Direct Observation and Semiquantitative Analysis of Hierarchical Structures in Graft-Type Polymer Electrolyte Membranes Using the AFM Technique.

The structural features of a polymer electrolyte membrane (PEM), consisting of polystyrene sulfonic acid (PSSA) grafted onto poly(ethylene-co-tetrafluoroethylene) (ETFE), can be characterized semiquantitatively by atomic force microscopy (AFM). The cross-sectional AFM phase images are converted to the binarized image by fitting two Gaussian functions. The domains correspond to hydrophilic PSSA domains and hydrophobic ETFE crystalline and amorphous regions, respectively, at lower and higher phase shift values. The area fraction of PSSA domains was consistent with the volume fraction determined by the grafting degree (GD). The dependence of the radius and interdomain distance of the PSSA domains on the GDs of PEMs shows discontinuous features at the threshold GD (39%). The former slightly increased from 10 to 12 nm and significantly increased to 17 nm at a GD greater than 39%; the latter decreased from 140 to 54 nm with increases in GDs up to 39% but inversely increased to 78 nm at a GD of 46%. This discontinuous change in radius and interdomain distance should be caused by the fusion of adjacent PSSA domains to form a larger size and spacing and thus less connectivity between each large domain, thereby lowering the conductivity at GD greater than 39%. We were able to demonstrate the existence of an ion-conducting hydrophilic path with a radius of approximately 10 nm. Even though it has received little attention in the past, it is expected to enable the design of electrolyte membrane functions in the future.

Physical Properties and Reactivity of Microdomains in Phosphatidylinositol-Containing Supported Lipid Bilayer.

We characterized the size, distribution, and fluidity of microdomains in a lipid bilayer containing phosphatidylinositol (PI) and revealed their roles during the two-dimensional assembly of a membrane deformation protein (FBP17). The morphology of the supported lipid bilayer (SLB) consisting of PI and phosphatidylcholine (PC) on a mica substrate was observed with atomic force microscope (AFM). Single particle tracking (SPT) was performed for the PI+PC-SLB on the mica substrate by using the diagonal illumination setup. The AFM topography showed that PI-derived submicron domains existed in the PI+PC-SLB. The spatiotemporal dependence of the lateral lipid diffusion obtained by SPT showed that the microdomain had lower fluidity than the surrounding region and worked as the obstacles for the lipid diffusion. We observed the two-dimensional assembly of FBP17, which is one of F-BAR family proteins included in endocytosis processes and has the function generating lipid bilayer tubules in vitro. At the initial stage of the FBP17 assembly, the PI-derived microdomain worked as a scaffold for the FBP17 adsorption, and the fluid surrounding region supplied FBP17 to grow the FBP17 domain via the lateral molecular diffusion. This study demonstrated an example clearly revealing the roles of two lipid microregions during the protein reaction on a lipid bilayer.

Substrate-Induced Structure and Molecular Dynamics in a Lipid Bilayer Membrane.

The solid-substrate-dependent structure and dynamics of molecules in a supported lipid bilayer (SLB) were directly investigated via atomic force microscopy (AFM) and single particle tracking (SPT) measurements. The appearance of either vertical or horizontal heterogeneities in the SLB was found to be strongly dependent on the underlying substrates. SLB has been widely used as a biointerface with incorporated proteins and other biological materials. Both silica and mica are popular substrates for SLB. Using single-molecule dynamics, the fluidity of the upper and lower membrane leaflets was found to depend on the substrate, undergoing coupling and decoupling on the SiO2/Si and mica substrates, respectively. The anisotropic diffusion caused by the locally destabilized structure of the SLB at atomic steps appeared on the Al2O3(0001) substrate because of the strong van der Waals interaction between the SLB and the substrate. Our finding that the well-defined surfaces of mica and sapphire result in asymmetry and anisotropy in the plasma membrane is useful for the design of new plasma-membrane-mimetic systems. The application of well-defined supporting substrates for SLBs should have similar effects as cell membrane scaffolds, which regulate the dynamic structure of the membrane.

Formation of Cell Membrane Component Domains in Artificial Lipid Bilayer.

The lipid bilayer environment around membrane proteins strongly affects their structure and functions. Here, we aimed to study the fusion of proteoliposomes (PLs) derived from cultured cells with an artificial lipid bilayer membrane and the distribution of the PL components after the fusion. PLs, which were extracted as a crude membrane fraction from Chinese hamster ovary (CHO) cells, formed isolated domains in a supported lipid bilayer (SLB), comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (Chol), after the fusion. Observation with a fluorescence microscope and an atomic force microscope showed that the membrane fusion occurred selectively at microdomains in the PC + PE + Chol-SLB, and that almost all the components of the PL were retained in the domain. PLs derived from human embryonic kidney 293 (HEK) cells also formed isolated domains in the PC + PE + Chol-SLB, but their fusion kinetics was different from that of the CHO-PLs. We attempted to explain the mechanism of the PL-SLB fusion and the difference between CHO- and HEK-PLs, based on a kinetic model. The domains that contained the whole cell membrane components provided environments similar to that of natural cell membranes, and were thus effective for studying membrane proteins using artificial lipid bilayer membranes.

