Cell-surface contacts determine volume and mechanical properties of human embryonic kidney 293 T cells.

Alterations in the organization of the cytoskeleton precede the escape of adherent cells from the framework of cell-cell and cell-matrix interactions into suspension. With cytoskeletal dynamics being linked to cell mechanical properties, many studies elucidated this relationship under either native adherent or suspended conditions. In contrast, tethered cells that mimic the transition between both states have not been the focus of recent research. Using human embryonic kidney 293 T cells we investigated all three conditions in the light of alterations in cellular shape, volume, as well as mechanical properties and relate these findings to the level, structure, and intracellular localization of filamentous actin (F-actin). For cells adhered to a substrate, our data shows that seeding density affects cell size but does not alter their elastic properties. Removing surface contacts leads to cell stiffening that is accompanied by changes in cell shape, and a reduction in cellular volume but no alterations in F-actin density. Instead, we observe changes in the organization of F-actin indicated by the appearance of blebs in the semi-adherent state. In summary, our work reveals an interplay between molecular and mechanical alterations when cells detach from a surface that is mainly dominated by cell morphology.

Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease.

MYH9-related disease patients with mutations in the contractile protein nonmuscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild-to-moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction in the presence of tPA and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in patients with MYH9-related disease, and treatment with tranexamic acid can improve the hemostatic function.

Specific domain V reduction of beta-2-glycoprotein I induces protein flexibility and alters pathogenic antibody binding.

Beta-2-glycoprotein I (ß2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). ß2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of ß2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of ß2GPI. Atomic force microscopy images suggested that reduced ß2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced ß2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of ß2GPI reduction on the protein structure and its implications for antibody binding in APS patients.

Enhancement of Intracellular Calcium Ion Mobilization by Moderately but Not Highly Positive Material Surface Charges.

Electrostatic forces at the cell interface affect the nature of cell adhesion and function; but there is still limited knowledge about the impact of positive or negative surface charges on cell-material interactions in regenerative medicine. Titanium surfaces with a variety of zeta potentials between -90 mV and +50 mV were generated by functionalizing them with amino polymers, extracellular matrix proteins/peptide motifs and polyelectrolyte multilayers. A significant enhancement of intracellular calcium mobilization was achieved on surfaces with a moderately positive (+1 to +10 mV) compared with a negative zeta potential (-90 to -3 mV). Dramatic losses of cell activity (membrane integrity, viability, proliferation, calcium mobilization) were observed on surfaces with a highly positive zeta potential (+50 mV). This systematic study indicates that cells do not prefer positive charges in general, merely moderately positive ones. The cell behavior of MG-63s could be correlated with the materials' zeta potential; but not with water contact angle or surface free energy. Our findings present new insights and provide an essential knowledge for future applications in dental and orthopedic surgery.

High-throughput cell and spheroid mechanics in virtual fluidic channels.

Microfluidics by soft lithography has proven to be of key importance for biophysics and life science research. While being based on replicating structures of a master mold using benchtop devices, design modifications are time consuming and require sophisticated cleanroom equipment. Here, we introduce virtual fluidic channels as a flexible and robust alternative to microfluidic devices made by soft lithography. Virtual channels are liquid-bound fluidic systems that can be created in glass cuvettes and tailored in three dimensions within seconds for rheological studies on a wide size range of biological samples. We demonstrate that the liquid-liquid interface imposes a hydrodynamic stress on confined samples, and the resulting strain can be used to calculate rheological parameters from simple linear models. In proof-of-principle experiments, we perform high-throughput rheology inside a flow cytometer cuvette and show the Young's modulus of isolated cells exceeds the one of the corresponding tissue by one order of magnitude.

Lysine residues control the conformational dynamics of beta 2-glycoprotein I.

One of the major problems in the study of the dynamics of proteins is the visualization of changing conformations that are important for processes ranging from enzyme catalysis to signaling. A protein exhibiting conformational dynamics is the soluble blood protein beta 2-glycoprotein I (beta2GPI), which exists in two conformations: the closed (circular) form and the open (linear) form. It is hypothesized that an increased proportion of the open conformation leads to the autoimmune disease antiphospholipid syndrome (APS). A characteristic feature of beta2GPI is the high content of lysine residues. However, the potential role of lysine in the conformational dynamics of beta2GPI has been poorly investigated. Here, we report on a strategy to permanently open up the closed protein conformation by chemical acetylation of lysine residues using acetic acid N-hydroxysuccinimide ester (NHS-Ac). Specific and complete acetylation was demonstrated by the quantification of primary amino groups with fluoraldehyde o-phthalaldehyde (OPA) reagent, as well as western blot analysis with an anti-acetylated lysine antibody. Our results demonstrate that acetylated beta2GPI preserves its secondary and tertiary structures, as shown by circular dichroism spectroscopy. We found that after lysine acetylation, the majority of proteins are in the open conformation as revealed by atomic force microscopy high-resolution images. Using this strategy, we proved that the electrostatic interaction of lysine residues plays a major role in stabilizing the beta2GPI closed conformation, as confirmed by lysine charge distribution calculations. We foresee that our approach will be applied to other lysine-rich proteins (e.g. histones) undergoing conformational transitions. For instance, conformational dynamics can be triggered by environmental conditions (e.g. pH, ion concentration, post-translational modifications, and binding of ligands). Therefore, our study may be relevant for investigating the equilibrium of protein conformations causing diseases.

