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Cell biology 2014 ( revised 4/2 -14 ). Lecture 6 & 7:. Life and death of cells part I & II . All diseases involve changes of cells. All cells come from cells. Cells of all life forms have a common ancestor. Rudolf Virchow, Die Cellularpathologie (1858) .

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Life and death of cells part i ii

Cell biology 2014 (revised 4/2 -14)

Lecture 6 & 7:

Life and death of cells

part I & II

All diseases involve changes of cells

All cells come

from cells

Cells of all life forms have a common ancestor

Rudolf Virchow, Die Cellularpathologie (1858)


Life and death of cells part i ii

Cell cycle control in multi-cellular eukaryotes

somatic mutations

&

chromosomal instability

Controlled and co-

ordinated divisions

Uncontrolled divisions

 Tumor (clonalorigin!)

1013

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 Uranus Pluto

Sun

Mars


Life and death of cells part i ii

“Simple” model systems for studies of eukaryotes

Budding yeast, S. cerevisiae

Polarized (bud & shmoo)

Primordial eukaryote

(polarity & sex)

Fission yeast, S pombe

Symmetric 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.


Life and death of cells part i ii

Principles of conditional mutants

Wild type protein

Mutant protein

Functional

(i.e. nophenotype)

26oC

Non-functional

(i.e. phenotype)

36oC

The mutant gene product is temperature sensitive (Ts)

OK at 26oC, butunfolds at 36oC

(i.e. the function of gene product can be switched off)


Life and death of cells part i ii

Missing!

Temperature sensitive yeast mutants

= Ts clone

26o C

36o C

Cell cycleTs mutant or not?

26o C

36o C

or

(if lucky!)

House keeping

gene mutated

Cell cycle control

gene mutated


Life and death of cells part i ii

Identification of cell division control (cdc) genes

ZZZ

Gene library

(i.e., wild type genes cloned

into bacterial plasmids)

ZZZ

ZZZ

+

36o C

OK

OK

OK

Yeast cell

cycle Ts mutant

Mutated gene:

(OK at 26oC but not 36oC)

ZZZ

Complementation by :


Life and death of cells part i ii

Cell cycle regulators are evolutionary conserved

Budding yeast with a temperature

sensitive mutation inan 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!


Life and death of cells part i ii

Three distinct cell cycle regulated events

DNA replication

Increased size

Nuclear division

followed by

cytoplasmic division


Life and death of cells part i ii

S

G2

GO

G1

Interphase and mitosis

Quiescent or

post-mitotic

Condensed chromatin

”thread-like”

Interphase (“between-phase(s)”)

(90100% of a cell population)

S (DNA-Synthesis)

Mitosis (Cell division phase)

(010% of a cell population)

G1 (Gap 1)

G2 (Gap 2)

Chromosome segregation

Cell division

Cell cycle exit  “G0”

Chromatin

“attract dyes”

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


Life and death of cells part i ii

2.

3.

2.

1.

3.

1.

Key events and checkpoints of the cell cycle

G1/S checkpoint (“Start”)

Sense: Surroundings

Cell size

Block: DNA replication

M

G1

G2/M checkpoint

Sense: DNA replication status

DNA damage

Cell size

G2

S

Block: Mitotic entry

Spindle assembly checkpoint

Sense: Chromosome attachment to the mitotic spindle

Block: Chromosome separation and cytoplasmic division


Life and death of cells part i ii

P

P

P

Ub

Ub

Ub

Cell cycle regulation of proteins

Amount of protein

Activity of protein

- Control of protein expression

- Binding partners

DNA

Transcription

+

=

mRNA

Translation

Protein

+

=

- Control of protein turnover

  • Phosphorylation

26S Proteosome


Life and death of cells part i ii

Cyclin dependent kinases (Cdk) – the controllers

Cdk

Cyclin

H

O

Serine or threonine

P

Phosphatase

Kinase

-

O

Kinase motif

-

P

O

O

P

Inhibitory domain (T-loop)

O

Both the cyclin and are required

to activate the Cdk

P

Serine or threonine

Cyclin  substrate specificity


Life and death of cells part i ii

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

Cdk 4/6

Cdk 2

Cdk 1

Diffuse border

M

G1

S

G2

M

G1

Cyclin:

(expression)

G1

(D)

S

(A)

G1/S

(E)

M

(B)

Interphase cyclins

Mitotic cyclin

v-SNARE

v-SNARE

Ci

NFAT

Ras

Src

v-SNARE

R7

cGMP

Hsp70

MHC

The cell can not reside in two cell cycle phases simultaneously

The cell can not reside in two cell cycle phases simultaneously

Wnt

cGMP

cGMP

Notch

COPII

ACTH

Retinoic acid

RelA

v-SNARE

Clathrin

v-SNARE


Life and death of cells part i ii

P

P

P

P

P

P

1.

