The Biophysics of Cell Membranes Biological Consequences

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Richard M. Epand • Jean-Marie Ruysschaert
Editors

The Biophysics of Cell

Membranes
Biological Consequences

123
Editors
Richard M. Epand Jean-Marie Ruysschaert
Biochemistry and Biomedical Sciences Sciences Faculty
McMaster University Université Libre de Bruxelles
Hamilton, ON, Canada Bruxelles, Belgium

ISSN 0932-2353 ISSN 1868-2561 (electronic)

Springer Series in Biophysics
ISBN 978-981-10-6243-8 ISBN 978-981-10-6244-5 (eBook)
DOI 10.1007/978-981-10-6244-5

Library of Congress Control Number: 2017952612

© Springer Nature Singapore Pte Ltd. 2017

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Preface

Chapters in this volume discuss several aspects of the physical properties of

biological membranes and how these properties influence their functioning. The
reviews emphasize the mechanisms that result in these changes in membrane
properties and function.
One of the rapidly developing areas in membrane biophysics in recent years has
been the role of transbilayer lipid asymmetry. It is known that the lipid composition
of the bilayer of a biological membrane is very different for the two monolayers that
compose this bilayer. Most studies of model membranes have employed membranes
with identical composition for the two monolayers. There are technical difficulties
in making model membranes with transbilayer asymmetry. Chapter 1 describes
the methods that are being developed to facilitate the preparation of asymmetric
model membranes and how the presence of this transbilayer lipid asymmetry affects
the physical properties of the membrane. The maintenance of transbilayer lipid
asymmetry is intimately connected with the rates of lipid flip-flop, i.e. the movement
of lipid from one face of the bilayer to the opposite side. In model membranes
devoid of protein, flip-flop rates of polar lipids are generally very slow. However,
in biological membranes these rates can be accelerated by specific proteins, in
some cases using an active transport mechanism, as well as through non-specific
disordering of membrane packing compared with a pure lipid membrane. Chapter
2 discusses how the flipping rate is dependent on both the chemical structure of the
lipid as well as on the physical state of the membrane. Results of studies of flip-flop
rates obtained both from experiments as well as computer simulations are presented.
The two principle components of biological membranes are proteins and lipids.
The function of membrane proteins is modulated by lipids, both by binding to
specific lipid binding sites on the protein as well as by modulating the general
biophysical properties of the membrane. Some of these properties, including the
formation of supercritical fluids as well as long range interactions involving
curvature stress, curvature elasticity and hydrophobicity play key roles in the
coupling of lipids and proteins. The mechanisms of this modulation of membrane
protein function through coupling with the physical properties of the membrane

v
vi Preface

are reviewed in Chap. 3. Chapter 4 describes how mechano-sensitive channels

can be gated by stretching of the bilayer(forces-from-lipids principle)and/or by the
forces conveyed to the channel from the cytoskeleton/extracellular matrix(force-
from filament). The final two Chapters deal with larger scale systems. Chapter 5
discusses mechanisms of changes in cell shape. The factors involved can include
the cytoskeleton, membrane-bending proteins and membrane biophysical properties
including a role for lipid domains in cell membranes. The final Chapter considers the
liposome as a minimal cellular model that can be used to simulate diverse processes
from the origin-of-life to a reconstituted biochemical pathway. The possibility of
applying such systems for future biotechnological applications is also considered.
This volume thus summarizes, from diverse points of view, the nature of
membrane biophysical properties and how these properties impinge on the various
functions of a biological membrane.

