A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans

Clustering neurotransmitter receptors at the synapse is crucial for efficient neurotransmission. Here we identify a Caenorhabditis elegans locus, lev-10, required for postsynaptic aggregation of ionotropic acetylcholine receptors (AChRs). lev-10 mutants were identified on the basis of weak resistance to the anthelminthic drug levamisole, a nematode-specific cholinergic agonist that activates AChRs present at neuromuscular junctions (NMJs) resulting in muscle hypercontraction and death at high concentrations. In lev-10 mutants, the density of levamisole-sensitive AChRs at NMJs is markedly reduced, yet the number of functional AChRs present at the muscle cell surface remains unchanged. LEV-10 is a transmembrane protein localized to cholinergic NMJs and required in body-wall muscles for AChR clustering. We also show that the LEV-10 extracellular region, containing five predicted CUB domains and one LDLa domain, is sufficient to rescue AChR aggregation in lev-10 mutants. This suggests a mechanism for AChR clustering that relies on extracellular protein-protein interactions. Such a mechanism is likely to be evolutionarily conserved because CUB/LDL transmembrane proteins similar to LEV-10, but lacking any assigned function, are expressed in the mammalian nervous system and might be used to cluster ionotropic receptors in vertebrates.     

Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo

Cell divisions that create daughter cells of different sizes are crucial for the generation of cell diversity during animal development. In such asymmetric divisions, the mitotic spindle must be asymmetrically positioned at the end of anaphase. The mechanisms by which cell polarity translates to asymmetric spindle positioning remain unclear. Here we examine the nature of the forces governing asymmetric spindle positioning in the single-cell-stage Caenorhabditis elegans embryo. To reveal the forces that act on each spindle pole, we removed the central spindle in living embryos either physically with an ultraviolet laser microbeam, or genetically by RNA-mediated interference of a kinesin. We show that pulling forces external to the spindle act on the two spindle poles. A stronger net force acts on the posterior pole, thereby explaining the overall posterior displacement seen in wild-type embryos. We also show that the net force acting on each spindle pole is under control of the par genes that are required for cell polarity along the anterior-posterior embryonic axis. Finally, we discuss simple mathematical models that describe the main features of spindle pole behaviour. Our work suggests a mechanism for generating asymmetry in spindle positioning by varying the net pulling force that acts on each spindle pole, thus allowing for the generation of daughter cells with different sizes.     

Centriole assembly in Caenorhabditis elegans

Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.     

A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans

Messenger RNA regulation is a critical mode of controlling gene expression. Regulation of mRNA stability and translation is linked to controls of poly(A) tail length. Poly(A) lengthening can stabilize and translationally activate mRNAs, whereas poly(A) removal can trigger degradation and translational repression. Germline granules (for example, polar granules in flies, P granules in worms) are ribonucleoprotein particles implicated in translational control. Here we report that the Caenorhabditis elegans gene gld-2, a regulator of mitosis/meiosis decision and other germline events, encodes the catalytic moiety of a cytoplasmic poly(A) polymerase (PAP) that is associated with P granules in early embryos. Importantly, the GLD-2 protein sequence has diverged substantially from that of conventional eukaryotic PAPs, and lacks a recognizable RRM (RNA recognition motif)-like domain. GLD-2 has little PAP activity on its own, but is stimulated in vitro by GLD-3. GLD-3 is also a developmental regulator, and belongs to the Bicaudal-C family of RNA binding proteins. We suggest that GLD-2 is the prototype for a class of regulatory cytoplasmic PAPs that are recruited to specific mRNAs by a binding partner, thereby targeting those mRNAs for polyadenylation and increased expression.     

Carboxy-terminal truncation activates glp-1 protein to specify vulval fates in Caenorhabditis elegans

The glp-1 and lin-12 genes encode homologous transmembrane proteins that may act as receptors for cell interactions during development. The glp-1 product is required for induction of germ-line proliferation and for embryogenesis. By contrast, lin-12 mediates somatic cell interactions, including those between the precursor cells that form the vulval hypodermis (VPCs). Here we analyse an unusual allele of glp-1, glp-1(q35), which displays a semidominant multivulva phenotype (Muv), as well as the typical recessive, loss-of-function Glp phenotypes (sterility and embryonic lethality). We find that the effects of glp-1(q35) on VPC development mimic those of dominant lin-12 mutations, even in the absence of lin-12 activity. The glp-1(q35) gene bears a nonsense mutation predicted to eliminate the 122 C-terminal amino acids, including a ProGluSerThr (PEST) sequence thought to destabilize proteins. We suggest that the carboxy terminus bears a negative regulatory domain which normally inactivates glp-1 in the VPCs. We propose that inappropriate glp-1(q35) activity can substitute for lin-12 to determine vulval fate, perhaps by driving the VPCs to proliferate.     

