RESUMEN
Myosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. Here, we identify a binding protein for Drosophila myosin-7a termed M7BP, and describe how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex's processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Miosina VIIa/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Movimiento Celular/genética , Movimiento Celular/fisiología , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Microscopía Fluorescente/métodos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Miosina VIIa/química , Miosina VIIa/genética , Unión Proteica , Multimerización de Proteína , Seudópodos/genética , Seudópodos/fisiología , Estereocilios/genética , Estereocilios/fisiologíaRESUMEN
Mutations in myosin-VIIa (MYO7A) cause Usher syndrome type 1, characterized by combined deafness and blindness. MYO7A is proposed to function as a motor that tensions the hair cell mechanotransduction (MET) complex, but conclusive evidence is lacking. Here we report that multiple MYO7A isoforms are expressed in the mouse cochlea. In mice with a specific deletion of the canonical isoform (Myo7a-ΔC mouse), MYO7A is severely diminished in inner hair cells (IHCs), while expression in outer hair cells is affected tonotopically. IHCs of Myo7a-ΔC mice undergo normal development, but exhibit reduced resting open probability and slowed onset of MET currents, consistent with MYO7A's proposed role in tensioning the tip link. Mature IHCs of Myo7a-ΔC mice degenerate over time, giving rise to progressive hearing loss. Taken together, our study reveals an unexpected isoform diversity of MYO7A expression in the cochlea and highlights MYO7A's essential role in tensioning the hair cell MET complex.
Asunto(s)
Células Ciliadas Auditivas Internas/metabolismo , Mecanotransducción Celular , Miosina VIIa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Eliminación de Gen , Células Ciliadas Auditivas Internas/ultraestructura , Pérdida Auditiva/metabolismo , Pérdida Auditiva/patología , Ratones Endogámicos C57BL , Miosina VIIa/química , Miosina VIIa/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Estereocilios/metabolismo , Estereocilios/ultraestructuraRESUMEN
Hair cell stereocilia tip-links function to sense mechanical forces generated by sound waves and maintain the structure of stereocilia by rooting the tail of cadherins to highly dense structures known as tip-link densities. Although the molecular components are largely known, the mechanisms underlying the tip-link density formation are unknown. Here, we show that Myosin VIIB (MYO7B), USH1C, and ANKS4B, which form a specific complex stabilizing tip-links in intestine microvilli, could form dense condensates via liquid-liquid phase separation in vitro and in cells. The MYO7A, USH1C, and USH1G complex also undergoes phase separation in cells. Formation of the MYO7A/USH1C/USH1G and MYO7B/USH1C/ANKS4B condensates requires strong and multivalent interactions between proteins in both tripartite complexes. Point mutations of MYO7A found in Usher syndrome patients weaken or even disrupt the multivalent interactions of the MYO7A/USH1C/USH1G complex and impair its phase separation. Thus, the stereocilia tip-link densities may form via phase separation of the MYO7A/USH1C/USH1G complex.