Registered and antiregistered phase separation of mixed amphiphilic bilayers

We derive a mean-field free energy for the phase behaviour of coupled bilayer leaflets, which is implicated in cellular processes and important to the design of artificial membranes. Our model accounts for amphiphile-level structural features, particularly hydrophobic mismatch, which promotes antiregistration (AR), in competition with the `direct' trans-midplane coupling usually studied, promoting registration (R). We show that the phase diagram of coupled leaflets allows multiple \textit{metastable} coexistences, then illustrate the kinetic implications with a detailed study of a bilayer of equimolar overall composition. For approximate parameters estimated to apply to phospholipids, equilibrium coexistence is typically registered, but metastable antiregistered phases can be kinetically favoured by hydrophobic mismatch. Thus a bilayer in the spinodal region can require nucleation to equilibrate, in a novel manifestation of Ostwald's `rule of stages'. Our results provide a framework for understanding disparate existing observations, elucidating a subtle competition of couplings, and a key role for phase transition kinetics in bilayer phase behaviour.Comment: Final authors' version. Important typo in Eq. A24 corrected. To appear in Biophysical Journa

Pattern formation in cell-sized membranes

The research presented in this dissertation follows in the tradition of experimental membrane biophysics. Our goal is to study the physical mechanisms underlying organization in the plasma membrane of living cells by using model systems. The central result from our experiments is that mixed-lipid membrane vesicles that are adhered by proteins to a solid-supported lipid membrane can dynamically form long-lived holes at the adhesion interface between the membranes. The first set of experiments we discuss exhibit the stable persistence of static patterns. The patterns are formed by adhering ternary-lipid vesicle membranes to a planar membrane supported on a solid, glass substrate \textit{via} biotin-avidin binding. The membrane and avidin are marked with spectrally distinct fluorescent dyes. We use fluorescence microscopy to acquire data. Adhesion causes half of adhered vesicles to form rough annular patterns with a central region that is devoid of membrane dye and protein binders. The peripheral region is dense in proteins and enriched in dye compared to the free, non-adhered portion of the same membrane. We measure the volume V and surface area A of adhered membranes. Using the measure 6[square root of pi]V/A[superscript 3/2] we find 0.84 for patterned and 0.98 for non-patterned membranes. Thus, adhered vesicles have two equilibrium states, one with annular patterns and one without, and the transition between them involves a loss of internal volume. Collectively our results suggest the annular patterns are holes. Finally, we report on a dynamic pattern that occurs in binary-lipid membranes adhered to a supported lipid bilayer. The pattern consists of finger-shaped holes that invade the protein-bound region. We show the characteristics of the fingers depend on the density [rho] of the protein binders in the adhered region: the width of static fingers [lambda] scales as [lambda] \sim\ [rho] and the rate of finger formation r, defined as the number of fingers that branch off from a boundary per unit time, scales as ln [r] \sim\ [rho]. Theoretically, we treat the formation of a finger as a thermally activated event occurring in a tense elastic film. The activation energy required to form a finger is approximately 3.5 kT, a biologically relevant energy scale.Physic

Protein Diffusion on Charged Membranes: A Dynamic Mean-Field Model Describes Time Evolution and Lipid Reorganization

As charged macromolecules adsorb and diffuse on cell membranes in a large variety of cell signaling processes, they can attract or repel oppositely charged lipids. This results in lateral membrane rearrangement and affects the dynamics of protein function. To address such processes quantitatively we introduce a dynamic mean-field scheme that allows self-consistent calculations of the equilibrium state of membrane-protein complexes after such lateral reorganization of the membrane components, and serves to probe kinetic details of the process. Applicable to membranes with heterogeneous compositions containing several types of lipids, this comprehensive method accounts for mobile salt ions and charged macromolecules in three dimensions, as well as for lateral demixing of charged and net-neutral lipids in the membrane plane. In our model, the mobility of membrane components is governed by the diffusion-like Cahn-Hilliard equation, while the local electrochemical potential is based on nonlinear Poisson-Boltzmann theory. We illustrate the method by applying it to the adsorption of the anionic polypeptide poly-Lysine on negatively charged lipid membranes composed of binary mixtures of neutral and monovalent lipids, or onto ternary mixtures of neutral, monovalent, and multivalent lipids. Consistent with previous calculations and experiments, our results show that at steady-state multivalent lipids (such as PIP2), but not monovalent lipid (such as phosphatidylserine), will segregate near the adsorbing macromolecules. To address the corresponding diffusion of the adsorbing protein in the membrane plane, we couple lipid mobility with the propagation of the adsorbing protein through a dynamic Monte Carlo scheme. We find that due to their higher mobility dictated by the electrochemical potential, multivalent lipids such as PIP2 more quickly segregate near oppositely charged proteins than do monovalent lipids, even though their diffusion constants may be similar. The segregation, in turn, slows protein diffusion, as lipids introduce an effective drag on the motion of the adsorbate. In contrast, monovalent lipids such as phosphatidylserine only weakly segregate, and the diffusions of protein and lipid remain largely uncorrelated

