RESUMEN
KEY POINTS: The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano-electrical transducer (MET) channels. The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli. In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles. We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop. We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role. ABSTRACT: Mutations in Myo6, the gene encoding the (F-actin) minus end-directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells' apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano-electrical transduction. We report that Ca(2+) -dependent adaptation of the mechano-electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca(2+) -dependent adaptation.
Asunto(s)
Células Ciliadas Auditivas Internas/fisiología , Células Ciliadas Auditivas Externas/fisiología , Mecanotransducción Celular/fisiología , Cadenas Pesadas de Miosina/fisiología , Animales , Calcio/fisiología , Ratones , Ratones Mutantes , Cadenas Pesadas de Miosina/genéticaRESUMEN
RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.
Asunto(s)
Membrana Celular/metabolismo , Canal de Potasio Kv1.5/metabolismo , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/fisiología , Miosina Tipo V/fisiología , Miosinas/fisiología , Transporte de Proteínas/fisiología , Citoesqueleto de Actina/fisiología , Animales , Arritmias Cardíacas/fisiopatología , Línea Celular , Conexina 43/análisis , Canal de Potasio ERG1 , Endocitosis , Canales de Potasio Éter-A-Go-Go/análisis , Uniones Comunicantes , Genes Reporteros , Sistema de Conducción Cardíaco/fisiopatología , Transporte Iónico , Canal de Potasio Kv1.5/genética , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos Cardiovasculares , Cadenas Pesadas de Miosina/deficiencia , Cadenas Pesadas de Miosina/genética , Miosina Tipo V/deficiencia , Miosina Tipo V/genética , Miosinas/deficiencia , Miosinas/genética , Potasio/metabolismo , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rab/fisiologíaRESUMEN
We investigated the effects of a specific mixture of amino acid (AA) supplements on the adaptation changes induced by aging in the soleus muscle of rats. Male Wistar rats were divided into 3 groups (n = 5 each): young control (YO), 3 months of age; elderly control (EL), 18 months of age; and elderly orally supplemented with an AA mixture (EL-AA), 18 months of age, given as 0.1 g/kg per day in drinking water for 8 weeks. Myosin heavy chain (MHC) composition was analyzed in all muscles. The total fiber number and fiber cross-sectional area of types 1 and 2A fibers were also measured in immunostained sections of the soleus muscle. The ratios between the sarcomere volume (Vsar) and the total volume (Vtot) and single muscle fibers were studied by electron microscopy. The expression of total and phosphorylated serine/threonine protein kinase mammalian target of rapamycin (mTOR), a potent regulator of messenger RNA translation initiation, was also determined in all groups. Aging was associated with an overall shift toward the expression of a slower MHC phenotype, atrophy of fast and slow fibers, a significant decrease in Vtot/Vsar, and no changes in total fiber number. AA supplementation antagonized the effects of aging. A shift toward the expression of faster MHC isoforms was observed. Fiber atrophy appeared to be partly counteracted by the AA supplements; we noted an increase in cross-sectional area fibers and Vtot/Vsar in EL-AAs. Total and phosphorylated mTOR expression appeared to decrease in EL and was restored by the AA supplements. Collectively, these results suggest that aging-induced muscle adaptations can be partly restored by AA supplementation. An mTOR signal pathway may mediate the effects on fiber trophism.
Asunto(s)
Aminoácidos/administración & dosificación , Suplementos Dietéticos , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Envejecimiento/patología , Envejecimiento/fisiología , Animales , Western Blotting , Inmunohistoquímica , Masculino , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Cadenas Pesadas de Miosina/efectos de los fármacos , Cadenas Pesadas de Miosina/fisiología , Ratas , Ratas WistarRESUMEN
This review, dedicated to the memory of Professor Setsuro Ebashi, focuses on our current work investigating the cellular functions and regulation of the unique unconventional motor, myosin VI. This myosin, unlike all the other myosins so far studied, moves towards the minus end of actin filaments and has been implicated in a wide range of cellular processes such as endocytosis, exocytosis, cell migration, cell division and cytokinesis. Myosin VI's involvement in these cellular pathways is mediated by its interaction with specific adaptor proteins and is regulated by multiple regulatory signals and modifications such as calcium ions, PtdIns(4,5)P(2) (PIP(2)) and phosphorylation. Understanding the functions of myosin VI within the cell and how it is regulated is now of utmost importance given the recent observations that it is associated with a number of human disorders such as deafness and cancers.
