Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an AFM to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, that involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from Mass Spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing 4 constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment.

The folding dynamics of proteins at the single-molecule level has been studied with single-molecule force spectroscopy experiments for 20 years, but a common standardized method for the analysis of the collected data and for sharing among the scientific community members is still not available. We have developed a new open-source tool-Fodis-for the analysis of the force-distance curves obtained in single-molecule force spectroscopy experiments, providing almost automatic processing, analysis, and classification of the obtained data. Our method provides also a classification of the possible unfolding pathways and the structural heterogeneity present during the unfolding of proteins.

The out-of-plane optical constants of monolayer two-dimensional materials have proven to be experimentally elusive. Owing to their reduced dimensionality, optical measurements have limited sensitivity to these properties which are hidden by the optical response of the substrate. Therefore, there remains an absence of scientific consensus on how to correctly model these crystals. Here we perform an experiment on the optical response of a single-layer two-dimensional crystal that addresses these problems. We successfully remove the substrate contribution to its optical response by a step deposition of a monolayer crystal inside a thick polydimethylsiloxane prism. This allows for a reliable determination of both the in-plane and the out-of-plane components of its surface susceptibility tensor. Our results prescribe one clear theoretical model for these crystals. This precise characterization of their optical properties will be relevant to future progresses in photonics and optoelectronics with two-dimensional materials.

Cell membranes separate the cell interior from the external environment. They are constituted by a variety of lipids; their composition determines the dynamics of membrane proteins and affects the ability of the cells to adapt. Even though the study of model membranes allows to understand the interactions among lipids and the overall mechanics, little is known about these properties in native membranes. To combine topology and nanomechanics analysis of native membranes, I designed a method to investigate the plasma membranes isolated from a variety of single cells. Five cell types were chosen and tested, revealing 20% variation in membrane thickness. I probed the resistance of the isolated membranes to indent, finding their line tension and spreading pressure. These results show that membranes isolated from neurons are stiffer and less diffusive than brain cancer cell membranes. This method gives direct quantitative insights on the mechanics of native cell membranes.

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