Identification and functional characterization of components of the OPA1 complexes targeted during apoptotic cristae remodeling

Mitochondria are cellular organelles participating actively in energy production, signal transduction and apoptosis. They are characterized by a sophisticated structure essential for hosting the multiple mitochondrial functions. An outer membrane defines the border of the organelle and the cytoplasm, while an inner membrane compartmentalizes the internal space into intermembrane space, matrix and cristae. Cristae are the sites of oxidative phosphorylation and are separated from the intermembrane space by narrow tubular cristae junctions (Frey and Mannella, 2000; Vogel et al., 2006). During programmed cell death mitochondrial shape and ultrastructure change. These morphological alterations that include the widening of the narrow cristae junctions – the so-called “cristae remodeling” – contribute to the mobilization of the cristae-endowed cytochrome c to the intermembrane space and its complete release to the cytosol (Scorrano et al., 2002). Genetic and biochemical research on the molecular mechanisms of mitochondrial structure modifications during apoptosis unveiled that the inner mitochondrial membrane dynamin-related protein, Optic atrophy 1 (OPA1) is a key regulator of cristae remodeling. OPA1 forms multiple complexes that maintain the cristae junctions close; thereby regulating how much cytochrome c is available in the intermembrane space for release in the cytosol upon permeabilization of the outer mitochondrial membrane. Upon apoptotic stimulus, the high molecular weight (H.M.W) OPA1-containing complexes are disrupted, with concomitant enlargement of the cristae junctions and cytochrome c release (Frezza et al., 2006; Cogliati et al., 2013). Interestingly, these assemblies are composed by the transmembrane (long) and soluble (short) OPA1 forms and their size ranges from 500 to 800 KDa, as judged by blue native gel electrophoresis and by size exclusion chromatography. This size suggests that they contain proteins other than OPA1, whose identity and function calls for investigation. The aim of my PhD thesis is to explore the composition of the OPA1-containing complexes, as well as to characterize the function of identified OPA1 partners in mitochondrial physiology and cell death. Three dimensional BlueNative-BlueNative-SDS PAGE (3D BN-BN-SDS PAGE) followed by semi-quantitative LC/MS analysis of normal and apoptotic mouse liver mitochondria, carried out in our lab, revealed a cohort of candidate proteins present in the OPA1-containing complexes whose distribution changes during programmed cell death. To further mark off the number of candidate OPA1 partners, we additionally performed a semi-quantitative LC/MS analysis in blue native PAGE of mouse heart mitochondria. Furthermore, we employed stable isotope labeling by amino acids (SILAC) (Ong and Mann, 2007) in mouse adult fibroblasts (MAFs), combined with blue native PAGE and LC-MS/MS. In order to distinguish the proteins that are regulated specifically during cristae remodeling, in the two latter approaches we compared a normal mitochondrial population with mitochondrial populations stimulated with the wild type pro-apoptotic caspase 8 cleaved BID (cBIDwt) or a cristae remodeling incompetent mutant of cBID (cBIDKKAA) (Cogliati et al., 2013). The resulted hits of all the three high throughput strategies were analyzed collectively to obtain the final lists of potential OPA1 interactors and the subset of proteins that significantly decrease or increase in the various multimeric OPA1 complexes in the course of cristae remodeling. The discovery of the same OPA1 interactors between the techniques indicated a tissue-independency and that our methods had the level of definition required to inspect OPA1 complexes in cell life and death. Among the identified proteins, our attention was firstly caught by two MICOS complex members: Mic60 and Mic19. “Mitochondrial contact site and cristae organizing system” (MICOS) is a large heterooligomeric protein structure, which regulates cristae junction biogenesis. Nevertheless, the master regulator of cristae shape OPA1 is not part of MICOS and its exact composition, architecture and functions in yeast and mammals are still under active investigation. In all the three proteomic approaches and after immunoblotting of Blue Native PAGE the core component of MICOS, Mic60, was retrieved in high molecular weight complexes with similar electrophoretic mobility as those of OPA1. In order to unravel the potential crosstalk between OPA1 and Mic60, we performed biochemical and genetic analysis in OPA1 or/and Mic60 ablation and demonstrated that the two IMM proteins interact and are indispensable for the Mic60/OPA1 complexes formation. Functionally, electron microscopy revealed that these complexes are essential for cristae junction formation, but not for specifying the width of the cristae lumen or the cristae junction mouth. Moreover, we showed that the Mic60/OPA1 complexes are eliminated by cBIDwt but not by its mutant cBIDKKAA, indicating a novel role of Mic60 that regards apoptotic cristae remodeling. Worth noting, bioinformatics analysis uncovered a structural divergence in the transmembrane and middle domains between yeast and mammalian Mic60 orthologues, potentially explaining the additional Mic60 functions at the outset of evolution. Similarly, we also demonstrated that Mic19 is part of high molecular weight complexes, which are targeted during cristae remodeling, raising the possibility that mammalian Mic19 might also regulate mitochondria biogenesis through the OPA1 pathway. Besides MICOS components, we, additionally, wished to investigate another identified protein that was significantly reduced in apoptosis. This protein is christened nitric oxide-associated 1 (NOA1). The co-existence of NOA1 and OPA1 in the same complexes was confirmed biochemically in BMH crosslinked mitochondria, as well as after co-immunoprecipitation. These initial data, along with the obscure exact function of this newly characterized protein, prompted us to further investigate its role in mitochondria and its relationship with OPA1 in the control of mitochondrial morphology and cristae remodeling. To shed light on the functional link between the two proteins of interest, we used NOA1 deficient MEFs where we surprisingly observed an upregulation of OPA1, both at the protein and at the transcript level. In addition, NOA1 was absent in Opa1-/- MEFs, while its level went back to normal when OPA1 was re-introduced, suggesting a genetic interaction between the two. Next, in order to gain an insight into the role of NOA1 in mitochondria, we analyzed NOA1 deficient MEFs by confocal and electron microscopy. Mitochondria of Noa1-/- cells were fragmented and displayed ultrastructural changes. Interestingly, overexpression of OPA1 corrected the aberrant mitochondrial ultrastructure at a similar extent as upon re-introduction of NOA1, further supporting the hypothesis of an OPA1-NOA1 functional interplay. Given the mitochondrial structural defects, we hypothesized that NOA1 ablation affected also mitochondrial functions. Indeed, Seahorse analysis showed impaired mitochondrial respiration in NOA1 lacking cells, consistent with previously published reports. SDS and blue native PAGE revealed a notable decrease of OXPHOS subunits protein levels and a defect on respiratory complexes assembly in NOA1 deficiency. Remarkably, mtDNA copy number was unaltered, while the protein reduction was not restricted to mtDNA encoded proteins. Consequently, we suggest that the general instability of OXPHOS proteins is probably due to the inability of the respiratory chain complexes to properly assemble, potentially explaining the bioenergetic dysfunction. Functionally, Noa1-/- cells grew slower than WT in galactose-containing medium and after two days they were unable to tolerate glucose deprivation, suggesting a potential role of NOA1 in mitochondrial-dependent cell growth. Due to the fact that OPA1 is an anti-apoptotic protein whose complexes are disrupted after apoptotic stimulation, we aimed to study its partner, NOA1, in the course of apoptosis. Like OPA1, NOA1 complexes that have the same electrophoretic motility after crosslinking as those of OPA1 are similarly disrupted upon cBID induced cristae remodeling. On top of this, during apoptosis, NOA1 is likely proteolyticallly cleaved, which could be a potential mechanism for the elimination of NOA1/OPA1-contining complexes early in cell death stimulation. In conclusion, our study unraveled the composition of OPA1 complexes that are eliminated during apoptosis. We uncovered a crosstalk between the core cristae biogenesis regulators, OPA1 and Mic60, and characterized the role of Mic60 during apoptotic cristae remodeling. Moreover, we discovered a novel player of cristae shape maintenance, cristae remodeling and mitochondrial respiration, NOA1, that acts in the same pathway as OPA1

