Han M et al. (1991) Genes Dev "Analysis of dominant-negative mutations of the Caenorhabditis elegans let-60 ras gene."

Sternberg PW, Han M

[Genes Dev, 1991]

The let-60 gene of Caenorhabditis elegans controls the choice between vulval and hypodermal differentiation in response to an inductive signal from the gonad. let-60 encodes a ras protein that acts downstream of the let-23 receptor tyrosine kinase in a signal transduction pathway. Dominant-negative mutations of let-60 [let-60(dn)] cause a reduction of the gene activity in let-60(dn)/+ heterozygotes and a vulva-less mutant phenotype. We have found that nine let-60(dn) mutations cause replacements of conserved residues. Four are in two novel positions; others are in positions known previously to cause dominant-negative mutations in mammalian cells. The locations of these lesions suggest that they disrupt the ability of the ras protein to bind guanine nucleotides. Four let-60(dn) mutant genes were introduced into wild-type animals in the form of extrachromosomal arrays and were found to generate three dominant phenotypes--lethality, vulva-less, or multivulva--depending on gene dose and alleles. The dominant lethality caused by high-dose transgenic let-60(dn) genes suggests a toxic effect of these mutant genes in early development. The dominant-negative effects of these mutations in heterozygotes are likely to be caused by competition between let-60(dn) and let-60(+) protein for a positive regulator. All let-60(dn) mutations interfere with let-60(+) activity, but some alleles have partial constitutive activity, suggesting that the ability to interact with the activator is separable from the ability to exert a physiological effect (stimulation of vulval differentiation). These dn mutations might be useful for interfering with ras-mediated signal transduction pathways in other multicellular organisms.

Trivedi, S. et al. (2019) International Worm Meeting "Polymethoxyflavone 5-demethylnobiletin promotes DR induced autophagy dependent longevity in Caenorhabditis elegans."

Pandey, R., Trivedi, S.

[International Worm Meeting, 2019]

Aging is a global concern that brings formidable socioeconomic and healthcare challenges. It is characterized as accumulation of deleterious changes in organelles, cells and tissues with expanding age that collectively responsible for the risk of several age associated diseases. In the last decade, various studies have shown evidences towards the utilization of plant derived molecules against aging and various neurological diseases. Here we report the identification of the polymethoxyflavone, 5-demethylnobiletin (DN) as a natural compound with anti-aging potential. We have utilized Caenorhabditis elegans as the model system to study anti-aging effects of DN in vivo (life span, lipofuscin accumulation, oxidative stress resistance, ROS levels lipid levels, pharyngeal pumping, reproductive span and total brood size) and pathway involved in underlying mechanism. DN is isolated from gum exudates of Gardenia lucida, which is known to possess rich anti-oxidant activity. We show that external administration of DN extends the lifespan and oxidative stress tolerance against artificial ROS inducer juglone, in a dose dependent manner. The studies revealed that lifespan extension is arbitrated through decreased levels of ROS (DCF-DA method), lipid levels (Nile red staining) and intestinal lipofuscin levels in both young adult and aged worms upon low dose administration of DN. Additionally, DN also reduces the pharyngeal pumping and enhances the reproductive span of worms. The interaction of DN with genes required for lifespan extension is deciphered by studying effect of DN on gene mutants and gene expression of these genes. DN activates dietary restriction (DR) pathway in nematodes mediated by pha-4, sir-2.1, skn-1 and eat-2 genes. We also show that DR induced lifespan requires the activation of autophagy genes viz. bec-1, unc-51 and lgg-1. This induction of both processes is fully required for lifespan extension in the worm. Moreover, mutant analysis is further validated by gene reporter assay (DA2123, LGG-1::GFP; SM481, PHA-4::GFP and LD1, SKN-1::GFP) and qPCR studies of relevant genes. Altogether, DN can enhance the lifespan of C. elegans via DR pathway which also requires the induction of autophagy mechanism.

Trivedi S et al. (2017) Neurosci Lett "5-Desmethylnobiletin augments synaptic ACh levels and nicotinic ACh receptor activity: a potential candidate for alleviation of cholinergic dysfunction."

Sammi SR, Pandey R, Gupta MM, Trivedi S, Maurya P

[Neurosci Lett, 2017]

Cholinergic function is compromised in plethora of neurodegenerative disorders especially Alzheimer's disease. Increasing acetylcholine (ACh) levels has been the mainstay in majority of the therapeutic regimens, accepted for management of disease. The present study investigates the efficacy of 5-Desmethylnobiletin (DN), a polymethoxyflavone in augmenting cholinergic function using Caenorhabditis elegans as a model organism. The studies revealed significant elevation in cholinergic transmission mediated through increased levels of ACh and activity of nicotinic acetylcholine receptors (nAChR). Further investigation in to the mechanistic aspects indicated that DN enhanced cholinergic function through down modulation of acetylcholinesterase activity at enzyme and transcript level along with upregulation of non alpha subunit, unc-29 which could be linked with enhanced nAChR activity as evident from levamisole assay. Additionally, studies on antioxidant properties, implicated significant potential of DN in curtailing ROS, both in vivo and in vitro. Our studies present DN as a phytomolecule with novel biological activities which could be exploited and researched upon for therapeutic avenues in terms of cholinergic function and antioxidant potential.

Gu T et al. (1995) International C. elegans Meeting "Suppressors of let-60 Dominant Negative Mutations"

Gu T, Han M

[International C. elegans Meeting, 1995]

A Ras mediated signal transduction pathway plays a key role in a genetic pathway that specifies the vulva cell fate. Each component acting in the pathway is likely to be regulated positively as well as negatively by other factors. To identify new negative regulators downstream of let-60 ras, we have screened and isolated suppressors that revert the Vul phenotype caused by a let-60 ras dominant-negative (dn) mutation. Seven different genes are identified from fourteen recessive extragenic suppressors. Of those seven genes, four are located on LGX, one is on LGII, one is on LGIII, and one is yet to be mapped to a linkage group. Phenotypic characterization of these suppressors show that the Vul phenotype caused by let-60(dn) is reverted from 97% to less than 20%, while VPC percent induction is elevated from 32% to greater than 74%. We are currently conducting further genetic analysis of ku105, one of the four alleles defining a gene on X chromosome. The suppression of let-60(dn) by ku105 is not allele specific since it can suppress two different let-60(dn) alleles: sy94, and sy101. ku105 homozygote suppresses let-60(dn)/+ better than ku105/uDf1, indicating that ku105 is possibly a recessive antimorph. We are currently cloing as well as conducting various genetic experiments on ku105.

Han M et al. (1991) International C. elegans Meeting "ANALYSIS OF DOMINANT NEGATIVE MUTATIONS OF let-60: MUTIPLE RAS MOLECULES MAY FUNCTION IN A COMPLEX"

Han M, Sternberg PW

[International C. elegans Meeting, 1991]

Genetic analysis of the let-60 gene has shown that its activity controls the decision between vulval and hypodermal cell fates for the six vulval precursor cells (P3.p-P8.p, or VPCs). The activity of let- 60, which normally is subject to the regulation by the inductive signal from anchor cell, can be forced to constitutively high or constitutively low levels by mutations within lel-60 gene or by misexpression of ectopic wild type gene. Iet-60 encodes a protein highly similar to mammalian ras proteins. Iet-60 appears to act after lin-3 (growth factor like molecule) and let-23 (EGF receptor like molecule) but before lin-l in the signal transduction pathway. Dominant negative mutations of let-60 [let-60(dn)] cause a dominant vulvaless phenotype, and all but one allele (sy93) also cause a recessive lethal phenotype. Determination of the molecular lesions of these let-60(dn) mutations, as well as of two intragenic revertants suggests that all the let-60(dn) mutations likely inactivate the protein by disrupting the binding ability of the ras protein to guanine nucleotides. Further genetic and molecular analysis of let-60(dn) has been carried out to study the mechanism of the dominant negative effect and the mechanism of ras protein function in the pathways. Four of the let- 60(dn mutant genes were introduced back into hermaphrodites in the form of extrachromosomal arrays. Our results suggest that: (l) the dominant negative effect is comman to all let-60-mediated signal transduction pathways, and the pathway involved in vulval development is more sensitive to alteration of let-60 activity than is the pathway( s) during early larval growth; (2) let-60(dn) mutant proteins may interfere with wild type ras activity prior to formation of the ras- GTP complex; (3) let-60 ras protein may interact with each other directly or indirectly to form a complex containing multiple ras molecules. The dominant negative effect may be caused by inactivation of a protein complex containing both let60(dn) and let-60(+) proteins.

