Mechanisms of neurotransmitter receptor packaging and delivery to the synapse

During development and learning, synapses increase their strength by adding
neurotransmitter receptors to the postsynapse. The current model of postsynaptic
receptor addition is the "diffusion-trap". In this model, receptors insert
extrasynaptically and then diffuse into the postsynapse, where they are trapped via
interaction with scaffold molecules. Here we propose a new model for postsynaptic
receptor addition: the "preassemble-insert" model. In this model, extrasynaptic
receptors endocytose, preassemble with scaffold, and then insert directly to the
postsynapse without a diffusion step. Hence receptor-scaffold packages are inserted
as immobile units in the postsynapse.
To investigate postsynaptic receptor diffusion-trapping and preassembly-insertion,
we use the agrin model of AChR clustering, live cell imaging, FRAP, and single
molecule tracking. We find that AChRs diffuse into clusters during formation, but
directly insert during strengthening. Inserted AChRs transcytose from the surface
plasma membrane via caveolin-3 t-tubules, and then exocytose via a Rab27a
vesicular compartment, in a MyosinVa dependant manner. During caveolin-3 ttubule
transcytosis, AChRs preassemble with the scaffold molecule rapsyn in a
transport package, and caveolin-3 trafficking is required for AChR-rapsyn
packaging. Insertion of preassembled AChR-rapsyn packages is required for AChR
cluster strengthening, but not formation.
a-Dystroglycan does not preassemble with AChR-rapsyn, and clusters
independently of the caveolin-3 t-tubule pathway. However, a-dystroglycan is
required for caveolin-3 t-tubule organelle recruitment and polarization towards the
cluster, which permits the insertion of AChR-rapsyn packages. Finally, we confirm
caveolin-3 and Rab27a trafficking in vivo, and correlate t-tubule recruitment with
NMJ strengthening. We also report prepackaging of Glur1-stargazin at CNS synapses
after chemical LTP.
The "diffusion-trap" has predominated as the model for postsynaptic receptor
addition, and is a generalized mechanism for the addition of transmembrane protein
to cell-cell junctions. These findings provide evidence for a new model of
postsynaptic receptor addition, in which preassembled protein complexes insert
directly, without a diffusion step.