«membranes ISSN 2077-0375 Review Asymmetric Lipid Membranes: Towards More Realistic Model Systems Drew Marquardt 1,2, ...»
Membranes 2015, 5, 180-196; doi:10.3390/membranes5020180
membranes ISSN 2077-0375
Asymmetric Lipid Membranes: Towards More Realistic
Drew Marquardt 1,2, *, Barbara Geier 1,2 and Georg Pabst 1,2, *
Institute of Molecular Biosciences, Biophysics Division, University of Graz, NAWI Graz,
Humboldtstr 50/III, Graz, 8010, Austria; E-Mail: firstname.lastname@example.org
BioTechMed-Graz, Graz, 8010, Austria
* Authors to whom correspondence should be addressed; E-Mails: email@example.com (D.M.);
firstname.lastname@example.org (G.P.); Tel.: +43-316-380-4989 (G.P.) Academic Editor: Maikel Rheinstadter Received: 6 April 2015 / Accepted: 28 April 2015 / Published: 6 May 2015 Abstract: Despite the ubiquity of transbilayer asymmetry in natural cell membranes, the vast majority of existing research has utilized chemically well-deﬁned symmetric liposomes, where the inner and outer bilayer leaﬂets have the same composition. Here, we review various aspects of asymmetry in nature and in model systems in anticipation for the next phase of model membrane studies.
Keywords: asymmetry; vesicles; model membranes; phospholipids
1. Asymmetry in Natural Membranes: A Brief Introduction Arguably the most notable year in the study of biological membranes was 1972. Not only the archetypal ﬂuid mosaic model of Singer and Nicolson  was published in 1972, but Mark Bretscher provided the ﬁrst report of partial lipid asymmetry in membranes [2,3]. Remarkably, only a year later, quantitative analysis of the asymmetric lipid distribution in various cell types, including human erythrocytes, was determined (Figure 1) , piloting membrane research already in its early days and, in particular, membrane biophysics toward deciphering the role of membrane asymmetry.
Like all eukaryotic cells, mammalian plasma membranes (PM) actively sequester nearly all of their sphingomyelin, (SM) and phosphatidylcholine (PC) within the outer monolayer of the membrane. The inner monolayer was determined to consist of phosphatidylethanolamine (PE) and the negatively-charged lipids phosphatidylserine (PS) and phosphatidylinositol Membranes 2015, 5 181 (PI) [4,5]. Cholesterol (Chol) is found in both membrane leaﬂets, but apparently enriched within the inner leaﬂet . Asymmetry is also observed in bacterial membranes, although it is more difﬁcult to quantify. Nevertheless, it has been reported that PI and PE are preferentially located in the inner leaﬂet, phosphatidylglycerol (PG) in the outer leaﬂet, while cardiolipin (CL) is distributed over both leaﬂets in plasma membranes in Gram-positive bacteria [7,8]. Membrane asymmetry is known to affect various bilayer properties, including membrane potential, surface charge, permeability, shape, as well as stability [5,9,10]. Loss of asymmetry has physiological consequences. For example, PS exposure occurs in mammalian cells during apoptosis and is an important signal for their disposal by macrophages (see, e.g., Fadok and Henson ). PS externalization has also been linked to blood coagulation and erythrocyte adhesion (see, e.g., Lentz or Wautier et al. [12,13]) and myotube formation  and has been reported recently for cancer cells .
Figure 1. Proposed distribution of phospholipids in human red blood cells put forward by Verkleij et al.
