Lensless Tomographic Imaging of Near Surface Structures of Frozen Hydrated Malaria-Infected Human Erythrocytes by Coherent X-Ray Diffraction Microscopy.

Lensless, coherent X-ray diffraction microscopy has been drawing considerable attentions for tomographic imaging of whole human cells. In this study, we performed cryogenic coherent X-ray diffraction imaging of human erythrocytes with and without malaria infection. To shed light on structural features near the surface, "ghost cells" were prepared by the removal of cytoplasm. From two-dimensional images, we found that the surface of erythrocytes after 32 h of infection became much rougher compared to that of healthy, uninfected erythrocytes. The Gaussian roughness of an infected erythrocyte surface (69 nm) is about two times larger than that of an uninfected one (31 nm), reflecting the formation of protein knobs on infected erythrocyte surfaces. Three-dimensional tomography further enables to obtain images of the whole cells with no remarkable radiation damage, whose accuracy was estimated using phase retrieval transfer functions to be as good as 64 nm for uninfected and 80 nm for infected erythrocytes, respectively. Future improvements in phase retrieval algorithm, increase in degree of coherence, and higher flux in combination with complementary X-ray fluorescence are necessary to gain both structural and chemical details of mesoscopic architectures, such as cytoskeletons, membraneous structures, and protein complexes, in frozen hydrated human cells, especially under diseased states.

Generic Role of Polymer Supports in the Fine Adjustment of Interfacial Interactions between Solid Substrates and Model Cell Membranes.

To understand the generic role of soft, hydrated biopolymers in adjusting interfacial interactions at biological interfaces, we designed a defined model of the cell-extracellular matrix contacts based on planar lipid membranes deposited on polymer supports (polymer-supported membranes). Highly uniform polymer supports made out of regenerated cellulose allow for the control of film thickness without changing the surface roughness and without osmotic dehydration. The complementary combination of specular neutron reflectivity and high-energy specular X-ray reflectivity yields the equilibrium membrane-substrate distances, which can quantitatively be modeled by computing the interplay of van der Waals interaction, hydration repulsion, and repulsion caused by the thermal undulation of membranes. The obtained results help to understand the role of a biopolymer in the interfacial interactions of cell membranes from a physical point of view and also open a large potential to generally bridge soft, biological matter and hard inorganic materials.

Quantitative determination of lateral concentration and depth profile of histidine-tagged recombinant proteins probed by grazing incidence X-ray fluorescence.

We have demonstrated that the complementary combination of grazing incidence X-ray fluorescence (GIXF) with specular X-ray reflectivity (XRR) can be used to quantitatively determine the density profiles of Ni(2)(+) ions complexed with chelator headgroups as well as S atoms in recombinant proteins anchored to lipid monolayers at the air/water interface. First, we prepared phospholipid monolayers incorporating chelator lipid anchors at different molar fractions at the air/water interface. The fine-structures perpendicular to the global plane of monolayers were characterized by XRR in the presence of Ni(2)(+) ions, yielding the thickness, roughness, and electron density of the stratified lipid monolayers. X-ray fluorescence intensities from Ni Kα core levels recorded at the incidence angles below and above the critical angle of total reflection allow for the determination of the position and lateral density of Ni(2)(+) ions associated with chelator headgroups with a high spatial accuracy (±5 Å). The coupling of histidine-tagged Xenopus cadherin 11 (Xcad-11) can also be identified by changes in the fines-structures using XRR. Although fluorescence intensities from S Kα level were much weaker than Ni Kα signals, we could detect the location of S atoms in recombinant Xcad-11 proteins.

Cell differentiation of pluripotent tissue sheets immobilized on supported membranes displaying cadherin-11.

Investigating cohesive tissue sheets in controlled cultures still poses a challenge since the complex intercellular interactions are difficult to mimic in in vitro models. We used supported lipid membranes functionalized by the adhesive part of the extracellular domain of the cell adhesion molecule cadherin-11 for the immobilization of pluripotent tissue sheets, the animal cap isolated from Xenopus laevis blastula stage embryos. Cadherin-11 was bound via histidine tag to lipid membranes with chelator head groups. In the first step, quantitative functionalization of the membranes with cadherin-11 was confirmed by quartz crystal microbalance and high energy specular X-ray reflectivity. In the next step, animal cap tissue sheets induced to neural crest cell fate were cultured on the membranes functionalized with cadherin-11. The adhesion of cells within the cohesive tissue was significantly dependent on changes in lateral densities of cadherin-11. The formation of filopodia and lamellipodia in the cohesive tissue verified the viability and sustainability of the culture over several hours. The expression of the transcription factor slug in externally induced tissue demonstrated the applicability of lipid membranes displaying adhesive molecules for controlled differentiation of cohesive pluripotent tissue sheets.

