Bottom Effect in Atomic Force Microscopy Nanomechanics

In this work, the influence of the rigid substrate on the determination of the sample Young''s modulus, the so-called bottom-effect artifact, is demonstrated by an atomic force microscopy force-spectroscopy experiment. The nanomechanical properties of a one-component supported lipid membrane (SLM) exhibiting areas of two different thicknesses are studied: While a standard contact mechanics model (Sneddon) provides two different elastic moduli for these two morphologies, it is shown that Garcia''s bottom-effect artifact correction yields a unique value, as expected for an intrinsic material property. Remarkably, it is demonstrated that the ratio between the contact radius (and not only the indentation) and the sample thickness is the key parameter addressing the relevance of the bottom-effect artifact. The experimental results are validated by finite element method simulations providing a solid support to Garcia''s theory. The amphiphilic nature of the investigated material is representative of several kinds of lipids, suggesting that the results have far reaching implications for determining the correct Young''s modulus of SLMs. The generality of Garcia''s bottom-effect artifact correction allows its application to every kind of supported soft film

Synthesis of Gb₃ glycosphingolipids with labeled head groups: Distribution in phase-separated giant unilamellar vesicles

The receptor lipid Gb3 is responsible for the specific internalization of Shiga toxin (STx) into cells. The head group of Gb3 defines the specificity of STx binding, and the backbone with different fatty acids is expected to influence its localization within membranes impacting membrane organization and protein internalization. To investigate this influence, a set of Gb3 glycosphingolipids labeled with a BODIPY fluorophore attached to the head group was synthesized. C24 fatty acids, saturated, unsaturated, α-hydroxylated derivatives, and a combination thereof, were attached to the sphingosine backbone. The synthetic Gb3 glycosphingolipids were reconstituted into coexisting liquid-ordered (lo )/liquid-disordered (ld ) giant unilamellar vesicles (GUVs), and STx binding was verified by fluorescence microscopy. Gb3 with the C24:0 fatty acid partitioned mostly in the lo phase, while the unsaturated C24:1 fatty acid distributes more into the ld phase. The α-hydroxylation does not influence its partitioning

Shiga toxin binding alters lipid packing and the domain structure of Gb₃-containing membranes: A solid-state NMR study

We studied the influence of globotriaosylceramide (Gb3) lipid molecules on the properties of phospholipid membranes composed of a liquid ordered (lo)/liquid disordered (ld) phase separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/N-palmitoyl-d-erythro-sphingosylphosphorylcholine (PSM)/cholesterol mixture (40/35/20, mol/mol/mol) supplemented with 5 mol% of either short acyl chain palmitoyl-Gb3 or long acyl chain lignoceryl-Gb3 using 2H solid-state NMR spectroscopy. To this end, both globotriaosylceramides were chemically synthesized featuring a perdeuterated lipid acyl chain. The solid-state 2H NMR spectra support the phase separation into a POPC-rich ld phase and a PSM/cholesterol-rich lo phase. The long chain lignoceryl-Gb3 showed a rather unusual order parameter profile of the acyl chain, which flattens out for the last ∼6 methylene segments. Such an odd chain conformation can be explained by partial chain interdigitation and/or a very fluid midplane region of the membrane. Possibly, the Gb3 molecules may thus preferentially be localized at the lo/ld phase boundary. In contrast, the short chain palmitoyl-Gb3 was well associated with the PSM/cholesterol-rich lo phase. Gb3 molecules act as membrane receptors for the Shiga toxin (STx) produced by Shigella dysenteriae and by enterohemorrhagic strains of Escherichia coli (EHEC). The B-subunits of STx (STxB) forming a pentameric structure were produced recombinantly and incubated with the membrane mixtures leading to alterations in the lipid packing properties and lateral organization of the membranes. Typically, STxB binding led to a decrease in lipid chain order in agreement with partial immersion of protein segments into the lipid-water interface of the membrane. In the presence of STxB, Gb3 preferentially partitioned into the lo membrane phase. In particular the short acyl chain palmitoyl-Gb3 showed very similar chain order parameters to PSM. In the presence of STxB, all lipid species showed isotropic contributions to the 2H NMR powder spectra; this was most pronounced for the Gb3 molecules. Such isotropic contributions are caused by highly curved membrane structures, which have previously been detected as membrane invaginations in fluorescence microscopy. Our analysis estimated that STxB induced highly curved membrane structures with a curvature radius of less than ∼10 nm likely related to the insertion of STxB segments into the lipid-water interface of the membrane