ATP/O ratio, reflecting efficiency of ATP synthesis, was higher in shCLSHepaRG cells (+30% p b 0.05) without modification of ATP synthesisrate (using succinate as substrate). Mechanisms involved in thesemodifications are under investigation. In conclusion, our work clearlydemonstrated, for the first time, that the reduction of cardiolipincontent regulates directly ATP synthesis in human hepatic cells.

doi:10.1016/j.bbabio.2014.05.300

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Effects of reaction probability and occupation area in the electrontransfer kinetics of the mitochondrial chain segment involvingcomplexes I, II and ubiquinone: A Monte-Carlo simulation studyJorge MartinsIBB-CBME, DCBB-FCT, Universidade do Algarve, PortugalE-mail address: jmartin@ualg.pt

The components of mitochondrial chain were conceived to movefreely and interact by random collisions, e.g. ubiquinone movingmore rapidly and shuttling electrons from complexes I and II tocomplex III [1]. The behavior of ubiquinone and cytochrome c poolsfailed to support the diffusion model of independent complexesrandomly distributed [2]. Given the molecular crowding (and likelyheterogeneous distribution of reactants), lateral diffusion is expectedto be restricted in this membrane. So, the kinetic analysis shouldtake on theories considering irregular lateral diffusion, reaction-controlled and domain-limited reactivity [1]. In this work, the effectsof reaction probability varying upon collision and two-dimensionalconcentration, involving complexes I, II and ubiquinone, are studiedusing Monte-Carlo techniques. The simulations are based on con-tinuous Pearson-type random walk [3], considering the reactants asnon-overlapping disks, whose diameters mimic the molecular areasof the lipid and proteins. The probability varied between 1 (thediffusion-control limit considers the entire perimeter as active),0.5 (half the perimeter is inactive), 0.25, 0.125 and 0.0625. Theregular lateral diffusion of ubiquinone is equivalent to phospholipidsand proteins are much less mobile (10−10 cm2/s). The stoichiometryis taken as 1:2:30, for complex I, complex II and ubiquinone,respectively. The analysis is based on the half-time for the decayof initial concentration of reactants (within μs time range). For adisk occupation area of 17.28%, bellow what is expected in vivo,the outcomes indicate obstructed diffusion effects, comparing withsimulations with overlapping particles. The results emphasize theimportance of reaction kinetics and molecular crowding in modelsof mitochondrial electron transport. This work was supported bynational Portuguese funding through FCT — Fundação para a Ciênciae a Tecnologia, project ref. PEst-OE/EQB/LA0023/2013.

References[1] E.Melo, J. Martins, Kinetics of bimolecular reactions inmodel bilayers

and biological membranes. A critical review, Biophys. Chem. 123(2006) 77–94.

[2] H. Boumans, L.A. Grivell, J.A. Berden, The respiratory chain in yeastbehaves as a single functionalunit, J. Biol. Chem.273 (1998)4872–4877

[3] J. Martins, K. Razi Naqvi, E. Melo, Kinetics of two-dimensionaldiffusion-controlled reactions: a Monte Carlo simulation of hard-disk reactants undergoing a Pearson-type random walk, J. Phys.Chem. B 104 (2000) 4986–4991.

doi:10.1016/j.bbabio.2014.05.301

S2.P14

Targetingmitochondria throughmodulation of eIF5A hypusinationprotects from anoxia-induced cell deathNicolas MelisLP2M — CNRS-UMR 7370, FranceE-mail address: nmelis@unice.fr

eIF5A, for eukaryotic initiation factor 5 A, is a highly conservedprotein throughout evolution. Firstly described as a translationinitiation factor, it became more noteworthy for its unique post-translational activation through hypusination. This modification, a4-aminobutyl moiety from spermidine transferred onto a lysyl residue,is sequentially catalyzed by two enzymatic steps involving respectivelythe deoxyhypusine synthase (DHPS) and the deoxyhypusine hydrox-ylase (DOHH). We took advantage of this unique characteristic anduse the highly specific DHPS competitive inhibitor GC7 (N-guanyl-1,7-diaminoheptane) to demonstrate an unsuspected link between eIF5Ahypusination, mitochondria activity and the cellular resistance toanoxia. To investigate the involvement of this pathway in the resistanceto low oxygen level, renal proximal tubular cells (PCT) were exposed toanoxia (b0.1% O2, 24 h). GC7 pre-treatment (30 μM) or RNA silencing-mediated inhibition of DHPS or DOHH was largely protected fromthe anoxia-induced cell death. This tolerance to anoxia is paralleledby a marked increase in glucose consumption and lactate productionreflecting a reversible metabolic shift from aerobic OXPHOS toanaerobic glycolysis, preserving the cellular energetic status. We alsostudied the effect of GC7 on mitochondrial status and showed thatGC7 induced a reversible “mitochondrial silencing” characterized by adecrease inmitochondrial potential (ΔΨm) and a drasticmitochondrialstructure remodeling associated to a down-regulation of respiratorychain complex expression. The resulting effect is a decrease in O2

