Force Mapping Study of Actinoporin Effect in Membranes Presenting Phase Domains

Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins.

Interplay Between Receptor-Ligand Binding and Lipid Domain Formation Depends on the Mobility of Ligands in Cell-Substrate Adhesion

Cell-cell adhesion and the adhesion of cells to extracellular matrix are mediated by the specific binding of receptors on the cell membrane to their cognate ligands on the opposing surface. The adhesion receptors can exhibit affinity for nanoscale lipid clusters that form in the cell membrane. Experimental studies of such adhesion systems often involve a cell adhering either to a solid surface with immobile ligands or a supported lipid bilayer with mobile ligands. A central question in these cell-substrate adhesions is how the mobility of the ligands physically affects their binding to the adhesion receptors and thereby the behavior of the nanoscale lipid clusters associated with the receptors. Using a statistical mechanical model and Monte Carlo simulations for the adhesion of cells to substrates with ligands, we find that, for mobile ligands, binding to adhesion receptors can promote the formation of mesoscale lipid domains, which in turn enhances the receptor-ligand binding. However, in the case of immobile ligands, the receptor-ligand binding and the tendency for the nanoscale lipid clusters to further coalesce depend on the distribution of the ligands on the substrate. Our findings help to explain why different adhesion experiments for identifying the interplay between receptor-ligand binding and heterogeneities in cell membranes led to contradictory results.

Quercetin Induces Lipid Domain-Dependent Permeability

In this work, we present our results on quercetin interaction with distinct model membranes exploring the importance of lipid phases, ld, ld/lo and ld+lo+so, to the action of this flavonoid in bilayers and possibly contributing to clarifying some controversial aspects related to quercetin multiple activities. We found out that quercetin is able to increase membrane permeability in a manner dependent on the presence and characteristics of lipid domains. In the presence of sphingomyelin, we found the greatest increase in mean membrane permeability (at least 10 times higher than the other lipid compositions). We also observed the presence of micrometric domains whose shape and size were disturbed by the action of quercetin. The presence of cholesterol increased membrane rigidity. This effect was enhanced with the presence of quercetin, but for chol-sphingomyelin combination, the bilayers became more flaccid at low quercetin/lipid proportions (< 1/5) and moderately rigid at proportions of the 1/1 order. The affinity parameters were higher for the most homogeneous systems and with larger areas and extensions of disordered liquid phase than for those systems of higher heterogeneity.

Quercetin Induces Lipid Domain-Dependent Permeability

In this work, we present our results on quercetin interaction with distinct model membranes exploring the importance of lipid phases, ld, ld/lo and ld+lo+so, to the action of this flavonoid in bilayers and possibly contributing to clarifying some controversial aspects related to quercetin multiple activities. We found out that quercetin is able to increase membrane permeability in a manner dependent on the presence and characteristics of lipid domains. In the presence of sphingomyelin, we found the greatest increase in mean membrane permeability (at least 10 times higher than the other lipid compositions). We also observed the presence of micrometric domains whose shape and size were disturbed by the action of quercetin. The presence of cholesterol increased membrane rigidity. This effect was enhanced with the presence of quercetin, but for chol-sphingomyelin combination, the bilayers became more flaccid at low quercetin/lipid proportions (< 1/5) and moderately rigid at proportions of the 1/1 order. The affinity parameters were higher for the most homogeneous systems and with larger areas and extensions of disordered liquid phase than for those systems of higher heterogeneity.

Quercetin Induces Lipid Domain-Dependent Permeability

In this work, we present our results on quercetin interaction with distinct model membranes exploring the importance of lipid phases, ld, ld/lo and ld+lo+so, to the action of this flavonoid in bilayers and possibly contributing to clarifying some controversial aspects related to quercetin multiple activities. We found out that quercetin is able to increase membrane permeability in a manner dependent on the presence and characteristics of lipid domains. In the presence of sphingomyelin, we found the greatest increase in mean membrane permeability (at least 10 times higher than the other lipid compositions). We also observed the presence of micrometric domains whose shape and size were disturbed by the action of quercetin. The presence of cholesterol increased membrane rigidity. This effect was enhanced with the presence of quercetin, but for chol-sphingomyelin combination, the bilayers became more flaccid at low quercetin/lipid proportions (< 1/5) and moderately rigid at proportions of the 1/1 order. The affinity parameters were higher for the most homogeneous systems and with larger areas and extensions of disordered liquid phase than for those systems of higher heterogeneity.

Cytoplasmic physical state governs the influence of oxygen on Pinus densiflora seed ageing

During desiccation, the cytoplasm of orthodox seeds solidifies in a glass with highly restricted diffusion and molecular mobility, which extend longevity. Temperature and moisture determine seed cellular physical state, and O2 can promote deteriorative reactions of seed ageing. However, whether seed physical state affects O2-mediated biochemical reactions during ageing remains unknown. Here, we answered this question using oil-rich Pinus densiflora seeds aged by controlled deterioration (CD) at 45 °C and distinct relative humidities (RHs), resulting in a glassy (9 and 33% RH) or fluid (64 and 85% RH) cytoplasm. Regardless of CD regimes, the cellular lipid domain remained always fluid. Hypoxia (0.4% O2) prevented seed deterioration only in the glassy state, limiting non-enzymatic lipid peroxidation, consumption of antioxidants (glutathione, tocopherols) and unsaturated fatty acids, accompanied by decreased lipid melt enthalpy and lower concentrations of aldehydes and reactive electrophile species (RES). In contrast, a fluid cytoplasm promoted faster seed deterioration and enabled the resumption of enzymatic activities implicated in glutathione metabolism and RES detoxification, regardless of O2 availability. Furthermore, seeds stored under dry/cold seed bank conditions showed biochemical profiles similar to those of CD-aged seeds with glassy cytoplasm under normoxia. These findings are discussed in the context of germplasm management.