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Atomic force microscopy (AFM) is emerging as an innovative tool to phenotype the brain. This study demonstrates the utility of AFM to determine nanomechanical and nanostructural features of the murine dorsolateral frontal cortex from weaning to adulthood. We found an increase in tissue stiffness of the primary somatosensory cortex with age, along with an increased cortical mechanical heterogeneity. To characterize the features potentially responsible for this heterogeneity, we applied AFM scan mode to directly image the topography of thin sections of the primary somatosensory cortical layers II/III, IV and V/VI. Topographical mapping of the cortical layers at successive ages showed progressive smoothing of the surface. Topographical images were also compared with histochemically derived morphological information, which demonstrated the deposition of perineuronal nets, important extracellular components and markers of maturity. Our work demonstrates that high-resolution AFM images can be used to determine the nanostructural properties of cortical maturation, well beyond embryonic and postnatal development. Furthermore, it may offer a new method for brain phenotyping and screening to uncover topographical changes in early stages of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. [ABSTRACT FROM AUTHOR]