Probing Membrane Viscosity and Interleaflet Friction of Supported Lipid Bilayers by Tracking Electrostatically Adsorbed, Nano-Sized Vesicles

Particle tracking is used to measure the diffusional motion of nanosized (≈100 nm), lipid vesicles that are electrostatically adsorbed onto a solid supported lipid bilayer. It is found that the motion of membrane-adhering vesicles is Brownian and depends inversely on the vesicle size, but is insensitive to the vesicle surface charge. The measured diffusivity agrees well with the Evans–Sackmann model for the diffusion of inclusions in supported, fluidic membranes. The agreement implies that the vesicle motion is coupled to that of a nanoscopic lipid cluster in the upper leaflet, which slides over the lower leaflet. The diffusivity of membrane-adhering vesicles is therefore predominantly governed by the interleaflet friction coefficient, while the diffusivity of single lipids is mainly governed by the membrane viscosity. Combined with fluorescence recovery after photobleaching analysis, the interleaflet friction coefficient and the membrane viscosity are determined by applying the Evans–Sackmann model to the measured diffusivity of membrane adhering vesicles and that of supported membrane lipids. This approach provides an alternative to existing methods for measuring the interleaflet friction coefficient and the membrane viscosity

Effect of Glucose on the Mobility of Membrane-Adhering Liposomes

Enclosed lipid bilayer structures, referred to as liposomes or lipid vesicles, have a wide range of biological functions, such as cellular signaling and membrane trafficking. The efficiency of cellular uptake of liposomes, a key step in many of these functions, is strongly dependent on the contact area between a liposome and a cell membrane, which is governed by the adhesion force w, the membrane bending energy κ, and the osmotic pressure Δp. Herein, we investigate the relationship between these forces and the physicochemical properties of the solvent, namely, the presence of glucose (a nonionic osmolyte). Using fluorescence microscopy, we measure the diffusivity D of small (∼50 nm radius), fluorescently labeled liposomes adhering to a supported lipid bilayer or to the freestanding membrane of a giant (∼10 μm radius) liposome. It is observed that glucose in solution reduces D on the supported membrane, while having negligible effect on D on the freestanding membrane. Using well-known hydrodynamic theory for the diffusivity of membrane inclusions, these observations suggest that glucose enhances the contact area between the small liposomes and the underlying membrane, while not affecting the viscosity of the underlying membrane. In addition, quartz crystal microbalance experiments showed no significant change in the hydrodynamic height of the adsorbed liposomes, upon adding glucose. This observation suggests that instead of osmotic deflation, glucose enhances the contact area via adhesion forces, presumably due to the depletion of the glucose molecules from the intermembrane hydration layer

A model derived from hydrodynamic simulations for extracting the size of spherical particles from the quartz crystal microbalance

One challenging aspect of quartz crystal microbalance (QCM) measurements is the characterization of adsorbed particles as the change in resonance frequency (Δf) is proportional not only to the inertia of the adsorbed layer but also to that of the hydrodynamically coupled fluid. Herein, by solving numerically the Navier–Stokes equations, we scrutinize Δf for sparsely deposited, rigid spherical particles that are firmly attached to an oscillating surface. The analysis is shown to be applicable to adsorbed, small unilamellar vesicles (SUVs) of controlled size under experimental conditions in which adhesion-induced vesicle deformation is negligible. The model supports a hydrodynamic explanation for the overtone dependence of Δf, and was fitted to experimental data concerning three monodisperse populations of SUVs with different average sizes ranging between 56 and 114 nm diameter. Using this procedure, we determined the average size of adsorbed vesicles to be within 16% of the size that was measured by dynamic light scattering experiments in bulk solution. In conclusion, this model offers a means to extract the particle size from QCM-D measurement data, with applications to biological and synthetic nanoparticles

Hydrodynamic Propulsion of Liposomes Electrostatically Attracted to a Lipid Membrane Reveals Size-Dependent Conformational Changes

