To identify novel lipid genes that influence the UPR^ER, we carried out an RNAi screen under conditions of proteotoxic stress. We utilized the hsp-4 (C. elegans Hsp70/BiP) transcriptional GFP reporter (hsp-4p::GFP) to assess the UPR^ER induction, and sec-11 (an ER serine-peptidase) RNAi to generate ER stress and induce the GFP reporter (Calfon et al., 2002). In combination with the sec-11 RNAi, we individually knocked down each candidate gene to perform a double RNAi screen (Figure 1A). We focused on proteins associated with LDs instead of general lipid synthesis genes due to the LD’s central role in lipid regulation and their contribution to ER proteostasis (Moldavski et al., 2015; Vevea et al., 2015). By cross-referencing two independent C. elegans LD proteomic datasets, we identified 163 high-confidence LD proteins (Zhang et al., 2012; Na et al., 2015; Vrablik et al., 2015). We found that RNA interference of 49 genes led to reduced induction of the UPR^ER reporter (Supplementary file 1). While a large portion of these genes are annotated as functioning in general translation (e.g. ribosomal subunits), potentially affecting global gene expression, a subset of 11 belonged to other functional groups and were therefore more likely to specifically affect the UPR^ER (Figure 1—figure supplement 1A). From this subset of genes only let-767, an acyl-CoA reductase, was noted as having lipid enzymatic activity, while the other genes function as ATP synthases, GTPases, heat shock proteins, ATPases, methyltransferases, and tubulin.

We focused on let-767 of the screen subset due to its direct role in lipid metabolism, which has been previously characterized (Entchev et al., 2008; Desnoyers et al., 2007; Kuervers et al., 2003). let-767 has been implicated in long-chain fatty acid (LCFA) and monomethyl branched-chain fatty acid (mmBCFA) synthesis as an acyl-CoA reductase, and in steroid metabolism as a 17-beta hydroxysteroid dehydrogenase that directly acts on steroid molecules. Within the fatty acid elongation pathway, after addition of a malonyl-CoA, acyl-CoA reductase is responsible for reduction of the 3-ketoacyl-CoA into a 3-hydroxyacyl-CoA which is then further processed into a fatty acid with 2 more carbon molecules. Knockdown of let-767 suppressed the UPR^ER induction caused by RNAi of two different ER genes, sec-11, which would impact ER signal peptide cleavage, and tag-335, a GDP-mannose pyrophosphorylase, which would reduce protein glycosylation (Figure 1B–C; Bar-Ziv et al., 2020). Furthermore, qPCR analysis confirmed that let-767 RNAi also resulted in reduced splicing of xbp-1u to xpb-1s while under stress, suggesting that loss of let-767 suppresses the UPR^ER induction by preventing IRE-1’s splicing of xbp-1 (Figure 1—figure supplement 1B). Next, we utilized the transcriptional reporters of the mitochondrial unfolded protein response (UPR^mt, hsp-6p::GFP) and the heat shock response (HSR, hsp-16.2::GFP) to determine the effect of let-767 knockdown on other stress responses. We observed that let-767 RNAi does not suppress the heat shock response and only has minor effects on the mitochondrial stress response in comparison to the highly suppressed UPR^ER (Figure 1D–G). Together, these results show that the function of let-767 is specifically important for ER function and homeostasis.

Both fatty acid elongation and steroid processing is carried out by steroid hydrogenases within mammals (Sakurai et al., 2006). To determine whether reduced UPR^ER induction was a general phenotype of steroid dehydrogenase knockdown, we performed our double RNAi protocol on the four most closely related (steroid dehydrogenase family) genes found in C. elegans, stdh-1,2,3 and 4. Knockdown of any of the stdh genes had only mild effects on the UPR^ER induction, showing that the diminished UPR^ER induction phenotype was more specific to let-767 and not a general phenomenon of decreased steroid dehydrogenase function (Figure 1—figure supplement 1C–D). We then tested whether RNAi of other genes implicated in the LCFA and mmBCFA pathways also affected the UPR^ER induction (Zhang et al., 2011; Kniazeva et al., 2004; Kniazeva et al., 2012). Of these genes, acs-1 had the most similar phenotype to let-767, while elo-5 and hpo-8 had a significant, but less severe effects on the UPR^ER induction without stalling development, like pod-2 (Figure 1—figure supplement 1E–F). acs-1 functions in the first step of fatty acid processing by attaching a CoA to the fatty acid, particularly for the mmBCFA isoC17 (Kniazeva et al., 2012; Zhang et al., 2021). While elo-5 and hpo-8 function as a fatty acid elongase and a 3-hydroxyacyl-CoA dehydratase, the steps before and after let-767 in the fatty elongation pathway, respectively. These results indicate that perturbation of the LCFA/mmBCFA pathways negatively impacts the UPR^ER, with let-767 and acs-1 being the most critical.

