Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information.

Galactosylceramides (GalCer) are glycosphingolipids bound to a monosaccharide group, responsible for inducing extensive hydrogen bonds that yield their alignment and accumulation in the outer leaflet of the biological membrane together with cholesterol (Chol) in rafts. In this work, the influence of GalCer on the nanomechanical properties of supported lipid bilayers (SLBs) based on DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DLPC (1,2-didodecanoyl-sn-glycero-3-phosphocoline) as model systems was assessed. Phosphatidylcholine (PC):GalCer SLBs were characterized by means of differential scanning calorimetry (DSC) and atomic force microscopy (AFM), in both imaging and force spectroscopy (AFM-FS) modes. Comparing both PC systems, we determined that the behaviour of SLB mixtures is governed by the PC phase-like state at the working temperature. While a phase segregated system is observed for DLPC:GalCer SLBs, GalCer are found to be dissolved in DPPC SLBs for GalCer contents up to 20 mol%. In both systems, the incorporation of GalCer intensifies the nanomechanical properties of SLBs. Interestingly, segregated domains of exceptionally high mechanical stability are formed in DLPC:GalCer SLBs. Finally, the role of 20 mol% Chol in GalCer organization and function in the membranes was assessed. Both PC model systems displayed phase segregation and remarkable nanomechanical stability when GalCer and Chol coexist in SLBs.

Cell processes like endocytosis, membrane resealing, signaling and transcription involve conformational changes which depend on the chemical composition and the physicochemical properties of the lipid membrane. The better understanding of the mechanical role of lipids in cell membrane force-triggered and sensing mechanisms has recently become the focus of attention. Different membrane models and experimental methodologies are commonly explored. While general approaches involve controlled vesicle deformation using micropipettes or optical tweezers, due to the local and dynamic nature of the membrane, high spatial resolution atomic force microscopy (AFM) has been widely used to study the mechanical compression and indentation of supported lipid bilayers (SLBs). However, the substrate contribution remains unkown. Here, we demonstrate how pulling lipid tubes with an AFM out of model SLBs can be used to assess the nanomechanics of SLBs through the evaluation of the tube growing force (F_tube), allowing for very local evaluation with high spatial and force resolution of the lipid membrane tension. We first validate this approach to determine the contribution of different phospholipids, by varying the membrane composition, in both one-component and phase-segregated membranes. Finally, we successfully assess the contribution of the underlying substrate to the membrane mechanics, demonstrating that SLB models may represent an intermediate scenario between a free membrane (blebs) and a cytoskeleton supported membrane.

We present a novel and user-friendly setup for GIXD characterization of hydrated individual lipid bilayers, differentiating the coexisting domains in DPPC : chol membranes.

A fast atomic force microscope (AFM) has been developed that can be installed as a sample holder for grazing-incidence X-ray experiments at solid/gas or solid/liquid interfaces. It allows a wide range of possible investigations, including soft and biological samples under physiological conditions (hydrated specimens). The structural information obtained using the X-rays is combined with the data gathered with the AFM (morphology and mechanical properties), providing a unique characterization of the specimen and its dynamicsin situduring an experiment. In this work, lipid monolayers and bilayers in air or liquid environment have been investigated by means of AFM, both with imaging and force spectroscopy, and X-ray reflectivity. In addition, this combination allows the radiation damage induced by the beam on the sample to be studied, as has been observed on DOPC and DPPC supported lipid bilayers under physiological conditions.