Transcription

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

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

4 The Bcl-2 Family in Mammals antiapoptotic members proapoptotic members

5 Cory and Adams, NRC 2002

6 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

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

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

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

10 FISSION & FUSION - MOMP & Cytochrome C

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

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

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

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

15 FISSION & FUSION - MOMP & Cytochrome C

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

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

18 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

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

20 Is there life after MOMP? IAPs and caspases

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

22 Death Receptor Signalling Bahardwaj & Aggarwal; J Clin Immunol 2003

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

24 Micheau & Tschopp, Cell 2004 TNF-R signalling

25 Modulation of DR-signalling by lipids

26 Modulation of DR-signalling by location

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

28 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

29 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)

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

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

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

33 AnnexinV/PI-stain in hepatocytes II untreated treated

34 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

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

36 Do not draw conclusions based on caspase substrate assays! WT C2-ko RAIDD-ko 15 WT C2-ko RAIDD-ko 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

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

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

39 A clean genetic back-ground is vital! Cell viability [%] Thymocytes Cell viability [%] Splenic T-cells Time [hr] Time [hr] wt (SV129:C57BL/6) wt (C57BL/6)

40 Genetic back-ground differences may screw your day! γ-irradiation [1.25 Gy] Velcade [50 ng/ml] PMA [10 ng/ml] specific killing % wt (SV129:C57BL/6) wt (C57BL/6)

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