RESUMEN
Stereocilia are actin-based protrusions on auditory sensory hair cells that are deflected by sound waves to initiate the conversion of mechanical energy to neuronal signals. Stereocilia maintenance is essential because auditory hair cells are not renewed in mammals. This process requires both ß-actin and γ-actin as knock-out mice lacking either isoform develop distinct stereocilia pathology during aging. In addition, stereocilia integrity may hinge on immobilizing actin, which outside of a small region at stereocilia tips turns over with a very slow, months-long half-life. Here, we establish that ß-actin and the actin crosslinking protein fascin-2 cooperate to maintain stereocilia length and auditory function. We observed that mice expressing mutant fascin-2 (p.R109H) or mice lacking ß-actin share a common phenotype including progressive, high-frequency hearing loss together with shortening of a defined subset of stereocilia in the hair cell bundle. Fascin-2 binds ß-actin and γ-actin filaments with similar affinity in vitro and fascin-2 does not depend on ß-actin for localization in vivo. Nevertheless, double-mutant mice lacking ß-actin and expressing fascin-2 p.R109H have a more severe phenotype suggesting that each protein has a different function in a common stereocilia maintenance pathway. Because the fascin-2 p.R109H mutant binds but fails to efficiently crosslink actin filaments, we propose that fascin-2 crosslinks function to slow actin depolymerization at stereocilia tips to maintain stereocilia length.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Células Ciliadas Auditivas/citología , Proteínas de Microfilamentos/metabolismo , Estereocilios/fisiología , Estimulación Acústica , Actinas/deficiencia , Actinas/genética , Envejecimiento/genética , Animales , Benzofuranos , Cadherinas/genética , Proteínas Portadoras/genética , Electroencefalografía , Receptor alfa de Estrógeno/genética , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Femenino , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestructura , Pérdida Auditiva de Alta Frecuencia/genética , Pérdida Auditiva de Alta Frecuencia/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Microscopía de Fuerza Atómica , Mutación/genética , Fenotipo , Unión Proteica/genética , Quinolinas , Estereocilios/ultraestructuraRESUMEN
We have used time-resolved spectroscopy to investigate the structural dynamics of actin interaction with dystrophin and utrophin in relationship to the pathology of muscular dystrophy. Dystrophin and utrophin bind actin in vitro with similar affinities, but the molecular contacts of these two proteins with actin are different. It has been hypothesized that the presence of two low-affinity actin-binding sites in dystrophin allows more elastic response of the actin-dystrophin-sarcolemma linkage to muscle stretches, compared with utrophin, which binds via one contiguous actin-binding domain. We have directly tested this hypothesis by determining the effects of dystrophin and utrophin on the microsecond rotational dynamics of a phosphorescent dye attached to C374 on actin, as detected by transient phosphorescence anisotropy (TPA). Binding of dystrophin or utrophin to actin resulted in significant changes in the TPA decay, increasing the final anisotropy (restricting the rotational amplitude) and decreasing the rotational correlation times (increasing the rotational rates and the torsional flexibility). This paradoxical combination of effects on actin dynamics (decreased amplitude but increased rate) has not been observed for other actin-binding proteins. Thus, when dystrophin or utrophin binds, actin becomes less like cast iron (strong but brittle) and more like steel (stronger and more resilient). At low levels of saturation, the binding of dystrophin and utrophin has similar effects, but at higher levels, utrophin caused much greater restrictions in amplitude and increases in rate. The effects of dystrophin and utrophin on actin dynamics provide molecular insight into the pathology of muscular dystrophy.