Studying apoptosis in DT40 cells - Events
Studying apoptosis in DT40 cells - Events
Studying apoptosis in DT40 cells - Events
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<strong>Study<strong>in</strong>g</strong> <strong>apoptosis</strong> <strong>in</strong> <strong>DT40</strong> <strong>cells</strong><br />
Sandr<strong>in</strong>e Ruchaud<br />
Acrid<strong>in</strong>e Orange!<br />
Role of <strong>apoptosis</strong> <strong>in</strong> sculpt<strong>in</strong>g the mouse paw<br />
E12.5! E13.5!<br />
E14.5!<br />
gift of William Wood & Paul Mart<strong>in</strong>!<br />
University College, London!
Normal nucleus Apoptotic nucleus<br />
Which prote<strong>in</strong>s are <strong>in</strong>volved<br />
<strong>in</strong> apoptotic execution?
Key players responsible for nuclear apoptotic execution<br />
Initiator<br />
Caspases<br />
(-8, -10, etc.)<br />
Effector<br />
execution phase<br />
Caspases<br />
(-3, -7<br />
-6)<br />
Active CAD<br />
CAD/ICAD<br />
PARP<br />
P ARP<br />
Caspase-6<br />
I<br />
CAD<br />
CAD<br />
Lam<strong>in</strong>s<br />
(Lam<strong>in</strong> A/C)
Caspase-6 gene target<strong>in</strong>g<br />
5ʼ!<br />
GgPro-caspase 6!<br />
Prodoma<strong>in</strong>!<br />
N!<br />
KpnI! XbaI!<br />
XbaI! ApaI!<br />
E1! E2! E3! E4! E5! E6! E7! E8!<br />
Knockout construct!<br />
Wild type allele!<br />
Targeted allele (puro)!<br />
Targeted allele (hisD)!<br />
Large subunit! Small subunit!<br />
Start! BamHI!<br />
Stop!<br />
resistance!<br />
3 kb! cassette!<br />
2.1 kb!<br />
BamHI!<br />
BamHI!<br />
7.7 kb!<br />
puromyc<strong>in</strong>!<br />
hisD!<br />
5.3 kb!<br />
BamHI!<br />
2.7 kb!<br />
ApaI!<br />
ApaI!<br />
ApaI!<br />
C!<br />
BamHI!<br />
BamHI!<br />
BamHI!<br />
BamHI!<br />
3ʼ!<br />
Probe!
Southern blot<br />
wt<br />
Casp6 (+/-) puro<br />
Casp6 (-/-) puro/hisD<br />
7.7 kb wt allele<br />
5.3 kb puro allele<br />
2.7 kb hisD allele<br />
Northern and western blot<br />
wt (+/-) (-/-)<br />
wt (+/-)<br />
(-/-)<br />
Casp-6 mRNA<br />
Casp-3 mRNA<br />
Casp-7 mRNA<br />
β-act<strong>in</strong> mRNA<br />
Pro Casp-6<br />
α-tubul<strong>in</strong><br />
⇒ what is the specific role of caspase-6 dur<strong>in</strong>g apoptotic execution?
Apoptosis <strong>in</strong> caspase 6 -/- <strong>cells</strong> proceeds normally<br />
Et (h)<br />
wt<br />
wt<br />
(-/-)<br />
(-/-)<br />
0 2 3 4 5 0 2 3 4 5<br />
M<br />
(kb)<br />
4.0<br />
3.0<br />
2.0<br />
1.6<br />
1.0<br />
0.5<br />
No lam<strong>in</strong> A/C expression <strong>in</strong> <strong>DT40</strong> <strong>cells</strong><br />
- Lam<strong>in</strong> A is the only known substrate cleaved only by<br />
caspase 6.<br />
- Lymphoid tissues have been shown not to express<br />
lam<strong>in</strong> A<br />
Lam<strong>in</strong> A<br />
Lam<strong>in</strong> C<br />
Chicken<br />
muscle<br />
<strong>DT40</strong><br />
HeLa<br />
Jurkat<br />
anti chicken lam<strong>in</strong> A<br />
Used an apoptotic cell free system<br />
on isolated Hela nuclei
Sequential events happen<strong>in</strong>g dur<strong>in</strong>g <strong>in</strong> vitro <strong>in</strong>duced <strong>apoptosis</strong> of<br />
isolated Hela nuclei<br />
DAPI sta<strong>in</strong>ed<br />
HeLa nuclei<br />
1 2 3 4 5<br />
6 7 8 9 10
Caspase-6 deficient apoptotic extracts block apoptotic chromat<strong>in</strong> condensation<br />
%! 100!<br />
75!<br />
50!<br />
25!<br />
0!<br />
Genotype!<br />
VEID!<br />
DAPI!<br />
Lam<strong>in</strong> A!<br />
Lam<strong>in</strong> C!<br />
α tubul<strong>in</strong>!<br />
Control extracts! Apoptotic extracts!<br />
wt! (-/-)! wt! (+/-)! (-/-)! (-/-)! wt!<br />
-! -! -! -! -! +! +!<br />
Control!<br />
Apoptotic!<br />
stages!<br />
MW!<br />
(kDa)!<br />
62! 62.5!<br />
47.5!<br />
Caspase-6 is required for completion of chromat<strong>in</strong> condensation and<br />
formation of apoptotic bodies <strong>in</strong> nuclei conta<strong>in</strong><strong>in</strong>g lam<strong>in</strong> A<br />
I!<br />
II!<br />
III!<br />
IV!
