Stationary shapes of axisymmetric vesicles beyond lowest-energy configurations.

We conduct a systematic exploration of the energy landscape of vesicle morphologies within the framework of the Helfrich model. Vesicle shapes are determined by minimizing the elastic energy subject to constraints of constant area and volume. The results show that pressurized vesicles can adopt higher-energy spindle-like configurations that require the action of point forces at the poles. If the internal pressure is lower than the external one, multilobed shapes are predicted. We utilize our results to rationalize experimentally observed spindle shapes of giant vesicles in a uniform AC electric field.

Assessing membrane material properties from the response of giant unilamellar vesicles to electric fields.

Knowledge of the material properties of membranes is crucial to understanding cell viability and physiology. A number of methods have been developed to probe membranes in vitro, utilizing the response of minimal biomimetic membrane models to an external perturbation. In this review, we focus on techniques employing giant unilamellar vesicles (GUVs), model membrane systems, often referred to as minimal artificial cells because of the potential they offer to mimick certain cellular features. When exposed to electric fields, GUV deformation, dynamic response and poration can be used to deduce properties such as bending rigidity, pore edge tension, membrane capacitance, surface shear viscosity, excess area and membrane stability. We present a succinct overview of these techniques, which require only simple instrumentation, available in many labs, as well as reasonably facile experimental implementation and analysis.

Bending Rigidity, Capacitance, and Shear Viscosity of Giant Vesicle Membranes Prepared by Spontaneous Swelling, Electroformation, Gel-Assisted, and Phase Transfer Methods: A Comparative Study.

Closed lipid bilayers in the form of giant unilamellar vesicles (GUVs) are commonly used membrane models. Various methods have been developed to prepare GUVs, however it is unknown if all approaches yield membranes with the same elastic, electric, and rheological properties. Here, we combine flickering spectroscopy and electrodefomation of GUVs to measure, at identical conditions, membrane capacitance, bending rigidity and shear surface viscosity of palmitoyloleoylphosphatidylcholine (POPC) membranes formed by several commonly used preparation methods: thin film hydration (spontaneous swelling), electroformation, gel-assisted swelling using poly(vinyl alcohol) (PVA) or agarose, and phase-transfer. We find relatively similar bending rigidity value across all the methods except for the agarose hydration method. In addition, the capacitance values are similar except for vesicles prepared via PVA gel hydration. Intriguingly, membranes prepared by the gel-assisted and phase-transfer methods exhibit much higher shear viscosity compared to electroformation and spontaneous swelling, likely due to remnants of polymers (PVA and agarose) and oils (hexadecane and mineral) in the lipid bilayer structure.

A vesicle microrheometer for high-throughput viscosity measurements of lipid and polymer membranes.

Viscosity is a key property of cell membranes that controls mobility of embedded proteins and membrane remodeling. Measuring it is challenging because existing approaches involve complex experimental designs and/or models, and the applicability of some methods is limited to specific systems and membrane compositions. As a result there is scarcity of systematic data, and the reported values for membrane viscosity vary by orders of magnitude for the same system. Here, we show how viscosity of membranes can be easily obtained from the transient deformation of giant unilamellar vesicles. The approach enables a noninvasive, probe-independent, and high-throughput measurement of the viscosity of membranes made of lipids or polymers with a wide range of compositions and phase state. Using this novel method, we have collected a significant amount of data that provides insights into the relation between membrane viscosity, composition, and structure.

Electromechanical characterization of biomimetic membranes using electrodeformation of vesicles.

We describe a facile method to simultaneously measure the bending rigidity and capacitance of biomimetic lipid bilayers. Our approach utilizes the ellipsoidal deformation of quasi-spherical giant unilamellar vesicles induced by a uniform AC electric field. Vesicle shape depends on the electric field frequency and amplitude. Membrane bending rigidity can be obtained from the variation of the vesicle elongation on either field amplitude at fixed frequency or frequency at fixed field amplitude. Membrane capacitance is determined from the frequency at which the vesicle shape changes from prolate to oblate ellipsoid as the frequency is increased at a given field amplitude.

Torsional fracture of viscoelastic liquid bridges.

Short liquid bridges are stable under the action of surface tension. In applications like electronic packaging, food engineering, and additive manufacturing, this poses challenges to the clean and fast dispensing of viscoelastic fluids. Here, we investigate how viscoelastic liquid bridges can be destabilized by torsion. By combining high-speed imaging and numerical simulation, we show that concave surfaces of liquid bridges can localize shear, in turn localizing normal stresses and making the surface more concave. Such positive feedback creates an indent, which propagates toward the center and leads to breakup of the liquid bridge. The indent formation mechanism closely resembles edge fracture, an often undesired viscoelastic flow instability characterized by the sudden indentation of the fluid's free surface when the fluid is subjected to shear. By applying torsion, even short, capillary stable liquid bridges can be broken in the order of 1 s. This may lead to the development of dispensing protocols that reduce substrate contamination by the satellite droplets and long capillary tails formed by capillary retraction, which is the current mainstream industrial method for destabilizing viscoelastic liquid bridges.

EPA and DHA differentially modulate membrane elasticity in the presence of cholesterol.

