New Modes of GPCR Signalling

ABSTRACT
Spatial Control of Synaptic Transmission at a Cholinergic Synapse in
C. elegans
Jean-Louis BESSEREAU
Ecole Normale Superieure, INSERM U789, Paris, France
Chemical synapses are specialized junctions that ensure precise and efficient signal
transduction between excitable cells. Specifically, synaptic vesicles fuse with the
pre-synaptic membrane at the active zone, in register with a specialized domain of the
post-synaptic membrane containing a high density of neurotransmitter receptors.
To analyze the subcellular organization of synapses at high resolution in a near to
living-state, we physically immobilized freely moving nematodes C. elegans by instant
freezing under high-pressure. After freeze-substitution and resin embedding, we used
electron microscopy (EM) tomography to improve 3D resolution as compared to
classical transmission-EM. We observed that at cholinergic neuromuscular junctions
synaptic vesicles were interconnected by filaments similar to the cytomatrix of
vertebrates. Furthermore, vesicles docked at the plasma membrane were retained at the
active zone by physical contacts with the presynaptic dense projection. In unc-10/RIM
and syd-2/liprin mutants, docked vesicles mislocalized, which provided structural basis
for the functional defects observed in these mutants.
On the post-synaptic membrane, ionotropic acetylcholine receptors (AChRs) are
clustered at high density in front of ACh release sites. We previously demonstrated that
clustering depend on the muscle-secreted protein LEV-9 and the transmembrane protein
LEV-10 that assemble in an extracellular scaffold with AChRs. In a forward genetic
screen, we identified a gene coding for a small secreted protein containing only one
immunoglobulin domain. This protein localizes at cholinergic neuromuscular junctions
and is necessary to stabilize the AChR/LEV-9/LEV-10 complex at the synapse.