Characterizing agrin-dependent MuSK trafficking

Neuromuscular junctions form when a motor axon reaches a muscle fiber. Acetylcholine receptors become concentrated at the site of innervation and processes at the molecular and cellular level lead to the development of a mature and functional neuromuscular synapse. The receptor tyrosine kinase MuSK is the key signaling molecule at the neuromuscular synapse whose activity is required for the formation of a mature and functional neuromuscular synapse. Signal transduction downstream of MuSK regulates acetylcholine receptor clustering but to understand the exact mechanism of action further studies are required. Within this project I propose to address the important questions about how MuSK is localized to the postsynaptic membrane and how the level of MuSK protein is maintained. We intend to use a combination of imaging techniques, cell and molecular biology methods to analyze MuSK trafficking. Thereby we will focus on the molecular mechanisms that control MuSK endocytosis and their role during MuSK signaling. Secondly, we aim at dissecting the pathways that regulate MuSK transport to postsynaptic sites. The proposed study tackles so far poorly understood aspects of MuSK function and will therefore provide valuable novel insights into the molecular mechanisms, which control the formation of a functional neuromuscular junction.

Neuromuscular junctions form when a motor axon reaches a muscle fiber. Acetylcholine receptors become concentrated at the site of innervation and processes at the molecular and cellular level lead to the development of a mature and functional neuromuscular synapse. The receptor tyrosine kinase MuSK is the key signaling molecule at the neuromuscular synapse whose activity is required for the formation of a mature and functional neuromuscular synapse. Signal transduction downstream of MuSK regulates acetylcholine receptor clustering but to understand the exact mechanism of action further studies are required. Within this project we addressed the important questions about how MuSK is localized to the postsynaptic membrane and how the level of MuSK protein is maintained. We used a combination of imaging techniques, cell and molecular biology methods to analyze MuSK trafficking. Thereby we have focused on the molecular mechanisms that control MuSK endocytosis and their role during MuSK signaling. We found that MuSK endocytosis occurs independent of kinase activity and independent of Lrp4. Blocking endocytosis did not alter MuSK signaling suggesting that MuSK endocytosis does not influence MuSK signaling. Secondly, we performed experiments to study MuSK transport to postsynaptic sites. We found that MuSK is transported to synaptic sites via targeted insertion. Moreover, MuSK is highly mobile within synaptic sites. We established an assay to immobilize agrin via micro-contact printing. With this new approach we will be able to dissect the involved mechanisms in future experiments. Taken together, our study has tackled so far poorly understood aspects of MuSK function and has provided valuable novel insights into the molecular mechanisms, which control the formation of a functional neuromuscular junction.