Session B40: Lipid Bilayers and Biological Membranes: Dynamics and Thermodynamics

Correlating Anomalous Diffusion with Membrane Obstacle Structure Using Single Molecule Tracking and AFM

Anomalous diffusion has been observed abundantly in the plasma membrane, but the underlying mechanisms are still unclear. In general, it has not been possible to directly image the obstacles to diffusion in membranes, so the dynamics of diffusing particles are used to deduce the obstacle characteristics. We present a supported lipid bilayer system in which we characterized the anomalous diffusion of lipid molecules using single molecule tracking, while at the same time imaging the obstacles to diffusion with atomic force microscopy. To explain our experimental results, we performed lattice Monte Carlo simulations of tracer diffusion in the presence of the experimentally determined obstacle configurations. We correlate the observed anomalous diffusion with obstacle area fraction, fractal dimension and correlation length. We further discuss our results in the context of confinement models and the generating stochastic process.   

Tracking single Kv2.1 channels in live cells reveals anomalous subdiffusion and ergodicity breaking

The dynamic organization of the plasma membrane is responsible for essential cellular processes, such as receptor trafficking and signaling. By studying the dynamics of transmembrane proteins a greater understanding of these processes as a whole can be achieved. It is broadly observed that the diffusion pattern of membrane protein displays anomalous subdiffusion. However, the mechanisms responsible for this behavior are not yet established. We explore the dynamics of the voltage gated potassium channel Kv2.1 by using single-particle tracking. We analyze Kv2.1 channel trajectories in terms of the time and ensemble distributions of square displacements. Our results reveal that all Kv2.1 channels experience anomalous subdiffusion and we observe that the Kv2.1 diffusion pattern is non-ergodic. We further investigated the role of the actin cytoskeleton in these channel dynamics by applying actin depolymerizing drugs. It is seen that with the breakdown of the actin cytoskeleton the Kv2.1 channel trajectories recover ergodicity.   

Studies of molecular diffusion in single-supported bilayer lipid membranes at high hydration by quasielastic neutron scattering

Bilayer lipid membranes supported on a solid surface are attractive model systems for understanding the structure and dynamics of more complex biological membranes that form the outer boundary of living cells. We have recently obtained quasielastic neutron spectra from single-supported bilayer lipid membranes using the backscattering spectrometer BASIS at the Spallation Neutron Source. Protonated DMPC membranes were deposited onto SiO$_{2}$-coated Si(100) substrates and characterized by AFM. Analysis of their neutron spectra shows evidence of a relatively broad Lorentzian component that we associate with bulk-like water above a freezing temperature of $\sim $267 K. At lower temperatures, the spectra differ qualitatively from that of bulk supercooled water, a behavior that we attribute to water bound to the membrane. We also find evidence of a narrow Lorentzian component that we tentatively identify with a slower motion (time scale $\sim $1 ns) associated with conformational changes of the alkyl tails of the lipid molecules.   

Studies of molecular diffusion in single-supported bilayer lipid membranes at low hydration by quasielastic neutron scattering

We have extended our investigation of the quasielastic neutron scattering from single-supported bilayer lipid membranes to a sample of lower hydration using the backscattering spectrometer BASIS at the SNS of ORNL. To focus on the diffusive motion of the water, tail-deuterated DMPC membranes were deposited onto SiO$_{2}$-coated Si(100) substrates and characterized by AFM. Compared to a sample of higher hydration, the dryer sample does not have a step-like freezing transition at $\sim $267 K and shows less intensity at higher temperatures of a broad Lorentzian component representing bulk-like water. However, the broad component of the ``wet'' and ``dry'' samples behaves similarly at lower temperatures. The dryer sample also shows evidence of a narrow Lorentzian component that has a different temperature dependence than that attributed to conformational changes of the alkyl tails of the lipid molecules in the wet sample. We tentatively identify this slower diffusive motion (time scale $\sim $1 ns) with water more tightly bound to the membrane.   

Diffusion in Single Supported Lipid Bilayers

Despite their potential relevance for the development of functionalized surfaces and biosensors, the study of single supported membranes using neutron scattering has been limited by the challenge of obtaining relevant dynamic information from a sample with minimal material. Using state of the art neutron instrumentation we have, for the first time, modeled lipid diffusion in single supported lipid bilayers.\footnote{C.L. Armstrong, M.D. Kaye, M. Zamponi, E. Mamontov, M. Tyagi, T. Jenkins and M.C. Rheinst\"{a}dter, Soft Matter Communication, 2010, Advance Article, DOI: 10.1039/C0SM00637H } While we find that the diffusion coefficient for the single bilayer system is comparable to a multi-lamellar lipid system, the molecular mechanism for lipid motion in the single bilayer is a continuous diffusion process with no sign of the flow-like ballistic motion reported in the stacked membrane system. In the future, these membranes will be used to hold and align proteins, mimicking physiological conditions enabling the study of protein structure, function and interactions in relevant and highly topical membrane/protein systems with minimal sample material.   