How Do Cytokines Trigger Genomic Instability?

Inflammation is a double-edged sword presenting a dual effect on cancer development, from one hand promoting tumor initiation and progression and from the other hand protecting against cancer through immunosurveillance mechanisms. Cytokines are crucial components of inflammation, participating in the interaction between the cells of tumor microenvironment. A comprehensive study of the role of cytokines in the context of the inflammation-tumorigenesis interplay helps us to shed light in the pathogenesis of cancer. In this paper we focus on the role of cytokines in the development of genomic instability, an evolving hallmark of cancer

Optic Atrophy 1 Is Epistatic to the Core MICOS Component MIC60 in Mitochondrial Cristae Shape Control

The mitochondrial contact site and cristae organizing system (MICOS) and Optic atrophy 1 (OPA1) control cristae shape, thus affecting mitochondrial function and apoptosis. Whether and how they physically and functionally interact is unclear. Here, we provide evidence that OPA1 is epistatic to MICOS in the regulation of cristae shape. Proteomic analysis identifies multiple MICOS components in native OPA1-containing high molecular weight complexes disrupted during cristae remodeling. MIC60, a core MICOS protein, physically interacts with OPA1, and together, they control cristae junction number and stability, OPA1 being epistatic to MIC60. OPA1 defines cristae width and junction diameter independently of MIC60. Our combination of proteomics, biochemistry, genetics, and electron tomography provides a unifying model for mammalian cristae biogenesis by OPA1 and MICOS.We thank Drs. F. Caicci and F. Boldrin (EM Facility, Department of Biology, University of Padova) for EM and ALEMBIC, San Raffaele Scientific Institute, for tomography. L.S. is a senior scientist of the Dulbecco-Telethon Institute. Support was provided by Telethon-Italy (GGP15091 and GGP14187), AIRC Italy (ERC FP7-282280), FP7 CIG (PCIG13-GA-2013-618697), the Italian Ministry of Research (FIRB RBAP11Z3YA\_005), the Italian Ministry of Health (GR-2009-1600051 to L.S.), a University of Padua grant for a postdoctoral fellowship (2015 to M.E.S.), and an International Brain Research Organization-International Society for Neurochemistry research fellowship (2016 to A.M.).S

