Targeting Mcl-1 and Bak as a Therapeutic Tool to Selectively Induce Apoptosis in Hepaptocellualr Carcinoma Wednesday, February 6, 2013 655a Mitochondria in Cell Life and Death 3363-Pos Board B518 A Step Forwardin Understanding the Mechanismof VDAC Voltage-Gating Oscar Teijido Hermida1, Shay Rappaport1, Rachna Ujwal2, Jeff Abramson2, Vicente M. Aguilella3, Sergey M. Bezrukov1, Tatiana K. Rostovtseva1. 1NICHD, National Institutes of Health (NIH), Bethesda, MD, USA, 2David Geffen School of Medicine, UCLA, Los Angeles, CA, USA, 3 Universitat Jaume I, Castello de la Plana, Spain. The voltage-dependent anion channel (VDAC) governs the exchange of ions and metabolites between the mitochondria and the rest of the cell. In its open state VDAC exhibits high conductance and selectivity for anions that facilitates the passage of ADP, ATP, and other metabolites. At increased voltages (>30mV) VDAC switches to lower conducting states, termed as ‘‘closed’’ states. Closed states are cation-selective and impermeable for ATP. The voltage-induced transition from the open to closed states is referred to as voltage-gating. Although it is well established that VDAC voltage-gating in- volves large structural rearrangements, the precise molecular mechanism of this process is still under debate. We investigated VDAC voltage-gating by sys- tematically titrating VDAC charge residues and by using thermodynamic and kinetic approaches to study opening and closing of the channel. All the models proposed so far agree that N-terminal region plays a key role in VDAC voltage- gating. According to the original idea, the N-terminal region is a part of a mo- bile voltage sensor domain, which slides in and out of the channel lumen in re- sponse to the applied voltage. The alternative models consider independent movement of the N-terminal region upon gating. In order to test the role of VDAC N-terminal region in voltage-gating, we engineered a double Cys mu- tant of murine VDAC1 that cross-links the a-helix to the b-strand 11 of the pore wall. The cross-linked VDAC1 reconstituted into planar lipid membranes exhibited typical voltage gating, which suggests that the N-terminal a-helix is located inside the pore of VDAC in the open state and remains associated with the pore wall during voltage gating. Our findings support a model where b-bar- rel is not rigid but undergoes a conformational change that leads to a partial constriction upon transition to the closed states. 3364-Pos Board B519 Novel Mechanism of Mitochondrial Respiration Control through Competition between Hexokinase-2 and Tubulin for VDAC Binding Kely L. Sheldon1, Coert J. Zuurbier2, Sergey M. Bezrukov1, Tatiana K. Rostovtseva1. 1The Natl. Institutes of Health, Bethesda, MD, USA, 2University of Amsterdam, Amsterdam, Netherlands. The voltage dependent anion channel (VDAC) is involved in regulation of me- tabolite flux across the mitochondrial outer membrane (MOM). Hexokinse II (HK2) is known to bind the MOM where it phosphorylates glucose into glucose-6-phosphate (G6P). High expression of HXK2 is a common phenotype of many cancers, where its concentration can be 200 times of that in noncan- cerous cells, and is implicated in the Warburg effect. It is believed that VDAC serves as a HXK2 binding site in the MOM. The 15 amino acid N-ter- minal sequence of HXK2 is responsible for mitochondrial binding and, when conjugated to TAT (TAT-HK2), binds to mitochondria with higher affinity than native HXK2, causing HXK2 detachment. We have previously found that dimeric tubulin reversibly binds and partially blocks VDAC inhibiting me- tabolite flux across the MOM. Now we show that this binding can be attenuated by TAT-HXK2 peptide as well as by full length HXK2. We have found that TAT-HXK2 and recombinant full length HXK2 inhibit tubulin blockage of VDAC reconstituted into planar lipid bilayers without altering characteristic channel properties such as single channel conductance and selectivity. Binding of HXK2 to VDAC is verified by the generation of high-frequency excess cur- rent noise without channel closure. HXK2 bound to VDAC prevents subse- quent tubulin binding, but only when added before tubulin, and inhibits tubulin-induced VDAC blockage in a dose dependent manner. Moreover, G6P, which is known to cause HXK2 detachment from the MOM, fully re- verses the inhibition of tubulin-VDAC binding. This suggests that HXK2 de- tachment from VDAC (and hence the MOM) is caused by a HXK2 conformational change upon G6P binding. Thus we propose a novel mecha- nism of mitochondrial respiration control in cancer cells through the competi- tion between HXK2 and tubulin for VDAC binding. 