Mitochondria in apoptosis. Jean-Claude Martinou, MD, Ph.D Department of cell biology University of Geneva Geneva, Switzerland

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1 Mitochondria in apoptosis Jean-Claude Martinou, MD, Ph.D Department of cell biology University of Geneva Geneva, Switzerland

2 The dual role of mitochondria in life and death QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. 1) Oxydative phosphorylation ATP production 2) Apoptosis Frey and Mannella, Trends Biochem. Sci. 2000, 25:

3 Signaling pathways poptosis triggered by Death receptors Fas R Caspase 8 Growth factor deprivation Dexametasone UV irradiation etc. Bim,, Puma Fadd BAX Caspase 8 Bid tbid BCL-2 Cyt-c Caspase 3/7 Death Caspase 9 Cytochrome c Pro-caspase 9 Apaf-1 datp

4 Early observations on the role of mitochondria in cell death Suppression of oxidative phosphorylation in radiation-induced induced cell death (Ashwell & Hickman, 1952) Suppression of oxidative phosphorylation is associated with appearance of pyknotic nuclei (van Bekkum et al,.1964) Impairment of mitochondrial electron transport is linked to controlled release of mitochondrial cytochrome c in radiosensitive tissues ( cytochrome( c effect ) ) (Scaife( Scaife,, 1964) Release of cytochrome c in radiosensitive tissues precedes the appearance of pyknotic bodies (Hanson et al., 1965) Exogenous cytochrome c is able to restore oxidative phosphorylation in mitochondria isolated from radiosensitive tissues (van Bekkum et al., 1967; Hanson et al., 1969)

5 Early observations on the role of mitochondria in cell death Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis.cell Oct 22;75(2): Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature Nov 22;348(6299): Hennet T, Bertoni G, Richter C, Peterhans E. Expression of BCL-2 protein enhances the survival of mouse fibrosarcoid cells in tumor necrosis factor-mediated cytotoxicity.cancer Res Mar 15;53(6): Newmeyer DD, Farschon DM, Reed JC. Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria.cell Oct 21;79(2): Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A, Daugas E, Geuskens M, Kroemer G. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.j Exp Med Oct 1;184(4): Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for datp and cytochrome c.cell Jul 12;86(1):

6 Mitochondria: the Pandora s box Apoptotic stimuli Mitochondrial intermembrane space Omi

7 Release of cytochrome c during apoptosis

8 Analysis of cytochrome c release by Western blot QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

9 Goldstein et al. CDD The release of cytochrome c precedes the loss of mitochondrial membrane potential QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

10 Release of cytochrome c QuickTime and a Motion JPEG A decompressor are needed to see this picture.

11 How does Pandora s box open? Apoptotic stimuli Outer mitochondrial membrane Bcl-2 Bax Omi

12 Bcl-2 family Antiapoptotic proteins N- BH4 BH3 BH1 BH2 TM -C Pro-Apoptotic proteins Bax-like N- BH3 BH1 BH2 TM -C Bcl-2 Bcl-xL Bcl-w Mcl-1 Etc Bax Bak Bok BH3-only proteins N- BH3 TM -C TM Bid Bad Bim Noxa Puma Etc

13 Three dimensional structure N C a6 a5 Bax a6 a5 Bcl-x L

14 An hydrophobic pocket for interactions with BH3 domains QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. C N Bcl-X L -Bad

15 In Bax the hydrophobic pocket is occupied by the TM domain QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

16 Two functional classes of BH3-only proteins Cell death stimulus Bim-like proteins Bid-like proteins Bcl-2-like proteins Bax-like proteins Terradillos et al., FEBS Lett. 2002, 522: Death

17 Bax under resting conditions Cytosolic retention factors (Humanin, proteins, Ku70, ARC, αa- and αb-crystallin, Hsp70/dj1, Hsp70/dj2, Hsp60) C N MOM MIM Lucken-Ardjomande and Martinou, J Cell Sci. 2005, 118(Pt 3):473-83

18 Bax during apoptosis 3 N C Bax Hydrophobic groove tbid N C X N caspase activation Cardiolipin OMM IMM Apoptogenic factors Lucken-Ardjomande and Martinou, C R Biol Jul;328(:616-3

19 Exposure of N-terminal region QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

20 Bax insertion into membranes: resistance to alkali treatment QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

21 Gel filtration analysis of Bax complexes QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

22 Bax oligomers: the use of cross linkers QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

23 How do antiapoptotic proteins protect? 3 N C Bax Hydrophobic groove tbid N C X N Bcl-2 caspase activation OMM IMM Apoptogenic factors

24 Conclusion 1 1) Bax, when oligomerized, permeabilizes the outer mitochondrial membrane. Irreversible except in neurons 2) Bax requires BH3-only proteins of the Bid-like subgroup 3) Specific phospholipids such as cardiolipin and other proteins (as yet unknown) are also required for Bax oligomerization

25 Two fundamental questions What are the mechanisms responsible for Bax/Bak activation? By which mechanisms Bax like proteins render the outer mitochondrial membrane permeable?

