Mitochondrial alterations in apoptosis

Chemistry and Physics of Lipids 181 (2014) 62–75

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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip

Review

Mitochondrial alterations in apoptosis Katia Cosentino a,b , Ana J. García-Sáez a,b,c,∗ a

German Cancer Research Center, Heidelberg, Germany Max-Planck Institute for Intelligent Systems, Stuttgart, Germany c Interfaculty Institute of Biochemistry (IFIB), University of Tübingen, Tübingen, Germany b

a r t i c l e

i n f o

Article history: Received 31 January 2014 Received in revised form 20 March 2014 Accepted 2 April 2014 Available online 13 April 2014 Keywords: Apoptosis Mitochondrial outer membrane permeabilization Mitochondrial fission Bcl-2 family proteins Cardiolipin Cristae remodeling

a b s t r a c t Besides their conventional role as energy suppliers for the cell, mitochondria in vertebrates are active regulators of apoptosis. They release apoptotic factors from the intermembrane space into the cytosol through a mechanism that involves the Bcl-2 protein family, mediating permeabilization of the outer mitochondrial membrane. Associated with this event, a number of additional changes affect mitochondria during apoptosis. They include loss of important mitochondrial functions, such as the ability to maintain calcium homeostasis and to generate ATP, as well as mitochondrial fragmentation and cristae remodeling. Moreover, the lipidic component of mitochondrial membranes undergoes important alterations in composition and distribution, which have turned out to be relevant regulatory events for the proteins involved in apoptotic mitochondrial damage. © 2014 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

3.

4. 5.

6. 7.

Introduction: mitochondrial regulation of apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipids and apoptosis: changes in the mitochondrial membrane composition and lipid distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. CL recruitment to MOM: lipid transfer from MIM to MOM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Mitochondria-ER associated membranes and microdomains in apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Permeability alteration of the MOM: a key point in apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Bcl-2 interaction network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. MOMP by the Bcl-2 proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Role of lipids in MOMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Bax/Bak pore formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Loss of mitochondrial function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Toolbox of the mitochondrial morphology machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Fission of mitochondria: a constant in apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cristae remodeling and release of cytochrome c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Correlation between mitochondrial alterations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

63 63 65 66 67 68 68 68 69 70 70 70 70 71 71

Abbreviations: CL, cardiolipin; Drp1, dynamin-related protein 1; ER, endoplasmic reticulum; GTPase, guanosine triphosphatases; Mfn, mitofusin; MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane; MOMP, mitochondrial outer membrane permeabilization; MPTP, mitochondrial permeability transition pores; mtDNA, mitochondrial DNA; NAO, 10-N-nonyl acridine orange; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; ROS, reactive oxygen species; VDAC, voltage-dependent anion channel. ∗ Corresponding author at: Interfaculty Institute of Biochemistry (IFIB), Universität Tübingen, Tübingen, Germany. Tel.: +49 7071 29 73318; fax: +49 7071 29 35296. E-mail address: [email protected] (A.J. García-Sáez). http://dx.doi.org/10.1016/j.chemphyslip.2014.04.001 0009-3084/© 2014 Elsevier Ireland Ltd. All rights reserved.

K. Cosentino, A.J. García-Sáez / Chemistry and Physics of Lipids 181 (2014) 62–75

8.

Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transparency document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction: mitochondrial regulation of apoptosis Mitochondria are organelles that play an essential role in the life and death of the cell. Their importance is mainly attributed to energy production in the form of ATP, but they are also involved in pathogenetic mechanisms leading to neurodegenerative diseases (e.g. autosomal dominant optic atrophy, Parkinson, Charcot-MarieTooth, Alzheimer (Green, 2004; Knott et al., 2008) and cancer (Green, 2004; Juin et al., 2013)). Mitochondria are believed to originate from prokaryotic cells that invaded mammalian cells resulting in symbiosis. They are located in the cytosol and have different shapes, tubular or vesicular, with sizes ranging between 1 and 10 ␮m in length, depending on the cell type. These organelles are surrounded by a double membrane system, the mitochondrial outer and inner membranes (MOM and MIM, respectively), which differ in morphology and lipid composition (reviewed in Horvath and Daum, 2013). The MIM is folded into invaginations called cristae, which contain proteins involved in important mitochondrial functions, such as cytochrome c (Ow et al., 2008), and is rich in the anionic phospholipid cardiolipin (CL), which in eukaryotic cells is exclusively found in mitochondria. The MOM, instead, is smooth and the CL content is reduced compared to the MIM (Ardail et al., 1990). On the other hand, the MOM is fluid and permeable to small polar molecules (up to 3–5 KDa) due to the presence of protein transmembrane channels, while the MIM has restricted metabolite permeability. In healthy conditions, mitochondria participate in important metabolic pathways, the most prominent being the production of energy by generating an electrochemical potential used to drive oxidative phosphorylation of ADP to ATP. Another key aspect of healthy mitochondria is their dynamic morphology. The regulated combination of mitochondrial fission and fusion determine the shape and number of mitochondria in the cell. In addition, these processes are important to preserve the health of the mitochondrial network by segregating damaged mitochondria (fission) or sharing and distributing damaged components (fusion) (Youle and van der Bliek, 2012). A dysfunction in the mechanisms that controls mitochondrial networks is an early step in neurodegeneration (Knott et al., 2008). Under apoptotic stimuli, mitochondria undergo dramatic changes in their structure and function (Suen et al., 2008; Martinou and Youle, 2011; Ugarte-Uribe and Garcia-Saez, 2013). While this aspect is valid for both invertebrates and vertebrates, only in the latter mitochondria actively contribute to cell death by taking part in the so-called intrinsic pathway of apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins by inducing mitochondrial outer membrane permeabilization (MOMP) (García-Sáez et al., 2010; Shamas-Din et al., 2013a,b). Pore formation in the membrane allows the release of cytochrome c and other apoptotic factors from the inter-membrane space into the cytosol, which activate caspases and induce cell death (Wei, 2001). The dysregulation of this mitochondrial function has an important role in tumorigenesis and in the cellular responses to anti-cancer therapies (Juin et al., 2013; Czabotar et al., 2014). Associated to MOMP, many other alterations of mitochondria occur during apoptosis. These include lipid transfer between mitochondria and other organelles (Hoppins and Nunnari, 2012) as well as between the MIM and the MOM (Kagan et al., 2005),

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loss of mitochondrial functions, such as loss of the transmembrane potential required to drive oxidative phosphorylation, and loss of the ability to maintain calcium homeostasis (Wang, 2001; Green, 2004). In addition, fragmentation of mitochondria and cristae remodeling constantly coincide with MOMP (Suen et al., 2008; Wasilewski and Scorrano, 2009; Martinou and Youle, 2011; UgarteUribe and Garcia-Saez, 2013). Here, we present an overview of the different alterations in mitochondria morphology and functionality during apoptosis (Fig. 1 and Table 1). We review the recent understanding of the mechanisms that induce mitochondrial damages and discuss the relevance of lipids in executing these processes, which finally lead to cell death. In addition we discuss the influence of each process on the others, providing a network (Fig. 2) which reports some links so far never reviewed in the literature.

