Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro-fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy-lysosome and the ubiquitin-proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial-dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme.

SummaryHow signaling dynamics encode information is a central question in biology. During vertebrate development, dynamic Notch signaling oscillations control segmentation of the presomitic mesoderm (PSM). In mouse embryos, this molecular clock comprises signaling oscillations of several pathways, i.e., Notch, Wnt, and FGF signaling. Here, we directly address the role of the relative timing between Wnt and Notch signaling oscillations during PSM patterning. To this end, we developed a new experimental strategy using microfluidics-based entrainment that enables specific control of the rhythm of segmentation clock oscillations. Using this approach, we find that Wnt and Notch signaling are coupled at the level of their oscillation dynamics. Furthermore, we provide functional evidence that the oscillation phase shift between Wnt and Notch signaling is critical for PSM segmentation. Our work hence reveals that dynamic signaling, i.e., the relative timing between oscillatory signals, encodes essential information during multicellular development.

Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denervation. We hypothesize that neuronal ligands trigger local activation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, myosin Va mediates correct positioning of PKA in a postsynaptic microdomain, presumably by tethering PKA to the actin cytoskeleton.cAMP | muscle | neuromuscular junction | microdomain | synaptic plasticity R ecently, myosin V motor proteins were found to be crucial for the plasticity of central and peripheral synapses by facilitating, respectively, the recycling of AMPA (1-3) and acetylcholine receptors (AChRs) (4). The neuromuscular junction (NMJ) is the synapse between motor neurons and skeletal muscle fibers. Even though it is formed during ontogenesis and then maintained over the years (5), half-lives (t 1/2 ) of a few days are typical for its protein constituents, such as the AChR (6). The high enrichment of AChRs in the postsynapse is regulated by accurate targeting and turnover of this receptor. Vesicular transport is employed for this purpose, using different routes of exocytosis, endocytosis, and recycling (7-9). We identified myosin Va as the first motor protein involved in AChR trafficking and have found it to facilitate its recycling (4). Down-regulation of myosin Va led to a reduced t 1/2 of AChRs (4) similar to pathological conditions, such as denervation (6, 10-13) or dystrophy (14). A shortened t 1/2 of AChRs accompanies morphological changes of the NMJ: while NMJs in mouse muscles are normally elliptic and pretzel-shaped (15), they elongate and fragment under pathological conditions (16,17).Previous studies have revealed a major role of cAMP signaling in controlling the t 1/2 of AChRs (12, 18). cAMP is formed upon activation of G protein-coupled receptors and mediates its effects mainly through protein kinase A (PKA). Notably, stimulation of cAMP synthesis rescued AChR lifetime in denervated (12,19) and dystrophic muscles (14). Several groups have described antagonistic functions of protein kinase C and PKA on AChR persistence and synaptic strength (18,20,21): although activation of protein kinase C destabilizes AChRs, PKA activity protects this receptor from such destabilization (18). In this context, the neuropeptide and cAMP agonist, α-calcitonin gene-rel...

BackgroundMyosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ).Methodology/Principal Findings Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins.Conclusions/SignificanceIn summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to the postsynaptic membrane.

SummaryThe stabilisation of acetylcholine receptors (AChRs) at the neuromuscular junction depends on muscle activity and the cooperative action of myosin Va and protein kinase A (PKA) type I. To execute its function, PKA has to be present in a subsynaptic microdomain where it is enriched by anchoring proteins. Here, we show that the AChR-associated protein, rapsyn, interacts with PKA type I in C2C12 and T-REx293 cells as well as in live mouse muscle beneath the neuromuscular junction. Molecular modelling, immunoprecipitation and bimolecular fluorescence complementation approaches identify an a-helical stretch of rapsyn to be crucial for binding to the dimerisation and docking domain of PKA type I. When expressed in live mouse muscle, a peptide encompassing the rapsyn a-helical sequence efficiently delocalises PKA type I from the neuromuscular junction. The same peptide, as well as a rapsyn construct lacking the a-helical domain, induces severe alteration of acetylcholine receptor turnover as well as fragmentation of synapses. This shows that rapsyn anchors PKA type I in close proximity to the postsynaptic membrane and suggests that this function is essential for synapse maintenance.