RESUMO
Repeated rounds of fusion between apposing myoblasts allow muscles to become multinucleated. New research finds that myoblasts undergoing fusion in the Drosophila embryo respond to hormone signaling from a nearby tissue, resulting in the activation of a myoblast-specific gene necessary for the fusion process.
Assuntos
Fusão Celular , Mioblastos , Animais , Mioblastos/metabolismo , Mioblastos/fisiologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Transdução de Sinais , Comunicação CelularRESUMO
Many techniques exist for the identification of protein interaction networks. We present a protocol that relies on an affinity purification-mass spectrometry (AP-MS) approach to detect proteins that co-purify with a tagged bait of interest from Drosophila melanogaster larval muscles using the GAL4/upstream activating sequence (UAS) expression system. We also describe steps for the isolation and identification of protein complexes, followed by streamlined bioinformatics analysis for rapid and reproducible results. This protocol can be extended to investigate protein interactions in other tissues. For complete details on the use and execution of this protocol, please refer to Guo et al.1.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Larva , Espectrometria de Massas , Animais , Drosophila melanogaster/metabolismo , Larva/metabolismo , Espectrometria de Massas/métodos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cromatografia de Afinidade/métodos , Mapeamento de Interação de Proteínas/métodos , Biologia Computacional/métodosRESUMO
Skeletal muscle metabolism has implications for swine feed efficiency (FE); however, it remains unclear if the metabolic profile of skeletal muscle changes during postnatal growth. To assess the metabolic changes, samples were collected from the longissimus dorsi (LD, glycolytic muscle), latissimus dorsi (LAT, mixed muscle), and masseter (MS, oxidative muscle) at 20, 53, 87, 120, and 180 days of age from barrows. Muscles were assessed to determine the abundance of several metabolic enzymes. Lactate dehydrogenase (LDHα) decreased in all muscles from 20 to 87 d (p < 0.01), which may be attributed to the muscles being more glycolytic at weaning from a milk-based diet. Pyruvate carboxylase (PC) increased in all muscles at 53 d compared to the other time points (p < 0.01), while pyruvate dehydrogenase α 1 (PDHα1) increased at 87 and 180 d in MS compared to LD (p < 0.05), indicating that potential changes occur in pyruvate entry into the tricarboxylic acid (TCA) cycle during growth. Isolated mitochondria from each muscle were incubated with 13C-labeled metabolites to assess isotopomer enrichment patterns of TCA intermediates. Citrate M + 2 and M + 4 derived from [13C3]-pyruvate increased at 87 d in LAT and MS mitochondria compared to LD mitochondria (p < 0.05). Regardless of the muscle, citrate M+3 increased at 87 d compared to 20, 53, and 120 d, while 180 d showed intermediate values (p < 0.01). These data support the notion that pyruvate metabolism is dynamic during growth. Our findings establish a metabolic fingerprint associated with postnatal muscle hypertrophy.