Publications by authors named "Marta Giacomello"

39 Publications

Mitochondria-rough-ER contacts in the liver regulate systemic lipid homeostasis.

Cell Rep 2021 Mar;34(11):108873

Mitochondria Biology Laboratory, Brain Research Center, Quebec, QC, Canada; Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Quebec, QC, Canada. Electronic address:

Contacts between organelles create microdomains that play major roles in regulating key intracellular activities and signaling pathways, but whether they also regulate systemic functions remains unknown. Here, we report the ultrastructural organization and dynamics of the inter-organellar contact established by sheets of curved rough endoplasmic reticulum closely wrapped around the mitochondria (wrappER). To elucidate the in vivo function of this contact, mouse liver fractions enriched in wrappER-associated mitochondria are analyzed by transcriptomics, proteomics, and lipidomics. The biochemical signature of the wrappER points to a role in the biogenesis of very-low-density lipoproteins (VLDL). Altering wrappER-mitochondria contacts curtails VLDL secretion and increases hepatic fatty acids, lipid droplets, and neutral lipid content. Conversely, acute liver-specific ablation of Mttp, the most upstream regulator of VLDL biogenesis, recapitulates this hepatic dyslipidemia phenotype and promotes remodeling of the wrappER-mitochondria contact. The discovery that liver wrappER-mitochondria contacts participate in VLDL biology suggests an involvement of inter-organelle contacts in systemic lipid homeostasis.
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http://dx.doi.org/10.1016/j.celrep.2021.108873DOI Listing
March 2021

Deletion of the mitochondria-shaping protein Opa1 during early thymocyte maturation impacts mature memory T cell metabolism.

Cell Death Differ 2021 Jul 1;28(7):2194-2206. Epub 2021 Mar 1.

Veneto Institute of Molecular Medicine, Padua, Italy.

Optic atrophy 1 (OPA1), a mitochondria-shaping protein controlling cristae biogenesis and respiration, is required for memory T cell function, but whether it affects intrathymic T cell development is unknown. Here we show that OPA1 is necessary for thymocyte maturation at the double negative (DN)3 stage when rearrangement of the T cell receptor β (Tcrβ) locus occurs. By profiling mitochondrial function at different stages of thymocyte maturation, we find that DN3 cells rely on oxidative phosphorylation. Consistently, Opa1 deletion during early T cell development impairs respiration of DN3 cells and reduces their number. Opa1-deficient DN3 cells indeed display stronger TCR signaling and are more prone to cell death. The surviving Opa1 thymocytes that reach the periphery as mature T cells display an effector memory phenotype even in the absence of antigenic stimulation but are unable to generate metabolically fit long-term memory T cells. Thus, mitochondrial defects early during T cell development affect mature T cell function.
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http://dx.doi.org/10.1038/s41418-021-00747-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8257785PMC
July 2021

A new target for an old DUB: UCH-L1 regulates mitofusin-2 levels, altering mitochondrial morphology, function and calcium uptake.

Redox Biol 2020 10 7;37:101676. Epub 2020 Aug 7.

Obesity Research Center, Molecular Medicine, Boston University School of Medicine, Boston, MA, 02111, USA; UCLA Section of Endocrinology, Department of Medicine, David Geffen School of Medicine, UCLA, CA, 9095-7073, USA. Electronic address:

UCH-L1 is a deubiquitinating enzyme (DUB), highly abundant in neurons, with a sub-cellular localization dependent on its farnesylation state. Despite UCH-L1's association with familial Parkinson's Disease (PD), the effects on mitochondrial bioenergetics and quality control remain unexplored. Here we investigated the role of UCHL-1 in mitochondrial dynamics and bioenergetics. We demonstrate that knock-down (KD) of UCH-L1 in different cell lines reduces the levels of the mitochondrial fusion protein Mitofusin-2, but not Mitofusin-1, resulting in mitochondrial enlargement and disruption of the tubular network. This was associated with lower tethering between mitochondria and the endoplasmic reticulum, consequently altering mitochondrial calcium uptake. Respiratory function was also altered, as UCH-L1 KD cells displayed higher proton leak and maximum respiratory capacity. Conversely, overexpression of UCH-L1 increased Mfn2 levels, an effect dramatically enhanced by the mutation of the farnesylation site (C220S), which drives UCH-L1 binding to membranes. These data indicate that the soluble cytosolic form of UCH-L1 regulates Mitofusin-2 levels and mitochondrial function. These effects are biologically conserved, since knock-down of the corresponding UCH-L1 ortholog in D. melanogaster reduces levels of the mitofusin ortholog Marf and also increases mitochondrial respiratory capacity. We thus show that Mfn-2 levels are directly affected by UCH-L1, demonstrating that the mitochondrial roles of DUBs go beyond controlling mitophagy rates.
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http://dx.doi.org/10.1016/j.redox.2020.101676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509235PMC
October 2020

Protein electrostatics: From computational and structural analysis to discovery of functional fingerprints and biotechnological design.

