The AFD and FLP sensory neurons mediate Tav response in the head.

<p>(A) Laser ablation of AFD, FLP and AIB led to severe defects in the head Tav response compared to mock-ablated animals (n>8). Individual neurons were identified by GFP-labeling and the success of the ablations was visualized by the disappearance of the GFP label in these neurons. Tav responses of the mock-ablated animals slightly differed, which is perhaps due to different GFP transgenes (see Methods). (B) AFD-laser-ablated and two transgenic lines [expressing <i>Diphtheria</i> Toxin A (DT-A) under the control of the <i>gcy-8</i> promoter] in which AFD was genetically ablated showed defective head Tav response, whereas DT-A killing of five pairs of sensory neurons (from the <i>odr-3</i> promoter), but not AFD, behaved like wild-type (n>80). (**P<0.001). Error bars indicate SD.</p

YFP/CFP ratio change (calcium influx) after noxious heat stimuli in different sensory neurons.

<p>Five seconds after recording, the animals were exposed to the noxious heat stimuli which reached 38°C maximal temperature after seven seconds. Values reported are mean % ± SD % (sample size) of the average maximal FRET ratio change amplitude in each population post stimuli. ND: not determined.</p

AFD, FLP, and PHC function as primary sensory neurons in the Tav response.

<p>(A) Average ratio changes in AFD, PLM, PVD and ALM upon noxious heat. (**P<0.001 different from ratio changes in AFD in wild-type). (B), (C) Average ratio changes in FLP and PHC upon heat stimuli in wild-type, <i>unc-13</i> and <i>unc-31</i> mutant backgrounds. Data are shown in box plot (n>4). Error bars indicate SD.</p

OCR-2 and OSM-9 contribute to the Tav response in the FLP neurons in the head and in the PHC neurons in the tail of <i>C. elegans</i>.

<p>(A), (B) The head and tail Tav of <i>osm-9</i> and <i>ocr</i> single and double mutants are shown. (C) The defective Tav response in the head of the <i>ocr-2 osm-9</i> double mutant was rescued by the expression of <i>ocr-2</i> and/or <i>osm-9</i> full length genomic DNA as well as by expression of their respective cDNAs under the control of the <i>mec-3</i> promoter. (D) The expression of either <i>ocr-2</i> or <i>osm-9</i> full-length genomic DNA was sufficient for at least partial rescue of the defective Tav response in the tail of the <i>ocr-2 osm-9</i> double mutant. (*P<0.01; **P<0.001, n>80). Error bars indicate SD.</p

The PVC and DVA interneurons mediate thermonociception in the tail of <i>C. elegans</i>.

<p>The PVC and DVA interneurons mediate thermonociception in the tail of <i>C. elegans</i>.</p

A cGMP signaling contributes to the Tav response in the AFD neurons.

<p>(A) The head Tav responses of mutants in CNG channel genes are shown. (B) Genetic-ablation of AFD did not further decrease the head Tav response in <i>tax-2</i>;<i>tax-4</i> double mutant. (C), (D) The defective Tav phenotype of both <i>tax-2</i> and <i>tax-4</i> single mutant was rescued by expressing a wild-type copy of the respective gene (<i>tax-2</i> or <i>tax-4</i>). Expression of <i>tax-4</i> or <i>tax-2</i> cDNA under the control of the AFD-specific <i>gcy-8</i> promoter rescued the respective mutant phenotype. No rescue was obtained when the <i>tax-4</i> or <i>tax-2</i> cDNA was expressed from the <i>odr-4</i> promoter (expressed in 12 neurons, but not in AFD). (E) The head Tav responses of the <i>gcy</i> mutants are shown. (*P<0.01; **P<0.001, n>50). Error bars indicate SD.</p

cSADDs inhibits paraquat-mediated signaling to <i>hsp-6</i> through KGB-1.

<p>A–B. In <i>kgb-1(um3)</i> mutant animals, which are cSADDs deficient, paraquat induced <i>hsp-6</i> induction is not blocked by <i>elt-2</i> RNAi. Thus, ROS induced UPR^mt is activated in the absence of functional cSADDs. In contrast, <i>kgb-1(um3)</i> does not prevent inhibition of <i>hsp-6</i> induction by <i>afts-1</i> and <i>pifk-1</i> knockdown, suggesting that they function downstream of <i>kgb-1</i> and the cSADDs. Columns represent normalized values plus standard error of the mean (SEM). Numbers in or on columns indicate the number of analyzed animals (n_total = 248). ***: p<0.001, *: p<0.05; Kruskal-Wallis test plus Dunn's Multiple Comparison Test (A). Equal optical settings, scale bar 400 µm. <i>(i)</i>: RNAi; L4440: empty vector control; +: wild type allele (B). C. Model: Genes activating the cSADDs (cellular surveillance system) inhibit the paraquat-triggered induction of the UPR^mt.</p

Surveillance-Activated Defenses Block the ROS–Induced Mitochondrial Unfolded Protein Response