Gu T et al. (1995) Worm Breeder's Gazette "Suppressors of let-60 Dominant Negative Mutations."

Gu T, Han M

[Worm Breeder's Gazette, 1995]

Suppressors of let-60 Dominant Negative Mutations Trent Gu and Min Han Department of MCD Biology, University of Colorado, Boulder The gene let-60 ras plays a key role in a genetic pathway that specifies the vulva cell fate. Loss of function (lf) mutations of let-60 cause Vul (and therefore is egg laying defective), in which all of the VPCs take on the hyperdermal cell fate. Hyperactive mutations or gain of function (gf) of let-60 cause Muv, in which more than three VPCs are differentiated to become vulva cells. Ras is used to transmit cell growth signals in many organisms from yeast to human. Studying the role of let-60 ras pathway in C. elegans will help in understanding the Ras signal transduction pathway in other organisms. How the lef-60 ras pathway is negatively controlled is not well understood. For example, we don't know how the kinases downstream of let-60 ras are kept inactive. To isolate the negative regulators downstream of let-60 ras, we have screened and isolated suppressors that revert the Vul phenotype caused by a let-60 ras dominant-negative (dn) mutation. Seven different genes are identified from fourteen recessive extragenic suppressors. The preliminary mapping and complementation results of nine recessive extragenic suppressors are shown below The suppression of let-60 (dn) by ku105 is not allele specific since ku105 can suppress three different let-60 (dn) alleles: sy94, syl01,and syl00. ku105 homozygous suppress let-60(dn) better than ku105/Df, indicating that ku105 is possibly a recessive antimorph. Since previous genetic study suggests that the let-60 ras (dn) mutation decreases the pathway's activity (thus leading to the vulvaless phenotype), potential suppressors are expected to increase or restore the normal activity of the pathway.

Broadbent ID et al. (2002) Curr Biol "The C. elegans hmr-1 gene can encode a neuronal classic cadherin involved in the regulation of axon fasciculation."

Pettitt J, Broadbent ID

[Curr Biol, 2002]

Nervous system morphogenesis is characterized by extensive interactions between individual axon growth cones and their cellular environments. Selective cell adhesion is one mechanism by which the growth of an axon can be modulated, and members of the classic cadherin group of cell adhesion molecules have been shown to play a role in this process in both vertebrates and Drosophila [1-4]. In Drosophila, there are two classic cadherins: one involved primarily in regulating the morphogenesis of epithelia [5, 6], and the other, DN-cadherin [3], required almost exclusively in neuronal development. In contrast, C. elegans has a single classic cadherin gene, hmr-1, whose function is required for epithelial morphogenesis [7]. We show here that hmr-1 also encodes a second classic cadherin via a novel mechanism involving an alternative, neuron-specific promoter, coupled with alternative splicing. This novel HMR-1 isoform is very similar to DN-cadherin, and a mutant strain that specifically lacks the function of this isoform displays defects in the fasciculation and outgrowth of a subset of motor neuron processes; a phenotype that resembles loss of DN-cadherin function in Drosophila. These results indicate that Drosophila and C. elegans share a conserved, cadherin-dependent mechanism involved in regulating axonal patterning and fasciculation.

Jang H et al. (2017) Proc Natl Acad Sci U S A "Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans."

Mende F, Jang H, Levy S, Flavell SW, Bargmann CI, Zimmer M, Latham R

[Proc Natl Acad Sci U S A, 2017]

A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9-containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9-based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits.

