From single-molecule to living mammalian cells
The Alsteens’ team, part of the Nanobiophysics lab at ISV, aims at studying biological processes from the single-molecule to the cellular level. To this end, we develop and apply new nanobiophysical methods to investigate and quantitate the molecular interactions driving biological processes. Using atomic force microscopy (AFM), a microscopy technique using a very sharp tip as a force sensor, we image at high-resolution (sub-nanometer range) the architecture of complex biosystems and simultaneously force-probe the molecular interactions involved in crucial dynamic processes such as signal transduction, mechanosensing or specific interactions.
At the single-molecule level, we are interested in measuring the molecular interactions established either within a protein (intramolecular interactions) or between proteins (intermolecular interactions). Intramolecular interactions maintain the global three-dimensional structure of the polypeptide and at the same time allow the protein to adopt different conformations. Using AFM, we force-probe these interactions, by stretching the polypeptide and extracting for each sub-domains the force involved in the folded structure. Combining these measurements with molecular biology we decipher how individual amino acids are involved in the stabilization of the 3D-structure while enabling conformational variation. Often complex biological processes are triggered by intermolecular interactions such as ligand binding to a surface cellular receptor. Using functionalized AFM tips, we force-probe these interactions on a quantitative manner under physiological conditions. Extracting the interaction energy landscape allows us to better understand how a single ligand triggers a cellular response.
Imaging single membrane receptors reconstituted in lipid membranes. (a) Cartoon showing an AFM tip contouring membrane receptors under physiological conditions. (b) AFM topographical image showing single G-protein coupled receptors (small white dots) within a lipid bilayer (brown) deposited on mica (dark brown).
At the cellular level, many biological processes are established at the cells surface, e.g. cell adhesion, cell deformation, sensing or infection by pathogens. Studying the biophysical properties of cell surfaces allows us to better understand how a cell responds to an extracellular stimulus. Using AFM tips derivatized with biological molecules (from individual ligand to viruses or cells), we can measure interactions directly on the cellular context and better understand how cell surface receptors are involved in these biological processes. Recently, we measured the first binding step of a single rabies virus to living mammalian cells. We were able to quantitate the number of bonds established between a single virus particle and cell surface receptors and even understand the molecular details of this interaction. All the AFM measurements are simultaneously recorded with fluorescence images giving insight into the cellular state.
Imaging living mouse fibroblast cells. (a) Superimposition of wide field and fluorescence images (eGFP-histone (green) and mCherry-actin (orange) and (b) AFM topographic of the cells surfaces recorded in the dashed square in the fluorescence image.