3365-Pos Board B520 Reprogramming of Mitochondrial Ca 2D Handling in MICU1-Deficient HeLa Cells Tünde Golenár1, György Csordás1, Erin L. Seifert1, Cynthia Moffat1, Fabiana Perocchi2, Yasemin Sancak2, David Weaver1, Vamsi K. Mootha2, György Hajnóczky1. 1 Thomas Jefferson University, Philadelphia, PA, USA, 2 Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA. Recent studies have revealed MCU as the pore forming domain and MICU1 as a critical Ca2þ-sensitive regulator of the mitochondrial Ca2þ uniporter. How- ever, the exact role of MICU1 in Ca2þ transport remains to be addressed. Our previous studies showed that prolonged down-regulation of MICU1 in HeLa cells (shMICU1) promotes mitochondrial Ca 2þ uptake at low [Ca 2þ ], which unexpectedly, fails to effectively increase matrix [Ca 2þ ]. To determine the source of discrepancy between the mitochondrial Ca 2þ uptake and the ma- trix [Ca2þ] phenotypes, first we simultaneously monitored ruthenium red- sensitive clearance of added Ca2þ from the cytoplasm and the corresponding matrix [Ca2þ] response in permeabilized shMICU1 cells. Under conditions of similar cytoplasmic Ca 2þ clearance, shMICU1 cells showed a smaller matrix [Ca 2þ ] increase than the control, indicating enhanced buffering of Ca 2þ in the matrix. Enhanced Ca 2þ binding in the matrix likely reflects alkalinization and enhanced phosphate transport. To test if upregulation of Ca2þ buffering is di- rectly linked to MICU1 depletion, we also assessed mitochondrial Ca2þ han- dling after 72hr silencing of MICU1 (siMICU1). In siMICU1cells both mitochondrial Ca 2þ uptake and the matrix [Ca 2þ ] rise were effectively stimu- lated at low Ca 2þ levels. Thus, upregulation of matrix Ca 2þ buffering seems to be a component of an adaptive response to sensitization of mitochondrial Ca 2þ uptake in shMICU1. The adaptive response is likely to be important to attenuate some MICU1-depletion induced cellular impairments that we found to manifest as attenuated mitochondrial ATP production and cell proliferation. 3366-Pos Board B521 MICU1-dependent Threshold and Cooperativity of Mitochondrial Ca 2D Uptake in the Liver György Csordás1, Erin L. Seifert1, Tünde Golenár1, Cynthia Moffat1, Sergio de la Fuente Perez1, David Weaver1, Roman Bogorad2, Victor Koteliansky2, Vamsi K. Mootha3, György Hajnóczky1. 1Thomas Jefferson University, Philadelphia, PA, USA, 2Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA, 3 Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA. Recent studies have revealed MCU as the pore forming domain and MICU1 as a critical Ca2þ-sensitive regulator of the mitochondrial Ca2þ uniporter. How- ever, the mechanism of the complex Ca2þ dependence of the uniporter activity remains elusive. Our previous studies showed that prolonged down-regulation of MICU1 in HeLa cells causes lower threshold and decreased cooperativity of mitochondrial Ca 2þ uptake. To study the functional significance of the effects of MICU1 we used hepatocytes harvested from the liver of mice exposed to in vivo silencing (4 weeks). Silencing of MICU1 or MCU resulted in >80% decrease in their respective mRNA levels. Silencing of MICU1 caused a left- ward-shifted dose response and decreased cooperativity of mitochondrial Ca2þ uptake in both permeabilized and intact hepatocytes. By contrast, silenc- ing of MCU resulted in slower Ca 2þ uptake in the entire range of Ca 2þ concen- trations without change in threshold. Mitochondrial respiration and cellular ATP content were unaffected in media containing both