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.

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.

Ian D Broadbent et al. (2002) European Worm Meeting "Classical cadherin-catenin complexes and the developing C. elegans nervous system."

Ian D Broadbent, Jonathan Pettitt

[European Worm Meeting, 2002]

The cadherin superfamily of molecules has been implicated in many cellular developmental processes including adhesion, proliferation and morphogenesis. However, the mechanisms by which cadherins affect neuronal development remain enigmatic, particularly with respect to axon guidance and fasciculation, and the formation and maintenance of synapses. The C. elegans cadherin superfamily is comprised of 12 loci, yet only the hmr-1 locus is capable of encoding a "classical" cadherin, as defined by conserved regions in the cytoplasmic C- terminus responsible for binding to a set of cytoplasmic proteins, the catenins (1,2). In contrast, Drosophila possesses two distinct classical cadherin loci, encoding DE- and DN-cadherin. DE- cadherin is expressed in epithelial tissues, and is required for the maintenance of adherens junctions, whereas DN-cadherin is expressed in neuronal tissues, and is involved in fasciculation of axon bundles and pathfinding (3,4,5). The hmr-1 locus has been shown previously to encode an epithelial cadherin, HMR-1A, which is essential for epidermal morphogenesis during embryonic development (2). However, we have demonstrated recently that the hmr-1 locus can also encode a second isoform, HMR-1B, via an unusual mechanism involving alternative splicing and an alternative promoter (6). Recent information from the C. briggsae genome project suggests that this mechanism is also conserved in the C. briggsae hmr-1 locus. In contrast to the mainly epithelial HMR-1A isoform, HMR-1B is expressed exclusively in the nervous system in a particular subset of neurons, and shows a striking degree of sequence similarity to DN-cadherin. We have selectively disrupted the functions of HMR-1B using a hmr- 1 null allele in conjunction with a HMR-1A rescuing construct, and a "snapback" RNAi construct designed to specifically target HMR-1B. Animals lacking HMR-1B function assayed by either of these approaches display defects in motorneuron pathfinding and ventral nerve cord fasciculation. We have also observed similar defects in animals carrying a null mutation in the hmp-1 _-catenin gene that have been rescued for epidermal (but not neuronal) HMP-1 function. We are currently using this epidermally-rescued hmp-1 background to identify molecules that act in concert with the cadherin-catenin complex to regulate nervous system development in C. elegans.

Gu T et al. (1996) West Coast Worm Meeting "SUPPRESSORS OF LET-60 DOMINANT NEGATIVE MUTATIONS"

Han M, Gu T

[West Coast Worm Meeting, 1996]

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 for and isolated suppressors that revert the Vul phenotype caused by a let-60 ras dominant-negative (dn) mutation. One of the suppressor loci , sur-5 (suppressor of ras), is defined by four recessive alleles on LGX between unc-6 and dpy-7. sur-5 mutants have no phenotype other than suppression of the vulvaless phenotype caused by the let-60 (dn). sur-5 (ku74) does not suppress let-23 (sy1), and it does not enhance the multivulva phenotype caused by heterozygous let-60 (n1046)-the gf allele of let-60. We have cloned sur-5. The cDNA is 2.1 kb in size, and it seems to be expressed in all stages of development. SUR-5 has some limited homology to known acetyl CoA synthetases-22% aa sequence identity. However, SUR-5 may have a more closely related human homolog although its function is unknown. We have identified the lesions in two sur-5 alleles. One of the mutations, ku74, is a 295 bp in frame deletion, and another mutation, ku103, is a Pro to Ser aa change. When sur-5 is overexpressed in a stable array, it suppresses the multivulva phenotype caused by let-60(n1046). Currently, we are trying to study the function of sur-5 in the ras signal pathway using both classical genetics and molecular biology approaches.

Gu T et al. (1997) International C. elegans Meeting "SUR-5, A NEW NEGATIVE FACTOR THAT SPECIFICALLY REGULATES LET-60 RAS"

Gu T, Han M

[International C. elegans Meeting, 1997]

A let-60 ras mediated signal transduction pathway is involved in specifying the vulva cell fate. It is likely that each component acting in the pathway is under both positive and negative regulation. To identify new negative regulators of the pathway, we screened for mutants that suppress the dominant vulvaless phenotype caused by the let-60 (dn). sur-5 is one of the genes we identified from these mutants. sur-5 mutants are wild type in let-60 (+) background. Our genetic study indicates that sur-5 is a negative regulator of let-60. We show that sur-5 mutations specifically suppress only one group of dominant negative mutations of let-60 but not other let-60 dn or lf mutations, indicating its specific role on ras. sur-5 maps between dyp-7 and unc-6 on LGX. We have cloned sur-5. Its cDNA is 2.1 kb, and the SUR-5::GFP fusion protein is expressed in the nucleus of most cells in all stages of development. SUR-5 has some limited homology to known acetyl CoA synthetases (22% identical). However, SUR-5 may have a more closely related mammalian homolog although its function is unknown. We have identified the lesions in two sur-5 alleles. One of the mutations, ku74, is a in frame deletion of 295 bp, and another mutation, ku103, is a Pro to Ser change on a conserved a.a. Recessive sur-5 mutations fail to enhance the multivulva (Muv) phenotype caused by heterozygous let-60 (n1046)-the gf allele of let-60. However, when sur-5 is overexpressed in a stable array, it partially suppresses the Muv phenotype caused by homozygous let-60(n1046). Currently we are trying to study sur-5 function using molecular biology approaches.