Membrane stress relaxation by transbilayer cholesterol exchange

Fusion and fission events in the cell membrane play a crucial role in many biological processes, yet the mechanism for inducing the membrane bending deformations required for such events remains poorly understood. In particular, standard membrane elastic models predict a problematically high energy barrier for the strongly curved ``neck'' region formed during fusion and fission. These models assume that the exchange of lipids between membrane leaflets is negligible. While this is valid for phospholipids, other amphiphilic molecules such as cholesterol undergo rapid flip-flop between leaflets. Such exchange can relax bending stresses in the membrane: By flipping from the compressed to the expanded leaflet, cholesterol can reduce the energy required to bend the membrane. In this talk, we present a coarse-grained energetic model (derived from a simple microscopic description of the membrane) for a two-component lipid bilayer that contains a lipid species that can undergo rapid transbilayer exchange. Using this model, we show that lipid flip-flop dramatically reduces the energetic barriers encountered during membrane fusion and fission events and also plays an important role in determining the deformations induced by external forces such as osmotic pressure.   

Ethanol enhances collective dynamics of lipid membranes

Lipid bilayers have long been considered simple homogeneous passive barriers. However, there is a growing consensus that bilayer composition and properties impact their role in membrane function. One molecule which participates in lipid bilayers is ethanol. Ethanol is principally known to increase membrane permeability, serving as a model drug enhancer. While bilayer permeability was thought to depend solely on structural properties such as the area per lipid, this may be supported by thermal fluctuations in the bilayer core. Thermal motion results in the formation of small voids in the hydrocarbon chains, which may play a role in the transport small molecules through the membrane core. In both inelastic neutron scattering experiments and molecular dynamics simulations we find evidence for a new low-energy dynamic mode in the fluid phase of DMPC bilayers immersed in a 5{\%} water/ethanol solution [1]. The molecular motion associated with this phonon corresponds to coherent displacements of the carbon atoms in the lipid tails both in, and partially normal to, the plane of the membrane. This finding supports the possibility of a fluctuation supported trans-membrane transport process in lipid bilayers. \\[4pt] [1] ``Ethanol enhances collective dynamics of lipid membranes'', M. D. Kaye, M. Tarek, K. Schmalzl, M. C. Rheinst\"{a}dter, submitted to Physical Review Letters   

Vesicle Shape Transformations Driven by Active and Spontaneous Lipid Flip-flop

The lipid composition of cell membranes is created and maintained in part by flippases, enzymes that translocate lipid molecules from one layer of the bilayer membrane to the other. We study how lipid translocation can affect membrane shape, using a cylindrical vesicle as a simple model system. For a short pulse of flippase activity, in which a fraction of lipids are flipped from one layer to the other, we calculate the fraction of flipped lipids that makes the cylinder unstable to a periodic modulation in its radius, as well as the growth rate of perturbations of different wavenumber. We also study the cases of continuous flippase activity and spontaneous flip-flop.   

\textit{In vitro} approach to the mechanics of lipid membrane area regulation: vesicle absorption and tube formation

We have designed an experimental approach that allows us to study the response of supported lipid bilayers to cycles of biaxial expansion and compression. We observed that the bilayer effectively adjusts its area during dilatational or compressive strains in order to reduce its tension. For example, if there is a sufficient lipid reservoir in the form of attached vesicles, then a lipid bilayer may accommodate strains tens of times larger than the critical strain for rupture by expanding its area. Additionally, upon compression the bilayer reduces its area by expelling lipid tubes out of its plane. These observations offer new insights into how cells regulate their surface area in response to various mechanical stimuli, i.e. during physiological volume changes, locomotion, cyclic expansion and compression of the uro- and the alveolar- epithelium, etc.   

Structural Phase Diagram for Multi-lamellar Tubular Deformations of Lipid Mesophases

Stable multi-lamellar cylindrical tubules protrude readily from concentrated mass of amphiphilic molecules in response to a variety of external stresses. Using energetic considerations, we have developed an phase diagram, predicting various types of morphologies of equilibrium multilamellar tubular deformations that stabilize for a broad range of their bending rigidity and surface tension values. Tubular morphologies are described in terms of core radius(rc) and number of lamellae(N). Results of the calculations reveal that emergent tubular morphologies can be classified into three major classes: (1) thin tethers (small rc and low N); (2) solid tubes (high N); and (3) hollow tubes (large rc and and low N). Experimental validation of these predictions is obtained in experiments involving hydration of dry stack lipids Here, tubular deformations, referred to as myelin figures, of all predicted morphologies form in separate populations. Furthermore, the phase diagram also sheds light on a long-standing question of the determinants of the thickness of such myelin figures.   