glycolytic and mito- chondrial fuels in either MICU1 or MCU-deficient hepatocytes. However, si- lencing of MICU1 caused an augmented loss of ATP when the cells were confined to oxidative metabolism and an enhanced sensitivity to mitochondrial Ca 2þ overload and permeabilization. During stimulation with vasopressin, a Ca 2þ mobilizing hormone, both MICU1 and MCU-deficient cells displayed an attenuated mitochondrial matrix [Ca 2þ ] increase and stimulation of respira- tion. Collectively, these results show that keeping the gate of MCU closed by MICU1 at low [Ca2þ] is required to maintain healthy mitochondria, and MICU1-mediated control of MCU (cooperativity?) is required to support the propagation of short-lasting calcium spikes and oscillations to the mitochondria and the ensuing physiological stimulation of oxidative metabolism. 3367-Pos Board B522 Targeting Mcl-1 and Bak as a Therapeutic Tool to Selectively Induce Apoptosis in Hepaptocellualr Carcinoma Nima Niknejad, Soumya Sinha Roy, Eric Knudsen, György Hajnóczky. Thomas Jefferson University, Philadelphia, PA, USA. In this study we seek to identify novel drug targets to induce apoptosis in he- patocellular carcinoma (HCC) cells thus providing opportunities to develop novel treatments to improve the prognosis of liver cancer patients. Several ap- optotic pathways are mediated through cleavage of Bid (a BH3 domain-only, pro-apoptotic protein) to produce truncated Bid (tBid). tBid induces apoptosis through induction of outer mitochondrial membrane (OMM) permeabilization by activation of pro-apoptotic Bak that resides in the OMM or cytoplasmic Bax. Due to its localization, Bak can mediate the early phase of the response to tBid. We have recently demonstrated that OMM targeting of Bak and the sensitivity to tBid-induced OMM permeabilization is dependent on the expression of 656a Wednesday, February 6, 2013 voltage-dependent anion channel isoform 2 (VDAC2). Here, we show that HCC induced by carcinogenic compounds, aflatoxin or DEN is highly sensi- tized to tBid-induced OMM permeabilization and cyto c release when com- pared to normal liver. Expression levels of VDAC2 and Bak were also higher in HCC than in control liver. The role of the VDAC2-Bak pathway in the differential tBid sensitivity was validated by overexpression of VDAC2 in primary hepatocytes. In HCC cells, elevation of Bak was also associated with an increased level of Mcl-1, an inhibitor of Bak. Furthermore, both constitutive and drug-induced genetic deletion of Mcl-1 in mouse embryonic fibroblasts caused sensitization to tBid-induced OMM permeabilization in the absence of a change in Bcl-xL or Bax . Based on these results, we are evaluating the possibility of selective killing of HCC cells by the combination of a Bak activator and an Mcl-1 inhibitor. 3368-Pos Board B523 Which Domain of VDAC2 is Necessary for Bak Insertion to the Outer Mitochondrial Membrane and tBid - Induced Cytochrome C Release? Shamim Naghdi1, Peter Varnai2, Soumya Sinha Roy1, Laszlo Hunyady2, Gyorgy Hajnoczky1. 1 Thomas jefferson university, philadlephia, PA, USA, 2 Semmelweis University, Budapest, Hungary. VDAC proteins represent a main component of the outer mitochondrial membrane (OMM). The VDAC family is composed of 3 isoforms with more than 70 percent similarity. Although their primary role was known to be in ion and metabolite transport between mitochondria and cytosol, it has been dis- covered that VDACs are also involved in apoptotic pathways. We have recently found that specifically the VDAC2 isoform is needed for tBid-induced cyto- chrome c release, due to its role in supporting Bak insertion to the OMM (Roy et al. 2009. EMBO Rep.10:1341-7). To determine the domain(s) of VDAC2 which supports Bak insertion, VDAC1 and VDAC2 amino acid composition compared and the unique attributes of VDAC2sequencewereconsideredformutationalanalysis.Totestfunctionalsig- nificance of the changes in VDAC2 or VDAC1, VDAC2-/- MEF cells that are resistant to tBid, were transfected with the mutants and insertion of Bak to the OMM and cytochrome c release were