Versatile Endogenous Editing of GluRIIA in Drosophila melanogaster.

Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the GluRIIA gene have been utilized. Furthermore, mutated and tagged versions of GluRIIA have been examined in the past, but most of these constructs were not expressed under endogenous regulatory control or involved the mentioned imprecise GluRIIA knockouts. We performed CRISPR/Cas9-assisted gene editing at the endogenous locus of GluRIIA. This enabled the investigation of the endogenous expression pattern of GluRIIA using tagged constructs with an EGFP and an ALFA tag for super-resolution immunofluorescence imaging, including structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). All GluRIIA constructs exhibited full functionality and PHP could be induced by philanthotoxin at control levels. By applying hierarchical clustering algorithms to analyze the dSTORM data, we detected postsynaptic receptor cluster areas of ~0.15 µm2. Consequently, our constructs are suitable for ultrastructural analyses of GluRIIA.

Drosophila ammonium transporter Rh50 is required for integrity of larval muscles and neuromuscular system.

Rhesus glycoproteins (Rh50) have been shown to be ammonia transporters in many species from bacteria to human. They are involved in various physiological processes including acid excretion and pH regulation. Rh50 proteins can also provide a structural link between the cytoskeleton and the plasma membranes that maintain cellular integrity. Although ammonia plays essential roles in the nervous system, in particular at glutamatergic synapses, a potential role for Rh50 proteins at synapses has not yet been investigated. To better understand the function of these proteins in vivo, we studied the unique Rh50 gene of Drosophila melanogaster, which encodes two isoforms, Rh50A and Rh50BC. We found that Drosophila Rh50A is expressed in larval muscles and enriched in the postsynaptic regions of the glutamatergic neuromuscular junctions. Rh50 inactivation by RNA interference selectively in muscle cells caused muscular atrophy in larval stages and pupal lethality. Interestingly, Rh50-deficiency in muscles specifically increased glutamate receptor subunit IIA (GluRIIA) level and the frequency of spontaneous excitatory postsynaptic potentials. Our work therefore highlights a new role for Rh50 proteins in the maintenance of Drosophila muscle architecture and synaptic physiology, which could be conserved in other species.

A Presynaptic Regulatory System Acts Transsynaptically via Mon1 to Regulate Glutamate Receptor Levels in Drosophila.

Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. We have identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. We generated mutants in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels, suggesting its involvement in a presynaptic mechanism that regulates postsynaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants, suggesting that homeostatic mechanisms are not affected in these mutants. We propose that DMon1 is part of a presynaptically regulated transsynaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction.