Regulation of postsynaptic acetylcholine receptor clustering by neural agrin

Summary

This project uses advanced fluorescence imaging techniques to study the regulation of postsynaptic clustering of neurotransmitter receptors.

Supervisor(s)

Associate Professor William (Bill) Phillips

Research Location

Bosch Institute (Physiology)

Program Type

Masters/PHD

Synopsis

The agrin/MuSK signalling system is vital for the formation and maintenance of the neuromuscular junction (NMJ), but we don’t yet understand exactly how it works. Understanding how agrin regulates its effector proteins offers the potential to improve our understanding and treatment of neuromuscular disorders such as myasthenia gravis (Loosen et al. 2005, Brain 128: 2327-37). Agrin is a proteoglycan that is released by the nerve terminal. When agrin is added to cultured muscle cells (myotubes) a receptor tyrosine kinase called Muscle Specific Kinase (MuSK) in the postsynaptic membrane is activated. Activation of MuSK initiates clustering of acetylcholine receptors (AChRs) but the intermediate cytoplasmic signalling pathways remain uncertain. The cytoplasmic receptor-associated protein, rapsyn, is an essential final effector of AChR clustering. Mice lacking rapsyn fail to form AChR clusters, their NMJs don’t work properly and they and die at birth. More subtle mutations in the human rapsyn gene cause a rare form of childhood myasthenia gravis. In myotubes lacking rapsyn, agrin can activate MuSK, which assembles into clusters on the muscle cell membrane, but AChRs don’t get recruited to the MuSK clusters. This shows that rapsyn acts downstream of MuSK to help recruit the AChR. Agrin/MuSK signalling acts to increase the number of rapsyn molecules bound to each AChR. In this way agrin can enhance AChR aggregation and slow the recycling of the AChR (Brockhausen et al, 2008 Dev Neurobiol 68:1153-69). Thus, the agrin/MuSK/rapsyn system at the adult NMJ helps to stabilize postsynaptic AChR (Gervasio & Phillips 2007 Dev Biol 305:262-75). This project will use immunoprecipitation and immunoblotting techniques to study the way in which agrin regulates rapsyn and MuSK. We will also investigate how agrin modifies the sub-cellular trafficking of rapsyn and MuSK in cultured muscle cells using rapsyn and MuSK tagged with green fluorescent protein. The involvement of protein kinases and other second messenger signaling systems will be investigated using drugs and interfering RNA techniques.

Additional Information

The Phillips lab has considerable experience in quantitative fluorescence imaging techniques such as FRET and fluorescence recovery after photobleaching (FRAP) as well as the immunoblotting and immunoprecipitation experiments needed for this project. Additional training and advice in these techniques is available from the experienced staff of the Advanced Imaging and Molecular Biology facilities of the Bosch Institute, and the nearby Electron Microscope Unit. Please see the lab publications list for examples of some recent studies using these techniques (for example the FRET study by Gervasio et al 2007). To learn more about this project and about scholarship opportunities that may be current at the time of your enquiry please contact the advisor.

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Keywords

Neuromuscular junction, Myasthenia gravis, confocal imaging, developmental neurobiology, fluorescence microscopy, Cell biology, Synaptic plasticity, Protein-protein interactions, cell culture

Opportunity ID

The opportunity ID for this research opportunity is: 673

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