A nervous system-specific subnuclear organelle in Caenorhabditis elegans.

We describe here phase-separated subnuclear organelles in the nematode Caenorhabditis elegans, which we term NUN (NUclear Nervous system-specific) bodies. Unlike other previously described subnuclear organelles, NUN bodies are highly cell type specific. In fully mature animals, 4-10 NUN bodies are observed exclusively in the nucleus of neuronal, glial and neuron-like cells, but not in other somatic cell types. Based on co-localization and genetic loss of function studies, NUN bodies are not related to other previously described subnuclear organelles, such as nucleoli, splicing speckles, paraspeckles, Polycomb bodies, promyelocytic leukemia bodies, gems, stress-induced nuclear bodies, or clastosomes. NUN bodies form immediately after cell cycle exit, before other signs of overt neuronal differentiation and are unaffected by the genetic elimination of transcription factors that control many other aspects of neuronal identity. In one unusual neuron class, the canal-associated neurons, NUN bodies remodel during larval development, and this remodeling depends on the Prd-type homeobox gene ceh-10. In conclusion, we have characterized here a novel subnuclear organelle whose cell type specificity poses the intriguing question of what biochemical process in the nucleus makes all nervous system-associated cells different from cells outside the nervous system.

Mapping functional domains of chloride intracellular channel (CLIC) proteins in vivo.

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A<70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the ''PTM'' domain, named after an amphipathic putative transmembrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.

Caenorhabditis elegans ABL-1 antagonizes p53-mediated germline apoptosis after ionizing irradiation.

c-Abl, a conserved nonreceptor tyrosine kinase, integrates genotoxic stress responses, acting as a transducer of both pro- and antiapoptotic effector pathways. Nuclear c-Abl seems to interact with the p53 homolog p73 to elicit apoptosis. Although several observations suggest that cytoplasmic localization of c-Abl is required for antiapoptotic function, the signals that mediate its antiapoptotic effect are largely unknown. Here we show that worms carrying an abl-1 deletion allele, abl-1(ok171), are specifically hypersensitive to radiation-induced apoptosis in the Caenorhabditis elegans germ line. Our findings delineate an apoptotic pathway antagonized by ABL-1, which requires sequentially the cell cycle checkpoint genes clk-2, hus-1 and mrt-2; the C. elegans p53 homolog, cep-1; and the genes encoding the components of the conserved apoptotic machinery, ced-3, ced-9 and egl-1. ABL-1 does not antagonize germline apoptosis induced by the DNA-alkylating agent ethylnitrosourea. Furthermore, worms treated with the c-Abl inhibitor STI-571 (Gleevec; used in human cancer therapy), two newly synthesized STI-571 variants or PD166326 had a phenotype similar to that generated by abl-1(ok171). These studies indicate that ABL-1 distinguishes proapoptotic signals triggered by two different DNA-damaging agents and suggest that C. elegans might provide tissue models for development of anticancer drugs.

A conserved postsynaptic transmembrane protein affecting neuromuscular signaling in Caenorhabditis elegans.

For a motor unit to function, neurons and muscle cells need to adopt their correct cell fate, form appropriate cellular contacts, and assemble a specific repertoire of signaling proteins into presynaptic and postsynaptic structures. In the nematode Caenorhabditis elegans, a disruption of any of these steps causes uncoordinated locomotory behavior (unc phenotype). We report here the positional cloning of a new unc gene, unc-122, which we show by mosaic analysis and tissue-specific rescue experiments to act in muscle to affect locomotory behavior. unc-122 codes for a phylogenetically conserved type II transmembrane protein with collagen repeats and a cysteine-rich olfactomedin domain. Together with uncharacterized proteins in C. elegans, Drosophila, and vertebrates, UNC-122 defines a novel family of proteins that we term "Colmedins." UNC-122 protein is expressed exclusively in muscle and coelomocytes and localizes to the postsynaptic surface of GABAergic and cholinergic neuromuscular junctions (NMJs). Presynaptic and postsynaptic structures are present and properly aligned in unc-122 mutant animals, yet the animals display neurotransmission defects characterized by an altered sensitivity toward drugs that interfere with cholinergic signaling. Moreover, unc-122 mutant animals display anatomical defects in motor axons that are likely a secondary consequence of neurotransmission defects. Both the neuroanatomical and locomotory defects worsen progressively during the life of an animal, consistent with a role of unc-122 in acute signaling at the NMJ. On the basis of motifs in the UNC-122 protein sequence that are characteristic of extracellular matrix proteins, we propose that UNC-122 is involved in maintaining a structural microenvironment that allows efficient neuromuscular signaling.

A C. elegans CLIC-like protein required for intracellular tube formation and maintenance.

The Caenorhabditis elegans excretory canal is composed of a single elongated and branched cell that is tunneled by an inner lumen of apical character. Loss of the exc-4 gene causes a cystic enlargement of this intracellular tube. exc-4 encodes a member of the chloride intracellular channel (CLIC) family of proteins. EXC-4 protein localizes to various tubular membranes in distinct cell types, including the lumenal membrane of the excretory tubes. A conserved 55-amino acid domain enables EXC-4 translocation from the cytosol to the lumenal membrane. The tubular architecture of this membrane requires EXC-4 for both its formation and maintenance.

Heparan sulfate proteoglycan-dependent induction of axon branching and axon misrouting by the Kallmann syndrome gene kal-1.

Kallmann syndrome is a neurological disorder characterized by various behavioral and neuroanatomical defects. The X-linked form of this disease is caused by mutations in the KAL-1 gene, which codes for a secreted molecule that is expressed in restricted regions of the brain. Its molecular mechanism of action has thus far remained largely elusive. We show here that expression of the Caenorhabditis elegans homolog of KAL-1 in selected sensory and interneuron classes causes a highly penetrant, dosage-dependent, and cell autonomous axon-branching phenotype. In a different cellular context, heterologous C. elegans kal-1 expression causes a highly penetrant axon-misrouting phenotype. The axon-branching and -misrouting activities require different domains of the KAL-1 protein. In a genetic modifier screen we isolated several loci that either suppress or enhance the kal-1-induced axonal defects, one of which codes for an enzyme that modifies specific residues in heparan sulfate proteoglycans, namely heparan-6O-sulfotransferase. We hypothesize that KAL-1 binds by means of a heparan sulfate proteoglycan to its cognate receptor or other extracellular cues to induce axonal branching and axon misrouting.