2. Lipids and apoptosis: changes in the mitochondrial membrane composition and lipid distribution Mitochondria have a complex membrane structure, due to the presence of two distinct bilayers. The lipid composition in each membrane is highly characteristic and differs between the outer and the inner membranes. The smooth mitochondrial outer membrane of most mammalian cells is mainly rich in phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) and presents relatively small amounts of phosphatidylserine (PS) and CL (<1–10 mol%) (Colbeau et al., 1971; Hovius et al., 1993; de Kroon et al., 1997). In contrast, the highly folded MIM, is enriched in non-bilayer forming lipids, like CL (14–23 mol%) and PE (Colbeau et al., 1971; de Kroon et al., 1997). MIM and MOM communicate with each other by formation of contact sites. It has been speculated that these contact sites enhance the exchange of lipids and proteins between the two mitochondrial membranes (Reichert and Neupert, 2002). In addition, these sites, being particularly rich in non-lamellar lipids CL and PE, may form hexagonal HII structures (van Venetië and Verkleij, 1982; Aguilar et al., 1999; Unsay et al., 2013) and may provide a suitable place for the targeting of Bcl-2 proteins to the mitochondrial membrane (Lutter et al., 2000; Gonzalvez et al., 2005). Although the existence of contact sites has been debated for a long time, new evidence implicates the presence of specific proteins, or complexes of proteins, in their formation (Maniti et al., 2009; Harner et al., 2011). Nevertheless, the exact mechanisms involved in such process are not yet understood. The lipid distribution within the two membranes is not arbitrary but rather organized for supporting specific mitochondrial functions (Claypool and Koehler, 2012; Horvath and Daum, 2013; Tatsuta et al., 2013). PE and CL, for example, associate with the TOM and SAM protein complexes to promote the insertion of proteins in a particular orientation into the MOM (Gebert et al., 2009; Becker et al., 2013). In addition, CL in the outer and inner membranes associates with several proteins of the apoptotic machinery (Schug and Gottlieb, 2009; Crimi and Esposti, 2011): it promotes caspase-8 translocation (Gonzalvez et al., 2008) and the targeting of Bcl2 proteins (Lutter et al., 2000; Kuwana et al., 2002) to the MOM. Also, reduced levels of CL affect the binding of cytocrome c to the MIM and its oxidation promotes the release of this protein from the inter-membrane space (Ott et al., 2002; Kagan et al., 2005).

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Table 1 Main mitochondrial alterations during apoptosis. Type

Proteins involved

Effect/Function

Mechanism

References

Changes in lipid composition/distribution

tBid

Translocation of lipids to mitochondria

Through activation of PLA2 (phospholipase A 2), hydrolysis of CL and binding with MonoCL and DiCL

Degli Esposti et al. (2001), Tyurin et al. (2006), Goonesinghe et al. (2005), Chen et al. (2009)

Reorganization of CL in the membrane and formation of CL microdomains

Possible involvement of contact sites

Gonzalvez et al. (2005)

Ups1 (yeast)

PA translocation between IMM and OMM Conversion of PA to CL in the inner membrane

Dynamic assembly of Ups1/Mdm35

Connerth et al. (2012)

ERMES (yeast) Mfn2 (mammals)

Exchange of lipids between ER-mitochondria

Formation of tethering

de Brito and Scorrano (2008), Kornmann et al. (2009), Helle et al. (2013), Tatsuta et al. (2013)

Bax, Bak

Execution of MOMP and release of apoptotic factors into the cytosol

Pore formation in the MOM (mechanism of pore formation still unknown)

Wei (2001), Kuwana et al. (2002), Lovell et al. (2008), Bleicken et al. (2010), Leshchiner et al. (2013), Moldoveanu et al. (2013)

Alteration in lipid curvature

Basanez (2002), García-Sáez et al. (2007)

Mechanism mediated by membrane lipids and involving Bax/Bak conformational changes

Kuwana et al. (2002), Lovell et al. (2008), Letai et al. (2002), Kim et al. (2009), Kuwana et al. (2005), Leber et al. (2007), Du et al. (2011), Llambi et al. (2011), Leshchiner et al. (2013), Weber et al. (2013), Moldoveanu et al. (2013)

Preferential interaction of Bim with Bax and tBid with Bak

Sarosiek et al. (2013)

Momp

Bid (tBid), Bim, Noxa, Bmf, PUMA

Loss of mitochondrial function

Recruitment and activation of Bax at the MOM Activation of Bak

Bad, Noxa, Bmf, Bik, PUMA

Inhibition of antiapoptotic proteins

Mechanism mediated by membrane lipids

Letai et al. (2002), Lovell et al. (2008), Willis et al. (2005), Leber et al. (2007), Du et al. (2011), Llambi et al. (2011), Weber et al. (2013)

Bcl-xL, Bcl-2, Bcl-w, Mcl-1, A1

Inhibition of MOMP

Inhibition of Bax activation. Inhibition of BH3-only proteins function

Kuwana et al. (2002), Billen et al. (2008), Lovell et al. (2008), Weber et al. (2013), Bleicken et al. (2013a,b)

MTCH2/MIMP

Recruitment of tBid at the membrane

It behaves as a receptor of tBid

Zaltsman et al. (2010)

VDAC2

Proapoptotic function.