Comput Struct Biotechnol J 2020 30;18:1774-1789. Epub 2020 Jun 30.

Synthetic Biology and Biotechnology Unit, Department of Biology, University of Padua, Italy.

Computationally driven engineering of proteins aims to allow them to withstand an extended range of conditions and to mediate modified or novel functions. Therefore, it is crucial to the biotechnological industry, to biomedicine and to afford new challenges in environmental sciences, such as biocatalysis for green chemistry and bioremediation. In order to achieve these goals, it is important to clarify molecular mechanisms underlying proteins stability and modulating their interactions. So far, much attention has been given to hydrophobic and polar packing interactions and stability of the protein core. In contrast, the role of electrostatics and, in particular, of surface interactions has received less attention. However, electrostatics plays a pivotal role along the whole life cycle of a protein, since early folding steps to maturation, and it is involved in the regulation of protein localization and interactions with other cellular or artificial molecules. Short- and long-range electrostatic interactions, together with other forces, provide essential guidance cues in molecular and macromolecular assembly. We report here on methods for computing protein electrostatics and for individual or comparative analysis able to sort proteins by electrostatic similarity. Then, we provide examples of electrostatic analysis and fingerprints in natural protein evolution and in biotechnological design, in fields as diverse as biocatalysis, antibody and nanobody engineering, drug design and delivery, molecular virology, nanotechnology and regenerative medicine.
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http://dx.doi.org/10.1016/j.csbj.2020.06.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355722PMC
June 2020

Chemical Modulation of Mitochondria-Endoplasmic Reticulum Contact Sites.

Cells 2020 07 7;9(7). Epub 2020 Jul 7.

Department of Biology, University of Padua, Via U. Bassi 58/B, 35121 Padua, Italy.

Contact sites between mitochondria and endoplasmic reticulum (ER) are points in which the two organelles are in close proximity. Due to their structural and functional complexity, their exploitation as pharmacological targets has never been considered so far. Notwithstanding, the number of compounds described to target proteins residing at these interfaces either directly or indirectly is rising. Here we provide original insight into mitochondria-ER contact sites (MERCs), with a comprehensive overview of the current MERCs pharmacology. Importantly, we discuss the considerable potential of MERCs to become a druggable target for the development of novel therapeutic strategies.
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http://dx.doi.org/10.3390/cells9071637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408517PMC
July 2020

Interaction Between Mitochondrial DNA Variants and Mitochondria/Endoplasmic Reticulum Contact Sites: A Perspective Review.

DNA Cell Biol 2020 Aug 26;39(8):1431-1443. Epub 2020 Jun 26.

Department of Biology, University of Padova, Padova, Italy.

Mitochondria contain their own genome, mitochondrial DNA (mtDNA), essential to support their fundamental intracellular role in ATP production and other key metabolic and homeostatic pathways. Mitochondria are highly dynamic organelles that communicate with all the other cellular compartments, through sites of high physical proximity. Among all, their crosstalk with the endoplasmic reticulum (ER) appears particularly important as its derangement is tightly implicated with several human disorders. Population-specific mtDNA variants clustered in defining the haplogroups have been shown to exacerbate or mitigate these pathological conditions. The exact mechanisms of the mtDNA background-modifying effect are not completely clear and a possible explanation is the outcome of mitochondrial efficiency on retrograde signaling to the nucleus. However, the possibility that different haplogroups shape the proximity and crosstalk between mitochondria and the ER has never been proposed neither investigated. In this study, we pose and discuss this question and provide preliminary data to answer it. Besides, we also address the possibility that single, disease-causing mtDNA point mutations may act also by reshaping organelle communication. Overall, this perspective review provides a theoretical platform for future studies on the interaction between mtDNA variants and organelle contact sites.
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http://dx.doi.org/10.1089/dna.2020.5614DOI Listing
August 2020

Mitochondrial Function in Enamel Development.

Front Physiol 2020 29;11:538. Epub 2020 May 29.

College of Dentistry, Department of Molecular Pathobiology, New York University, New York, NY, United States.

Enamel is the most calcified tissue in vertebrates. Enamel formation and mineralization is a two-step process that is mediated by ameloblast cells during their secretory and maturation stages. In these two stages, ameloblasts are characterized by different morphology and function, which is fundamental for proper mineral growth in the extracellular space. Ultrastructural studies have shown that the mitochondria in these cells localize to different subcellular regions in both stages. However, limited knowledge is available on the role/s of mitochondria in enamel formation. To address this issue, we analyzed mitochondrial biogenesis and respiration, as well as the redox status of rat primary enamel cells isolated from the secretory and maturation stages. We show that maturation stage cells have an increased expression of PGC1α, a marker of mitochondrial biogenesis, and of components of the electron transport chain. Oxygen consumption rate (OCR), a proxy for mitochondrial function, showed a significant increase in oxidative phosphorylation during the maturation stage, promoting ATP production. The GSH/GSSG ratio was lower in the maturation stage, indicative of increased oxidation. Because higher oxidative phosphorylation can lead to higher ROS production, we tested if ROS affected the expression of and genes that are essential for enamel formation. The ameloblast cell line LS8 treated with HO to promote ROS elicited significant expression changes in and . Our data highlight important metabolic and physiological differences across the two enamel stages, with higher ATP levels in the maturation stage indicative of a higher energy demand. Besides these metabolic shifts, it is likely that the enhanced ETC function results in ROS-mediated transcriptional changes.
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http://dx.doi.org/10.3389/fphys.2020.00538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274036PMC
May 2020

Developmental and Tumor Angiogenesis Requires the Mitochondria-Shaping Protein Opa1.

Cell Metab 2020 05 20;31(5):987-1003.e8. Epub 2020 Apr 20.

Department of Biology, University of Padova, Via U. Bassi 58B, 35121 Padova, Italy; Veneto Institute of Molecular Medicine, Via Orus 2, 35129 Padova, Italy. Electronic address:

While endothelial cell (EC) function is influenced by mitochondrial metabolism, the role of mitochondrial dynamics in angiogenesis, the formation of new blood vessels from existing vasculature, is unknown. Here we show that the inner mitochondrial membrane mitochondrial fusion protein optic atrophy 1 (OPA1) is required for angiogenesis. In response to angiogenic stimuli, OPA1 levels rapidly increase to limit nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling, ultimately allowing angiogenic genes expression and angiogenesis. Endothelial Opa1 is indeed required in an NFκB-dependent pathway essential for developmental and tumor angiogenesis, impacting tumor growth and metastatization. A first-in-class small molecule-specific OPA1 inhibitor confirms that EC Opa1 can be pharmacologically targeted to curtail tumor growth. Our data identify Opa1 as a crucial component of physiological and tumor angiogenesis.
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http://dx.doi.org/10.1016/j.cmet.2020.04.007DOI Listing
May 2020

The cell biology of mitochondrial membrane dynamics.

Nat Rev Mol Cell Biol 2020 04 18;21(4):204-224. Epub 2020 Feb 18.

Department of Biology, University of Padua, Padua, Italy.

Owing to their ability to efficiently generate ATP required to sustain normal cell function, mitochondria are often considered the 'powerhouses of the cell'. 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.
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http://dx.doi.org/10.1038/s41580-020-0210-7DOI Listing
April 2020

MFN2 mutations in Charcot-Marie-Tooth disease alter mitochondria-associated ER membrane function but do not impair bioenergetics.

Hum Mol Genet 2019 06;28(11):1782-1800

Department of Biology, University of Padova 35131, Italy.

Charcot-Marie-Tooth disease (CMT) type 2A is a form of peripheral neuropathy, due almost exclusively to dominant mutations in the nuclear gene encoding the mitochondrial protein mitofusin-2 (MFN2). However, there is no understanding of the relationship of clinical phenotype to genotype. MFN2 has two functions: it promotes inter-mitochondrial fusion and mediates endoplasmic reticulum (ER)-mitochondrial tethering at mitochondria-associated ER membranes (MAM). MAM regulates a number of key cellular functions, including lipid and calcium homeostasis, and mitochondrial behavior. To date, no studies have been performed to address whether mutations in MFN2 in CMT2A patient cells affect MAM function, which might provide insight into pathogenesis. Using fibroblasts from three CMT2AMFN2 patients with different mutations in MFN2, we found that some, but not all, examined aspects of ER-mitochondrial connectivity and of MAM function were indeed altered, and correlated with disease severity. Notably, however, respiratory chain function in those cells was unimpaired. Our results suggest that CMT2AMFN2 is a MAM-related disorder but is not a respiratory chain-deficiency disease. The alterations in MAM function described here could also provide insight into the pathogenesis of other forms of CMT.
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http://dx.doi.org/10.1093/hmg/ddz008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522073PMC
June 2019

Shipping Calpastatin to the Rescue: Prevention of Neuromuscular Degeneration through Mitofusin 2.