Hamilton, ON, USA Richard M. Epand

Bruxelles, Belgium Jean-Marie Ruysschaert
Contents

1 Preparation and Physical Properties of Asymmetric Model

Membrane Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Johnna R. St. Clair, Qing Wang, Guangtao Li, and Erwin London
2 Spontaneous Lipid Flip-Flop in Membranes: A Still Unsettled
Picture from Experiments and Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Maria Maddalena Sperotto and Alberta Ferrarini
3 Membrane Lipid-Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Michael F. Brown, Udeep Chawla, and Suchithranga M.D.C. Perera
4 Principles of Mechanosensing at the Membrane Interface . . . . . . . . . . . . . . 85
Navid Bavi, Yury A. Nikolaev, Omid Bavi, Pietro Ridone,
Adam D. Martinac, Yoshitaka Nakayama, Charles D. Cox,
and Boris Martinac
5 Lipid Domains and Membrane (Re)Shaping: From Biophysics
to Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Catherine Léonard, David Alsteens, Andra C. Dumitru,
Marie-Paule Mingeot-Leclercq, and Donatienne Tyteca
6 Minimal Cellular Models for Origins-of-Life Studies and
Biotechnology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Pasquale Stano

vii
Chapter 1
Preparation and Physical Properties
of Asymmetric Model Membrane Vesicles

Johnna R. St. Clair, Qing Wang, Guangtao Li, and Erwin London

Abstract Model biomembrane vesicles composed of lipids have been widely used
to investigate the principles of membrane assembly and organization. A limitation of
these vesicles has been that they do not mimic the transbilayer lipid asymmetry seen
in many natural membranes, most notably the asymmetry in the plasma membrane
of eukaryotic cells. Recently, a number of approaches have been developed to
prepare asymmetric membranes and study their properties. This review describes
methods to prepare asymmetric model membranes, and the physical properties
of asymmetric lipid vesicles. Emphasis is placed on the vesicles prepared by
cyclodextrin-catalyzed exchange, which has proven to be a versatile and powerful
tool, including for studies manipulating lipid asymmetry in living cells.

Keywords Membrane domains • Liquid ordered state • Sphingolipids •

Phospholipids • Cyclodextrins

1.1 Lipid Asymmetry: Definition and Origin

When studying biological membrane organization and function, one important

aspect to consider is lipid asymmetry. Lipid asymmetry refers to the difference in
lipid composition in the outer (exoplasmic, exofacial) leaflet vs. the inner (cytoplas-
mic, cytofacial) leaflet of a membrane. Many cell membranes possess lipid asym-
metry. In mammalian cells, the outer leaflet of the plasma membrane is enriched
in sphingomyelin (SM), glycosphingolipids (GSL) and phosphatidylcholine (PC),
while the inner leaflet is composed mainly of phosphatidylethanolamine (PE),
and anionic lipids such as phosphatidylserine (PS) and phosphatidylinositol (PI)
(Fig. 1.1) [1]. Cholesterol is present in both leaflets, but its distribution is still in
dispute [2, 3].

J.R. St. Clair • Q. Wang • G. Li • E. London ()

Department of Biochemistry and Cell Biology, Stony Brook University,
Stony Brook, NY, 11794-5215, USA
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2017 1

R.M. Epand, J.-M. Ruysschaert (eds.), The Biophysics of Cell Membranes,
Springer Series in Biophysics 19, DOI 10.1007/978-981-10-6244-5_1
2 J.R.St. Clair et al.

Fig. 1.1 Representation of lipid asymmetry in natural biomembranes. In mammalian cells the
outer, or exofacial, leaflet is enriched in saturated acyl-chain sphingomyelin and in phosphatidyl-
choline, while the inner, or cytofacial, leaflet is composed primarily of phosphatidylethanolamine
and phosphatidylserine. Cholesterol, shown in gray is present in both leaflets

In cells, lipid asymmetry is maintained by flippases and floppases, enzymes that

control the movement of lipids across the bilayer, and by enzymes that synthesize
and degrade lipids in one or the other leaflet [4]. Lipid flip-flop, or the transverse
diffusion of lipids from one leaflet to another, counteracts asymmetry. The rate of
spontaneous phospholipid flip-flop in the absence of proteins is generally slow,
and can take days [5–9]. In contrast, cholesterol with its small and weakly polar
headgroup, can cross the lipid bilayer in a minute or less [10].