requirements associated with a reduction of the deleterious reactiveoxygen species produced during anoxia. Thus, targeting mitochondriathrough modulation of eIF5A hypusination pathway may offer aninnovative therapeutic strategy for ischaemic human diseases—e.g.,stroke or myocardial infarction—or organ transplantation.

doi:10.1016/j.bbabio.2014.05.302

S2.P15

Mitochondrial nucleoid visualization in isolated pancreatic β-cellsTomas Olejara, Lukas Alanb, Monika Cahovac, Zuzana Berkovac,Frantisek Saudekc, Petr JežekdaDepartment of Membrane Transport Biophysics, Institute of Physiology,Academy of Sciences of the Czech Republic, Czech RepublicbMembrane Transport Biophysics, Institute of Physiology, AS CR, v.v.i.,Czech RepubliccInstitute of Clinical and Experimental Medicine, Czech RepublicdDepartment of Membrane Transport Biophysics, Institute of Physiology,ASCR, Czech RepublicE-mail address: olejar@biomed.cas.cz

Quality of mitochondrial (mt) network and mtDNA is widelydiscussed in relationship to reactive oxygen species (ROS) productionand development of the type 2 diabetes mellitus. Photoactivatablefluorescence proteins like Green fluorescence protein (GFP) and Eostagged to different lentiviral protein vectors are utilized to studyrespective targets in vivo. As an approach to study mt morphology andmt nucleoid distribution in isolated pancreatic β-cells, we implementedlentiviral transfection of matrix-addressed GFP (Mito-GFP) to visualizemt network and Eos-tagged Single-strand DNA binding protein-1(SSBP1-Eos) to image nucleoids. Diabetic Goto-Kakizaki (GK) rats,

Abstracts e29

respective Wistar (W) control rat, or hereditary hypertriglyceridemic(HHTg) rats with or without high sucrose diet (HSD) aged 14, 9 and3 monthswere investigated. Lentiviral particles containingMito-GFP orSSBP1-Eos were used to transfect beta cells separated by acutasefrom the isolated Langerhans islets [1]. β-cells isolated from W andHHTg (3 and 9 months) without HSD were easily susceptible for trans-duction byMito-GFP, while GK showedminimal transduction rate withfragmentedmitochondria.β-cells fromHHTg+ HSD (14 months)werenot transducible by Mito-GFP at all. Only β-cells from W and HHTg(3 and 9 months) were susceptible for transduction by SSBP1-Eos. Ourpilot study shows certain difficulties with lentiviral transduction ofMito-GFP and SSBP1-Eos in elder animals with diabetic or pre-diabeticpathology. Based on these results, certain level of relationship betweenmetabolic state in β-cells with turnover of mitochondrial and nucleoidcomponents is discussed. Supported by GACR grant no. 13-06666.

Reference[1] L. Alán, T. Špaček, J. Zelenka, J. Tauber, Z. Berková, K. Zacharovová,

et al., Assessment of mitochondrial DNA as an indicator of isletquality: an example inGoto Kakizaki rats., Transplant. Proc. 43 (2011)3281–4. http://dx.doi.org/10.1016/j.transproceed.2011.09.055.

doi:10.1016/j.bbabio.2014.05.303

S2.P16

Silencing of mitochondrial Lon protease deeply altersmitochondrial proteome and functionality in RKOcolorectal carcinoma cellsMarcello Pintia, Lara Gibellinib, Federica Boraldia, Valentina Giorgioc,Paolo Bernardic, Milena Nasib, Sara De Biasib, Paolo Pintond,Daniela Quaglinoa, Andrea CossarizzabaDepartment of Life Sciences, University of Modena and Reggio Emilia,ItalybDepartment of Surgery, Medicine, Dentistry andMorphological Sciences,University of Modena and Reggio Emilia, ItalycDepartment of Biomedical Sciences, University of Padova, ItalydDepartment of Morphology, Surgery and Experimental Medicine,University of Ferrara, ItalyE-mail address: marcello.pinti@unimore.it