Cell Metab 2018 10;28(4):536-538

Department of Biology, University of Padua, Via U Bassi 58B, 35121 Padua, Italy; Venetian Institute of Molecular Medicine, Via Orus 2, 35129 Padua, Italy. Electronic address:

How neuromuscular junctions (NMJs) are lost in disease and aging is unclear. Recently in Cell Metabolism, Wang et al. (2018) discovered that endoplasmic reticulum-mitochondria tethering by Mitofusin 2 is required to organize a cleft between these two organelles, which, like a lorry, traffics down the axon to distribute calpastatin to terminals where it blocks NMJ degradation.
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http://dx.doi.org/10.1016/j.cmet.2018.09.017DOI Listing
October 2018

Regulation of ER-mitochondria contacts by Parkin via Mfn2.

Pharmacol Res 2018 12 13;138:43-56. Epub 2018 Sep 13.

Department of Biology, University of Padova, Padova, Italy; Fondazione Ospedale San Camillo, IRCCS, Lido di Venezia, Venezia, Italy. Electronic address:

Parkin, an E3 ubiquitin ligase and a Parkinson's disease (PD) related gene, translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy. Mitochondrial pro-fusion protein Mitofusins (Mfn1 and Mfn2) were found to be a target for Parkin mediated ubiquitination. Mfns are transmembrane GTPase embedded in the outer membrane of mitochondria, which are required on adjacent mitochondria to mediate fusion. In mammals, Mfn2 also forms complexes that are capable of tethering mitochondria to endoplasmic reticulum (ER), a structural feature essential for mitochondrial energy metabolism, calcium (Ca) transfer between the organelles and Ca dependent cell death. Despite its fundamental physiological role, the molecular mechanisms that control ER-mitochondria cross talk are obscure. Ubiquitination has recently emerged as a powerful tool to modulate protein function, via regulation of protein subcellular localization and protein ability to interact with other proteins. Ubiquitination is also a reversible mechanism, which can be actively controlled by opposing ubiquitination-deubiquitination events. In this work we found that in Parkin deficient cells and parkin mutant human fibroblasts, the tether between ER and mitochondria is decreased. We identified the site of Parkin dependent ubiquitination and showed that the non-ubiquitinatable Mfn2 mutant fails to restore ER-mitochondria physical and functional interaction. Finally, we took advantage of an established in vivo model of PD to demonstrate that manipulation of ER-mitochondria tethering by expressing an ER-mitochondria synthetic linker is sufficient to rescue the locomotor deficit associated to an in vivo Drosophila model of PD.
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http://dx.doi.org/10.1016/j.phrs.2018.09.006DOI Listing
December 2018

The INs and OUTs of mitofusins.

J Cell Biol 2018 02 18;217(2):439-440. Epub 2018 Jan 18.

Department of Biology, University of Padua, Padua, Italy

Mitofusins are outer membrane proteins essential for mitochondrial fusion. Their accepted topology posits that both N and C termini face the cytoplasm. In this issue, Mattie et al. (2018. https://doi.org/10.1083/jcb.201611194) demonstrate instead that their C termini reside in the intermembrane space. These findings call for a revision of the current models of mitochondrial fusion.
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http://dx.doi.org/10.1083/jcb.201801042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800822PMC
February 2018

Protein Localization at Mitochondria-ER Contact Sites in Basal and Stress Conditions.

Front Cell Dev Biol 2017 12;5:107. Epub 2017 Dec 12.

Department of Biology, University of Padova, Padova, Italy.

Mitochondria-endoplasmic reticulum (ER) contacts (MERCs) are sites at which the outer mitochondria membrane and the Endoplasmic Reticulum surface run in parallel at a constant distance. The juxtaposition between these organelles determines several intracellular processes such as to name a few, Ca and lipid homeostasis or autophagy. These specific tasks can be exploited thanks to the enrichment (or re-localization) of dedicated proteins at these interfaces. Recent proteomic studies highlight the tissue specific composition of MERCs, but the overall mechanisms that control MERCs plasticity remains unclear. Understanding how proteins are targeted at these sites seems pivotal to clarify such contextual function of MERCs. This review aims to summarize the current knowledge on protein localization at MERCs and the possible contribution of the mislocalization of MERCs components to human disorders.
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http://dx.doi.org/10.3389/fcell.2017.00107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733094PMC
December 2017

SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition.