1.2 Biological Function of Asymmetry

The full significance of lipid asymmetry remains elusive, but is known to be

important in several biological processes. For example, the loss of PS asymmetry
and the resulting display of PS in the outer leaflet of cell membranes is a signal
which leads to the consumption of apoptotic cells by phagocytes [11], and is also
a signal indicating that the membrane has been damaged, promoting blood clotting
[12]. Some viruses even display PS in their outer leaflet to encourage engulfment
by macrophages and achieve host infection [13, 14].
Asymmetry may also affect lipid-protein interaction. Transmembrane (TM)
proteins typically have a higher positive charge at the cytofacial end of their
TM helices relative to their exofacial end. This is known as the positive-inside
rule [15]. Lipid charge asymmetry, with a higher negative surface charge at the
inner leaflet/cytofacial surface, may help determine TM protein orientation, as
well as influence the conformation of positively charged cytofacial juxtamembrane
sequences [16]. Importantly, it has recently been observed that plasma membrane
TM segments also have an orientational preference as judged from their abundance
in natural sequences. The segments of TM helices having amino acids with smaller
side chains exhibit a preference to be located in the outer leaflet relative to the inner
leaflet [17].
1 Preparation and Physical Properties of Asymmetric Model Membrane Vesicles 3

1.3 The Physical State of Membranes: Membrane Domains

and How They Might Be Influenced by Asymmetry

Another important aspect of membranes that is likely to be affected by asymmetry

is lipid physical state. In a lipid bilayer composed of only phospholipids or
sphingolipids there are two common physical states: the gel state and the liquid
disordered state (Ld). As the name implies, lipids in the Ld state are disordered
and loosely packed. Lipids in the gel state are tightly packed and solid-like, with
much less lateral mobility/diffusion than Ld state lipids. In a pure lipid bilayer each
individual lipid has its own characteristic melting, or transition temperature (Tm),
at which the gel state will reversibly melt to form the Ld state. In lipid bilayers with
mixtures of both high Tm lipids and low Tm lipids, gel and Ld states can co-exist
in the same bilayer. The addition of cholesterol to gel state lipids generally changes
the bilayer’s physical state to what is known as the liquid ordered (Lo) state [18,
19]. Since sphingolipids and other lipids with saturated acyl chains have high Tm
values, they readily form the Lo state. The Lo state has lipids that are tightly packed
and ordered, as in the gel state, but with fast lateral diffusion, as in the Ld state.
By light microscopy it is possible to observe co-existing Lo and Ld lipid phases in
giant unilamellar vesicles (GUVs) composed of mixtures of high Tm lipids, low Tm
lipids and cholesterol, for example, sphingolipids, unsaturated phospholipids and
cholesterol [20, 21], and in giant plasma membrane vesicles (GPMV) derived from
eukaryotic plasma membranes, which are rich in sphingolipids and cholesterol [22].
The lipid raft model posits that sphingolipid and cholesterol-rich Lo domains
(lipid rafts) co-exist with Ld domains in living cells [18]. Lipid rafts may be
an important feature of lipid organization in natural membranes. They have been
proposed to play an important role in many cellular processes such as amyloid
formation, protein and lipid sorting, cell signal transduction, and pathogen invasion
[23–30].
If lipid rafts are an integral component of the machinery for transmitting
information through the bilayer from the outside of the cell to the inside, then an
interesting question arises: since the inner leaflet of the bilayer contains little to
no raft-forming sphingolipids, then how can Lo domains form in the inner leaflet?
The answer to this may be that outer leaflet lipids influence the physical properties
of the inner leaflet: the physical properties of the two leaflets could be ‘coupled’
[31, 32]. This coupling could transfer information across membranes via the lipids
themselves. For example, it is possible that domains are induced in the inner leaflet
and these domains could then concentrate cytosolic-anchored proteins which have a
high affinity for ordered domains, such as proteins anchored by saturated acyl chains
[33, 34]. This could then lead to formation of specific protein-protein interactions
(Fig. 1.2). Hints that interleaflet coupling may occur come from studies by the
Mayor group which have shown that transmembrane interactions between outer
leaflet long acyl-chain lipids and inner leaflet phosphatidylserine are crucial in
generating actin dependent clustering of cytofacial lipid-anchored proteins [35]. It
should be noted that this would represent a very different mechanism for signal
4 J.R.St. Clair et al.

