Session B36: Biomembranes

Specific Ion Effects and Solvation at Metastable Biomimetic Interfaces

Interfaces host a range of phenomena found in a diverse range of fields spanning chemical separations, soft matter electronics and biological function. While it is known that these interfaces are important, there is a surprising lack of insight into how ionic and interfacial constituents self-assemble into emergent structures capable of these functions. Here we show, using surface specific vibrational spectroscopy, how specific ion effects and associated changes to interfacial hydrogen bonding networks can play a key role in the formation of metastable assemblies responsible for memcapacitive behaviors and long-term potentiation in biomimetic membranes.   

Coherent lipid acyl tail correlation dynamics measured using polarized quasi-elastic neutron scattering

Lipid molecules self-assemble in aqueous environment to form bilayers. Although the elastic and viscous properties of the bilayers are controlled by molecular orientations and dynamics, detailed mechanisms of how macroscopic membrane properties relate to the molecular scale structure and dynamics. In a previous study, the acyl tail correlation dynamics represented two characteristic relaxation processes in the fluid phase, where the fast dynamics related to the density fluctuations of acyl tails of a lipid molecule while the slower dynamics was considered to the structural relaxation of lipid molecules. The relaxation times were used to estimate membrane viscosity and the extracted values were close to the average of the reported values in literature. In order to further understanding the microscopic origins of membrane viscosity, more detailed analysis of neutron scattering data were performed. Namely, components of distinct and self-correlations of the acyl tail dynamics are separated by employing polarized quasi-elastic neutron scattering techniques.   

Physical Response of Pseudomonas Aeruginosa Outer Membrane to Signaling Molecule Insertion

Gram-negative bacteria utilize various signaling molecules to increase their chances of survival. Involved in quorum sensing, these small molecules help bacteria with nutrient acquisition, virulence factor production, and inter-cell communication. Moreover, signaling molecules can enhance cells’ ability to resist antibiotics. The Pseudomonas Quinolone Signal (PQS), a prominent signaling molecule for the Pseudomonas aeruginosa bacteria, enables additional protection by facilitating outer membrane vesicles’ formation, which contributes to a defensive barrier for the bacterium. Using an accurate in silico model, this work explores how PQS interaction varies physical properties of P. aeruginosa outer membrane. The model membrane is constructed using all-atom molecular dynamics simulation with an asymmetrical composition, a distinct feature for gram-negative bacteria. The inner leaflet comprises a POPE and POPG phospholipids mixture, while the outer leaflet contains PA14 Lipid A exclusively. PQS spontaneously intercalates into this membrane due to its strong hydrophobicity and specific interactions with Lipid A. We investigate the membrane properties post-intercalation as a function of PQS concentration in the outer leaflet. The structural analysis shows that while PQS does not vary the order parameter of inner leaflet acyl chains, it does enhance the outer leaflet ordering. This enhancement correlates with membrane differential stress, indicating the crowding of the outer leaflet after the PQS intercalation. In addition, we probe the influence of PQS addition on membrane deformability, particularly the effect of PQS concentration on mechanical attributes like compressibility and bending moduli. These insights provide a deeper understanding of PQS’s role and impact on membrane physical properties, contributing valuable information to the field.   

Biological tuning of the membrane phase transition facilitates plasma membrane organization and function.

Isolated cell plasma membranes are biologically tuned to be in a single phase at growth temperature but close to a critical point of the membrane phase transition.  This talk will explore several consequences of this biological tuning through experiments in model and intact cell membranes. For example, near-critical membranes have a high compositional susceptibility, meaning that stable membrane domains can assemble in response to membrane proximal forces.  This is demonstrated in live B cells through quantitative super-resolution nanoscopy measurements that detect the emergence of functional domains upon B cell receptor clustering.  Near-critical tuning of the membrane phase transition can also enhance the stability of proteins condensed at membranes, and this is demonstrated through simulation and experiments in model and cellular systems.   

