A central event in synapse development is formation of the presynaptic active zone in response to positional cues. Three active zone proteins, RIM, ELKS (also known as ERC or CAST) and Liprin-alpha, bind each other and are implicated in linking active zone formation to synaptic vesicle release. Loss of function in Caenorhabditis elegans syd-2 Liprin-alpha alters the size of presynaptic specializations and disrupts synaptic vesicle accumulation. Here we report that a missense mutation in the coiled-coil domain of SYD-2 causes a gain of function. In HSN synapses, the syd-2(gf) mutation promotes synapse formation in the absence of syd-1, which is essential for HSN synapse formation. syd-2(gf) also partially suppresses the synaptogenesis defects in syg-1 and syg-2 mutants. The activity of syd-2(gf) requires elks-1, an ELKS homolog; but not unc-10, a RIM homolog. The mutant SYD-2 shows increased association with ELKS. These results establish a functional dependency for assembly of the presynaptic active zone in which SYD-2 plays a key role.

C. elegans RPM-1 (for Regulator of Presynaptic Morphology) is a member of a conserved protein family that includes Drosophila Highwire and mammalian Pam and Phr1. These are large proteins recently shown to regulate synaptogenesis through E3 ubiquitin ligase activities. Here, we report the identification of an RCC1-like guanine nucleotide exchange factor, GLO-4, from mass spectrometry analysis of RPM-1-associated proteins. GLO-4 colocalizes with RPM-1 at presynaptic terminals. Loss of function in glo-4 or in its target Rab GTPase, glo-1, causes neuronal defects resembling those in rpm-1 mutants. We show that the glo pathway functions downstream of rpm-1 and acts in parallel to fsn-1, a partner of RPM-1 E3 ligase function. We find that late endosomes are specifically disorganized at the presynaptic terminals of glo-4 mutants. Our data suggest that RPM-1 positively regulates a Rab GTPase pathway to promote vesicular trafficking via late endosomes.

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.

Leukocyte-common antigen related (LAR)-like phosphatase receptors are conserved cell adhesion molecules that function in multiple developmental processes. The Caenorhabditis elegans ptp-3 gene encodes two LAR family isoforms that differ in the extracellular domain. We show here that the long isoform, PTP-3A, localizes specifically at synapses and that the short isoform, PTP-3B, is extrasynaptic. Mutations in ptp-3 cause defects in axon guidance that can be rescued by PTP-3B but not by PTP-3A. Mutations that specifically affect ptp-3A do not affect axon guidance but instead cause alterations in synapse morphology. Genetic double-mutant analysis is consistent with ptp-3A acting with the extracellular matrix component nidogen, nid-1, and the intracellular adaptor ␣-liprin, syd-2. nid-1 and syd-2 are required for the recruitment and stability of PTP-3A at synapses, and mutations in ptp-3 or nid-1 result in aberrant localization of SYD-2. Overexpression of PTP-3A is able to bypass the requirement for nid-1 for the localization of SYD-2 and RIM. We propose that PTP-3A acts as a molecular link between the extracellular matrix and ␣-liprin during synaptogenesis.