SUMMARY Changes in neuronal activity create local and transient changes in energy demands at synapses. Here we discover a metabolic compartment that forms in vivo near synapses to meet local energy demands and support synaptic function in Caenorhabditis elegans neurons. Under conditions of energy stress, glycolytic enzymes redistribute from a diffuse localization in the cytoplasm to a punctate localization adjacent to synapses. Glycolytic enzymes colocalize, suggesting the ad hoc formation of a glycolysis compartment, or a ‘glycolytic metabolon’, that can maintain local levels of ATP. Local formation of the glycolytic metabolon is dependent on presynaptic scaffolding proteins, and disruption of the glycolytic metabolon blocks the synaptic vesicle cycle, impairs synaptic recovery, and affects locomotion. Our studies indicate that under energy stress conditions, energy demands in C. elegans synapses are met locally through the assembly of a glycolytic metabolon to sustain synaptic function and behavior.

Intracellular membrane fusion is mediated by the concerted action of N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. During fusion, SM proteins bind the N-terminal peptide (N-peptide) motif of the SNARE subunit syntaxin, but the function of this interaction is unknown. Here, using FRET-based biochemical reconstitution and Caenorhabditis elegans genetics, we show that the N-peptide of syntaxin-1 recruits the SM protein Munc18-1/nSec1 to the SNARE bundle, facilitating their assembly into a fusion-competent complex. The recruitment is achieved through physical tethering rather than allosteric activation of Munc18-1. Consistent with the recruitment role, the Npeptide is not spatially constrained along syntaxin-1, and it is functional when translocated to another SNARE subunit SNAP-25 or even when simply anchored in the target membrane. The N-peptide function is restricted to an early initiation stage of the fusion reaction. After association, Munc18-1 and the SNARE bundle together drive membrane merging without further involving the N-peptide. Thus, the syntaxin N-peptide is an initiation factor for the assembly of the SNARE-SM membrane fusion complex.I ntracellular membrane fusion is the basis of a wide range of fundamental biological processes, including organelle maintenance, hormone secretion, and inside-outside distribution of receptors and transporters. The merging of intracellular membrane bilayers is mediated by a fusion complex comprised of SNAREs and Sec1/Munc18 (SM) proteins (1). The core of the fusion machinery is the trans-SNARE complex (SNAREpin) formed by the pairing of the vesicle-rooted SNARE (v-SNARE) with the target membrane-associated SNAREs (t-SNAREs) (2-5). N-to C-terminal zippering of the trans-SNARE complex brings two membranes into close apposition and helps to overcome the energy barrier for fusion (6-10). SM proteins are soluble factors of 60-70 kDa that directly interact with their cognate trans-SNARE complexes to promote the speed and specificity of a fusion reaction (11)(12)(13)(14).Each fusion pathway in the cell requires a specific subset of SNAREs and SM proteins (15). The most intensely studied form of intracellular membrane fusion is calcium-triggered neurotransmitter release at the chemical synapse, which serves as the brain's major form of cell-cell communication (15)(16)(17)(18)(19). Neurotransmitter secretion is mediated by the fusion of exocytic vesicles with the plasma membrane and requires the v-SNARE vesicle-associated membrane protein 2 (VAMP2; also known as synaptobrevin-2), the t-SNAREs syntaxin-1 and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-25, and the SM protein Munc18-1/ nSec1 (UNC-18 in nematodes and ROP in flies) (20-28).The interaction between SNAREs and SM proteins involves multiple binding modes. The primary target of SM proteins is believed to be the four-helix SNARE bundle (29-31). Assembled from the SNARE motifs and the transmembrane domains of t-and vSNAREs (4, 5), the SNARE bun...

Conventional genetic testing of individuals with neurodevelopmental presentations and congenital anomalies (ND/CAs), i.e., the analysis of sequence and copy number variants, leaves a substantial proportion of them unexplained. Some of these cases have been shown to result from DNA methylation defects at a single locus (epi-variants), while others can exhibit syndrome-specific DNA methylation changes across multiple loci (epi-signatures). Here, we investigate the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved ND/CAs. We generate a computational model enabling concurrent detection of 14 syndromes using DNA methylation data with full accuracy. We demonstrate the ability of this model in resolving 67 individuals with uncertain clinical diagnoses, some of whom had variants of unknown clinical significance (VUS) in the related genes. We show that the provisional diagnoses can be ruled out in many of the case subjects, some of whom are shown by our model to have other diseases initially not considered. By applying this model to a cohort of 965 ND/CA-affected subjects without a previous diagnostic assumption and a separate assessment of rare epi-variants in this cohort, we identify 15 case subjects with syndromic Mendelian disorders, 12 case subjects with imprinting and trinucleotide repeat expansion disorders, as well as 106 case subjects with rare epi-variants, a portion of which involved genes clinically or functionally linked to the subjects' phenotypes. This study demonstrates that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of epigenomic testing in the routine clinical assessment of ND/CAs.