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 excitatory 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 Drosophila NMJ can thus be used to analyze and model defects in the structural and physiological plasticity of glutamatergic synapses, which are associated with a variety of human pathologies from learning, memory deficits to autism. The similarity in gross architecture, function, and molecular machinery supports the notion that studying the assembly and development of fly glutamatergic synapses will shed light on their vertebrate counterparts. In flies as in humans, synapse strength and plasticity is determined by the interplay between different iGluRs subtypes. The subunits that form the Drosophila glutamate-gated ion channels (iGluRs) are known, but only the NMJ receptor complexes have been well studied in vivo. They NMJ receptors consist of four different subunits: GluRIIC, -IID and -IIE, and either GluRIIA (in type-A receptors) or IIB (type-B). Various mechanisms regulating the extent of type-A and type-B receptors accumulation at synaptic sites have been described but the molecular mechanisms for the initial localization and clustering of receptors at synaptic sites remained unclear. We have recently discovered an obligatory auxiliary protein, Neto (Neuropillin and Tolloid-like), absolutely required for the iGluRs clustering and NMJ functionality. Neto belongs to a family of highly conserved proteins sharing an ancestral role in formation and modulation of glutamatergic synapses. Our investigations uncovered essential roles for Neto during synapse development and strongly support the notion that trafficking of both iGluR subtypes on the muscle membrane, their synaptic recruitment and stabilization, and their function are tightly regulated by Neto. Our results further suggest that the fly Neto isoforms (alpha and beta) directly engage iGluRs as well as other intracellular and extracellular proteins to selectively regulate the distribution of iGluRs subtypes, the recruitment of postsynaptic proteins, and the organization of postsynaptic structures. Since iGluRs gating properties control the distribution and trafficking of these receptors in vivo, Neto could influence the synaptic recruitment of iGluRs by simultaneously controlling multiple steps in receptor trafficking and clustering and/or receptor function. Until recently, our investigations were limited by the inability to reconstitute functional Drosophila NMJ receptors in heterologous systems and identify the structural elements and the auxiliary subunits important for receptor assembly, surface delivery, synaptic recruitment and function. We have recently solved this problem by accomplishing the first functional reconstitution of NMJ iGluRs in Xenopus oocytes. Furthermore, we have succeeded in expressing functional receptors in HEK293 cells, where the biophysical properties of these receptors can be further dissected using fast glutamate applications. The ability to examine the functional characteristics of iGluRs in heterologous systems opens up tremendous opportunities to study the modulation of iGluRs function and parse out a role for Neto and/or other auxiliary proteins in the receptor function vs. receptor assembly, surface expression, synaptic trafficking and/or stabilization.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2017
Total Cost
Indirect Cost
Name
U.S. National Inst/Child Hlth/Human Dev
Department
Type
DUNS #
City
State
Country
Zip Code
Wang, Qi; Han, Tae Hee; Nguyen, Peter et al. (2018) Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction. Elife 7:
Meyerson, Joel R; Chittori, Sagar; Merk, Alan et al. (2016) Structural basis of kainate subtype glutamate receptor desensitization. Nature 537:567-571
Sulkowski, Mikolaj J; Han, Tae Hee; Ott, Carolyn et al. (2016) A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction. PLoS Genet 12:e1005810
Li, Yan; Dharkar, Poorva; Han, Tae-Hee et al. (2016) Novel Functional Properties of Drosophila CNS Glutamate Receptors. Neuron 92:1036-1048
Horstick, Eric J; Jordan, Diana C; Bergeron, Sadie A et al. (2015) Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish. Nucleic Acids Res 43:e48
Kim, Young-Jun; Igiesuorobo, Oghomwen; Ramos, Cathy I et al. (2015) Prodomain removal enables neto to stabilize glutamate receptors at the Drosophila neuromuscular junction. PLoS Genet 11:e1004988
Han, Tae Hee; Dharkar, Poorva; Mayer, Mark L et al. (2015) Functional reconstitution of Drosophila melanogaster NMJ glutamate receptors. Proc Natl Acad Sci U S A 112:6182-7
Serpe, Mihaela (2015) What does it take to get the job done? Mol Biol Cell 26:3684-6
Ramos, Cathy I; Igiesuorobo, Oghomwen; Wang, Qi et al. (2015) Neto-mediated intracellular interactions shape postsynaptic composition at the Drosophila neuromuscular junction. PLoS Genet 11:e1005191
Sulkowski, Mikolaj; Kim, Young-Jun; Serpe, Mihaela (2014) Postsynaptic glutamate receptors regulate local BMP signaling at the Drosophila neuromuscular junction. Development 141:436-47

Showing the most recent 10 out of 12 publications