Kools, W. | bioRxiv Science

bioRxiv2026

Myosin-X sorts actin filaments into parallel bundles and tunes barbed-end dynamics

Whether a molecular motor can spontaneously organize actin filaments into bundles has remained an open question despite decades of in vitro investigation. Here, we show that the dimeric myosin-X, essential for filopodia initiation and extension in cells, is capable of sorting actin filaments into parallel bundles, gathering barbed ends within 1-2 micrometers. We observe that myosin-X processivity is comparable on single filaments and on bundles induced by myosin-X or fascin. Upon reaching barbed ends, myosin-X slows down the addition or removal of actin subunits, in a myosin density-dependent manner. Furthermore, the funneling of myosin-X towards the remaining filaments at the bundle tip increases motor density and triggers dynamic clustering. Together, we propose that the motor activity of myosin-X is sufficient to initiate filopodia, independently of passive crosslinkers such as fascin or fimbrin.

biochemistry↗

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↗