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Apoptosis in Mammalian Cells

7.16 2-10-05

Apoptosis is an important factor in many human diseases

• Cancer – malignant cells evade death by suppressing apoptosis (too little apoptosis)

• Stroke – damaged neurons commit apoptosis, leading to more extensive damage (too much apoptosis)

• Autoimmune disorders – immune cells survive for too long, leading to over-reactive immune response (too little apoptosis)

The death receptor pathway

• In mammals, there is a family of death ligands that bind to receptors on the cell surface, sending a pro-apoptotic signal to the cell

Y YYYDie!

Death ligand family members

• FasL – very important in the immune system

• TNF (Tumor necrosis factor) – controls inflammatory responses

• TRAIL –selectively kills tumor cells; currently in clinical trials as an anti-cancer agent

FasL

FAD

D

FAD

D

casp8

casp3

IAP

Bid

Bax Bax

casp3

casp9

cyto c

IAP

SmacSmac

Tools for observing apoptosis

• Immunofluorescence mircroscopy

• Flow cytometry

• Live cell microscopy

Principles of Fluorescence

Two diagrams removed for copyright reasons.

Immunofluorescence can detect specific proteins in cells

Immunofluorescence can detect specific proteins in cells

Fix with formaldehyde

Immunofluorescence can detect specific proteins in cells

Fix with formaldehyde

Permeablize with detergent

Immunofluorescence can detect specific proteins in cells

Antibodies bind to specific proteins.

We can generate antibodies to nearly any protein we’re interested in.

We can label antibodies with fluorescent molecules.

Fluorescently labeled antibodies can reveal the location of specific proteins in the cell.

Detecting apoptosis with immunofluorescence microscopyDNA Cleaved cytokeratin Cleaved caspase-3

Blue: DNA Green: cleaved cytokRed: cleaved casp3

Determining the order of apoptotic eventsBlue: H33342

Green: cytochromecRed: cleaved casp3

Healthy cells

Early apoptotic cells

Late apoptotic cells

Red fluorescence

Gre

en fl

uore

scen

ce

Quantitating apoptosis using flow cytometry

Cyt

oker

atin

clea

vage

Caspase 3 cleavage

Combining flow cytometrywith RNAi

No RNAiCaspase 8

RNAi

+TNF

Bid RNAi

Cyt

oker

atin

clea

vage

Caspase 3 cleavage

Green Fluorescent Protein –a genetically encoded fluorescent tag

Diagram removed for copyright reasons.

The fluorescent protein family

Jellyfish Aequoria victoria Reef coral

Green fluorescent

protein

Engineered mutations

Red fluorescent

protein

Blue fluorescent

protein

Cyan fluorescent

protein

Yellow fluorescent

protein

GFP has been engineered to make blue and yellow variants; red fluorescent proteins

have been isolated from other sea creatures

Two photos removed for copyright reasons.

GFP can be spliced into any gene

DNA sequence atgactgacagtccattaccggactttga atgttcagggatcccataattagtga

Unfolded protein

Folded protein

atgactgacagtccattaccggacttatgttcagggatcccataattagtga

cfp yfp cfp yfp

FRET – a phenomenon that occurs when 2 fluorescent molecules are very close

FRET!

FRET: Forster Resonance Energy Transfer

No FRET

< 6 nm > 6 nm

DEVD

cfp yfp

casp

3

DEVD

cfp yfp

DEVD

cfp yfp

Excitation with UV light. Energy transfer from CFP to YFP.

Emission of yellow light.Cleavage of substrate by

caspase-3.

Excitation with UV light. Emission of cyan light.

A FRET-based caspase activity reporter

FRET!

Monitoring caspase activity over time in living cells

0.9

1

1.1

1.2

1.3

1.4

1.5

0 50 100 150

healthy celldying cell

FRET ratio

FRET reporters can be modified to detect different caspase activities

DEVD

cfp yfp Caspase-3

IETD

cfp yfp Caspase-8

LEHD

cfp yfp Caspase-9

Much of the apoptotic pathway is conserved

Eiger

Dredd

DroncDcpDrice

Debcl

RprGrim Hid

Buffy

cytoc

FADD

DIAP

Fas

cIAP

casp8

casp9casp3

Bid

Bax

Smac

Bcl2

cytoc

FADD

XIAP

Mammals FliesDeath signals

Death signals

But the importance of certain pathways changesEiger

Dredd

DroncDcp

Drice

Debcl

Rpr Grim Hid

Buffy

cytoc

FADD

DIAP

FliesFas

cIAP

casp8

casp9casp3

Bid

Bax

Smac

Bcl2

cytoc

FADD

XIAP

Mammals

Recent development – XIAP is important in human cancer cells

See Li, L. et al. “A Small Molecule Smac Mimic Potentiates TRAIL- and TNF-αMediated Cell Death.” Science 305 no. 5689 (2004 Sep 3): 1411-3.

Even within a species, differences are observedFas

cIAP

casp8

casp9casp3

Bid

Bax

Smac

Bcl2

cytoc

FADD

XIAP

Human Cancer cell 1Fas

cIAP

casp8

casp9casp3

Bid

Bax

Smac

Bcl2

cytoc

FADD

XIAP

Human Cancer cell 2

A computational model of caspase activation

Studying apoptosis to learn how cells make decisions

Internal state

Death signals Diagram removed for

copyright reasons. Die?Survival signals

Quantitating apoptosis using flow cytometry

Effe

ctor

Cas

pase

Act

ivity

(c

ytok

erat

incl

eava

ge)

Untreated TNF treated TNF treated, Bax -/-

Initiator Caspase Activity (caspase 3 cleavage)

The order of apoptotic events varies between human cancer cells

Hct-116 HeLa

Blue: H33342 Green: cleaved cytok Red: cleaved casp3