monitored in permeabilized cells by immunoblotting. Previously, we showed that the VDAC2-specific N terminal tail and cysteins are dispensable for tBid-induced OMM permeabilization. To approach the remaining differences four chimeras were prepared: VDAC1 (1-185)VDAC2(198-295), VDAC2(1-12)VDAC1(1-185)VDAC2(198-295), VDAC2(1-188)VDAC1(177-283) and VDAC2(13-188)VDAC1(177-283). These studies revealed that tBid dependent OMM permeabilization is only sup- ported by VDAC2(1-188)VDAC1(177-283) and VDAC2(13-188)VDAC1(177- 283), indicating that the first two third of VDAC2 is required and sufficient for Bak targeting to the OMM and tBid induced cytochrome c release. 3369-Pos Board B524 Outer Mitochondrial Membrane Protein Distribution and Function Depend on Mitochondrial Fusion David Weaver1, Xingguo Liu1, Veronica Eisner1, Peter Varnai2, Laszlo Hunyady2, Gyorgy Hajnoczky1. 1Thomas Jefferson University, Philadelphia, PA, USA, 2Semmelweis University, Budapest, Hungary. Cells and tissues deficient in mitochondrial fusion have been shown to have defects linked to the exchange of inner membrane (IMM) and matrix compo- nents, particularly mitochondrial DNA. Outer membrane (OMM) constituents originate in the cytoplasm, thus the role of intermitochondrial transfer of OMM components by fusion remained unexplored. Here we show that fibro- blasts lacking the GTPases responsible for OMM fusion, Mfn1/2, have a more heterogeneous distribution of OMM proteins than wild-type cells, and in partic- ular that heterogeneity of pro-apoptotic Bak leads to dysregulation of Bid- dependent apoptotic signaling. Homogeneous distribution of Bak is partially rescued by introduction of Mfn2 into Mfn1/2-/- cells. Furthermore, fusions be- tween mitochondria lacking and containing Bak result in hybrids sensitive to Bid. Proteins with different modes of OMM association display varying degrees of heterogeneity in Mfn1/2 /- cells and different kinetics of transfer during fusion in fusion-competent cells. Efficient coupling of OMM to IMM fusion depends on the presence of both Mfn isoforms and is antagonized by the mitochondrial fission protein, Drp1. Thus, OMM function depends on mitochondrial fusion and is a locus of dysfunction in conditions of fusion deficiency. 3370-Pos Board B525 Mitochondrial Fusion Dynamics in Skeletal Muscle of Healthy and Diseased Rat Veronica Eisner1, Guy Lenaers2, Gyorgy Hajnóczky1. 1 Thomas Jefferson University, Philadelphia, PA, USA, 2 INSERM U-583, Institut des Neurosciences de Montpellier, Montpellier, France. Mitochondria are highly mobile and dynamic organelles in many cell types. However, in the muscle, mitochondria are crammed into the narrow space among the myofilaments. Until now, it remained unclear if mitochondrial fusion occurs in and supports the contractile activity of skeletal muscle (SM). Here we applied mitochondria matrix-targeted DsRed and photoactiv- able GFP to study mitochondrial fusion in rat FDB SM fibers. In vivo electro- porated fibers were imaged by confocal microscopy. When we tagged the mitochondria in ~5% of cell area with twophoton photoactivation of GFP, rapid spreading of fluorescence showed subsets of interconnected mitochondria, mostly in longitudinal direction. Matrix fusion occurred with a rate of 0.5 and 6.4 events/min in adult fibers and skeletal myotubes, respectively. Expres- sion of Autosomal Dominant Optical Atrophy-causing mutants of the fusion protein Opa1 or ethanol exposure caused suppression of fusion, which is fol- lowed by mitochondrial dysfunction and late onset myopathies. When chal- lenged by repetitive tetanic stimulation, ethanol exposed cells displayed intracellular Ca2þ dysregulation, appearing as a fatigue pattern. Ethanol also induced a decrease in Mfn1 protein levels, without significantly altering Mfn2, Opa1 or deltapsi. Depletion of Mfn1 alone was sufficient to suppress mi- tochondrial fusion in FDB fibers. Fusion inhibition was apparent before any cell dysfunction, suggesting that suppression of fusion is not secondary to other problems in the cells. To directly test the role of Mfn1 in Ca 2þ regulation, RyR1-transfected control and Mfn1KO MEFs were stimulated with caffeine. Mfn1KO cells showed oscillatory Ca2þ transients that decayed faster than that in the control. Thus, fusion dynamically connects skeletal muscle mito- chondria and serves as a target mechanism of both mutations and environmen- tal cues to cause impaired excitation-contraction coupling. 