http://cwp.embo.org/w09-13/index.html

Roads to Ruin is s o t p o ap healthy cell autophagic cell death ne cr os is Thanks to Seamus Martin!

Evolution of apoptosis signalling cascades Inhibitor Adopted from B. Hay, NRG 2004

The Bcl-2 Family in Mammals antiapoptotic members proapoptotic members

Cory and Adams, NRC 2002

Limited conservation of the AA sequence within the BH3-domain I A R K L Q C I A D Q F H R L I A Q E L R R I G D E F N A Y I G S K L A A M C D D F D A Q V G R Q L A I I G D D I N R L S E C L K R I G D E L D S L A L R L A C I G D E M D V L A L R L A C I G D E M D V R N S S BMF BIM EGL1 BAK BAX BID BIK T A A R L K A L G D E L H Q Y G R E L R R M S D E F V D R S HRK BAD

The pro-apoptotic function of Bmf relies on a functional BH3-domain viable GFP + cells (%) 100 50 Jurkat T cells 24h after transfection n=3 Hygromycin-resistant colonies 300 250 200 150 100 50 L929 fibroblasts 10 GFP Bmf Bmf Bcl-2 Bcl-2 p35 BimL ΔBH3 mut1 0 ve+ Bmf Bmf Bmf BimL neg. ΔBH3 L138A +Bmf

How do BH3-only proteins trigger cell death? Cory & Adams, Oncogene 2007

BH3-only proteins integrate various stress signals FISSION FUSION? MOMP? Cyt. C adopted from Willis et al. 2005

FISSION & FUSION - MOMP & Cytochrome C

FISSION & FUSION - MOMP & Cytochrome C Chipuk&Green CDD, 2006

FISSION & FUSION - MOMP & Cytochrome C Chipuk&Green CDD, 2006

FISSION & FUSION - MOMP & Cytochrome C Suen et al, G&D, 2008

FISSION & FUSION - MOMP & Cytochrome C Mfn OPA1 mitopld Bak Cyt. C Suen et al, G&D, 2008

FISSION & FUSION - MOMP & Cytochrome C

CASPASES Executioners of Apoptosis DISC DED Death effector domain CARD Caspase activation & recruitment domain Apoptosome Riedl and Shi,2004 RAIDD PIDD-CC PIDDosome

CASPASES Executioners of Apoptosis pro-domain large subunit small subunit DED Death effector domain CARD Caspase recruitment domain modified from Riedl and Shi,2004

Processing vs. Activation of Initiator Caspases Caspase-2 activation p18 p12 CARD large small 5h UV 5h Eto 5h control [25 mj/cm²] [10 µg/ml] wt ko wt ko wt ko Caspase-2 p50 p32 p18 Activated caspase-3 GAPDH p17 38 kda

Effector caspases: Activation = Processing Time min Slee et al. JCB, 1999

Is there life after MOMP? IAPs and caspases

IAP-antagonists: A smac in the face of caspase-inhibitors? IBM: IAP-binding motive Smac/DIABLO HtrA2/Omi Silke & Vaux, NRCB 2005

Death Receptor Signalling Bahardwaj & Aggarwal; J Clin Immunol 2003

CD95/CD95L signalling TYPE II CELL BID TYPE I CELL tbid Caspase-9 Caspase-3/7 MOMP adopted from P. Krammer, NRI 2007

Micheau & Tschopp, Cell 2004 TNF-R signalling

Modulation of DR-signalling by lipids

Modulation of DR-signalling by location

Things to consider when investigating apoptotic signalling events alive dead Trypan-blue is for U

Investigating apoptotic signalling events the cell surface DISC analysis upstream of/at mitochondria/er Bcl-2 family interactions the level of mitochondria Fission/Fusion & MOMP post MOMP Release of apopt. factors, loss of ΔΨm cytosolic events Caspase activation/substrate cleavage the nucleus DNase activity, endog, AIF suicidal cell not yet committed to die committed to die past the point of no return

Possible Pittfalls Viability assay choice (MTT, Annexin V, sub G1) Overexpression systems (non-physiological protein-protein interactions) Antibody quality (lack of specificity) Lack of substrate specificity (caspase-assays) sirna (off-target effects, lack of controls) Genetic background differences (mouse strain)

MTT, XTT, MTS... Apoptosis vs. Necrosis Cytostatic vs. Cytotoxic Differentiated vs. Proliferating Cells

AnnexinV/PI-stain in BaF3-pro B cells SSC R2 SSC R2 90% 50% FL-3H FL-3H

AnnexinV/PI-stain in hepatocytes I FL-3H FL-3H SSC SSC untreated treated

AnnexinV/PI-stain in hepatocytes II untreated treated

Different methods should yield comparable results! B 4,6 % wt 0h 2,5 % 39,7 % wt 10h 50,0 % 3,4 % wt 10h + QVD 15,7 % 37,8 % 47,8 % bim -/- 10h 34,2 % * bmf -/- 10h 46,7 % sub G1 activated caspase-3

Ordering the caspase cascade ( C2) (C3/7) (C6) (C8/10) (C9) McStay et al CDD 2008

Do not draw conclusions based on caspase substrate assays! 20 20 18 16 14 WT C2-ko RAIDD-ko 15 WT C2-ko RAIDD-ko 12 10 10 8 6 4 5 DEVDase activity Ac-DEVD-AM 2 0 cont-0h cont-6h ETO-6h cont-24h ETO-24h VDVADase activity Ac-VDVAD-A 0 cont-0h cont-6h ETO-6h cont-24h ETO-24h

Never trust an antibody! wt wt puma-/- wt puma-/- puma-/- wt puma-/- * 0 3 6 9 γ-radiation [hr] Orginal Blot * Non specific band

Working with knock-out mice can be tricky, too!

A clean genetic back-ground is vital! Cell viability [%] 110 90 70 50 30 Thymocytes Cell viability [%] 110 90 70 50 30 Splenic T-cells 10 10 0 12 24 48 72 96 0 12 24 48 72 96 Time [hr] Time [hr] wt (SV129:C57BL/6) wt (C57BL/6)

Genetic back-ground differences may screw your day! γ-irradiation [1.25 Gy] Velcade [50 ng/ml] PMA [10 ng/ml] 110 110 110 specific killing % 90 70 50 30 10 90 70 50 30 10 90 70 50 30 10 0 12 24 48 0 12 24 48 0 12 24 48 wt (SV129:C57BL/6) wt (C57BL/6)