Picture from Singh K et al. (2011) Curr Biol "C. elegans Notch signaling regulates adult chemosensory response and larval ...."

glp-1 is likely expressed in neurons. A GFP reporter was constructed containing 2.5 kb of putative regulatory sequence upstream and downstream of the glp-1 translation start site (glp-1p::gfp; see Supplemental methods for details). GFP expression was observed near the excretory pore, anus, in vulval precursor cells, intestinal cells, and in a few head neurons that could not be identified due to low expression levels. Previous studies indicated that glp-1 activation increases glp-1 transcription [11,12,13,14]. As the glp-1 reporter construct described above contains numerous binding sites for LAG-1, a C. elegans homolog of the CSL/Su(H) transcription factor homolog that is critical for Notch transcriptional activation of target genes, we reasoned that over-expressing osm-11 might lead to increased glp-1p::gfp expression. Within hours heat shock induction of OSM-11 expression, GFP expression was clearly observed in many neurons of hsp::osm-11; glp-1p::gfp transgenic animals. These neurons included ASG, ASH, ASI, ADL, ASJ, ADF, and ASE sensory neurons, but not the RIG interneurons that express lin-12. The ASH, ADL, and AWB sensory neurons detect octanol semi-redundantly [4,15] suggesting that glp-1 might act to modulate octanol response in these sensory neurons. Shown here is glp-1p::gfp expression in head neurons (arrow indicates ASHR ). Scale bar represents 10 m.

Picture from Shaye DD et al. (2005) Development "LIN-12/Notch trafficking and regulation of DSL ligand activity during vulval ...."

Figure 4. Trans-acting factors required for LIN-12 downregulation. (A) Beta-Galactosidase filter lift assay showing that ALX-1 interacts with LIN-12(region 1) in the yeast two-hybrid system. Four independent transformants carrying the activation domain (AD) and DNA-binding domain (DBD) fusions indicated are shown. (B) Domain structure of Ce ALX-1, Saccharomyces cerevisiae (Sc) Bro1p and H. sapiens (Hs) Alix, showing the N-terminal BRO1 domain, which mediates localization to late endosomes (Kim et al., 2005), a highly conserved Src kinase phosphorylation consensus site, one or two coiled-coils and a C-terminal proline-rich domain (PRD). (C) A GFP::ALX-1 translational reporter is highly and widely expressed (data not shown), and is seen in all the VPCs. This is a lateral view, with AJM-1 (red) detected by immunofluorescence, while native fluorescence of fixed GFP::ALX-1 is visualized without the need for anti-GFP staining. Note that GFP::ALX-1 appears to accumulate in vesicular structures (arrowheads) that are probably LE/MVEs. (D) Domain structure of Ce WWP-1, D. melanogaster (Dm) Su(dx) and Mus musculus (Mm) Itch, showing the C2 phospholipid-binding motif, WW motifs, and the ubiquitin-conjugating HECT domain (reviewed by Ingham et al., 2004). (E) Phylogenetic relationship among Nedd4-family members from C. elegans, Drosophila and mouse. Neighbor-joining analysis of the full-length proteins was done with MacVector (Accelrys), using an uncorrected p' setting for distance calculation and 1000 bootstrap repetitions to calculate the percent branch support, shown above each branch. CeD2085.4 (HECT domain only) and CeF45H7.6 (WW repeats and HECT domain) were used to anchor the tree. Scale bar: 10 μm.

Picture from Haynes CM et al. (2010) Mol Cell "The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the ...."

Figure 1. Impaired UPRmt in haf-1 Mutant Animals (A) Representative fluorescent photomicrographs of hsp-60pr::gfp transgenic worms (reporting on the UPRmt) with a temperature-sensitive mutation (zc32) that activates the UPRmt in an otherwise WT or haf-1(ok705)-deleted background. Where indicated, WT or haf-1(ok705) worms were raised on spg-7(RNAi) for 3 days to induce mitochondrial misfolded protein stress. (B) A corresponding immunoblot of GFP expressed by hsp-60pr::gfp transgenic worms in whom mitochondrial unfolded protein stress was induced (as in A). The endogenous 55 kDa ER protein, detected with an anti-HDEL monoclonal antibody (lower panel), serves as a loading control. (C) Quantitative analysis (by quantitative RT-PCR) of endogenous hsp-60 mRNA in WT or two different haf-1-deleted strains (ok705 and tm843) subjected to mitochondrial unfolded protein stress by hsp-6(RNAi) or spg-7(RNAi). Displayed is the mean ±SEM, n = 3, *p < 0.05. (D) Immunoblot of proteins reactive with an anti-HDEL monoclonal antibody in extracts of WT and haf-1 mutant worms in which ER stress was induced by exposure to elevated temperature (30C) for the indicated time. The upper band, BiP (HSP-4), is a UPRER target gene, whereas the lower invariant 55 kDa band (*) serves as a loading control. (E) Fluorescent photomicrographs of CHO cells expressing GFP (upper panels) or GFP fused to aa 1-75 of HAF-1 (HAF-11-75::GFP, lower panels). The cells were costained with the vital dye MitoTracker, which stains mitochondria (middle panels). (F) Immunoblot of extracts from HEK293T cells expressing GFP (as a cytosolic marker) and C-terminally tagged HAF-1::FLAG following cellular fractionation into total lysate (T), postmitochondrial supernatant (S), and mitochondrial pellet (M). Lanes 4-8 are from mitochondria treated with hypotonic buffer to generate mitoplasts and further treated with digitonin and Proteinase K where indicated. Lanes 7 and 8 are mitoplasts incubated in Na2CO3 followed by centrifugation at 150,000 X g and separated into the pellet (P) and supernatant (Su).

