Expression of Multiple UNC-13 Proteins in theCaenorhabditis elegansNervous System

Abstract

The Caenorhabditis elegans UNC-13 protein and its mammalian homologues are important for normal neurotransmitter release. We have identified a set of transcripts from the unc-13 locus in C. elegans resulting from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but that vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. We have analyzed the sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. We have also isolated a deletion allele that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products. INTRODUCTIONTransfer of information between neurons or between neurons and muscles requires the Ca 2ϩ -dependent release of neurotransmitters from synaptic vesicles. According to the SNARE hypothesis, specific synaptic vesicle proteins interact with specific plasma membrane proteins at docking sites at the active zone (Sö llner et al., 1993a,b). Interacting proteins include synaptobrevin (also known as VAMP) and synaptotagmin in the synaptic vesicle and syntaxin and SNAP-25 in the presynaptic membrane (Hayashi et al., 1993). Soluble factors, including N-ethylmaleimide-sensitive factor and ␣-and ␤-soluble N-ethylmaleimide-sensitive factor attachment proteins (Sö llner et al., 1993b), are involved in the formation and/or regulation of these proteins. Although many components of synaptic vesicle targeting and neurotransmitter release have been identified, the regulation of these processes cannot be completely explained.unc-13 was originally identified in Caenorhabditis elegans as a gene important for normal locomotion. Mutations in this gene result in severely uncoordinated movement, increased accumulation of the neurotransmitter acetylcholine, and resistance to acetylcholinesterase inhibitors (Brenner, 1974;Rand and Russell, 1985;Hosono et al., 1989;Nguyen et al., 1995). These phenotypes suggest a decrease in the synaptic release of acetylcholine, and other studies have indicated that unc-13 mutants are deficient in the release of most or all neurotransmitters (Miller et al., 1996).The unc-13 gene encodes a protein (UNC-13) containing C1 and C2 homology domains (Maruyama and Brenner, 1991). Mammalian homologues of UNC-13, Munc13-1, Munc13-2, and Munc13-3 in rat brain (Brose et al., 1995) and Hmunc13 in human kidney (Song et al., 1999), also contain one C1 and two or three C2 domains. C1 and C2 r...

Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction

“…To confirm that light-evoked currents result from the canonical synaptic vesicle fusion machinery, we tested whether these currents required the unc-13 gene. UNC-13 is a protein essential for synaptic vesicle exocytosis (23)(24)(25). Consistent with the absence of electrically evoked currents in unc-13(s69) mutants (25), light stimuli failed to evoke detectable currents in unc-13(s69) Punc-17::ChR2::mCherry animals ( Fig.…”

Mutations That Rescue the Paralysis of Caenorhabditis elegans ric-8 (Synembryn) Mutants Activate the Gαs Pathway and Define a Third Major Branch of the Synaptic Signaling Network

“…Together with the accompanying study dropping. (Reynolds et al 2005), these results suggest that three Screens were performed as described above, in weekly cyhighly conserved G␣ signaling pathways form the synapcles, alternating between 3 successive cycles of each screen, for 16 cycles for the ric-8 suppressor screen and 12 cycles tic signaling network, an integrated molecular circuit for the N2 hyperactive screen. We estimate that each cycle that is likely to be a major substrate for behavioral modiscreened 3000 mutagenized genomes for a total of 48,000 fication, learning, and memory.…”

“…However, given the central importance of (G␣ q ) pathway have begun to shed light on some of the core G␣ q pathway with respect to neurotransmitter these questions by revealing a large network of proteins release (Reynolds et al 2005, accompanying article in that regulates neurotransmitter release ( Figure 1). As this issue), we hypothesized that there could be other in vertebrates, G␣ q 's action in the C. elegans nervous components, in addition to RIC-8, that positively regusystem appears to be mediated by phospholipase C␤ late, or otherwise impinge upon, the EGL-30 (G␣ q ) path-(EGL-8), although the C. elegans studies also point to way (Figure 1).…”

“…and yet activating the G␣ s pathway in ric-8(md303) seems This is not caused by "leaking" of the muscle promoter to restore steady-state neurotransmitter release to levels in nervous system tissue, because control experiments, in excess of wild type ( Figure 9C). done as part of a separate study, showed that the myo-3 Suppression of ric-8(md303) occurs via the neuronal promoter, even at high levels, cannot drive rescue of a G␣ s pathway, but both the muscle and nervous system nervous-system-specific mutant (Reynolds et al 2005). G␣ s pathways contribute to the locomotion rate and However, unlike the nervous-system-specific acy-1 (P260S) drug sensitivity phenotypes: The C. elegans GSA-1 (G␣ s ) transgene, the muscle-specific acy-1 (P260S) transgene pathway is expressed in both nervous system and bodywas unable to cause any rescue of the paralysis of ricwall muscle cells.…”

A Conserved Mechanism of Synaptogyrin Localization

“…SV proteins such as RAB-3, SNB-1, and SNT-1 all require unc-104 for synaptic localization (Nonet et al, 1993. In contrast, localization of nonvesicular localized proteins such as SYD-2, RIM, UNC-11, and UNC-13 does not require unc-104 (Nonet et al, 1999;Zhen and Jin, 1999;Kohn et al, 2000; Hadwiger and Nonet, unpublished observations). In unc-104(e1265) mutants, SNG-1::GFP was concentrated in neuronal cell bodies in both the head ganglia and the ventral nerve cord (Figure 2, C and D).…”