Gauthier, K. et al. (2019) International Worm Meeting "LIN-2/7/10 complex revisited: Complex-independent function of LIN-10 and LIN-7 in LET-23 EGFR signaling."

Rocheleau, C. E., Gauthier, K.

[International Worm Meeting, 2019]

Spatiotemporal organization of cellular signaling components is essential to regulate signal transduction cascades, and to prevent developmental disorders and cancer. This can be modeled in C. elegans vulval cell fate induction, which requires basolateral localization of LET-23 (Epidermal Growth Factor Receptor), mediated by the LIN-2/LIN-7/LIN-10 complex, to activate the canonical LET-60 Ras/MPK-1 ERK pathway. Disruption of the complex results in exclusive apical localization of LET-23, loss of signaling, and a vulvaless phenotype. The complex has been defined biochemically and is evolutionarily conserved, but where and when the complex forms in vivo, or how it regulates LET-23 localization remains unknown. Using CRISPR/Cas9, we fluorescently tagged endogenous LIN-2, LIN-7, and LIN-10. We found that LIN-2 and LIN-7 are cytoplasmic, occasionally punctate, and colocalized in the VPCs. LIN-10 localizes to cytoplasmic foci independently of LIN-2 and LIN-7, and likely localizes to Golgi or recycling endosomes. There is infrequent overlap between LIN-2 and LIN-10 at cytoplasmic foci. Unexpectedly, we found that overexpression of LIN-10 rescues lin-2 and lin-7 vulvaless phenotypes and recovers some basolateral LET-23 localization in a lin-2 mutant. LIN-7 overexpression also rescues lin-2 and lin-10, but LIN-2 cannot rescue its complex components. This suggests that LIN-10 and LIN-7 have a complex-independent function in promoting LET-23 signaling. Our observations are consistent with a model in which cytoplasmic LIN-7 and LIN-2 likely form a stable interaction, and transiently interact with LIN-10 at Golgi or recycling endosomes. Our results indicate that complex formation with LIN-2 is only one of multiple ways in which LIN-7 and LIN-10 promote LET-23 signaling and trafficking. This also suggests the existence of unidentified interactors that help mediate these complex-independent functions. The results will offer new insight into how scaffolding components regulate cellular events by controlling the spatial organization of signalling proteins in polarized epithelial cells.

Meras, I. et al. (2017) International Worm Meeting "Endosomal/autophagic regulation of FOXO transcription factors."

Rocheleau, C., E., Chotard, L., Meras, I.

[International Worm Meeting, 2017]

FOXO proteins are a conserved family of transcription factors that regulate metabolism, stress responses, and lifespan. Loss of FOXO proteins is associated with cancer, neurodegenerative and metabolic diseases. Insulin/IGF signaling (IIS) pathway negatively regulate FOXO by activating the Akt1/2 kinase, which phosphorylates FOXO. The 14-3-3 proteins bind phospho-FOXO (pFOXO), sequestering it in the cytoplasm. FOXO proteins were first identified as downstream targets of IIS in the nematode C. elegans which has a single FOXO, DAF-16, that is most closely related to human FOXO3a. During fed conditions, C. elegans IIS is constitutive which keeps DAF-16 in the cytoplasm. Under starvation conditions (and other stresses), there is no IIS, and DAF-16 enters the nucleus to regulate gene expression. We found that in the intestine of fed C. elegans, DAF-16 localizes to a subset of RAB-5-positive early endosomes. DAF-16 endosomes are lost in rab-5(RNAi) animals and significantly expanded in tbc-2 mutants that result in increased RAB-5 activity. In starved animals, DAF-16 endosomes are lost, and DAF-16 localizes mainly to the nucleus, whereas re-feeding results in relocalization of DAF-16 onto endosomal membranes. These results suggest that IIS promotes DAF-16 localization to endosomes. Consistent with this hypothesis, loss of daf-18 pten, a negative regulator of IIS, results in an increase in the number of animals with DAF-16 endosomes, while knockdown of the 14-3-3 protein, FTT-2, results in loss of DAF-16 endosomes. Furthermore, we found that FTT-2 14-3-3 protein colocalizes with DAF-16 on endosomes. This suggests that the endosomal pool of DAF-16 is phosphorylated. This is conserved in human cells as we discovered that pFOXO1/3a colocalizes with activated Rab5 on the endosomes in HEK293 cells. We do not know the ultimate fate of pFOXO on endosomes, but our data shows that LGG-1 engulfs DAF-16 endosomes and knockdown of lgg-1/2 increased the number of DAF-16 endosomes, suggesting that they are degraded by autophagy. We hypothesize that IIS pathway regulates FOXO proteins on endosomes where it can be released if needed or degraded via selective autophagy. Relevance: Here we are proposing a new mechanism of FOXO regulation on signaling endosomes and turnover by selective autophagy. We believe this project will provide new insights into the regulation of FOXO proteins and open new avenues for drug development to regulate FOXO in cancer, neurodegenerative and metabolic diseases.

