Boxer, Steven G.

Hughes, Laura D.

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DNA-Based Patterning of Tethered Membrane Patches 

Sunday, February 3, 2013 33abilayers. These probably transient microstructures indicate that the micelliza-
tion of detergent super-saturated vesicles occurs via a series of three stages:
(i) bending of detergent-rich monolayers into curved thread-like cover of the
perimeter of the holes, (ii) formation of thread-like micelles attached to the ves-
icles due to the line tension of the holes and (iii) detachment of the (most stable)
mixed micelles from the vesicles. All the available data (spectroscopic, micro-
scopic and calorimetric) are consistent with this mechanistic model.
168-Plat
Measuring Lipid Bilayer Bending Energy in a Dual-Beam Optical Trap
Mehmet E. Solmaz, Shalene Sankhagowit, Roshni Biswas,
James R. Thompson, Camilo A. Mejia, Michelle L. Povinelli,
Noah Malmstadt.
University of Southern California, Los Angeles, CA, USA.
While cell membrane bending is central to many physiological processes, tech-
niques for measuring the bending energy of lipid bilayers have reported widely
divergent results. Here, we show that a dual-beam optical trap (DBOT) can be
used to apply finely controlled tensions to lipid bilayer membranes in a giant
unilamellar vesicle (GUV) format. Optical force from the trap stretches the
GUV; video microscopy is used to measure the change in membrane area dur-
ing stretching. As laser power is increased, the surface area of the GUV also
increases. Laser power is translated to membrane tension using a ray optics ap-
proach. The resulting tension-area relationship is fit to a model of membrane
mechanics to yield a bending modulus.
The entire DBOT system is integrated with a microfluidic flow channel in such
a manner as to facilitate the high-throughput analysis of large populations of
GUVs. Performing such an analysis, we have shown that the presence of cho-
lesterol has no effect on the bending modulus of bilayers made from the unsat-
urated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Both
pure POPC bilayers and bilayers consisting of 80% POPC, 20% cholesterol
have a bending modulus around 8 kT.
This technique is a promising route to detailed, accurate data relating lipid
bilayer composition to membrane mechanical properties.
169-Plat
DNA-Based Patterning of Tethered Membrane Patches
Laura D. Hughes, Steven G. Boxer.
Stanford University, Stanford, CA, USA.
Solid-supported lipid bilayers are useful model systems for mimicking cellular
membranes; however, the interaction of the bilayer with the surface can disrupt
the function of integral membrane proteins. As a result, many groups have in-
troduced tethered lipid bilayers, which retain the proximity to the surface, en-
abling surface-sensitive techniques, but physically distance the bilayer from the
surface. We have recently developed a method for spatially separating a lipid
bilayer from a solid support using DNA lipids (Chung and Boxer et al., J.
Struct. Biol., 2009). In this system, a DNA strand is covalently attached to a si-
lane-modified glass slide or SiO2 wafer. The complementary DNA strand con-
jugated to a lipid moiety is inserted into giant unilamellar vesicles (GUVs), and
the DNA-modified GUVs hybridize to the strands on the surface, inducing flat-
tening and rupture of the GUV to a planar tethered lipid bilayer. However, the
location of the patch is random, determined by where the DNA-GUV initially
binds with its complement. To allow greater versatility and control, we sought
a way to pattern tethered membrane patches. We present a method for creating
spatially distinct tethered membrane patches on a glass slide using microarray
printing. Surface-reactive DNA sequences are spotted onto the slide, incubated
to covalently link the DNA to the surface, and DNA-GUVs patches are formed
selectively on the printed DNA. Different DNA sequences can be printed on the
same slide, creating a unique handle on each GUV patch. This handle enables
the creation of patches of different lipid compositions, dyes, and/or DNA-lipid
sequences in adjacent but distinct areas, and the control over the placement of
the tethered lipid bilayer potentially allows interfacing with devices. This ap-
proach would also enable rapid screening of different patches in protein binding
assays and as targets for membrane fusion.
170-Plat
Sleeping Bubbles: Effects of Volatile Anesthetics in the Lateral Structure
of Giant Unilamellar Vesicles
Leonel S. Malacrida1, Arturo Briva1, Luis A. Bagatolli2, Pablo Aguilar3,
Ana Denicola4.
1A´rea de Investigacio´n Respiratoria, Departamento de Fisiopatologı´a,
Hospital de Clı´nicas, Universidad de la Repu´blica, Montevideo, Uruguay,
2Membrane Biophysics and Biophotonics group/MEMPHYS - Center for
Biomembrane Physics, Department of Biochemistry and Molecular Biology,
University of Southern Denmark, Odense, Denmark, 3Laboratorio deBiologı´a Celular de Membranas, Instituto Pasteur de Montevideo,