A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans

How left/right functional asymmetry is layered on top of an anatomically symmetrical nervous system is poorly understood. In the nematode Caenorhabditis elegans, two morphologically bilateral taste receptor neurons, ASE left (ASEL) and ASE right (ASER), display a left/right asymmetrical expression pattern of putative chemoreceptor genes that correlates with a diversification of chemosensory specificities. Here we show that a previously undefined microRNA termed lsy-6 controls this neuronal left/right asymmetry of chemosensory receptor expression. lsy-6 mutants that we retrieved from a genetic screen for defects in neuronal left/right asymmetry display a loss of the ASEL-specific chemoreceptor expression profile with a concomitant gain of the ASER-specific profile. A lsy-6 reporter gene construct is expressed in less than ten neurons including ASEL, but not ASER. lsy-6 exerts its effects on ASEL through repression of cog-1, an Nkx-type homeobox gene, which contains a lsy-6 complementary site in its 3' untranslated region and that has been shown to control ASE-specific chemoreceptor expression profiles. lsy-6 is the first microRNA to our knowledge with a role in neuronal patterning, providing new insights into left/right axis formation.     

Caenorhabditis elegans gene ced-9 protects cells from programmed cell death.

The gene ced-9 of the nematode Caenorhabditis elegans acts to protect cells from programmed cell death. A mutation that abnormally activates ced-9 prevents the cell deaths that occur during normal C. elegans development. Conversely, mutations that inactivate ced-9 cause cells that normally live to undergo programmed cell death; these mutations result in embryonic lethality, indicating that ced-9 function is essential for development. The ced-9 gene functions by negatively regulating the activities of other genes that are required for the process of programmed cell death.     

MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans

The germ line is an immortal cell lineage that is passed indefinitely from one generation to the next. To identify the genes that are required for germline immortality, we isolated Caenorhabditis elegans mutants with mortal germ lines--worms that can reproduce for several healthy generations but eventually become sterile. One of these mortal germline (mrt) mutants, mrt-2, exhibits progressive telomere shortening and accumulates end-to-end chromosome fusions in later generations, indicating that the MRT-2 protein is required for telomere replication. In addition, the germ line of mrt-2 is hypersensitive to X-rays and to transposon activity. Therefore, mrt-2 has defects in responding both to damaged DNA and to normal double-strand breaks present at telomeres. mrt-2 encodes a homologue of a checkpoint gene that is required to sense DNA damage in yeast. These results indicate that telomeres may be identified as a type of DNA damage and then repaired by the telomere-replication enzyme telomerase.     

Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans

Chromosomes are divided into domains of open chromatin, where genes have the potential to be expressed, and domains of closed chromatin, where genes are not expressed. Classic examples of open chromatin domains include 'puffs' on polytene chromosomes in Drosophila and extended loops from lampbrush chromosomes. If multiple genes were typically expressed together from a single open chromatin domain, the position of co-expressed genes along the chromosomes would appear clustered. To investigate whether co-expressed genes are clustered, we examined the chromosomal positions of the genes expressed in the muscle of Caenorhabditis elegans at the first larval stage. Here we show that co-expressed genes in C. elegans are clustered in groups of 2-5 along the chromosomes, suggesting that expression from a chromatin domain can extend over several genes. These observations reveal a higher-order organization of the structure of the genome, in which the order of the genes along the chromosome id correlated with their expression in specific tissues.     

Induction of gut in Caenorhabditis elegans embryos.