The cristae modulator Optic atrophy 1 requires mitochondrial ATP synthase oligomers to safeguard mitochondrial function

It is unclear how the mitochondrial fusion protein Optic atrophy 1 (OPA1), which inhibits cristae remodeling, protects from mitochondrial dysfunction. Here we identify the mitochondrial F1Fo-ATP synthase as the effector of OPA1 in mitochondrial protection. In OPA1 overexpressing cells, the loss of proton electrochemical gradient caused by respiratory chain complex III inhibition is blunted and this protection is abolished by the ATP synthase inhibitor oligomycin. Mechanistically, OPA1 and ATP synthase can interact, but recombinant OPA1 fails to promote oligomerization of purified ATP synthase reconstituted in liposomes, suggesting that OPA1 favors ATP synthase oligomerization and reversal activity by modulating cristae shape. When ATP synthase oligomers are genetically destabilized by silencing the key dimerization subunit e, OPA1 is no longer able to preserve mitochondrial function and cell viability upon complex III inhibition. Thus, OPA1 protects mitochondria from respiratory chain inhibition by stabilizing cristae shape and favoring ATP synthase oligomerization

Mitophagy Promotes Resistance to BH3 Mimetics in Acute Myeloid Leukemia

BH3 mimetics are used as an efficient strategy to induce cell death in several blood malignancies, including acute myeloid leukemia (AML). Venetoclax, a potent BCL-2 antagonist, is used clinically in combination with hypomethylating agents for the treatment of AML. Moreover, MCL1 or dual BCL-2/BCL-xL antagonists are under investigation. Yet, resistance to single or combinatorial BH3-mimetic therapies eventually ensues. Integration of multiple genome-wide CRISPR/Cas9 screens revealed that loss of mitophagy modulators sensitizes AML cells to various BH3 mimetics targeting different BCL-2 family members. One such regulator is MFN2, whose protein levels positively correlate with drug resistance in patients with AML. MFN2 overexpression is sufficient to drive resistance to BH3 mimetics in AML. Insensitivity to BH3 mimetics is accompanied by enhanced mitochondria-endoplasmic reticulum interactions and augmented mitophagy flux, which acts as a prosurvival mechanism to eliminate mitochondrial damage. Genetic or pharmacologic MFN2 targeting synergizes with BH3 mimetics by impairing mitochondrial clearance and enhancing apoptosis in AML

defective mitochondrial trna taurine modification activates global proteostress and leads to mitochondrial disease

Summary: A subset of mitochondrial tRNAs (mt-tRNAs) contains taurine-derived modifications at 34U of the anticodon. Loss of taurine modification has been linked to the development of mitochondrial diseases, but the molecular mechanism is still unclear. Here, we showed that taurine modification is catalyzed by mitochondrial optimization 1 (Mto1) in mammals. Mto1 deficiency severely impaired mitochondrial translation and respiratory activity. Moreover, Mto1-deficient cells exhibited abnormal mitochondrial morphology owing to aberrant trafficking of nuclear DNA-encoded mitochondrial proteins, including Opa1. The mistargeted proteins were aggregated and misfolded in the cytoplasm, which induced cytotoxic unfolded protein response. Importantly, application of chemical chaperones successfully suppressed cytotoxicity by reducing protein misfolding and increasing functional mitochondrial proteins in Mto1-deficient cells and mice. Thus, our results demonstrate the essential role of taurine modification in mitochondrial translation and reveal an intrinsic protein homeostasis network between the mitochondria and cytosol, which has therapeutic potential for mitochondrial diseases. : Taurine modification of mitochondrial tRNA is associated with mitochondrial disease. Fakruddin et al. find that taurine modification is indispensable for mitochondrial protein translation. The authors also find that deficiency of taurine modification impairs a mitochondrial-cytosolic proteostatic network through an Opa1-dependent mechanism and demonstrate the therapeutic potential of chemical chaperones. Keywords: tRNA, modification, taurine, mitochondria, Opa

Identification and functional characterization of components of the OPA1 complexes targeted during apoptotic cristae remodeling

