At the Drosophila NMJ, 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 pharmacological properties of selective kainate-type receptors. Neto1/Neto2 double knockout mice have defects in long term potentiation, and in learning and memory. The neto genes code for transmembrane proteins that contain CUB repeats in their extracellular part. CUB domains are developmentally important protein-protein interaction motifs, which are also present in Tld/BMP-1. To study the function of Drosophila Neto, we generated an allelic series using transposable elements located within the neto locus. Several imprecise excisions were isolated and were molecularly characterized. neto36 is likely a genetic null;neto36 hemizygous embryos do not hatch into the larval stage and die as late embryos that are completely paralyzed, and lack any body wall peristalsis and hatching movements. neto109 lacks the start codon-containing exon and shows 50% lethality. The adult 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. Intriguingly, muscle expression of Neto rescued the completely paralyzed neto36 embryos to viable, fertile adults. Neto is present at two distinct locations in the striated muscle on the muscle surface, 1) in a striped pattern reminiscent of the T-tubules, and 2) concentrated at the NMJ. At the NMJ, Neto co-localizes with the ionotropic glutamate receptors (iGluRs) in puncta juxtaposed to sites of neurotransmitter/ glutamate release, the active zones. The iGluRs of the Drosophila NMJ are hetero-tetrameric complexes composed of three essential subunits IIC, IID, IIE, and either IIA or IIB. Embryos without any of the essential subunits, or without IIA and IIB together, are paralyzed and cannot hatch into the larval stages. The essential subunits are not only required for viability, but also for the clustering of the other iGluRs at the NMJ. The similarities in subcellular localizations and loss-of-function phenotypes of Neto and iGluRs raised the possibility that Neto may be important for iGluRs clustering at the NMJ. Through histological and physiological studies we found that Neto is required for clustering of iGluRs at the onset of synaptogenesis, as well as during later developmental stages. iGluRs begin to concentrate at the synaptic cleft soon after contact between the motoneuron growth cone and muscle. However, iGluRs do not cluster in the absence of Neto and remain scattered in small, extrasynaptic aggregates. These defects were not caused by inappropriate axon guidance or lack of adhesion since the pre- and postsynaptic partners appeared properly aligned. The synapse pre-patterning is also normal, as seen by the accumulation of pre-synaptic Bruchpilot (Brp), an active zone protein, and post-synaptic P21 activating kinase (PAK) clusters. The lethality and iGluRs clustering defects in neto null mutants are completely rescued when Neto activity is provided in the striated muscle. The extent of iGluRs clustering at the NMJ was also markedly reduced later in development in third instar larvae for neto hypomorphs. In these larvae, Neto itself forms a drastically reduced number of junctional clusters that co-localized with iGluRs. In neto mutants the level of receptors appeared unchanged by Western analysis, but their immunoreactivity at the NMJ is much decreased and instead shifted to extrajunctional locations. The PAK signals at individual PSDs were also strongly reduced in neto109 larvae. The presence of PAK clusters at the pre-patterning stage in neto null embryos and lack thereof in larval synapses with suboptimal Neto suggest a deficit in the maintenance of mature PSDs in neto mutants. A similar deficit was reported for NMJ synapses developing in the near absence of iGluRs. In contrast, the presynaptic release sites appeared normal at suboptimal levels of Neto or iGluRs. 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. To understand how Neto controls the clustering of iGluRs, we explored the timing of Neto clustering at the NMJ using live imaging of embryos with muscle expressed Neto-eGFP. Neto accumulates and clusters at the NMJ as early as 14 hours after egg laying, at the time when the iGluR receptors begin to accumulate and cluster at the synapse. Most importantly, Neto positive puncta could no longer be detected in gluRIID-/- null embryos. Thus, Neto and iGluRs depend on each other for clustering at the NMJ. Basically, Neto functions as a non-channel, essential subunit of the iGluR complexes. 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. A newly formed PSD grows by a continuous incorporation of iGluRs likely derived from cell-wide plasma membrane pools via lateral diffusion. Several lines of evidence point to a rate-limiting role of iGluR levels in NMJ formation. By controlling the extent of the iGluR clustering, Neto itself is rate limiting and appears to directly impact synapse formation. Thus Netos constitute a family of proteins conserved from flies to humans that influence the function of glutamatergic synapses, and have acquired species- and tissue-specific roles during evolution.
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