Membrane undulations play a vital role in many biological processes, including the regulation of membrane protein activity. The asymmetric lipid composition of most biological membranes complicates theoretical description of these bending fluctuations, yet experimental data that would inform any such a theory is scarce. Here, we used neutron spin-echo (NSE) spectroscopy to measure the bending fluctuations of large unilamellar vesicles (LUV) having an asymmetric transbilayer distribution of high- and low-melting lipids. The asymmetric vesicles were prepared using cyclodextrin-mediated lipid exchange, and were composed of an outer leaflet enriched in egg sphingomyelin (ESM) and an inner leaflet enriched in 1-palmitoyl-2-oleoyl-phosphoethanolamine (POPE), which have main transition temperatures of 37 °C and 25 °C, respectively. The overall membrane bending rigidity was measured at three temperatures: 15 °C, where both lipids are in a gel state; 45 °C, where both lipids are in a fluid state; and 30 °C, where there is gel-fluid co-existence. Remarkably, the dynamics for the fluid asymmetric LUVs (aLUVs) at 30 °C and 45 °C do not follow trends predicted by their symmetric counterparts. At 30 °C, compositional asymmetry suppressed the bending fluctuations, with the asymmetric bilayer exhibiting a larger bending modulus than that of symmetric bilayers corresponding to either the outer or inner leaflet. We conclude that the compositional asymmetry and leaflet coupling influence the internal dissipation within the bilayer and result in membrane properties that cannot be directly predicted from corresponding symmetric bilayers.
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Determining the Bending Rigidity of Free-Standing Planar Phospholipid Bilayers
We describe a method to determine membrane bending rigidity from capacitance measurements on large area, free-standing, planar, biomembranes. The bending rigidity of lipid membranes is an important biological mechanical property that is commonly optically measured in vesicles, but difficult to quantify in a planar, unsupported system. To accomplish this, we simultaneously image and apply an electric potential to free-standing, millimeter area, planar lipid bilayers composed of DOPC and DOPG phospholipids to measure the membrane Young’s (elasticity) modulus. The bilayer is then modeled as two adjacent thin elastic films to calculate bending rigidity from the electromechanical response of the membrane to the applied field. Using DOPC, we show that bending rigidities determined by this approach are in good agreement with the existing work using neutron spin echo on vesicles, atomic force spectroscopy on supported lipid bilayers, and micropipette aspiration of giant unilamellar vesicles. We study the effect of asymmetric calcium concentration on symmetric DOPC and DOPG membranes and quantify the resulting changes in bending rigidity. This platform offers the ability to create planar bilayers of controlled lipid composition and aqueous ionic environment, with the ability to asymmetrically alter both. We aim to leverage this high degree of compositional and environmental control, along with the capacity to measure physical properties, in the study of various biological processes in the future.
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- Award ID(s):
- 1942581
- NSF-PAR ID:
- 10397136
- Date Published:
- Journal Name:
- Membranes
- Volume:
- 13
- Issue:
- 2
- ISSN:
- 2077-0375
- Page Range / eLocation ID:
- 129
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-
sn -glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure–property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach—combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (2H NMR) spectroscopy, and molecular dynamics (MD) simulations—we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer’s packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure–property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol’s role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid–protein interactions. -
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Phosphatidic acid (PA) is a signaling lipid that is produced enzymatically from phosphatidylcholine (PC), lysophosphatidic acid, or diacylglycerol. Compared to PC, PA lacks a choline moiety on the headgroup, making the headgroup smaller than that of PC and PA, and PA has a net negative charge. Unlike the cylindrical geometry of PC, PA, with its small headgroup relative to the two fatty acid tails, is proposed to support negatively curved membranes. Thus, PA is thought to play a role in a variety of biological processes that involve bending membranes, such as the formation of intraluminal vesicles in multivesicular bodies and membrane fusion. Using supported tubulated lipid bilayers (STuBs), the extent to which PA localizes to curved membranes was determined. STuBs were created via liposome deposition with varying concentrations of NaCl (500 mM to 1 M) on glass to form supported bilayers with connected tubules. The location of fluorescently labeled lipids relative to tubules was determined by imaging with total internal reflection or confocal fluorescence microscopy. The accumulation of various forms of PA (with acyl chains of 16:0-6:0, 16:0-12:0, 18:1-12:0) were compared to PC and the headgroup labeled phosphatidylethanolamine (PE), a lipid that has been shown to accumulate at regions of curvature. PA and PE accumulated more at tubules and led to the formation of more tubules than PC. Using large unilamellar liposomes in a dye-quenching assay, the location of the headgroup labeled PE was determined to be mostly on the outer, positively curved leaflet, whereas the tail labeled PA was located more on the inner, negatively curved leaflet. This study demonstrates that PA localizes to regions of negative curvature in liposomes and supports the formation of curved, tubulated membranes. This is one way that PA could be involved with curvature formation during a variety of cell processes.