Mitochondria are cellular organelles participating actively in energy production, signal transduction and apoptosis. They are characterized by a sophisticated structure essential for hosting the multiple mitochondrial functions. An outer membrane defines the border of the organelle and the cytoplasm, while an inner membrane compartmentalizes the internal space into intermembrane space, matrix and cristae. Cristae are the sites of oxidative phosphorylation and are separated from the intermembrane space by narrow tubular cristae junctions (Frey and Mannella, 2000; Vogel et al., 2006). During programmed cell death mitochondrial shape and ultrastructure change. These morphological alterations that include the widening of the narrow cristae junctions – the so-called “cristae remodeling” – contribute to the mobilization of the cristae-endowed cytochrome c to the intermembrane space and its complete release to the cytosol (Scorrano et al., 2002). Genetic and biochemical research on the molecular mechanisms of mitochondrial structure modifications during apoptosis unveiled that the inner mitochondrial membrane dynamin-related protein, Optic atrophy 1 (OPA1) is a key regulator of cristae remodeling. OPA1 forms multiple complexes that maintain the cristae junctions close; thereby regulating how much cytochrome c is available in the intermembrane space for release in the cytosol upon permeabilization of the outer mitochondrial membrane. Upon apoptotic stimulus, the high molecular weight (H.M.W) OPA1-containing complexes are disrupted, with concomitant enlargement of the cristae junctions and cytochrome c release (Frezza et al., 2006; Cogliati et al., 2013). Interestingly, these assemblies are composed by the transmembrane (long) and soluble (short) OPA1 forms and their size ranges from 500 to 800 KDa, as judged by blue native gel electrophoresis and by size exclusion chromatography. This size suggests that they contain proteins other than OPA1, whose identity and function calls for investigation. The aim of my PhD thesis is to explore the composition of the OPA1-containing complexes, as well as to characterize the function of identified OPA1 partners in mitochondrial physiology and cell death. Three dimensional BlueNative-BlueNative-SDS PAGE (3D BN-BN-SDS PAGE) followed by semi-quantitative LC/MS analysis of normal and apoptotic mouse liver mitochondria, carried out in our lab, revealed a cohort of candidate proteins present in the OPA1-containing complexes whose distribution changes during programmed cell death. To further mark off the number of candidate OPA1 partners, we additionally performed a semi-quantitative LC/MS analysis in blue native PAGE of mouse heart mitochondria. Furthermore, we employed stable isotope labeling by amino acids (SILAC) (Ong and Mann, 2007) in mouse adult fibroblasts (MAFs), combined with blue native PAGE and LC-MS/MS. In order to distinguish the proteins that are regulated specifically during cristae remodeling, in the two latter approaches we compared a normal mitochondrial population with mitochondrial populations stimulated with the wild type pro-apoptotic caspase 8 cleaved BID (cBIDwt) or a cristae remodeling incompetent mutant of cBID (cBIDKKAA) (Cogliati et al., 2013). The resulted hits of all the three high throughput strategies were analyzed collectively to obtain the final lists of potential OPA1 interactors and the subset of proteins that significantly decrease or increase in the various multimeric OPA1 complexes in the course of cristae remodeling. The discovery of the same OPA1 interactors between the techniques indicated a tissue-independency and that our methods had the level of definition required to inspect OPA1 complexes in cell life and death. Among the identified proteins, our attention was firstly caught by two MICOS complex members: Mic60 and Mic19. “Mitochondrial contact site and cristae organizing system” (MICOS) is a large heterooligomeric protein structure, which regulates cristae junction biogenesis. Nevertheless, the master regulator of cristae shape OPA1 is not part of MICOS and its exact composition, architecture and functions in yeast and mammals are still under active investigation. In all the three proteomic approaches and after immunoblotting of Blue Native PAGE the core component of MICOS, Mic60, was retrieved in high molecular weight complexes with similar electrophoretic mobility as those of OPA1. In order to unravel the potential crosstalk between OPA1 and Mic60, we performed biochemical and genetic analysis in OPA1 or/and Mic60 ablation and demonstrated that the two IMM proteins interact and are indispensable for the Mic60/OPA1 complexes formation. Functionally, electron microscopy revealed that these complexes are essential for cristae junction formation, but not for specifying the width of the cristae lumen or the cristae junction mouth. Moreover, we showed that the Mic60/OPA1 complexes are eliminated by cBIDwt but not by its mutant cBIDKKAA, indicating a novel role of Mic60 that regards apoptotic cristae remodeling. Worth noting, bioinformatics analysis uncovered a structural divergence in the transmembrane and middle domains between yeast and mammalian Mic60 orthologues, potentially explaining the additional Mic60 functions at the outset of evolution. Similarly, we also demonstrated that Mic19 is part of high molecular weight complexes, which are targeted during cristae remodeling, raising the possibility that mammalian Mic19 might also regulate mitochondria biogenesis through the OPA1 pathway. Besides MICOS components, we, additionally, wished to investigate another identified protein that was significantly reduced in apoptosis. This protein is christened nitric oxide-associated 1 (NOA1). The co-existence of NOA1 and OPA1 in the same complexes was confirmed biochemically in BMH crosslinked mitochondria, as well as after co-immunoprecipitation. These initial data, along with the obscure exact function of this newly characterized protein, prompted us to further investigate its role in mitochondria and its relationship with OPA1 in the control of mitochondrial morphology and cristae remodeling. To shed light on the functional link between the two proteins of interest, we used NOA1 deficient MEFs where we surprisingly