Picture from Singh K et al. (2011) Curr Biol "C. elegans Notch signaling regulates adult chemosensory response and larval ...."

Figure S2. Notch Receptors Act in Neurons to Regulate Octanol Response, Related to Figure 2 Time to respond to octanol is reported as mean time to initiate backward locomotion (s) +/- SEM. Sample size for each genotype or treatment is given on each bar. (A) glp-1 is likely expressed in neurons. A GFP reporter was constructed containing 2.5 kb of putative regulatory sequence upstream and downstream of the glp-1 translation start site (glp-1p::gfp; see Supplemental methods for details). GFP expression was observed near the excretory pore, anus, in vulval precursor cells, intestinal cells, and in a few head neurons that could not be identified due to low expression levels. Previous studies indicated that glp-1 activation increases glp-1 transcription [11,12,13,14]. As the glp-1 reporter construct described above contains numerous binding sites for LAG-1, a C. elegans homolog of the CSL/Su(H) transcription factor homolog that is critical for Notch transcriptional activation of target genes, we reasoned that over-expressing osm-11 might lead to increased glp-1p::gfp expression. Within hours heat shock induction of OSM-11 expression, GFP expression was clearly observed in many neurons of hsp::osm-11; glp-1p::gfp transgenic animals. These neurons included ASG, ASH, ASI, ADL, ASJ, ADF, and ASE sensory neurons, but not the RIG interneurons that express lin-12. The ASH, ADL, and AWB sensory neurons detect octanol semi-redundantly [4,15] suggesting that glp-1 might act to modulate octanol response in these sensory neurons. Shown here is glp-1p::gfp expression in head neurons (arrow indicates ASHR ). Scale bar represents 10 m. (B) glp-1(RNAi) in subsets of neurons had modest impact on octanol response (osm-10p::glp-1(RNAi) in ASH, ASI, PHA, PHB sensory neurons; gpa-11p::glp-1(RNAi) in ASH and ADL only). *p < 10-13 vs. WT, #p < 0.025 vs. lin-12(null), @p < 0.004 vs. lin-12(null);gpa-11p::gfp. Germline morphology, fertility and vulval morphology were not visibly altered by neuronal glp-1(RNAi) constructs, suggesting that no dsRNA spreading occurred (data not shown). (C) osm-11 acts via Notch receptors. The two Notch gain of function alleles: lin-12(n137n460), a non-constitutive, cold-sensitive gain of function (csgf) allele, and glp-1(ar202), a temperature-sensitive, gain of function (tsgf) allele were used to determine if osm-11 acts via Notch receptors. The gain of function alleles had little or no effect on octanol response in otherwise normal animals regardless of temperature. Conditional gain-of-function alleles lin-12(csgf) and glp-1(tsgf) (reared at restrictive temperature of 15oC and 25C, respectively) partially suppressed the octanol response defects of osm-11(null) animals. p < 0.01 vs. WT. p < 0.05 vs. osm-11(null) reared at 15C; &p < 0.05 vs. osm-11(null) reared at 25C. (D) lag-2p::gfp is expressed in many head neurons of adult C. elegans including the AVA, one of the command interneurons (arrow) that regulates locomotion and helps initiate reversals in response to aversive stimuli. Scale bar represents 10 m. (E) egl-4 and lst-1, are two candidate genes that act downstream of Notch and express in neurons. egl-4 encodes a cGMP-dependent protein kinase (PKG)[16] that is expressed in many head neurons and has been previously implicated in several C. elegans behaviors. The gain-of-function egl-4(ad450) allele partially suppressed the octanol response defect of osm-11(null) animals suggesting that egl-4 likely acts downstream of Notch signaling pathway in the regulation of octanol response. As no obvious changes in egl-4p::gfp expression were observed in Notch receptor mutant backgrounds (data not shown), either the reporter gene was not sensitive enough to detect modest changes in gene expression, the regulation occurs at a post-transcriptional level, or egl-4 is not directly downstream. Next, the role of lst-1 was tested. If lst-1 [17] is a direct target of Notch receptors in neurons, then increasing Notch receptor signaling should have no effect on octanol response in animals lacking lst-1. However, the gain-of-function lin-12(n137n460csgf) allele suppressed the octanol response defects of lst-1(null) animals, as did over-expression of osm-11. Thus, lst-1 likely works in parallel with other genes downstream of Notch signaling in octanol response. *p < 10-3 vs. WT; p < 0.001 vs. osm-11(null); #p < 0.01 vs. lst-1(null).