Dos Santos, T. | bioRxiv Science

bioRxiv2025

Probing protein-protein interactions with drag flow: A case study of F-actin and tropomyosin

Tropomyosin are central regulators of the actin cytoskeleton, controlling the binding and activity of the other actin binding proteins. The interaction between tropomyosin and actin is quite unique: single tropomyosin dimers bind weakly to actin filaments but get stabilised by end-to-end attachment with neighbouring tropomyosin dimers, forming clusters which wrap around the filament. Force spectroscopy is a powerful approach for studying protein-protein interactions, but classical methods which usually pull with pN forces on a single protein pair, are not well adapted to tropomyosins. Here, we propose a method in which a hydrodynamic drag force is applied directly to the proteins of interest, by imposing a controlled fluid flow inside a microfluidic chamber. The breaking of the protein bonds is directly visualised with fluorescence microscopy. Using this approach, we reveal that very low forces from 0.01 to 0.1 pN per tropomyosin dimer trigger the detachment of entire tropomyosin clusters from actin filaments. We show that the tropomyosin cluster detachment rate depends on the cytoplasmic tropomyosin isoform (Tpm1.6, 1.7, 1.8) and increases exponentially with the applied force. These observations lead us to propose a cluster detachment model which suggests that tropomyosins dynamically explore different positions over the actin filament. Our experimental setup can be used with many other cytoskeletal proteins, and we show, as a proof-of-concept, that the velocity of myosin-X motors is reduced by an opposing fluid flow. Overall, this method expands the range of protein-protein interactions that can be studied by force spectroscopy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/653996v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@19298b4org.highwire.dtl.DTLVardef@16766beorg.highwire.dtl.DTLVardef@18a70c6org.highwire.dtl.DTLVardef@1f1fad5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Biomimetic Virus-Like Particles to control cell functions

Biomimetic cues from the extracellular matrix (ECM) are essential for optimizing cell microenvironments and biomaterials. While native ECM proteins or synthetic peptides offer potential solutions, challenges such as production cost, solubility, and conformational stability limit their use. Here, we present the development of virus-like particles (VLPs) derived from the AP205 RNA phage displaying peptides from key ECM proteins and evaluate their biological activity in a variety of assays. We show that our engineered VLPs can effectively stimulate cell adhesion, migration, proliferation and differentiation. By comparing focal adhesions formed by RGD VLPs with their parent protein, fibronectin, we elucidate both similarities and differences in cell interactions. In addition, we construct heterodimeric particles co-expressing RGD with differentiation peptides and demonstrate retention of bioactivity in a multi-peptide context. This study establishes AP205 VLPs as versatile nanoscale platforms capable of tuning cell functions, with promising applications in nanomedicine and biomaterials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/612851v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@191b2c2org.highwire.dtl.DTLVardef@6edf5corg.highwire.dtl.DTLVardef@10a134aorg.highwire.dtl.DTLVardef@7864ef_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

bioengineering↗