CJ Malone et al. (1999) International C. elegans Meeting "Characterization of the role of rho-1 and nuclear migration during P cell migration"

S Orita, CJ Malone, M Han

[International C. elegans Meeting, 1999]

The small GTPase rhoA has been implicated in a variety of cellular movements. To address the role of rhoA during C. elegans development we have performed a reverse genetics approach. RNAi of the C. elegans rhoA homolog, rho-1 , produces embryonic lethality, indicating that it is an essential gene. To identify specific requirements for rho-1 we expressed dominant negative (dn) and gain-of-function (gf) rho-1 under the control of the col-10 promoter, which drives expression in hypodermal cells. Expression of this rho-1(dn) construct causes less than the normal 12 P cells to occupy the ventral cord. Analysis of these animals suggests that P cells are missing from the ventral cord due to failed P cell migration. unc-73 , which encodes a protein capable of acting as an exchange factor for the mammalian small GTPase, Rac1, also causes a P cell migration defect. To test whether UNC-73 acts through rho-1 during P cell migration, we expressed rho-1(gf) in animals homozygous for a weak unc-73(e936) allele. This construct was able to rescue the P cell migration defect of unc-73 , suggesting that UNC-73 may act as an exchange factor for RHO-1, activating it during P cell migration. We have shown previously that the nuclear envelope protein UNC-84 is required for nuclear migration during P cell migration. To identify other genes that may be involved in the P cell nuclear migration, we have undertaken RNAi studies of the C. elegans homologs of NUDC and NUDF, two proteins required for Aspergillus nuclear migration. Both C. elegans genes appear to be essential, because RNAi of either can cause complete embryonic lethality in the progeny of injected animals. Although severely deformed, these embryos form a pharynx, develop gut granules and express the MH27 epitope. Interestingly, escapers from the RNAi injections are Unc, Pvl and sterile. The Unc and Pvl phenotypes are similar to those observed in animals mutant for unc-83 or unc-84 , which cause P cell nuclear migration defects. We are currently determining if a P cell nuclear migration defect is the cause of the Unc and Pvl phenotypes of the NUDC/F RNAi animals.

Jason M Kinchen et al. (2003) International Worm Meeting "Understanding the role of CED-5/DOCK180 in cell migration and engulfment in C. elegans"

Doris Klingele, Kodimangalam S Ravichandran, Jason M Kinchen, Michael O Hengartner

[International Worm Meeting, 2003]

The distal tip cells (DTCs) migrate in a defined pattern to specify the shape of the hermaphrodite germ line; only rarely is there deviation in this migration. Unlike many other cells, the path of DTCs migration can be readily visualized without the aid of time-lapse (or 4D) microscopy, as it forms the shape of the developed hermaphrodite gonad. Along with a clear structure that makes fixation irrelevant, the ability to assay movement without time-lapse microscopy makes the DTCs a very nice model system to study cell migration. The migration of the DTCs depends on the redundant activity of two Rac orthologues, CED-10 and MIG-2, as well as a central signaling pathway that involves CED-2, CED-5, and CED-12. In contrast, the engulfment of apoptotic cells requires only ced-2, -5, -10, and -12 and does not require the second Rac isoform, mig-2. It has been a mystery for quite some time as to how ced-10 is activated in response to an eat me signal without a GEF. Recently, however, it has been published that members of the CDM (ced-5 Dock180 and myoblast city) family of assembly proteins have an intrinsic RacGEF activity that is mediated by a novel (but conserved) domain in the C-terminus of the molecule called the DOCKER domain. This domain preferentially binds to nucleotide-free Rac and probably stabilizes the transition state so that it can acquire a GTP molecule. Thus, it is quite possible that CED-5 provides the GEF activity for CED-10 during the process of engulfment and DTC migration. We have used the CED-5 homologue DOCK180 to assay the involvement of this DOCKER domain in cell migration in vivo. While DOCK180(WT) partially rescues the DTC migration defect of ced-5 mutant worms, overexpressing a DOCK180 that had a mutant DOCKER domain caused a dominant negative effect. To further characterize this dominant negative (dn) effect, the DOCK180(dn) array was crossed into several mutant backgrounds. Enhanced mismigration was seen in ced-10 and ced-12 mutant worms, but not mig-2 mutants. This is an interesting result because it suggests that DOCK180 mediates rescue of DTC migration solely via the Rac orthologue MIG-2 rather than both MIG-2 and CED-10, and provides a reasoning for the inability of DOCK180 to rescue the engulfment defect of ced-5 mutant worms.

