RESUMEN
Translational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent satellite cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2α (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with the small molecule sal003 maintains P-eIF2α and permits the expansion of satellite cells ex vivo Paradoxically, P-eIF2α also increases the translation of specific mRNAs, which is mediated by P-eIF2α-dependent read-through of inhibitory upstream open reading frames (uORFs). Here, we ask whether P-eIF2α-dependent mRNA translation enables expansion of satellite cells. Using transcriptomic and proteomic analyses, we show a number of genes associated with the assembly of the spindle pole to be upregulated at the level of protein, without corresponding change in mRNA levels, in satellite cells expanded in the presence of sal003. We show that uORFs in the 5' UTR of mRNA for the mitotic spindle stability gene Tacc3 direct P-eIF2α-dependent translation. Satellite cells deficient for TACC3 exhibit defects in expansion, self-renewal and regeneration of skeletal muscle.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas Fetales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Biosíntesis de Proteínas , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , Animales , Diferenciación Celular/genética , Proliferación Celular , Autorrenovación de las Células , Células Cultivadas , Regulación hacia Abajo/genética , Ratones Endogámicos C57BL , Factor de Transcripción PAX7/metabolismo , Fosforilación , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regeneración , Transcriptoma/genética , Regulación hacia Arriba/genéticaRESUMEN
Myosin X forms an antiparallel dimer and moves processively on actin bundles. How the antiparallel dimer affects the stepping mechanism of myosin X remains elusive. Here, we generated several chimeras using domains of myosin V and X and performed single-molecule motility assays. We found that the chimera containing the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X has multiple forward step sizes and moves processively, similar to full-length myosin X. The chimera containing the motor domain and lever arm from myosin X and the parallel coiled-coil from myosin V takes steps of â¼40 nm at lower ATP concentrations but was nonprocessive at higher ATP concentrations. Furthermore, mutant myosin X with four mutations in the antiparallel coiled-coil domain failed to dimerize and was nonprocessive. These results imply that the antiparallel coiled-coil domain is necessary for multiple forward step sizes of myosin X.