2.

3.

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Mechanisms for interphase cyclin degradation

Cdk

Cdk

G1

High intrinsic

turnover

of G1 cyclin

+

Kinase X

-dependent

ubiquitination of G1/S cyclin

P

Cdk

Cdk

Cdk

G1/S

G1/S

+

Cdk

M

Cdk/M-cyclin

mediated 

ubiquitination

of S cyclin

P

Cdk

Cdk

Cdk

S

S

+


Life and death of cells part i ii

Cdk

Cdk

G1

G1/S

Cdk/cyclin control of progression and transitions

Active:

Cdk

Cdk

X

S

Cdk

S

Cdk

M

Transitions:

M

G1

S

G2

M

G1

G0


Life and death of cells part i ii

Wnt

XGF

External signals (mitogens)G1 cyclin expression

Hedgehog

Receptor

Tyrosine

Kinase

GTP

Ras

= DNA

Myc

Myc

G1

myc gene

G1 cyclin gene


Life and death of cells part i ii

P

P

Cdk

Cdk

G1/S

G1

2.

1.

2.

3.

3.

1.

Rb

Rb phosphorylation by Cdk/G1 cyclin  E2F activation

Positive feedback loop

G1/S

DNA

replisome

E2F

E2F

Rb

S

activator

repressor

= DNA binding

protein

Phosphorylation by Cdk-G1 cyclin dissociates Rb from E2F

Transcription of E2F regulated genes

Enhancement by newly formed Cdk-G1/S cyclin


Life and death of cells part i ii

P

P

S-phase components

DNA

replisome

E2F

Rb

Transcriptional control of S phase components

E2F

E2F

E2F

E2F

Rb

S-phase components

Non-dividing cell:

Dominantrepressionby the E2F/Rb complex

G1/S

S

Proliferating cell:

E2F mediated transcription of “S phase genes”

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


Life and death of cells part i ii

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


Life and death of cells part i ii

ORC

ORC

ORC

Cdc6

Cdc6

Cdc6

ORC

ORC

ORC

Cdc6

Cdc6

Cdc6

Mcm

Mcm

Mcm

ORC

ORC

ORC

Mcm

Mcm

Mcm

Licensing of DNA for a single round of replication

Early G1

Cdc6 is recruited to ORCs

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


Life and death of cells part i ii

P

P

P

Cdk

S

DNA

replisome

Firing of pre-RC during S-phase

DNA

replisome

Cdc6

Mcm

Late G1

ORC

Mcm

Firing of the first ORC

point of no return!

Phosphorylates Cdc6 and Mcm

Cdc6

ORC

DNA

replisome

S

Mcm

Mcm

ORC

DNA strand separation by helicase activity of Mcm proteins


Life and death of cells part i ii

P

P

P

P

1.

4.

1.

2.

4.

3.

2.

3.

DNA

replisome

Ub

Ub

Ub

A fool-proof system for prevention of re-replication

ORC

DNA

replisome

Mcm

Mcm

ORC

Phosphorylated Cdc6

dissociates from ORC

Cdc6

Phosphorylated Cdc6 is

recognized by SCF

S

C

F

Cdc6

Ubiquitylation of Cdc6

by SCF (an E3-ligase)

Proteosomal degradation

of Cdc6

Proteosome


Life and death of cells part i ii

P

P

P

Cdk

Cdk

M

S

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Cdk

S

Prevention of DNA licensing until the next G1-phase

Degradation of “free” Cdc6

G1: “free” Cdc6 is available

Cdc6

Cdc6

Cdc6

Cdc6

Cdc6

Cdc6

Cdc6

Cdc6

Mcm

ORC

S

G2

M

G1

Cdc6 levels


Life and death of cells part i ii

P

P

Cdk

Cdk

S

G1/S

Cell cycle entry and DNA replication

Externalsignal  Activationof the Rb pathway!

Cdk

G1

G1

Myc

Growth

factor

Positive

feedback loop

Rb

Rb/E2F = dominant repressor of E2F

E2F

Cdc6

ORC

Mcm

DNA

replisome


Life and death of cells part i ii

Mdm2

4.

3.

1.

3.

2.

3.

4.

1.

2.

