The actin cytoskeleton mediates mechanical coupling between cells and their tissue microenvironments. The architecture and composition of actin networks are modulated by force, but it is unclear how interactions between actin filaments (F-actin) and associated proteins are mechanically regulated. Here, we employ both optical trapping and biochemical reconstitution with myosin motor proteins to show single piconewton forces applied solely to F-actin enhance binding by the human version of the essential cell-cell adhesion protein αE-catenin, but not its homolog vinculin. Cryo-electron microscopy structures of both proteins bound to F-actin reveal unique rearrangements that facilitate their flexible C-termini refolding to engage distinct interfaces. Truncating α-catenin's C-terminus eliminates force-activated F-actin binding, and addition of this motif to vinculin confers force-activated binding, demonstrating that α-catenin's C-terminus is a modular detector of F-actin tension. Our studies establish that piconewton force on F-actin can enhance partner binding, which we propose mechanically regulates cellular adhesion through a-catenin.

ATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation1–3. In turn, force4,5 and actin filament (F-actin) nucleotide state6 regulate actin dynamics by tuning F-actin’s engagement of actin-binding proteins through mechanisms that are unclear. Here we show that the nucleotide state of actin modulates F-actin structural transitions evoked by bending forces. Cryo-electron microscopy structures of ADP–F-actin and ADP-Pi–F-actin with sufficient resolution to visualize bound solvent reveal intersubunit interfaces bridged by water molecules that could mediate filament lattice flexibility. Despite extensive ordered solvent differences in the nucleotide cleft, these structures feature nearly identical lattices and essentially indistinguishable protein backbone conformations that are unlikely to be discriminable by actin-binding proteins. We next introduce a machine-learning-enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain-level detail. Bent F-actin structures reveal rearrangements at intersubunit interfaces characterized by substantial alterations of helical twist and deformations in individual protomers, transitions that are distinct in ADP–F-actin and ADP-Pi–F-actin. This suggests that phosphate rigidifies actin subunits to alter the bending structural landscape of F-actin. As bending forces evoke nucleotide-state dependent conformational transitions of sufficient magnitude to be detected by actin-binding proteins, we propose that actin nucleotide state can serve as a co-regulator of F-actin mechanical regulation.

Polycomb repressive complex 2 (PRC2) installs and spreads repressive histone methylation marks on eukaryotic chromosomes. Because of the key roles that PRC2 plays in development and disease, how this epigenetic machinery interacts with DNA and nucleosomes is of major interest. Nonetheless, the mechanism by which PRC2 engages with native-like chromatin remains incompletely understood. In this work, we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the behavior of PRC2 on polynucleosome arrays. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides reproducing known binding modes in which PRC2 interacts with bare DNA, mononucleosomes, and adjacent nucleosome pairs, our data also provide direct evidence that PRC2 can bridge pairs of distal nucleosomes. In particular, the “1–3” bridging mode, in which PRC2 engages two nucleosomes separated by one spacer nucleosome, is a preferred low-energy configuration. Moreover, we show that the distribution and stability of different PRC2–chromatin interaction modes are modulated by accessory subunits, oncogenic histone mutations, and the methylation state of chromatin. Overall, these findings have implications for the mechanism by which PRC2 spreads histone modifications and compacts chromatin. The experimental and computational platforms developed here provide a framework for understanding the molecular basis of epigenetic maintenance mediated by Polycomb-group proteins.

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and mutations in myosin-15 cause hereditary deafness. In addition to transporting actin regulatory machinery to stereocilia tips, myosin-15 directly nucleates actin filament (“F-actin”) assembly, which is disrupted by a progressive hearing loss mutation (p.D1647G, “ jordan ”). Here, we present cryo–electron microscopy structures of myosin-15 bound to F-actin, providing a framework for interpreting the impacts of deafness mutations on motor activity and actin nucleation. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin’s D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. Adenosine diphosphate–bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin’s structural plasticity in a myosin nucleotide state–dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.

SummaryThe motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and myosin-15 mutations cause profound deafness. In a companion study, we report that myosin-15 nucleates actin filament (“F-actin”) assembly and identify a progressive hearing loss mutation (p.D1647G, “jordan”) which disrupts stereocilia elongation by inhibiting actin polymerization. Here, we present cryo-EM structures of myosin-15 bound to F-actin, providing a framework for interpreting deafness mutations and their impacts on myosin-stimulated actin assembly. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin’s D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. ADP-bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin’s structural plasticity in a myosin nucleotide-state dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.