Interaction with Bax/Bak and Bcl-xL

Malia and Wagner (2006)

Antiapoptotic function

It facilitates Bak insertion in the membrane, but inhibits its activation

Cheng et al. (2003)

Drp1

Enhances tBid induced Bax oligomerization

Formation of hemifusion intermediates

Frank et al. (2001), Montessuit et al. (2010)

Mfn2

Involved in apoptosis Transfer of lipids involved in MOMP

It helps Bax translocation to the mitochondria. Formation of ER-mitochondria tethering

Karbowski et al. (2002), Karbowski et al. (2006), Wasilewski and Scorrano (2009)

Growth factor proteins

Stop of electron transfer. Change in the transmembrane potential

Deprivation of growth factors

Vander Heiden et al. (1999)

tBid

Blockage of electron transferoxidative phosphorylation coupling

Not specified

Wang (2001)

Loss of calcium homeostasis

Not specified

Wang (2001)

Formation of mitochondrial permeability transition pores (MPTP)

Interaction of cyclophilin with calcium

Basso et al. (2005)

Cyclophilin D

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Table 1 (Continued) Type

Proteins involved

Effect/Function

Mechanism

References

Fragmentation

Drp1 (mammals)

Mitochondrion from filiform to puntiform

Assembling into spirals around the mitochondria. Fission mediated by Mff and MIEF1

Ingerman et al. (2005), Otera et al. (2010), Palmer et al. (2011)

Dnm1 (yeast)

Mitochondrion from filiform to puntiform

Fission mediated by Fis1

Tieu and Nunnari (2000), Karren et al. (2005), Naylor et al. (2006)

Mff

Drp1 recruitment

Colocalization with DRP1 on the MOM. Interaction by N-terminal region

Gandre-Babbe and van der Bliek (2008), Otera et al. (2010)

MIEF1 (MiD49 and MiD51)

Drp1 recruitment

Alteration of Drp1 assembly and membrane constriction

Palmer et al. (2011), Koirala et al. (2013)

Fis1

Dnm 1 recruitment

Transient interaction mediated by Mdv1/Caf4

Tieu and Nunnari (2000), Dohm et al. (2004), Karren et al. (2005), Naylor et al. (2006)

hFis1/Bap31

Stimulation of Drp1 fission and cytochrome release

hFis1 interaction with Bap31 and consequent transmission of ER Ca2+ signal to mitochondria

Breckenridge et al. (2003)

Mtp18

Antiapoptotic function: silencing leads to cyt c release

Inhibition of Drp1 recruitment to the OMM

Tondera et al. (2004)

Bif-1

Involved in mitochondrial dynamics and Bax activation

Change in membrane curvature

Karbowski et al. (2004), Peter et al. (2004)

SUMO1

Promotion of membrane fragmentation

Bax/Bak -dependent sumoylation of Drp1

Wasiak et al. (2007)

Bax, Bak

Inhibition of mitochondrial fusion

Formation of Bax-Mfn2 and Bak-Mfn1 homotypic complex

Hoppins et al. (2007), Brooks et al. (2011), Karbowski et al. (2002)

Opa 1

Its inhibition induces morphological alteration, loss of integrity of cristae and release of cytochrome c

Loss of OPA 1 function

Scorrano et al. (2002), Cipolat et al. (2006), Frezza et al. (2006)

tBid

It widens the cristae junctions

Disruption of OPA1 complex

Scorrano et al. (2002), Frezza et al. (2006)

Parl

Its absence increases cristae remodeling

Its inhibition prevents assembly of OPA oligomers

Cipolat et al. (2006)

Cristae remodeling

During apoptosis, a series of variations occur in the membrane lipid composition, some involving changes in the chemistry of lipids (for a complete review see Crimi and Esposti, 2011), and others concerning inter-membrane lipid transfer, based on the communication between the MOM and MIM, or between mitochondria and other organelles (such as the endoplasmic reticulum, ER). In this section, we focus on the components that govern the import of lipids and regulate their redistribution between membranes, emphasizing the important role played by lipids in the mitochondrial apoptotic pathway.

2.1. CL recruitment to MOM: lipid transfer from MIM to MOM Cardiolipin is a lipid almost exclusively present in mitochondrial membranes, mainly in the MIM. In the early stage of apoptosis, redistribution of CL is observed between the inner and the outer membranes (Fig. 1a) (Garcia Fernandez et al., 2002; Kagan et al., 2005). This event precedes any other alteration in mitochondria during apoptosis, except the generation of reactive oxygen species (ROS) (Garcia Fernandez et al., 2002) (Fig. 2). Due to its high number of unsaturated fatty acids, CL is oxidized by ROS (Tyurin et al., 2008), which is considered essential for the release of proapoptotic factors (Kagan et al., 2005) (Fig. 2). The process that induces CL translocation is still elusive, and although there is no evidence yet, it has been proposed that the presence of contact sites between the two membranes, identified

by electron microscopy, might allow for CL transfer and recruitment to the MOM (Fig. 1a) (Reichert and Neupert, 2002). At these zones, both membranes get enriched in PE and CL (Ardail et al., 1990; Horvath and Daum, 2013). Studies in membrane models from mitochondrial lipids, as well as in isolated mitochondria, have suggested that the propensity of these two non-lamellar lipids to form membranes with negative curvature favors the formation of hexagonal HII phases between the two membranes (Cullis and De Kruijff, 1979; van Venetië and Verkleij, 1982; Unsay et al., 2013). The resulting membrane fusion might facilitate CL redistribution from the inner to the outer membrane (Reichert and Neupert, 2002). There is additional evidence that the Bcl2 family member tBid may play a role in the translocation of lipids to the MOM (Esposti et al., 2001). The lipid transfer activity of tBid, and of recombinant Bid, has been proven in vesicles mimicking mitochondrial membranes as well as in isolated mitochondria (Degli Esposti et al., 2001; Degli Esposti, 2002; Esposti et al., 2003). Additionally a role has been proposed for tBid in the reorganization of CL in the membrane (Gonzalvez et al., 2005; Tyurin et al., 2006). tBid preferentially interacts with CL at the contact sites (Lutter et al., 2001; Gonzalvez et al., 2005) and it has been speculated that this could facilitate the redistribution of CL into microdomains. The presence of CL-rich domains and local alterations in the membrane curvature may facilitate the recruitment of additional tBid to mitochondria and thus promote the release of cytochrome c (Fig. 2).

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Fig. 1. Mitochondrial alterations during apoptosis. (a) Lipid transfer occurs either at the contact sites between the MOM and the MIM or between the MOM and the ER. (b) MOMP: activation of Bax/Bak at the MOM by tBid induces Bax/Bak oligomerization and pore formation with consequent release of apoptotic factors. Bcl-xL inhibits this process by either blocking tBid (in solution as well as at the membrane) or active Bax. (c) The loss of mitochondrial function is a direct consequence of MOMP. Pore formation induces mitochondrial swelling and loss of inner transmembrane potential, m . (d) Fragmentation of mitochondria is thought to occur at constriction sites created by ER tubules wrapping around mitochondria (left) which facilitate the formation of Drp1 and Mfn2 foci and mitochondrial division. These points are likely to be the place of MOMP due to the presence of Bax foci (right). (e) Cristae remodeling: the OPA1 complex is responsible for the maintenance of the cristae junction integrity and for the confinement of cytochrome c located in the cristae. Disassembly of the Opa1 complex leads to cristae remodeling and cyt c release.

