Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons.

Identification of the genes orchestrating neurogenesis would greatly enhance our understanding of this process. Genes have been identified that specify neuron type (for example cut and numb in Drosophila and mec-3 in Caenorhabditis elegans) and process guidance (for example, unc-5, unc-6 and unc-40 in C. elegans and the fas-1 gene of Drosophila). We sought genes defining synaptic specificity by identifying mutations that alter synaptic connectivity in the motor circuitry in the nematode C. elegans. We used electron microscopy of serial sections to reconstruct the ventral nerve-cords of uncoordinated (unc) mutants that have distinctive locomotory choreographies. Here we describe the phenotype of mutations in the unc-4 gene in which a locomotory defect is correlated with specific changes in synaptic input to a subset of the excitatory VA motor neurons, normally used in reverse locomotion. The circuitry alterations do not arise because of the inaccessibility of the appropriate synaptic partners, but are a consequence of changes in synaptic specificity. The VA motor neurons with altered synaptic inputs are all lineal sisters of VB motor neurons; the VA motor neurons without VB sisters have essentially the same synaptic inputs as in wild-type animals. The normal function of the wild-type allele of unc-4 may thus be to invoke the appropriate synaptic specificities to VA motor neurons produced in particular developmental contexts.     

C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains.

The induction of the hermaphrodite vulva and the migration of the sex myoblasts in the nematode Caenorhabditis elegans are both controlled by intercellular signalling. The gonadal anchor cell induces formation of the vulva from nearby hypodermal cells, and a set of somatic gonadal cells attract the migrating sex myoblasts to their final positions. Many genes required for vulval induction have been identified, including the let-23 receptor tyrosine kinase gene and the let-60 ras gene. We report here the identification and characterization of a new gene, sem-5 (sem, sex muscle abnormal), that acts both in vulval induction and in sex myoblast migration. On the basis of its DNA sequence, sem-5 encodes a novel 228-amino-acid protein which consists almost entirely of one SH2 (SH, src homology region) and two SH3 domains. SH2 and SH3 domains are present in many signalling proteins regulated by receptor and non-receptor tyrosine kinases. Mutations that impair sem-5 activity alter residues that are highly conserved among different SH2 and SH3 domains. Our results indicate that the sem-5 gene encodes a novel protein that functions in at least two distinct cell-signalling processes.     

Transposon-induced deletions in unc-22 of C. elegans associated with almost normal gene activity

The unc-22 gene of Caenorhabolitis elegans encodes a protein which is a component of the myosin-containing A-band of the worm's striated body-wall muscle. Among 51 revertants of a transposon-induced mutant, we have identified four which retain a barely detectable mutant phenotype. Molecular analysis shows that three of these have in-frame deletions of 1.0, 1.3 and 2.0 kilobases, whereas the fourth partial revertant and two other apparently complete revertants have small insertions. All these rearrangements involve coding sequence and, in the case of the deletions, result in polypeptides that are shorter than the wild-type protein. The region of the gene containing these rearrangements contains 10 copies of a motif recognized in other regions of the gene (our unpublished data). We suggest that one explanation for the minimally mutant phenotype associated with the deletions is that the size and the repeated nature of the unc-22 protein structure make it relatively tolerant of substitutions or deletions involving one or a small number of repeated motifs. These results could explain why in some human genetic diseases, such as Duchenne's muscular dystrophy, deletions can be associated with only mild forms of the disease.     

The Caenorhabditis elegans heterochronic gene lin-14 encodes a nuclear protein that forms a temporal developmental switch

During wild-type development, a protein product of the Caenorhabditis elegans heterochronic gene lin-14 is localized to nuclei of specific somatic cells in embryos and early larvae, but is absent in late larvae and adult soma. Gain-of-function lin-14 mutations cause the level of lin-14 protein to remain high throughout development, resulting in developmental reiterations of early cell lineages. The normal down-regulation of the lin-14 nuclear protein level encodes a temporal switch between early and late cell fates.     