An asymmetric membrane is a system not at equilibrium, which would prefer a symmetric distribution of membrane lipids. Thus, maintaining membrane asymmetry in live cells is an active process (requiring ATP) carried out by proteins, known as ﬂipases or ﬂoppases [16,17]. In addition to transmembrane asymmetry, lateral inhomogeneities in membranes, and, in particular, the formation of functional platforms (domains), such as membrane rafts, have attracted signiﬁcant interest and research efforts in the past few decades . Membrane rafts are thought to enable the assembly of signaling proteins or transbilayer transport and are enriched in SM and Chol ; similarly, recent experiments suggested signiﬁcant inﬂuences originating from the cytoskeleton . Interestingly, several laboratories have reported that lipid-only membranes with a symmetric distribution of outer leaﬂet lipids (SM, PC) and Chol readily separate into coexisting liquid-disordered (Ld ) and liquid-ordered (Lo ) phases over a wide range of compositions and temperatures (for a review, see, e.g., ). Moreover, Lo domains were found to be enriched in high-melting lipids, such as SM and Chol , thus encouraging their application as simple models to study the properties of membrane rafts. On the contrary, inner leaﬂet lipid mixtures (e.g., PE, PS and Chol) were found to exhibit complete miscibility, i.e., PE/PS/Chol Membranes 2015, 5 182 mixtures form a uniform Ld phase in symmetric bilayers . However, membrane domain formation in the outer leaﬂet somehow inﬂuences the organization of the inner leaﬂet-associated proteins during signal transduction [24,25]. Understanding the origin of this coupling mechanism is a major challenge in understanding the role of rafts in membrane function. Moreover, these observations suggest an ability of inner leaﬂet components to sense and respond to the physical state of the outer leaﬂet components, implying the existence of interleaﬂet communication. Whether such communication manifests itself in coupling of domain formation across the bilayer or induces other characteristic structural and dynamic changes in the lipids of the two leaﬂets remains unclear despite research efforts in this direction [26–29].
Lipid-only model membranes offer unique insight into such interactions from well-deﬁned systems. However, with a few exemptions detailed below, lipids, including complex lipid mixtures, self-assemble into symmetric bilayers, and asymmetry is difﬁcult to establish experimentally without being able to resort to ﬂipases or ﬂoppases. Thus, the majority of lipid-only studies have been performed using symmetric lipid membranes. Only recently has there been a new and strong impetus toward studying asymmetric membranes brought about by new and easy to follow protocols. At this dawn of a new era of membrane biophysics, we highlight the progress made and early insights achieved from such model systems, including a brief account of membrane asymmetry originating either from lipid properties or external constraints.
2. Asymmetry in Model Membranes
2.1. Geometric Asymmetry The most common source of asymmetry, and often overlooked, in model vesicles is the non-equal number of lipid molecules between bilayer leaﬂets as a result of vesicle size. As the vesicle diameter decreases, the difference in leaﬂet surface area increases. This difference in surface area is reﬂected in the number of lipid molecules that exist in each leaﬂet, which can be calculated based on the structural details of unstressed bilayers (Figure 2). A recent coarse-grained MD simulation demonstrates membrane asymmetry by increasing the lipid density in one leaﬂet . Lipid number density asymmetry is most easily observed experimentally by means of nuclear magnetic resonance spectroscopy [31,32]. The asymmetry has been shown via the use of a paramagnetic shift reagent, which interacts with the outer monolayer only, creating two separate signals (i.e., the inner and outer leaﬂet signals separate).
In the special case of small unilamellar vesicles (SUV), 50 nm, the asymmetric distribution can be qualitatively observed directly, as the packing of the inner and outer monolayers is different [31,32].
This directly affects the melting transition, which is distinct from unstressed bilayers .
A further driving force for membrane asymmetry results from lipid intrinsic curvature leading to lateral and transverse lipid separation . This effect may be also coupled to vesicle size, but does in general not depend on it, as it originates from an intrinsic lipid property. In the original postulation by Chapman, Willaims and Ladbrooke, Equation (1), the angle between the hydrocarbon chain axes and the phospholipid/water interface (τ) is described by the lateral space occupied by the fatty acid chains Membranes 2015, 5 183
Figure 2. Asymmetric lipid distribution due to vesicle size.
Data were generated using the area per lipid and bilayer thickness of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as determined by Kuˇ erka and co-workers .
c Although headgroup and tail composition both play a role in determining the shape parameter, a general rule is that PC lipids, as well as SM form regions of neutral or positive curvature, whereas PS and PE form neutral to negative regions of curvature, which explains the predominance of PS and PE on the inner monolayer of the PM [4,5], at least from a plain physical point of view. Quantitative assessment of intrinsic curvature for different phospholipids has been reported recently by Kollmitzer et al. .
Membranes 2015, 5 184 Figure 3. Cartoon illustration of differently-shaped lipids and the associated curvature.