Background: Lon is a nuclear-encoded, mitochondrial ATP-dependent protease that assists protein folding, degrades oxidized/damaged proteins and participates in maintaining mitochondrialDNA (mtDNA) levels. Here we show that Lon is upregulated inseveral human cancers, including the colon cancer derived cell lineRKO, and that its silencing in these cells causes profound alterationsof mitochondrial proteome and massive cell death. Materials andmethods: Lon mRNA was downregulated by RNA interference usingtwo vectors for constitutive and inducible (doxycycline-regulated)expression of Lon short hairpin RNA. Mitochondrial proteome wasanalysed by 2-dimensional gel electrophoresis and mass spectrom-etry. Mitochondrial DNA and RNA were quantified by real timePCR. Apoptosis, content of mitochondrial anion superoxide andcellular hydrogen peroxide, and mitochondrial membrane potentialwere measured by flow cytometry. Mitochondrial morphology wasanalysed by confocal and transmission electron microscopy. Resultsand discussion: Lon silencing in RKO colon cancer cells causedprofound alterations of mitochondrial proteome, and massive celldeath. Mitochondria of Lon-silenced cells displayed levels of mtDNAcomparable to those of control cells, but low levels ofmtDNA transcripts, reduced levels of several subunits of oxidative

phosphorylation complexes (complex I being the most affected), amarked reduction of oxygen consumption rate, and impairedcapability to synthetize ATP. Higher levels of hydrogen peroxideand mitochondrial superoxide anion were also observed. Mitochon-drial network appeared fragmented, with mitochondria heteroge-neous in size and shape, dilated cristae, vacuoles and electron-denseinclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, an inhibitor of Lon proteolytic activity, was able to partiallymimic Lon silencing. Conclusion: Lon is essential for maintainingmitochondrial shape and functions, and for the survival of RKO coloncancer cells.

doi:10.1016/j.bbabio.2014.05.304

S2.P17

Mitochondrial cristae remodeling in HepG2 cells adaptedto hypoxiaLydie Plecita-Hlavataa, Hana Engstovab, Lukas Alanc, Tomas Spacekb,Vendula Stradalovad, Andrea Dlaskováe, Jan Taubera, Jitka Spackovaa,Katarina Smolkovaa, Mark Lessardf, Jan Malinskyd,Joerg Bewersdorfg, Petr JežekeaInstitute of Physiology, Czech Academy of Sciences, Czech RepublicbInstitute of Physiology, Dept. 75, Prague, Czech RepubliccInstitute of Physiology, Czech Academy of Sciences v.v.i., Dept. 75,Czech RepublicdInstitute of Experimental Medicine, Czech Academy of Sciences,Czech RepubliceDepartment of Membrane Transport Biophysics, Institute of Physiology,ASCR, Czech RepublicfJackson Laboratory, United StatesgYale University, United StatesE-mail address: plecita@biomed.cas.cz

HepG2 cells cultivated in glucose media adapted to hypoxia (5%oxygen, 72 h) show metabolic shift to glycolysis at the expense ofmitochondrial bioenergetics, called Warburg effect. This is accompa-nied by a drop of mitochondrial superoxide release into the matrix.We found that mitochondrial morphology of those cells is changed,i.e., the mitochondrial intermembrane space and mostly intracristalspace are expanded, using 3D superresolution (~30 nm) BiplaneFPALM microscopy [1] in combination with Eos-conjugated markersof desired mitochondrial compartments. In parallel, cryo-preservedelectron microscopy has been performed, indicating cristae widthincrease from 18 ± 5 to 24 ± 10 nm at 5 mM glucose. The observedcristae expansion was accompanied by 20–30% downregulation ofcristae-shaping protein mitofilin and enhanced OPA1 processing intoshorter isoforms. To explain such hypoxia-adapted cristae inflation,we propose that the intracristal space volume increase releasesproton motive force control of proton pumping into expandedcristae compartments and thus diminishes superoxide formation.Supported by GACR grant nos. 13-02033 and P302/10/0346.

Reference[1] M.J. Mlodzianoski, J.M. Schreiner, S.P. Callahan, K. Smolková, A.

Dlasková, J. Šantorová, P. Ježek, J. Bewersdorf. Sample driftcorrection in 3D fluorescence photoactivation localization mi-croscopy. Opt. Express. 19 (2011) 15009–15019.

doi:10.1016/j.bbabio.2014.05.305

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