The efficiency of lipid nanoparticle uptake across cellular membranes is strongly dependent on the very first interaction step. Detailed understanding of this step is in part hampered by the large heterogeneity in the physicochemical properties of lipid nanoparticles, such as liposomes, making conventional ensemble-averaging methods too blunt to address details of this complex process. Here, we contribute a means to explore whether individual liposomes become deformed upon binding to fluid cell-membrane mimics. This was accomplished by using hydrodynamic forces to control the propulsion of nanoscale liposomes electrostatically attracted to a supported lipid bilayer. In this way, the size of individual liposomes could be determined by simultaneously measuring both their individual drift velocity and diffusivity, revealing that for a radius of ∼45 nm, a close agreement with dynamic light scattering data was observed, while larger liposomes (radius ∼75 nm) displayed a significant deformation unless composed of a gel-phase lipid. The relevance of being able to extract this type of information is discussed in the context of membrane fusion and cellular uptake

Quartz Crystal Microbalance Model for Quantitatively Probing the Deformation of Adsorbed Particles at Low Surface Coverage

Characterizing the deformation of nanoscale, soft-matter particulates at solid− liquid interfaces is a demanding task, and there are limited experimental options to perform quantitative measurements in a nonperturbative manner. Previous attempts, based on the quartz crystal microbalance (QCM) technique, focused on the high surface coverage regime and modeled the adsorbed particles as a homogeneous film, while not considering the coupling between particles and surrounding fluid and hence resulting in an underestimation of the known particle height. In this work, we develop a model for the hydrodynamic coupling between adsorbed particles and surrounding fluid in the limit of a low surface coverage, which can be used to extract shape information from QCM measurement data. We tackle this problem by using hydrodynamic simulations of an ellipsoidal particle on an oscillating surface. From the simulation results, we derived a phenomenological relation between the aspect ratio r of the absorbed particles and the slope and intercept of the line that fits instantaneous, overtone-dependent QCM data on (δ/a, −Δf/n) coordinates where δ is the viscous penetration depth, a is the particle radius, Δf is the QCM frequency shift, and n is the overtone number. The model was applied to QCM measurement data pertaining to the adsorption of 34 nm radius, fluid-phase and gel-phase liposomes onto a titanium oxide-coated surface. The osmotic pressure across the liposomal bilayer was varied to induce shape deformation. By combining these results with a membrane bending model, we determined the membrane bending energy for the gel-phase liposomes, and the results are consistent with literature values. In summary, a phenomenological model is presented and validated in order to show for the first time that QCM experiments can quantitatively measure the deformation of adsorbed particles at low surface coverage

Sugarcane genes associated with sucrose content

<p>Abstract</p> <p>Background -</p> <p>Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants.</p> <p>Results -</p> <p>We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways.</p> <p>Conclusion -</p> <p>Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.</p

Transcriptional responses of winter barley to cold indicate nucleosome remodelling as a specific feature of crown tissues

We report a series of microarray-based comparisons of gene expression in the leaf and crown of the winter barley cultivar Luxor, following the exposure of young plants to various periods of low (above and below zero) temperatures. A transcriptomic analysis identified genes which were either expressed in both the leaf and crown, or specifically in one or the other. Among the former were genes responsible for calcium and abscisic acid signalling, polyamine synthesis, late embryogenesis abundant proteins and dehydrins. In the crown, the key organ for cereal overwintering, cold treatment induced transient changes in the transcription of nucleosome assembly genes, and especially H2A and HTA11, which have been implicated in cold sensing in Arabidopsis thaliana. In the leaf, various heat-shock proteins were induced. Differences in expression pattern between the crown and leaf were frequent for genes involved in certain pathways responsible for osmolyte production (sucrose and starch, raffinose, γ-aminobutyric acid metabolism), sugar signalling (trehalose metabolism) and secondary metabolism (lignin synthesis). The action of proteins with antifreeze activity, which were markedly induced during hardening, was demonstrated by a depression in the ice nucleation temperature