Cell Death Differ 2018 06 11;25(6):1131-1145. Epub 2017 Dec 11.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8-10 nm) and wide (40-50 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER-mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER-mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses of organelle juxtaposition in the whole animal.
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http://dx.doi.org/10.1038/s41418-017-0033-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988678PMC
June 2018

The prion protein regulates glutamate-mediated Ca entry and mitochondrial Ca accumulation in neurons.

J Cell Sci 2017 Aug 12;130(16):2736-2746. Epub 2017 Jul 12.

Department of Biomedical Science, University of Padova, 35131 Padova, Italy

The cellular prion protein (PrP) whose conformational misfolding leads to the production of deadly prions, has a still-unclarified cellular function despite decades of intensive research. Following our recent finding that PrP limits Ca entry via store-operated Ca channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca probes and expressing, or not expressing, PrP Our investigation demonstrated that PrP downregulates Ca entry through each specific agonist-stimulated iGluR and after stimulation by glutamate. We found that, although PrP-knockout (KO) mitochondria were displaced from the plasma membrane, glutamate addition resulted in a higher mitochondrial Ca uptake in PrP-KO neurons than in their PrP-expressing counterpart. This was because the increased Ca entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca-induced Ca release via ryanodine receptor channels. These data thus suggest that PrP takes part in the cell apparatus controlling Ca homeostasis, and that PrP is involved in protecting neurons from toxic Ca overloads.
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http://dx.doi.org/10.1242/jcs.196972DOI Listing
August 2017

L-OPA1 regulates mitoflash biogenesis independently from membrane fusion.

EMBO Rep 2017 03 7;18(3):451-463. Epub 2017 Feb 7.

Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland

Mitochondrial flashes mediated by optic atrophy 1 (OPA1) fusion protein are bioenergetic responses to stochastic drops in mitochondrial membrane potential (Δψ) whose origin is unclear. Using structurally distinct genetically encoded pH-sensitive probes, we confirm that flashes are matrix alkalinization transients, thereby establishing the pH nature of these events, which we renamed "mitopHlashes". Probes located in cristae or intermembrane space as verified by electron microscopy do not report pH changes during Δψ drops or respiratory chain inhibition. ablation does not alter Δψ fluctuations but drastically decreases the efficiency of mitopHlash/Δψ coupling, which is restored by re-expressing fusion-deficient OPA1 and preserved in cells lacking the outer-membrane fusion proteins MFN1/2 or the OPA1 proteases OMA1 and YME1L, indicating that mitochondrial membrane fusion and OPA1 proteolytic processing are dispensable. pH/Δψ uncoupling occurs early during staurosporine-induced apoptosis and is mitigated by OPA1 overexpression, suggesting that OPA1 maintains mitopHlash competence during stress conditions. We propose that OPA1 stabilizes respiratory chain supercomplexes in a conformation that enables respiring mitochondria to compensate a drop in Δψ by an explosive matrix pH flash.
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http://dx.doi.org/10.15252/embr.201642931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331265PMC
March 2017

Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum-mitochondria tether.

Proc Natl Acad Sci U S A 2016 10 19;113(40):11249-11254. Epub 2016 Sep 19.

Department of Biology, University of Padua, 35121 Padua, Italy; Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine, 35129 Padua, Italy;

The discovery of the multiple roles of mitochondria-endoplasmic reticulum (ER) juxtaposition in cell biology often relied upon the exploitation of Mitofusin (Mfn) 2 as an ER-mitochondria tether. However, this established Mfn2 function was recently questioned, calling for a critical re-evaluation of Mfn2's role in ER-mitochondria cross-talk. Electron microscopy and fluorescence-based probes of organelle proximity confirmed that ER-mitochondria juxtaposition was reduced by constitutive or acute Mfn2 deletion. Functionally, mitochondrial uptake of Ca released from the ER was reduced following acute Mfn2 ablation, as well as in Mfn2 cells overexpressing the mitochondrial calcium uniporter. Mitochondrial Ca uptake rate and extent were normal in isolated Mfn2 liver mitochondria, consistent with the finding that acute or chronic Mfn2 ablation or overexpression did not alter mitochondrial calcium uniporter complex component levels. Hence, Mfn2 stands as a bona fide ER-mitochondria tether whose ablation decreases interorganellar juxtaposition and communication.
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http://dx.doi.org/10.1073/pnas.1606786113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056088PMC
October 2016

(Neuro)degenerated Mitochondria-ER contacts.