Ub

Ub

Ub

p53: The guard against tumors

p53

p53

p53

p53

p53

ATM

p53 gene

ARF

p53 is constitutively expressed but is normally…

degraded through ubiquitination by Mdm2 (E3-ligase)

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

ATM

ARF

Transcriptional activation of p53 responsive genes

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


Life and death of cells part i ii

p21

DNA

repair

proteins

Target genes of p53 dependent transcription

p53

PUMA

Bax

Cell cycle block

DNA repair

Apoptosis

Loss of p53 function

I´m gonna

live forever

Genetic instability


Life and death of cells part i ii

p16

Two distinct CdKInhibitor (CKI) families

Specificity: G1, G1/S and S G1 only

p21

Cdk

G1

Cyclin

Cyclin

Cdk

4/6

Inhibits: Catalytic activity Cyclin association

(Cip/Kip family) (Ink4 family)

In both cases, the Cdk activity is abolished

Molecular_models: 17.1-Cdk2


Life and death of cells part i ii

Rb & p53 pathways cell cycle and apoptosis

P

Cdk

Cdk

Cdk

G1

S

G1/S

p21

1.

2.

1.

DNA

replisome

Myc

p16

Growth

factor

Ras

PI3K

  • p53 pathway detects:

  • 1. Unbalanced/ excessive

  • proliferation signals (+++)

  • 2. DNA damage

  • Cell cycle block

  • Apoptosis

Survival

signals

+++

ARF

PKB/Akt

Rb

ATM

+++

p53

E2F

Cdc6

ORC

Apoptosis

G1/S block


Life and death of cells part i ii

Three distinct cell cycle regulated events

DNA replication

Size growth

Nuclear division

followed by

cytoplasmic division


Life and death of cells part i ii

Cell division and cell growth

Oocytes grow without dividing

Fertilized eggs replicate and divide without growing


Life and death of cells part i ii

P

P

P

P

P

P

P

P

P

P

PI3-kinase signaling regulates protein synthesis

RTK

P.M.

3

3

PKB/

Akt

PI-3 K

PKB/Akt

PDK1

PKB/Akt

Proteins

Translation

initiation

factor

Cellular

size growth

mRNA

=

+

+

Ribosomes


Life and death of cells part i ii

P

P

P

P

P

P

P

P

P

P

Ras

Divergent (cooperating) RTK receptor signals

RTK

”Mitogen” signaling

(Rb-pathway)

”Growth factor” signaling

PTEN

G1

3

3

G1

G1

PI-3 K

Cell cycle

entry

PKB/Akt

Increased

cell size

Bad

Survival


Life and death of cells part i ii

DNA replication

Sizegrowth

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

Translation

Initiation factor

”Mitogen” signaling:

G1

+

=

G1

G1

DNA

replisome

G1

G1

G1

Ras

G1

G1

G1

G1

Bad

”Growth factor” signaling:

PI-3 K


Life and death of cells part i ii

The end of the cell cycle

Cell division = nuclear division + cytoplasmic division

M-phase

Mitosis

Cytokinesis

Sorting of 2 x 46 sister chromatids

Defines the division plane/symmetry

Protein sorting (if asymmetric division)

Topics of case 13: The cytoskeleton


Life and death of cells part i ii

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

Interphase (G2)

Prophase

Telophase/

cytokinesis

Prometaphase

Transition points

 Checkpoint

control

Anaphase

Metaphase

Video: 17.4

Animal_cell_division


Life and death of cells part i ii

P

P

P

G2  M transition

Event

Cause

Consequence

Change in

MT dynamics

Mitotic spindle formation

MAPs

DNA packaging

Chromosome condensation

Condensin

Breakdown of

nuclear envelope

Disassembly of

nuclear lamina

Nuclear lamins


Life and death of cells part i ii

P

P

P

Cdk

Cdk

Cdk

Cdk

M

M

M

M

P

P

P

Checkpoint control of Cdk/M-cyclin activity

G2/M checkpoint control and regulation of Cdc25 activity:

DNA replication stall

DNA damage

Cdc25

Insufficient cell size

Dual feedback loops

 activity

burst!

P

P

Inhibitory sites:

Activating site:

CAK

wee1

Cdc25

P

Cdk

M

+

Inactive

Inactive

Inactive

Active

G2

G2M


Life and death of cells part i ii

Chromosome segregation

Ub

Ub

Ub

Ub

Ub

Ub

Anaphase

initiation

+

Attached

during

S-phase

Sister chromatid

separation

(anaphase)

+

Securin

Cohesin

Separase


Life and death of cells part i ii

Ub

Ub

Ub

Checkpoint control of metaphase-anaphase transition

Cdc20

APC/C

APC/C

Cdc20

Inactive

Active

+

 1 unattached kinetochore

(spindle assembly checkpoint)

Sorting of 2 x 46 chromosomes

Metaphase

Anaphase


Life and death of cells part i ii

P

Cdk

M

Ub

Ub

Ub

Mitotic exit and initiation of cytokinesis

Checkpoint

Active

Cdk

M

Inactivation of

Mitotic exit and

cytokinesis

Cdk/M activity block initiation of cytokinesis!


