Motomu Tanaka

Polymer-supported membranes as models of the cell surface

Lipid-bilayer membranes supported on solid substrates are widely used as cell-surface models that connect biological and artificial materials. They can be placed either directly on solids or on ultrathin polymer supports that mimic the generic role of the extracellular matrix. The tools of modern genetic engineering and bioorganic chemistry make it possible to couple many types of biomolecule to supported membranes. This results in sophisticated interfaces that can be used to control, organize and study the properties and function of membranes and membrane-associated proteins. Particularly exciting opportunities arise when these systems are coupled with advanced semiconductor technology.

Functional Incorporation of Integrins into Solid Supported Membranes on Ultrathin Films of Cellulose: Impact on Adhesion

Biomimetic models of cell surfaces were designed to study the physical basis of cell adhesion. Vesicles bearing reconstituted blood platelet integrin receptors alpha(IIb)beta(3) were spread on ultrathin films of cellulose, forming continuous supported membranes. One fraction of the integrin receptors, which were facing their extracellular domain toward the aqueous phase, were mobile, exhibiting a diffusion constant of 0.6 micro m(2) s(-1). The functionality of receptors on bare glass and on cellulose cushions was compared by measuring adhesion strength to giant vesicles. The vesicles contained lipid-coupled cyclic hexapeptides that are specifically recognized by integrin alpha(IIb)beta(3). To mimic the steric repulsion forces of the cell glycocalix, lipids with polyethylene glycol headgroups were incorporated into the vesicles. The free adhesion energy per unit area deltag(ad) was determined by micro-interferometric analysis of the vesicles contour near the membrane surface in terms of the equilibrium of the elastic forces. By accounting for the reduction of the adhesion strength by the repellers and from measuring the density of receptors one could estimate the specific receptor ligand binding energy. We estimate the receptor-ligand binding energy to be 10 k(B)T under bioanalogue conditions.

Chemical functionalization of GaN and AlN surfaces

The covalent functionalization of GaN and AlN surfaces with organosilanes is demonstrated. Both octadecyltrimethoxysilane and aminopropyltriethoxysilane form self-assembled monolayers on hydroxylated GaN and AlN surfaces, confirmed by x-ray photoelectron spectroscopy and atomic force microscopy. The monolayer thickness on GaN was determined to 2.5±0.2nm by x-ray reflectivity. Temperature-programmed desorption measurements reveal a desorption enthalpy of 240kJ∕mol. The realization of micropatterned self-assembled monolayers and the hybridization of deoxyribonucleic acid molecules on biofunctionalized GaN surfaces are shown.

Stripes of partially fluorinated alkyl chains: Dipolar Langmuir monolayers

Stripelike domains of Langmuir monolayers formed by surfactants with partially fluorinated lipid anchors (F-alkyl lipids) are observed at the gas-liquid phase coexistence. The average periodicity of the stripes, measured by fluorescence microscopy, is in the micrometer range, varying between 2 and 8 microm. The observed stripelike patterns are stabilized due to dipole-dipole interactions between terminal- CF(3) groups. These interactions are particularly strong as compared with nonfluorinated lipids due to the low dielectric constant of the surrounding media (air). These long-range dipolar interactions tend to elongate the domains, in contrast to the line tension that tends to minimize the length of the domain boundary. This behavior should be compared with that of the lipid monolayer having alkyl chains, and which form spherical microdomains (bubbles) at the gas-liquid coexistence. The measured stripe periodicity agrees quantitatively with a theoretical model. Moreover, the reduction in line tension by adding traces (0.1 mol %) of cholesterol results, as expected, in a decrease in the domain periodicity.

Quantitative Evaluation of Mechanosensing of Cells on Dynamically Tunable Hydrogels

Thin hydrogel films based on an ABA triblock copolymer gelator [where A is pH-sensitive poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) and B is biocompatible poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC)] were used as a stimulus-responsive substrate that allows fine adjustment of the mechanical environment experienced by mouse myoblast cells. The hydrogel film elasticity could be reversibly modulated by a factor of 40 via careful pH adjustment without adversely affecting cell viability. Myoblast cells exhibited pronounced stress fiber formation and flattening on increasing the hydrogel elasticity. As a new tool to evaluate the strength of cell adhesion, we combined a picosecond laser with an inverted microscope and utilized the strong shock wave created by the laser pulse to determine the critical pressure required for cell detachment. Furthermore, we demonstrate that an abrupt jump in the hydrogel elasticity can be utilized to monitor how cells adapt their morphology to changes in their mechanical environment.