2.2. Cholesterol Distribution Most of the currently available data suggest that cholesterol is asymmetrically distributed in membranes; however, which leaﬂet Chol resides in is still debated. Several studies on natural plasma membranes, using quenching of dehydroergosterol (a natural ﬂuorescent sterol found in sponge and yeast ) suggest that Chol mainly resides in the inner leaﬂet (see, e.g.,  for a review). Chol was observed to be distributed asymmetrically in mono-unsaturated PC lipids. Unfortunately, the authors could only speculate that Chol partitions preferentially to the inner monolayer , which was based on work by Huang and co-workers demonstrating that Chol concentrates in regions of high curvature .
Giang and Schick  summarize the corresponding theoretical framework explaining the afﬁnity of Chol for regions of high curvature. The theory suggests that PE, which is known to predominantly exist in the inner leaﬂet of the PM [4,5] and forms regions of high negative curvature (S 1), draws Chol to the inner leaﬂet to lower the bending free energy. Interestingly, when applied to biologically-relevant systems, such as human erythrocytes, a nearly symmetric Chol distribution, that is only 50%–60% of the Chol should reside in the inner monolayer of the PM, was observed .
Chol sequestered to the inner half of the bilayer is contrary to numerous biophysical studies on lipid-only bilayers. These studies report a tight interaction of Chol with SM , which locates almost exclusively to the outer leaﬂet [42–44]. In fact, coupling of Chol and SM is one of the foundations of the raft hypothesis . To relieve this disparity, it has been suggested that Chol might interact equally well with the saturated acyl chains of inner monolayer lipids .
Interesting insight comes from a highly-detailed coarse-grain MD simulation , studying Chol location and dynamics in a number of asymmetric bilayers of differing leaﬂet compositions. The simulations demonstrate that Chol adopts an asymmetric distribution upon reaching equilibrium after up to 10 µs (all of the systems were simulated for 12–15 µs) . The equilibrium location of Chol was found to depend not only on the leaﬂet on which it resides, but also on the composition of the other leaﬂet, demonstrating that an asymmetric bilayer must be viewed as one entity and not as being composed of two non-communicating leaﬂets (discussed further in Section 3.2). When reconciling model system data with Membranes 2015, 5 185 PM observations, one must keep in mind that the PM is far more complex, and cholesterol asymmetry could be organized differently compared to large unilamellar vesicles (LUVs) and MD simulations.
2.3. Charge Small angle X-ray scattering (SAXS) experiments on liposomes composed of PS lipids have reported that the scattered intensity does not approach zero between the second and third lobe, indicating an asymmetric electron density proﬁle [47,48]. This feature in the scattering intensity could in principle also originate from constraints from vesicle size, as discussed in Section 2.1. However, zwitterionic PC vesicles of the same size, that is LUVs, exhibit a minimum with zero scattered intensity in this regime, revealing that the asymmetry is not imposed by bilayer curvature . Furthermore, the coexistence of an interdigitated and a non-interdigitated gel phase, which was reported for PG lipids by Pabst et al. , can be ruled out for ﬂuid PS bilayers. Instead, asymmetry must originate from some lipid property. Brzustowicz et al. have demonstrated from ﬁtting their SAXS data of 100 nm 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (SOPS) LUV that the inner leaﬂet is more disordered, suggesting that SOPS vesicles are rougher on the inner leaﬂet compared to the outer .
On the other hand, it is well known that the charge state of a lipid depends strongly on the pH. For example, the relative charge goes from +1 at a pH value of one to−2 for a pH value of 13 for PS [50,51].
A different charge state leads to a change in headgroup sizes, which again cause a change of the shape (see Figure 3) . In support of this idea, Hope et al. reported that a transbilayer pH gradient can induce an asymmetric lipid distribution between the inner and outer leaﬂet .
3. Synthesized Asymmetry
3.1. Construction of Asymmetric Vesicles Aside from the aforementioned special conditions, several techniques have been developed to realize free-ﬂoating asymmetric lipid-only membranes [10,28,54–59], opening up several ways to study the role of membrane asymmetry.
Most promising in this respect appears to be important progress made in the laboratory of Erwin London (Stony Brook, NY, USA) using cyclodextrin-mediated lipid exchange, as depicted in Figure 4 (left) [10,28,57–59]. Recently, the protocol has been adapted for the construction of asymmetric supported bilayers . The technique can be applied to construct free-ﬂoating asymmetric lipid vesicles with various headgroup and acyl chain compositions containing Chol without the removal of Chol from the model membrane by cyclodextrin [58,59,61]. This has opened a new window to several biophysical studies, including the reconstitution of membrane proteins, as discussed further below.