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.

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Polyelectrolyte hydrogel fibers can mimic the extracellular matrix and be used for tissue scaffolding. Mechanical properties of polyelectrolyte nanofibers are crucial in manipulating cell behavior, which metal ions have been found to enable tuning. While metal ions play an important role in manipulating the mechanical properties of the fibers, evaluating the mechanical properties of a single hydrated hydrogel fiber remains a challenging task and a more detailed understanding of how ions modulate the mechanical properties of individual polyelectrolyte polymers is still lacking. In this study, dark-field microscopy and persistence length analysis help directly evaluate fiber mechanics using electrospun fibers of poly(acrylic acid) (PAA), chitosan (CS), and ferric ions as a model system. By comparing the persistence length and estimated Young’s modulus of different nanofibers, we demonstrate that persistence length analysis is a viable approach to evaluate mechanical properties of hydrated fibers. Ferric ions were found to create shorter and stiffer nanofibers, with Young’s modulus estimated at a few kilopascals. Ferric ions, at low concentration, reduce the Young’s modulus of PAA and PAA/CS fibers through the interaction between ferric ions and carboxylate groups. Such interaction was further supported by nanoscale infrared spectroscopy studies of PAA and PAA/CS fibers with different concentrations of ferric ions.

Highlights d TDAExplore combines topological data analysis with machine learning classification d As few as 20-30 high-resolution images can be used to train TDAExplore models d TDAExplore is robust to different microscopy modes, dataset size, image features d TDAExplore quantifies where and how much each image resembles the training data