26 Models for the release of cytochrome c Desagher and Martinou,

27 The permeability transition pore

28 Swelling of mitochondria induced by opening of PTP Ctrl +Ca ++ +Bax QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

29 Opening of the PTP into question 1) PT opening can form in the absence of ANT 2) Bax can trigger apoptosis in the absence of major components of the PTP (ANT and Cyclophilin D)

30 What about VDAC? 1) Bax can trigger VDAC opening in synthetic liposomes 2) Bax is inefficient in inducing cytochrome c release in yeast deficient in VDAC isoforms

31 Models for the release of cytochrome c

32 Structural similarities between Bcl-2 family proteins and bacterial toxins a6 a5 a6 a5 Bax Pore forming domain of colicins

33 QuickTime and a Bax channels in lipid planner bilayers TIFF (Uncompressed) decompressor are needed to see this picture.

34 Bax pores 1) Bax can form pores in lipid planar bilayers and synthetic liposomes 2) Oligomerized Bax combined with tbid can form very large pores that can release 2000 kda size dextran molecules

35 Models for the release of cytochrome c

36 Lipids and mitochondrial membrane permeabilization

37 Structure of phospholipids Flat (ex: PC, PG, PS, PI, ) Positive curvature (ex: lysophospholipids, ) Negative curvature (ex: PE, DAG, ) Bilayer Lipid pore Hexagonal II phase Lucken-Ardjomande and Martinou,

38 Bax lipidic pores 1) Bax pores in planar lipid bilayers are unstable and can result in membrane rupture 2) In contrast the pores formed by Bcl-xL are stable and do not result in membrane rupture 3) Formation of lipidic pores by Bax would be facilitated by lipids that impose a positive curvature to membranes 4) Formation of pores by Bcl-x L is facilitated by lipids with a negative curvature

39 Summary 1) The mechanism of mitochondrial membrane permeabilization is still unclear 2) The PTP does not appear to be essential for mitochondrial membrane permeabilization 3) Modification of the lipid structure is the most likely explanation

40 Bax lipidic pores 1) Bax pores in planar lipid bilayers are unstable and can result in membrane rupture 2) In contrast the pores formed by Bcl-xL are stable and do not result in membrane rupture 3) Formation of lipidic pores by Bax would be facilitated by lipids that impose a positive curvature to membranes 4) Formation of pores by Bcl-x L is facilitated by lipids with a negative curvature

41 The role of mitochondrial fission in apoptosis

42 QuickTime and a Sorenson Video decompressor are needed to see this picture.

43 Mitochondria dynamics usion proteins : Mfn1&2 OPA1 ission proteins : Drp1 (Dlp1) hfis

44 mitochondrial fission

45 Bax triggers mitochondrial fission QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

46 Differential release of cytochrome c and Smac/DIABLO F1 F1 a-cyto c a-smac/diablo Ctrl D1

47 Mitochondrial fission and cytochrome c release Bcl-X L Apoptotic signalling Bax activation Fission Machinery hfis1, Drp1 Drp K38A hfis1, Drp1 RNAi Permeabilisation of OMM (pore formation) Fission and cristae Remodelling (loss of OPA1 function) Cytochrome c (20%) Smac/DIABLO Cytochrome c release (80%) Cytochrome c pool available for release

48 The role of mitochondrial fission during apoptosis Q u i c k T i m e a n d a T I F F ( U n c o m p r e s s e d ) d e c o m p r e s s o r a r e n e e d e d t o s e e t h i s p i c t u r e. Q u i c k T i m e a n d a T I F F ( U n c o m p r e s s e d ) d e c o m p r e s s o r a r e n e e d e d t o s e e t h i s p i c t u r e. Q u i c k T i m e a n d a T I F F ( U n c o m p r e s s e d ) d e c o m p r e s s o r a r e n e e d e d t o s e e t h i s p i c t u r e. Bax/Bak dependent death stimuli 25 Bax Drp1 Endophilin OPA1 Cyt c A? A > 25 Smac/ DIABLO and others A+B B... 25

49 A different view on mitochondria Frey and Mannella, Trends Biochem. Sci. 2000, 25: QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

50 Loss of OPA1 function and mobilization of the cytochrome c OPA1 OPA1