Stocker, Stephanie et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Regulation of cell-cycle control in the C. elegans intestine"

Stocker, Stephanie, Schneider, Robin, Bossinger, Olaf

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The C. elegans intestine consists of only 20 cells, which clonally derive from the E-cell, one of five somatic founder cells in the early embryo (Sulston et al., 1983). In the 4-cell-stage, the P2-cell induces the EMS-cell to divide asymmetrically into a larger MS- and a smaller E-cell that differ with regard to cell-cycle, number of progeny and cell-fate (Goldstein, 1992; Schierenberg, 1987). The involvement of a Wnt-pathway (Rocheleau et al., 1997; Thorpe et al., 1997) provided a significant step of understanding the basic strategies to control determination of the E-cell fate (Korswagen, 2002). In addition, the intestine is one of the few cell-lineages where a plausible sequence of direct molecular interactions can be proposed throughout the life-cycle (Maduro, 2009; McGhee, 2007). We have started to elucidate the rol e of different signaling pathways that on the one hand are involved in the correct specification of the E-cell (i.e. Wnt-, Src-, MAPK- and APC-like signaling) and on the other hand might also control proliferation of intestinal cells. Using RNAi by feeding against APR-1 (Hoier et al., 2000; Rocheleau et al., 1997), the C. elegans homolog of the human tumor suppressor APC, we found the number of E-cells approximately doubled. Hyperplasia starts with the onset of polarization of the intestinal primordium and hence disturbs the typical alignment of intestinal cells. Intestinal hyperplasia after apr-1RNAi and in cdc-25.1(gof) embryos is presumably generated in an independent manner (Segref et al., 2010). We are currently testing the role of the cyclin-dependent kinase inhibitor CKI-1 (Fukuyama et al., 2003) during intestinal hyperplasia.

Saiki R et al. (2008) Aging Cell "Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q."

Saiki R, Clarke CF, Furukawa S, Larsen PL, Bixler T, Lunceford AL, Lee W, Dang P

[Aging Cell, 2008]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. C. elegans fed E. coli devoid of Q(8) have a significant life span extension when compared to C. elegans fed a standard "Q-replete"E. coli diet. Here we examine possible mechanisms for the life span extension caused by the Q-less E. coli diet. A bioassay for Q uptake shows that a water-soluble formulation of Q(10) is effectively taken up by both clk-1 mutant and wild-type nematodes, but does not reverse life span extension mediated by the Q-less E. coli diet, indicating that life span extension is not due to the absence of dietary Q per se. The enhanced longevity mediated by the Q-less E. coli diet cannot be attributed to dietary restriction, different Qn isoforms, reduced pathogenesis or slowed growth of the Q-less E. coli, and in fact requires E. coli viability. Q-less E. coli have defects in respiratory metabolism. C. elegans fed Q-replete E. coli mutants with similarly impaired respiratory metabolism due to defects in complex V also show a pronounced life span extension, although not as dramatic as those fed the respiratory deficient Q-less E. coli diet. The data suggest that feeding respiratory incompetent E. coli, whether Q-less or Q-replete, produces a robust life extension in wild-type C. elegans. We believe the fermentation-based metabolism of the E. coli diet is an important parameter of C. elegans longevity.

Govindan JA et al. (2015) Proc Natl Acad Sci U S A "Dialogue between E. coli free radical pathways and the mitochondria of C. elegans."

Mylonakis E, Govindan JA, Zhang X, Ruvkun G, Jayamani E

[Proc Natl Acad Sci U S A, 2015]

