Studying apoptosis in DT40 cells

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Studying apoptosis in DT40 cells Sandrine Ruchaud E12.5! Acridine Orange! Role of apoptosis in sculpting the mouse paw E13.5! E14.5! gift of William Wood & Paul Martin! University College, London!

Normal nucleus Apoptotic nucleus Which proteins are involved in apoptotic execution?

Key players responsible for nuclear apoptotic execution Initiator Caspases (-8, -10, etc.) PARP P ARP Effector execution phase Caspases (-3, -7-6) CAD/ICAD Caspase-6 I CAD CAD Lamins (Lamin A/C) Active CAD

Caspase-6 gene targeting GgPro-caspase 6! Prodomain! N! Large subunit! Small subunit! C! 5ʼ! KpnI! XbaI! XbaI! ApaI! E1! E2! E3! E4! E5! E6! E7! E8! Knockout construct! Start! BamHI! resistance! Stop! 3 kb! cassette! 2.1 kb! BamHI! 3ʼ! Wild type allele! BamHI! 7.7 kb! ApaI! BamHI! Probe! Targeted allele (puro)! BamHI! puromycin! 5.3 kb! ApaI! BamHI! Targeted allele (hisd)! hisd! BamHI! ApaI! BamHI! 2.7 kb!

Southern blot Northern and western blot wt Casp6 (+/-) puro Casp6 (-/-) puro/hisd wt (+/-) (-/-) Casp-6 mrna Casp-3 mrna Casp-7 mrna 7.7 kb wt allele β-actin mrna 5.3 kb puro allele 2.7 kb hisd allele wt (+/-) (-/-) Pro Casp-6 α-tubulin what is the specific role of caspase-6 during apoptotic execution?

Apoptosis in caspase 6 -/- cells proceeds normally wt (-/-) No lamin A/C expression in DT40 cells - Lamin A is the only known substrate cleaved only by caspase 6. - Lymphoid tissues have been shown not to express lamin A Et (h) wt (-/-) 0 2 3 4 5 0 2 3 4 5 M (kb) 4.0 3.0 2.0 1.6 Lamin A Lamin C Chicken muscle DT40 HeLa Jurkat anti chicken lamin A 1.0 0.5 Used an apoptotic cell free system on isolated Hela nuclei

Sequential events happening during in vitro induced apoptosis of isolated Hela nuclei 1 2 3 4 5 DAPI stained HeLa nuclei 6 7 8 9 10

Caspase-6 deficient apoptotic extracts block apoptotic chromatin condensation %! 100! 75! 50! Control! Apoptotic! stages! I! II! 25! 0! Genotype! VEID! Control extracts! wt! (-/-)! -! -! Apoptotic extracts! wt! (+/-)! (-/-)! (-/-)! wt! -! -! -! +! +! III! IV! DAPI! Lamin A! Lamin C! MW! (kda)! 62! 62.5! 47.5! α tubulin! Caspase-6 is required for completion of chromatin condensation and formation of apoptotic bodies in nuclei containing lamin A

Is Lamin-A cleavage critical for nuclear apoptotic execution? Control extracts Apoptotic extracts wt (-/-) wt (-/-) Lamin A expression HeLa nuclei No lamin A expression Jurkat nuclei DAPI Jurkat GFP-lamin A nuclei Lamin A is the critical substrate that, when present, must be cleaved by caspase-6 in order for nuclear apoptosis to go to completion * GFP Merged

Caspase-7 gene targeting GgPro-caspase-7! Prodomain! N! Large subunit! Small subunit! C! 5ʼ! Start! Knockout construct! EcoRI! 5ʼ arm! Ev! Ew!Ex! Ey! Ez! Stop! resistance! 3ʼ arm! 3 kb! cassette! 2 kb! EcoRI! 3ʼ! probe! Wild type allele! EcoRI! EcoRI! 11 kb! 11 kb! Targeted allele! (HisD or BsR)! EcoRI! EcoRI! 4.3 kb! 4.3 kb! what is the role of caspase-7 in apoptosis? Korfali N.

Caspase-7 / cells show an increased resistance to apoptosis Cell viability (% of control)! 100! 80! 60! 40! 20! 0!.1! casp 7 +/- a! casp 7 +/- b! casp 7 --/- a! casp 7 -/- b! 1! 10! 100! Etoposide concentration (µm)! Cell viability (% of control)! 100! 80! 60! 40! 20! 0!.1! casp 7 --/-! casp 7 -/- :casp-7:egfp! 1! 10! 100! Etoposide concentration (µm)! Korfali N.

Caspase-7 deficient cells show a delay in DNA fragmentation A! bp 12.0! 5.0! 3.0! 2.0! 1.6! 1.0! 0.5! MW! 0 h! 2 h! 3 h! -/-! -/-! -/-! -/-! -/-! -/-! B! Tunel positive cells (% of total)! 100! 90! 80! 70! 60! 50! 40! 30! 20! 10! 0! casp7 -/-! casp7 --/- :casp-7:egfp! 0 1 2 3 4 5 Time (hours)! Korfali N.

Caspase-7-deficient cells show delayed loss of plasma membrane asymmetry during apoptotic execution An emerging view of the role of Caspase-7 in apoptosis. Caspase-7 may function closer to the apex of the Caspase cascade. Korfali N. A! B! Annexin V positive cells (% of total)! Annexin V positive cells (% of total)! 80! 70! 60! 50! 40! 30! 20! 10! 0! 35! 30! 25! 20! 15! 10! 5! 0! casp 7 -/-! casp 7 -/- :casp-7:egfp! etoposide! 0! 1! 2! 3! 4! 5! 6! Time (hours)! casp 7 -/-! casp 7 -/- :casp-7:egfp! staurosporine! 0! 1! 2! 3! 4! 5! 6! 7! 8! 9! Time (hours)!

CAD knockout strategy Samejima K

Inter-nucleosomal DNA cleavage is absent in CAD -/- cells and chromatin condensation is blocked at an early stage Samejima K

ICAD and CAD/ICAD double knockout strategy Ageichik A.

Inter-nucleosomal DNA cleavage is absent and chromatin condensation is blocked at early stages in both ICAD and CAD/ICAD double knockouts Ageichik A.

Nadia Korfali Alexander Ageichik Kumiko Samejima Bill Earnshaw

Caspase Activation Is Delayed in Caspase-7 / Cells A! B! LEHD-AFC cleavage! (pmol/min/mg prtotein) DEVD-AFC cleavage! (pmol/min/mg prtotein)! VEID-AFC cleavage! (pmol/min/mg prtotein)! 0 h! 3 h! 2000 1500 1000 500 0 2000 1500 1000 500 0 2000 1500 1000 500 Time (h) 0 6 h! 0 h! casp-7 -/-! 3 h! 6 h! 0 3 6 0 3 6 WT Caspase-7-/- zek(bio)d-aomk! labeling! Biotenylated proteins loading control! C! D! LEHD-AFC cleavage! (pmol/min/mg prtotein! DEVD-AFC cleavage)! (pmol/min/mg prtotein! VEID-AFC cleavage! (pmol/min/mg prtotein! 0 h! 2000 1500 1000 500 0 2000 1500 1000 500 0 2000 1500 1000 500 0 Time (h) 0.5 h! 1 h! 0 h! casp-7 -/-! 0.5 h! 1 h! 0 0.5 1 0 0.5 1 WT Caspase-7-/- zek(bio)d-aomk! labeling! Biotenylated proteins loading control!

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