aex-3 mutants show multiple defects in their behavior.
aex-3 mutants were originally isolated based on abnormal defecation: two motor program steps in defecation are frequently missing, anterior body wall muscle contraction and expulsion 1. We found that
aex-3 mutants are also weakly defective in some sensory behavior and male mating. Finally,
aex-3 mutants are resistant to the acetylcholinesterase inhibitor, aldicarb (our data and E. Jorgensen, per. comm.) but are as sensitive as wild type to the nicotinic acetylcholine receptor agonist, levamisole. These observations indicate that
aex-3 functions widely in the nervous system. In particular, the pharmacological results suggest that
aex-3 is involved in controlling presynaptic activity. It was previously shown that
aex-3 mutations cause a dauer formation constitutive (Daf-c) phenotype and a pharyngeal pumping defect when combined with an
unc-31 mutation 1,2. We made all combinations of double mutants between five
aex-3 alleles (
ad418,
sa5,
ad696,
n2166,
y255; thank Jorgensen for
n2166) and three
unc-31 alleles (
e169,
e928,
u280). All the double mutants are strongly Daf-c at 25C. We also found that double mutants between
aex-3 (five alleles) and
unc-64 (
e246,
md130) are Daf-c at 25C.
unc-31 and
unc-64 encode
p145 (D. Livingstone, per. comm) and syntaxin (M. Nonet, unpublished data), respectively. Both function in Ca++ dependent exocytosis in the mammalian nervous system. These interactions suggest that
aex-3 function contributes to Ca++ dependent exocytosis in the nervous system. To determine how
aex-3 mutations impair presynaptic activity, we examined
aex-3 mutants using antibodies specific for the synaptic vesicle components, SNT-1 (a nematode synaptotagmin 3) and RAB-3 (a nematode
rab3a homologue). In the wild type and
aex-3 mutants anti-SNT-1 antiserum stained only axons and not cell bodies. Anti-RAB-3 antiserum stained mainly axons in the wild type and faintly stained cell bodies. In the
aex-3 mutant, anti-RAB-3 antiserum heavily stained cell bodies in addition to axons of all neurons. This observation suggests that
aex-3 is required for proper localization of RAB-3 protein in axons. The synaptic defects caused by
aex-3 mutations could be due, in part, to improper localization of RAB-3 at the presynaptic terminal. We cloned
aex-3 by transformation rescue of the Aex phenotype (cosmids provided by A. Coulson). Using the smallest rescuing fragment as a probe, we identified a 5 kb mRNA on a Northern blot and isolated a cDNA clone from three hundred thousand cDNA library plaques (a gift from R. Barstead). Combining the sequence of this cDNA and RT-PCR products, we identified an
aex-3 message that is 4.7 kb long, contains an SL1 leader sequence, and encodes a novel protein (1410 animo acid long) with a weak similarity to a HeLa tumor suppressor gene (HTS-1). We conclude that
aex-3 encodes a novel protein that controls presynaptic activity in C. elegans, perhaps by interacting with RAB-3. 1. J. H. Thomas (1990) Genetics 124:855. 2. L. Avery (1993) Genetics 133: 897 3. Nonet et al. (1993) Cell 73: 1291