Enhancing the Cell-Free Expression of Native Membrane Proteins by In Silico Optimization of the Coding Sequence-An Experimental Study of the Human Voltage-Dependent Anion Channel.

Membrane proteins are involved in many aspects of cellular biology; for example, they regulate how cells interact with their environment, so such proteins are important drug targets. The rapid advancement in the field of immune effector cell therapy has been expanding the horizons of synthetic membrane receptors in the areas of cell-based immunotherapy and cellular medicine. However, the investigation of membrane proteins, which are key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, which is of unpredictable yield.

AFM and Fluorescence Microscopy of Single Cells with Simultaneous Mechanical Stimulation via Electrically Stretchable Substrates.

We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber.

Baseline correction of AFM force curves in the force-time representation.

This note reports on the proper correction of force data acquired with an atomic force microscope (AFM). The force-time representation is hereby used to obtain the correction factors for the overall offset and slope for a single force-time curve, as the initial force, F  = F(t ), and the rate of change in the force per unit of time, dF/dt, respectively. The report shows that a complete set of force data, including the approach, delay and retraction regions, can be simultaneously corrected in the force-time representation by subtracting the line CL = F  + dF/dt·t to the experimental data.

Inward multivesiculation at the basal membrane of adherent giant phospholipid vesicles.

Adherent giant vesicles composed of phosphatidylcholine, phosphatidylserine and biotinylated lipids form clusters of inward spherical buds at their basal membrane. The process is spontaneous and occurs when the vesicles undergo a sequence of osmotic swelling and deswelling. The daughter vesicles have a uniform size (diameter ≈ 2-3 μm), engulf small volumes of outer fluid and remain attached to the region of the membrane from which they generate, even after restoring the isotonicity.

A new automatic contact point detection algorithm for AFM force curves.

A new method for estimating the contact point in AFM force curves, based on a local regression algorithm, is presented. The main advantage of this method is that can be easily implemented as a computer algorithm and used for a fully automatic detection of the contact points in the approach force curves on living cells. The estimated contact points have been compared to those obtained by other published methods, which were applied either for materials with an elastic response to indentation forces or for experiments at high loading rates.

Nanostructure of polysaccharide complexes.

The interaction of gum arabic (GA) with chitosan (Ch) of different degree of deacetylation was studied by turbidity measurements, dynamic light scattering and atomic force microscopy. The structure of the complexes was found to be directly related to the charge density of chitosan molecules. Gum arabic and chitosan with a degree of deacetylation of 75% form soluble complexes with a loosely globular structure of about 250 nm, at weight ratios up to 1.

Stress relaxation and creep on living cells with the atomic force microscope: a means to calculate elastic moduli and viscosities of cell components.

In this work we present a unified method to study the mechanical properties of cells using the atomic force microscope. Stress relaxation and creep compliance measurements permitted us to determine, the relaxation times, the Young moduli and the viscosity of breast cancer cells (MCF-7). The results show that the mechanical behaviour of MCF-7 cells responds to a two-layered model of similar elasticity but differing viscosity.

The new future of scanning probe microscopy: Combining atomic force microscopy with other surface-sensitive techniques, optical microscopy and fluorescence techniques.

Atomic force microscopy (AFM) is in its thirties and has become an invaluable tool for studying the micro- and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. Still, the technique has limitations to overcome.

Rationalized approach to the determination of contact point in force-distance curves: application to polymer brushes in salt solutions and in water.

In this work, we present two methods to determine the contact point in force-distance curves obtained with the atomic force microscope. These procedures are compared with the typical determination of contact point by a visual assessment of the data. One method, based on the assumption that the sample shows linear elastic behavior, provides results similar to those obtained by a visual assessment of the data, and will be suitable for determining the contact point in cases where ionic repulsion is not significant.

Physical attachment of fluorescent protein particles to atomic force microscopy probes in aqueous media: implications for surface pH, fluorescence, and mechanical properties studies.

Transfer of a fluorescently labeled protein particle from a surface to a microsized scanning probe has been induced by repetitive scanning in aqueous medium. The so-attached particle can in turn act as a probing tool to study particle-substrate and particle-particle interactions. Attachment of the fluorescent particle occurs at the apical region of an atomic force microscope (AFM) cantilever tip and it endures repetitive loading-unloading cycles against the sample surface.

Surface dependence of protein nanocrystal formation.

The self-assembly kinetics and nanocrystal formation of the bacterial surface-layer-protein SbpA are studied with a combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Silane coupling agents, aminopropyltriethoxysilane (APTS) and octadecyltrichlorosilane (OTS), are used to vary the protein-surface interaction in order to induce new recrystallization pathways. The results show that the final S-layer crystal lattice parameters (a = b = 14 nm, gamma = 90 degrees ), the layer thickness (15 nm), and the adsorbed mass density (1700 ng cm(-2)) are independent of the surface chemistry.

Evaporation dynamics of microdroplets on self-assembled monolayers of dialkyl disulfides.

We present a study of the static wettability and evaporation dynamics of sessile microdroplets of water on self-assembled monolayers (SAMs) prepared with unsymmetric dialkyl disulfides CH(3)-(CH(2))(11+m)-S-S-(CH(2))(11)-OH (m = 0, +/- 2, +/- 4, +/- 6) on gold-covered mica. The advancing and receding contact angles decrease linearly with increasing hydrophilicity of the SAM. The latter was changed either via the molar ratio or via the chain length of the hydroxyl-terminated alkyl chains in the monolayer.

Stress relaxation microscopy: imaging local stress in cells.

Biomechanics is gaining relevance as complementary discipline to structural and cellular biology. The response of cells to mechanical stimuli determines cell type and function, while the spatial distribution of mechanical forces within the cells is crucial to understand cell activity. The experimental methodologies to approach cell mechanics are diverse but either they are effective in few cases or they rule out the innate cell complexity.

Quantitative characterization of nanoadhesion by dynamic force spectroscopy.

We present a method for the characterization of adhesive bonds formed in nanocontacts. Using a modified atomic force microscope, the nanoadhesion between a silicon nitride tip and a self-assembled monolayer of 1-nonanethiol on gold(111) was measured at different loading rates. Adhesion force-versus-loading rate curves could be fitted with two logarithmic terms, indicating a two step (two energy barrier) process.

Structure, surface interactions, and compressibility of bacterial S-layers through scanning force microscopy and the surface force apparatus.

Two-dimensional crystalline bacterial surface layers (S-layers) are found in a broad range of bacteria and archaea as the outermost cell envelope component. The self-assembling properties of the S-layers permit them to recrystallize on solid substrates. Beyond their biological interest as S-layers, they are currently used in nanotechnology to build supramolecular structures.

Chemical and thermal denaturation of crystalline bacterial S-layer proteins: an atomic force microscopy study.

Crystalline monomolecular cell surface layers, S-layers, are one of the most common outermost cell envelope components of the prokaryotic organisms (bacteria and archaeda) that protects them from competitive habitats. Since isolated S-protein subunits are able to re-assemble into crystalline arrays on lipid films and solid supports making biomimetic surfaces, S-layer technology is currently used in nanobiotechnology. An important aspect of the biomimetic surfaces built with S-layers is their stability under extreme solvent conditions or temperature.