Two types of developmental events can cause an embryonic cell to adopt a fate different from that of its neighbours: during a cell division particular contents may be segregated to only one daughter cell and cells may experience different external cues, commonly in the form of inductive cell interactions. Work on development in the nematode Caenorhabditis elegans suggests that most cell fates are specified without a need for cell interactions. In particular, the gut cell lineage of C. elegans has been used as a primary example of specification by differential segregation of determinants. Here I re-examine the role of induction in gut specification by isolating early blastomeres. In C. elegans, the gut derives from all the progeny of a single blastomere (E) of the eight-cell stage. When a gut precursor cell (EMS) is isolated during the first half of the four-cell stage, gut does not differentiate. Gut differentiation is rescued by recombining EMS with its posterior neighbour (P2), but not by recombining EMS with one or both of the other two cells of the four-cell stage. These results demonstrate that P2 induces EMS to form gut in C. elegans.     

Lateral inhibition during vulval induction in Caenorhabditis elegans

During Caenorhabditis elegans vulval induction the anchor cell of the gonad specifies a spatial pattern of three cell types among a set of six multipotent epidermal cells, the vulval precursor cells (VPCs). Previous studies suggested that the anchor cell produces a graded inductive signal which can directly stimulate VPCs away from a ground state (type 3) to become type 1 or type 2 depending on their distance from the anchor cell. Here, we investigate the interactions among VPCs in a mutant, lin-15, in which VPC fates are rendered partially independent of the inductive signal, and show that type 1 cells actively inhibit adjacent cells from also becoming type 1 cells. The fate of each VPC therefore depends on the combined action of two intercellular signals: a graded inductive signal from the anchor cell, and a lateral inhibitory signal from at least some of its neighbours. Pattern formation among the VPCs lin-15 mutant is analogous to the establishment of the pattern of neuroblasts and dermatoblasts during early insect neurogenesis, suggesting that the similarities in inferred molecular structure of the lin-12 and Notch gene products, which are involved in these two instances of pattern formation, might extend to similarities in function.     

Somatic support cells restrict germline stem cell self-renewal and promote differentiation

Stem cells maintain populations of highly differentiated, short-lived cell-types, including blood, skin and sperm, throughout adult life. Understanding the mechanisms that regulate stem cell behaviour is crucial for realizing their potential in regenerative medicine. A fundamental characteristic of stem cells is their capacity for asymmetric division: daughter cells either retain stem cell identity or initiate differentiation. However, stem cells are also capable of symmetric division where both daughters remain stem cells, indicating that mechanisms must exist to balance self-renewal capacity with differentiation. Here we present evidence that support cells surrounding the stem cells restrict self-renewal and control stem cell number by ensuring asymmetric division. Loss of function of the Drosophila Epidermal growth factor receptor in somatic cells disrupted the balance of self-renewal versus differentiation in the male germline, increasing the number of germline stem cells. We propose that activation of this receptor specifies normal behaviour of somatic support cells; in turn, the somatic cells play a guardian role, providing information that prevents self-renewal of stem cell identity by the germ cell they enclose.     

Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans

Chromatin modifiers regulate lifespan in several organisms, raising the question of whether changes in chromatin states in the parental generation could be incompletely reprogrammed in the next generation and thereby affect the lifespan of descendants. The histone H3 lysine 4 trimethylation (H3K4me3) complex, composed of ASH-2, WDR-5 and the histone methyltransferase SET-2, regulates Caenorhabditis elegans lifespan. Here we show that deficiencies in the H3K4me3 chromatin modifiers ASH-2, WDR-5 or SET-2 in the parental generation extend the lifespan of descendants up until the third generation. The transgenerational inheritance of lifespan extension by members of the ASH-2 complex is dependent on the H3K4me3 demethylase RBR-2, and requires the presence of a functioning germline in the descendants. Transgenerational inheritance of lifespan is specific for the H3K4me3 methylation complex and is associated with epigenetic changes in gene expression. Thus, manipulation of specific chromatin modifiers only in parents can induce an epigenetic memory of longevity in descendants.     

An antidepressant that extends lifespan in adult Caenorhabditis elegans

The mechanisms that determine the lifespan of an organism are still largely a mystery. One goal of ageing research is to find drugs that would increase lifespan and vitality when given to an adult animal. To this end, we tested 88,000 chemicals for the ability to extend the lifespan of adult Caenorhabditis elegans nematodes. Here we report that a drug used as an antidepressant in humans increases C. elegans lifespan. In humans, this drug blocks neural signalling by the neurotransmitter serotonin. In C. elegans, the effect of the drug on lifespan is reduced or eradicated by mutations that affect serotonin synthesis, serotonin re-uptake at synapses, or either of two G-protein-coupled receptors: one that recognizes serotonin and the other that detects another neurotransmitter, octopamine. In vitro studies show that the drug acts as an antagonist at both receptors. Testing of the drug on dietary-restricted animals or animals with mutations that affect lifespan indicates that its effect on lifespan involves mechanisms associated with lifespan extension by dietary restriction. These studies indicate that lifespan can be extended by blocking certain types of neurotransmission implicated in food sensing in the adult animal, possibly leading to a state of perceived, although not real, starvation.     

