Mechanisms of synapse assembly
Synapse development is a highly orchestrated process that requires bidirectional communication between two synaptic partners across the synaptic cleft. Before a muscle is innervated, low levels of ionotropic glutamate receptors (iGluRs) are present diffusely in the muscle membrane at the fly NMJ. Once the motor neuron growth cone arrives at its target muscle, iGluRs begin to concentrate at the synaptic cleft. To date, the mechanism by which neuronal arrival triggers postsynaptic receptor clustering remains a mystery. Live studies demonstrated that receptor clusters are practically immobilized at postsynaptic densities (PSDs), which grow up to a limited, mature size. Using the Drosophila NMJ as a model for the development of glutamatergic synapses, we focus on several key questions in synaptogenesis: 1) how do glutamate receptors traffic and cluster at the synapse; 2) what is the neuronal cue that triggers receptor aggregation; 3) how cellular communication balances synapse growth and maturation.
In vertebrates, the cytoplasmic domains of iGluRs are involved in receptor localization and regulation. Also, the amino terminal domains of selective iGluR subunits are engaged in binding proteins secreted from or tethered to the presynaptic compartment. The fly iGluR subunits have relatively short intracellular domains and no known extracellular binding partner. Several mechanisms regulating the abundance of synaptic glutamate receptors have been previous described, but none appear to be essential for localizing iGluRs.
We have recently characterized Neto as a novel molecule that directly binds to the iGluR complexes and is absolutely required for clustering of the receptor complexes at the onset of synaptogenesis. Neto co-localizes with the iGluRs at the PSDs in puncta juxtaposing the active zones. We generated a neto allelic series and found that neto loss-of-function phenotypes parallel the loss-of-function defects described for iGluR complexes. First, neto null embryos lack any body wall peristalsis and hatching movements and have no detectable iGluR clusters at the NMJ. Second, animals with suboptimal Neto levels have dramatically reduced number of synaptic iGluR clusters and reduced postsynaptic sensitivity. Finally, Neto-deprived animals exhibit a deficit in the maintenance of mature PSDs. During synapse formation, iGluRs incorporation into the postsynaptic membrane is critical to enlarge PSDs. By clustering in concert to iGluRs, Neto is essential for functional iGluR complexes and directly controls synapse formation at the Drosophila NMJ. Neto does not contain any catalytic domain; instead, it has a number of extracellular protein interaction domains, and two alternative intracellular domains rich in putative phosphorylation sites and docking motifs. Therefore, Neto likely functions in iGluRs synaptic targeting and PSD stabilization via binding to iGluRs and/or other interaction partners. Ongoing structure-function studies and characterization of interaction partners are expected to provide insights on mechanisms underlying the multiple Neto functions at glutamatergic synapses.
Modulation of BMP signaling
A recurrent theme in biology is that cells utilize a limited toolbox to establish their communication networks. BMPs modulate long-range signaling during patterning, but also ensure short-range communication at specialized cell-cell junctions, such as the NMJ. Through studies on extracellular modulators of BMP signaling, such as Short gastrulation (Sog) and Crossveinless-2 (Cv-2), we aim to elucidate mechanisms that shape cell-cell communication during early patterning and at NMJ synapses.
BMPs have the ability to function as morphogens, that is to specify cell fate in a concentration dependent manner. In the early Drosophila embryo, Decapentaplegic (Dpp), a homologue of the vertebrate BMP2/4, is key in assigning identity to all dorsal structures. Dpp is transcribed uniformly throughout the dorsal domain, yet it forms an activity gradient via a cascade of extracellular regulation that restricts Dpp availability laterally, while simultaneously amplifying Dpp activity near the dorsal midline. The BMP shuttling mechanism is highly conserved throughout the animal kingdom and relies on the spatial distribution of a BMP ligand (Dpp/ BMP2/4) and a BMP antagonist (Sog/ Chordin). The BMP signaling domains are further refined by positive feedback and additional secreted BMP modulators, such as Cv-2, which binds to the cell surface and acts over a short range. However, the biochemical properties of pathway components can vary according to species- and stage-specific developmental requirements. For example, Tolloid (Tld)-like metalloproteases cleave vertebrate Chordins constitutively, while the Drosophila ortholog Sog is only cleaved efficiently when bound to BMPs. We have identified Sog characteristics responsible for making its cleavage dependent on BMP binding. ‘‘Chordin-like’’ variants that are processed independently of BMPs changed the steep BMP gradient found in Drosophila embryos to a shallower profile, analogous to that observed in some vertebrate embryos. This change affected cell fate allocation and tissue size and resulted in increased variability of patterning. Thus, the acquisition of BMP-dependent Sog processing during evolution facilitated long-range ligand diffusion and formation of the robust, bi-stable BMP morphogen gradients required for early Drosophila patterning.
Download the dpp movie.