Quantitative determination of ion distributions in bacterial lipopolysaccharide membranes by grazing-incidence X-ray fluorescence

A model of the outer membrane of Gram-negative bacteria was created by the deposition of a monolayer of purified rough mutant lipopolysaccharides at an air/water interface. The density profiles of monovalent (K+) and divalent (Ca2+) cations normal to the lipopolysaccharides (LPS) monolayers were investigated using grazing-incidence X-ray fluorescence. In the absence of Ca2+, a K+ concentration peak was found in the negatively charged LPS headgroup region. With the addition of CaCl2, Ca2+ ions almost completely displaced K+ ions from the headgroup region. By integrating the experimentally reconstructed excess ion density profiles, we obtained an accurate measurement of the effective charge density of LPS monolayers. The experimental findings were compared to the results of Monte Carlo simulations based on a coarse-grained minimal model of LPS molecules and showed excellent agreement.

Polymer-tethered membranes as quantitative models for the study of integrin-mediated cell adhesion

Here we report a remarkable enhancement in the adhesion strength of transmembrane cell receptors, human platelet integrin, in a new class of supported lipid membranes, which are separated from the solid substrates by linear polymer spacers. The amphiphilic polymer tether consists of linear hydrophilic poly(2-oxazoline) chains of defined length (degree of polymerization n = 104, MW/Mn = 1.30), whose chain termini are functionalized with the tri-functional silane surface coupling group and hydrophobic n-alkyl chains as membrane anchors (lipopolymers). As a model of test cells, giant lipid vesicles were functionalized with synthetic ligand molecules containing the RGD sequence, and the free energy of adhesion Δgad between the integrin-doped tethered membrane and the vesicle was measured using a micro-interferometry technique. It has been demonstrated that the adhesion function of integrin receptors in these polymer-tethered membranes is 30 times stronger than those incorporated into membranes directly deposited onto solid substrates (solid-supported membranes). The obtained results demonstrate that linear lipopolymer spacers provide a fluid and non-denaturing environment for the incorporated cell receptors and allow quantitative modelling of cell adhesion processes.

Calcium ions induce collapse of charged O-side chains of lipopolysaccharides from Pseudomonas aeruginosa

Lipopolysaccharide (LPS) monolayers deposited on planar, hydrophobic substrates were used as a defined model of outer membranes of Pseudomonas aeruginosa strain dps 89. To investigate the influence of ions on the (out-of-plane) monolayer structure, we measured specular X-ray reflectivity at high energy (22 keV) to ensure transmission through water. Electron density profiles were reconstructed from the reflectivity curves, and they indicate that the presence of Ca2+ ions induces a significant change in the conformation of the charged polysaccharide head groups (O-side chains). Monte Carlo simulations based on a minimal computer model of LPS molecules allow for the modelling of 100 or more molecules over 10−3 s and theoretically explained the tendency found by experiments.

Electrochemical passivation of gallium arsenide surface with organic self-assembled monolayers in aqueous electrolytes

Self-assembled monolayers of octadecylthiol (ODT) were reconstituted on freshly etched gallium arsenide (n-GaAs) for the electrochemical stabilization against decomposition of the surfaces (passivation) in aqueous buffers. The surface composition was evaluated by x-ray photoelectron spectroscopy to optimize the surface treatment before ODT deposition. Electrochemical properties of the monolayers were monitored by cyclic voltammetry and impedance spectroscopy. The impedance spectrum of the photoetched n-GaAs after the deposition of the ODT monolayer was stable in an aqueous electrolyte at pH=7.5 for more than 24 h within the sensitivity of our experimental technique. The effective passivation of GaAs surfaces is an essential step towards biosensor applications.

Orientation selective immobilization of human erythrocyte membranes on ultrathin cellulose films

We report the orientation selective immobilization of human erythrocyte membranes on planar solid supports. The orientation of the immobilized membrane was identified with selective fluorescence labels. When the right-side-out (RSO) ghosts were incubated with planar glass cover slides, no adsorption or rupture of erythrocytes could be observed. To increase the interfacial attraction between cells and the surface, two types of hydrated polymer films were deposited on the glass cover slides; (a) physisorbed films of cationic polylysine, and (b) Langmuir–Blodgett (LB) films of cellulose derivatives. On polylysine films, patches of the ruptured membranes could be observed, but the surface coverage still remained poor. On the other hand, RSO ghosts were likely to coat the surface of cellulose films more continuously. The fluorescence labeling demonstrated that immobilized erythrocyte membranes selectively inverted their native orientation. Tentatively, we interpreted this larger surface coverage on the cellulose film in terms of the “wetting affinity” between the cell surface glycocalix and the polysaccharides. Such ultrathin (thickness 5–10 nm), biological polysaccharide films have a large potential to immobilize native cell membranes without denaturing their structure, membrane orientation, and functions.