbUb

Cdk MS Cdk

Cdc6

ORCMcm

Cdc6

Degradation of “free” Cdc6G1: “free” Cdc6 is

available

Cdc6Cdc6

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Cdk G1

Cdk S

Rb

E2F

Cell cycle entry and DNA replication

Cdk G1/S

MycGrowth factor G1

External signal Activation of the Rb pathway!

DNAreplisome

Mcm

ORCCdc6

P

P

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Positive feedback loop

Rb/E2F = dominant repressor of E2F

p53: The guard against tumors

p53Mdm2

Ub

UbUb

p53

Transcriptional activation of p53 responsive genes

2.3.

4.

1.

2.

3.

4.

DNA damage ( ) or unbalanced/excessive proliferation signaling ( ) inhibits Mdm2 stabilization of p53

degraded through ubiquitination by Mdm2 (E3-ligase)

p53 is constitutively expressed but is normally…

p53p53ATM

ARF

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p531.

p53 gene

3.

ATM

ARF

(ATM: ataxia telangiectasia-mutated, ARF: Alternative Reading Frame)

Target genes of p53 dependent transcription

p21DNA repairproteins

Cell cycle block DNA repair Apoptosis

Loss of p53 function

I´m gonna live foreverGenetic instability

p53

PUMABax

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Two distinct CdK Inhibitor (CKI) families

p

16

Cyclin

p21

CdkG1

CyclinCdk 4/6

Inhibits: Catalytic activity Cyclin association (Cip/Kip family) (Ink4 family)

Specificity: G1, G1/S and S G1 only

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In both cases, the Cdk activity is abolished

Molecular_models: 17.1-Cdk2

Cdk G1

Cdk S

Rb

DNAreplisome ORC

Cdc6P

E2F

p16

Rb & p53 pathways cell cycle and apoptosis

p53

Cdk G1/S

Myc

p53 pathway detects:1. Unbalanced/ excessive proliferation signals (+++)2. DNA damage

Cell cycle block Apoptosis

ARF

ATM

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PI3K

Apoptosisp21

G1/S block

PK

B/A

kt

Survivalsignals

+++

+++

Growth factor

Ras

1.

2.

1.

Three distinct cell cycle regulated events

Size growth

Nuclear division followed by cytoplasmic division

DNA replication

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Cell division and cell growth

Oocytes grow without dividing

Fertilized eggs replicate and divide without growing30

PI3-kinase signaling regulates protein synthesis

P.M.

PP

P

PP

P

PDK1 PKB/Akt

3 3

PKB/AktP

P

PKB/ Akt

Ribosomes

Translation initiation factor

= ProteinsmRNA+ Cellular size growth+

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RTK

PPI-3 K

P

Divergent (cooperating) RTK receptor signals

P3 3 P

PP

P

PKB/AktP

RTK

PPI-3 K

Ras PTEN

Survival

Bad

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”Mitogen” signaling(Rb-pathway)

”Growth factor” signaling

PPP

Increasedcell size

Cell cycleentry

G1 G1G1

DNA replication

Summary: G1-, S- and G2-phase (interphase)

G1 G1 G1 G1G1 G1 G1

G1 G1G1

DNA replisome

”Mitogen” signaling:

Size growth

+ =Translation

Initiation factor”Growth factor” signaling:

Bad

Ras

PI-3 K33

The end of the cell cycle

Cell division = nuclear division + cytoplasmic division

M-phase Mitosis Cytokinesis

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Sorting of 2 x 46 sister chromatids

Defines the division plane/symmetryProtein sorting (if asymmetric division)

Topics of case 13: The cytoskeleton

Progression of cell division: two points of “No return”

Prometaphase

MetaphaseAnaphase

Telophase/cytokinesis

ProphaseInterphase (G2)

Transition points Checkpoint control

Video: 17.4Animal_cell_division

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G2 M transition

MAPsP

Mitotic spindle formation

Event Cause

Change in MT dynamics

CondensinP

Chromosome condensation DNA packaging

Nuclear laminsP Breakdown of

nuclear envelopeDisassembly of nuclear lamina

Consequence

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Checkpoint control of Cdk/M-cyclin activity

+ Cdk MCdk M Cdk M Cdk MP P

P Cdk MP

wee1 CAK Cdc25

Inactive Inactive Inactive Active

G2 G2M

PPP

Inhibitory sites:Activating site:

P P

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Dual feedback loops activity burst!