Is Lam<strong>in</strong>-A cleavage critical for nuclear apoptotic execution ?<br />
Lam<strong>in</strong> A expression<br />
No lam<strong>in</strong> A expression<br />
HeLa<br />
nuclei<br />
Jurkat<br />
nuclei<br />
Jurkat<br />
GFP-lam<strong>in</strong> A<br />
nuclei<br />
Lam<strong>in</strong> A is the critical substrate that,<br />
when present, must be cleaved by<br />
caspase-6 <strong>in</strong> order for nuclear <strong>apoptosis</strong><br />
to go to completion<br />
Control extracts Apoptotic extracts<br />
wt (-/-) wt (-/-)<br />
*<br />
DAPI<br />
GFP<br />
Merged
Caspase-7 gene target<strong>in</strong>g<br />
GgPro-caspase-7!<br />
Prodoma<strong>in</strong>!<br />
N!<br />
Large subunit! Small subunit!<br />
5ʼ!<br />
EcoRI! Ev! Ew!Ex! Ey! Ez!<br />
Start! Stop!<br />
Knockout construct!<br />
5ʼ arm!<br />
3 kb!<br />
resistance!<br />
cassette!<br />
3ʼ arm!<br />
2 kb!<br />
Wild type allele!<br />
Targeted allele !<br />
(HisD or BsR)!<br />
EcoRI!<br />
11 kb!<br />
4.3 kb!<br />
C!<br />
EcoRI! EcoRI!<br />
EcoRI!<br />
3ʼ!<br />
probe!<br />
EcoRI!<br />
⇒ what is the role of caspase-7 <strong>in</strong> <strong>apoptosis</strong>?<br />
11 kb!<br />
4.3 kb!<br />
Korfali N.
Caspase-7 –/– <strong>cells</strong> show an <strong>in</strong>creased resistance to <strong>apoptosis</strong><br />
Cell viability (% of control)!<br />
100!<br />
80!<br />
60!<br />
40!<br />
20!<br />
0!<br />
.1!<br />
WT!<br />
casp 7 +/- a!<br />
casp 7 +/- b !<br />
casp 7 --/- a!<br />
casp 7 -/- b !<br />
1!<br />
10! 100!<br />
Etoposide concentration (µM)!<br />
Cell viability (% of control)!<br />
100!<br />
80!<br />
60!<br />
40!<br />
20!<br />
0!<br />
.1!<br />
1!<br />
WT!<br />
casp 7 --/- !<br />
casp 7 -/- :casp-7:EGFP !<br />
10!<br />
Etoposide concentration (µM)!<br />
100!<br />
Korfali N.
Caspase-7 deficient <strong>cells</strong> show a delay <strong>in</strong> DNA fragmentation<br />
A!<br />
bp<br />
12.0!<br />
5.0!<br />
3.0!<br />
2.0!<br />
1.6!<br />
1.0!<br />
0.5!<br />
MW!<br />
WT!<br />
0 h! 2 h! 3 h!<br />
-/-!<br />
-/-!<br />
WT!<br />
-/-!<br />
-/-!<br />
WT!<br />
-/-!<br />
-/-!<br />
B!<br />
Tunel positive <strong>cells</strong> (% of total) !<br />
100!<br />
90!<br />
80!<br />
70!<br />
60!<br />
50!<br />
40!<br />
30!<br />
20!<br />
10!<br />
0!<br />
WT!<br />
casp7 -/- !<br />
casp7 --/- :casp-7:EGFP !<br />
0 1 2 3 4 5<br />
Time (hours)!<br />
Korfali N.
