The Dynamics of the Acetylcholine Receptor and the Receptor-Associated Rapsyn Scaffolding Protein Studied In Vitro and In Vivo.

Essential for proper synaptic transmission is the establishment and maintenance of a high density of receptors which are clustered and anchored in place in direct apposition to pre-synaptic nerve terminals. At the neuromuscular junction (NMJ), acetylcholine receptors (AChRs) bind directly to rapsyn, which links AChRs to the intracellular scaffold. While this static structure of the receptor cluster and associated scaffold is relatively well-characterized, many gaps exist in our understanding of receptor dynamics and very little is known about the dynamics of scaffolding proteins. Using muscle cell cultures and the sternomastoid muscle of living mice, I have characterized a number of aspects of the dynamic processes involved in the post-synaptic machinery. First, I used a novel labeling method to identify and characterize an activity-dependent receptor recycling pathway active in the neuromuscular junction of live mice, but not present in cultured muscle cells. These results debunk a long-held assumption that AChRs do not recycle, and demonstrates a novel mechanism which allows rapid AChR turnover. Next, using a novel laser imaging technique to induce the dispersal of AChRs from individual receptor clusters, I showed that receptor dissipation resulted in the removal of scaffolding proteins specifically from the laser illuminated region, indicating that AChRs are necessary for the accumulation of new scaffolding proteins and the maintenance of the intracellular scaffold. Finally, by introducing a rapsyn-GFP fusion protein into muscle cells with transfection or electroporation and using fluorescent recovery after photobleaching, I showed that the rapsyn scaffolding protein is more dynamic and regulated differently than the AChRs to which they bind, challenging the assumption that the scaffold is a stable and static structure at the mature synapse. The dynamics of receptors and the stability of the scaffold anchoring them in place are key to our greater understanding of the development and maintenance of post-synaptic clusters, and greater relevance of this process is illustrated by the fact that receptor dynamics in the central nervous system are essential for synaptic plasticity, which underlies the processes of cellular learning and memory.Ph.D.NeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/58403/1/ebruneau_1.pd