Role of Myosin Va in the Plasticity of the Vertebrate Neuromuscular Junction In Vivo

Ira Verena Röder,Veit Witzemann,Rüdiger Rudolf,Kyeong Rok Choi,Yvonne Petersen,John A Hammer,Katrina Gwinn

doi:10.1371/journal.pone.0003871

Ira Verena Röder, Veit Witzemann + Show 5 more

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https://doi.org/10.1371/journal.pone.0003871

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Abstract

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.

Highlights

Vertebrate neuromuscular junctions (NMJs) are the synapses between motoneurones and skeletal muscle fibres and mediate any kind of voluntary movement [for review, 1,2]
We provide in vivo and in vitro evidence for a crucial role of the molecular motor protein, myosin Va, in the postsynaptic plasticity of the mammalian nerve-muscle synapse
We show that NMJs in dilute lethal mice lacking functional myosin Va exhibit a postnatal, time-dependent morphological deterioration that correlates with the dramatic aetiopathology leading to lethal seizures at around P21 (Fig. 1, Fig S1, Movies S1, S2, S3)

Summary

Introduction

Vertebrate neuromuscular junctions (NMJs) are the synapses between motoneurones and skeletal muscle fibres and mediate any kind of voluntary movement [for review, 1,2]. NMJs form during embryogenesis and are maintained after a perinatal period of synapse rearrangements [6,7] in an essentially stable manner for long time periods [8,9], presumably for the entire life span of a muscle fibre. Despite this long persistence of the overall structure, individual NMJ components such as AChRs have much shorter life spans, usually in the range of days [10]. We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ)

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