2.2. Mitochondria-ER associated membranes and microdomains in apoptosis The endoplasmic reticulum (ER) is the organelle where most of the membrane lipid synthesis takes place (Lev, 2012; Tatsuta et al., 2013). The production of CL in the MIM starts from the generation of phosphatidic acid (PA) in the ER, which is then transferred to the mitochondrial membrane (Fig. 1a) (Tatsuta et al., 2013). The subsequent transfer between mitochondrial membranes, which in yeast is mediated by Ups1 assembled with Mdm35 (Connerth et al.,

2012), is followed by conversion of PA to CL in the inner membrane (Connerth et al., 2012). Contact sites between mitochondria and the ER are supported by tethering-forming protein complexes, such as the ER-mitochondria encounter structure (ERMES) in yeast (Siskind et al., 2008) or by the protein Mfn2 in mammals (Ganesan et al., 2010). These systems, together with lipid-transfer proteins, are responsible for the exchange of lipids between the ER and the mitochondria (Ganesan et al., 2010; Helle et al., 2013; Tatsuta et al., 2013). The proapoptotic protein Bid also exhibits lysolipid transfer activity between the

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Fig. 2. Scheme correlating the main mitochondrial alterations during apoptosis according to data from the literature (see notes and text). Boxes of the same color belong to the same mitochondrial-alteration category: yellow for lipid transfer, pink for MOMP, green for loss of function, blue for fragmentation, orange for cristae remodeling. The arrows are experimentally proved (black) or hypothetical (red) connections between two effects (boxes). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

ER and the mitochondrial membranes (Cristea and Degli Esposti, 2004; Goonesinghe et al., 2005). This property of Bid has been confirmed using truncated oxidized phospholipid compounds, which accumulate at the mitochondria depending on the concentration of Bid (Chen et al., 2009). Chipuk et al. have recently proven the importance of the ERmitochondria membrane association and revealed the role of lipid trafficking in the MOMP pathway (Chipuk et al., 2012). Concretely, they proposed that the ER provides sphingomyelin to mitochondria, which is converted by hydrolysis first into ceramide and subsequently to sphingosine-1-phosphate and hexadecenal, both involved in the activation of the proapoptotic proteins Bak and Bax, respectively (Chipuk et al., 2012). Moreover, the contact sites between the ER and mitochondria generate lipid microdomains (Csordás et al., 2010) implicated in the recruitment of proteins involved in mitochondrial fission ((Friedman et al., 2011) and discussed in Section 5.2), as well as of lipids and calcium. Sudden increases of Ca2+ can be fatal and

trigger apoptosis by inducing the formation of CL microdomains, generation of ROS and CL peroxidation ((Grijalba et al., 1999); for a discussion on the loss of calcium homeostasis during apoptosis see Section 4). 3. Permeability alteration of the MOM: a key point in apoptosis The recruitment and reorganization of lipids in the mitochondrial membranes prepares the ground for the key event in the mitochondrial pathway of apoptosis: the permeabilization of the MOM, which leads to the release of intermembrane apoptotic factors into the cytosol. In healthy conditions, the permeability of the MOM is restricted to small molecules (less than 5 KDa) due to the presence of membrane channels made by a transmembrane protein known as mitochondrial porin or VDAC (voltage-dependent anion channel) (Colombini, 1979; Dolder et al., 1999). VDAC channels are only 2–3 nm in size and are not sufficiently big to allow the

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release of apoptotic factors into the cytosol, such as cytochrome c (12 KDa) and SMAC/DIABLO (27 KDa in its intact structure but mature Smac/DIABLO exists as a dimer which behaves as an ∼100 KDa molecule), which are required for the downstream activation of caspases and induction of cell death. During apoptosis, the proteins of the Bcl-2 family come into play to alter the MOM permeability. in vitro studies show that these proteins can induce the formation of bigger pores (up to 100 nm in diameter) (Schafer et al., 2009; Bleicken et al., 2010, 2013a). In this section we describe how the different members of the Bcl2 family interact with each other during the MOMP, and the special role of lipids in Bcl2 protein recruitment, activation and pore formation. 3.1. Bcl-2 interaction network The proteins of the Bcl-2 family are classified into three groups (Youle and Strasser, 2008; García-Sáez, 2012; Shamas-Din et al., 2013a,b): (1) the pro-survival (or anti-apoptotic) Bcl-2 proteins, like Bcl-2, Bcl-xL or Mcl-1, which inhibit apoptosis by interacting with the pro-apoptotic members of the family; (2) the executioners Bax and Bak, which promote MOMP, likely by direct pore formation and (3) the pro-apoptotic BH3-only proteins, which are initiators of apoptosis and induce Bax/Bak activation and/or block the inhibitory activity of the antiapoptotic family members (Table 1). Historically, the BH3-only proteins were further divided in two sub-groups: the “activators” (e.g. Bid and Bim), which bind to pro-apoptotic Bax and Bak to activate them, and the “sensitizers/derepressors” (e.g. Bad and Bik), which interact with anti-apoptotic proteins to inhibit their functions (Letai et al., 2002). Recently, this strict classification has been challenged by studies showing the ability of some BH3-only proteins to behave either as activators or as sensitizers (Kim et al., 2009; Du et al., 2011). When inactive, BH3-only proteins remain in the cytosol or associated to the outer mitochondrial membrane. However, in the presence of apoptotic stimuli, intracellular signals activate the BH3only proteins, which then bind to the executioners Bax/Bak and activate them. Upon activation by conformational changes, Bax and Bak (which is constitutively associated to the MOM) insert into the membrane, oligomerize and induce MOM permeabilization (Wei, 2001; Lovell et al., 2008). The consequent release of intermembrane space proteins, like cytochrome c, into the cytoplasm and activation of the cascade of proteases (caspases), complete the mitochondrial apoptotic pathway (Wei, 2001). Antiapoptotic Bcl-2 proteins inhibit this process by interacting with the proapoptotic members (Fig. 1b) (Billen et al., 2008; Lovell et al., 2008; Bleicken et al., 2013b; Weber et al., 2013). The network of interactions between the Bcl-2 family members is very intricate (García-Sáez, 2012; Westphal et al., 2013) and several models have been suggested to recapitulate the events responsible for MOMP (for a review on these models see Bogner et al., 2010; García-Sáez, 2012; Shamas-Din et al., 2013b). In the direct model, Bax and Bak need to be activated by “direct activator” BH3-only proteins in order to promote MOMP (Letai et al., 2002; Kuwana et al., 2005), while in the indirect or “displacement” model, Bax and Bak are constitutively active and inhibited by the antiapoptotic Bcl-2 members. In this latter model, the BH3-only proteins sequester the antiapoptotic Bcl-2 proteins and block their function, thus releasing Bax and Bak and inducing apoptosis (Willis et al., 2005). The embedded together (Leber et al., 2007, 2010) and unified models (Llambi et al., 2011) combine the two previous models. In the embedded together model, BH3-only proteins support MOMP by either sequestering the antiapoptotic Bcl-2 proteins and blocking their interaction with Bax/Bak (as considered in the indirect model) or by directly triggering Bax and Bak activation (as described in the direct model). In addition, it emphasizes the important role of the membrane inducing conformational changes

