Session B41: Lipid Bilayers and Biological Membranes

Computer Simulation of Cytoskeleton-Induced Blebbing of Lipid Membranes

Blebs are balloon-shaped membrane protrusions that form during many physiological processes such as cytokinesis, cell motility and apoptosis. Using computer simulation of a particle-based model for self-assembled lipid bilayers coupled to an elastic meshwork, we investigated the phase behavior and kinetics of blebbing. We found that for small values of the mismatch parameter, defined as the ratio between the area of the lipid bilayer divided by the rest area of the cytoskeleton, the equilibrium state is that of a homogeneous vesicle with the cytoskeleton conforming to the bilayer. However, for large values of a mismatch parameter, the equilibrium state is that of a blebbed vesicle. We also found that blebbing can be induced when the cytoskeleton is subject to a localized ablation or a uniform compression. The obtained results are qualitatively in agreement with the experimental evidence and the model opens up the possibility to study the kinetics of bleb formation in detail. For more information see Spangler {\em et al.}, Phys. Rev. E {\bf 84}, 051906 (2011).   

Coarse-grained simulation of lipid vesicles with ``n-atic'' orientational order

We perform coarse-grained simulation studies of fluid lipid vesicles with in-plane ``n-atic'' orientational order associated with the shape of lipid head group, to test the theoretical predictions of Park, Lubensky and MacKintosh [1] for resulting vesicle shape and defect structures. Our simulation model uses a single layer coarse-grained implicit-solvent approach proposed by Yuan et al [2], with addition of an extra vector degree of freedom representing in-plane orientational order. We carry out simulation studies for n=1 to 6, examining in each case the spatial distribution of defects and resulting deformation of the vesicle. An initially spherical vesicle (genus zero) with n-atic order has a ground state with 2n vortices of strength 1/n, as expected, but the observed equilibrium shapes are sometimes quite different from those predicted theoretically. For the n=1 case, we find that the vesicle may become trapped in a disordered, long-lived metastable state with extra +/- defects whose pair-annihilation is inhibited by local changes in membrane curvature, and thus may never reach its predicted ground state. \\[4pt] [1] J. Park, T. C. Lubensky, and F. C. MacKintosh, Europhys. Lett. 20, 279 (1992)\\[0pt] [2] H. Yuan, C. Huang, Ju Li, G. Lykotrafitis, and S. Zhang, Phys. Rev. E 82, 011905 (2010)   

Morphologies of Elastic Membranes with Fluctuating Connectivity

We numerically investigate the effects of topological defects in single-component two-dimensional elastic membranes with spherical topology allowing changes in shape. The membrane is simulated as a closed, triangulated elastic surface in three dimensions, where the vertices are permitted to move in space and the connectivity of the triangulation is able to fluctuate. Fluctuations in connectivity allow the creation of topological defects. A Monte Carlo simulated annealing method is utilized to explore optimal shapes and connectivities. The familiar defect-driven buckling transition [Seung \& Nelson, PRA 1988, 38:1005] from a sphere to an icosahedron is shifted as a result of the fluctuating connectivity.   

Cooperative Motion in Lipid Bilayer Membranes

Lipid bilayer membranes, like the cell membrane, are complex biological systems. Transport of specific molecules in and out of cells are controlled by these membranes. Therefore, it is vital to understand the detailed dynamics that ultimately control membrane transport. We use molecular dynamics simulations of a coarse-grained and solvent-free lipid model that has been previously shown to spontaneously assemble a bilayer structure. Approaching the crossover to the gel-like state of the bilayer, the lipid dynamics become extremely slow. We analyze the cooperativity of the lipid motion and compare it with the cooperativity that has been well-characterized in liquids nearing a glass transition. Future simulations will examine the generality of this behavior using more realistic models, and comparing with experiments.   

How the antimicrobial peptides destroy bacteria cell membrane: Translocations vs. membrane buckling

In this study, coarse grained Dissipative Particle Dynamics simulation with implementation of electrostatic interactions is developed in constant pressure and surface tension ensemble to elucidate how the antimicrobial peptide molecules affect bilayer cell membrane structure and kill bacteria. We find that peptides with different chemical-physical properties exhibit different membrane obstructing mechanisms. Peptide molecules can destroy vital functions of the affected bacteria by translocating across their membranes via worm-holes, or by associating with membrane lipids to form hydrophilic cores trapped inside the hydrophobic domain of the membranes. In the latter scenario, the affected membranes are strongly corrugated (buckled) in accord with very recent experimental observations [G. E. Fantner \textit{et al.}, Nat. Nanotech., 5 (2010), pp. 280-285].   