Phenotypes caused by a reduction in the LET-767 enzyme could likely result from insufficient production of key metabolites, i.e., mmBCFAs or LCFAs. The UPR^ER induction might then be restored to wild-type levels by supplementation of these lipids. To this end, we supplemented animals with a crude lysate composed of homogenized N2 (wild type) adult animals to provide a complete panel of lipids. We observed that supplementation of lysate was sufficient to rescue the phenotypes of acs-1 RNAi, including suppression of the UPR^ER upon ER stress (Figure 2—figure supplement 1A–B). Phenotypes of acs-1 RNAi have been shown to be a results of insufficient lipid species, suggesting that the lysate is sufficient to rescue deficiencies in lipids (Zhang et al., 2021). However, lysate supplementation of let-767 RNAi treated animals resulted in a significant improvement in organismal size, but only a slight improvement in the UPR^ER induction (Figure 2A–B). The ability of lysate supplementation to rescue a known essential lipid phenotype, but not suppression of the UPR^ER from let-767 RNAi, would suggest a potential mechanistic difference between the UPR^ER and size phenotypes. Although the reduced animal size is likely the result of insufficient lipids, the suppression of the UPR^ER is potentially due to other complications caused by knockdown of the let-767 pathway.

To investigate whether lysate supplementation had an impact on ER and lipid subcellular phenotypes of let-767 RNAi, we further characterized the impact of let-767 RNAi and lysate supplementation on the LD and ER morphologies. We found that knockdown of let-767 caused an extreme reduction in LD size, from large spheres to small points (Figure 2C). let-767 knockdown also resulted in substantial changes to the ER morphology, practically eliminating the wider structures of the ER. However, unlike the UPR^ER induction, supplementation of lysate improved the organelle morphology appearance by restoring the presence of wide ER structures and lipid droplets, albeit at considerably lower abundance and size (Figure 2C–D). These observations mark a distinction between the ability of supplementation to rescue morphological and functional phenotypes of the ER caused by let-767 RNAi. The rescue of animal size and ER morphology phenotypes by lysate supplementation hint that they are a result of insufficient lipids, while the abilities of the ER to induce the UPR^ER and expand lipid droplets are potentially not due to the depleted levels of lipids.

One potential reason for the ineffective rescue of the UPR^ER induction by lysate could be insufficient levels of lipids within the lysate. We sought to determine whether excess supplementation of two lipids associated with let-767, mmBCFAs or LCFAs (Entchev et al., 2008), could rescue the UPR^ER induction of animals with reduced let-767. We supplemented animals grown on let-767 and sec-11 RNAi with exogenous isoC17, an essential mmBCFA, or oleic acid, a LCFA known to be a significant component of the C. elegans fatty acid content and to increase lifespan (Imanikia et al., 2019; Kniazeva et al., 2004; Henry et al., 2016). mmBCFA supplementation was not sufficient to rescue induction of the UPR^ER in let-767 knockdown animals, showing only a slight improvement in the stress response induction (Figure 2—figure supplement 1C–D). However, mmBCFA supplementation was sufficient to rescue phenotypes of acs-1 RNAi, providing evidence that our supplementation effectively delivered the essential mmBCFA and was also sufficient to rescue functional phenotypes of mmBCFA insufficiency (Figure 2—figure supplement 1E–F). Similar to isoC17, supplementation of oleic acid showed a very minor improvement in the ER stress response of let-767 knockdown animals (Figure 2—figure supplement 1G–H). These data show that while the UPR^ER dysfunction caused by let-767 knockdown is likely not due to the specific loss of the essential mmBCFA or LCFA (oleic acid), isoC17 is indeed essential to the UPR^ER in the absence of acs-1.

Since supplementation of a WT mixture of lipids was not sufficient to recover the UPR^ER induction, but was able to rescue ER morphology, we considered whether let-767 knockdown could be compromising the ER membrane through unbound LET-767 partners or accumulation of upstream metabolic intermediates. A disrupted membrane could hinder ER membrane protein function for ER stress signaling as well as for lipid droplet and lipid synthesis enzymes, explaining both phenotypes and why the restored ER morphology did not coincide with restored UPR^ER function. Indeed, a disrupted ER membrane affecting ER function was suggested as the mechanistic cause of acs-1 phenotypes, where insufficient isoC17 disrupted the ER membrane quality and hindered lipid droplet production (Zhang et al., 2021). Furthermore, ER stress through sec-11 RNAi alone caused a reduction in let-767 transcript levels (Figure 1—figure supplement 1B), suggesting that a reduction of let-767 itself is not directly suppressing the UPR^ER induction, but possibly dependent on the levels of other factors. Instead, we hypothesized that disequilibrium within the let-767 pathway could be the cause of membrane disruption and ultimately the loss of UPR^ER induction. To identify a lipid that could disrupt the ER membrane, we performed untargeted complex lipidomic analysis of worms treated with let-767 RNAi (Supplementary file 3, Source data 1). We observed a reduction in most lipids, including nearly all triglycerides, which would be expected from the reduction in lipid droplets. However, we were not able to identify a potentially accumulated lipid, with many unknown features unable to be accurately identified.