The Caenorhabditis elegans lin-12 gene encodes a transmembrane protein with overall similarity to Drosophila Notch

The lin-12 gene seems to control certain binary decisions during Caenorhabditis elegans development, from genetic and anatomical studies of lin-12 mutants that have either elevated or reduced levels of lin-12 activity. We report here the complete DNA sequence of lin-12: 13.5 kilobases (kb) derived from genomic clones and 4.5 kb from complementary DNA clones. It is of interest that the predicted product is a putative transmembrane protein, given that many of the decisions controlled by lin-12 activity require cell-cell interactions for the correct choice of cell fate. In addition, the predicted lin-12 product may be classified into several regions, based on amino acid sequence similarities to other proteins. These include extensive overall sequence similarity to the Drosophila Notch protein, which also is involved in cell-cell interactions that specify cell fate; a repeated motif found in proteins encoded by the yeast cell-cycle control genes cdc10 (Schizosaccharomyces pombe) and SWI6 (Saccharomyces cerevisiae); and a repeated motif exemplified by epidermal growth factor, found in many mammalian proteins.     

SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer.

Non-clathrin coated vesicles have been implicated in early steps of intercompartmental transport. A distinct set of coat proteins are peripherally associated with the exterior of purified mammalian intra-Golgi transport vesicles. The 'coatomer', a cytosolic complex containing a similar subunit composition to and sharing at least one subunit (beta-COP) with the coat found on vesicles, has been postulated to be the precursor of this non-clathrin coat. Here we describe the characterization of SEC21, an essential gene required for protein transport from the endoplasmic reticulum to the Golgi in the yeast Saccharomyces cerevisiae. The 105K product of this gene, Sec21p, participates in a cytosolic complex that we show to be a yeast homologue of the mammalian coatomer. These observations demonstrate that a non-clathrin coat protein plays an essential role in intercompartmental transport.     

The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration.

Three dominant mutations of mec-4, a gene needed for mechanosensation, cause the touch-receptor neurons of Caenorhabditis elegans to degenerate. With deg-1, another C. elegans gene that can mutate to induce neuronal degeneration and that is similar in sequence, mec-4 defines a new gene family. Cross-hybridizing sequences are detectable in other species, raising the possibility that degenerative conditions in other organisms may be caused by mutations in similar genes. All three dominant mec-4 mutations affect the same amino acid. Effects of amino-acid substitutions at this position suggest that steric hindrance may induce the degenerative state.     

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

The mechanosensory protein MEC-6 is a subunit of the C. elegans touch-cell degenerin channel

Mechanosensory transduction in touch receptor neurons is believed to be mediated by DEG/ENaC (degenerin/epithelial Na+ channel) proteins in nematodes and mammals. In the nematode Caenorhabditis elegans, gain-of-function mutations in the degenerin genes mec-4 and mec-10 (denoted mec-4(d) and mec-10(d), respectively) cause degeneration of the touch cells. This phenotype is completely suppressed by mutation in a third gene, mec-6 (refs 3, 4), that is needed for touch sensitivity. This last gene is also required for the function of other degenerins. Here we show that mec-6 encodes a single-pass membrane-spanning protein with limited similarity to paraoxonases, which are implicated in human coronary heart disease. This gene is expressed in muscle cells and in many neurons, including the six touch receptor neurons. MEC-6 increases amiloride-sensitive Na+ currents produced by MEC-4(d)/MEC-10(d) by approximately 30-fold, and functions synergistically with MEC-2 (a stomatin-like protein that regulates MEC-4(d)/MEC-10(d) channel activity) to increase the currents by 200-fold. MEC-6 physically interacts with all three channel proteins. In vivo, MEC-6 co-localizes with MEC-4, and is required for punctate MEC-4 expression along touch-neuron processes. We propose that MEC-6 is a part of the degenerin channel complex that may mediate mechanotransduction in touch cells.     

Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir.

Human cytomegalovirus (HCMV, a betaherpes virus) is the cause of serious disease in immunologically compromised individuals, including those with acquired immunodeficiency syndrome. One of the compounds used in the chemotherapy of HCMV infections is the nucleoside analogue 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (ganciclovir). The mechanism of action of this drug is dependent on the formation of the nucleoside triphosphate, which is a strong inhibitor of the viral DNA polymerase. Thymidine kinase, which is encoded by many of the herpesviruses, catalyses the initial phosphorylation of ganciclovir. But there is no evidence for the coding of this enzyme by HCMV, and DNA sequence analysis of the HCMV genome has shown that there is no open reading frame characteristic of a herpesvirus thymidine kinase. Here we present biochemical and immunological evidence that the HCMV UL97 open reading frame codes for a protein capable of phosphorylating ganciclovir. This protein seems to be responsible for the selectivity of ganciclovir and will be useful tool in the understanding and refinement of the antiviral activity of new selective anti-HCMV compounds.     

