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
Y
Y
Y
Die!
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
FADD
FADD
FasL
Bid
casp8
Bax
casp3
casp3
IAP
IAP
casp9
cyto Smac
cSmac
Bax
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 microscopy
DNA
Cleaved cytokeratin
Cleaved caspase-3
Blue: DNA
Green: cleaved cytok
Red: cleaved casp3
Determining the order of apoptotic events
Blue: H33342
Green: cytochromec
Red: cleaved casp3
Green fluorescence
Cytokeratin cleavage
Quantitating apoptosis
using flow cytometry
Late
apoptotic
cells
Healthy
cells
Early
apoptotic
cells
Red fluorescence
Caspase 3 cleavage
Combining flow cytometry
with RNAi
Cytokeratin cleavage
+TNF
No RNAi
Caspase 8
RNAi
Caspase 3 cleavage
Bid RNAi
Green Fluorescent Protein –
a genetically encoded fluorescent tag
Diagram removed for copyright reasons.
The fluorescent protein family
Jellyfish Aequoria victoria
Green
fluorescent
protein
Red
fluorescent
protein
Engineered mutations
Blue
fluorescent
protein
Cyan
fluorescent
protein
Reef coral
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
FRET – a phenomenon that occurs when 2
fluorescent molecules are very close
No FRET
FRET!
cfp
yfp
< 6 nm
yfp
cfp
> 6 nm
FRET: Forster Resonance Energy Transfer
A FRET-based caspase activity reporter
FRET!
DEVD
yfp
cfp
yfp
DEVD
casp3
cfp
Excitation with UV light.
Energy transfer from CFP to YFP.
Emission of yellow light.
Cleavage of substrate by
caspase-3.
cfp
yfp
DEVD
Excitation with UV light.
Emission of cyan light.
Monitoring caspase activity over
time in living cells
1.5
healthy cell
1.4
dying cell
1.3
FRET
ratio
1.2
1.1
1
0.9
0
50
100
150
FRET reporters can be modified to
detect different caspase activities
cfp
yfp
Caspase-3
DEVD
cfp
yfp
Caspase-8
yfp
Caspase-9
IETD
cfp
LEHD
Much of the apoptotic pathway is conserved
Mammals
Death
signals
Fas
Flies
FADD
cIAP
FADD
casp8
Bid
Dredd
Bax
Death
signals
Eiger
Debcl
Bcl2
Buffy
cytoc
cytoc
Rpr
Grim
Hid
casp3
casp9
XIAP
Smac
Dcp
Drice
Dronc
DIAP
But the importance of certain pathways changes
Mammals
Flies
Fas
FADD
cIAP
FADD
Eiger
Dredd
Bid
casp8
Debcl
Buffy
Bax
Bcl2
cytoc
Rpr Grim Hid
cytoc
Dcp
Drice
Dronc
casp3
casp9
Smac
DIAP
XIAP
Recent development – XIAP is
important in human cancer cells
See Li, L. et al. “A Small Molecule Smac Mimic Potentiates TRAIL- and TNF-α
Mediated Cell Death.” Science 305 no. 5689 (2004 Sep 3): 1411-3.
Even within a species, differences are observed
Fas
Human Cancer cell 1
Human Cancer cell 2
Fas
FADD
cIAP
cIAP
FADD
Bid
Bid
casp8
casp8
Bax
Bcl2
Bax
Bcl2
cytoc
casp3
cytoc
casp9
casp3
Smac
XIAP
casp9
XIAP
Smac
A computational model of
caspase activation
Studying apoptosis to learn how
cells make decisions
Internal
state
Death
signals
Survival
signals
Diagram removed for
copyright reasons.
Die?
Effector Caspase Activity
(cytokeratin cleavage)
Quantitating apoptosis
using flow cytometry
Untreated
TNF treated
Initiator Caspase Activity
(caspase 3 cleavage)
TNF treated, Bax -/-
The order of apoptotic events varies between
human cancer cells
Hct-116
HeLa
Blue: H33342 Green: cleaved cytok Red: cleaved casp3