Myosin forces elicit an F-actin structural landscape that mediates mechanosensitive protein recognition

Abstract

Cells mechanically interface with their surroundings through cytoskeleton-linked adhesions, allowing them to sense physical cues that instruct development and drive diseases such as cancer. Contractile forces generated by myosin motor proteins mediate these mechanical signal transduction processes through unclear protein structural mechanisms. Here, we show that myosin forces elicit structural changes in actin filaments (F-actin) that modulate binding by the mechanosensitive adhesion protein α-catenin. Using correlative cryo-fluorescence microscopy and cryo-electron tomography, we identify F-actin featuring domains of nanoscale oscillating curvature at cytoskeleton-adhesion interfaces enriched in zyxin, a marker of actin-myosin generated traction forces. We next introduce a reconstitution system for visualizing F-actin in the presence of myosin forces with cryo-electron microscopy, which reveals morphologically similar superhelical F-actin spirals. In simulations, transient forces mimicking tugging and release of filaments by motors produce spirals, supporting a mechanistic link to myosin's ATPase mechanochemical cycle. Three-dimensional reconstruction of spirals uncovers extensive asymmetric remodeling of F-actin's helical lattice. This is recognized by α-catenin, which cooperatively binds along individual strands, preferentially engaging interfaces featuring extended inter-subunit distances while simultaneously suppressing rotational deviations to regularize the lattice. Collectively, we find that myosin forces can deform F-actin, generating a conformational landscape that is detected and reciprocally modulated by a mechanosensitive protein, providing a direct structural glimpse at active force transduction through the cytoskeleton.

Keywords: Actin; cryo-electron microscopy; mechanobiology; mechanotransduction; myosin.

Fig. 1:. Myosin forces evoke oscillatory domains in F-actin.

a, Top: Low-magnification cryo-light microscopy (LM) image of Ptk2 cell, highlighting targeted adhesion. Middle: Medium magnification correlation between cryo-electron microscopy (EM) and LM, highlighting site of tomogram acquisition. Bottom: Fluorescence intensity scan along dashed line. Blue box indicates acquisition area, which is enriched in zyxin. b, Left: segmented tomogram. Right: False colored 5.1 nm thick projection of boxed area, highlighting F-actin oscillatory domain. c, Schematic of myosin force reconstitution assay. d, False-colored cryo-EM images of dual motor reconstitution in the presence and absence of ATP. Oscillatory domains, magenta; canonical F-actin, blue; carbon film, orange; ice contamination, yellow. e, Cryo-EM images of oscillatory domains formed in single-motor conditions, false colored as in d. f, Quantification of oscillatory domain wavelengths in the pointed end directed (n = 35) and barbed end directed (n = 63) conditions, from N = 2 independent experiments. Distributions were compared with an unpaired two-tailed Mann-Whitney test.

Fig. 2:. Transient force produces superhelical F-actin spirals.

a, Left: projection and right: serial slices of an F-actin oscillatory domain tomogram (pointed end directed force condition). b, Orthogonal views of denoised density from tomogram in a, highlighting spiraling oscillations in both XY and XZ dimensions. c, Aligned projection views along principal components (PC) of filament traces with spiral character in pointed end directed (purple, top, n = 7) and barbed end directed (green, bottom, n = 9) force conditions. Transparent lines represent individual filament traces, while solid lines represent averages of aligned traces. d, Quantitation of data from c. Top and middle: Analysis of filament trace amplitudes in PC1-PC2 vs. PC1-PC3 planes from pointed-end (top) and barbed-end force conditions (middle), compared by paired t-test. Bottom: fractional wavelength offset between trace projections in PC1-PC2 vs. PC1-PC3 planes; force conditions were compared by unpaired t-test. e, Schematic of potentials used in coarse-grained molecular dynamics simulations. f, Representative simulation snapshots and principal component analyses of key frames in indicated conditions (constant force protocol, or apply then release force protocol, for both tension and compression). BE: barbed end; PE: pointed end. g, Quantification of oscillatory domain wavelengths from the apply then release force protocol. Conditions were compared by an unpaired t-test.

Fig. 3:. Superhelical F-actin features unique architectural remodeling and subunit deformations

a, Left: cryo-EM density map of superhelical F-actin reconstructed from the dual motor condition. Strands are colored in alternating shades of blue. BE: barbed end; PE: pointed end. Right: Stitch of five copies of the map, recapitulating the morphology of oscillatory domains. b, Diagrams (left) and plots (right) of instantaneous helical parameters. Strands are colored as in a. Shaded regions represent 95% CI from 3 independent analyses. Vertical dashed lines indicate parameters of canonical F-actin (ref. 56). c, Left: ribbon diagram of an actin subunit, with subdomains colored in varying shades. Subdomain centroids are indicated by connected gray spheres. Right: averaged subdomain displacements (scaled 15X for visualization) after MDFF analysis relative to a canonical F-actin subunit (PDB 8D13) for the indicated conditions.

Fig. 4:. α-catenin detects and reciprocally modulates force-evoked structural changes in F-actin.

a, Frames from 3DVA variability analysis of the α-catenin ABD–F-actin complex reconstructed in the dual motor force condition, highlighting alternating binding along F-actin strands (arrowheads) and varying intensity of α-catenin density across the trajectory. b, Quantification of average filament curvature (top) and average α-catenin intensity (bottom) across the 3DVA trajectory. Boxes indicate regions with high filament curvature and either low (blue) or high (orange) α-catenin intensity. c, Plots of instantaneous helical rise from trajectory regions indicated in b. Vertical dashed lines indicate canonical F-actin rise. d, Docking analysis (PDB: 6UPV) of consensus reconstruction highlights inter-ABD contacts mediated by α-catenin’s C-terminal extension (arrows). e, Quantification of α-catenin intensity versus instantaneous rise of the consensus reconstruction (Fig. S9b). Vertical dashed line indicates canonical F-actin rise. f, Averaged subdomain displacements (scaled 15X for visualization) after MDFF analysis of the consensus map versus canonical F-actin (top, PDB: 8D13) and superhelical F-actin (bottom, MDFF model from Fig. 3f).

Fig. 5:. Conceptual model of myosin force transduction through mechanosensitive F-actin binding.

Cartoon schematizing generation of destabilized superhelical F-actin by myosin forces, which is detected and stabilized through cooperative binding contacts by α-catenin.