Biochem Biophys Res Commun 2017 02 11;483(4):1096-1109. Epub 2016 Jul 11.

Venetian Institute of Molecular Medicine, Via Orus 2, 35129, Padova, Italy; Department of Biology, University of Padova, Via U. Bassi 58B, 35121, Padova, Italy. Electronic address:

In the last years, a considerable amount of experimental evidence has highlighted the association between neurodegenerative disorders (NDD) and the biology of mitochondria-Endoplasmic Reticulum contacts (MERCs). In this review, we summarize the most recent findings on this topic. We underline that dysregulation of MERCs can contribute to the neurodegenerative process either by altering directly the functionality of neurons and their response to stress stimuli and metabolic shifts or by indirectly influencing the neuroinflammatory response that accompanies NDD. Our overview of the current literature suggest that defective MERCs could be a common determinant to the "hypergeneration" and "neurodegeneration" programs, leading respectively to tumours and NDD.
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http://dx.doi.org/10.1016/j.bbrc.2016.07.056DOI Listing
February 2017

Interplay between hepatic mitochondria-associated membranes, lipid metabolism and caveolin-1 in mice.

Sci Rep 2016 06 6;6:27351. Epub 2016 Jun 6.

Integrin Signaling Laboratory, Cell &Developmental Biology Area, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain.

The mitochondria-associated membrane (MAM) is a specialized subdomain of the endoplasmic reticulum (ER) which acts as an intracellular signaling hub. MAM dysfunction has been related to liver disease. We report a high-throughput mass spectrometry-based proteomics characterization of MAMs from mouse liver, which portrays them as an extremely complex compartment involved in different metabolic processes, including steroid metabolism. Interestingly, we identified caveolin-1 (CAV1) as an integral component of hepatic MAMs, which determine the relative cholesterol content of these ER subdomains. Finally, a detailed comparative proteomics analysis between MAMs from wild type and CAV1-deficient mice suggests that functional CAV1 contributes to the recruitment and regulation of intracellular steroid and lipoprotein metabolism-related processes accrued at MAMs. The potential impact of these novel aspects of CAV1 biology on global cell homeostasis and disease is discussed.
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http://dx.doi.org/10.1038/srep27351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894368PMC
June 2016

Calcium Handling by Endoplasmic Reticulum and Mitochondria in a Cell Model of Huntington's Disease.

PLoS Curr 2016 Jan 6;8. Epub 2016 Jan 6.

Biomedical Sciences, University of Padova, Padova, Italy; Venetian Institute of Molecular Medicine, Padova, Italy.

Huntington disease (HD) is caused by the CAG (Q) expansion in exon 1 of the IT15 gene encoding a polyglutamine (poly-Q) stretch of the Huntingtin protein (Htt). In the wild type protein, the repeats specify a stretch of up 34 Q in the N-terminal portion of Htt. In the pathological protein (mHtt) the poly-Q tract is longer. Proteolytic cleavage of the protein liberates an N-terminal fragment containing the expanded poly-Q tract becomes harmful to cells, in particular to striatal neurons. The fragments cause the transcriptional dysfunction of genes that are essential for neuronal survival. Htt, however, could also have non-transcriptional effects, e.g. it could directly alter Ca2+ homeostasis and/or mitochondrial morphology and function. Ca2+ dyshomeostasis and mitochondrial dysfunction are considered important in the molecular aetiology of the disease. Here we have analyzed the effect of the overexpression of Htt fragments (18Q, wild type form, wtHtt and 150Q mutated form, mHtt) on Ca2+ homeostasis in striatal neuronal precursor cells (Q7/7). We have found that the transient overexpression of the Htt fragments increases Ca2+ transients in the mitochondria of cells stimulated with Ca2+-mobilizing agonists. The bulk Ca2+ transients in the cytosol were unaffected, but the Ca2+ content of the endoplasmic reticulum was significantly decreased in the case of mHtt expression. To rule out possible transcriptional effects due to the presence of mHtt, we have measured the mRNA level of a subunit of the respiratory chain complex II, whose expression is commonly altered in many HD models. No effects on the mRNA level was found suggesting that, in our experimental condition, transcriptional action of Htt is not occurring and that the effects on Ca2+ homeostasis were dependent to non-transcriptional mechanisms.
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http://dx.doi.org/10.1371/currents.hd.37fcb1c9a27503dc845594ee4a7316c3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720599PMC
January 2016

Nicotine mediates oxidative stress and apoptosis through cross talk between NOX1 and Bcl-2 in lung epithelial cells.