Fig. 1.2 Schematic illustration of interleaflet coupling giving rise to lipid-mediated signal trans-
duction across membranes. Lo forming outer leaflet lipids are shown inducing Lo domains in the
inner leaflet. This in turn may cause protein clustering in inner leaflet Lo domains which regulates
signal transduction

transduction across membranes than that mediated by transmembrane proteins, and

might provide a new target for biomedical applications.

1.4 Symmetric Model Membrane Vesicles

Techniques employing artificial membrane vesicles for the exploration of the princi-
ples governing natural membrane structure and function have been well-established.
The most commonly used vesicles have lipid symmetry, i.e. the lipid composition
in both leaflets is identical, or near-identical. Symmetric model membrane vesicles
can be made from a variety of phospholipids and sphingolipids, with or without
sterols, and in a variety of sizes. Multi-lamellar vesicles (MLV) can be prepared by
adding buffer to a dried lipid film and then agitating. As the name implies MLV are
many-layered, and they can be highly variable in size, with 8–15 concentric bilayers
enclosed in a vesicle ranging from 0.5 to several microns in diameter [36]. Small
unilamellar vesicles (SUV) are a single bilayer and are generally less than 50 nm in
diameter. SUVs can be made by the sonication of dried lipids in an aqueous medium,
or by dilution from ethanol [37, 38]. Generating SUVs with ethanol injection allows
control over the size of SUVs by adjusting the concentration of lipid in ethanol
before dilution [37]. Due to their small size, a feature of SUVs is a high level of
curvature stress on the lipids and this has been shown to impact lipid packing [39].
Large unilamellar vesicles (LUV), commonly with a diameter of 100–200 nm, are
1 Preparation and Physical Properties of Asymmetric Model Membrane Vesicles 5

relatively free from the curvature stress of SUVs. They can often be prepared by
subjecting MLVs to multiple cycles of freeze-thaw followed by extrusion through
membrane filters with pores of desired size, to obtain vesicles with a carefully
controlled diameter [40]. Giant unilamellar vesicles (GUV) can approximate the
size of living eukaryotic cells, ranging from 10,000 to 50,000 nm in diameter. GUV
can be generated by electroformation [41], gentle hydration [42], and a number of
oil-in-water techniques [43–51]. They are particularly useful for light microscopy
studies.
Using NMR, interleaflet coupling was investigated in sonicated, symmetric SM
SUV in one early pioneering study [52]. It was found that ion-induced changes
in transition (melting) temperatures in outer leaflet could be partly transmitted to
inner leaflet, indicating some level of interleaflet coupling, and it was proposed that
interleaflet coupling could be a mechanism to transmit information across bilayers.
Furthermore, it was found that the SM chain length influenced coupling, which was
detected when SM acyl chain length was long. This could reflect some effect of acyl
chain interdigitation.

1.5 Asymmetric Model Membranes: Planar Bilayers

Several different methods have been developed for the construction of asymmet-
ric planar supported bilayers. The Langmuir/Blodgett (LB) [53] and Langmuir–
Schaeffer (LS) [54] methods involve first depositing a monolayer of lipids onto
a silicon substrate, and then adding a second monolayer by dipping the substrate
again through an aqueous phase containing second leaflet lipids. Watanabe et al.
recently modified supported planar bilayer techniques to develop a system with
a capacity for simultaneously generating over 10,000 asymmetric supported lipid
bilayers [55].
Fluorescence Interference Contrast Microscopy (FLIC) was used to compare
the stability of asymmetry in planar supported bilayers constructed with several
techniques [56]. Lipid asymmetry was measured in bilayers with and without
cholesterol in Lo and Ld phases. Lipid asymmetry remained stable at 80% for up
to 6 h, as judged by fluorescent probe partitioning in supported bilayers generated
using the Langmuir-Blodgett/vesicle fusion (LB/VF) method.
Sum frequency vibrational spectroscopy also was used to examine the kinetics
and thermodynamics of lipid asymmetry. In cholesterol-free supported planar
LB/LS bilayers, asymmetry stability was limited to several hours and was variably
dependent on headgroup structure [57], acyl chain length, lateral surface pressure
[58], and acyl chain saturation [59]. Later work in LB/LS supported bilayers inves-
tigated the effects of sterol structural variation on flip-flop rates [60]. Vibrational
spectroscopy revealed that in distearoyl PC (DSPC) bilayers the stability of lipid
asymmetry varied considerably with sterol concentration and structure. The lack of
stability seen in these studies may be due to the presence of bilayer defects specific
to supported membranes [5].
6 J.R.St. Clair et al.

