Andrews, David W.

Bindner, Scott

Fradin, Cecile

Leber, Brian

Shamas-Din, Aisha

Elsevier - Publisher Connector 

Binding of the Pro-Apoptotic protein Bid to Mitochondrial Membranes is a Two Step Process 

Tuesday, March 3, 2009 425amechanism behind the recognition of the mitochondrial membrane system by
the Bax-a1 sequence.
2190-Pos Board B160
Direct Interactions Between Tbid And Bcl-xlDXl Are Enhanced In Lipid
Membranes
Ana J. Garcia-Saez1, Jonas Ries1, Mar Orzaez2, Enrique Perez-Paya2,
Petra Schwille1.
1TU Dresden, Dresden, Germany, 2Instituto de Investigacion Principe Felipe,
Valencia, Spain.
The proteins of the Bcl-2 family are key regulators of apoptosis, but their mo-
lecular mechanisms remains controversial. Two important aspects that center
the debate involve the interaction network between the pro- and antiapoptotic
family members and the role of their translocation to the mitochondrial outer
membrane (MOM) during apoptosis. We have used FCCS to examine quanti-
tatively the dynamic interactions of Bid and tBid with Bcl-xLDCt in solution
and in lipid membranes. We found that only the active form tBid binds to
Bcl-xLDCt and that the membrane strongly promotes binding between them.
Importantly for drug design, a BH3 peptide from Bid disrupts the tBid/Bcl-
xL complex in solution but not in lipid bilayers. Our findings convincingly sug-
gest that the most relevant interaction between tBid and Bcl-xL happens in the
membrane and reveal its significance as an additional regulatory stage for
MOM permeabilization.
2191-Pos Board B161
Membrane Changes during Apoptosis: Part of the Process or Characteris-
tics of the Corpse?
Elizabeth Gibbons, Caitlin E. Askew, Katalyn R. Griffith,
Michael C. Streeter, Ashley O. Warcup, Celestine H-Y Yeung,
Allan M. Judd, John D. Bell.
Brigham Young University, Provo, UT, USA.
Thapsigargin-induced apoptosis in S49 lymphoma cells causes biophysical
changes in the plasma membrane. Thapsigargin (TG) is a sarco/endoplasmic
reticulum Ca2þ ATPase inhibitor that causes depletion of intracellular calcium
stores and increased cytosolic calcium levels. It is used to model endoplasmic
reticulum stress-induced apoptosis. This study focuses on membrane physical
changes involved in this specific apoptotic pathway and possible cellular mech-
anisms that could account for these alterations. The fluorescent probes mero-
cyanine 540 (MC540), laurdan, patman, and diphenylhexatriene (DPH) were
used to assess lipid spacing, order, and fluidity using fluorescence spectroscopy,
two-photon excitation microscopy, and confocal microscopy. MC540 fluores-
cence intensity increased throughout the apoptotic process, suggesting an in-
crease in interlipid spacing. Two-photon microscopy images with laurdan
showed a transient reduction in membrane order. Alexa Fluor-labeled annexin
V was used to assess phosphatidylserine exposure in the outer leaflet of the
plasma membrane. Flow cytometry experiments showed a sharp increase in
the population showing this flip-flop after 2.5 h incubation with TG. Suscepti-
bility of the cells to secretory phospholipase A2 (sPLA2), a hydrolytic enzyme
that can distinguish apoptotic membranes from healthy ones, was also evalu-
ated by measuring membrane permeability to propidium iodide. Two hours af-
ter addition of TG, a small population of cells became susceptible to sPLA2,
and that population increased steadily with longer incubations. All of these al-
terations in the plasma membrane were compared temporally with caspase ac-
tivation using a fluorescently labeled caspase inhibitor, FAM-VAD-fmk. This
comparison suggested that caspase activation, susceptibility to sPLA2, and de-
creased lipid order detected by laurdan precede PS exposure, elevated MC540
fluorescence, and eventual cellular demise. Experiments are in progress with
multiple pharmacological agents to assess cause and effect relationships among
these events and a possible role of ceramide.
2192-Pos Board B162
Chemotherapeutic Apoptosis: Who Assailed The Membrane, The Inducer
Or The Induced?
Jennifer Nelson, Kristen Barlow, D. Olin Beck, Amanda Berbert,
Kelly Damm, Nathan Eschenroder, Kristina Neeley, Mark Pruitt,
Kyle Thompson, Brian Thurber, Celestine H-Y Yeung, Allan M. Judd,
John D. Bell.
Brigham Young University, Provo, UT, USA.
The ability of certain chemotherapeutic agents to partition into the cell mem-
brane raises the possibility that some of their effects on cells may involve direct
disruption of normal bilayer function. Moreover, previous studies on hormone-
stimulated apoptosis indicate a variety of indirect membrane changes that ac-
company the death process including changes in membrane fluidity and order,
increases in interlipid spacing, and susceptibility to hydrolysis by secretory
phospholipase A2 (sPLA2). To compare the relative roles of potential direct
and indirect effects of chemotherapeutic agents on cell membrane properties,we treated S49 lymphoma cells with daunorubicin (partitions in membrane)
