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7 e colloque du réseau MeetOchondrie 4 – 7 mai 2014 – Evian Table Ronde Dynamique Mitochondriale Manuel Rojo Institut de Biochimie et Génétique Cellulaires UMR 5095 CNRS – Université de Bordeaux Dynamique fonctionnelle : acteurs et pertinence physiologique de la dynamique mitochondriale

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7e colloque du réseau MeetOchondrie 4 – 7 mai 2014 – Evian Table Ronde Dynamique Mitochondriale

Manuel Rojo Institut de Biochimie et Génétique Cellulaires UMR 5095 CNRS – Université de Bordeaux

Dynamique fonctionnelle : acteurs et pertinence physiologique de la dynamique mitochondriale

Ultrastructure,!Overall Morphology!Position & Mobility!

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matrice!

membrane!interne!

membrane!externe!

espace intermembranaire!Alberts et al. 1983!

Les mitochondries: ultrastructure!

(1999)!

(1985)!

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mouse heart mouse hepatocyte snail epithelial cell

Morphologie, Distribution et Ultrastructure mitochondriales

(Pierre Favard dans Alberts, Molecular Biology of the Cell)

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mitochondrial dynamics <-> a futile cycle of fusion & fission

Intracellular distribution - HeLa: mtDsRed & !tubulin!

cont

rol!

+ no

coda

zole!

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JCB 2009

- M, Safiulina D, Szabadkai G, Das S, Fransson A, Aspenstrom P, Rizzuto R, Hajnóczky G. (2008) Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase.Saotome . Proc Natl Acad Sci U S A.105(52):20728-33. - MacAskill, A.F., Rinholm, J.E., Twelvetrees, A.E., Arancibia-Carcamo, I.L., Muir, J., Fransson, Å., Aspenström, P., Attwell, D. and Kittler, J. (2009) Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses. Neuron 61, 541–555. - Wang, X. and Schwarz, T.L. (2009) The mechanism of Ca2+-dependent regulation of kinesin-mediated mitochondrial motility. Cell 136, 163–174.

Miro forms a complex with Milton to recruit kinesin to the mitochondria Miro–Milton complex supports mitochondrial motility along microtubules Miro–Milton complex confers Ca2+ sensitivity to mitochondrial motility

JCB 2006

microtubule

Miro/Gem1p

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JCB 2009

- M, Safiulina D, Szabadkai G, Das S, Fransson A, Aspenstrom P, Rizzuto R, Hajnóczky G. (2008) Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase.Saotome . Proc Natl Acad Sci U S A.105(52):20728-33. - MacAskill, A.F., Rinholm, J.E., Twelvetrees, A.E., Arancibia-Carcamo, I.L., Muir, J., Fransson, Å., Aspenström, P., Attwell, D. and Kittler, J. (2009) Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses. Neuron 61, 541–555. - Wang, X. and Schwarz, T.L. (2009) The mechanism of Ca2+-dependent regulation of kinesin-mediated mitochondrial motility. Cell 136, 163–174.

Miro forms a complex with Milton to recruit kinesin to the mitochondria Miro–Milton complex supports mitochondrial motility along microtubules Miro–Milton complex confers Ca2+ sensitivity to mitochondrial motility

JCB 2006

microtubule

Miro/Gem1p

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2007

anterograde: Arp2/3+Jsn1p/Puf1p+Puf3p

retrograde: Arp2/3 + typeII Myosin (Myo2)

bud

actin filament

2011

actin filament

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2007

anterograde: Arp2/3+Jsn1p/Puf1p+Puf3p

retrograde: Arp2/3 + typeII Myosin (Myo2)

bud

actin filament

2011

actin filament

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Fusion and Fission Dynamics!

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Drp1, Mff, Fis1, ….

Mfn1, Mfn2, OPA1, ….

* dynamin-related proteins

mitochondrial dynamics <-> a futile cycle of fusion & fission

cell fusion &

mitochondrial fusion

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Drp1, Mff, Fis1, ….

Mfn1, Mfn2, OPA1, ….

mitochondrial fusion: a "#m-dependent process

cell fusion + cccp (no "#m) - cccp

"$m is essential for inner membrane (IM) fusion

"$m is dispensable for outer membrane (OM) fusion

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De-energized mitochondria are excluded from the network of energized, fusogenic mitochondria This allows their selective targeting for degradation.

Drp1 Fis1 Mff

Drp1 Fis1 Mff

Mfn1 Mfn2 OPA1

Mfn1 Mfn2 OPA1

However, for such models to apply, fusion inhibition must be: - dominant - occur with physiological/pathological OXPHOS defects.

Twig et al 2008

Main Actors !