Life and death of cells part i ii

P

P

P

P

P

Cdk

Cdk

M

M

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Molecular events during mitotic exit

Active APC/C  cyclin degradation  inactive Cdk  mitotic exit

APC/C

Cdc20

+

Cdk

+

Constitutively active phosphatases drives the cell out of mitosis

MAPs

MAPs

Phosphatase

Mitotic exit

Nuclear lamins

Condensin

Condensin

Nuclear lamins

Mitosisversusmeiosis:

Animation: 21.2-meiosis


Life and death of cells part i ii

P

P

P

Cdk

Cdk

M

M

P

Checkpoints function: block of premature transitions

Spindle assembly checkpoint

Spindle attachment to chromosomes

APC/C

Cdc20

G1/S checkpoint

The surroundings

M

G1

G2/M checkpoint

DNA status and

cell size

Mitogen

G2

S

E2F

Cdc25

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

P

DNA

replisome

Mcm


Life and death of cells part i ii

Cell death in multi-cellular eukaryots

Cell death

The word apoptosis is

greek for fallen leaves

Necrosis

Apoptosis

- ”Suicide”

- ”Murder”

  • Death for the benefit

  • of the organism

  • Associated with

  • inflammation

  • (and vice versa)

  • No inflammatory response


Life and death of cells part i ii

Importance of apoptosis

Immune

survelliance

Homeostasis

Development

x

x

Elimination of

superfluous cells

between the

developing fingers

Killing of virus

infected cells

Apoptosis and cell

proliferation must

balance each other


Life and death of cells part i ii

Cellular changes during apoptosis

Different stages of apoptosis

Normal cell

Cell shrinkage

Membrane ruffling

DNA condensation

DNA fragmentation

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

Burp!

Video: 18.1 Apoptosis


Life and death of cells part i ii

Apoptosis – a protease cascade

Initiator

caspase

monomers

Apoptotic signal

multimers

Active initiator

caspase

Cytoskeleton

DNA

Other things

Inactive effector

caspase

Active effector

caspase

o

A

s

C

=

D

y

t

k

N


Life and death of cells part i ii

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

Anti-apoptotic

Pro-apoptotic

Cell survival

Apoptosis


Life and death of cells part i ii

The intrinsic apoptotic pathway

Anti-

apoptotic

Pro-

apoptotic

Cyt. C

Cyt. C

Bcl-2

BH123

Cyt. C

Cyt. C

Pore former

Inhibitor of

pore formation

Cyt. C

Pore formation in the outer

mitochondrion membrane

release

Note - Bcl2 has all four BH domains

(Albert et al: Fig. 18-9)

Cyt. C


Life and death of cells part i ii

Cytochrome C in the cytosol triggers apoptosis

Apoptosome

Apaf1

: APoptosisActivating Factor

Caspase 9

Cyt. C

Cyt. C

Apaf1

Apaf1

Caspase 9

Cyt. C

Caspase 3

Caspase 9

Target proteins

a g t r t i s

T r e p o e n

Caspase 3


Life and death of cells part i ii

Regulation of the intrinsic apoptotic pathway

Anti-

apoptotic

Pro-

apoptotic

p53

p53 pathway: Pro-apoptotic

downstream mediators

Bcl-2

BH123 (family)

Bax

PUMA

BH3-only (family)

ATM

ARF

Note - Bcl2 has all four BH domains

 per definition! (Albert et al: Fig. 18-9)


Life and death of cells part i ii

- DNA

- Cytoskeleton

- Other things

Summary on apoptosis

Extrinsic apoptosis

Intrinsic apoptosis

Plasma membrane

Ligand

Survival

signals

BH123

Bcl-2

Death

receptor

Death

signals

Cyt. C

Initiatorcaspase

Initiatorcaspase

BH3-only

Effector caspases

A

o

s

C

D

y

t

k

N


Life and death of cells part i ii

CKI

x

x

Determinants of survival and proliferation

Anti-

apoptotic

G1 cyclin

Pro-

apoptotic

Regulation of cell survival

Regulation of cell proliferation

Apoptosis

or

+

+

Cell proliferation

or

+

Quiescent cell (G0)


Life and death of cells part i ii

A single external signal may serve multiple functions

Growth factor

Anti-

apoptotic

Pro-

apoptotic

Mitogen

Survival factor

Ribosome

Translation

initiation factor

G1 cyclin

+

One specific ligand/receptor complex may transduce several distinct signals

G1

Translation

Initiation factor

+


Life and death of cells part i ii

Recommended reading

Chapter 18

1115-1128

Chapter 17

1053-1080

1092-1094

1101-1112

Alberts et al

5th edition


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