observed an upregulation of OPA1, both at the protein and at the transcript level. In addition, NOA1 was absent in Opa1-/- MEFs, while its level went back to normal when OPA1 was re-introduced, suggesting a genetic interaction between the two. Next, in order to gain an insight into the role of NOA1 in mitochondria, we analyzed NOA1 deficient MEFs by confocal and electron microscopy. Mitochondria of Noa1-/- cells were fragmented and displayed ultrastructural changes. Interestingly, overexpression of OPA1 corrected the aberrant mitochondrial ultrastructure at a similar extent as upon re-introduction of NOA1, further supporting the hypothesis of an OPA1-NOA1 functional interplay. Given the mitochondrial structural defects, we hypothesized that NOA1 ablation affected also mitochondrial functions. Indeed, Seahorse analysis showed impaired mitochondrial respiration in NOA1 lacking cells, consistent with previously published reports. SDS and blue native PAGE revealed a notable decrease of OXPHOS subunits protein levels and a defect on respiratory complexes assembly in NOA1 deficiency. Remarkably, mtDNA copy number was unaltered, while the protein reduction was not restricted to mtDNA encoded proteins. Consequently, we suggest that the general instability of OXPHOS proteins is probably due to the inability of the respiratory chain complexes to properly assemble, potentially explaining the bioenergetic dysfunction. Functionally, Noa1-/- cells grew slower than WT in galactose-containing medium and after two days they were unable to tolerate glucose deprivation, suggesting a potential role of NOA1 in mitochondrial-dependent cell growth. Due to the fact that OPA1 is an anti-apoptotic protein whose complexes are disrupted after apoptotic stimulation, we aimed to study its partner, NOA1, in the course of apoptosis. Like OPA1, NOA1 complexes that have the same electrophoretic motility after crosslinking as those of OPA1 are similarly disrupted upon cBID induced cristae remodeling. On top of this, during apoptosis, NOA1 is likely proteolyticallly cleaved, which could be a potential mechanism for the elimination of NOA1/OPA1-contining complexes early in cell death stimulation. In conclusion, our study unraveled the composition of OPA1 complexes that are eliminated during apoptosis. We uncovered a crosstalk between the core cristae biogenesis regulators, OPA1 and Mic60, and characterized the role of Mic60 during apoptotic cristae remodeling. Moreover, we discovered a novel player of cristae shape maintenance, cristae remodeling and mitochondrial respiration, NOA1, that acts in the same pathway as OPA1.I mitocondri sono organelli cellulari che partecipano attivamente alla produzione di energia, la trasduzione del segnale e l'apoptosi. Essi sono caratterizzati da una struttura sofisticata indispensabile per ospitare le molteplici funzioni mitocondriali. Una membrana esterna definisce il confine del organello e il citoplasma, mentre una membrana interna compartimentalizza lo spazio interno in spazio intermembrane, matrice e cristae. Le cristae sono i siti di fosforilazione ossidativa e sono separate dallo spazio intermembrana da strette giunzioni tubolari (Frey and Mannella, 2000; Vogel et al., 2006). Durante la morte cellulare programmata la forma e l’ ultrastruttura mitocondriale cambiano. Queste alterazioni morfologiche che comprendono l'ampliamento delle giunzioni strette delle cristae - il cosiddetto "rimodellamento di cristae" - contribuisce alla mobilitazione del citocromo c, e il suo rilascio completo dallo spazio intermembrana al citoplasma (Scorrano et al., 2002). Ricerche genetiche e biochimiche sui meccanismi molecolari della modificazione mitocondriale durante l'apoptosi hanno svelato che la proteina della membrana mitocondriale interiore, optic atrophy (OPA1) è un regolatore chiave del rimodellamento delle cristae. OPA1 forma vari complessi che mantengono le giunzioni delle cristae regolando così la quantità del citocromo c che è disponibile nello spazio intermembrana per il suo rilascio nel citoplasma dopo la permeabilizzazione della membrana mitocondriale esterna. Su stimolo apoptotico, i complessi di alto peso molecolare (high molecular weight, HMW) contenenti OPA1 sono interrotti, con l'allargamento concomitante delle giunzioni e il rilascio del citocromo c (Frezza et al., 2006; Cogliati et al., 2013). È interessante notare che questi gruppi sono composti da forme transmembrana (lunghe) e forme solubili (corte) di OPA1 e la loro dimensione è compresa tra i 500 e gli 800 KDa, come giudicato da elettroforesi su gel blu nativo e per dimensione cromatografia di esclusione. Questa dimensione suggerisce che essi contengono anche altre proteine oltre che OPA1, la cui identità e funzione deve essere esaminata. Lo scopo della mia tesi di dottorato è di esplorare la composizione dei complessi-OPA1 contenenti, e di caratterizzare la funzione delle parti di OPA1 identificate nella fisiologia mitocondriale e nella morte cellulare. Elettroforesi tridimensionale su gel di poliacrilammide blu nativo (BN-PAGE) (3D BN-BN-SDS PAGE) seguita da semi-quantitativa LC/MS analisi di mitocondri normali e apoptotici di fegato di topo, effettuata nel nostro laboratorio, ha rivelato una coorte di proteine putative presenti nei complessi OPA1–contenenti la cui distribuzione cambia durante la morte cellulare programmata. Per identificare meglio i partner putativi per OPA1 abbiamo inoltre eseguito un semi-quantitativa LC/MS analisi in su gel BlueNative dei mitocondri murini cardiaci. Inoltre, abbiamo impiegato etichettatura a isotopo stabile con aminoacidi (SILAC) (Ong and Mann, 2007) in fibroblasti di topo adulto (MAFS), in combinazione con 2D BN-BN PAGE e LC/MS. Per distinguere le proteine che sono regolate particolarmente durante il rimodellamento delle cristae, con questi due approcci abbiamo confrontato la popolazione mitocondriale normale con le popolazioni mitocondriali stimolate con BID, un fattore pro-apoptotico tagliato proteoliticamente dal caspasi 8, in forma wild type (cBIDwt) o con mutazione che compromette il rimodellamento delle cristae (cBIDKKAA) (Cogliati et al., 2013). I risultati ottenuti dalle tre strategie high throughput sono stati analizzati per ottenere gli elenchi definitivi dei potenziali