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yeast S. cerevisiae ! ! !mammals!! ! !!

facultative anaerob ! ! ! !obligate aerobic !!linear mtDNA molecules ! ! !circular mtDNA molecules!~ 80 kb ; 20 - 50 copies / cell ! ! !16.5 kb ; 1000s of copies / cell !4 respiratory complexes (no complex I) ! !5 respiratory complexes!cell ~ 3-6 µm ! ! ! !cell ≥ ~ 30 - 50 µm !!

Fzo1p* ! ! ! ! !Mfn1*, Mfn2* !!Mgm1p* ! ! ! ! !OPA1* ! !!Ugo1p ! ! ! ! !--------!-------! ! ! ! ! !Mito-PLD!-------! ! ! ! ! !Bcl2-family proteins !!

Dnm1p* ! ! ! ! !Drp1/Dlp1*!!Fis1p! ! ! ! ! !Fis1!----- ! ! ! ! ! !Mff!----- ! ! ! ! ! !MiD49 & MiD51 !!Mdv1p/Net2p ! ! ! !------ ! !!Caf4p ! ! ! ! !------!------ ! ! ! ! ! !GDAP1!------ ! ! ! ! ! !MTP18!

Mdm10p/Mdm12p/Mmm1p ! ! !------!Mdm31/Mdm32 ! ! ! !------!Gem1p ! ! ! ! !------!

Mitochondrial fusion/fission machineries

m.rojo / * GTP-binding proteins of the dynamin-superfamily

fusion

fission

“tubulation” or otherwise unknown…

Sesaki H, Jensen RE (1999) Division versus Fusion: Dnm1p and Fzo1p Antagonistically Regulate Mitochondrial Shape JCell Biol 147:699-706

Sesaki H, Southard SM, Yaffe MP, Jensen RE (2003) Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane MolBiolCell 14:2342-256.

WT mgm1"% dnm11"% mgm1"/dnm11"%

Antagonistic activity fusion and fission determine mitochondrial morphology S. cerevisiae

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Wakabayashi et al. (2009). The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice. The Journal of Cell Biology, 186(6), 805–816.

Antagonistic activity fusion and fission determine mitochondrial morphology mammals

DRP1

Koshiba, et al. (2004). Structural basis of mitochondrial tethering by mitofusin complexes. Science (New York, NY), 305(5685), 858–862. doi:10.1126/science.1099793

MFN1 MFN2

wt DKO

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mitochondria!secretory and endocytic organelles!

ER!

Golgi!

PM!

endosomes!

lysosomes!

nucleus!

budding/fission!

fusion!movement!

budding/fission!

maturation!

maturation, budding and cargo selection vectorial vesicular traffic

heterotypic fusion rabs & SNAREs

mitochondria!

peroxisomes!

fusion/fission!

fusion/fission!

?!?!

?!

?!

homotypic fusion & fission no rabs & no SNAREs

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Interaction(s) of mitochondria with other organelles?

2009

Mmm1, Mdm10, Mdm12 + Gem1p - “tubulation” mutants of mitochondrial distribution and morphology (mdm) (1994) - association with actin cables (2003) -  mtDNA-nucleoid maintenance (2004) - assembly of OM proteins (2004 – 2007)

ERMES: ER-mitochondria Encounter Structure -> non-vesicular lipid exchange -> Ca2+ exchange

2011

ER-mitochondrial connections/interactions (S.cerevisiae)

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2008

ER-mitochondrial connections/interactions (mammals)

2012

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Physio(patho)logical relevance!

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What is the physio(patho)logical relevance of mitochondrial dynamics? (animal models: KO and mutations)

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Sauvanet et al. (2010) Dynamique et morphologie mitochondriales. Acteurs, mécanismes et pertinence fonctionnelle Med Sci (Paris). 26(10):823-9.

KO Fzo1/Mfn: severe physiological and developmental consequences

KO Mgm1/OPA1: severe physiological and developmental consequences

KO Dnm1/DRP1: severe physiological and developmental consequences

What is the physio(patho)logical relevance of mitochondrial dynamics? (animal models: KO and mutations)

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Sauvanet et al. (2010) Dynamique et morphologie mitochondriales. Acteurs, mécanismes et pertinence fonctionnelle Med Sci (Paris). 26(10):823-9.

