The membrane transporter lactose permease increases lipid bilayer bending rigidity.

Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC/DOPE/DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.

Recurrent dynamics of rupture transitions of giant lipid vesicles at solid surfaces.

Single giant unilamellar vesicles (GUVs) rupture spontaneously from their salt-laden suspension onto solid surfaces. At hydrophobic surfaces, the GUVs rupture via a recurrent, bouncing ball rhythm. During each contact, the GUVs, rendered tense by the substrate interactions, porate, and spread a molecularly transformed motif of a monomolecular layer on the hydrophobic surface from the point of contact in a symmetric manner. The competition from pore closure, however, limits the spreading and produces a daughter vesicle, which re-engages with the substrate. At solid hydrophilic surfaces, by contrast, GUVs rupture via a distinctly different recurrent burst-heal dynamics; during burst, single pores nucleate at the contact boundary of the adhering vesicles, facilitating asymmetric spreading and producing a "heart"-shaped membrane patch. During the healing phase, the competing pore closure produces a daughter vesicle. In both cases, the pattern of burst-reseal events repeats multiple times, splashing and spreading the vesicular fragments as bilayer patches at the solid surface in a pulsatory manner. These remarkable recurrent dynamics arise, not because of the elastic properties of the solid surface, but because the competition between membrane spreading and pore healing, prompted by the surface-energy-dependent adhesion, determine the course of the topological transition.

Coupling Phase Behavior of Fatty Acid Containing Membranes to Membrane Bio-Mechanics.

Biological membranes constantly modulate their fluidity for proper functioning of the cell. Modulation of membrane properties via regulation of fatty acid composition has gained a renewed interest owing to its relevance in endocytosis, endoplasmic reticulum membrane homeostasis, and adaptation mechanisms in the deep sea. Endowed with significant degrees of freedom, the presence of free fatty acids can alter the curvature of membranes which in turn can alter the response of curvature sensing proteins, thus defining adaptive ways to reconfigure membranes. Most significantly, recent experiments demonstrated that polyunsaturated lipids facilitate membrane bending and fission by endocytic proteins - the first step in the biogenesis of synaptic vesicles. Despite the vital roles of fatty acids, a systematic study relating the interactions between fatty acids and membrane and the consequent effect on the bio-mechanics of membranes under the influence of fatty acids has been sparse. Of specific interest is the vast disparity in the properties of cis and trans fatty acids, that only differ in the orientation of the double bond and yet have entirely unique and opposing chemical properties. Here we demonstrate a combined X-ray diffraction and membrane fluctuation analysis method to couple the structural properties to the biophysical properties of fatty acid-laden membranes to address current gaps in our understanding. By systematically doping pure dioleoyl phosphatidylcholine (DOPC) membranes with cis fatty acid and trans fatty acid we demonstrate that the presence of fatty acids doesn't always fluidize the membrane. Rather, an intricate balance between the curvature, molecular interactions, as well as the amount of specific fatty acid dictates the fluidity of membranes. Lower concentrations are dominated by the nature of interactions between the phospholipid and the fatty acids. Trans fatty acid increases the rigidity while decreasing the area per lipid similar to the properties depicted by the addition of saturated fatty acids to lipidic membranes. Cis fatty acid however displays the accepted view of having a fluidizing effect at small concentrations. At higher concentrations curvature frustration dominates, leading to increased rigidity irrespective of the type of fatty acid. These results are consistent with theoretical predictions as detailed in the manuscript.

Permeability and Line-Tension-Dependent Response of Polyunsaturated Membranes to Osmotic Stresses.