Okano H et al. (2000) Japanese Worm Meeting "PSA-7 is involved in Ras and Wnt signaling pathways in C. elegans"

Kouike H, Yoda A, Sawa H, Okano H

[Japanese Worm Meeting, 2000]

It has been shown that vulva is induced to form by activating Ras signaling pathway. psa-7 mutants exhibit a multivulva (Muv) phenotype, suggesting that psa-7 negatively regulates this pathway. Although mutations in lin-3(lf), let-23(lf), let-60(dn) and mpk-1(lf) result in a vulvaless (Vul) phenotype, we observed that double mutants containing psa-7 and mutation of any of these genes exhibited Muv phenotype. This result shows that psa-7 acts downstream of Ras signaling pathway. It has been reported that transcriptional regulators; LIN-1, SUR-2 and LIN-35 regulate vulval induction. We observed that loss-of-function mutants of these transcriptional regulators enhanced the Muv phenotype of psa-7. The strong genetic interactions of PSA-7 and these transcriptional regulators indicate that PSA-7 may play important roles in vulval signaling pathway by regulating transcription of vulval specific genes. In psa-7 mutants, normally asymmetric T cell division becomes symmetric. It has been reported that lin-44/wnt and lin-17/frizzled regulate asymmetric T cell division. But the genes that act downstream of lin-17 remain to be identified. On the analogy of genetic roles of psa-7 in Ras signaling pathway, PSA-7 may also function as a transcriptional regulator in Wnt signaling pathway. We are currently trying to rescue the phenotype of psa-7 by injecting cosmids from the corresponding region of the physical map.

Herman MA et al. (2000) European Worm Meeting "Separate C. elegans beta-catenins mediate POP-1/Tcf transcriptional coactivation and HMR-1/cadherin adhesion"

Clevers HC, Korswagen HC, Herman MA

[European Worm Meeting, 2000]

Armadillo/beta-catenin plays an essential role in both Wnt signaling and adhesion. beta-catenin mediates Wnt signaling by serving as an essential transcriptional coactivator upon binding to Tcf/LEF-1 transcription factors. beta-catenin also associates with the intracellular domain of cadherins to mediate adhesion. Three putative beta-catenin homologs have been identified in C. elegans: WRM-1, BAR-1 and HMP-2. WRM-1 functions in a Wnt pathway that is different from the pathway in flies and vertebrates. Instead of interacting directly with the Tcf homolog POP-1, WRM-1 interacts with a kinase, LIT-1. The activities of WRM-1 and LIT-1 ensure that POP-1, which probably acts as a repressor in this pathway, is downregulated in the posterior daughter cells of many anterior/posterior cell divisions. Differences in POP-1 expression levels may subsequently specify differences in fate. In addition to this putative repressor function, we show that POP-1 can also promote target gene transcription. POP-1 binds to the consensus Tcf target site and activates transcription of a Tcf reporter gene in combination with Armadillo. We asked which of the three worm beta-catenins can bind directly to POP-1. Only BAR-1 binds to POP-1 in yeast-two-hybrid and co-immunoprecipitation experiments. Furthermore, only the combination of POP-1 and BAR-1 activates transcription of a Tcf reporter gene. This suggests that POP-1 and BAR-1 have a function that is similar to that of Tcf and beta-catenin. To investigate this further, we asked if the POP-1/BAR-1 interaction is required for the expression of a putative Wnt target gene, mab-5. The Hox gene mab-5 is required for the posterior migration of the descendants of QL (QL.d) and mutation of mab-5 results in the anterior migration of these cells. Mutations in the Wnt pathway components egl-20/Wnt, lin-17/Fz and bar-1 also result in QL.d migration defects. These mutations show reduced or absent mab-5 expression, suggesting that a Wnt pathway regulates the expression of this Hox gene. To test if the interaction between POP-1 and BAR-1 is also required for the expression of mab-5, we inducibly expressed a pop-1 mutant (DN-pop-1) that lacks the BAR-1 interaction domain and acts as a strong dominant negative mutation in a Tcf reporter gene assay. We find that overexpression of DN-pop-1 results in the anterior migration of the QL.d and the inhibition of mab-5 expression. This demonstrates that BAR-1 and POP-1 are indeed required for the expression of a Wnt target gene, which suggests that BAR-1 and POP-1 function in a Wnt pathway that is similar to that in flies and vertebrates. We find that the third worm beta-catenin, HMP-2, is the only beta-catenin homolog that interacts with the single cadherin, HMR-1. The interaction of HMP-2 with HMR-1 was anticipated from the previously reported colocalization of HMP-2 with the alpha-catenin HMP-1 and HMR-1 in adherens junctions. The absence of an apparent adhesion function for BAR-1 and a signaling function for HMP-2 is however unexpected. We propose that the functions of Armadillo/beta-catenin have been distributed over three different beta-catenins in C. elegans: WRM-1 and BAR-1 function in Wnt signaling, whereas HMP-2 functions specifically in adhesion