of Bcl-2 proteins, required for their insertion in the membrane, and affecting the binding affinity between Bcl-2 family members, thus contributing to regulate MOMP (Lovell et al., 2008; Garcia-Saez et al., 2009). The unified model builds on the embedded together model and attributes different weights to the inhibitory actions of the Bcl-2 proteins: their interaction with activator BH3-only proteins is less efficient than their direct inhibition of Bax and Bak. This latter aspect restores the importance of sensitizer BH3-only proteins in inhibiting the Bcl-2 antiapoptotic function. Recently, a new insight has arisen from the study of Weber et al. (2013). They have shown that the displacement of Bcl-xL by the BH3 mimetic ABT-737 was sufficient to induce Bak oligomerization, but not membrane permeabilization, which required the presence of the direct activator Bim (Weber et al., 2013). 3.2. MOMP by the Bcl-2 proteins In healthy conditions, inactive Bax is constantly retrotranslocating between the MOM and the cytosol in a process that seems to be mediated by transient interactions with Bcl-xL (Edlich et al., 2011; Todt et al., 2013). The equilibrium between cytosolic and mitochondrial Bax is destroyed by the presence of BH3-only proteins which inhibit retro-translocation and allow Bax accumulation on the membrane (Edlich et al., 2011; Schellenberg et al., 2013). Interestingly, the interaction between the direct activator tBid and Bax occurs almost exclusively when a membrane is present (Billen et al., 2008; Lovell et al., 2008). tBid is the active product of Bid cleavage by caspase 8 and, once formed, it translocates to the MOM. There, it activates Bax by promoting its insertion into the membrane (Lovell et al., 2008; Shamas-Din et al., 2013a) concomitant with a series of conformational changes (reviewed in Westphal et al., 2013). Bax inserted in the membrane recruits other Bax proteins, oligomerizes and induces MOMP (Lovell et al., 2008; Bleicken et al., 2010). Unlike Bax, Bak is constitutively associated to the membrane but it also needs structural changes induced by the BH3-only proteins Bid or Bim to be activated and initiate MOMP (Aluvila et al., 2013; Leshchiner et al., 2013; Moldoveanu et al., 2013; Weber et al., 2013). In particular, it has recently been shown that the activation of Bak is induced preferentially by Bid, while Bim activates preferentially Bax, but the molecular mechanisms behind such preferences remain unknown (Sarosiek et al., 2013). The anti-apoptotic Bcl-2 proteins inhibit this process by either sequestering BH3-only proteins and then preventing the activation of Bax and Bak (Billen et al., 2008; Llambi et al., 2011; Aranovich et al., 2012), or sequestering embedded Bax/Bak (Billen et al., 2008; Llambi et al., 2011). Additional experiments with tBid have shown a strong affinity between Bcl-xL and Bax in model membranes. Both these proteins are recruited by tBid to the membrane (Bleicken et al., 2013a,b), show conformational changes when binding to it (Desagher et al., 1999; Garcia-Saez et al., 2009) and both bind to other Bax proteins. Nonetheless their function is completely opposed and whereas Bax recruits more Bax molecules to oligomerize and induce MOM permeabilization, Bcl-xL inhibits such process by sequestering embedded Bax (Bogner et al., 2010). In addition, recent findings have stressed the fundamental differences in the membrane organization of these proteins: although both these proteins show membrane pore activity, pores formed by Bax are stable and big enough to release apoptotic factors, while pores formed by BclxL are transient and small and do not affect MOM permeability (Bleicken et al., 2013a,b). 3.3. Role of lipids in MOMP An in vitro study has shown that the presence of membranes is necessary for tBid to interact with Bax before its insertion into the