Asunto(s)
Fenómenos Fisiológicos Celulares , Modelos Biológicos , Proteínas Motoras Moleculares/fisiología , Cadenas Pesadas de Miosina/fisiología , Transporte de Proteínas/fisiología , Retroalimentación/fisiologíaRESUMEN
The recently solved structure of the myosin VI motor demonstrates that the unique insert at the end of the motor is responsible for the reversal of the normal myosin directionality. A second class-specific insert near the nucleotide-binding pocket contributes to myosin VI's unique kinetic tuning, allowing it to function either as an actin-based transporter or as an anchoring protein. Recent biochemical and biophysical studies have shown that the native molecule can form dimers upon clustering, and cell biological studies have demonstrated that it clearly does play both transport and anchoring roles in cells. These mechanistic insights allow us to speculate on how unusual aspects of myosin VI structure and function allow it to fill unique niches in cells.
Asunto(s)
Citoesqueleto de Actina/fisiología , Cadenas Pesadas de Miosina/fisiología , Animales , Dimerización , Drosophila melanogaster/fisiología , Endocitosis/fisiología , Modelos Moleculares , Cadenas Pesadas de Miosina/química , Miosina Tipo V/química , Miosina Tipo V/fisiología , Unión Proteica , Conformación ProteicaRESUMEN
Myosin VII is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. Here we present the first study of the mechanism of action of a myosin VII isoform. We have expressed a truncated single-headed Drosophila myosin VIIB construct in the baculovirus-Sf9 system that bound calmodulin light chains. By using steady-state and transient kinetic methods, we showed that myosin VIIB exhibits a fast release of phosphate and a slower, rate-limiting ADP release from actomyosin. As a result, myosin VIIB will be predominantly strongly bound to actin during steady-state ATP hydrolysis (its duty ratio will be at least 80%). This kinetic pattern is in many respects similar to that of the single-molecule vesicle transporters myosin V and VI. The enzymatic properties of myosin VIIB provide a kinetic basis for processivity upon possible dimerization via the C-terminal domains of the heavy chain. Our experiments also revealed conformational heterogeneity of the actomyosin VIIB complex in the absence of nucleotide.
Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/fisiología , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/fisiología , Miosinas/química , Actinas/química , Actomiosina/química , Adenosina Difosfato/química , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Animales , Baculoviridae/metabolismo , Adhesión Celular , Línea Celular , ADN Complementario/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Drosophila melanogaster , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Insectos , Cinética , Sustancias Macromoleculares/química , Modelos Químicos , Miosinas/metabolismo , Fagocitosis , Fosfatos/química , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
Myosin VI (Myo6) is an actin-based motor protein implicated in clathrin-mediated endocytosis in nonneuronal cells, though little is known about its function in the nervous system. Here, we find that Myo6 is highly expressed throughout the brain, localized to synapses, and enriched at the postsynaptic density. Myo6-deficient (Snell's waltzer; sv/sv) hippocampus exhibits a decrease in synapse number, abnormally short dendritic spines, and profound astrogliosis. Similarly, cultured sv/sv hippocampal neurons display decreased numbers of synapses and dendritic spines, and dominant-negative disruption of Myo6 in wild-type hippocampal neurons induces synapse loss. Importantly, we find that sv/sv hippocampal neurons display a significant deficit in the stimulation-induced internalization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPARs), and that Myo6 exists in a complex with the AMPAR, AP-2, and SAP97 in brain. These results suggest that Myo6 plays a role in the clathrin-mediated endocytosis of AMPARs, and that its loss leads to alterations in synaptic structure and astrogliosis.