or methotrexate (non-membrane perturbing). An additional difference between
the two drugs relates to their involvement of caspase-3 in the apoptotic process;
daunorubicin requires it, and methotrexate does not. Membrane properties were
assessed over time after addition of the drugs by fluorescence spectroscopy and
microscopy using merocyanine 540, laurdan, diphenylhexatriene, and patman.
The preliminary results of these studies showed commonalities between dauno-
rubicin and methotrexate. For example, their effects on susceptibility to sPLA2
were nearly identical. Initially, the membrane remained resistant to hydrolysis
for several hours. Thereafter, a sharp increase in sPLA2 activity was observed.
These results suggested that changes that render the membrane vulnerable to
hydrolytic attack are controlled by biochemical processes associated with apo-
ptosis rather than reflecting direct effects of a chemotherapeutic drug on the cell
membrane. Interestingly, activation of caspase-3 appeared not to be part of
those processes.
2193-Pos Board B163
Designing Single Fluorescent Protein Based Caspase Sensor For Monitor-
ing Apoptosis In Living Cells
Shenghui Xue1, Ning Chen2, Yun Huang2, Jenny J. Yang2.
1Department of Chemistry, Department of Biology, Georgia State University,
Atlanta, GA, USA, 2Department of Chemistry,Georgia State University,
Atlanta, GA, USA.
Intracellular apoptotic signals regulated by caspase-cascade systems are closely
associated with human diseases such as cancer and neurodegenerative diseases.
Monitoring the activation and inhibition of caspase 3 and other caspases with
fluorescence spectrum changes in living cells is essential for further under-
standing these processes. Here, we report progress in the development of
caspase sensors based on a single fluorescent protein. These developed sensors
exhibit strong enzymatic selectivity as well as high sensitivity based on ob-
served ratiometric fluorescence changes. Additionally, our sensors can be tar-
geted to different subcellular locations, such as the ER and mitochondria.
We have further applied these sensors to monitor caspase-dependant apoptosis
in different cells. Our results indicate that different inducers and drugs have
diversified effects on triggering cell death pathways.
2194-Pos Board B164
Binding of the Pro-Apoptotic protein Bid to Mitochondrial Membranes
is a Two Step Process
Aisha Shamas-Din, Scott Bindner, Brian Leber, David W. Andrews,
Cecile Fradin.
McMaster University, Hamilton, ON, Canada.
Apoptosis is an essential process for the development of all multi-cellular or-
ganisms, and Bcl-2 family proteins critically regulate most pathways of apopto-
sis at the level of mitochondria. Bid is a pro-apoptotic member of Bcl-2 family
proteins that regulates the integrity of OMM (Outer Mitochondrial Membrane).
Upon induction of apoptosis, Bid is cleaved into a p15 and a p7 fragment,
a complex that is held together by strong non-covalent interactions. The sepa-
rated p15 fragment, also known as tBid (truncated Bid) is a potent inducer of
cell death, however, the mechanism of the fragments’ separation and activation
of Bid to tBid is unknown. The focus of this work was to develop an in vitro
fluorescent assay system to elucidate the mechanism of activation of Bid using
a recombinant liposomal system bearing physiological relevance along with
isolated mitochondria. Single cysteine mutants of Bid were created, and la-
belled with fluorescent thiol-reactive molecules to study the individual steps
of the activation of Bid. Using size exclusion chromatography, FCS (Fluores-
cent Correlation Spectroscopy), and FRET (Fo¨rster Resonance Energy Trans-
fer), it was quantitatively determined that the two cleaved fragments of Bid
spontaneously separate upon binding to the membrane without any additional
post-translational modifications. After the initial binding to the membrane,
p15 fragment undergoes a conformational change to adopt its active form. Tak-
ing together, we found that the activation of Bid is a two step process encom-
passing the separation of the cleaved fragments and a conformational change in
the membrane.
2195-Pos Board B165
Protein BAX During Detergent Activation: Characterization by Fluores-
cence Correlation Spectroscopy and Fluorescence Intensity Distribution
Analysis
Olena Ivashyna1, Ana J. Garcı´a-S´ez2, Jonas Ries2, Eric Christenson1,
Petra Schwille2, Paul H. Schlesinger1.
1Washington University in St. Louis, St. Louis, MO, USA.
2BIOTEC, TU Dresden, Dresden, Germany.
BAX is a pro-apoptotic member of the BCL-2 protein family. During apoptosis
in mammalian cells cytoplasmic BAX is activated and translocates to the outer
mitochondrial membrane (OMM), where it participates in formation of an


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Binding of the Pro-Apoptotic protein Bid to Mitochondrial Membranes is a Two Step Process

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