Myosin-driven mechanical stress triggers curvature-dependent membrane phase separation within cell-like model blebs

The membrane phase behavior in the cell plasma membrane plays pivotal physical and biochemical roles in controlling membrane stability, rigidity, viral infection, and ion channel functions. The physical interaction between the plasma membrane and the underlying cytoskeleton has been hypothesized to be a crucial regulator of membrane phase behaviors; however, the impact of membrane deformation on membrane phase behaviors, coupled with cytoskeleton-generated mechanical stress, remains unclear. Here, by constructing a membrane-bound actomyosin layer within liposomes, we demonstrate that myosin contractility drives cortical actin flow and cell-like blebbing, leading to membrane phase separation within blebs. Through laser ablation of the actin cortex and physical modeling, we established the mechanical link between the actomyosin cortex and the membrane. Remarkably, laser ablation-induced blebbing revealed that cell-like blebs accumulate more cholesterol than the mother liposome. Furthermore, we observed that cholesterol accumulation increases as bleb size decreases, indicating a curvature-dependent redistribution of cholesterol within the membrane. These results provide valuable mechanical insights into the coordination among cytoskeleton-generated stress, membrane curvature, and membrane phase separation. This study sheds light on membrane phase behaviors regulated by cytoskeletal forces in mammalian cells.   

Localization of lipid density-sensing proteins in fluctuating biomembranes

Proteins like septins that can sense biomembrane curvature on the micron-scale have amphipathic helices that embed themselves into the membrane bilayer. These proteins may be sensing curvature by detecting packing defects in the arrangement of lipids in the membrane. As an initial step to model this lipid defect sensing, we model a diffusing membrane protein with an energetic preference for a particular local lipid density, as well as bending and compressibility moduli that differ from the host membrane. This extends our earlier work on continuum simulations of fluctuating membrane height and lipid density using Fourier Space Brownian Dynamics. We focus on a membrane adhered to a sinusoidal substrate, which induces curvature and leads to differences in the lipid number densities projected by the upper and lower leaflets of the bilayer. Using our simulations, we obtain probability distributions of the protein's position relative to the substrate. We study the dependence of protein localization on various physical parameters of the membrane-protein system. In particular, we investigate how a protein's area compressibility modulus relative to the membrane's can change the protein's ability to localize to regions of the membrane with its preferred lipid density.   

Membrane fusion as a pathway to fission

Remodeling of biological membranes, such as fusion and fission, is involved in a variety of basic, cellular processes. This work investigates the mechanisms and pathways for the fission of phospholipid membranes, in particular double-membrane fission as it occurs in mitochondrial division. We employ self-consistent field theory and utilize the string method to find the Minimum Free Energy Path (MFEP) in order to determine the most likely pathway for the transition. The complex landscape of possible rearrangements gives rise to multiple possible mechanisms for double membrane fission. The simplest pathway involves the local constriction, hemifusion and fission of the inner membrane, without contact with the outer membrane. Intriguingly, we also uncover a new mechanism whereby local fusion contact between the inner and outer membrane can catalyze the fission of the inner membrane. Not only does the new mechanism have a lower total free energy barrier, but also an intermediate metastable state, allowing the system to ratchet its way up the rate-limiting step.   

Interplay between elastic and structural properties in cholesterol-rich lipid membranes

Cholesterol is a vital component of cell membranes and a common additive in artificial lipid membranes. Structurally, cholesterol is known to increase lipid ordering and decrease the area per lipid, A_L, in fluid lipid membranes. Despite these unified structural effects, cholesterol’s influence on the membrane bending modulus, κ,<!--[if gte msEquation 12]> style='mso-bidi-font-style:normal'>_κ, has been reported to vary with lipid chain unsaturation – presenting a dilemma for structure-property relations. To address this, we examined the interdependence of cholesterol-driven changes in structural and elastic membrane properties on mesoscopic scales using neutron spin-echo spectroscopy, combined with MD simulations and solid-state ²H NMR spectroscopy. Our results clearly show that cholesterol stiffens all studied membranes, irrespective of chain unsaturation. More importantly, by mapping these observations to those from small-angle X-ray scattering studies, we find that the measured changes in κ<!--[if gte msEquation 12]> style='mso-bidi-font-style:normal'>_κ, normalized against cholesterol-free membranes, correlate with changes in A_L via a universal scaling law. Such scaling relations are transformative on biological and technological fronts, enabling predictions of elastic properties of complex biological membranes and accelerating the design rules of engineered membranes with tunable functionalities.   

