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Apoptosis in Mammalian Cells
7.16 2-10-05
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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)
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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!
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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
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FasL
FAD
D
FAD
D
casp8
casp3
IAP
Bid
Bax Bax
casp3
casp9
cyto c
IAP
SmacSmac
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Tools for observing apoptosis
• Immunofluorescence mircroscopy
• Flow cytometry
• Live cell microscopy
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Principles of Fluorescence
Two diagrams removed for copyright reasons.
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Immunofluorescence can detect specific proteins in cells
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Immunofluorescence can detect specific proteins in cells
Fix with formaldehyde
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Immunofluorescence can detect specific proteins in cells
Fix with formaldehyde
Permeablize with detergent
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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.
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Detecting apoptosis with immunofluorescence microscopyDNA Cleaved cytokeratin Cleaved caspase-3
Blue: DNA Green: cleaved cytokRed: cleaved casp3
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Determining the order of apoptotic eventsBlue: H33342
Green: cytochromecRed: cleaved casp3
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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
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Combining flow cytometrywith RNAi
No RNAiCaspase 8
RNAi
+TNF
Bid RNAi
Cyt
oker
atin
clea
vage
Caspase 3 cleavage
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Green Fluorescent Protein –a genetically encoded fluorescent tag
Diagram removed for copyright reasons.
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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
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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.
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GFP can be spliced into any gene
DNA sequence atgactgacagtccattaccggactttga atgttcagggatcccataattagtga
Unfolded protein
Folded protein
atgactgacagtccattaccggacttatgttcagggatcccataattagtga
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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
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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!
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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
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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
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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
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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
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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.
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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
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A computational model of caspase activation
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Studying apoptosis to learn how cells make decisions
Internal state
Death signals Diagram removed for
copyright reasons. Die?Survival signals
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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)
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The order of apoptotic events varies between human cancer cells
Hct-116 HeLa
Blue: H33342 Green: cleaved cytok Red: cleaved casp3