The spread of Na+ spikes determines the pattern of dendritic Ca2+ entry into hippocampal neurons.

The dendrites of many types of neurons contain voltage-dependent Na+ and Ca2+ conductances that generate action potentials (see ref. 1 for review). The function of these spikes is not well understood, but the Ca2+ entry stimulated by spikes probably affects Ca(2+)-dependent processes in dendrites. These include synaptic plasticity, cytotoxicity and exocytosis. Several lines of evidence suggest that dendritic spikes occur within subregions of the dendrites. To study the mechanism that govern the spread of spikes in the dendrites of hippocampal pyramidal cells, we imaged Ca2+ entry with Fura-2 (ref. 9) and Na+ entry with a newly developed Na(+)-sensitive dye. Our results indicate that Ca2+ entry into dendrites is triggered by Na+ spikes that actively invade the dendrites. The restricted spatial distribution of Ca2+ entry seems to depend on the spread of Na+ spikes in the dendrites, rather than on a limited distribution of Ca2+ channels. In addition, we have observed an activity-dependent process that modulates the invasion of spikes into the dendrites and progressively restricts Ca2+ entry to more proximal dendritic regions.     

The let-23 gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily

The let-23 gene is required for induction of the Caenorhabditis elegans vulva. It is shown that let-23 encodes a putative tyrosine kinase of the epidermal growth factor receptor subfamily. Thus, let-23 might encode the receptor for the inductive signal required for vulval development. Because let-23 acts upstream of let-60 ras in the vulval determination pathway, the identification of the let-23 product provides support for a link in vivo between tyrosine kinase growth factor receptors and ras proteins in a pathway of cell-type determination.     

Cholecystokinin induces a decrease in Ca2+ current in snail neurons that appears to be mediated by protein kinase C

Three distinct classes of protein kinases have been shown to regulate Ca2+ current in excitable tissues. Cyclic AMP-dependent protein kinase mediates the action of noradrenaline on the Ca2+ current of cardiac muscle cells. Cyclic GMP-dependent protein kinase mediates the serotonin-induced modulation of the Ca2+ current in identified snail neurons. The Ca2+/diacylglycerol-dependent protein kinase (protein kinase C) has also been found to regulate Ca2+ currents of neurons. However, no neurotransmitter has yet been shown to regulate Ca2+ current through the activation of protein kinase C. We now report that cholecystokinin, a widely occurring neuropeptide which is present in molluscan neuron, modulates the Ca2+ current in identified neurons of the snail Helix aspersa, and that this effect appears to be mediated by protein kinase C. Specifically, sulphated cholecystokinin octapeptide 26-33 (CCK8), activators of protein kinase C, and intracellular injection of protein kinase C, all shorten the Ca2+-dependent action potential and decrease the amplitude of the Ca2+ current in these cells. All these effects are not reversible within the duration of the experiments. Moreover, intracellular injections of low concentrations of protein kinase C, which are ineffective by themselves, enhance the effectiveness of low concentrations of CCK8 on the Ca2+ current.     

The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans.

At synaptic junctions, specialized subcellular structures occur in both pre- and postsynaptic cells. Most presynaptic termini contain electron-dense membrane structures, often referred to as active zones, which function in vesicle docking and release. The components of those active zones and how they are formed are largely unknown. We report here that a mutation in the Caenorhabditis elegans syd-2 (for synapse-defective) gene causes a diffused localization of several presynaptic proteins and of a synaptic-vesicle membrane associated green fluorescent protein (GFP) marker. Ultrastructural analysis revealed that the active zones of syd-2 mutants were significantly lengthened, whereas the total number of vesicles per synapse and the number of vesicles at the prominent active zones were comparable to those in wild-type animals. Synaptic transmission is partially impaired in syd-2 mutants. syd-2 encodes a member of the liprin (for LAR-interacting protein) family of proteins which interact with LAR-type (for leukocyte common antigen related) receptor proteins with tyrosine phosphatase activity (RPTPs). SYD-2 protein is localized at presynaptic termini independently of the presence of vesicles, and functions cell autonomously. We propose that SYD-2 regulates the differentiation of presynaptic termini in particular the formation of the active zone, by acting as an intracellular anchor for RPTP signalling at synaptic junctions.