3371-Pos Board B526 BCL-2 Proapoptotic Proteins Distribution in U-87 MG Glioma Cells before and after Hypericin Photodynamic Action Katarina Stroffekova, Lucia Balogová, Mária Maslaňáková, Lenka Dzurová, Pavol Mi�skovský Mi�skovský. Safarik University, Kosice, Slovakia. Apoptosis is a key process in the development and maintenance of tissue homeostasis. This process of controlled cell death is tightly regulated by a balance between cell survival and damage signals. We focused our attention towards the intrinsic mitochondrial apoptotic pathway, where Bcl-2 family of proteins plays the major role. We were particularly interested in two pro- apoptotic players Bak and Bax. Here we investigated their role in apoptosis triggered by photodynamic action. We show the localization of Bax and Bak in U-87 MG human glioma cells incubated with photosensitizer hypericin (Hyp) before and after photodynamic action. Apoptotic stimulus by Hyp photodynamic action caused Bax translocation into mitochondria. However our results suggest that under these condi- tions there are two populations of mito- chondria: one which contains Bax and Bak simultaneously, and is almost exclu- sively localized near the plasma mem- brane; the other which contains Bax only and is distributed throughout the cell. The different protein content and spatial distri- bution of these two populations suggest that they can play different roles in re- sponse to apoptotic stimuli. 3372-Pos Board B527 Stimulation of Bax Mitochondrial Localization by Bcl-xL Thibaud Renault1, Oscar Teijido2, Gisele Velours1, Yogesh Tengarai Ganesan2, Florent Missire1, Nadine Camougrand1, Bruno Antonsson3, Laurent Dejean4, Stephen Manon1. 1IBGC-CNRS, Bordeaux, France, 2NYU College of Dentistry, New York, NY, USA, 3Merck-Serono SA, Geneva Research Center, Geneva, Switzerland, 4California State University of Fresno, Fresno, CA, USA. Cytochrome c release, the commitment step of apoptosis, is regulated at the mitochondria through protein-protein interactions between the Bcl-2 family proteins. An imbalance of this interaction network due to the up-regulation of the proto-oncogenes Bcl-2 and/or Bcl-xL lead to a resistance to apoptosis and is associated with tumor formation. Bcl-xL overexpression act at the level of the mitochondrial outer membrane (MOM) by inhibiting Bax-mediated apoptosis; more particularly MAC formation and cytochrome c release. How- ever, the molecular mechanisms through which Bcl-xL affect earlier steps of Bax-mediated apoptosis are not fully understood. Surprisingly, we found that mitochondrial Bax redistribution and change of conformation were not inhibited but rather spontaneously increased in response of Bcl-xL overexpres- sion. In order to further investigate the molecular mechanisms involved in this A Step Forward in Understanding the Mechanism of VDAC Voltage-Gating Novel Mechanism of Mitochondrial Respiration Control through Competition between Hexokinase-2 and Tubulin for VDAC Binding Reprogramming of Mitochondrial Ca2+ Handling in MICU1-Deficient HeLa Cells MICU1-dependent Threshold and Cooperativity of Mitochondrial Ca2+ Uptake in the Liver Targeting Mcl-1 and Bak as a Therapeutic Tool to Selectively Induce Apoptosis in Hepaptocellualr Carcinoma Which Domain of VDAC2 is Necessary for Bak Insertion to the Outer Mitochondrial Membrane and tBid - Induced Cytochrome C Re ... Outer Mitochondrial Membrane Protein Distribution and Function Depend on Mitochondrial Fusion Mitochondrial Fusion Dynamics in Skeletal Muscle of Healthy and Diseased Rat BCL-2 Proapoptotic Proteins Distribution in U-87 MG Glioma Cells before and after Hypericin Photodynamic Action Stimulation of Bax Mitochondrial Localization by Bcl-xL