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membrane (Lovell et al., 2008). Instead, Bcl-xL can interact with tBid both in solution and within lipid membranes. Interestingly, the membrane plays an active role by increasing complex formation between the two proteins (Garcia-Saez et al., 2009). Bcl-xL can also heterodimerize with Bax in the membrane environment and block its oligomerization, thus inhibiting Bax pore formation (Billen et al., 2008). These findings emphasize the important role of the membrane in the MOMP and are considered in the “embedded together” model (Leber et al., 2007; Bogner et al., 2010). The membrane role as a modulator of the interactions between Bcl-2 proteins can be explained by the increased concentration of proteins that a 2D confinement implies compared to the 3D cytosolic environment, as well as by the specific orientation and conformational changes imposed to the proteins in their membrane-bound state, which may expose and/or create new interaction sites (García-Sáez, 2012). Nonetheless, the molecular reasons that explain this membrane effect are not well understood. It has been shown by many studies that specific lipids play a key role in the MOMP process (summarized in reviews from Crimi and Esposti, 2011; Lindsay et al., 2011). CL, for example, is associated with many steps that finally induce MOMP (Schug and Gottlieb, 2009). First, upon FAS receptor activation, caspase-8 activation is promoted by CL at the MOM (Gonzalvez et al., 2008). In agreement with this, in Barth syndrome patients lacking mature CL this process of caspase-8 activation is inhibited (Gonzalvez et al., 2008). Based on this, it was proposed that active caspase-8 at CL-enriched regions constitute activation platforms at the mitochondrial contact sites, where Bid is recruited (Lutter et al., 2000) and activated (Gonzalvez et al., 2005). Second, tBid targeting at the mitochondrial contact sites is again mediated by the presence of CL (Lutter et al., 2000; Kuwana et al., 2002). Nonetheless, some studies have challenged the role of CL as essential promoter of MOMP, attributing the role of tBid receptors to some MOM associated proteins, like MTCH2/MIMP (Schafer et al., 2009; Zaltsman et al., 2010). CL seems also to play a role in Bax activation at the MOM. An in vitro study with liposomes formed from extracted ER and mitochondrial lipids has shown that dextran release by Bax activated with BH3 peptides is less efficient in the absence of CL, whereas it is increased in mitochondrial vesicles (Kuwana et al., 2002; Gonzalvez et al., 2005). One hypothesis behind the effect of CL on membrane permeabilization is that its ability to exert membrane curvature stress can facilitate pore formation in the membrane (Basanez, 2002). Finally, also Bax oligomerization is affected by the lipid environment. Chipuk et al. have recently reported that two sphingolipid metabolites, sphingosine-1-phosphate and hexadecenal, promote Bak and Bax oligomerization (Chipuk et al., 2012). In line with these results, very recently it has been reported that suppression of SMSr, an ER-resident ceramide phosphoethanolamine (CPE) synthase, induces accumulation of ER ceramides, provokes mistargeting to the mitochondria and triggers apoptosis (Tafesse et al., 2013). Increased levels of ceramides have been observed in several studies (Bose et al., 1995; Kroesen et al., 2001; Dai et al., 2004) early during apoptosis. Furthermore, addition of ceramides resulted in an enhanced permeability of the MOM to cytochrome c suggestive of an involvement of these sphingolipids in the MOMP (Siskind et al., 2002). This effect can be reversed by addition of fatty acid depleted albumin (Siskind et al., 2002), sphingosine (Elrick et al., 2006) as well as by interaction with Bcl-xL (Siskind et al., 2008). More recently, it has been found that the synergic action of low levels of ceramides and activated Bax induces a greater level of mitochondrial permeabilization compared to the action of the single agents (Ganesan et al., 2010). These findings suggest that the mechanism of permeabilization by ceramides involves their well-known property of forming large channels in the membrane (6–10 nm in diameter) (Siskind and Colombini, 2000) and that Bcl-2 proteins affect the

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ability of ceramides to form channels (Siskind et al., 2008; Ganesan et al., 2010) (for a detailed description on ceramide channels and their role in apoptosis we invite the reader to see the reviews by Colombini, 2010; Tirodkar and Voelkel-Johnson, 2012).

3.4. Bax/Bak pore formation The activity attributed to Bax (and to its fragment corresponding to helix ␣5) to form stable pores has been verified in vitro in large (Terrones et al., 2004; Schafer et al., 2009; Fuertes et al., 2010) and giant (Fuertes et al., 2010; Bleicken et al., 2013a,b) unilamellar vesicles, in planar lipid bilayers (Epand et al., 2002; Lin et al., 2011), as well as in isolated mitochondria (Roucou et al., 2002; Ganesan et al., 2012). Ionic strength modulation has turned out to be a useful tool to study MOMP kinetics: after an initial fast pore formation, detected by cytochrome c release from the intermembrane space, long-lasting channels evolve slowly over time (Ganesan et al., 2012). In model membranes, pores become larger immediately after formation and then relax to a smaller size that changes between 3 and 8 nm depending on the protein concentration (Fuertes et al., 2010; Bleicken et al., 2013a). Conductance measurements on planar membranes in the presence of full-length Bax have revealed that this protein destabilizes the membrane and reduces its lifetime by decreasing the line tension of the membrane, or in other words, by lowering the energetic barrier for ˜ pore formation (Basanez et al., 1999). In line with these results, electrophysiological studies on full-length Bax have reported the presence of two types of Bax pores: one voltage-gated, small, and weakly selective to cations, but unable to permeabilize proteins, and another one that is voltage-independent and, likely, big enough to allow translocations of proteins, such cytochrome c, across the membrane (Lin et al., 2011). In the presence of agents which affect the membrane tension (such as lanthanides), these latter channels can be converted to the small impermeable ones, showing that regulatory mechanisms can be responsible for protein release by Bax channels (Lin et al., 2011). How the number of Bax and Bak units is related to the pore size is still an open question, and although the oligomerization mechanism is starting to be understood, the molecular details of that process are still elusive. It has been reported that the properties of Bax-induced pores depend on the lipid composition (Basanez, 2002). For this reason, a lipidic/proteinic nature of the pores formed by Bax has been ˜ proposed (Basanez et al., 1999; Basanez, 2002). This hypothesis is supported by a cryo-EM study on cardiolipin-liposomes revealing the formation of Bax-induced pores of various sizes and shapes (Schafer et al., 2009). Indeed, the pores formed by both Bax and Bak appear to be tunable in size (Bleicken et al., 2013a). This model assumes a toroidal arrangement where pores are delimited by both lipids and proteins. At the edge of the pore the two lipid monolayers are in contact forming a continuous sheet (Yang et al., 2001). X-ray studies of membranes in the presence of the pore-forming helix ␣5 of Bax have confirmed the lipidic nature of the pores (Qian et al., 2008). Nonetheless, the location of the peptides in respect to the pore could not be revealed using this technique. According to some theoretical models, the action of these molecules should affect some physical properties of the membrane and stabilize the open pores (Huang et al., 2004). These models have also been supported by experimental studies in lipid bilayers, where it has been shown that pore formation induced by Bax/Bak or helix 5 from Bax occurs via a mechanism related to the intrinsic monolayer curvature and that the charge of lipids affects the ˜ et al., pore ion selectivity (García-Sáez et al., 2005, 2006; Basanez 1999; Basanez, 2002; Kuwana et al., 2002; García-Sáez et al., 2007;