<div><p>Disturbance of cellular functions results in the activation of stress-signaling pathways that aim at restoring homeostasis. We performed a genome-wide screen to identify components of the signal transduction of the mitochondrial unfolded protein response (UPR^mt) to a nuclear chaperone promoter. We used the ROS generating complex I inhibitor paraquat to induce the UPR^mt, and we employed RNAi exposure post-embryonically to allow testing genes whose knockdown results in embryonic lethality. We identified 54 novel regulators of the ROS–induced UPR^mt. Activation of the UPR^mt, but not of other stress-signaling pathways, failed when homeostasis of basic cellular mechanisms such as translation and protein transport were impaired. These mechanisms are monitored by a recently discovered surveillance system that interprets interruption of these processes as pathogen attack and depends on signaling through the JNK-like MAP-kinase KGB-1. Mutation of <i>kgb-1</i> abrogated the inhibition of ROS–induced UPR^mt, suggesting that surveillance-activated defenses specifically inhibit the UPR^mt but do not compromise activation of the heat shock response, the UPR of the endoplasmic reticulum, or the SKN-1/Nrf2 mediated response to cytosolic stress. In addition, we identified PIFK-1, the orthologue of the <i>Drosophila</i> PI 4-kinase four wheel drive (FWD), and found that it is the only known factor so far that is essential for the unfolded protein responses of both mitochondria and endoplasmic reticulum. This suggests that both UPRs may share a common membrane associated mechanism.</p> </div

Knockdown of <i>rpl-36</i>, <i>atfs-1</i>, and <i>pifk-1</i> suppresses the <i>isp-1(qm150)</i>–mediated induction of the <i>hsp-6</i> reporter.

<p>The <i>isp-1(qm150)</i> mutant of mitochondrial superoxide <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003346#pgen.1003346-Schuster1" target="_blank">[17]</a> constitutively activated the <i>Phsp-6</i> reporter (<i>Phsp-6::gfp</i>). RNAi of all three tested genes suppressed (p<0.001) the constitutive <i>hsp-6</i> reporter gene induction. Representative micrographs (A) and quantification of GFP fluorescence intensity (B). <i>hsp-6</i> reporter worms carrying the <i>qm150</i> allele were analyzed for GFP expression after one week on the respective RNAi plates. Columns represent pooled values of three independent experiments plus standard error of the mean (SEM). Numbers in columns indicate the number of analyzed animals (n_total = 317). <i>***:</i> p<0.001; Kruskal-Wallis test plus Dunn's Multiple Comparison Test. Equal optical settings per row, scale bar 100 µm. <i>(i)</i>: RNAi; L4440: empty vector control.</p

The knockdown of <i>rpl-36</i> and <i>atfs-1</i> does not prevent non-mitochondrial stress responses.

<p>Worms were grown from L1 larval stage on the respective RNAi plates before being exposed to the respective stress and analyzed four days after L1. A. A reporter strain for the SKN-1 dependent phase II response <i>(Pgst-4</i>::<i>gfp)</i> was exposed to 2.1 mM acrylamide starting at early L3 stage. RNAi of <i>rpl-36</i> and <i>atfs-1</i> did not prevent reporter gene induction as compared to vector control. Columns represent pooled normalized values of four independent experiments plus standard error of the mean (SEM). Numbers in columns indicate the number of analyzed animals (n_total = 613). ***: p<0.001; Kruskal-Wallis test plus Dunn's Multiple Comparison Test; Mann Whitney test (comparison of vector with and without acrylamide). Equal optical settings, scale bar 200 μm. B. Cytosolic UPR (heat shock) reporter worms <i>(Phsp-16</i>.<i>2</i>::<i>gfp)</i> were exposed to 34°C for 4h at L4. The knockdown of <i>rpl-36</i> and <i>atfs-1</i> significantly decreased heat stress induced reporter expression (p<0.001). Columns represent pooled normalized values of two independent experiments plus standard error of the mean (SEM). Numbers in or on columns indicate the number of analyzed animals (n_total = 266). <i>***</i>: p<0.001; Kruskal-Wallis test plus Dunn's Multiple Comparison Test; Mann Whitney test (comparison of vector with and without heat shock). Equal optical settings, scale bar 100 μm. C. The UPR^ER reporter strain (<i>Phsp-4</i>::<i>gfp</i>) was raised from L1 stage RNAi plates (with 7.2 μg/ml tunicamycin). UPR^ER induction was not blocked by any RNAi tested here, but <i>rpl-36</i> (RNAi) strongly impaired its induction (p<0.001). Columns represent pooled normalized values of four independent experiments plus standard error of the mean (SEM). Numbers in or on columns indicate the number of analyzed animals (n_total = 363). <i>***</i>: p<0.001; Kruskal-Wallis test plus Dunn's Multiple Comparison Test; Mann Whitney test (comparison of vector with and without tunicamycin). Equal optical settings, scale bar 100 μm. <i>(i)</i>: RNAi; L4440: empty vector control.</p