Physical chemistry of biological interfaces: generic and specific roles of soft interlayers.

Nature defines the spatial boundaries between different phases using membranes, and the interfacial interactions are mediated by soft biopolymer interlayers that contain various carbohydrates. This Review provides a comprehensive overview on the interplay of generic and specific interactions at cell-cell and cell-tissue interfaces. A focus will be put on the combination of defined model systems, experimental techniques in real- and reciprocal space, and theoretical simulations.

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.

Native supported membranes on planar polymer supports and micro-particle supports.

To bridge soft biological materials and hard inorganic materials is an interdisciplinary scientific challenge. Despite of experimental difficulties, the deposition of native biological membranes on supports is a straightforward strategy. This review provides an overview of advances in the fabrication and characterization of native biological membranes on planar polymer supports and micro-particles.

Formation of supported lipid bilayers on indium tin oxide for dynamically-patterned membrane-functionalized microelectrode arrays.

Supported lipid bilayers (SLBs) are ideal platforms for the study of membrane proteins and function. Assembly of functional SLBs in an array format would lead to a breakthrough in high-throughput screening of membrane-associated processes, e.g., drugs binding to transmembrane proteins. We report the formation of SLBs from the rupture of anionic vesicles in the presence of Ca(2+) ions on ITO-coated surfaces and characterise the assembly and SLB properties. Furthermore, the formation, manipulation and regeneration of SLBs adsorbed on ITO microelectrode array spots using an electric potential switch are demonstrated. This platform enables addressable assembly and the study of electrochemically mediated membrane processes in a microarray format which can be regenerated in situ.

Orientation-selective incorporation of transmembrane F0F1 ATP synthase complex from micrococcus luteus in polymer-supported membranes.

We report the vectorial incorporation of a highly asymmetric F0F1 ATP synthase complex from Micrococcus luteus into polymer-supported membranes. Dynamic light scattering and cryo electron microscopy confirm that the use of weak surfactants (bile acid) allows for the non-disruptive protein incorporation into lipid vesicles. Spreading of vesicles with ATP synthase onto a cellulose support results in a homogeneous distribution of proteins, in contrast to a patchy image observed on bare glass slides. The orientation of ATP synthase can be identified using an antibody to the ATP binding site as well as from topographic profiles of the surface. The method to "align" transmembrane proteins in supported membranes would open a possibility to quantify protein functions in biomimetic model systems.

Structures of regenerated cellulose films revealed by grazing incidence small-angle x-ray scattering.

The characteristic in-plane length scales of ultrathin films of regenerated cellulose are measured using noncontact atomic force microscopy (NC-AFM) and grazing incidence small-angle x-ray scattering (GISAXS) in ambient atmosphere and under various humidity conditions. The aim is to elucidate the structural basis for the excellent compatibility of cellulose supports to planar lipid membranes. Films are deposited on silicon wafers by Langmuir-Blodgett (LB) transfer and spin coating. NC-AFM height profiles and the resulting calculated power spectral density functions indicate that both kinds of cellulose films have almost identical root-mean-square roughness values (0.7-0.8 nm) and very similar characteristic length scales (32 nm), respectively. GISAXS measurements, both above and below the critical angle of total external reflection, show that the dominant length scales in the bulk and near the surface of the films are comparable ( approximately 50 nm). The origin of these length scales can be attributed to the bundle of rodlike molecules of cellulose that result during the regeneration process (i.e., as a consequence of the cleavage of the silyl side chains of trimethylsilylcellulose). Exposure of the cellulose samples to various humidities shows that above a relative humidity of 97% a significant swelling of the films occurs, which is consistent with our previous findings. The swelling of films with more than 30 LB monolayers of cellulose induces a remarkable out-of-plane rearrangement of the cellulose bundles, due to a reduced influence of the solid substrate compared to thinner films with only eight to ten LB monolayers.

Asymmetric distribution of phosphatidyl serine in supported phospholipid bilayers on titanium dioxide.