Lateral Diffusion and Molecular Interaction in a Bilayer Membrane Consisting of Partially Fluorinated Phospholipids.

Fluorinated lipids and surfactants are attractive biomimetic materials for the extraction and reorganization of membrane proteins because of the biological inertness of fluorocarbons. We investigated the fundamental physical properties of a partially fluorinated phospholipid (F4-DMPC), such as phase transition, area thermal expansion, and lateral lipid diffusion, to evaluate the intermolecular interaction of F4-DMPC in the hydrophobic region quantitatively on the basis of free-volume theory. Fluorescence microscope observation of the supported lipid bilayer (SLB) of F4-DMPC showed that the phase transition between the liquid crystalline and gel phases occurred at 5 °C and that the area thermal expansion coefficient was independent of the temperature near the phase transition temperature. We performed a single particle tracking of the F4-DMPC-SLB on a SiO2/Si substrate, to measure the diffusion coefficient and its temperature dependence. The apparent activation energy (E'a) of lateral lipid diffusion, which is an indicator of intermolecular interaction, was 39.1 kJ/mol for F4-DMPC, and 48.2 kJ/mol for a nonfluorinated 1,2-dioleoyl-sn-glycero-3-phosphocholine as a control. The difference of 9 kJ/mol in E'a was significant compared with the difference due to the acyl chain species among nonfluorinated phosphatidylcholine and also that caused by the addition of cholesterol and alcohol in the bilayer membranes. We quantitatively evaluated the attenuation of intermolecular interaction, which results from the competition between the dipole-induced packing effect and steric effect at the fluorocarbon segment in F4-DMPC.

Effective Brownian ratchet separation by a combination of molecular filtering and a self-spreading lipid bilayer system.

A new molecular manipulation method in the self-spreading lipid bilayer membrane by combining Brownian ratchet and molecular filtering effects is reported. The newly designed ratchet obstacle was developed to effectively separate dye-lipid molecules. The self-spreading lipid bilayer acted as both a molecular transport system and a manipulation medium. By controlling the size and shape of ratchet obstacles, we achieved a significant increase in the separation angle for dye-lipid molecules compared to that with the previous ratchet obstacle. A clear difference was observed between the experimental results and the simple random walk simulation that takes into consideration only the geometrical effect of the ratchet obstacles. This difference was explained by considering an obstacle-dependent local decrease in molecular diffusivity near the obstacles, known as the molecular filtering effect at nanospace. Our experimental findings open up a novel controlling factor in the Brownian ratchet manipulation that allow the efficient separation of molecules in the lipid bilayer based on the combination of Brownian ratchet and molecular filtering effects.

Single-molecule observations for determining the orientation and diffusivity of dye molecules in lipid bilayers.

The molecular orientation and diffusion of dye molecules in artificial lipid bilayers were observed using total internal reflection fluorescence microscopy. An artificial lipid bilayer composed of a ternary lipid mixture of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and cholesterol was used. The molecular orientation, which was obtained through defocused imaging, clarified the microscopic features, including cholesterol-induced changes in the local packing structure. Diffusion analysis gave insights into the macroscopic aspects of phase distribution in the heterogeneous bilayer system. Combining these two independent investigations, we revealed the effect of cholesterol addition on microscopic local packing and macroscopic phase structures. Our observations showed a transition from a DLPC-network-like structure to a DPPC-network-like structure upon the addition of cholesterol, which was not evident from previous domain shape observations. The present single-molecule observations yielded the actual phase structure that controls the motion of molecules in the membrane. The results imply that the orientation and diffusivity of molecules offer useful information regarding the phase distribution, which may be hindered by the apparent phase structure in a heterogeneous lipid bilayer that contains cholesterol.

Enhanced Brownian ratchet molecular separation using a self-spreading lipid bilayer.

A new approach is proposed for two-dimensional molecular separation based on the Brownian ratchet mechanism by use of a self-spreading lipid bilayer as both a molecular transport and separation medium. In addition to conventional diffusivity-dependence on the ratchet separation efficiency, the difference in the intermolecular interactions between the target molecules and the lipid bilayer is also incorporated as a new separation factor in the present self-spreading ratchet system. Spreading at the gap between two ratchet obstacles causes a local change in the lipid density at the gap. This effect produces an additional opportunity for a molecule to be deflected at the ratchet obstacle and thus causes an additional angle shift. This enables the separation of molecules with the same diffusivity but with different intermolecular interaction between the target molecule and surrounding lipid molecules. Here we demonstrate this aspect by using cholera toxin subunit B (CTB)-ganglioside GM1 (GM1) complexes with different configurations. The present results will unlock a new strategy for two-dimensional molecular manipulation with ultrasmall devices.