ArticleProceedings of the National Academy of Sciences of the United States of America2014
Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 52 papers.
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Who cites it
52 citing papers in PubMed, 76 citations in OpenAlex.
- Myosin-9b Controls Epithelial Brush Border Architecture through Motility-Dependent RhoA Signaling.bioRxiv : the preprint server for biology · 2026Article
- Tonotopic specialization of MYO7A isoforms in auditory hair cells.Nature communications · 2026Article
- Molecular features of myosin F adapted for driving actin flows in Toxoplasma gondii.Journal of cell science · 2026Article
- Molecular features of Myosin F adapted for driving actin flows inbioRxiv : the preprint server for biology · 2025Article
- Influence of Myosin Regulatory Light Chain and Myosin Light Chain Kinase on the Physiological Function of Inner Ear Hair Cells.Journal of the Association for Research in Otolaryngology : JARO · 2025Article
- Myosin-based nucleation of actin filaments contributes to stereocilia development critical for hearing.Nature communications · 2025Article
- Expression, Purification, and In Vitro Analysis of Myosin.Methods in molecular biology (Clifton, N.J.) · 2025Article
- MultiBac System-based Purification and Biophysical Characterization of Human Myosin-7a.Journal of visualized experiments : JoVE · 2024Article
- Motor properties of Myosin 5c are modulated by tropomyosin isoforms and inhibited by pentabromopseudilin.Frontiers in physiology · 2024Article
- Insect Cell-Based Expression of Cytoskeletal Motor Proteins for Single-Molecule Studies.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Pathophysiology of human hearing loss associated with variants in myosins.Frontiers in physiology · 2024Review
- Nonmuscle myosin 2 filaments are processive in cells.Biophysical journal · 2023Article
- The actin cytoskeleton in hair bundle development and hearing loss.Hearing research · 2023Review
- Non-muscle myosin 2 filaments are processive in cells.bioRxiv : the preprint server for biology · 2023Article
- Ensembles of human myosin-19 bound to calmodulin and regulatory light chain RLC12B drive multimicron transport.The Journal of biological chemistry · 2023Article
- Protrusion growth driven by myosin-generated force.Developmental cell · 2023Article
- Myosin motors in sensory hair bundle assembly.Current opinion in cell biology · 2022Review
- Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia.Science advances · 2022Article
- Myosin II proteins are required for organization of calcium-induced actin networks upstream of mitochondrial division.Molecular biology of the cell · 2022Article
- Human deafness-associated variants alter the dynamics of key molecules in hair cell stereocilia F-actin cores.Human genetics · 2022Review
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Authors and funding
6 authors at 3 institutions in 2 countries.
Funding
Abstract
Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (∼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) ∼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.
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