Supported phospholipid bilayers (SPBs) are useful for studying cell adhesion, cell-cell interactions, protein-lipid interactions, protein crystallization, and applications in biosensor and biomaterial areas. We have recently reported that SPBs could be formed on titanium dioxide, an important biomaterial, from vesicles containing anionic phospholipid phosphatidyl serine (PS) in the presence of calcium. Here, we show that the mobility of the fluorescently labeled PS present in these bilayers is severely restricted, whereas that of the zwitterionic phosphatidyl choline is not affected. Removal of calcium alleviated the restriction on the mobility of PS. Both components were found to be mobile in SPBs of identical compositions prepared in the presence of calcium on silica. To explain these results, we propose that, on TiO2, PS is trapped in the proximal leaflet of the bilayers. This proposal is supported by the results of protein adsorption experiments carried out on bilayers containing various amounts of PS prepared on silica and titania.

Interactions between titanium dioxide and phosphatidyl serine-containing liposomes: formation and patterning of supported phospholipid bilayers on the surface of a medically relevant material.

Titanium is widely used in biomedical applications. Its mechanical properties and biocompatibility, conferred by a layer of oxide present on its surface, make titanium the material of choice for various implants (artificial hip and knee joints, dental prosthetics, vascular stents, heart valves). Furthermore, the high refractive index of titanium oxide is advantageous in biosensor applications based on optical detection methods. In both of the above fields of application, novel surface modification strategies leading to biointeractive interfaces (that trigger specific responses in biological systems) are continuously sought. In this report, we investigate the interactions between TiO2 and phosphatidyl serine-containing liposomes, present a novel approach for preparing supported phospholipid bilayers (SPBs) of various compositions on TiO2, and use the unique ability of liposomes to distinguish between different surfaces to create SPB corrals on SiO2/TiO2 structured substrates. These results represent an important first step toward the design of biointeractive interfaces on titanium oxide surfaces that are based on a cell membrane-like environment.

Investigating the properties of supported vesicular layers on titanium dioxide by quartz crystal microbalance with dissipation measurements.

Adsorption of phospholipid vesicles on titanium dioxide was studied by a combination of quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy techniques. Vesicle size, concentration in solution, and bilayer composition were systematically varied. A strong dependence of the QCM-D response (magnitude of the frequency and dissipation factor shifts) on the vesicle concentration in solution was observed. QCM-D data were compared with a linear viscoelastic model based on the Voight element to determine layer thickness, density, elastic modulus, and viscosity. Based on the results of this comparison, it is proposed that (i) layer thickness and density, as sensed by QCM-D, saturate much earlier (in time) than the actual surface coverage of the vesicles (number of vesicles per unit area); (ii) changes in surface coverage that occur after the density and thickness, as sensed by QCM-D, have saturated, are interpreted by the model as changes in the layer's viscoelastic properties. This is caused by the replacement of the viscous media (water) between the vesicles by viscoelastic media of similar density (vesicles); (iii) viscoelastic properties of layers formed at different vesicle concentrations differ significantly, while the vesicle surface coverage in those layers does not. Based on the comparison between the atomic force microscopy images and QCM-D data acquired at various vesicle concentrations it is proposed that QCM-D response is not directly related to the surface coverage of the vesicles.

Interaction of poly(L-lysine)-g-poly(ethylene glycol) with supported phospholipid bilayers.

Interactions between the graft copolymer poly(L-lysine)-g-poly(ethylene glycol), PLL-g-PEG, and two kinds of surface-supported lipidic systems (supported phospholipid bilayers and supported vesicular layers) were investigated by a combination of microscopic and spectroscopic techniques. It was found that the application of the copolymer to zwitterionic or negatively charged supported bilayers in a buffer of low ionic strength led to their decomposition, with the resulting formation of free copolymer-lipid complexes. The same copolymer had no destructive effect on a supported vesicular layer made up of vesicles of identical composition. A comparison between poly(L-lysine), which did not induce decomposition of supported bilayers, and PLL-g-PEG copolymers with various amounts of PEG side chains per backbone lysine unit, suggested that steric repulsion between the PEG chains that developed upon adsorption of the polymer to the nearly planar surface of a supported phospholipid bilayer (SPB) was one of the factors responsible for the destruction of the SPBs by the copolymer. Other factors included the ionic strength of the buffer used and the quality of the bilayers, pointing toward the important role defects present in the SPBs play in the decomposition process.