Worm Breeder's Gazette 12(4): 68 (October 1, 1992)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The C. elegans gene ric-2 was originally defined genetically by a set of mutations that conferred recessive resistance to inhibitors of cholinesterase. ric-2 mutants are small shrinker-uncs - when the plate is tapped, the anterior portion of the animal shrinks and then relaxes, while the posterior of the animal coils. The mutants are very resistant to cholinesterase inhibitors (e.g. aldicarb and trichlorfon); they are small, slow-growing, and impaired in pharyngeal pumping and defecation. Nevertheless, the animals are active and capable of propagating limited body waves. Cholinergic enzymes (choline acetyltransferase and all three forms of acetylcholinesterase) are present at normal levels, but acetylcholine is elevated approximately three-fold. We initially believed that ric-2 animals were defective in the release of acetylcholine, but we now believe that they are deficient in the release of most (or all) neurotransmitters.
Eleven alleles of ric-2 have been isolated so far: 9 in screens for aldicarb resistance, 1 by Leon Avery in a screen for Eat mutants, and 1 by Erik Jorgensen in a screen for defecation-defective mutants. They have very similar phenotypes, and the phenotypes are not enhanced in animals containing a ric-2 allele in trans to a deficiency. We therefore cautiously propose that many of the mutations are nulls or near-nulls, a notion supported by the molecular analysis (see below). Three-factor mapping and deficiency mapping located ric-2 in the 0.09 map unit interval between dpy-2 and tra-2 on LG 11.
We have recently demonstrated that ric-2 encodes a C. elegans homolog of synaptotagmin ( p65 ),an abundant synaptic vesicle protein which is believed to bind calcium and trigger vesicle fusion and exocytosis (Sudhof and Jahn, Neuron 6:665, 1991). Our evidence is based on the following arguments. 1) A 19 kb subclone of a cosmid containing the synaptotagmin structural gene (WBG 12(3):71) rescues all of the behavioral phenotypes of a ric-2 mutant. 2) 4 independent ric-2 mutations are associated with DNA rearrangements in the synaptotagmin genomic region. A portion of the coding region is deleted in one allele. 3) Immunohistochemical staining of synaptotagmin is absent or is greatly reduced in the 3 ric-2 strains examined (using mouse polyclonal anti-C. elegans synaptotagmin antibodies), while staining remains normal for another synaptic vesicle associated protein, rab3 (using mouse polyclonal anti-C. elegans rab3 antibodies). We believe that these are the first mutants characterized in any organism with a defect in a known synaptic vesicle protein.
To document that synaptotagmin is associated with synaptic vesicles in C. elegans, we utilized unc-104 mutants in which synaptic vesicles are not transported out to synapses, but instead remain associated with neuronal cell bodies (Hall and Hedgecock, Cell 65:837,1991). Synaptotagmin immunohistochemical staining is restricted to processes in wild-type animals (WBG 12(3):71). In contrast, synaptotagmin staining is localized in irregular punctate patterns in the cell bodies of many neurons in unc-104 ( rh43 )animals. Additionally, staining of the processes of the ventral cord, dorsal cord, and nerve ring disappears. Thus, synaptotagmin is probably present in most C. elegans neurons and is associated with synaptic vesicles.
The strongest ric-2 mutant alleles retain some synaptic function since mutations that eliminate synaptic development ( unc-104 nulls) or cholinergic function ( cha-1 nulls) are lethal. We therefore propose that synaptotagmin is not absolutely required for synaptic release, although it is clearly necessary for normal synaptic function.
Hall and Hedgecock, Cell 65:837,1991