Other efforts have concentrated on preparation of unsupported or cushioned

planar bilayers in order to avoid the influence of a supporting substrate upon lipid
behavior. Unsupported asymmetric planar bilayers can be made via the Montal-
Mueller method [61]. This entails forming two separate lipid monolayers on each
side of a moveable Teflon partition containing a small hole. Carefully moving the
Teflon partition allows the formation of a bilayer at the hole in the partition where
the monolayers make contact. Using asymmetric unsupported bilayers, the Keller
group observed that in mixtures of diphytanoyl PC, dipalmitoylPC (DPPC) and
cholesterol, Lo domains can form in one leaflet independently of the other and that
by adjusting the lipid composition of one leaflet, domain formation in the opposing
leaflet could be suppressed [62]. However, the use of hexadecane to prepare the
monolayers raises the issue of residual solvent, which if present between the lipid
leaflets could influence interleaflet coupling.
Avoiding issues of solvent and support effects by adapting the LB and LS
methods, the Tamm and Naumann groups have used asymmetric bilayers cushioned
by polymer tethered to lipids. The Tamm group generated a tethered double
bilayer system and used single particle tracking to compare the mobility of the
transmembrane protein syntaxin-1A in tethered vs. supported bilayers. The work
demonstrated that there was no significant difference in syntaxin-1A mobility in
tethered vs. supported bilayers [63]. Using the same approach the Naumann group
examined the influence of lipid asymmetry on the sequestering and oligomerization
behavior of integrins ’v“3 and ’5“1 in bilayers [64]. The behavior of these
TM proteins in asymmetric bilayers differed significantly from that in symmetric
bilayers.

1.6 Asymmetric Lipid Vesicles

Lipid vesicles closely resemble natural biological membranes in that they are
unsupported, fully hydrated and the bilayer is unbounded. Because of this, the
results of work using lipid vesicles may be most informative for comparison to
biological systems. Even more closely resembling natural biomembranes, would
be asymmetric vesicles that reflected both the lipid diversity and asymmetry found
in the membranes of living cells.
Early work in the development of asymmetric lipid vesicles examined sponta-
neous fluorescent lipid analog exchange between vesicle populations [65]. It was
demonstrated that NBD-PC fluorescent lipid analogs could spontaneously transfer
from the outer leaflet of ‘donor’ lipid vesicles to outer leaflet of ‘acceptor’ lipid
vesicles. This would result in asymmetric vesicles in the sense that the fluorescent
lipid would be restricted to a single leaflet after exchange. In some cases, as much
as 50% of fluorescent donor lipids could be transferred to the acceptor vesicles.
However, the method was limited in that it required use of fluorescent lipids in
which one acyl chain was modified with a somewhat polar group.
1 Preparation and Physical Properties of Asymmetric Model Membrane Vesicles 7