Free Radic Biol Med 2014 Nov 20;76:173-84. Epub 2014 Aug 20.

Department of Pediatrics, Children's Hospital, 1211 Geneva, Switzerland; Department of Pathology and Immunology, Medical School, and University of Geneva, Geneva, Switzerland. Electronic address:

Nicotine contributes to the onset and progression of several pulmonary diseases. Among the various pathophysiological mechanisms triggered by nicotine, oxidative stress and cell death are reported in several cell types. We found that chronic exposure to nicotine (48h) induced NOX1-dependent oxidative stress and apoptosis in primary pulmonary cells. In murine (MLE-12) and human (BEAS-2B) lung epithelial cell lines, nicotine acted as a sensitizer to cell death and synergistically enhanced apoptosis when cells were concomitantly exposed to hyperoxia. The precise signaling pathway was investigated in MLE-12 cells in which NOX1 was abrogated by a specific inhibitor or stably silenced by shRNA. In the early phase of exposure (1h), nicotine mediated intracellular Ca(2+) fluxes and activation of protein kinase C, which in its turn activated NOX1, leading to cellular and mitochondrial oxidative stress. The latter triggered the intrinsic apoptotic machinery by modulating the expression of Bcl-2 and Bax. Overexpression of Bcl-2 completely prevented nicotine's detrimental effects, suggesting Bcl-2as a downstream key regulator in nicotine/NOX1-induced cell damage. These results suggest that NOX1 is a major contributor to the generation of intracellular oxidative stress induced by nicotine and might be an important molecule to target in nicotine-related lung pathologies.
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http://dx.doi.org/10.1016/j.freeradbiomed.2014.08.002DOI Listing
November 2014

The plasma membrane calcium pump: new ways to look at an old enzyme.

J Biol Chem 2014 Apr 25;289(15):10261-10268. Epub 2014 Feb 25.

Venetian Institute of Molecular Medicine, University of Padova, 35129 Padova, Italy. Electronic address:

The three-dimensional structure of the PMCA pump has not been solved, but its basic mechanistic properties are known to repeat those of the other Ca(2+) pumps. However, the pump also has unique properties. They concern essentially its numerous regulatory mechanisms, the most important of which is the autoinhibition by its C-terminal tail. Other regulatory mechanisms involve protein kinases and the phospholipids of the membrane in which the pump is embedded. Permanent activation of the pump, e.g. by calmodulin, is physiologically as harmful to cells as its absence. The concept is now emerging that the global control of cell Ca(2+) may not be the main function of the pump; in some cell types, it could even be irrelevant. The main pump role would be the regulation of Ca(2+) in cell microdomains in which the pump co-segregates with partners that modulate the Ca(2+) message and transduce it to important cell functions.
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http://dx.doi.org/10.1074/jbc.O114.555565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036151PMC
April 2014

Mitochondrial Ca2+-handling in fast skeletal muscle fibers from wild type and calsequestrin-null mice.

PLoS One 2013 3;8(10):e74919. Epub 2013 Oct 3.

Department of Biomedical Sciences and Interuniversity Institute of Myology (IIM), University of Padova, Padua, Italy.

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca(2+)-binding protein inside SR, calsequestrin. Mitochondrial free Ca(2+)-concentration ([Ca(2+)]mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca(2+) release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca(2+)]mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca(2+) concentration. The rise in [Ca(2+)]mito during electrical stimulation occurred in 20-30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s(-1). Accordingly, frequency-dependent increase in [Ca(2+)]mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca(2+)]mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca(2+)]mito transients were increased by inhibition of mitochondrial Na(+)/Ca(2+) exchanger (mNCX). These results provide direct evidence for fast Ca(2+) accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca(2+)-handling and a dependence of mitochondrial Ca(2+)-handling on intracellular (SR) and external Ca(2+) stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca(2+)-leakage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074919PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789688PMC
June 2014

OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange.

EMBO J 2013 Jul 28;32(13):1927-40. Epub 2013 May 28.

Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion-free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro-fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro-fission Dynamin-related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria.
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http://dx.doi.org/10.1038/emboj.2013.124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3981180PMC
July 2013

Neuronal Ca(2+) dyshomeostasis in Huntington disease.

Prion 2013 Jan-Feb;7(1):76-84. Epub 2013 Jan 1.

University of Geneva, Geneva, Switzerland.