The microbial world presents a complex palette of opportunities and dangers to animals, which have developed surveillance and response strategies to hints of microbial intent. We show here that the mitochondrial homeostatic response pathway of the nematode Caenorhabditis elegans responds to Escherichia coli mutations that activate free radical detoxification pathways. Activation of C. elegans mitochondrial responses could be suppressed by additional mutations in E. coli, suggesting that C. elegans responds to products of E. coli to anticipate challenges to its mitochondrion. Out of 50 C. elegans gene inactivations known to mediate mitochondrial defense, we found that 7 genes were required for C. elegans response to a free radical producing E. coli mutant, including the bZip transcription factor atfs-1 (activating transcription factor associated with stress). An atfs-1 loss-of-function mutant was partially resistant to the effects of free radical-producing E. coli mutant, but a constitutively active atfs-1 mutant growing on wild-type E. coli inappropriately activated the pattern of mitochondrial responses normally induced by an E. coli free radical pathway mutant. Carbonylated proteins from free radical-producing E. coli mutant may directly activate the ATFS-1/bZIP transcription factor to induce mitochondrial stress response: feeding C. elegans with H2O2-treated E. coli induces the mitochondrial unfolded protein response, and inhibition of a gut peptide transporter partially suppressed C. elegans response to free radical damaged E. coli.

Yuen, Grace J. et al. (2013) International Worm Meeting "Enterococcus infection of C. elegans as a model of innate immunity."

Yuen, Grace J., Ausubel, Frederick M.

[International Worm Meeting, 2013]

Enterococcus is a Gram-positive commensal that is also an important opportunistic pathogen. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both species. We have previously shown that infection with either species leads to gut distention, but only E. faecalis is able to establish a persistent and lethal infection in the nematode. We now provide evidence that at least three canonical C. elegans immune signaling pathways are important for survival during infection with E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by E. faecium infection, mutations in the PMK-1, FSHR-1, and BAR-1 immune signaling pathways lead to an immunocompromised phenotype. This new finding suggests that an active host response is required to keep E. faecium infection "in check" in the worm intestine. To further characterize the C. elegans host response to Enterococcus infections, we used genome-wide transcriptional profiling of nematodes feeding on E. faecalis and E. faecium, as well as two controls, heat-killed E. coli and live Bacillus subtilis, a non-pathogenic Gram-positive. We found that relative to B. subtilis, E. faecalis and E. faecium caused the upregulation of 249 and 166 genes, respectively, of which 105 genes were common to both, comprising the Enterococcus gene signature. Shared by both Enterococcus infection signatures were genes relating to oxidation/reduction, acyl-CoA dehydrogenase/oxidase activity, fatty acid metabolism, and C-type lectins. Additionally, the Enterococcus infection gene signature is fairly distinct from the P. aeruginosa, S. aureus, and C. albicans infection signatures. Furthermore, of the 91 genes that are upregulated in E. faecalis (more virulent) relative to E. faecium (less virulent), 22 are shared with the 77 genes upregulated in worms infected with virulent Microbacterium nematophilum relative to avirulent M. nematophilum (O'Rourke et al., 2006), suggesting that these genes may comprise a "virulence response signature." Studies are underway in understanding the biology of Enterococcus infection in C. elegans and identifying novel Enterococcus-activated pathways.

Harrington LA et al. (1988) Mech Ageing Dev "Effect of vitamin E on lifespan and reproduction in Caenorhabditis elegans."

Harrington LA, Harley CB

[Mech Ageing Dev, 1988]

Vitamin E extends the lifespan of many animals, including the nematode Caenorhabditis elegans. Our results confirm previous studies that 200 micrograms/ml vitamin E significantly prolonged C. elegans survival (17-23%, P less than 0.05) when added from hatching to day 3, while continuous exposure, either at hatching or from 4 days prior to hatching, had little additional effect. Treatment with 100 or 400 micrograms/ml vitamin E, or with other antioxidants (80 micrograms/ml vitamin C, either alone or in combination with vitamin E, or 120 micrograms/ml N,N'-diphenyl-1,4-diphenylenediamine (DPPD] did not significantly affect lifespan. All treatments with 200 micrograms/ml vitamin E moderately reduced fecundity (total progeny) and increased the mean day of reproduction. At 400 micrograms/ml, vitamin E had severe effects, while DPPD, vitamin C, and 100 micrograms/ml vitamin E had slight effects on both these parameters of reproduction. These data suggest that vitamin E increases lifespan in C. elegans in part by slowing development in the same manner that metabolic-depressant or mildly cytotoxic drugs increase lifespan, decrease fecundity, and delay the timing of reproduction.

Ryoichi Saiki et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Caenorhabditis elegans lifespan is profoundly modulated by its diet of E. coli: What are the roles of E. coli metabolism and coenzyme Q?"