Therefore, we tested whether reducing potentially accumulated upstream metabolites of let-767 would improve the UPR^ER activation. Since the complete enzymatic pathway for let-767 has yet to be definitively identified and previous works have implicated the enzyme in multiple pathways, we accomplished this by knocking down the ortholog of human SREBP, sbp-1, a major transcriptional regulator of numerous lipogenic enzymes and pathways (Nomura et al., 2010). Analysis of a published RNAseq dataset of sbp-1 RNAi treated nematodes showed downregulation of numerous lipid synthesis genes including the fatty elongation pathway genes, such as hpo-8, elo-1/2/4/5/6/9, and let-767, the latter of which we confirmed through qPCR (Figure 3A; Lee et al., 2015). We found that animals grown on let-767 and sbp-1 RNAi appeared larger and healthier than on let-767 RNAi alone. More importantly, knockdown of sbp-1 was able to significantly improve the UPR^ER reporter induction in let-767 knockdown animals under ER stress from sec-11 RNAi and effectively restore splicing of xbp-1 to xbp-1s (Figure 3A–C). To test whether the improved UPR^ER signaling was indeed a functional restoration of the ER stress response, we performed an ER stress survival assay. As expected, animals treated with let-767 RNAi have a severe defect in survival when exposed to the proteotoxic stress of Tunicamycin, an inhibitor of N-linked glycosylation. Correlating with our observations of the UPR^ER reporter, sbp-1 knockdown rescued the survival of let-767 RNAi treated animals to the level of sbp-1 RNAi alone (Figure 3D–E, Supplementary file 2). Interestingly, sbp-1 RNAi itself reduced the induction level of the UPR^ER reporter and the survival rate of animals on Tunicamycin, suggesting that while a reduction in the entire let-767 pathway could indeed rescue the UPR^ER suppression caused by let-767 RNAi, a global reduction in lipid synthesis enzymes limits the maximal induction of the UPR^ER.

Finally, we determined whether sbp-1 RNAi would also rescue the defects in LD and ER morphology caused by let-767 knockdown. Congruent with SREBP’s central role in promoting lipogenesis, we observed a depletion of lipid droplets and a slight perturbation of the ER morphology with sbp-1 RNAi (Figure 3F). In combination with let-767 RNAi, sbp-1 knockdown did not rescue the ER and lipid droplet morphology to WT conditions, highlighting that restoring the UPR^ER signaling is not completely dependent on restoring ER lipid levels and morphology. However, in the presence of lysate supplementation, sbp-1 RNAi alone or combined with let-767 RNAi resulted in ER and lipid droplet morphology resembling that of WT animals with the presence of wide sheet-like structures and larger lipid droplets. Therefore, a more complete rescue of the phenotypes exhibited by let-767 knockdown animals could be achieved by a combination of (1) the reduction of the let-767 pathway through knockdown of sbp-1 to reduce global lipid synthesis, and (2) exogenous supplementation of the lipids to restore lipids lost by sbp-1 and let-767 RNAi.

Proper UPR^ER signaling is dependent on dimerization of IRE-1 at the ER membrane to splice xbp-1 mRNA to its active xbp-1s isoform. Therefore, one possible mechanism by which loss of let-767 can impact the UPR^ER is by impeding IRE-1 activity through altered membrane dynamics. Indeed, knockdown of let-767 results in altered ER membrane dynamics where the mobile fraction of an ER transmembrane protein (SPCS-1) is significantly reduced when measured by Fluorescence Recovery After Photobleaching (FRAP) (Figure 4A–B). The percent mobile being the calculated fraction of fluorophore that can move within the bleached area during the FRAP process. Supplementation of lysate improved the ER membrane mobility to 62% of WT levels. Comparatively, ER luminal mRuby dynamics, which would likely be indirectly affected by the global changes in ER 3D structure, were less severely impacted by let-767 RNAi. These effects were rescued to a greater extent by lysate supplementation, potentially due to the recovered ER structure, restoring the mobile fraction of let-767 RNAi treated animals to 84% of WT levels (Figure 4C–D). While specific changes in dynamics are likely dependent on the individual protein being observed, our results provide evidence that membrane dynamics have been altered when let-767 is knocked down.