Cdc25DNA damageDNA replication stall

G2/M checkpoint control and regulation of Cdc25 activity:

Insufficient cell size

Chromosome segregation

Ub

UbUb

+

Ub

UbUb

Cohesin Separase Securin

AttachedduringS-phase

+Sister chromatid separation (anaphase)

Anaphase initiation

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Checkpoint control of metaphase-anaphase transition

APC/CCdc20

Active

APC/C

Inactive

1 unattached kinetochore(spindle assembly checkpoint)

Cdc20

+

Ub

UbUb

Metaphase Anaphase39

Sorting of 2 x 46 chromosomes

Mitotic exit and cytokinesis

Mitotic exit and initiation of cytokinesis

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Cdk MActive P

Cdk MInactivation of

Ub

UbUb

Checkpoint

Cdk/M activity block initiation of cytokinesis!

Molecular events during mitotic exit

APC/CCdc20

+

Ub

UbUb

Cdk M Cdk M

Ub

UbUb

Cdk

Active APC/C cyclin degradation inactive Cdk mitotic exit

CondensinP

MAPs

Nuclear laminsP

P

Mitotic exit

Condensin

Nuclear lamins

MAPs

Constitutively active phosphatases drives the cell out of mitosis

Phosphatase

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Mitosis versus meiosis:Animation: 21.2-meiosis

Ub

UbUb

+

P P

Cdc25

G1

SG2

MG2/M checkpointDNA status and cell size

Spindle assembly checkpointSpindle attachment to chromosomes

G1/S checkpointThe surroundings

APC/CCdc20

Checkpoints function: block of premature transitions

DNAreplisome

Mcm

Mitogen

PCdk M Cdk MP PPP

E2F

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Checkpoints verify that all processes at each cell cycle phase have been completed before transition into the next

Cell death in multi-cellular eukaryots

Cell death

Necrosis Apoptosis

- ”Murder”- Associated with inflammation (and vice versa)

- ”Suicide”- Death for the benefit of the organism- No inflammatory response

The word apoptosis is greek for fallen leaves

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Development Homeostasis Immune survelliance

x x

Importance of apoptosis

Apoptosis and cell proliferation must balance each other

Killing of virus infected cells

Elimination ofsuperfluous cells

between the developing fingers

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Cellular changes during apoptosisNormal cell Different stages of apoptosis

Cell shrinkageMembrane ruffling

DNA condensationDNA fragmentation

Phagocytic cells recognizes phosphatidyl serine ( ) which becomes exposed on the surface during apoptosis

Video: 18.1 Apoptosis45

Burp!

Apoptosis – a protease cascade

DNACytoskeleton

Other things

AD

NC

y t

os k

Active effector caspase

Inactive effector caspase

Active initiator caspase

Initiatorcaspase

monomers

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Apoptotic signal multimers

=

Control of apoptosis

Anti-apoptoticPro-apoptotic Cell survival

Apoptosis

External and internal signals

• 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

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The intrinsic apoptotic pathway

Cyt. C

Cyt. C Cyt. C

Cyt. C

Bcl-2 BH123

Cyt. C

Pro-apoptotic

Pore formation in the outer mitochondrion membrane

release

Pore former Inhibitor of pore formation

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Note - Bcl2 has all four BH domains(Albert et al: Fig. 18-9)

Anti-apoptotic

Cyt. C

Cytochrome C in the cytosol triggers apoptosis

Caspase 3

Caspase 9Apaf1

Cyt. C

Apaf1Cyt. C

Caspase 9

Apaf1Cyt. C

Caspase 9

Caspase 3

Apoptosome

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Target proteins

a g t r t i sT r e p o e n

: APoptosis Activating Factor

Regulation of the intrinsic apoptotic pathway

Bcl-2 BH123 (family)

Pro-apoptotic

BH3-only (family)

Bax

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Note - Bcl2 has all four BH domains per definition! (Albert et al: Fig. 18-9)

PUMA p53

Anti-apoptotic

ARFATM

p53 pathway: Pro-apoptotic downstream mediators

BH3-only

Effector caspases

- DNA- Cytoskeleton- Other things

AD

NC

y t

os k

Survival signals Death

signalsCyt. C

Deathreceptor

Ligand

Extrinsic apoptosisPlasma membrane

Summary on apoptosis

Intrinsic apoptosis

BH123Bcl-2

Initiator caspaseInitiator caspase

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Determinants of survival and proliferation

Pro-apoptotic

G1 cyclin CKI

Regulation of cell survival Regulation of cell proliferation

+

x x

or

+

+

Apoptosis

Cell proliferation

Quiescent cell (G0)

or

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Anti-apoptotic

A single external signal may serve multiple functions

Growth factor

Survival factorMitogen

Anti-apoptotic

Pro-apoptoticG1 cyclin +

+

G1

One specific ligand/receptor complex may transduce several distinct signals

Ribosome Translation initiation

factor

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Translation Initiation

factor

Recommended reading

Alberts et al5th edition

Chapter 171053-10801092-10941101-1112

Chapter 181115-1128

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