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Bleicken et al., 2013a,b). Moreover, as helix 5 of Bax has been shown to induce a decrease of the line tension at the edges of the membrane (García-Sáez et al., 2007), the pore forming molecules might be localized at the polar surface of the membrane, variably inserted in the hydrocarbon region. It is worth mentioning that most of the conclusions on the structure of Bax pores are based, so far, on studies carried out using Bax peptides (–␣5–␣6 motifs), which are believed to be responsible for protein insertion and pore formation (García-Sáez et al., 2005, 2006); however, it is not definitely clear whether these results are applicable as well to full length Bax and only further experiments will definitely elucidate this issue. 4. Loss of mitochondrial function MOMP inevitably leads to loss of mitochondrial functions (Wang, 2001; Ricci et al., 2003, 2004). One of them is the loss of inner transmembrane potential,  m (Fig. 1c) (Ly et al., 2003) that in physiological conditions is created by the respiratory chain, thanks to the high impermeability of the MIM. MOMP induces the release of cytochrome c and caspase activation, which in turn interrupts the respiratory electron transfer chain and affects the maintenance of the mitochondrial membrane potential (Ricci, 2003; Ricci et al., 2004). In growth-factor deprived cells, the loss of mitochondrial ATP synthesis causes a hyperpolarization of the inner mitochondrial membrane and matrix swelling. This effect is inhibited by the antiapoptotic Bcl-2 proteins (Vander Heiden et al., 1999). Similarly, the interruption of mitochondrial fusion and consequent fragmentation associated to apoptosis is the result of mitochondrial depolarization (Cereghetti et al., 2008). In addition, tBid is sufficient to inhibit the ability of mitochondria to buffer calcium (Wang, 2001). Calcium homeostasis is an important requirement for cell functionality. It has been found that at ER-mitochondria contact sites, Ca2+ accumulates in heterogeneous microdomains of variable size and shape (Csordás et al., 2010). Ca2+ , accumulated at the mitochondrial membranes, easily interacts with the negatively charged CL and such interaction provides the right in-plane configuration of lipids and proteins to promote ROS production and lipid peroxidation (Fig. 2) (Grijalba et al., 1999). In addition, CL-Ca2+ interaction might be a first step toward Ca2+ -induced MOMP via CL microdomain formation and enrichment of CL-binding proteins. Another mechanism in which calcium is involved is the opening of mitochondrial permeability transition pores (MPTP) in the mitochondrial matrix upon interaction with cyclophilin D (Basso et al., 2005). This process, mediated by the Bcl-2 proteins (Scorrano et al., 2003), causes loss of the inner transmembrane potential, matrix swelling and consequent rupture of the membrane and cytochrome c release (Feldmann et al., 2000; Basso et al., 2005). 5. Mitochondrial fragmentation Mitochondria are very dynamic organelles, continuously undergoing fusion and fission processes. During apoptosis, the mitochondrial network experiences dramatic rearrangements, which result in mitochondrial fragmentation and remodeling of cristae. The first process occurs at the level of the MOM and is discussed in this section, while the second one, which clearly involves the MIM, is discussed in Section 6. 5.1. Toolbox of the mitochondrial morphology machinery The key components for mitochondrial fusion and fission are large guanosine triphosphatases (GTPase) belonging to the

dynamin family (Hoppins et al., 2007; Ugarte-Uribe and GarciaSaez, 2013). They act at the level of both the MOM and MIM and are regulated by proteolysis and post-translational modifications (Hoppins et al., 2007). Fusion between MOMs is regulated by two membrane-anchored proteins called mitofusins, Mfn1 and Mfn2 (Chen et al., 2003). These proteins localize in the external membrane leaflet toward the cytosol and induce fusion by formation of trans-homo- and hetero-oligomers. Fusion between MIMs is mediated by the dynamin family member OPA1 (Olichon et al., 2002). This protein faces the mitochondrial inter-membrane space and creates complexes in the MIM comprising membrane-bound long forms and soluble short forms of the protein (Frezza et al., 2006). Only one protein is responsible for MOM and MIM fission, the dynamin-related protein Drp1 in mammals (and Dnm1 in yeast) (Table 1) (Frank et al., 2001). This protein, after translocation from the cytosol to the MOM, is thought to induce membrane fission by assembling into spirals around the mitochondria (Fig. 1d) (Ingerman et al., 2005). Some studies proving that fission requires membrane tension have challenged this model: fission occurs not only by Drp1 mediated membrane constriction, but by its combined action with lipid remodeling (Bashkirov et al., 2008; Pucadyil and Schmid, 2008). Drp1 recruitment to the mitochondrial membrane is mediated by membrane receptors, such as Fis1 (in yeast) (Tieu and Nunnari, 2000; Dohm et al., 2004), and the recently discovered mammalian receptors Mid49 and Mid51, which induce alteration in Drp1 assembly (Palmer et al., 2011; Koirala et al., 2013), and Mff, which has been found to colocalize with Drp1 into foci at the MOM (Otera et al., 2010). Fis1 is also present in mammals and coimmunoprecipitation together with crosslinking studies have shown that it interacts with Drp1 (Yoon et al., 2003); however its mechanism of action in mammals remains to be elucidated. Other proteins involved in mitochondrial dynamics are Mtp18, which transmits signal division to the MIM (Tondera et al., 2004), and Endophilin B1 (or Bif-1), which regulates membrane curvature (Karbowski et al., 2004; Peter et al., 2004) (Table 1). 5.2. Fission of mitochondria: a constant in apoptosis Mitochondrial membrane fission is always associated to apoptosis (Suen et al., 2008; Martinou and Youle, 2011; Elgass et al., 2013). The link between Bax/Bak activation and membrane fission has been suggested by the detection of Bax foci colocalizing with Drp1 and Mfn2 foci at mitochondrial constriction sites (Frank et al., 2001; Karbowski et al., 2002; Montessuit et al., 2010). These sites seem to be created by ER tubules wrapping around mitochondria, which create “target points” for fission/fusion protein recruitment and mitochondrial division (Fig. 1d) (Friedman et al., 2011). Possibly, the contact sites between the MOM and MIM are generated here. In correspondence to these microdomains, local alterations in the membrane curvature (and lipid composition, as discussed above) may provide the right environment for Drp1 spiral assembly and formation of Bax pores (Fig. 1d) (Yuan et al., 2007). Accordingly, disruption of lipid domains, mimicking the mitochondrial lipid composition, leads to the impairment of Drp1 recruitment and, consequently, blocks fission and apoptosis (Ciarlo et al., 2010). An in vitro study has shown that Drp1 promotion of non-lamellar membrane structures triggers tBid-induced Bax oligomerization (Montessuit et al., 2010). Accordingly, the authors claim that CL (which is a non-bilayer lipid) is required for promoting Drp1 function (Montessuit et al., 2010). Mitochondrial fragmentation is an upstream event with respect to the formation of Bax foci (Yuan et al., 2007) and may help Bax insertion and activation during apoptosis (Brooks et al., 2011). Overexpression of Mfns or inhibition of Drp1 by a negative dominant mutant reduced mitochondrial fragmentation and cytochrome c release by blocking Bax insertion, but not its

