Rim1 was previously identified as a Rab3 effector localized to the presynaptic active zone in vertebrates. Here we demonstrate that C. elegans unc-10 mutants lacking Rim are viable, but exhibit behavioral and physiological defects that are more severe than those of Rab3 mutants. Rim is localized to synaptic sites in C. elegans, but the ultrastructure of the presynaptic densities is normal in Rim mutants. Moreover, normal levels of docked synaptic vesicles were observed in mutants, suggesting that Rim is not involved in the docking process. The level of fusion competent vesicles at release sites was reduced fivefold in Rim mutants, but calcium sensitivity of release events was unchanged. Furthermore, expression of a constitutively open form of syntaxin suppressed the physiological defects of Rim mutants, suggesting Rim normally acts to regulate conformational changes in syntaxin. These data suggest Rim acts after vesicle docking likely via regulating priming.

Little is known of mechanisms regulating presynaptic differentiation. We identified rpm-1 in a screen for mutants with defects in patterning of a presynaptic marker at certain interneuronal synapses. The predicted RPM-1 protein contains zinc binding, RCC1, and other conserved motifs. In rpm-1 mutants, mechanosensory neurons fail to accumulate tagged vesicles, retract synaptic branches, and ectopically extend axons. Some motor neurons branch and overgrow; others show altered synaptic organization. Expression of RPM-1 in the presynaptic mechanosensory neurons is sufficient to rescue phenotypes in these cells. Certain rpm-1 phenotypes are temperature sensitive, revealing that RPM-1 function can be bypassed by maintaining mutants at the permissive temperature at stages commensurate with synapse formation in wild-type animals. These results indicate that RPM-1 functions cell autonomously during synaptogenesis to regulate neuronal morphology.

Sec1-related proteins function in most, if not all, membrane trafficking pathways in eukaryotic cells. The Sec1-related protein required in neurons for synaptic vesicle exocytosis is UNC-18. Several models for UNC-18 function during vesicle exocytosis are under consideration. We have tested these models by characterizing unc-18 mutants of the nematode Caenorhabditis elegans. In the absence of UNC-18, the size of the readily releasable pool is severely reduced. Our results show that the near absence of fusion-competent vesicles is not caused by a reduction in syntaxin levels, by a mislocalization of syntaxin, by a defect in fusion or by a failure to open syntaxin during priming. Rather, we found a reduction of docked vesicles at the active zone in unc-18 mutants, suggesting that UNC-18 functions, directly or indirectly, as a facilitator of vesicle docking.Synaptic vesicles must be tethered to sites of release and then become competent for fusion with the plasma membrane. These processes are called docking and priming, respectively 1 . The molecular processes of docking are not well understood. Priming appears to involve the formation of trans-SNARE complexes, in which the synaptic vesicle protein synaptobrevin (also called VAMP) forms a helical bundle with the plasma membrane proteins syntaxin and . The formation of SNARE complexes is sufficient for membrane fusion in vitro 3 , but may require other components in vivo 4,5 .Another protein that is involved in synaptic vesicle exocytosis is . unc-18 mutants were first isolated in C. elegans on the basis of their severe locomotion defects 7 . The UNC-18 protein is similar to the yeast SEC1 protein 8 , and orthologs of UNC-18 function at the synapses of fruit flies (Rop) 9 and vertebrates (Muncl8-1; also known as Stxbpl, N-secl, Rb-secl) 10 . Genetic studies of UNC-18 in various organisms suggest that UNC-18 is required for synaptic vesicle exocytosis. For example, unc-18 mutants in C. elegans are severely paralyzed and are resistant to inhibitors of cholinesterase but sensitive to acetylcholine (ACh) receptor agonists 11 , implicating UNC-18 in the exocytosis of ACh.

Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function. INTRODUCTIONNeurotransmitter release is accomplished by the fusion of neurotransmitter-filled synaptic vesicles at the presynaptic nerve terminal. This process occurs through a series of highly regulated steps that include synaptic vesicle transport, docking/tethering, priming, fusion, endocytosis, recycling, and neurotransmitter refilling (Sudhof, 2004). Several genes have been assigned roles in the various steps of the synaptic vesicle cycle. Rab3 (termed RAB-3 in Caenorhabditis elegans), a member of the Rab family of small GTPases, regulates synaptic transmission, possibly through the docking, priming, or fusion steps Schluter et al., 2004).Rabs act in a variety of cell types and regulate vesicular transport between organelles. Rabs cycle on and off membranes via a GTP-dependent mechanism (Zerial and McBride, 2001). Rab activity is regulated by two proteins, which act in an antagonistic manner. The guanine nucleotide exchange factor (GEF) exchanges GTP for GDP, and the GTPase activating protein activates the intrinsic GTPase activity of a Rab (Bernards, 2003). GDP bound Rabs are held off of the membrane by a GDP dissociation inhibitor (Wu et al., 1996). The GTP/membrane-bound form of Rabs typically binds to a variety of effectors that regulate particular steps of membrane transport and cell signaling (Zerial and McBride, 2001;Spang, 2004).Rab3 was once thought to play a central role in regulating release. However, recent work has shown that a quadruple knockout of all four isoforms of Rab3 in mice only results in a 30% reduction in evoked synaptic response (Schluter et al., 2004). This is consistent with work in C. elegans showing that mutations in the single rab-3 gene cause only mild defects in synaptic transmission . Surprisingly, more dramatic phe...

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.