Mitochondrial dynamics in cell life and death

Programmed cell death is a well-conserved pathway whose basic tenets appear common to all metazoans. Key components identified by genetic and biochemical approaches regulate the commitment step and/or participate in effecting cell demise. Mitochondria are the most well documented intracellular organelle that participate in apoptosis following a variety of death stimuli. The manifold aspects of mitochondrial involvement in apoptosis include two crucial events, the release of proteins normally stored in the intermembrane space, such as cytochrome c, and the development of multiple parameters of mitochondrial dysfunction. Cytochrome c triggers a post-mitochondrial pathway essential to activate the effector caspases that accomplish cell demise. We have shown that to grant complete release of cytochrome c and mitochondrial dysfunction mitochondria must undergo complex changes in their ultrastructure in response to apoptotic stimuli. Moreover, the release of proapoptotic proteins from mitochondria is associated with a transition towards fragmentation of the tubular network. The ultrastructure and the shape changes of the organelle result from the equilibrium between fusion and fission events, controlled by a family of "mitochondria-shaping" proteins that include dynamin related proteins. These are large GTPases that participate in the tubulation, vesiculation, scission and fusion of biological membranes and that therefore participate in crucial processes such as endo- and exocytosis. The main interest of our lab is to investigate the function and the regulation of these mitochondriashaping proteins. We use an integrated approach of genetics, advanced imaging, biochemistry, physiology and electron tomography to unravel the role of these proteins in cell life and death. We are currently focusing on three main projects.

Molecular mechanisms of OPA1 functions

Figure 1. Cartoon representing the role of OPA1 oligomerization in keeping the cristae in shape and in the regulation of cytochrome c release during apoptosis. Artwork: Eric Smith, Boston.

Figure 1. Cartoon representing the role of OPA1 oligomerization in keeping the cristae in shape and in the regulation of cytochrome c release during apoptosis. Artwork: Eric Smith, Boston.
[click image to enlarge]

We have shown that the inner mitochondrial membrane dynamin-related protein OPA1 has multiple, genetically distinguishable functions in mitochondrial fusion and in the control of cristae remodelling and cytochrome c release. While the pro-fusion effect depends on the outer membrane protein of the same family Mitofusin 1 (Cipolat et al, PNAS, 101, 15927-15932, 2004), the remodelling of the cristae depends on the inner mitochondrial membrane rhomboid protease PARL (Cipolat et al., Cell 126, 163- 175, 2006). This is required for the complete processing of OPA1 into a soluble, intermembrane space form that participates together with the membrane bound one in the formation of a high molecular weight oligomer. OPA1-containing oligomers are early targets during remodelling of the cristae in the course of apoptosis (Frezza et al., Cell 126, 177-189, 2006). We now plan to extend our analysis to understand at the molecular mechanism the function of OPA1 in these two genetically distinguishable pathways. We will capitalize on mouse models of OPA1 overexpression and conditional ablation in order to identify the partners of OPA1 in these high-molecular weight complexes in normal and apoptotic mitochondria and to identify their relative role in one of the two processes controlled by OPA1. These mouse models will also be instrumental to elucidate the role of OPA1 in retinal ganglion cells, which are selectively affected in Dominant Optic Atrophy, caused by mutations in OPA1. Moreover, we wish to pharmacologically target OPA1 to verify if this can augment the susceptibility to antineoplastic drugs. These approaches are expected to increase our knowledge of the molecular mechanisms controlling the shape of mitochondria and their participation in the decisional phase of apoptosis; and to shed new light on the pathogenesis of dominant optic atrophy.

Functional divergence between Mitofusin 1 and 2

Figure 2. representative images of mitochondria (red)-ER (green) tethering (yellow) in wt (A) and Mfn2-/- (B) mouse embryonic fibroblasts.

Figure 2. representative images of mitochondria (red)-ER (green) tethering (yellow) in wt (A) and Mfn2-/- (B) mouse embryonic fibroblasts.
[click image to enlarge]

Mammals possess two outer membrane proteins involved in mitochondrial fusion, originated probably by gene duplication of the yeast ancestor Fzo1. These two mitofusins are apparently not redundant, since they differentially participate in the regulation of mitochondrial fusion. Moreover, Mitofusin 2 is mutated in Charcot-Marie-Tooth IIa and has multiple roles in controlling mitochondrial metabolism and cell cycle. We therefore extended our studies on the functional divergence of Mitofusins and found that MFN2 is unexpectedly required for the correct morphogenesis of the endoplasmic reticulum. We found that this protein is highly enriched at the level of "mitochondria-associated membranes", patches of endoplasmic reticulum in close contact with mitochondria. This further supports the novel role for this protein in keeping endoplasmic reticulum in close juxtaposition with mitochondria. This novel function impact on Ca²⁺ transfer from the endoplasmic reticulum to mitochondria, providing the first experimental evidence for the hypothesis of the high Ca²⁺ microdomains that are required for mitochondrial Ca²⁺ uptake in the course of physiological Ca²⁺ signaling. We now wish to explore the molecular mechanism and the regulation of the extramitochondrial function of Mfn2.