KO Fzo1/Mfn: severe physiological and developmental consequences

KO Mgm1/OPA1: severe physiological and developmental consequences

KO Dnm1/DRP1: severe physiological and developmental consequences

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mitochondrial distribution, morphology & dynamics 2014

autophagy-mitophagy

mitochondrial DNA transmission & segregation

cellular & mitochondrial Bioenergetics

OXPHOS-diseases

proliferation apoptosis

Ca2+-signalling ROS production & consumption

senescence

signaling regulation

innate immunity

neuronal function - neuropathies morphogenesis of spines and synapses

Lymphocyte chemotaxis

Alzheimer Huntington Parkinson

obesity diabetes Insulin release

viral infection

Cardiomyocite development

function of pancreatic beta-cells Insulin secretion

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mitochondrial distribution, morphology & dynamics 2014

cellular & mitochondrial Bioenergetics

OXPHOS-diseases

proliferation apoptosis

neuronal function - neuropathies morphogenesis of spines and

synapses

Alzheimer Huntington Parkinson

Mitochondrial dynamics and hereditary neuro(muscular) diseases

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MFN2: maladie de Charcot-Marie-Tooth de type 2A (CMT2A)

OPA1: maladie de Kjer, atrophie autosomique dominante de type 1 (DOA)

DRP1: syndrome néonatal létal

GDAP1: maladie de Charcot-Marie-Tooth de type 4A et 2K (CMT4A et CMT2K)

Mitochondrial dynamics and hereditary neuro(muscular) diseases

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MFN2: maladie de Charcot-Marie-Tooth de type 2A (CMT2A)

OPA1: maladie de Kjer, atrophie autosomique dominante de type 1 (DOA)

DRP1: syndrome néonatal létal

GDAP1: maladie de Charcot-Marie-Tooth de type 4A et 2K (CMT4A et CMT2K)

Lenaers et al. (2010). De la levure aux maladies neurodégénératives. Dix ans d’exploration des pathologies de la dynamique mitochondriale. Médecine Sciences : M/S, 26(10), 836–841.

m.rojo /

mitochondrial distribution, morphology & dynamics 2014

cellular & mitochondrial Bioenergetics

OXPHOS-diseases

proliferation apoptosis

neuronal function - neuropathies morphogenesis of spines and

synapses

Alzheimer Huntington Parkinson

Mitochondrial fusion / fission and bioenergetics

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This inhibition enables exclusion of defective mitochondria and their selective targeting to mitophagy.

2012

2008

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mitochondrial distribution, morphology & dynamics 2014

cellular & mitochondrial Bioenergetics

OXPHOS-diseases

proliferation apoptosis

neuronal function - neuropathies morphogenesis of spines and

synapses

Alzheimer Huntington Parkinson

m.rojo /

fly muscle mtGFP

Marf = mitofusin

2008

Mitochondrial dynamics and parkinson disease

m.rojo /

mitochondrial distribution, morphology & dynamics 2014

cellular & mitochondrial Bioenergetics

OXPHOS-diseases

proliferation apoptosis

neuronal function - neuropathies morphogenesis of spines and

synapses

Alzheimer Huntington Parkinson

2001

2006

Mitochondrial dynamics and apoptosis

m.rojo /

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Voire la dynamique mitochondriale

autophagy-mitophagy

mitochondrial DNA transmission & segregation

cellular & mitochondrial Bioenergetics

OXPHOS-diseases

proliferation apoptosis

Ca2+-signalling ROS production & consumption

senescence

signaling regulation

innate immunity

neuronal function - neuropathies morphogenesis of spines and synapses

Lymphocyte chemotaxis

Alzheimer Huntington Parkinson

obesity diabetes Insulin release

viral infection

Cardiomyocite development

function of pancreatic beta-cells Insulin secretion

m.rojo /

Voire la dynamique mitochondriale

m.rojo /

Lenaers et al. (2010). De la levure aux maladies neurodégénératives. Dix ans d’exploration des pathologies de la dynamique mitochondriale. Médecine Sciences : M/S, 26(10), 836–841.

Agier et al. (2012). Defective mitochondrial fusion, altered respiratory function, and distorted cristae structure in skin fibroblasts with heterozygous OPA1 mutations. Biochimica Et Biophysica Acta, 1822(10), 1570–1580.

Voire la morphologie mitochondriale – mutations d’OPA1 - mitochondrial morphology varies between alleles -

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Mishra et al. (2014). Proteolytic cleavage of opa1 stimulates mitochondrial inner membrane fusion and couples fusion to oxidative phosphorylation. Cell Metabolism, 19(4), 630–641

Guillery et al. (2008). Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts. Neuromuscular Disorders : NMD, 18(4), 319–330.

WT

ND1

glucose deoxy-glucose

Voire la morphologie mitochondriale – deficits de l’OXPHOS - specific morphology requires specific culture conditions -

IBGC – Bordeaux Jean-Paul di Rago

Stéphane Duvezin-Caubet

Patrick Paumard!Marie-France Giraud!

Stéphen Manon Muriel Priault

Bénédicte Salin, Corinne Blancard!

IBGC – Bordeaux Organisation et Dynamique Mitochondriales

Marine Bouhier Claudine David

Manuel Rojo

Cécile Sauvanet (2008 – 2011)

Institut Jacques Monod David Tareste

notre équipe collaborations

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Bordeaux, a nice place to work and to live. Contact: [email protected]