doi:10.1016/j.bpj.2009.12.2046


380a Monday, February 22, 2010
mechanisms through which upregulation of Bcl2 affects earlier steps of BAX-
mediated apoptosis are not fully understood. We found that BAX insertion
into the MOM was the earliest apoptotic step inhibited by Bcl2 overexpression.
Paradoxically, we also found that BAX translocation to the mitochondria was not
inhibited but rather spontaneously increased in this same genetic context. This
increase in mitochondrial associated BAX required a physical interaction be-
tween BAX and Bcl2. We therefore propose that, at least when upregulated,
Bcl2 behaves as a ‘decoy receptor’ which sequestrates BAX at the mitochondria
but inhibits its insertion into the MOM, committing the cell to survive.
Supported by NYU Research challenge Funds to LD.

1961-Pos
Compartmentalization of BCL2 Family Proteins Mediated by Organelle
Lipid Membranes
Rebecca J. Boohaker, Ge Zhang, Adina Loosley, Kathleen Nemec,
Annette R. Khaled.
University of Central Florida, Orlando, FL, USA.
Cancer is defined by a pronounced inhibition of cell death. The BCL2 family of
proteins tightly regulates the delicate balance between life and death. One
method of regulation is the compartmentalization of antagonistic members.
For example, Bax, a pro-apoptotic member of this family, acts as the penulti-
mate factor in the apoptotic cascade by releasing apoptogenic factors such as
Cytochrome C from the mitochondrial lumen. The normally cytosolic protein
translocates from one internal compartment to another through an elusive
mechanism. Individual organelles are defined not only by function (mediated
by specific membrane bound proteins), but by the unique composition of their
phospholipid membranes. In this work, we have evaluated the contribution of
organelle lipids to the localization of of BCL2 proteins.

1962-Pos
In Search of the Structure of MAC in the Mitochondrial Outer Membrane
Pablo M. Peixoto1, Christian Renken2, Shefali Haldar3, Carmen Mannella2,
Kathleen W. Kinnally1.
1
New York University College of Dentistry, New York, NY, USA,

2Wadsworth Center, Albany, NY, USA, 3Academy of the Holy Names,
Albany, NY, USA.
Several groups have tried to determine the structure of the channel (MAC)
formed in mitochondrial outer membranes (OM) of apoptotic cells or in syn-
thetic membranes by Bax and related proteins/peptides, using electron micros-
copy (EM), atomic force microscopy and x-ray diffraction. Here, pore-like
structures ~3-10 nm were handpicked from transmission EM images of ura-
nyl-acetate-stained OMs isolated from control and apoptotic (IL3-deprived)
FL5.12 cells. These ‘‘candidate pores’’ were aligned by correlation procedures,
and class averages defined by principal component and K-means analyses.
Main differences in the class averages were (1) the presence of one or more
dark (stain-filled) pores, and (2) the nature of white (stain excluding) features
around the pores. A class average consisting of a single 3-nm pore with pro-
nounced white rim was rotationally averaged and used as a reference for
cross-correlation searches of 50 OM images from control and apoptotic cells.
Searches using this 3-nm ‘‘donut’’ motif and the same motif doubled in size
(6-nm ‘‘donut’’) yielded thousands of ‘‘hits’’ in both control and apoptotic
membranes, which were subsequently aligned and classified as before. The pre-
dominant stain-filled structures found with both motifs were not circular but
elongated (up to ~4x6 nm), extending away from stain-excluding crescent-
shaped features. The radial anisotropy ruled out reference bias and was in-
consistent with pores formed by rings of evenly spaced protein subunits. We
hypothesize that the different classes of structures detected represent stages
in formation of MAC as an increasingly large membrane bilayer defect (or
‘‘cleft’’) induced by successive aggregation or clustering of Bax/Bak molecules.
A progressive assembly mechanism for MAC has been recently suggested by
real-time monitoring of MAC conductances in isolated mitochondria by patch
clamping (Martinez-Caballero et al. J Biol Chem 284: 12235-45). Supported
by NIH grant GM57249.

1963-Pos
Effects of MAC Formation on Mitochondrial Morphology
Pablo M. Peixoto, Shin-Young Ryu, Robert Range, Kathleen W. Kinnally.
New York University College of Dentistry, New York, NY, USA.
Accumulating literature associate mitochondrial dynamics with apoptosis,
since regulation of either process has reciprocal effects. These processes
seem to converge in formation of the mitochondrial apoptosis induced channel,
MAC, which releases cytochrome c and triggers the degradation phase of apo-
ptosis. While Bax and Bak, core components of MAC, were shown to interact
with fusion and fission proteins, some studies also suggest proteins from the in-
termembrane space could leak to the cytosol and further promote mitochondrial
fission during apoptosis. The temporal relationship between apoptosis induc-
tion, MAC formation and mitochondrial fragmentation was investigated by
time lapse microscopy. MAC function was induced through staurosporine treat-
ment and microinjection of tBid or cytochrome c. MAC formation and mito-
chondrial dynamics under these conditions were monitored in Hela cells (clone
10) that stably express low levels of GFP-Bax and were transiently transfected
with a pDsRed-2 plasmid. GFP-Bax relocation to mitochondria only during
apoptosis signals MAC formation, while pDsRed-2 expression shows mito-
chondrial structure as red fluorescence. Treatment with staurosporine and
microinjection with tBid or cytochrome c induced relocation of Bax and col-
lapse of the mitochondrial network. The temporal relationship between these
two events was further analyzed. Interestingly, pretreatment with iMAC2, a spe-
cific MAC blocker, protected against cell death and prevented mitochondrial
fragmentation after tBid injection. Our results suggest a link exists between
MAC formation and collapse of the mitochondrial network during apoptosis.
Supported by NIH grant GM57249.

