The long-term goal of this project is to define molecular mechanisms that control synapse assembly and growth. Drosophila NMJ is a glutamatergic synapse, similar in structure and physiology to mammalian central AMPA/Kainate synapses. In flies each NMJ is unique and identifiable, synapses are large and accessible for electrophysiological and optical analysis, making the Drosophila NMJ a powerful genetic system to study synapse development. The subunits that form the glutamate-gated ion channels (iGluRs) are known and relatively well studied. However the mechanisms that control iGluRs clustering and stabilization at the postsynaptic densities, a key step that confers functionality to the nascent synapse, remain a mystery. Several mechanisms have been identified that regulate the subunit compositions and the extent of iGluRs synaptic localization, but no molecules other than the receptors themselves were shown to be absolutely required for clustering of the receptor complexes. In recent studies we have discovered that the neuropillin and tolloid-like protein, Neto, is an essential component for the clustering of the iGluRs at the Drosophila NMJ. Drosophila Neto has two vertebrate homologs called Neto-1 and -2, which have been recently shown to modulate the properties of selective kainate-type receptors. Neto1/Neto2 double knockout mice have defects in long term potentiation, and in learning and memory. To study the function of Drosophila Neto, we generated an allelic series and performed behavioral, anatomical, and physiological analyses. We found that neto null mutant animals do not hatch into the larval stage and die as completely paralyzed late embryos. Suboptimal Neto levels induced partial lethality;such escapers do not fly and have locomotor defects. Neto activity is essential in the striated muscle, since the lethality and the locomotor defects of neto mutant animals can be fully rescued when Neto is expressed in the muscle. In the striated muscle, endogenous Neto is concentrated at the NMJ and co-localizes with the iGluRs at postsynaptic densities in puncta juxtaposed to sites of neurotransmitter/ glutamate release, the active zones. We found that Neto is required for clustering of iGluRs at the onset of synaptogenesis: in the absence of Neto iGluRs do not cluster and remain scattered in small, extra synaptic aggregates. Moreover, Neto localization at the NMJ depends on iGluRs: in the absence of an essential subunit (i.e. GluRIID) Neto and the other iGluRs subunits do not cluster at the NMJ. Together these data indicate that Neto and iGluRs depend on each other for targeting and clustering at the NMJ at the onset of synaptogenesis. The extent of iGluRs clustering at the NMJ was also markedly reduced later in development at suboptimal Neto levels, such as in neto hypomorphs. During larval stages, the level of iGluRs appeared unchanged by Western analysis, but their immunoreactivity at the NMJ is decreased and shifted to extrajunctional locations. Physiological studies confirmed that neto mutations significantly impair the number and density of postsynaptic iGluRs without an apparent effect on presynaptic release. In collaboration with Bing Zhang at University of Oklahoma, we recorded evoked excitatory junctional potentials (EJPs) and spontaneous miniature potentials (mEJPs) at body-wall muscles of the third instar larvae. Both the frequency and amplitude of miniature synaptic potentials are reduced in neto mutants, but with no apparent defects in presynaptic release. Furthermore, both the quantity and the morphology of the synaptic boutons were altered at reduced Neto levels. Under electron microscopy, active zones in normal synapses are marked by electron-dense membranes and presynaptic specialization called T bars. The electron-dense membrane regions are greatly reduced or absent in neto mutants. This phenotype is restricted to the synaptic cleft and postsynaptic compartment, since normal T bars are present presynaptically at mutant synapses lacking electron-dense membranes. Additional histological analyses further showed that suboptimal Neto levels do not impact the presynaptic compartment, but have a profound effect on the organization and stabilization of PSDs. Our data fit best with the model in which Neto and the iGluRs are engaged in targeting each other to the PSDs via a direct interaction. This implies that Neto must associate with the iGluR complexes in vivo, and by co-immunoprecipitation experiments we found that is indeed the case. A newly formed PSD grows by a continuous incorporation of iGluRs likely derived from cell-wide plasma membrane pools via lateral diffusion. By controlling the extent of the iGluR clustering, Neto itself is rate limiting and appears to directly impact synapse assembly, organization of PSDs, and synapse functionality.
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