Janet S. Duerr

The cat-1 Gene of Caenorhabditis elegans Encodes a Vesicular Monoamine Transporter Required for Specific Monoamine- Dependent Behaviors

We have identified the Caenorhabditis eleganshomolog of the mammalian vesicular monoamine transporters (VMATs); it is 47% identical to human VMAT1 and 49% identical to human VMAT2.C. elegans VMAT is associated with synaptic vesicles in ∼25 neurons, including all of the cells reported to contain dopamine and serotonin, plus a few others. When C. elegans VMAT is expressed in mammalian cells, it has serotonin and dopamine transport activity; norepinephrine, tyramine, octopamine, and histamine also have high affinity for the transporter. The pharmacological profile of C. elegans VMAT is closer to mammalian VMAT2 than VMAT1. The C. elegans VMAT gene iscat-1; cat-1 knock-outs are totally deficient for VMAT immunostaining and for dopamine-mediated sensory behaviors, yet they are viable and grow relatively well. Thecat-1 mutant phenotypes can be rescued by C. elegans VMAT constructs and also (at least partially) by human VMAT1 or VMAT2 transgenes. It therefore appears that the function of amine neurotransmitters can be completely dependent on their loading into synaptic vesicles.

Identification of major classes of cholinergic neurons in the nematode Caenorhabditis elegans

The neurotransmitter acetylcholine (ACh) is specifically synthesized by the enzyme choline acetyltransferase (ChAT). Subsequently, it is loaded into synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). We have generated antibodies that recognize ChAT or VAChT in a model organism, the nematode Caenorhabditis elegans, in order to examine the subcellular and cellular distributions of these cholinergic proteins. ChAT and VAChT are found in the same neurons, including more than one‐third of the 302 total neurons present in the adult hermaphrodite. VAChT is found in synaptic regions, whereas ChAT appears to exist in two forms in neurons, a synapse‐enriched form and a more evenly distributed possibly cytosolic form. We have used antibodies to identify the cholinergic neurons in the body of larval and adult hermaphrodites. All of the classes of putative excitatory motor neurons in the ventral nerve cord appear to be cholinergic: the DA and DB neurons in the first larval stage and the AS, DA, DB, VA, VB, and VC neurons in the adult. In addition, several interneurons with somas in the tail and processes in the tail or body are cholinergic; sensory neurons are generally not cholinergic. Description of the normal pattern of cholinergic proteins and neurons will improve our understanding of the role of cholinergic neurons in the behavior and development of this model organism. J. Comp. Neurol. 506:398–408, 2008.

Neurogenetics of vesicular transporters in C. elegans

The nematode Caenorhabditis elegans has a number of advantages for the analysis of synaptic molecules. These include a simple nervous system in which all cells are identified and synaptic connectivity is known and reproducible, a large collection of mutants and powerful methods of genetic analysis, simple methods for the generation and analysis of transgenic animals, and a number of relatively simple quantifiable behaviors. Studies in C. elegans have made major contributions to our under‐standing of vesicular transmitter transporters. Two of the four classes of vesicular transporters so far identified (VAChT and VGAT) were first described and cloned in C. elegans; in both cases, the genes were first identified and cloned by means of muta¬tions causing a suggestive phenotype (1, 2). The phenotypes of eat‐4 mutants and the cell biology of the EAT‐4 protein were critical in the identification of this protein as the vesicular glutamate transporter (3, 4). In addition, the unusual gene structure asso¬ciated with the cholinergic locus was first described in C. elegans (5). The biochemical properties of the nematode transporters are surprisingly similar to their vertebrate counterparts, and they can be as¬sayed under similar conditions using the same types of mammalian cells (6, 7). In addition, mild and severe mutants (including knockouts) are available for each of the four C. elegans vesicular transporters, which has permitted a careful evaluation of the role(s) of vesicular transport in transmitter‐specific behaviors. Accordingly, it seems appropriate at this time to present the current status of the field. In this review, we will first discuss the properties of C. elegans vesicular transporters and transporter mu¬tants, and then explore some of the lessons and insights C. elegans research has provided to the field of vesicular transport.—Rand, J. B., Duerr, J. S., Frisby, D. L. Neurogenetics of vesicular transporters in C. elegans. FASEB J. 14, 2414–2422 (2000)

A genetic interaction between the vesicular acetylcholine transporter VAChT/UNC-17 and synaptobrevin/SNB-1 in C. elegans.

Acetylcholine, a major excitatory neurotransmitter in Caenorhabditis elegans, is transported into synaptic vesicles by the vesicular acetylcholine transporter encoded by unc-17. The abnormal behavior of unc-17(e245) mutants, which have a glycine-to-arginine substitution in a transmembrane domain, is markedly improved by a mutant synaptobrevin with an isoleucine-to-aspartate substitution in its transmembrane domain. These results suggest an association of vesicular soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) components with vesicular neurotransmitter transporters.