1964-Pos
Mechanism of the Mitochondrial Cytochrome C Release Wave in
Bid-Induced Apoptosis
Soumya Sinha Roy, Cecilia Garcia-Perez, Erika Davies, Xuena Lin,
György Hajnóczky.
Thomas Jefferson University, Philadelphia, PA, USA.
Bid, a BH3-only Bcl2 family protein, plays a central role in apoptosis. Bid is
cleaved by caspase-8 and other enzymes forming tBid that induce mitochon-
drial outer membrane (OMM) permeabilization and cytochrome c (cyto-c) re-
lease. However a mystery remains how Bid synchronizes the function of a large
number of discrete organelles, particularly in mitochondria-rich liver or muscle
cells. Here we showed that tBid (0.5-50nM) elicited progressive OMM perme-
abilization and complete cyto-c release with a dose-dependent lag time and rate
in H9c2 cell populations. Once started, the OMM permeabilization was not pre-
vented by tBid washout. In contrast, the dose-response for digitonin-induced
OMM permeabilization displayed quantal behavior. In single cell imaging
studies, permeabilized H9c2 and primary human cardiac cells transfected
with cyto-c-GFP showed complete tBid-induced cyto-c-GFP release closely
followed by mitochondrial depolarization. The cyto-c-GFP release started at
discrete sites and propagated through the mitochondria with a constant velocity
and a relatively stable kinetic of release in each organelle. Similar tBid-induced
cyto-c-GFP release wave was documented in intact H9c2 cells transfected with
tBid. The waves were not dependent on Ca

2þ
, caspase activation or permeabil-

ity transition pore opening. However, treatment with MnTMPyP, a ROS scav-
enger or overexpression of mitochondrial superoxide dismutase suppressed the
coordinated cyto-c release and also inhibited tBid-induced cell death. On the
other hand, both superoxide and hydrogen peroxide sensitized mitochondria
to the tBid-induced permeabilization. Thus, tBid engages a ROS-dependent
inter-mitochondrial signaling mechanism for spatial amplification of the
apoptotic signal by mitochondrial waves.

1965-Pos
Role of Milton Domains in the Calcium-Dependent Regulation of Mito-
chondrial Motility
Sudipto Das, Gyorgy Hajnoczky.
Thomas Jefferson University, Philadelphia, PA, USA.
The mammalian GRIF1 and OIP106, and the drosophila Milton are kinesin-
binding proteins that form a complex with the Miro GTPase, an outer mitochon-
drial membrane EF-hand protein, to support the movement of mitochondria
along the microtubules. Our study demonstrates that in H9c2 cells overexpress-
ing OIP106 or GRIF1, the basal motility of mitochondria is increased, whereas
the sensitivity to calcium-induced movement inhibition is decreased. To dissect
the interaction between Milton, kinesin and Miro, three different Milton
constructs were tested: Milton (1-450), the soluble domain of Milton; Milton
(750-1116), lacking the kinesin heavy chain binding site and Milton
(847-1116) that lacks additional ~100 amino acid presumably containing part
of the Miro binding site. Immunohistochemistry revealed that the overexpressed
Milton (1-450) was cytoplasmic, whereas the other two Milton constructs
showed mitochondrial localization. The basal mitochondrial motility was in-
creased by Milton (750-1116) but was not altered by Milton (847-1116) or Mil-
ton (1-450). A plot of mitochondrial motility against slowly rising cytoplasmic
[Ca2þ] induced by thapsigargin (2mM), shows that overexpression of Milton
(750-1116) significantly reduced the calcium sensitivity of mitochondrial motil-
ity reminiscent of OIP106 and GRIF1. By contrast, Milton (847-1116) or Milton
(1-450) did not have any effect. The thapsigargin-induced cytoplasmic calcium
signal was not affected by any of the Milton constructs. These data indicate that


	Compartmentalization of BCL2 Family Proteins Mediated by Organelle Lipid Membranes
	In Search of the Structure of MAC in the Mitochondrial Outer Membrane
	Effects of MAC Formation on Mitochondrial Morphology
	Mechanism of the Mitochondrial Cytochrome C Release Wave in Bid-Induced Apoptosis
	Role of Milton Domains in the Calcium-Dependent Regulation of Mitochondrial Motility