interattori di OPA1 e il coorte delle proteine che sono ridotte o aumentate in modo significativo nei vari complessi multimerici di OPA1 durante il rimodellamento delle cristae. La scoperta degli stessi interattori di OPA1 con le varie tecniche hanno dimostrato indipendenza dal tipo di tessuto, cosa che mostra che i nostri metodi hanno il livello di specificità necessario per l’ ispezione dei complessi OPA1 nella vita e morte cellulare. Tra le proteine identificate, la nostra attenzione è stata catturata da due componenti del complesso MICOS: Mic60 e Mic19. Il “Mitochondrial contact site and cristae organizing system” (MICOS) è una grande struttura proteica eterooligomerica, che regola la biogenesi delle giunzioni delle cristae. Tuttavia, il regolatore principale delle cristae OPA1 non fa parte del complesso MICOS e la sua esatta composizione, l'architettura e le funzioni nel lievito e nei mammiferi sono ancora sotto esame. In tutti e tre gli approcci di proteomica e dopo immunoblotting con gel Blue Native, il componente principale di MICOS, Mic60, è stato individuato in complessi ad alto peso molecolare con simile mobilità elettroforetica a quelli di OPA1. Al fine di svelare il potenziale crosstalk tra OPA1 e Mic60, abbiamo effettuato analisi biochimiche e genetiche in situazione di ablazione di OPA1 o/e Mic60 e dimostrato che le due proteine interagiscono nelle membrane mitocondriali interne (inner mitochondrial membranes-IMM) e sono essenziali nella formazione dei complessi OPA1/Mic60. Esperimenti di microscopia elettronica hanno rivelato che questi complessi sono essenziali nella formazione della giunzione delle cristae, ma non per specificare la larghezza del loro lumen o la giunzione del loro foro. Inoltre, abbiamo dimostrato che i complessi Mic60/OPA1 sono eliminati da cBIDwt ma non dal suo mutante cBIDKKAA, indicando un nuovo ruolo di Mic60 che riguarda il rimodellamento apoptotico delle cristae. E’ da notare che l'analisi bioinformatica ha mostrato una divergenza strutturale tra gli ortologi di Mic60 tra lievito e mammiferi, spiegando potenzialmente le ulteriori funzioni Mic60 sin dall'inizio dell'evoluzione. Allo stesso modo, abbiamo anche dimostrato che Mic19 fa parte di complessi ad alto peso molecolare presenti durante la ristrutturazione delle cristae, aumentando la possibilità che il Mic19 dei mammiferi potrebbe anche regolare la biogenesi dei mitocondri attraverso il pathway di OPA1. Oltre ai componenti di MICOS, abbiamo voluto, inoltre, indagare un'altra proteina trovata significativamente ridotta durante l’ apoptosi. Questa proteina è chiamata ossido nitrico-associato 1 (nitric oxide-associated 1, NOA1). La coesistenza di NOA1 e OPA1 negli stessi complessi è stata confermata con metodi biochimici in BMH mitocondri crosslinked, così come dopo co-immunoprecipitazione. Questi dati iniziali, insieme al fatto che la funzione di questa proteina era all’ oscuro sino ad ora, ci hanno spinto a studiare ulteriormente il suo ruolo nei mitocondri e la sua relazione con OPA1 nel controllo della morfologia mitocondriale e nel rimodellamento delle criste. Per far luce al collegamento funzionale tra le due proteine di interesse, abbiamo utilizzato MEF carenti di NOA1 dove abbiamo sorprendentemente osservato un aumento di OPA1, sia a livello di proteine che a livello di trascrizione. Inoltre, mentre NOA1 era assente nelle Opa1-/- MEF, il suo livello è tornato alla normalità quando è stata reintrodotta OPA1, suggerendo un interazione genetica tra le due. Successivamente, al fine di ottenere informazioni sul ruolo di NOA1 nei mitocondri, abbiamo analizzato Noa1-/- MEF mediante microscopia confocale ed elettronica. I mitocondri di cellule Noa1-/- sono frammentati e hanno alterazioni ultrastrutturali. È interessante notare che l' iperespressione di OPA1 ha corretto l’ ultrastruttura mitocondriale aberrante in misura simile a quella di reintroduzione di NOA1, sostenendo ulteriormente l'ipotesi di una interazione funzionale OPA1-NOA1. Dati i difetti strutturali mitocondriali, abbiamo ipotizzato che l’ ablazione di NOA1 colpisca anche funzioni mitocondriali. Infatti, analisi Seahorse ha mostrato una respirazione mitocondriale compromessa in cellule mancanti NOA1, coerente con quello che era già stato pubblicato. Analisi SDS e Blue Native hanno rivelato una notevole diminuzione nei livelli delle proteine delle varie subunità di OXPHOS e un difetto nel assemblaggio dei complessi respiratori mancanti NOA1. Sorprendentemente, il numero di copie di DNA mitocondriale (mtDNA) era inalterato, mentre la riduzione della proteina non era limitata alle proteine codificate da DNA mitocondriale. Di conseguenza, si suggerisce che l'instabilità generale delle proteine OXPHOS è probabilmente dovuta all’ incapacità dei complessi della catena respiratoria ad essere assemblati correttamente, potenzialmente spiegando le disfunzioni bioenergetiche. Funzionalmente, le cellule Noa1-/- crescevano più lentamente rispetto a quelle WT con medio contenente galattosio, e dopo 2 giorni non erano in grado di tollerare deprivazione di glucosio, suggerendo un ruolo potenziale di NOA1 nella crescita cellulare dipendente da mitocondri. A causa del fatto che OPA1 è una proteina antiapoptotica e i suoi complessi sono interrotti dopo stimoli apoptotici, abbiamo voluto studiare il suo partner, NOA1, nel corso dell’ apoptosi. Come per OPA1, i complessi di NOA1 che hanno la stessa motilità elettroforetica dopo crosslink come quelli di OPA1 sono similmente interrotti su rimodellamento delle criste indotto da cBID. Oltre a ciò, durante l'apoptosi, è probabile una scissione proteolitica di NOA1, che potrebbe essere un potenziale meccanismo per l'eliminazione di complessi NOA1-OPA1 subito dopo la stimolazione della morte cellulare. In conclusione, il nostro studio ha svelato la composizione dei complessi OPA1 che vengono eliminati durante l'apoptosi. Abbiamo scoperto un crosstalk tra i principali regolatori della biogenesi delle cristae, OPA1 e Mic60 e caratterizzato il ruolo di Mic60 durante il rimodellamento apoptotico delle cristae. Inoltre, abbiamo scoperto un nuovo giocatore nella manutenzione della forma delle cristae, nel loro rimodellamento e nella respirazione mitocondriale, NOA1, che agisce nello stesso pathway di OPA1