Interaction of PLGA and trimethyl chitosan modified PLGA nanoparticles with mixed anionic/zwitterionic phospholipid bilayers studied using molecular dynamics simulations

Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable polymer. Nanoparticles of PLGA are commonly used for drug delivery applications. The interaction of the nanoparticles with the cell membrane may influence the rate of their uptake by cells. Both PLGA and cell membranes are negatively charged, so adding positively charged polymers such as trimethyl chitosan (TMC) which adheres to the PLGA particles improves their cellular uptake. The interaction of 3 nm PLGA and TMC-modified-PLGA nanoparticles with lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine lipids was studied using molecular dynamics simulations. The free energy profiles as function of nanoparticles position along the normal direction to the bilayers were calculated, the distribution of phosphatidylserine lipids as a function of distance of the particle from the bilayer was calculated, and the time scale for particle motion in the directions parallel to the bilayer surface was estimated.   

Atomic Force Microscopy Study of Changes to the Mechanical Properties of \textit{Pseudomonas aeruginosa }Cells Induced By Antimicrobial Peptides

The cell envelope of Gram-negative bacteria plays a key role in the maintenance of cell shape and the selective transfer of small molecules in and out of the cell. Both the inner and outer membranes of the cell envelope can be major targets for antimicrobial peptides, which can ultimately compromise the mechanical integrity of the cell. We have applied a new, AFM-based creep deformation technique (1) to study changes to the mechanical properties of individual \textit{Pseudomonas aeruginosa }cells as a function of time of exposure to polymyxin B (PMB), a well-known cyclic antimicrobial peptide. The results can be understood in terms of simple viscoelastic models of arrangements of springs and dashpots. These measurements provide a direct measure of the mechanical integrity of the bacterial cell, and time-resolved creep deformation experiments reveal that the time of action for PMB is very fast (of the order of a minute). This measurement provides new insight into the mechanism of action of antimicrobial peptides. (1) V. Vadillo-Rodriguez, T. J. Beveridge, and J. R. Dutcher, \textit{J. Bacteriol}. \textbf{190}, 4225-4232 (2008).   

Interactions between HIV-1 Neutralizing Antibodies and Model Lipid Membranes imaged with AFM

Lipid membrane interactions with rare, broadly neutralizing antibodies (NAbs), 2F5 and 4E10, play a critical role in HIV-1 neutralization. Our research is motivated by recent immunization studies that have shown that induction of antibodies that avidly bind the gp41-MPER antigen is not sufficient for neutralization. Rather, it is required that antigen designs induce polyreactive antibodies that recognize MPER antigens as well as the viral lipid membrane. However, the mechanistic details of how membrane properties influence NAb-lipid and NAb-antigen interactions remain unknown. Furthermore, it is well established that the native viral membrane is heterogeneous, representing a mosaic of lipid rafts and protein clustering. However, the size, physical properties, and dynamics of these regions are poorly characterized and their potential roles in HIV-1 neutralization are also unknown. To understand how membrane properties contribute to 2F5/4E10 membrane interactions, we have engineered biomimetic supported lipid bilayers (SLBs) and use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions at sub-nanometer z-resolution. Our results show that localized binding of HIV-1 antigens and NAbs occur preferentially with the most fluid membrane domain. This supports the theory that NAbs may interact with regions of low lateral lipid forces that allow antibody insertion into the bilayer.   

Modeling curvature-dependent subcellular localization of a small sporulation protein in Bacillus subtilis

Recent experiments suggest that in the bacterium, B. subtilis, the cue for the localization of small sporulation protein, SpoVM, that plays a central role in spore coat formation, is curvature of the bacterial plasma membrane. This curvature-dependent localization is puzzling given the orders of magnitude difference in lengthscale of an individual protein and radius of curvature of the membrane. Here we develop a minimal model to study the relationship between curvature-dependent membrane absorption of SpoVM and clustering of membrane-associated SpoVM and compare our results with experiments.   

Relationship between peptide amino acid sequence and membrane curvature generation

Amphipathic peptides and amphipathic domains in proteins can perturb and restructure biological membranes. For example, it is believed that the cationic, amphipathic motif found in membrane active antimicrobial peptides (AMPs) is responsible for their membrane disruption mechanisms of action. And ApoA-I, the main apolipoprotein in high density lipoprotein contains a series of amphipathic $\alpha $-helical repeats which are responsible for its lipid associating properties. We use small angle x-ray scattering (SAXS) to investigate the interaction of model cell membranes with prototypical AMPs and consensus peptides derived from the helical structural motif of ApoA-I. The relationship between peptide sequence and the peptide-induced changes in membrane curvature and topology is examined. By comparing the membrane rearrangement and corresponding phase behavior induced by these two distinct classes of membrane restructuring peptides we will discuss the role of amino acid sequence on membrane curvature generation.   

