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This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α, and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

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Summary• Primary carbohydrate metabolism plays a special role related to carbon ⁄ nitrogen exchange, as well as metabolic support of fruiting body development, in ectomycorrhizal macrofungi. In this study, we used information retrieved from the recently sequenced Tuber melanosporum genome, together with transcriptome analysis data and targeted validation experiments, to construct the first genomewide catalogue of the proteins supporting carbohydrate metabolism in a plantsymbiotic ascomycete.• More than 100 genes coding for enzymes of the glycolysis, pentose phosphate, tricarboxylic acid, glyoxylate and methylcitrate pathways, glycogen, trehalose and mannitol metabolism and cell wall precursor were annotated. Transcriptional regulation of these pathways in different stages of the T. melanosporum lifecycle was investigated using whole-genome oligoarray expression data together with real-time reverse transcription-polymerase chain reaction analysis of selected genes.• The most significant results were the identification of methylcitrate cycle genes and of an acid invertase, the first enzyme of this kind to be described in a plantsymbiotic filamentous fungus.• A subset of transcripts coding for trehalose, glyoxylate and methylcitrate enzymes was up-regulated in fruiting bodies, whereas genes involved in mannitol and glycogen metabolism were preferentially expressed in mycelia and ectomycorrhizas, respectively. These data indicate a high degree of lifecycle stage specialization for particular branches of carbohydrate metabolism in T. melanosporum.