Peter Dang, Tarra Bixler, Catherine Clarke, Pamela Larsen, Ryoichi Saiki, Adam Lunceford, Wendy Lee

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Q is an essential component of respiratory electron transport and a potent lipid soluble antioxidant. Extended lifespans are observed in C. elegans clk-1 mutants with defects in Q biosynthesis. Intriguingly, mice heterozygous for a clk-1 gene disruption also show increased lifespan. C. elegans fed E. coli devoid of Q8 have a significant life span extension when compared to C. elegans fed a standard Q-replete E. coli diet. These results initially suggested that the lack of Q is the important parameter affecting lifespan. However, recent results indicate that feeding respiratory incompetent E. coli, whether Q-replete or Q-less, produces a robust lifespan extension in C. elegans. C. elegans skn-1 mutants fed the respiratory incompetent E. coli diets also show lifespan extension, suggesting that the response is independent of a dietary restriction effect. The respiratory incompetent E. coli diet may be imposed well after the worms reach adulthood, including post-reproductive adults, indicating that lifespan extension operates independently of worm development. Curiously, C. elegans fed the Q-less E. coli diet seem to exhibit more pronounced lifespan extension than when fed respiratory incompetent E. coli that are Q-replete. C. elegans ttx-3 mutants, harboring defects in the gene encoding a LIM homeodomain transcription factor, fail to distinguish the Q-less from the Q-replete respiratory defective E. coli. The difference in life span extension mediated by the two respiratory deficient E. coli diets depends on normal food seeking behavior exhibited by N2 C. elegans. The data suggest that the life span extension of N2 worms fed Q-less E. coli diet is caused by a combination of respiratory deficiency of the E. coli and the food seeking behavior of C. elegans. This work was supported by NIA Grant AG19777, and by the Ellison Medical Foundation.

BC Prasad et al. (1999) International C. elegans Meeting "The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function."

BC Prasad, RR Reed

[International C. elegans Meeting, 1999]

The O/E family of rHLH transcription factors has been implicated in neurogenesis, axonal pathfinding, muscle formation and B cell maturation. The murine O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis and are expressed in olfactory neurons throughout development where they may regulate components of the olfactory signaling cascade. We have identified a C. elegans O/E homologue (CeO/E) that is encoded by the unc-3 locus. Unc-3 mutants display an uncoordinated phenotype attributed to a severe defasiculation and miswiring of the ventral cord (VNC). Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed transiently in the excitatory VNC motor neurons and throughout development in a pair of chemosensory neurons (ASI amphid neurons). Several observations suggest that the protein may have distinct roles in the two cell types. Although the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI, unc-3 mutants enter the dauer pathway under inappropriate conditions. Although CeO/E possesses highly conserved domains associated with DNA binding in the mammalian homologue, identification of DNA binding sites for the C. elegans protein has not been achieved. We have shown that CeO/E protein can homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. Several unc-3 target genes ( daf-7, unc-17/cha-1, unc-4 ) have been identified by other laboratories and each contains a mammalian binding site in the promoter region. Remarkably, we have been unable to demonstrate CeO/E binding at these sites. We are generating chimeras of O/E-1 and CeO/E to understand the DNA binding specificity of the CeO/E protein and utilizing a yeast-2-hybrid screen with both O/E-1 and CeO/E to identify interacting proteins in C. elegans . These studies should reveal the mechanism of CeO/E transcriptional activation and target gene regulation.

Garsin DA et al. (2001) Proc Natl Acad Sci U S A "A simple model host for identifying Gram-positive virulence factors."

Mylonakis E, Ausubel FM, Singh KV, Sifri CD, Calderwood SB, Murray BE, Qin X, Garsin DA

[Proc Natl Acad Sci U S A, 2001]

We demonstrate the use of the nematode Caenorhabditis elegans as a facile and inexpensive model host for several Gram-positive human bacterial pathogens. Enterococcus faecalis, Streptococcus pneumoniae, and Staphylococcus aureus, but not Bacillus subtilis, Enterococcus faecium, or Streptococcus pyogenes, kill adult C elegans. Focusing our studies on the enterococcal species, we found that both E. faecalis and E. faecium kill C elegans eggs and hatchlings, although only E. faecalis kills the adults. In the case of adults, a low inoculum of E, faecalis grows to a high titer in the C elegans intestine, resulting in a persistent infection that cannot be eradicated by prolonged feeding on E. faecium. Interestingly, a high titer of E. faecium also accumulates in the nematode gut, but does not affect the longevity of the worms. Two E. faecalis virulence-related factors that play an important role in mammalian models of infection, tsr, a putative quorum-sensing system, and cytolysin, are also important for nematode killing. We exploit the apparent parallels between Gram-positive infection in simple and more complex organisms by using the nematode to identify an E. faecalis virulence factor, ScrB, which is relevant to mammalian pathogenesis.