The lipidome of plant plasma membranes-enriched in cellular phospholipids containing at least one polyunsaturated fatty acid tail and a variety of phytosterols and phytosphingolipids-is adapted to significant abiotic stresses. But how mesoscale membrane properties of these membranes such as permeability and deformability, which arise from their unique molecular compositions and corresponding lateral organization, facilitate response to global mechanical stresses is largely unknown. Here, using giant vesicles reconstituting mixtures of polyunsaturated lipids (soy phosphatidylcholine), glucosylceramide, and sitosterol common to plant membranes, we find that the membranes adopt "janus-like" domain morphologies and display anomalous solute permeabilities. The former textures the membrane with a single sterol-glucosylceramide-enriched, liquid-ordered domain separated from a liquid-disordered phase consisting primarily of soy phosphatidylcholine. When subject to osmotic downshifts, the giant unilamellar vesicles (GUVs) respond by transiently producing well-known swell-burst cycles. In each cycle, the influx of water swells the GUV, rendering the membrane tense. Subsequent rupture of the membrane through transient poration, which localizes in the liquid-disordered phase or at the domain boundaries, reduces the osmotic stress by expelling some of the excess osmolytes (and solvent) before sealing. When subject to abrupt hypertonic stress, they deform by nucleating buds at the domain phase boundaries. Remarkably, this incipient vesiculation is reversed in a statistically significant fraction of GUVs because of the interplay with solute permeation timescales, which render osmotic stresses short-lived. This, then, suggests a novel control mechanism in which an interplay of permeability and deformability regulates osmotically induced membrane deformation and limits vesiculation-induced loss of membrane material. Interestingly, recapitulation of such dynamic morphological reconfigurability-switching between budded and nonbudded morphologies-due to the interplay of membrane permeability, which temporally reverses the osmotic gradient, and domain boundaries, which select modes of deformations, might prove valuable in endowing synthetic cells with novel morphological responsiveness.

Dependence of norfloxacin diffusion across bilayers on lipid composition.

Antibiotic resistance is a growing concern in medicine and raises the need to develop and design new drug molecules that can efficiently inhibit bacterial replication. Spurring the passive uptake of the drug molecules is an obvious solution. However our limited understanding of drug-membrane interactions due to the presence of an overwhelming variety of lipids constituting cellular membranes and the lack of facile tools to probe the bio-physical interactions between drugs and lipids imposes a major challenge towards developing new drug molecules that can enter the cell via passive diffusion. Here, we used a label-free micro-fluidic platform combined with giant unilamellar lipid vesicles to investigate the permeability of membranes containing mixtures of DOPE and DOPG in DOPC, leading to a label-free measurement of passive membrane-permeability of autofluorescent antibiotics. A fluoroquinolone drug, norfloxacin was used as a case study. Our results indicate that the diffusion of norfloxacin is strongly dependent on the lipid composition which is not expected from the traditional octanol-lipid partition co-efficient assay. The anionic lipid, DOPG, slows the diffusion process whereas the diffusion across liposomes containing DOPE increases with higher DOPE concentration. Our findings emphasise the need to investigate drug-membrane interactions with focus on the specificity of drugs to lipids for efficient drug delivery, drug encapsulation and targeted drug-delivery.

Influence of High Pressure on the Bending Rigidity of Model Membranes.

Curvature is a fundamental lipid membrane property that influences many membrane-mediated biological processes and dynamic soft materials. One of the key parameters that determines the energetics of curvature change is the membrane bending rigidity. Understanding the intrinsic effect of pressure on membrane bending is critical to understanding the adaptation and structural behavior of biomembranes in deep-sea organisms as well as soft material processing. However, it has not previously been possible to measure the influence of high hydrostatic pressure on membrane bending energetics, and this bottleneck has primarily been due to a lack of technology platforms for performing such measurements. We have developed a new high-pressure microscopy cell which, combined with vesicle fluctuation analysis, has allowed us to make the first measurements of membrane bending rigidity as a function of pressure. Our results show a significant increase in bending rigidity at pressures up to 40 MPa. Above 40 MPa, the membrane mechanics become more complex. Corresponding small and wide-angle X-ray diffraction shows an increase in density and thickness of the bilayer with increasing pressure which correlates with the micromechanical measurements. These results are consistent with recent theoretical predictions of the bending rigidity as a function of hydrocarbon chain density. This technology has the potential to transform our quantitative understanding of the role of pressure in soft material processing, the structural behavior of biomembranes, and the adaptation mechanisms employed by deep-sea organisms.

Measurements of the effect of membrane asymmetry on the mechanical properties of lipid bilayers.

We detail an approach for constructing asymmetric membranes and characterising their mechanical properties, leading to the first measurement of the effect of asymmetry on lipid bilayer mechanics. Our results demonstrate that asymmetry induces a significant increase in rigidity compared to symmetric membranes. Given that all biological membranes are asymmetric our findings have profound implications for the role of this phenomenon in biology.