α-Actinin Is Required for the Proper Assembly of Z-Disk/Focal-Adhesion-Like Structures and for Efficient Locomotion in Caenorhabditis elegans

The actin binding protein α-actinin is a major component of focal adhesions found in vertebrate cells and of focal-adhesion-like structures found in the body wall muscle of the nematode Caenorhabditis elegans. To study its in vivo function in this genetic model system, we isolated a strain carrying a deletion of the single C. elegans α-actinin gene. We assessed the cytological organization of other C. elegans focal adhesion proteins and the ultrastructure of the mutant. The mutant does not have normal dense bodies, as observed by electron microscopy; however, these dense-body-like structures still contain the focal adhesion proteins integrin, talin, and vinculin, as observed by immunofluorescence microscopy. Actin is found in normal-appearing I-bands, but with abnormal accumulations near muscle cell membranes. Although swimming in water appeared grossly normal, use of automated methods for tracking the locomotion of individual worms revealed a defect in bending. We propose that the reduced motility of α-actinin null is due to abnormal dense bodies that are less able to transmit the forces generated by actin/myosin interactions.

Differential expression and function of synaptotagmin 1 isoforms in Caenorhabditis elegans

Synaptotagmin 1, encoded by the snt-1 gene in Caenorhabditis elegans, is a major synaptic vesicle protein containing two Ca(2+)-binding (C2) domains. Alternative splicing gives rise to two synaptotagmin 1 isoforms, designated SNT-1A and SNT-1B, which differ in amino acid sequence in the third, fourth, and fifth beta-strands of the second C2 domain (C2B). We report here that expression of either SNT-1 isoform under control of a strong pan-neural promoter fully rescues the snt-1 null phenotype. Furthermore, C-terminal fusions of either isoform with GFP are trafficked properly to synapses and are fully functional, unlike synaptotagmin 1Colon, two colonsGFP fusions in mice. Analysis of isoform expression with genomic GFP reporter constructs revealed that the SNT-1A and-1B isoforms are differentially expressed and localized in the C. elegans nervous system. We also report molecular, behavioral, and immunocytochemical analyses of twenty snt-1 mutations. One of these mutations, md259, specifically disrupts expression of the SNT-1A isoform and has defects in a subset of synaptotagmin 1-mediated behaviors. A second mutation, md220, is an in-frame 9-bp deletion that removes a conserved tri-peptide sequence (VIL) in the second beta-strand of the C2B domain and disrupts the proper intracellular trafficking of synaptotagmin. Site-directed mutagenesis of a functional SNT-1Colon, two colonsGFP fusion protein was used to examine the potential role of the VIL sequence in synaptotagmin trafficking. Although our results suggest the VIL sequence is most likely not a specific targeting motif, the use of SNT-1Colon, two colonsGFP fusions has great potential for investigating synaptotagmin trafficking and localization.

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-of-function mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-to-arginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/VAChT and synaptobrevin.

Using Caenorhabditis elegans to study vesicular transport.

Publisher Summary This chapter explores the ways in which studies using the nematode Caenorhabditis elegans can add to knowledge of vesicular transporters and vesicular transport. It discusses four general areas where the use of C . elegans offers advantages over other approaches: the use of C . elegans mutants to determine cellular and behavioral requirements for transporter function, the use of C. elegans for structure–function studies of vesicular transporters, the use of antibodies and mutants to explore protein targeting, and the use of C. elegans genetics to identify interacting genes and proteins. These studies rely on the strengths of C. elegans as a research organism. Caenorhabditis elegans is particularly suited for studying neural function. Its nervous system has many similarities to those of mammals, both morphologically and biochemically. The genome is relatively small, consisting of 10 8 base pairs on six chromosomes, and has been extensively mapped and sequenced.

NEUROGENETICS OF SYNAPTIC TRANSMISSION IN CAENORHABDITIS ELEGANS

Publisher Summary Caenorhabditis elegans are widely used as powerful tools for the analysis of mutants and genes. From the perspective of a mammalian neurobiologist, there are two basic strategies that may profitably be employed using C. elegans . Firstly, the basic information derived from C. elegans biology and molecular genetics is applied to mammalian systems; that is, genes first characterized in C. elegans are used to identify mammalian homologues. The other powerful use of C. elegans is to start with known mammalian biology and genes and then identify the C. elegans homologues of these genes. The availability of a large collection of C. elegans mutants has permitted careful in vivo analysis of cholinergic regulation and the behavioral consequences of cholinergic hypofunction. Animals completely lacking the vesicular acetylcholine transporter (VAChT) protein cannot grow or survive after hatching. Animals with milder mutations in the unc-17 gene are small, slow-growing, and display a number of neurornuscular deficits. They are also quite strongly resistant to inhibitors of acetylcholinesterase. All of these phenotypes are shared by cha-1 mutants that argue that vesicular transport is necessary for cholinergic function. Three amine neurotransmitters have been identified thus far in C. elegans : Dopamine (DA), formaldehyde-induced fluorescence (FIF), and serotonin (5HT). The C. elegans Genome Sequencing Project has identified a genomic sequence that appeared to encode part of a vesicular monoamine transporter (VMAT)-like protein. The C. elegans VMAT homologue appears to be encoded by the previously identified cat-2 gene. Using these transporter genes and mutants, together with standard C. elegans tools, it can be now possible to analyze the differentiation of particular neuronal cell types and also the contribution of particular protein domains to cellular and overall behavioral function.