1.7 Making Asymmetric Vesicles with Phospholipid

Carrier Proteins

In other early work, the Zilversmit group used phospholipid exchange protein
isolated from beef heart cytosol to exchange lipids between erythrocyte ghosts
and PC/cholesterol liposomes [66]. In doing so they explored the natural lipid
asymmetry in red blood cells and confirmed that lipid asymmetry is stable with
sphingomyelin (SM) and PC largely limited to the outer leaflet. In studies using
phospholipid exchange protein isolated from beef liver, the asymmetric transbilayer
distribution of SM, PE, and PC was assessed [67]. The transfer of radiolabeled
phospholipids from intact rat erythrocytes to unilamellar vesicles was measured as
a function of time. It was found that all of the SM and more than half of the PC were
located in the outer leaflet of the erythrocytes. Since nearly no PE was transferred,
it appeared to be largely confined to the inner leaflet.
Also using lipid-exchange proteins, the Holloway group prepared asymmetric
membranes to examine the depth of insertion of transmembrane protein cytochrome
b5 in membranes. They generated model membranes in which only one leaflet
contained phospholipids with brominated acyl chains, which quench tryptophan
fluorescence. They found that cytochrome b5 orients in the bilayer with the
tryptophan in its hydrophobic tail 7 nm from outer leaflet surface [68].
Lipids bound to bovine serum albumin (BSA) were used to generate asymmetry
in rat liver endoplasmic reticulum vesicles for the study of lipid flip-flop rates [69].
A series of spin-labeled phospholipids were introduced into the outer leaflets of
rat liver ER vesicles by binding them to BSA. To determine lipid flip-flop rates,
ESR spectroscopy and kinetics assays were performed. Results showed that in
all observed cases, phospholipid flip-flop was fast, with a half time of 20 min at
37 ı C, and not dependent on lipid head group type. However, the spin-labelled
phospholipids lacked long acyl chains, so the effects of acyl chain length on flip-
flop rates could not be gauged.
Holzer et al. used pro-sterol carrier protein (pro-SCP2) to generate vesicles that
were asymmetric with regard to negatively charged egg phosphatidylglycerol (PG)
[70]. The degree of asymmetry in the vesicles was then measured using free-flow
electrophoresis. Membrane curvature was found to be important for lipid transfer
efficiency by pro-SCP2. By using both small donor and small acceptor lipid vesicles
(50 nm diameter) exchange efficiency was increased by 55% over that achieved with
larger vesicles.

1.8 Using pH Gradients to Make Vesicles with Anionic Lipid

Asymmetry

The Cullis group induced asymmetry in vesicles containing negatively charged

lipids by inducing a pH gradient across the bilayer. Following acidification of
the exterior of PG-containing vesicles relative to the luminal cavity, 50% of PG
8 J.R.St. Clair et al.

(5% of total outer leaflet lipids) moved to the inner leaflet in within 50 min [71].
Increased acyl chain saturation, chain length and inclusion of cholesterol signifi-
cantly decreased this effect by decreasing membrane permeability and therefore the
ability of PG to move across the bilayer. It was also shown that it was possible
to reversibly manipulate the distribution of the negatively charged egg PG and
egg phosphatidic acid (PA) in the bilayer [72]. EggPG and eggPA, but not the
zwitterionic lipid dioleoylPE (DOPE) could be driven to the inner or outer leaflet,
depending on the direction of the proton gradient. Cryoelectron microscopy was
used to examine morphological changes in the bilayers of dioleoyl PG (DOPG)-
containing LUVs with pH gradient-induced DOPG asymmetry. Flipping of DOPG
from the outer to inner to leaflet or vice versa generated inversions and tubular
protrusions [73]. This approach is limited to the movement of anionic lipids,
and by the small percentages of anionic lipid that could be used (10% of total).
Furthermore, the pH gradient necessary to induce movement of anionic lipids would
not often be suitable for use in studies of protein conformation or function in
asymmetric vesicles.