A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans

In many organisms, introducing double-stranded RNA (dsRNA) causes the degradation of messenger RNA that is homologous to the trigger dsRNA--a process known as RNA interference. The dsRNA is cleaved into short interfering RNAs (siRNAs), which hybridize to homologous mRNAs and induce their degradation. dsRNAs vary in their ability to trigger RNA interference: many mRNA-targeting dsRNAs show weak phenotypes, and nearly all mRNAs of the Caenorhabditis elegans nervous system are refractory to RNA interference. C. elegans eri-1 was identified in a genetic screen for mutants with enhanced sensitivity to dsRNAs. Here we show that eri-1 encodes an evolutionarily conserved protein with domains homologous to nucleic-acid-binding and exonuclease proteins. After exposure to dsRNA or siRNAs, animals with eri-1 mutations accumulate more siRNAs than do wild-type animals. C. elegans ERI-1 and its human orthologue degrade siRNAs in vitro. In the nematode worm, ERI-1 is predominantly cytoplasmic and is expressed most highly in the gonad and a subset of neurons, suggesting that ERI-1 siRNase activity suppresses RNA interference more intensely in these tissues. Thus, ERI-1 is a negative regulator that may normally function to limit the duration, cell-type specificity or endogenous functions of RNA interference.     

A blend of small molecules regulates both mating and development in Caenorhabditis elegans

In many organisms, population-density sensing and sexual attraction rely on small-molecule-based signalling systems. In the nematode Caenorhabditis elegans, population density is monitored through specific glycosides of the dideoxysugar ascarylose (the 'ascarosides') that promote entry into an alternative larval stage, the non-feeding and highly persistent dauer stage. In addition, adult C. elegans males are attracted to hermaphrodites by a previously unidentified small-molecule signal. Here we show, by means of combinatorial activity-guided fractionation of the C. elegans metabolome, that the mating signal consists of a synergistic blend of three dauer-inducing ascarosides, which we call ascr#2, ascr#3 and ascr#4. This blend of ascarosides acts as a potent male attractant at very low concentrations, whereas at the higher concentrations required for dauer formation the compounds no longer attract males and instead deter hermaphrodites. The ascarosides ascr#2 and ascr#3 carry different, but overlapping, information, as ascr#3 is more potent as a male attractant than ascr#2, whereas ascr#2 is slightly more potent than ascr#3 in promoting dauer formation. We demonstrate that ascr#2, ascr#3 and ascr#4 are strongly synergistic, and that two types of neuron, the amphid single-ciliated sensory neuron type K (ASK) and the male-specific cephalic companion neuron (CEM), are required for male attraction by ascr#3. On the basis of these results, male attraction and dauer formation in C. elegans appear as alternative behavioural responses to a common set of signalling molecules. The ascaroside signalling system thus connects reproductive and developmental pathways and represents a unique example of structure- and concentration-dependent differential activity of signalling molecules.     

A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) is a major pathogen in immunosuppressed individuals, including patients with acquired immune deficiency syndrome. The nucleoside analogue ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)-guanine) is one of the few drugs available to treat HCMV infections, but resistant virus is a growing problem in the clinic and there is a critical need for new drugs. The study of ganciclovir-resistant mutants has indicated that the selective action of ganciclovir depends largely on virus-controlled phosphorylation in HCMV-infected cells. The enzyme(s) responsible have not been identified. Here we report that the HCMV gene UL97, whose predicted product shares regions of homology with protein kinases, guanylyl cyclase and bacterial phosphotransferases, controls phosphorylation of ganciclovir in HCMV-infected cells. A four-amino-acid deletion of UL97 in a conserved region, which in cyclic AMP-dependent protein kinase participates in substrate recognition, causes impaired ganciclovir phosphorylation. The implications of these results for antiviral drug development and drug resistance are discussed.