Determination of refractive index and layer thickness of nm-thin films via ellipsometry.

Ellipsometric measurements give information on two film properties with high precision, thickness and refractive index. In the simplest case, the substrate is covered with a single homogenous, transparent film. Yet, with ellipsometry, it is only possible to determine the two film properties thickness and refractive simultaneously if the layer thickness exceeds 15 nm - a restriction well known for a century. Here we present a technique to cross this limitation: A series expansion of the ellipsometric ratio ρ to the second order of the layer thickness relative to the wavelength reveals the first and second ellipsometric moment. These moments are properties of the thin film and independent of incident angle. Using both moments and one additional reference measurement enables to determine simultaneously both thickness and refractive index of ultra-thin films down to 5 nm thickness.

Abrogated Cell Contact Guidance on Amino-Functionalized Microgrooves.

Topographical and chemical features of biomaterial surfaces affect the cell physiology at the interface and are promising tools for the improvement of implants. The dominance of the surface topography on cell behavior is often accentuated. Striated surfaces induce an alignment of cells and their intracellular adhesion-mediated components. Recently, it could be demonstrated that a chemical modification via plasma polymerized allylamine was not only able to boost osteoblast cell adhesion and spreading but also override the cell alignment on stochastically machined titanium. In order to discern what kind of chemical surface modifications let the cell forget the underlying surface structure, we used an approach on geometric microgrooves produced by deep reactive ion etching (DRIE). In this study, we systematically investigated the surface modification by (i) methyl-, carboxyl-, and amino functionalization created via plasma polymerization processes, (ii) coating with the extracellular matrix protein collagen-I or immobilization of the integrin adhesion peptide sequence Arg-Gly-Asp (RGD), and (iii) treatment with an atmospheric pressure plasma jet operating with argon/oxygen gas (Ar/O2). Interestingly, only the amino functionalization, which presented positive charges at the surface, was able to chemically disguise the microgrooves and therefore to interrupt the microtopography induced contact guidance of the osteoblastic cells MG-63. However, the RGD peptide coating revealed enhanced cell spreading as well, with fine, actin-containing protrusions. The Ar/O2-functionalization demonstrated the best topography handling, e.g. cells closely attached even to features such as the sidewalls of the groove steps. In the end, the amino functionalization is unique in abrogating the cell contact guidance.

Lipid monolayers and adsorbed polyelectrolytes with different degrees of polymerization.

Polystyrene sulfonate (PSS) of different molecular weight M(w) is adsorbed to oppositely charged DODAB monolayers from dilute solutions (0.01 mmol/L). PSS adsorbs flatly in a lamellar manner, as is shown by X-ray reflectivity and grazing incidence diffraction (exception: PSS with M(w) below 7 kDa adsorbs flatly disordered to the liquid expanded phase). The surface coverage and the separation of the PSS chains are independent of PSS M(w). On monolayer compression, the surface charge density increases by a factor of 2, and the separation of the PSS chains decreases by the same factor. Isotherms show that on increase of PSS M(w) the transition pressure of the LE/LC (liquid expanded/liquid condensed) phase transition decreases. When the contour length exceeds the persistence length (21 nm), the transition pressure is low and constant. For low-M(w) PSS (<7 kDa) the LE/LC transition of the lipids and the disordered/ordered transition of adsorbed PSS occur simultaneously, leading to a maximum in the contour length dependence of the transition enthalpy. These findings show that lipid monolayers at the air/water interface are a suitable model substrate with adjustable surface charge density to study the equilibrium conformation of adsorbed polyelectrolytes as well as their interactions with a model membrane.

Temperature-induced transition from odd-even to even-odd effect in polyelectrolyte multilayers due to interpolyelectrolyte interactions.

Within a liquid cell the linear growth of polyelectrolyte multilayers from poly(styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) is observed with multiple angle null ellipsometry. The salt content is varied between 1 and 4 mol/L NaCl and the temperature between 20 and 55 °C. In the linear growth regime, the outermost layer is investigated. At low temperature, a top PSS layer is twice as thick as a top PAH layer (odd-even effect), consistent with the respective monomer volumes and the same water content for both kinds of top polyelectrolyte layers as confirmed by refractive index measurements. On heating, the thickness of a polycation/polyanion bilayer increases. For temperatures exceeding a crossover temperature, a top PAH layer is thicker than a top PSS layer (even-odd effect). Simultaneously, the index of refraction of the respective top layers indicates a compact PSS and a swollen PAH layer. It is suggested that, at elevated temperature and high salt conditions, secondary forces gain importance in comparison to electrostatic forces: therefore, a transition from an odd-even to an even-odd effect occurs, as well as the decreased film stability on drying as described before (Cornelsen, M., et al. Macromolecules 2010, 43, 4300). The ellipsometric data indicate that PAH/PSS layer pairs exceeding 8.6 nm thickness in solution are unstable in air.