Dissecting the impact of mitochondrial shape changes in autophagy and organelle movement

Changes in mitochondrial morphology are important players in complex cellular processes, such as migration of lymphocytes (Campello et al., J. Exp. Med., 203,2879-2886, 2006). The shape of the organelle must change in order to grant their correct positioning as well as their engulfment by the nascent phagophore during autophagy. Our recent data show that fission induced by hFis1 might or might not be associated with mitochondrial dysfunction, depending on the integrity of the short intermembrane stretch of aminoacids of this protein (Alirol et al., Mol. Biol. Cell 17 4593-605, 2006). This opened up the possibility of investigating the relationship between mitochondrial fission, dysfunction and autophagy.

We are therefore using a genetic approach to investigate whether fission and/or dysfunction are required to induce elimination of the organelle by autophagy. Moreover, we are investigating the role of a novel keratin-binding protein that partially localizes to mitochondria in regulating shape, function and movement of the organelle. Our data point to a role for binding to intermediate filaments in fast mitochondrial movement and positioning.

Synoptic CV

Honours

Selected VIMM Publications

• de Brito OM, Scorrano L (2008) Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456:605-10.
• Cereghetti GM, Stangherlin A, Martins de Brito O, Chang CR, Blackstone C, Bernardi P, Scorrano L (2008) Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria. Proc. Natl. Acad. Sci. U.S.A. 105:15803-8.
• Frezza C, Cipolat S, Martins de Brito O, Micaroni M, Beznoussenko GV, Rudka T, Bartoli D, Polishuck RS, Danial NN, De Strooper B, Scorrano L (2006) OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126:177-89.
• Cipolat S, Rudka T, Hartmann D, Costa V, Serneels L, Craessaerts K, Metzger K, Frezza C, Annaert W, D'Adamio L, Derks C, Dejaegere T, Pellegrini L, D'Hooge R, Scorrano L, De Strooper B (2006) Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 126:163-75.
• Cipolat S, Martins de Brito O, Dal Zilio B, Scorrano L (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. U.S.A. 101:15927-32.