The expansion of the N-terminal poly-glutamine tract of the huntingtin (Htt) protein is responsible for Huntington disease (HD). A large number of studies have explored the neuronal phenotype of HD, but the molecular aethiology of the disease is still very poorly understood. This has hampered the development of an appropriate therapeutical strategy to at least alleviate its symptoms. In this short review, we have focused our attention on the alteration of a specific cellular mechanism common to all HD models, either genetic or induced by treatment with 3-NPA, i.e. the cellular dyshomeostasis of Ca(2+). We have highlighted the direct and indirect (i.e. transcriptionally mediated) effects of mutated Htt on the maintenance of the intracellular Ca(2+) balance, the correct modulation of which is fundamental to cell survival and the disturbance of which plays a key role in the death of the cell.
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http://dx.doi.org/10.4161/pri.23581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609054PMC
August 2013

Plasma membrane calcium ATPases and related disorders.

Int J Biochem Cell Biol 2013 Mar 4;45(3):753-62. Epub 2012 Oct 4.

Venetian Institute of Molecular Medicine, University of Padova, Padua, Italy.

The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia.
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http://dx.doi.org/10.1016/j.biocel.2012.09.016DOI Listing
March 2013

Estradiol effects on intracellular Ca(2+) homeostasis in bovine brain-derived endothelial cells.

Cell Tissue Res 2012 Oct 20;350(1):109-18. Epub 2012 Jul 20.

Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020 Legnaro-Agripolis, Italy.

Estrogens diversely affect various physiological processes by genomic or non-genomic mechanisms, in both excitable and non-excitable cells. Additional to the trophic effects of estrogens promoting cell growth and differentiation, recent experimental evidence highlights their involvement in the regulation of intracellular Ca(2+) homeostasis. The effects of estrogens on excitable cells are well documented. However, these steroids also influence numerous physiological events in non-excitable cells, such as fibroblasts or vascular endothelial cells. We have focused our attention on an immortalized endothelial-like cell line derived from fetal bovine cerebellum. Estradiol (E(2)) effects on intracellular Ca(2+) homeostasis were tested by varying the exposure time to the hormone (8, 24, 48 h). Calcium measurements were performed with genetically encoded Ca(2+) probes (Cameleons) targeted to the main subcellular compartments involved in intracellular Ca(2+) homeostasis (cytosol, endoplasmic reticulum, mitochondria). Mitochondrial Ca(2+) uptake significantly decreased after 48-h exposure to E(2), whereas cytosolic and endoplasmic reticulum responses were unaffected. The effect of E(2) on mitochondrial Ca(2+) handling was blocked by ICI 182,780, a pure estrogen receptor antagonist, suggesting that the effect was estrogen-receptor-mediated. To evaluate whether the decrease of Ca(2+) uptake affected mitochondrial membrane potential (ΔΨm), cells were monitored in the presence of tetra-methyl-rhodamine-methylester; no significant changes were seen between cells treated with E(2) and controls. To investigate a mechanism of action, we assessed the possibile involvement of the permeability transition pore (PTP), an inner mitochondrial membrane channel influencing energy metabolism and cell viability. We treated cells with CyclosporinA (CsA), which binds to the matrix chaperone cyclophilin-D and regulates PTP opening. CsA reversed the effects of a 48-h treatment with E(2), suggesting a possible transcriptional modulation of proteins involved in the mitochondrial permeability transition process.
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http://dx.doi.org/10.1007/s00441-012-1460-2DOI Listing
October 2012

Hair cells, plasma membrane Ca²⁺ ATPase and deafness.

Int J Biochem Cell Biol 2012 May 13;44(5):679-83. Epub 2012 Feb 13.

Venetian Institute of Molecular Medicine, Via G. Orus, 2, 35129 Padua, Italy.

Hearing relies on the ability of the inner ear to convert sound waves into electrical signals. The main actors in this process are hair cells. Their stereocilia contain a number of specific proteins and a scaffold of actin molecules. They are organized in bundles by tip-link filaments composed of cadherin 23 and protocadherin 15. The bundle is deflected by sound waves leading to the opening of mechano-transduction channels and to the influx of K(+) and Ca(2+) into the stereocilia. Cadherin 23 and the plasma membrane calcium ATPase isoform 2 (PMCA2) are defective in human and murine cases of deafness. While the involvement of cadherin 23 in deafness/hearing could be expected due to its structural role in the tip-links, that of PMCA2 has been discovered only recently. This review will summarize the structural and functional characteristics of hair cells, focusing on the proteins whose mutations may lead to a deafness phenotype.
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http://dx.doi.org/10.1016/j.biocel.2012.02.006DOI Listing
May 2012
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