Sorting of Amphiphile Membrane Components in Curvature and Composition Gradients

Phase and shape heterogeneities in biomembranes are of functional importance. However, it is difficult to elucidate the roles membrane heterogeneities play in maintaining cellular function due to the complexity of biomembranes. Therefore, investigations of phase behavior and composition/curvature coupling in lipid and polymer model membranes offer some advantages. In this thesis, phase properties in lipid and polymer giant vesicles were studied. Line tension at the fluid/fluid phase boundary of giant lipid unilamellar vesicles was determined directly by micropipette aspiration, and found to be composition-dependent. Dynamics of calcium-induced domains within polyanionic vesicles subject to chemical stimuli were investigated, which revealed the strength of molecular interaction and suggested applications in triggered delivery. In addition, curvature sorting of lipids and proteins was examined. Lipid membrane tethers were pulled from giant unilamellar vesicles using two micropipettes and a bead. Tether radius can be controlled and measured in this system. By examining fluorescence intensity of labeled molecules as a function of curvature, we found that DiI dyes (lipid analogues with spontaneous curvatures) had no curvature preference down to radii of 10 nm. Theoretical calculation predicted that the distribution of small lipids was dominated by entropy instead of bending energy. However protein Cholera toxin subunit B was efficiently sorted away from the high positive curvature due to its negative spontaneous curvature. Bending stiffness was determined to decrease as curvature increased in homogeneous membranes with ternary lipid mixtures near a critical consulate point, revealing the strong preferential intermolecular interactions of such mixtures. In addition, diffusion controlled domain growth was observed in tethers pulled from phase-separated vesicles, which provides a new dynamic sorting principle for lipids and proteins in curvature gradients

Attachment of Rod-Like (BAR) Proteins and Membrane Shape

Previous studies have shown that cellular function depends on rod-like membrane proteins, among them Bin/Amphiphysin/Rvs (BAR) proteins may curve the membrane leading to physiologically important membrane invaginations and membrane protrusions. The membrane shaping induced by BAR proteins has a major role in various biological processes such as cell motility and cell growth. Different models of binding of BAR domains to the lipid bilayer are described. The binding includes hydrophobic insertion loops and electrostatic interactions between basic amino acids at the concave region of the BAR domain and negatively charged lipids. To shed light on the elusive binding dynamics, a novel experiment is proposed to expand the technique of single-molecule AFM for the traction of binding energy of a single BAR domain

Interaction of cyclic antimicrobial hexapeptides with model lipid membranes

Antimikrobielle Peptide gelten als vielversprechende Wirkstoffe für die Entwicklung neuer Antibiotika. Ein potentieller Wirkmechanismus antimikrobieller Peptide liegt in der Fähigkeit eine Entmischung der Lipide in der Lipidmembranen zu induzieren, auch Lipid-Clustering genannt. In dieser Arbeit haben wir versucht, die Randbedingungen des Lipid-Clusterings zu identifizieren. Wir haben die thermotropen Eigenschaften von binären und ternären Mischungen aus Phosphatidylethanolaminen, Phosphatidylglycerolen und Cardiolipin mit und ohne gebundenen zyklischen Hexapeptiden bestimmt. Die Hexapeptide bestanden aus drei positiv geladenen Aminosäuren und drei hydrophoben Aminosäuren in unterschiedlicher Aminosäureanordnungen. Die Ergebnisse zeigen, dass die Fähigkeit der Peptide, Lipidcluster zu induzieren von der Aminosäuresequenz und dem Mischverhalten der Lipide abhängt. Die Wirksamkeit dieser verschiedenen Hexapeptide, Cluster zu induzieren, korreliert dabei mit ihrer antimikrobiellen Aktivität.Antimicrobial peptides are promising agents for the development of novel antibiotics. A potential mechanism of action of antimicrobial peptides is the ability to induce lipid segregation in lipid membranes, also known as lipid clustering. In this work we tried to identify the boundary conditions of lipid clustering. We determined the thermotropic properties of binary and ternary mixtures of phosphatidylethanolamines, phosphatidylglycerols and cardiolipin, with and without bound cyclic hexapeptides. The hexapeptides contained three positively charged amino acids and three hydrophobic amino acids with varying amino acid arrangements. The results show that the ability of peptides to induce lipid clusters depends on the amino acid sequence and the lipid mixing behavior. The efficacy of these different hexapeptides to induce clusters was found to be correlated with their antimicrobial activity