Structure-function analysis of endogenous lectin mind-the-gap in synaptogenesis

Mind-the-Gap (MTG) is required for neuronal induction of Drosophila neuromuscular junction (NMJ) postsynaptic domains, including glutamate receptor (GluR) localization. We have previously hypothesized that MTG is secreted from the presynaptic terminal to reside in the synaptic cleft, where it binds glycans to organize the heavily glycosylated, extracellular synaptomatrix required for transsynaptic signaling between neuron and muscle. In this study, we test this hypothesis with MTG structure-function analyses of predicted signal peptide (SP) and carbohydrate-binding domain (CBD), by introducing deletion and point-mutant transgenic constructs into mtg null mutants. We show that the SP is required for MTG secretion and localization to synapses in vivo. We further show that the CBD is required to restrict MTG diffusion in the extracellular synaptomatrix and for postembryonic viability. However, CBD mutation results in elevation of postsynaptic GluR localization during synaptogenesis, not the mtg null mutant phenotype of reduced GluRs as predicted by our hypothesis, suggesting that proper synaptic localization of MTG limits GluR recruitment. In further testing CBD requirements, we show that MTG binds N-acetylglucosamine (GlcNAc) in a Ca(2+)-dependent manner, and thereby binds HRP-epitope glycans, but that these carbohydrate interactions do not require the CBD. We conclude that the MTG lectin has both positive and negative binding interactions with glycans in the extracellular synaptic domain, which both facilitate and limit GluR localization during NMJ embryonic synaptogenesis.     

Specific heparan sulfate modifications stabilize the synaptic organizer MADD-4/Punctin at Caenorhabditis elegans neuromuscular junctions

Heparan sulfate (HS) proteoglycans contribute to the structural organization of various neurochemical synapses. Depending on the system, their role involves either the core protein or the glycosaminoglycan chains. These linear sugar chains are extensively modified by HS modification enzymes, resulting in highly diverse molecules. Specific modifications of glycosaminoglycan chains may thus contribute to a sugar code involved in synapse specificity. Caenorhabditis elegans is particularly useful to address this question because of the low level of genomic redundancy of these enzymes, as opposed to mammals. Here, we systematically mutated the genes encoding HS modification enzymes in C. elegans and analyzed their impact on excitatory and inhibitory neuromuscular junctions (NMJs). Using single chain antibodies that recognize different HS modification patterns, we show in vivo that these two HS epitopes are carried by the SDN-1 core protein, the unique C. elegans syndecan ortholog, at NMJs. Intriguingly, these antibodies differentially bind to excitatory and inhibitory synapses, implying unique HS modification patterns at different NMJs. Moreover, while most enzymes are individually dispensable for proper organization of NMJs, we show that 3-O-sulfation of SDN-1 is required to maintain wild-type levels of the extracellular matrix protein MADD-4/Punctin, a central synaptic organizer that defines the identity of excitatory and inhibitory synaptic domains at the plasma membrane of muscle cells.