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accumulation, in the membrane (Brooks et al., 2011). This also means that Drp1 acts downstream of Bax translocation (Karbowski et al., 2002; Brooks et al., 2011). Indeed, Bax translocation increases the binding of Drp1 to the mitochondria. During apoptosis, Drp1 undergoes a Bax/Bak-dependent SUMOylation (Wasiak et al., 2007) through a mechanism which is not well understood. This process occurs between fission and release of cytochrome c (Wasiak et al., 2007), suggesting that Drp1 can act independently in these two events. Indeed, knockout of Drp1 slows down, but does not prevent, apoptosis (Frank et al., 2001; Karbowski et al., 2002; Ishihara et al., 2009). Therefore, other mechanisms, independent of Drp1, are likely involved (Young et al., 2010). For example, the contribution of Fis1 during apoptosis is independent of Drp1. The Fis1 apoptosis pathway does not pass through MOMP but rather through Ca2+ -mediated ultrastructural changes and dysfunction in the MIM (Alirol et al., 2006). Mfn2, the protein involved in MOM fusion which also colocalizes at Bax/Bak and Drp1 foci (Fig. 1d), is affected by Bcl-2 proteins and has also an effect on apoptosis. Mfn2 does not colocalize in foci in the absence of Bak, while the absence of Mfn2 foci prevents/delays Bax translocation to the mitochondria (Karbowski et al., 2006). It has been speculated that the action of Mfn2 during apoptosis can be related to the formation of tethering between the ER and mitochondria, which allows the transfer of lipids required for MOMP (Wasilewski and Scorrano, 2009).

6. Cristae remodeling and release of cytochrome c MIM fusion is central for maintenance of mitochondrial metabolic functions and preservation of mitochondrial DNA (mtDNA). The formation of the OPA1 complex (described in Section 5.1) is responsible for the maintenance of the cristae junction integrity and for the confinement of cytochrome c located in the cristae (Scorrano et al., 2002). The dysfunction of OPA1 induces severe alterations in the cristae morphology, affecting the respiratory chain processes as well as the replication of mtDNA (Olichon et al., 2003). Disassembly of the OPA1 oligomer during apoptosis causes the release of cytochrome c through a process called cristae remodeling (Fig. 1e) (Cipolat et al., 2006; Frezza et al., 2006). This process can occur in response to several apoptotic stimuli, such as those involving members of the Bcl-2 family (Frezza et al., 2006). tBid, for example, widens the cristae junctions and disrupts the OPA1 complex (Frezza et al., 2006). Another mechanism responsible for the disassembly of the OPA1 oligomer is the destabilization of the rhomboid protease Parl, as documented by studies in which silencing of Parl resulted in higher sensibility to apoptosis (Cipolat et al., 2006). It has been also suggested that changes in the mitochondrial membrane potential control cristae reorganization, which anticipates cytochrome c release (Gottlieb et al., 2003). A decreased potential may induce degradation of the OPA complex, additionally blocking fusion processes and leading to mitochondrial fragmentation (Cereghetti et al., 2008). Finally, cristae reorganization and the consequent release of cytochrome c are related to Bid interaction with CL. Electrospray ionization mass spectrometry studies showed that the presence of 10-N-nonyl acridine orange (NAO), a cardiolipin specific dye, suppressed Bid binding to the mitochondrial contact sites and inhibited cristae remodeling and cytochrome release (Kim, 2004). CL strongly affects cytochrome c binding to the MIM, since decreased levels of CL, or its oxidation, promote cytochrome c release from the inter-membrane space (Ott et al., 2002; Kagan et al., 2005). This process is mediated by Bax and oxidation of CL alone is not sufficient to stimulate the release of cytochrome c (Ott et al., 2002). Whether cristae remodeling precedes cytochrome c

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release (and it is required for it) or vice versa, it is still a matter of debate (Sun et al., 2007; Yamaguchi et al., 2008). In support of the first hypothesis, Parl destabilization, and therefore remodeling of the cristae, occurs as an early stage in apoptosis, before cytochrome c release (Cipolat et al., 2006). In addition, mitochondria with genetically morphological alterations have shown inability to release cytochrome c (Arnoult et al., 2005; Cipolat et al., 2006; Frezza et al., 2006).

7. Correlation between mitochondrial alterations Although apoptosis-induced mitochondrial alterations are often correlated, defining the sequence of these events is very challenging and remains controversial. A simplified representation of the correlations among the main events characterizing mitochondrial damage in apoptosis is illustrated in Fig. 2, based on the literature reviewed here. In summary: under apoptotic stimuli, a change in the composition and distribution of lipids between the outer and inner mitochondrial membranes (involving also mitochondrial interactions with the ER), induces the formation of “target sites” for the recruitment of fission/fusion and apoptotic proteins where fragmentation and MOMP, the key step in mitochondrial apoptosis, most likely take place. Coincident with or following these events, cristae remodeling induces the release of apoptotic factors which finally lead to cell death. However, these correlations are currently highly speculative and only further research will confirm or reject them. Indeed, many contradictory studies challenge the correlations between mitochondrial alterations. One open question is whether the presence of CL in the MOM of apoptotic cells resembles the high concentrations required to regulate the activity of Bcl-2 proteins in vitro. However, the most active debate regards the relation between MOMP and mitochondrial fragmentation. These two events are often associated, but many studies point out that they are not strictly correlated (Parone et al., 2006; Breckenridge et al., 2008; Sheridan et al., 2008; Ishihara et al., 2009). Down regulation or silencing of Drp1 slows down the release of cytochrome c but does not prevent the release of other apoptotic factors (Estaquier and Arnoult, 2007; Ishihara et al., 2009), suggesting that apoptosis can follow other ways independent of mitochondrial fragmentation, or that other factors are involved in the two processes. Other studies even show no correlation between fission and cytochrome c release (Alirol et al., 2006; Sheridan et al., 2008). Antiapoptotic Bcl2 proteins failed to inhibit mitochondrial fragmentation associated with Bax/Bak activation, clearly pointing out that, even though the same proteins are involved, apoptosis and membrane remodeling are two separated events (Sheridan et al., 2008). Similarly, the role of Mfn2 during apoptosis has been dissociated from its action in mitochondrial fusion (Guo et al., 2007).

8. Concluding remarks There are several alterations happening in mitochondria during apoptosis and the mechanisms causing them are manifold. Extensive research in the last years has revealed the important role of membrane lipids, but there are still many open questions. Apart from a more comprehensive understanding of the mechanisms per se, a challenging question is to define the sequence(s) of events that characterize mitochondrial damage and clarify how, if at all, these events are correlated with each other. Elucidating these mechanisms in more detail will help defining therapeutic strategies in response to mitochondrial dysfunction-associated diseases and tumor onset.

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