1.9 Making Asymmetric Vesicles with Water-in-Oil

Techniques: Centrifugation Method

The Weitz group developed an oil-in-water technique to generate asymmetric giant

unilamellar vesicles (GUV) [74]. In the method, an emulsion of dodecane, inner
leaflet lipids and water was first prepared. This was layered over an intermediate
phase consisting of dodecane and outer leaflet lipids, which was directly supported
on an aqueous phase. As the droplets, which would have inner leaflet lipids
in an inverted micelle type of structure, passed via centrifugation through the
dodecane/outer leaflet layer into the aqueous phase, a monolayer of outer leaflet
lipids was deposited on the outside of the inner leaflet monolayer vesicles, forming
asymmetric bilayers. Using this technique, a controllable, high level (95%) of lipid
asymmetry was reported.
Studies using vesicles prepared by oil-in-water methods to examine the effect of
lipid asymmetry on the mechanical properties of bilayers showed that asymmetry
significantly increases membrane rigidity. Two types of fluorescently-labelled
asymmetric GUVs: dioleoyl PC (DOPC)in /1-palmitoyl-2-oleoyl PC (POPC)out and
POPCin /DOPCout (in D inner leaflet lipid, out D outer leaflet lipid) were generated.
Membrane rigidity was calculated by thermal fluctuation analysis of phase contrast
micrographs. Results revealed that both types of asymmetric membranes exhibited a
significantly higher bending rigidity compared to symmetric membranes consisting
of either DOPC, POPC, or 1:1 DOPC:POPC [75].
The Takagi group has developed a method to control the size of water-in-oil
asymmetric GUV. By adjusting the density of luminal contents with water-soluble
molecules such as sucrose and glucose, the rate of transfer and therefore the size
1 Preparation and Physical Properties of Asymmetric Model Membrane Vesicles 9

of vesicles could be controlled. Fluorescently labelled lipids were incorporated

into the cholesterol-containing asymmetric GUVs and facilitated the observance of
microdomain formation and confirmed lipid asymmetry [48].

1.10 Making Asymmetric Vesicles with Water-in-Oil

Techniques: Droplet Interface Bilayers

Hwang and colleagues examined the effect of lipid asymmetry on the behavior of
membrane proteins in droplet interface bilayers (DIB) [51]. In this method two types
of aqueous droplets surrounded by inverted lipid monolayers with different lipids
are deposited in an oil mixture. As the droplets meet, and monolayers come into
contact, and an asymmetric bilayer forms at their junction. Employing monolayer
droplets with differences in lipid charge, bilayers possessing a charge gradient were
constructed and used to measure charge gradient-induced changes in the insertion
and gating behavior of the outer membrane protein G (OmpG) from Escherichia
coli. This method is limited to use with lipids that are soluble in oil, but allows fine
control over mixtures of lipids for each monolayer.

1.11 Making Asymmetric Vesicles with Water-in-Oil

Techniques: Microfluidic Approaches

The introduction of microfluidic techniques to the production of asymmetric

lipid vesicles has enabled methods for the generation of water-in-oil GUV with
controllable size, lipid asymmetry, and lumenal content. The Malmstadt group used
a microfluidic flow-based layer-by-layer approach to produce asymmetric vesicles
with biologically-relevant PS limited to a single leaflet [46]. First sucrose-loaded
aqueous droplets were introduced to an oil stream containing dissolved inner leaflet
lipids to develop a lipid monolayer. The droplets were then released into a second
oil phase containing dissolved outer leaflet lipids floating over an aqueous phase.
Following centrifugation, the droplets passed through the aqueous phase and were
collected. Lipid asymmetry was confirmed via differential fluorescence quenching
and selective labeling with biotinylated lipids. The vesicles were size-selectable
because vesicles larger than 120 m do not survive the centrifugation step and those
smaller than 10 m don’t transfer through the second step. Lipid asymmetry was
confirmed with about 85% asymmetry. It was necessary to identify and select oil-
free vesicles visually.
The generation of asymmetric vesicles for drug delivery is a promising area
of research. In a study of drug cytotoxicity, microfluidics were employed to
generate GUVs with a cross-linked, chemically stable inner leaflet composed of
trichloro(1H,1H,2H,2H–perfluorooctyl)silane (TPS) and an outer leaflet containing
10 J.R.St. Clair et al.

DOPC. When loaded with the cancer therapeutic 5-fluorouacil, the asymmetric
vesicles exhibited a pH-dependent threefold higher cytotoxicity relative to the free
drug [76].
A high-throughput microfluidic process for generating water-in-oil GUV was
recently developed [47, 77]. In the method, water, oil and inner leaflet emulsions
were first formed. Outer leaflet lipids were then introduced via replacement of inner
leaflet lipid solution with a second solution. Water-in-oil double emulsions were
then formed and followed by the extraction of excess oil. Asymmetry and unil-
amellarity were confirmed with fluorescence quenching assays and transmembrane
protein insertion assays. In a second report using this same fabrication strategy,
the effects of lipid asymmetry on the mechanical properties of asymmetric bilayers
vs. symmetric bilayers were examined. In cholesterol-free symmetric vesicles
composed of dimyristoyl PC (DMPC), DOPC a 1:1 mixture of DMPC:DOPC, or
asymmetric vesicles with a DMPCin /DOPCout or DOPCin /DMPCout composition
results showed that the bending moduli and expansion moduli of asymmetric
bilayers are different that those of symmetric bilayers.