Family of pH-Low-Insertion-Peptides (pHLIPs)

pHLIP (pH (Low) Insertion Peptide) is a novel delivery system for targeting of acidic diseased tissue such as solid tumors, sites of inflammation, arthritis and other pathological states. The molecular mechanism of pHLIP action is based on pH-dependent insertion and folding of pHLIP in membrane. We performed sequence variation and investigated 16 pHLIP variants with main goals of understanding the main principles of peptide-lipid interactions and tune delivery capability of pHLIP. The biophysical studies including thermodynamics and kinetics of the peptides interaction with a lipid bilayer of liposomes and cellular membranes were carried out. We found that peptides association to membrane at neutral and low pH could be modulated by 3-4 times. The apparent pK of transition from surface bound to membrane-inserted state could be tuned from 6.5 to 4.5. The rate of peptide's insertion across a bilayer could be enhanced 100 times compared to parent pHLIP. As a result, blood clearance and tumor targeting were modulated in a significant degree. The work is supported by NIH grants CA133890 to OAA, DME, YRK.   

Membrane-associated peptide folding: pH triggered insertion and helical structure formation

We are interested in the molecular events that occur when a peptide inserts across a membrane or exits from it. pHLIP (pH (Low) Insertion Peptide) provides an opportunity to study membrane insertion/exit and folding/unfolding, since its insertion is modulated by pH and since it forms helical structure as it inserts. We found that pHLIP inserts across a POPC phospholipid bilayer in several steps: first is the rapid formation (100 ms) of an interfacial helix, which is then followed by a slow insertion pathway that contains several intermediates. We show that while the number of protonatable residues at the inserting end does not affect the formation of helical structure in the membrane, it correlates with the time for transmembrane insertion, the number of intermediate states on the folding pathway, and the rate of unfolding and exit. We conclude that particular intermediate states on the folding and unfolding pathways are not mandatory and, in the simple case of a polypeptide with a non-charged and non-polar inserting end, the folding and unfolding is well described as an all-or-none transition. A model for membrane-associated insertion/folding and exit/unfolding is proposed.   

Modulation of the transmembrane helix insertion pathway by polar cargo

In an earlier study, we found a series of kinetic steps in the pH-triggered insertion of the pHLIP{\textregistered} (pH (Low) Insertion Peptide). In the present work we observe that the polarity of the inserting end, including its cargo, modulates the number of intermediates, and that insertion can be described as a two state process for a simple case. Each investigated pHLIP variant preserve the pH-dependent properties of surface binding to membrane at neutral pH and insertion at low pH to form a transmembrane helix. However, there are thermodynamic and kinetic properties that are determined by the degree of cargo polarity. The presence of a polar cargo at the peptide's inserting end leads to the appearance of two additional intermediate states on the insertion pathway of the pHLIP-2E peptide, which itself (when no cargo is attached) shows an all-or-none transition from the partially unstructured membrane-surface to the transmembrane state described well by the two-state model at 800 ms timescale. We discuss the utility of our observations for the design of new delivery agents for the direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes. The work is supported by NIH grants CA133890 to OAA, DME, YRK.   

Exploration of phase separation in heterogeneous lipid monolayers

A Langmuir monolayer is a well established model of a single leaflet of a lipid membrane. In this work, we investigate the phase separation behavior of a model Langmuir monolayer as a function of both Langmuir surface pressure and ratio of saturated lipid : unsaturated lipid : cholesterol. The specifics of domain separation behavior, or ``rafting,'' in membranes are generally thought to be responsible for much of the behavior of living membranes, specifically in protein integration and transport. Off-specular x-ray scattering is used to probe in-plane structure of the membrane at the sub-micron scale. Additionally, atomic force microscopy imaging is taken on samples transferred to a rigid support. In-plane order is found to grow as a function of surface pressure. Also, the in-plane order is found to depend on cholesterol concentration in the monolayer. The phase space of the in-plane order as a function of lipid and cholesterol concentration is presented.   

Domain formation in multicomponent lipid bilayers coupled to elastic substrate

We will discuss the physics that governs the lipid localization and domain formation in multicomponent lipid bilayers coupled to an elastic substrate. Lipid localization and domain formation has been studied extensively in biological cell membranes. In this talk we will extend a previous model for membrane energetics to account for the coupling between the bending and the local lipid composition of the two leaflets. Our aim is to determine the relationship between the localization and domain formation in the presence of lipid flip-flops between the two leaflets and the effect of intrinsic curvature of the lipids. Using a lattice model for the membrane, we simulate the system and study the effect of lipid flip-flop on lipid organization in the membrane.