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Abstract Vesicles perform many essential functions in all living organisms. They respond like a transducer to mechanical stress in converting the applied force into mechanical and biological responses. At the same time, both biochemical and biophysical signals influence the vesicular response in bearing mechanical loads. In recent years, liposomes, artificial lipid vesicles, have gained substantial attention from the pharmaceutical industry as a prospective drug carrier which can also serve as an artificial cell‐mimetic system. The ability of these vesicles to enter through pores of even smaller size makes them ideal candidates for therapeutic agents to reach the infected sites effectively. Engineering of vesicles with desired mechanical properties that can encapsulate drugs and release as required is the prime challenge in this field. This requirement has led to the modifications of the composition of the bilayer membrane by adding cholesterol, sphingomyelin, etc. In this article, we review the manufacturing and characterization techniques of various artificial/synthetic vesicles. We particularly focus on the electric field‐driven characterization techniques to determine different properties of vesicle and its membranes, such as bending rigidity, viscosity, capacitance, conductance, etc., which are indicators of their content and mobility. Similarities and differences between artificial vesicles, natural vesicles, and cells are highlighted throughout the manuscript since most of these artificial vesicles are intended for cell mimetic functions.
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We use diamond nanoparticles (DNPs) wrapped in the cationic polyelectrolyte poly(allylamine) hydrochloride (PAH) and bilayers composed of either pure DOPC or a mixture of DOPC/DOPG to investigate the influence of membrane phospholipid composition and net surface charge on nanoparticle-membrane interactions and the extent of nanoparticle adhesion to supported phospholipid bilayers. Our results show that in all cases electrostatic attractions between the negatively charged bilayer and cationic PAH-DNP were responsible for the initial attachment of particles, and the lateral electrostatic repulsion between adsorbed particles on the bilayer surface determined the final extent of PAH-DNP attachment. Upon attachment, NPs attract lipids by the contact ion pairing between the ammonium groups on PAH and phosphate and glycerol groups on the lipids and acquire a lipid corona. Lipid corona formation on the PAH-DNP reduces the effective charge density of the particle and is in fact a key factor determining the final extent of NP attachment to the bilayer. Incorporation of DOPG to the bilayer leads to a decrease in efficiency and final extent of attachment compared to DOPC alone. The reduction in PAH-DNP attachment in the presence of DOPG is attributed to the adsorption of free PAH in equilibrium with bound PAH in the nanoparticle solution, thus reducing electrostatic attraction between PAH-DNPs and SLBs. This leads to an increase in hydrogen bonding interactions between lipid headgroups that limits extraction of phospholipids from the bilayer by PAH-DNP, lessening the reduction in interparticle repulsion achieved by acquisition of a lipid corona. Our results indicate that the inclusion of charged phospholipids in SLBs changes bilayer rigidity and stability and hinders the attachment of PAH-DNPs by preventing lipid extraction from the bilayer.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/membranes13020129