Zocher E et al. (2018) MicroPubl Biol "Dominant-negative VPS-4 disrupts ODR-10::GFP distribution but has limited effects on chemotaxis"

Ruth N, Dahlberg C, Zocher E

[MicroPubl Biol, 2018]

Accumulation of ODR-10 depends on the sex and fed state of C. elegans (Ryan et al. 2014), though the precise mechanism of its regulation is not known. We tested whether the ESCRT pathway is required to maintain normal accumulation and distribution of ODR-10::GFP in AWA neurons under well-fed and starved conditions. The conserved AAA ATPase VPS-4 is required as the final step of the ESCRT pathway, to dissociate the machinery as cargo is moved from endosomes to multivesicular bodies (Babst et al. 2011, Kim et al. 2011). Mutation of Glutamate 219 to Glutamine (E219Q) in VPS-4 results in a dominant negative enzyme (VPS-4 DN). VPS-4 DN disrupts normal accumulation of the glutamate receptor, GLR-1, in a cell autonomous manner (Chun et al. 2008). We investigated a potential role for VPS-4 in ODR-10 regulation using fluorescence microscopy and an attraction chemotaxis assay. We expressed VPS-4 DN under the odr-10 promoter in animals expressing ODR-10::GFP (Figure 1A). Using live-imaging (200X (total) magnification), ODR-10::GFP was not visible in over half of the wild type, well-fed animals. Using this baseline, we measured changes in ODR-10::GFP (abundance and localization) and found that VPS-4 DN increased the frequency with which we observed animals with aberrant ODR-10 accumulation (Figure 1B). In 8 animals (out of 58) there was observable fluorescence in the cell bodies of AWA neurons (1 with fluorescence exclusively in the cell body). Starvation exacerbated this effect: 26 out of 39 animals had fluorescence in the cell bodies of AWA neurons (7 with fluorescence exclusively in the cell body). In contrast to previously published data that relied on different imaging parameters (Ryan, et al., 2014) we also observed that starvation of animals led to increased frequency of visible fluorescence in the cilia of AWA in WT animals (Figure 1B). Expression of VPS-4 DN had no effect on the ability of animals to react to the chemoattractant diacetyl under well-fed conditions (Figure 1C). However, VPS-4 DN expression in the AWA neuron resulted in significantly lower chemotaxis indices, compared to wild type, fed animals (Figure 1C, asterisk). Starvation of animals has a subtle effect on the abundance of ODR-10 that accumulates in the cilia of AWA neurons, but that accumulation does not affect the efficiency of chemotaxis in wild type animals (Figure 1B and C). However, when food stress was paired with a disruption of the ESCRT pathway the aberrant accumulation of ODR-10::GFP correlated with a modest but significant decrease in chemotaxis index (Figure 1C). It is still unknown whether ODR-10 is a direct target of the ESCRT machinery or other ubiquitin-related pathways. These data could also represent indirect effects due to changes in availability or localization of cofactors that are required for normal receptor processing and/or localization within the AWA neuron.

Zhou T et al. (2010) Mol Biol Evol "Detecting positive and purifying selection at synonymous sites in yeast and worm."

Wilke CO, Zhou T, Gu W

[Mol Biol Evol, 2010]

We present a new computational method to identify positive and purifying selection at synonymous sites in yeast and worm. We define synonymous substitutions that change codons from preferred to unpreferred or vice versa as nonconservative synonymous substitutions and all other substitutions as conservative. Using a maximum-likelihood framework, we then test whether conservative and nonconservative synonymous substitutions occur at equal rates. Our approach replaces the standard rate of synonymous substitutions per synonymous site, dS, with two new rates, the conservative synonymous substitution rate (dS(C)) and the nonconservative synonymous substitution rate (dS(N)). Based on the ratio dS(N)/dS(C), we find that 0.05% of all yeast genes and none of worm genes show evidence of positive selection at synonymous sites (dS(N)/dS(C) > 1). On the other hand, 9.44% of all yeast genes and 5.12% of all worm genes show evidence of significant purifying selection on synonymous sites (dS(N)/dS(C) < 1). We also find that dS(N) correlates strongly with gene expression level, whereas the correlation between expression level and dS(C) is very weak. Thus, dS(N) captures most of the signal of selection for translational accuracy and speed, whereas dS(C) is not strongly influenced by this selection pressure. We suggest that the ratio dN/dS(C) may be more appropriate than the ratio dN/dS to identify positive or purifying selection on amino acids.