VIMM Publications

• [Pubmed ID: 20049710 * PubMed in process or DB hiatus]
• Wasilewski M, Scorrano L (2009) The changing shape of mitochondrial apoptosis. Trends Endocrinol. Metab. 20:287-94.
• Demaurex N, Scorrano L (2009) Reactive oxygen species are NOXious for neurons. Nat. Neurosci. 12:819-20.
• Lamarca V, Scorrano L (2009) When separation means death: killing through the mitochondria, but starting from the endoplasmic reticulum. EMBO J. 28:1681-3.
• Scorrano L, Liu D (2009) The SUMO arena goes mitochondrial with MAPL. EMBO Rep. 10:694-6.
• Scorrano L (2009) Opening the doors to cytochrome c: changes in mitochondrial shape and apoptosis. Int. J. Biochem. Cell Biol. 41:1875-83.
• Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, Bazan NG, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Castedo M, Cidlowski JA, Ciechanover A, Cohen GM, De Laurenzi V, De Maria R, Deshmukh M, Dynlacht BD, El-Deiry WS, Flavell RA, Fulda S, Garrido C, Golstein P, Gougeon ML, Green DR, Gronemeyer H, Hajnóczky G, Hardwick JM, Hengartner MO, Ichijo H, Jäättelä M, Kepp O, Kimchi A, Klionsky DJ, Knight RA, Kornbluth S, Kumar S, Levine B, Lipton SA, Lugli E, Madeo F, Malomi W, Marine JC, Martin SJ, Medema JP, Mehlen P, Melino G, Moll UM, Morselli E, Nagata S, Nicholson DW, Nicotera P, Nuñez G, Oren M, Penninger J, Pervaiz S, Peter ME, Piacentini M, Prehn JH, Puthalakath H, Rabinovich GA, Rizzuto R, Rodrigues CM, Rubinsztein DC, Rudel T, Scorrano L, Simon HU, Steller H, Tschopp J, Tsujimoto Y, Vandenabeele P, Vitale I, Vousden KH, Youle RJ, Yuan J, Zhivotovsky B, Kroemer G (2009) Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ. 16:1093-107.
• de Brito OM, Scorrano L (2009) Mitofusin-2 regulates mitochondrial and endoplasmic reticulum morphology and tethering: the role of Ras. Mitochondrion 9:222-6.
• Scorrano L (2008) Caspase-8 goes cardiolipin: a new platform to provide mitochondria with microdomains of apoptotic signals? J. Cell Biol. 183:579-81.
• [Pubmed ID: 18931701 * PubMed in process or DB hiatus]
• Spinazzi M, Cazzola S, Bortolozzi M, Baracca A, Loro E, Casarin A, Solaini G, Sgarbi G, Casalena G, Cenacchi G, Malena A, Frezza C, Carrara F, Angelini C, Scorrano L, Salviati L, Vergani L (2008) A novel deletion in the GTPase domain of OPA1 causes defects in mitochondrial morphology and distribution, but not in function. Hum. Mol. Genet. 17:3291-302.
• Gomes LC, Scorrano L (2008) High levels of Fis1, a pro-fission mitochondrial protein, trigger autophagy. Biochim. Biophys. Acta 1777:860-6.
• Frezza C, Cipolat S, Scorrano L (2007) Measuring mitochondrial shape changes and their consequences on mitochondrial involvement during apoptosis. Methods Mol. Biol. 372:405-20.
• de Brito OM, Scorrano L (2008) Mitofusin 2: a mitochondria-shaping protein with signaling roles beyond fusion. Antioxid. Redox Signal. 10:621-33.
• Scorrano L (2007) Multiple functions of mitochondria-shaping proteins. Novartis Found. Symp. 287:47-55; discussion 55.
• Dimmer KS, Navoni F, Casarin A, Trevisson E, Endele S, Winterpacht A, Salviati L, Scorrano L (2008) LETM1, deleted in Wolf-Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability. Hum. Mol. Genet. 17:201-14.
• Hausenloy DJ, Scorrano L (2007) Targeting cell death. Clin. Pharmacol. Ther. 82:370-3.
• Pellegrini L, Scorrano L (2007) A cut short to death: Parl and Opa1 in the regulation of mitochondrial morphology and apoptosis. Cell Death Differ. 14:1275-84.
• Frezza C, Cipolat S, Scorrano L (2007) Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2:287-95.
• Campello S, Lacalle RA, Bettella M, Mañes S, Scorrano L, Viola A (2006) Orchestration of lymphocyte chemotaxis by mitochondrial dynamics. J. Exp. Med. 203:2879-86.
• Alirol E, James D, Huber D, Marchetto A, Vergani L, Martinou JC, Scorrano L (2006) The mitochondrial fission protein hFis1 requires the endoplasmic reticulum gateway to induce apoptosis. Mol. Biol. Cell 17:4593-605.
• Cereghetti GM, Scorrano L (2006) The many shapes of mitochondrial death. Oncogene 25:4717-24.
• Dimmer KS, Scorrano L (2006) (De)constructing mitochondria: what for? 21:233-41.
• Cipolat S, Scorrano L (2006) To fuse and to protect. A novel role for CED-9 in mitochondrial morphology reveals an ancient function. Cell Death Differ. 13:1833-4.
• Letai A, Scorrano L (2006) Laying the foundations of programmed cell death. Cell Death Differ. 13:1245-7.
• Anesti V, Scorrano L (2006) The relationship between mitochondrial shape and function and the cytoskeleton. Biochim. Biophys. Acta 1757:692-9.
• Buytaert E, Callewaert G, Hendrickx N, Scorrano L, Hartmann D, Missiaen L, Vandenheede JR, Heirman I, Grooten J, Agostinis P (2006) Role of endoplasmic reticulum depletion and multidomain proapoptotic BAX and BAK proteins in shaping cell death after hypericin-mediated photodynamic therapy. FASEB J. 20:756-8.
• Scorrano L (2005) Proteins that fuse and fragment mitochondria in apoptosis: con-fissing a deadly con-fusion? J. Bioenerg. Biomembr. 37:165-70.
• Scorrano L (2005) Stanley J. Korsmeyer (1950-2005). J. Bioenerg. Biomembr. 37:109.

Additional Publications

• Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T, Korsmeyer SJ (2003) BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 300:135-9.
• Scorrano L, Ashiya M, Buttle K, Weiler S, Oakes SA, Mannella CA, Korsmeyer SJ (2002) A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Dev. Cell 2:55-67.

Selected Seminars

Contact

Luca Scorrano Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Tel.:  (+39) 049 7923 221 Fax:  (+39) 049 7923 271

Last updated: 28/03/2010, LS ·