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.

Gymnodinium acidotum Nygaard, a blue‐green dinoflagellate previously shown to contain cryptophycean chloroplasts and other organelles, was observed from water and soil samples and in culture. The dinoflagellate excysts from soil samples as a mononucleated colorless cell that is positively phototactic. Colorless cells in unialgal culture remain colorless and can only be maintained less than one week whereas pigmented cells cultured unialgally grow for 10 days but then decline rapidly. Colorless cells cultured with Chroomonas spp. regain chloroplasts and have been maintained in mixed cultures for nine months. Fifty‐seven percent of the dinoflagellates from mixed cultures are bi‐nucleated, and three cells have been observed possibly ingesting cryptophytes. We suggest that cryptophycean chloroplasts are retained and possibly utilized by G. acidotum for at least ten days and then digested.

The actin binding protein α-actinin is a major component of focal adhesions found in vertebrate cells and of the focal adhesion-like structures found 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 by EM but these dense body-like structures still contain the focal adhesion proteins integrin, talin and vinculin 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 locomotion of individual worms revealed a defect in bending. We propose that the reduced motility of the α-actinin null is due to abnormal dense bodies that are less able to transmit the forces generated by actin/myosin interactions.

Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in Caenorhabditis elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na ؉ and Cl ؊ dependent, has an EC 50 value of 168 M, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or postsynaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans and that the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.

In cluA- mutants of Dictyostelium, mitochondria are clustered near the cell center rather than being dispersed throughout the cytoplasm. We have examined two possible mechanisms that could account for this phenotype. First, we sought evidence that the cytoskeleton or a presumptive mitochondrion-cytoskeleton linkage was altered in mutant cells. We found that cytoskeletal structures in cluA- cells appeared normal by immunostaining, and that the distribution of peroxisomes in mutant cells was indistinguishable from that in wild type cells. Treatment of wild type cells with drugs that disrupted microtubules or actin filaments did not mimic the cluA- phenotype. Thus, cytoskeletal defects seemed unlikely to account for the mitochondrial clustering in cluA- cells. Observation of the movement of GFP-tagged mitochondria in wild type cells suggested that mitochondria are transported along microtubules, as in mammalian cells, rather than along actin filaments, as in budding yeast. Therefore, the similar phenotypes of cluA- Dictyostelium cells and clu1delta yeast cells argued against CluA/Clu1p acting as a mitochondrion-cytoskeleton linker. We next examined the ultrastructure of mitochondria in freeze-substituted, thin-sectioned cells. We found that the clustered mitochondria in cluA- cells are interconnected. Often, adjacent mitochondria are linked by narrow membranous strands, although sometimes the mitochondria are partially merged. The presence of narrow constrictions at presumptive division sites argues that the constriction step of division proceeds normally. Our data suggest that cluA- cells may be blocked at a very late step in fission of the outer mitochondrial membrane.