Οι πλατωνικές απόψεις για το κάλλος και τις τέχνες στον διάλογο «Φαίδρο»

Κανένας άλλος αρχαίος Έλληνας φιλόσοφος δεν αξιολόγησε το ωραίο και την τέχνη τόσο αρνητικά όσο ο Πλάτωνας. Κανένας άλλος δεν ύμνησε το ωραίο και την τέχνη όσο αυτός. Καταδίκασε την ποίηση, πολέμησε τη ρητορική, μείωσε τον γραπτό λόγο, προσέβαλε τις τέχνες εν γένει. Ταυτόχρονα εξήρε την ποίηση, συμφιλιώθηκε με τη ρητορική, αποδέχτηκε τον γραπτό λόγο, βρήκε το νόημα των τεχνών. Οι ακραίοι ισχυρισμοί του φιλοσόφου για το ωραίο και την τέχνη γίνονται καταφανείς στον διάλογο «Φαίδρο» φέρνοντάς μας έτσι στις παρυφές όχι μόνο της αισθητικής του θεωρίας αλλά και της ευρύτερης φιλοσοφική του σκέψης. Την αντινομία αυτή των απόψεων περί τέχνης του Πλάτωνα στον διάλογο «Φαίδρο» αποσκοπεί να αναδείξει η παρούσα μελέτη. Πιο συγκεκριμένα, αρχικά παρουσιάζεται το κάλλος ως το απόλυτο φως αλλά και ως μεταμορφωτική δύναμη που μαζί με τον έρωτα μπορούν να ανάγουν την ψυχή προς το θείο. Στη συνέχεια επιδιώκεται να ερμηνευτεί ο έκδηλα αντινομικός χαρακτήρας των πλατωνικών αποφάνσεων για την ποίηση και να τονιστεί η αξία της τέχνης μόνο όταν βασίζεται στην εσωτερική σχέση με τη μεταφυσική αρχή του κόσμου. Ως εκ τούτου, διακρίνεται ο καλός από τον κακό ποιητή, ο καλός από τον κακό ρήτορα, ο καλός από τον κακό μουσικό. Ο ίδιος μάλιστα ο συγγραφέας του διαλόγου, με τη χρήση του μύθου και τη εικόνας, χωρίς να χάνει την επιστημονική του φυσιογνωμία, αποδεικνύεται γνήσιος ποιητής και ο «Φαίδρος» γνήσιο καλλιτέχνημα. Παρατηρούμε να επιτίθεται κατά της σοφιστικής ρητορικής αλλά και να εισηγείται ένα πρόγραμμα «εξυγίανσης» της ρητορικής, έτσι ώστε να καταστεί η ίδια αληθινή τέχνη. Τέλος, επιχειρείται να εξεταστεί η σχέση του προφορικού και γραπτού λόγου, να παρουσιαστεί η πολύτιμη συνδρομή του μύθου και της εικόνας στο φιλοσοφικό σύστημα του Πλάτωνα αλλά και να καταγραφούν οι απόψεις του περί μουσικής. Απώτερος στόχος της παρούσας μελέτης είναι να τονιστεί η πλατωνική άποψη ότι η τέχνη και η φιλοσοφία όχι μόνο δεν είναι ασύμβατες αλλά αν συνεπικουρήσουν θα φέρουν πιο κοντά τον άνθρωπο στη σφαίρα της αλήθειας. Η ομορφιά έτσι μπορεί να σώσει τον κόσμο και τον άνθρωπο.No ancient Greek philosopher has evaluated fine art as negatively as Plato. And no one has praised fine art as much as he did. He condemned poetry, he fought rhetoric, he belittled written speech; he insulted the arts in general. At the same time, he praised poetry, reconciled with rhetoric, accepted the written word, found the meaning of the arts. The philosopher's extreme claims to fine art are evident in the ‘Phaedrus’ dialogue, bringing us to the brink of not only his aesthetic theory but also his broader philosophical thinking. The purpose of this study is to illustrate this antithesis of Plato's views on the ‘Phaedrus’ dialogue. More specifically, beauty is initially presented as the absolute light but also as a transformative force that combined with love, ‘eros’, can raise the soul to the divine. It is then sought to interpret the manifestly opposing nature of Platonic judgments on poetry and to emphasize the value of art only when it is based on the inner relationship with the metaphysical principle of the world. Therefore, the good poet is distinguished from the bad one, the good orator from the bad, the good musician from the bad. Indeed, the author of the dialogue himself, making use of myth and imagery, without losing his scientific character, proves to be a genuine poet and ‘Phaedrus’ a genuine artist. We see him attacking the sophisticated rhetoric and suggesting a program of rhetoric "rationalization", so that it becomes true art. Finally, the study attempts to examine the relationship between oral and written speech, to present the valuable contribution of myth and imagery to Plato's philosophical system and to record his views on music. The ultimate aim of the present study is to emphasize the Platonic view that art and philosophy are not only compatible but when used together, they will bring man closer to the realm of truth. Beauty can thus save the world and man