Polysaccharide functionalization reduces lipid vesicle stiffness

The biophysical properties of lipid vesicles are important for their stability and integrity; they are also important for controlling the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering of polymers to the membrane. Typically, synthetic polymers like polyethylene glycol are used to increase vesicle stability but polysaccharides are much less explored. Here, we report a general method to functionalize lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. Furthermore, we explore the effect of polysaccharide functionalization of lipid vesicles for drug delivery. We find that it increases the uptake of small unilamellar vesicles (SUVs) by cells and leads to an improved transfection. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the biophysical properties of drug delivery vehicles.   

X-ray Standing-Wave Fluorescence study of the transverse distribution of cholesterol within a supported phospholipid bilayer.

Abstract:

Standing wave x-ray fluorescence (SWXF) was used to measure the distribution of cholesterol transverse to the support plane of a phospholipid bilayer. In order to monitor the position of the cholesterol, the molecule was brominated near the region of the hydrophilic head group and the fluorescence intensity from the bromine was monitored as a function of the phase of the x-ray standing wave maxima relative to the support surface. The supporting surface was comprised of a Si/Mo multilayer with a repeat spacing chosen to be approximatley the bilayer thickness. Results revealed that in the gel phase, fluorescence is best described if the cholesterol is uniformly distributed throughout the bilayer rather than localized near the hydrophilic interfaces, which is in agreement with neutron scattering results of Marquardt³.   

The Effects of Propofol, Sevoflurane and Isoflurane on Lipid Membrane Fluidity at Clinic Concentrations

In this study, we compare the effects of three clinically significant anesthetic drugs: propofol, sevoflurane and isoflurane, on lipid membrane fluidity. To get complete picture, two representative model lipid membrane systems (pure DPPC and a 5-lipids mixture that mimics brain endothelial cell membrane) and red blood cells were chosen. Lipid membrane systems were labeled with Dipyrene-PC fluorescent probe, whose excimer/monomer (E/M) fluorescence peak ratio showed an immediate increase after adding the drugs, indicating a sharp increase of membrane fluidity. We studied clinical concentrations of 10µM propofol, 0.5mM sevoflurane and 1mM isoflurane. The fluidity increase at these concentrations on DPPC lipid bilayer are similar, and all three drugs are quite effective to loosen up the highly ordered lipid domains of saturated lipids. The supra-clinical concentrations of these drugs, 100µM propofol, 2mM sevoflurane and 5mM isoflurane, have also been examined. The magnitude of increases of E/M ratio in the 5-lipids system were smaller than that in DPPC bilayer. Furthermore, washed human red blood cells (RBC) were labeled with TMA-DPH fluorescent probe and fluorescence anisotropy measurements were carried out. At clinical concentrations, the decreases of TMA-DPH anisotropy were comparable for isoflurane and sevoflurane, and the effects are more than that of 174mM ethanol, which is ten times the legal alcohol limit level in human blood. However, the anisotropy increased after adding propofol, likely due the binding of propofol to certain proteins in RBC. All these findings depict that these anesthetic drugs at clinical concentrations have similar effects on a wide range of lipid membrane systems, and they significantly and rapidly increase lipid membrane fluidity.   

Scattering Simulations from Laterally Heterogeneous Lipid Vesicles

Lipid phase separation is a functional hallmark of cellular membranes and an attractive feature in lipid membrane applications. In cell membranes, lipids of different species partition into lateral heterogeneities, or domains, with contrasting structural and elastic properties. The manifestation of this phase separation on the nanoscale has remained rather elusive, with few techniques available for examination. Of these, small angle neutron scattering (SANS) has become an invaluable tool, sensing not only structural differences between the domains and matrix but also differences in lipid partitioning within emergent phases. Here, we expand on earlier scattering simulations employing circular domain morphology to more general domain configurations which have been previously observed in multicomponent lipid membranes. Combining SANS measurements with simulations, we determine the morphology and composition of lipid domains on spherical lipid vesicles composed of biological and engineered lipid species. Furthermore, we investigate how domain morphology influences the resulting scattering signals in response to compositional variations and molecular substitutions. These developed capabilities have far-reaching implications in artificial cell technologies and soft membrane designs.