Understanding molecular details about complex cellular processes

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 molecular interactions driving biological processes.  Using the last developments in atomic force microscopy (AFM), 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. We share facilities and staff with the Dufrêne’s team.

At the single-molecule level, we are interested in measuring the intra- and inter-molecular interactions involved in protein structure, structural dynamics, interactions and signaling. 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, we unfold individual proteins and we extract the folding energy landscape. This allows us to determine how receptors are stabilized and how they undergo conformational changes to induce signaling. 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 kinetic and thermodynamic parameters describing the binding free-energy landscape allows us to better understand how a single ligand interacts with its specific receptors and induce a cellular response.   

 Imaging single GPCRs reconstituted in lipid membranes. (a) Cartoon showing an AFM tip contouring membrane receptors under physiological conditions using force-distance based AFM. (b) Force vs time and force vs separation curves with quantitative parameters extracted from the force curves (c) Height and (d) adhesion images showing sparsely distributed GPCRs within the lipid bilayers with adhesion events localized on the receptors. For further details refer to the paper (Alsteens et al., Nature Methods, 2015).

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 acell responds to an extracellular stimulus. Using force-distance based AFM, we can image at high-resolution the topography of cellular surfaces and simultaneously extract the biophysical properties such as elasticity or adhesion. Using AFM tips derivatized with biological molecules (from individual ligand to viruses or cells), we can for example measure interactions directly in the cellular context and better understand how cell surface receptors are involved in various biological processes and better understand how biological systems change their structural and biophysical to adjust functionality. Recently, we measured the first binding steps 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 these interactions. All the AFM measurements are simultaneously recorded with fluorescence images giving insight into the cellular state. 

Probing virus-binding sites on living cells. (a) Cartoon showing an AFM tip functionalized with a single-virion on a confluent layer of mammalian cells. (b) Confocal image showing mCherry TVA receptors expressed at the cellular surface of MDCK cells (c) AFM height and adhesion images recorded with the functionalized tip. (d) Representative force vs time curves showing multiple unbinding events during the rupture of virus-surface interactions.  For further details please refer to the research article (Alsteens et al., Nature Nanotechnology, 2017).