Asunto(s)
Endocitosis/fisiología , Cadenas Pesadas de Miosina/fisiología , Receptores AMPA/metabolismo , Sinapsis/fisiología , Complejo 2 de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfato/farmacología , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Encéfalo/metabolismo , Encéfalo/ultraestructura , Química Encefálica , Dendritas/metabolismo , Dendritas/ultraestructura , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Homólogo 1 de la Proteína Discs Large , Dineínas/genética , Dineínas/metabolismo , Endocitosis/efectos de los fármacos , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Guanilato-Quinasas , Insulina/farmacología , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Neuronas/ultraestructura , Receptores AMPA/análisis , Receptores de Glutamato/metabolismo , Sacarosa/farmacología , Sinapsis/ultraestructura , Membranas Sinápticas/química , Membranas Sinápticas/efectos de los fármacos , Sinaptosomas/química , Sinaptosomas/efectos de los fármacos , Transferrina/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoAsunto(s)
Músculos/citología , Cadenas Pesadas de Miosina/fisiología , Miosinas/química , Miosinas/fisiología , Actinas/química , Adenosina Trifosfato/metabolismo , Adsorción , Animales , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas Fluorescentes Verdes , Hidrólisis , Insectos , Cinética , Proteínas Luminiscentes/metabolismo , Movimiento , Mutación , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/química , Miosinas/metabolismo , Distribución Normal , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
We evaluated the effects of testosterone overload on mitochondrial superoxide dismutase (MnSOD), cytochrome oxidase (COX) and citrate synthase (CS) activities of the rat superficial gastrocnemius both in non-exercised muscle and following moderate endurance training. Basal (bLPO) and stimulated (sLPO) lipid peroxidation was measured as an index of oxidative tissue damage. Furthermore, to assess the relationship between exercise and testosterone-induced metabolic adaptations and contractile protein expression, the distribution of myosin heavy chain (MHC) isoforms was analysed by SDS-PAGE. Samples were obtained from: controls (C), rats treated with testosterone propionate (Tp) (TP, 5 mg kg(-1) i.m. 6 days/week), trained rats (E, 5 days/week) and rats trained and treated with Tp (ETP). MnSOD significantly increased in E and TP in comparison with C and ETP. Training induced a significant increase in COX activity both in E and ETP whereas a statistical reduction was observed in TP in comparison with the other groups. Moreover, testosterone administration was associated with a significant reduction in CS activity which significantly increased in ETP. A reduction in lipid peroxidation was observed in E and ETP in comparison with controls both in basal and stimulated conditions, whereas TP showed a significant increase of bLPO. In trained rats enzymatic changes were correlated with an increase in the proportion of fast oxidative MHC-2A and MHC-2X with decrease of the proportion of fast MHC-2B. In contrast, Tp treatment induced an increase in the proportion of MHC-2B whereas MHC-2A and MHC-2X disappeared. Finally, ETP showed a reduction in MHC-2B and an increase in MHC-1 and MHC-2X. These data suggest that testosterone supplementation seems not to significantly modify the metabolic adaptation induced by exercise in gastrocnemius muscle. Furthermore, testosterone overload to non-exercised rats seems to reduce the mitochondrial function and increase the lipid peroxidation of the muscle.
Asunto(s)
Andrógenos/farmacología , Músculo Esquelético/metabolismo , Estrés Oxidativo/fisiología , Resistencia Física/fisiología , Testosterona/farmacología , Andrógenos/sangre , Animales , Citrato (si)-Sintasa/metabolismo , Isomerismo , Peroxidación de Lípido/fisiología , Masculino , Mitocondrias/enzimología , Músculo Esquelético/efectos de los fármacos , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/fisiología , Resistencia Física/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Testosterona/sangreRESUMEN
Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. Here, we report that the up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506.
Asunto(s)
Calcineurina/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Músculo Esquelético/fisiología , Animales , Inhibidores de la Calcineurina , Ciclosporina/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Masculino , Músculo Esquelético/crecimiento & desarrollo , Cadenas Pesadas de Miosina/fisiología , Ratas , Ratas Wistar , Regeneración , Tacrolimus/farmacología , Regulación hacia ArribaRESUMEN
Myosins and kinesins are molecular motors that hydrolyse ATP to track along actin filaments and microtubules, respectively. Although the kinesin family includes motors that move towards either the plus or minus ends of microtubules, all characterized myosin motors move towards the barbed (+) end of actin filaments. Crystal structures of myosin II (refs 3-6) have shown that small movements within the myosin motor core are transmitted through the 'converter domain' to a 'lever arm' consisting of a light-chain-binding helix and associated light chains. The lever arm further amplifies the motions of the converter domain into large directed movements. Here we report that myosin VI, an unconventional myosin, moves towards the pointed (-) end of actin. We visualized the myosin VI construct bound to actin using cryo-electron microscopy and image analysis, and found that an ADP-mediated conformational change in the domain distal to the motor, a structure likely to be the effective lever arm, is in the opposite direction to that observed for other myosins. Thus, it appears that myosin VI achieves reverse-direction movement by rotating its lever arm in the opposite direction to conventional myosin lever arm movement.