Mitochondrial Fusion Dynamics in Skeletal Muscle of Healthy and Diseased Rat


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


	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