Presynaptic establishment of the synaptic cleft extracellular matrix is required for post-synaptic differentiation

Abstract

Formation and regulation of excitatory glutamatergic synapses is essential for shaping neural circuits throughout development. In a Drosophila genetic screen for synaptogenesis mutants, we identified mind the gap (mtg), which encodes a secreted, extracellular N-glycosaminoglycan-binding protein. MTG is expressed neuronally and detected in the synaptic cleft, and is required to form the specialized transsynaptic matrix that links the presynaptic active zone with the post-synaptic glutamate receptor (GluR) domain. Null mtg embryonic mutant synapses exhibit greatly reduced GluR function, and a corresponding loss of localized GluR domains. All known post-synaptic signaling/scaffold proteins functioning upstream of GluR localization are also grossly reduced or mislocalized in mtg mutants, including the dPix-dPak-Dock cascade and the Dlg/PSD-95 scaffold. Ubiquitous or neuronally targeted mtg RNA interference (RNAi) similarly reduce post-synaptic assembly, whereas post-synaptically targeted RNAi has no effect, indicating that presynaptic MTG induces and maintains the post-synaptic pathways driving GluR domain formation. These findings suggest that MTG is secreted from the presynaptic terminal to shape the extracellular synaptic cleft domain, and that the cleft domain functions to mediate transsynaptic signals required for post-synaptic development.[Keywords: Glutamatergic synaptogenesis; post-synaptic density; glutamate receptor; secretion; Drosophila; neuromuscular junction] Supplemental material is available at http://www.genesdev.org.

N-glycosylation requirements in neuromuscular synaptogenesis

“…It will be of interest to perform immunoprecipitation on Mgat1 samples to identify changes in HRP bands. Importantly, MTG is crucial for synaptomatrix glycan patterning and functional synaptic development (Rohrbough et al, 2007). MTG regulates VVA synaptomatrix labeling (Rushton et al, 2009), suggesting a mechanistic link between the VVA and MTG changes in Mgat1 mutants.…”

“…Likewise, VVA lectin is a synaptic glycan marker at the Drosophila NMJ, which reportedly recognizes primarily postsynaptic Dystroglycan (Haines et al, 2007;Rushton et al, 2012). The endogenous MTG lectin patterns the extracellular glycosylated synaptomatrix (Rohrbough et al, 2007), modulates trans-synaptic signaling and is essential for functional synaptogenesis (Rushton et al, 2009;Rohrbough and Broadie, 2010). To begin to define synaptogenic roles of MGAT1, we labeled the NMJ with each of these lectins in genetic control, Mgat1 null mutants and Mgat1 rescue conditions (Fig.…”

Transmission, Development, and Plasticity of Synapses

“…Mutants of mtg exhibit a severe impairment in synaptic transmission, ultrastructural defects both in the synaptic cleft and in the postsynapse, and mislocalization of GluR puncta. Furthermore, Dlg, Pak, Dock, and Pix are either reduced or severely mislocalized from mtg mutant synapses, suggesting that MTG plays a critical role upstream of these proteins to regulate GluR localization (Rohrbough et al 2007). Interestingly, when mtg mutants are rescued with MTG lacking its carbohydrate-binding domain (CBD), postsynaptic GluR accumulation increases (Rushton et al 2012).…”

“…Mind-the-gap (MTG) is a presynaptically secreted lectin found in the synaptic cleft at the NMJ (Rohrbough et al 2007;Rushton et al 2009). Lectins are carbohydratebinding proteins with diverse cellular functions.…”

Stoned B mediates sorting of integral synaptic vesicle proteins

“…Mutant and wildtype control wandering third instar larvae were dissected, fixed, sectioned and visualized in parallel using standard transmission electron microscopy techniques, as reported previously (Rohrbough et al 2007), with only minor changes. Briefly, animals were dissected in PBS and subsequently fixed with 2.0% glutaraldehyde in 0.05M PBS for 15 mins; replaced with fresh 2.0% glutaraldehyde for 1 hr.…”