Montevideo, Uruguay, 4Laboratorio de Fisicoquı´mica Biolo´gica, Facultad de
Ciencias, Universidad de la Repu´blica, Montevideo, Uruguay.
Volatile anesthetics have been widely used for more than 170 years. However,
the mechanisms underlying the effects of anesthetics on membrane dynamics
and structure are still under debate. Herein we study this problem for first
time using fluorescence confocal microscopy experiments of giant Unilamellar
Vesicles (GUVs). This approach allows obtaining spatially resolved informa-
tion on membrane structure at a microscopic level.
GUVs were prepared using DLPC/DPPC 3:7 mol. This mixture showed a char-
acteristic gel (Lbeta)/liquid disordered (Lalfa) phase coexistence at room tem-
perature, with line-shape domains (Lbeta) of variable width depending on the
temperature[1]. The volatile anesthetic used was Sevoflurane. The administra-
tion strategies were two: high concentration with reduced exposure time; or low
concentration (clinically relevant) with long term exposition.
A dramatic alteration of the phase coexistence was observed, with a marked ef-
fect on the morphology of the gel phase domains. For the experiments with high
anesthetic concentration, the boundaries of the domains became diffuse with an
increment of the domain’s perimeter/area ratio. When low anesthetic concen-
trations were used, a complete loss of the domains structure was observed
with appearance of small circular shaped domains.
Sevoflurane dramatically affected the lateral structure of the studied mem-
branes, suggesting that similar mechanisms may occur in biological relevant
membranes. Particularly, at low concentration of the anesthetic our results
show some structural characteristic to that observed for cholesterol in canonical
raft ternary lipid mixtures (formation of round domains). More research is un-
derway to better understand the mechanisms underlying the sevoflurane effects
on membranes.
[1] L.A. Bagatolli and E. Gratton. 2000, Biophys J. 78:290-305.
171-Plat
The Influence of Noble Gases in Protein-FreeMembranes and the Pressure
Reversal Effect
Francisco J. Sierra Valdez.
Cinvestav, Apodaca, N.L., Mexico.
The knowledge gathered so far reports a surprising variety of different chemical
substances to induce anesthesia. The questions about how the noble gasesmodify
the excitability of nerve cells and even how such excitability can be recovered
under hyperbaric pressure remain open [1]. In the literature, one finds competing
theories relating anesthesia to their effect on lipid membranes or on their effect
on proteins, but the origin of the anesthetic effect is still not understood. Cur-
rently, the fashion of the biological mechanisms make us resort to proteins, how-
ever, in anesthesia, we must not neglect that anesthetics produce a melting point
depression in pure lipid systems [2]. Furthermore, the pressure reversal in the
effectproduced by some anesthetics is well-known in animals, so that it seems
hardly explainable regarding the protein receptors theory.Whereby, the intrinsic
physical properties of the noble gasesmay give us hints to understand the general
anesthesia mechanism. In the present work we show, for the first time, calori-
metric results of the melting point depression phenomenon in protein-free
membranes induced by noble gases, followed by a reversal effect of such depres-
sion with hydrostatic pressure. We finally correlate the electric polarizability of
noble gases with the shift in the melting transition of the lipid membranes. Our
results, in a pure lipid system, concur with other findings to underwrite the
idea that anesthesia does not need a specific binding site in a protein and allow
us to speculate that anesthesia only depends on the ability of certain atom ormol-
ecule to solubilized in lipids increasing the disorder of the membrane.
References
[1] E. Yamamoto et al. (2012). J. Phys. Chem. B. 116 (30), 8989-8995.
[2] T. Heimburg and A. D. Jackson. (2007). Biophys. J. 92 (9), 3159-3165.
172-Plat
Restructuring of Membrane Bilayers due to Osmotic Pressure: Deuterium
Solid-State NMR Study
K.J. Mallikarjunaiah1, Muwei Zheng1, Emma P. Myers2, Jacob J. Kinnun3,
Horia I. Petrache3, Michael F. Brown1,2.
1Department of Chemistry and Biochemistry, University of Arizona, Tucson,
AZ, USA, 2Department of Physics, University of Arizona, Tucson, AZ, USA,
3Department of Physics, Indiana University-Purdue University, Indianapolis,
IN, USA.
Lipid membrane composition and biophysical properties have substantial influ-
ences on cellular functions. Studies of environmental effects on membrane bi-
layers are a prerequisite for understanding membrane protein functions.
Experimental measures of structural parameters like cross-sectional area/lipid
of membrane bilayers are vital for molecular dynamics simulations [1,2]. We


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DNA-Based Patterning of Tethered Membrane Patches

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