Polyunsaturated fatty acids (PUFAs) modify the activity of a wide range of membrane proteins and are increasingly hypothesized to modulate protein activity by indirectly altering membrane physical properties. Among the various physical properties affected by PUFAs, the membrane area expansion modulus (Ka), which measures membrane strain in response to applied force, is expected to be a significant controller of channel activity. Yet, the impact of PUFAs on membrane Ka has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent Ka (Kapp) of phospholipid model membranes containing nonesterified fatty acids. First, we measured membrane Kapp as a function of the location of the unsaturated bonds and degree of unsaturation in the incorporated fatty acids and found that Kapp generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the Kapp of membranes containing cholesterol. In vesicles prepared with high amounts of cholesterol, which should increase the propensity of the membrane to phase segregate, we found that inclusion of DHA decreases the Kapp in comparison to EPA. We also measured how these ω-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane Kapp changes in relation to these other physical properties. Our study shows that PUFAs generally decrease the Kapp of membranes and that EPA and DHA have differential effects on Kapp when membranes contain higher levels of cholesterol. Our results suggest membrane phase behavior and the distribution of membrane-elasticizing amphiphiles impact the ability of a membrane to stretch.

Fluctuation spectroscopy of giant unilamellar vesicles using confocal and phase contrast microscopy.

A widely used method to measure the bending rigidity of bilayer membranes is fluctuation spectroscopy, which analyses the thermally-driven membrane undulations of giant unilamellar vesicles recorded with either phase-contrast or confocal microscopy. Here, we analyze the fluctuations of the same vesicle using both techniques and obtain consistent values for the bending modulus. We discuss the factors that may lead to discrepancies.

The Neutrally Charged Diarylurea Compound PQ401 Kills Antibiotic-Resistant and Antibiotic-Tolerant Staphylococcus aureus.

Resistance or tolerance to traditional antibiotics is a challenging issue in antimicrobial chemotherapy. Moreover, traditional bactericidal antibiotics kill only actively growing bacterial cells, whereas nongrowing metabolically inactive cells are tolerant to and therefore "persist" in the presence of legacy antibiotics. Here, we report that the diarylurea derivative PQ401, previously characterized as an inhibitor of the insulin-like growth factor I receptor, kills both antibiotic-resistant and nongrowing antibiotic-tolerant methicillin-resistant Staphylococcus aureus (MRSA) by lipid bilayer disruption. PQ401 showed several beneficial properties as an antimicrobial lead compound, including rapid killing kinetics, low probability for resistance development, high selectivity to bacterial membranes compared to mammalian membranes, and synergism with gentamicin. In contrast to well-studied membrane-disrupting cationic antimicrobial low-molecular-weight compounds and peptides, molecular dynamic simulations supported by efficacy data demonstrate that the neutral form of PQ401 penetrates and subsequently embeds into bacterial lipid bilayers more effectively than the cationic form. Lastly, PQ401 showed efficacy in both the Caenorhabditis elegans and Galleria mellonella models of MRSA infection. These data suggest that PQ401 may be a lead candidate for repurposing as a membrane-active antimicrobial and has potential for further development as a human antibacterial therapeutic for difficult-to-treat infections caused by both drug-resistant and -tolerant S. aureusIMPORTANCE Membrane-damaging antimicrobial agents have great potential to treat multidrug-resistant or multidrug-tolerant bacteria against which conventional antibiotics are not effective. However, their therapeutic applications are often hampered due to their low selectivity to bacterial over mammalian membranes or their potential for cross-resistance to a broad spectrum of cationic membrane-active antimicrobial agents. We discovered that the diarylurea derivative compound PQ401 has antimicrobial potency against multidrug-resistant and multidrug-tolerant Staphylococcus aureus PQ401 selectively disrupts bacterial membrane lipid bilayers in comparison to mammalian membranes. Unlike cationic membrane-active antimicrobials, the neutral form of PQ401 rather than its cationic form exhibits maximum membrane activity. Overall, our results demonstrate that PQ401 could be a promising lead compound that overcomes the current limitations of membrane selectivity and cross-resistance. Also, this work provides deeper insight into the design and development of new noncharged membrane-targeting therapeutics to combat hard-to-cure bacterial infections.

Bending rigidity of charged lipid bilayer membranes.

We experimentally investigate the effect of lipid charge on the stiffness of bilayer membranes. The bending rigidity of membranes with composition 0-100 mol% of charged lipids, in the absence and presence of salt at different concentrations, is measured with the flicker spectroscopy method, using the shape fluctuations of giant unilamellar vesicles. The analysis considers both the mean squared amplitudes and the time autocorrelations of the shape modes. Our results show that membrane charge increases the bending rigidity relative to the charge-free membrane. The effect is diminished by the addition of monovalent salt to the suspending solutions. The trend shown by the membrane bending rigidity correlates with zeta potential measurements, confirming charge screening at different salt concentrations. The experimental results in the presence of salt are in good agreement with existing theories of membrane stiffening by surface charge.

A selective membrane-targeting repurposed antibiotic with activity against persistent methicillin-resistant Staphylococcus aureus.

Treatment of Staphylococcus aureus infections is complicated by the development of antibiotic tolerance, a consequence of the ability of S. aureus to enter into a nongrowing, dormant state in which the organisms are referred to as persisters. We report that the clinically approved anthelmintic agent bithionol kills methicillin-resistant S. aureus (MRSA) persister cells, which correlates with its ability to disrupt the integrity of Gram-positive bacterial membranes. Critically, bithionol exhibits significant selectivity for bacterial compared with mammalian cell membranes. All-atom molecular dynamics (MD) simulations demonstrate that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterol-rich mammalian-mimic lipid bilayers. In addition to causing rapid membrane permeabilization, the insertion of bithionol increases membrane fluidity. By using bithionol and nTZDpa (another membrane-active antimicrobial agent), as well as analogs of these compounds, we show that the activity of membrane-active compounds against MRSA persisters positively correlates with their ability to increase membrane fluidity, thereby establishing an accurate biophysical indicator for estimating antipersister potency. Finally, we demonstrate that, in combination with gentamicin, bithionol effectively reduces bacterial burdens in a mouse model of chronic deep-seated MRSA infection. This work highlights the potential repurposing of bithionol as an antipersister therapeutic agent.