Caspase-7-deficient <strong>cells</strong><br />
show delayed loss of plasma<br />
membrane asymmetry dur<strong>in</strong>g<br />
apoptotic execution<br />
An emerg<strong>in</strong>g view of the role of<br />
Caspase-7 <strong>in</strong> <strong>apoptosis</strong>. Caspase-7<br />
may function closer to the apex of<br />
the Caspase cascade.<br />
Korfali N.<br />
A!<br />
B!<br />
Annex<strong>in</strong> V positive <strong>cells</strong> (% of total) !<br />
Annex<strong>in</strong> V positive <strong>cells</strong> (% of total) !<br />
80!<br />
70!<br />
60!<br />
50!<br />
40!<br />
30!<br />
20!<br />
10!<br />
0!<br />
35!<br />
30!<br />
25!<br />
20!<br />
15!<br />
10!<br />
5!<br />
0!<br />
WT!<br />
casp 7 -/- !<br />
casp 7 -/- :casp-7:EGFP !<br />
etoposide!<br />
0! 1! 2! 3! 4! 5! 6!<br />
Time (hours)!<br />
WT!<br />
casp 7 -/- !<br />
casp 7 -/- :casp-7:EGFP !<br />
staurospor<strong>in</strong>e!<br />
0! 1! 2! 3! 4! 5! 6! 7! 8! 9!<br />
Time (hours)!
CAD knockout strategy<br />
Samejima K
Inter-nucleosomal DNA cleavage is absent <strong>in</strong> CAD -/- <strong>cells</strong> and<br />
chromat<strong>in</strong> condensation is blocked at an early stage<br />
Samejima K
ICAD and CAD/ICAD double knockout strategy<br />
Ageichik A.
Inter-nucleosomal DNA cleavage is absent and chromat<strong>in</strong> condensation is<br />
blocked at early stages <strong>in</strong> both ICAD and CAD/ICAD double knockouts<br />
Ageichik A.
Nadia Korfali<br />
Alexander Ageichik<br />
Kumiko Samejima<br />
Bill Earnshaw
Caspase Activation Is Delayed <strong>in</strong> Caspase-7–/– Cells<br />
A! 3 h!<br />
B!<br />
LEHD-AFC cleavage!<br />
(pmol/m<strong>in</strong>/mg prtote<strong>in</strong>)<br />
0<br />
DEVD-AFC cleavage!<br />
(pmol/m<strong>in</strong>/mg<br />
prtote<strong>in</strong>)!<br />
VEID-AFC cleavage!<br />
(pmol/m<strong>in</strong>/mg<br />
prtote<strong>in</strong>)!<br />
0 h!<br />
2000<br />
1500<br />
1000<br />
500<br />
2000<br />
1500<br />
1000<br />
500<br />
0<br />
2000<br />
1500<br />
1000<br />
500<br />
Time (h) 0<br />
WT! casp-7<br />
-/- !<br />
6 h!<br />
0 h!<br />
3 h!<br />
6 h!<br />
0 3 6 0 3 6<br />
WT Caspase-7-/-<br />
zEK(bio)D-aomk !<br />
label<strong>in</strong>g!<br />
Biotenylated<br />
prote<strong>in</strong>s load<strong>in</strong>g<br />
control!<br />
C!<br />
D!<br />
LEHD-AFC cleavage!<br />
(pmol/m<strong>in</strong>/mg prtote<strong>in</strong>!<br />
DEVD-AFC cleavage)!<br />
(pmol/m<strong>in</strong>/mg prtote<strong>in</strong>!<br />
VEID-AFC cleavage!<br />
(pmol/m<strong>in</strong>/mg prtote<strong>in</strong>!<br />
0 h!<br />
0.5 h!<br />
1 h!<br />
2000<br />
1500<br />
1000<br />
500<br />
0<br />
2000<br />
1500<br />
1000<br />
500<br />
0<br />
2000<br />
1500<br />
1000<br />
500<br />
0<br />
Time (h)<br />
WT! casp-7<br />
-/- !<br />
0 h!<br />
0.5 h!<br />
1 h!<br />
0 0.51 0 0.5 1<br />
WT Caspase-7-/-<br />
zEK(bio)D-aomk !<br />
label<strong>in</strong>g!<br />
Biotenylated<br />
prote<strong>in</strong>s load<strong>in</strong>g<br />
control!
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