The cell biology of mitochondrial membrane dynamics

Owing to their ability to efficiently generate ATP required to sustain normal cell function, mitochondria are often considered the \u2018powerhouses of the cell\u2019. However, our understanding of the role of mitochondria in cell biology recently expanded when we recognized that they are key platforms for a plethora of cell signalling cascades. This functional versatility is tightly coupled to constant reshaping of the cellular mitochondrial network in a series of processes, collectively referred to as mitochondrial membrane dynamics and involving organelle fusion and fission (division) as well as ultrastructural remodelling of the membrane. Accordingly, mitochondrial dynamics influence and often orchestrate not only metabolism but also complex cell signalling events, such as those involved in regulating cell pluripotency, division, differentiation, senescence and death. Reciprocally, mitochondrial membrane dynamics are extensively regulated by post-translational modifications of its machinery and by the formation of membrane contact sites between mitochondria and other organelles, both of which have the capacity to integrate inputs from various pathways. Here, we discuss mitochondrial membrane dynamics and their regulation and describe how bioenergetics and cellular signalling are linked to these dynamic changes of mitochondrial morphology

How do cytokines trigger genomic instability?

Inflammation is a double-edged sword presenting a dual effect on cancer development, from one hand promoting tumor initiation and progression and from the other hand protecting against cancer through immunosurveillance mechanisms. Cytokines are crucial components of inflammation, participating in the interaction between the cells of tumor microenvironment. A comprehensive study of the role of cytokines in the context of the inflammation-tumorigenesis interplay helps us to shed light in the pathogenesis of cancer. In this paper we focus on the role of cytokines in the development of genomic instability, an evolving hallmark of cancer. © 2012 Ioannis L. Aivaliotis et al