1.12 Minimizing Residual Oil Contamination

A complication resulting from oil-in-water techniques used to generate asymmetric

GUV is residual oil contamination. This residual oil likely impacts the physical
properties and behavior of the bilayer. To circumvent this, methods to minimize oil
contamination have been developed.
A microfluidics approach in which lipid monolayers were brought together in
a fashion similar to that in droplet interface bilayers was employed to generate
asymmetric GUV likely to have only minimal oil and then used to examine the
effects of lipid composition and asymmetry on lipid vesicle fusion mediated by
SNARE-family transmembrane proteins [45].
In another study, a high-throughput microfluidics method was developed to
form asymmetric GUV with minimal oil contamination. In the study, cholesterol-
rich asymmetric GUVs with ‘ultrathin shell’ (residual oil) were generated for the
observation of lipid microdomain formation. Lipid asymmetry and the presence
of lipid domains were observed with the use of fluorescently labelled lipids and
fluorescent microscopy [44]. In another attempt to address the issues of solvent
contamination, an asymmetric planar lipid bilayer was formed into lipid tubules
which were then broken up by applying a microfluidic jet flow of aqueous
buffer. Unilamellarity and residual solvent content was evaluated using confocal
Raman scattering microscopy [78]. Although these methods may greatly reduce oil
contamination, it is not clear whether the residual oil that remains would not perturb
lipid behavior to a significant degree.
1 Preparation and Physical Properties of Asymmetric Model Membrane Vesicles 11

1.13 Early Studies Using Cyclodextrin Exchange to Alter

Lipid Composition

A different strategy that is becoming more widely used is to prepare asymmetric

lipid vesicles with cyclodextrins (CDs). CDs are water-soluble cyclic oligosac-
charides composed of glucose monomers linked together by ’-(1, 4) glycosidic
bonds, and are typically composed of 6(’), 7(“), or 8(”) units. They assume a
conical barrel shape with a non-polar cavity and hydrophilic exterior. Hydrophobic
guest molecules can complex with CDs. A wide range of functional groups can
be conjugated to hydroxyl groups at the edges of CDs to modify their binding
specificity [79, 80] (see Fig. 1.3). Changes the number of monomers comprising
a CD alters the volume of the hydrophobic cavity, which can be varied to exclude
or include larger or smaller guest molecules. For example, the hydrophobic cavity
of ’CDs is smaller than that of “CDs and therefore binds well to phospholipid acyl
chains, but binds cholesterol either not at all or very poorly [80–83].
Cyclodextrins have long been used to remove sterols from cells, introduce new
sterols into cells, or exchange one sterol in cells for another [84–88]. Similarly, CDs
have been used to exchange sterols between model membrane vesicles [10, 89].
Early work exploring the properties of CDs as lipid carriers involved interactions
of ”-CDs and fluorescent lipids. Carboxyethyl-”-CD accelerated fluorescent lipid
transfer from lipid vesicles to cells in culture [90]. Another study reported that
the large hydrophobic cavity of ”-CD could bind more than one lipid acyl chain
simultaneously [91]. In addition, stoichiometric measurements using fluorescent
binding assays showed that the association constants of pyrene-labeled PC/ -CD
complexes decreased strongly with increasing acyl chain length [91].

Fig. 1.3 ’-cyclodextrin

chemical structure. The 6-unit
1–4 linked
’-D-glucopyranoside ring
forms a barrel shape, with a
hydrophobic interior cavity
and a hydrophilic exterior
surface. Functional groups
can be conjugated to
hydroxyl groups to modify
their binding specificity.
R D OH or modified OH