Biomembranes that respond to specific triggers by phase separating

Lipid membranes are widely used as models for the cell membrane and for applications such as encapsulation, delivery, and controlled release. We have recently found that when membranes adhere nonspecifically, the adhesion site favors the nucleation and growth of more-ordered lipid phases. The physics behind this, which works by suppressing membrane fluctuations, should be applicable to specifically-adhering membranes as well. This will allow better experimental models for cell adhesion, which is mediated by transmembrane proteins and associated with lipid heterogeneities, and also indicates a new category of pathways for making `smart,' responsive materials out of lipid membranes. We are transforming our previous, non-specifically adhering systems into membranes that specifically adhere to a surface via binder molecules. We will determine the thresholds for forming ordered phases as a function of binder stiffness, length, and density, compatibility of the binder structure with the molecular packing of lipids in these phases, and membrane properties such as bending modulus and proximity to a phase transition.   

Lipid domains in supported SM-Chol membranes measured by GISANS

Cell membranes are known to contain regions (called lipid domains, or rafts) described as sphingolipid-cholesterol assemblies which also may contain a subset of membrane proteins. Currently, the main point of discussion is the methodology to study lipid domains and their sizes. We report on Grazing Incidence Small Angle Neutron Scattering (GISANS) measurements of lipid domains in supported sphingomyelin(SM)-cholesterol(Chol) bilayers in a fully aqueous environment. The model bilayers SM:Chol(2:1), SM:Chol(1:2), and a pure SM were deposited using Langmuir-Blodgett/Langmuir-Schaefer technique at a surface pressure of 10 mN/m and measured at 25 \r{ }C. First measurements revealed short range inhomogeneities of the order of 100 {\AA} in both binary systems. The control measurement of a pure SM bilayer exhibited nearly no GISANS indicating an absence of lipid domains in the SM bilayer. This observation is consistent with the notion that a single component system studied below the liquid-gel transition temperature will not produce lipid domains.   

Structure of the Stern layer in Phospholipid Systems

The structure of the Stern layer in Phospholipid Systems results from a subtle competition of salt concentration, ionic valence, specific ionic-phospolipid interactions and pH. It becomes very challenging to develop a rigorous theory that encompasses all these effects, yet its understanding is extremely relevant for both model and biological systems, as the structure of the Stern layer determines the interactions of phospholipids with proteins or electrostatic phase separation (rafts). In this talk we will present our theoretical model for the Stern Layer and discuss how all these effects are included. Particularly emphasis is made to Phosphoinositides and Phosphatidic acid.   

Modeling Signal Transduction and Lipid Rafts in Immune Cells

Experimental evidence increasingly suggests that lipid rafts are nanometer sized cholesterol dependent dynamic assemblies enriched in sphingolipids and associated proteins. Lipid rafts are dynamic structures that break-up and reform on a relatively short time-scale, and are believed to facilitate the interactions of raft-associated proteins. The role of these rafts in signaling has been controversial, partly due to controversies regarding the existence and nature of the rafts themselves. Experimental evidence has indicated that in several cell types, especially T cells, rafts do influence signal transduction and T cell activation. Given the emerging consensus on the biophysical character of lipid rafts, the question can be asked as to what roles they possibly play in signal transduction. Here we carry out simulations of minimal models of the signal transduction network that regulates Src-family kinase dynamics in T cells and other cell types. By separately treating raft-based biochemical interactions, we find that rafts can indeed putatively play an important role in signal transduction, and in particular may affect the sensitivity of signal transduction. This illuminates possible functional consequences of membrane heterogeneities on signal transduction and points towards mechanisms for spatial control of signaling by cells.   

Calcium-mediated rigidity in PIP2 lipid domains

In lipid mixtures containing the highly negatively charged lipid PIP2 (a crucial component in cell membrane mechanics) multivalent ions such as calcium can drive the formation of PIP2-rich domains by mediating attractions between the lipids. Although the existence of ion-mediated attractions is well known in macromolecular systems, their form is poorly understood because they result from strong correlations between the charged molecules and ions. Within a numerical model of a lipid monolayer, we analyze the mechanics of PIP2-rich domains. We show that they are liquid-like at moderate values of the PIP2-charge but rigid at higher PIP2-charge. We use a recently introduced method to extract the effective pair interaction between the charged lipids in the many-body system, in which the calcium ions and remaining lipids are integrated out.