Biology subjects

Valotteau, C.

Publications and source records attributed to Valotteau, C..

3 recordsLinked to original sources

bioRxiv2024

Modulation of SARS-CoV-2 spike binding to ACE2 throughconformational selection

The first step of SARS-CoV-2 infection involves the interaction between the trimeric viral spike protein (S) and the host angiotensin-converting enzyme 2 (ACE2). The receptor binding domain (RBD) of S adopts two conformations: open and closed, respectively, accessible and inaccessible to ACE2. Therefore, RBD motions are suspected to affect ACE2 binding; yet a quantitative description of the underlying mechanism has been elusive. Here, using single-molecule approaches, we visualize RBD opening and closing and probe the S/ACE2 interaction. Our results show that RBD dynamics affect ACE2 binding but not unbinding. The resulting modulation is quantitatively predicted by a conformational selection model in which each protomer behaves independently. Our work reveals a general molecular mechanism affecting binding affinity without altering binding strength, helping to understand coronavirus infection and immune evasion.

biophysics↗

bioRxiv2022

Coupled mechanical mapping and interference contrast microscopy reveal viscoelastic and adhesion hallmarks of monocytes differentiation into macrophages

Monocytes in the blood torrent, when activated by pro-inflammatory signals, adhere to the vascular endothelium and migrate into the tissue for ultimately differentiate into macrophages. Mechanics and adhesion play a crucial role in macrophage functions, such as migration and phagocytosis. However, how monocytes change their adhesion and mechanical properties upon differentiation into macrophages is still not well understood. In this work, we combined atomic force microscopy (AFM) viscoelastic mapping with interference contrast microscopy (ICM) to simultaneously probe, at the single-cell level, viscoelasticity and adhesion during monocyte differentiation. THP-1 monocytic cells were differentiated into macrophages through phorbol 12-myristate 13-acetate (PMA). Morphological quantification was achieved using holographic tomography imaging and the expression of integrin subunit CD11b was tracked as a marker of differentiation. Holographic tomography proved to be a quantitative in vivo technique, revealing a dramatic increase in macrophage volume and surface area and two subpopulations, spread and round macrophages. AFM viscoelastic mapping revealed an increased stiffness and more solid-like behavior of differentiated macrophages, especially in the lamellipodia and microvilli regions. Differentiated cells revealed an important increase of the apparent Youngs modulus (E0) and a decrease of cell fluidity ({beta}) on differentiated cells, which correlated with an increase in adhesion area. Macrophages with a spreading phenotype enhanced these changes. Remarkably, when adhesion was eliminated, differentiated macrophages remained stiffer and more solid-like than monocytes, suggesting a permanent reorganization of the cytoskeleton. We speculate that the more solid-like microvilli and lamellipodia might help macrophages to minimize energy dissipation during mechanosensitive activity, such as phagocytosis, making it more efficient. Our proposed approach revealed viscoelastic and adhesion hallmarks of monocyte differentiation that may be important for biological function.

biophysics↗

bioRxiv2022

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds

Single-molecule data are of great significance in biology, chemistry, and medicine. However, experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are needed. Acoustic force spectroscopy (AFS) is an emerging manipulation technique which generates acoustic waves to apply force in parallel on a large population of microbeads tethered to a surface. We have exploited this configuration on a recently developed modular Junctured-DNA (J-DNA) scaffold designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measured its unbinding kinetics under force at the single-bond level. Special effort was made in analyzing the data in order to identify potential pitfalls. We established a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy for measuring the force dependent rupture of a single domain antibody with its antigen. We get a good agreement with standard measurement at zero force. Our technique offers single molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

biophysics↗