To determine whether the UPR^ER was indeed being affected at the ER membrane, we examined the impact of let-767 knockdown on ectopic UPR^ER activation at two different points along the mechanistic pathway: (1) overexpression of ire-1, which would ectopically activate the UPR^ER at the membrane by constitutively splicing xbp-1 and (2) overexpression of the active xbp-1s isoform, which would bypass the requirement of IRE-1 splicing at the ER membrane (Li et al., 2010). In creating the necessary strains, we found that intestinal over-expression of the full-length ire-1a isoform proved to be lethal. However, overexpression of the ire-1b isoform lacking the luminal domain, fused with the mRuby fluorophore (mRuby::ire-1b), was viable and had constitutive activation of the UPR^ER. To ensure that our fusion protein was responsible for activating the UPR^ER, we knocked down ire-1 through an RNAi targeting the N-terminal region only found in ire-1a. The ire-1a RNAi was able to suppress the UPR^ER signaling in WT animals with and without ER stress but did not eliminate the basal UPR^ER signal in animals expressing mRuby::ire-1b. Conversely, RNAi targeting common sequences of both ire-1a and ire-1b suppressed the UPR^ER induction in both WT and animals expressing our mRuby::ire-1b construct, even when ER stress was induced by sec-11 RNAi. (Figure 5—figure supplement 1A–D). Therefore, our mRuby::ire-1b construct was sufficient to induce the intestinal UPR^ER without the endogenous ire-1a. Likewise, we confirmed that overexpression of our intestinal mRuby::xbp-1s construct was able to induce the UPR^ER in an xbp-1 (zc12) null-mutant background (Figure 5—figure supplement 1E–F). Beyond observing that our construct was sufficient to induce the UPR^ER, we also found that loss of endogenous xbp-1 resulted in an even higher level of basal UPR^ER induction, potentially due to a negative regulatory role for unspliced xbp-1, that has been previously suggested (Yoshida et al., 2006).

Next, we tested UPR^ER induction of our ire-1 and xbp-1s overexpression strains on let-767 RNAi. let-767 knockdown reduced the induction of the UPR^ER reporter in mRuby::ire-1b animals (Figure 5A–B). However, let-767 RNAi also reduced the UPR^ER reporter induction in the mRuby::xbp-1s, xbp-1(zc12) strain (Figure 5C–D). By utilizing the mRuby::xbp-1s, xbp-1 (zc12) null-mutant we eliminated the possibility that the reduced induction was due to the negative regulatory effects of unspliced xbp-1s. To ensure that the reduced UPR^ER induction was not due to altered expression of our mRuby::xbp-1s construct, we performed qPCR for xbp-1 in animals grown on let-767 RNAi. We observed a significant drop in the percent of xbp-1 splicing in animals expressing mRuby::ire-1b when they were grown on let-767 RNAi, but not in animals expressing mRuby::xbp-1s (Figure 5E–F). However, the reduced UPR^ER induction despite the similar levels of xbp-1s transcripts between EV and let-767 RNAi suggests that in addition to affecting xbp-1 splicing, let-767 RNAi is likely affecting xbp-1s activity downstream of its splicing.

To identify a node within the let-767 pathway that might be responsible for the disruptive metabolite or protein, we utilized mammalian cell culture to probe the effect of specific metabolites in the let-767 pathway on ER homeostasis. This would allow us to saturate cellular exposure to lipid intermediates and avoid the possibility of bacteria processing the intermediates prior to digestion by C. elegans animals. Previous works have provided evidence that the human ortholog of let-767 are HSD17B12 and HSD17B3 (Entchev et al., 2008). Interestingly, LET-767 shares the most protein sequence percent identity (39.87%) with HSD17B12, the 3-ketoacyl reductase, which caused similar phenotypes in mice when knocked out. HSD17B12 was found to be essential for development in mice and knockout of the gene in adult mice resulted in reduced body weight, reduced lipid content, and caused liver toxicity hypothesized to be from accumulation of toxic intermediates (Rantakari et al., 2010; Heikelä et al., 2020).

3-Ketoacyl reductases perform the second step in fatty acid synthesis/elongation, metabolizing 3-oxoacyl-CoA to 3-hydroxyacyl-CoA (Figure 6A; Moon and Horton, 2003). While the fatty acid elongation pathway has not been shown to affect the UPR^ER, use of the fatty acid synthase inhibitor, Cerulenin, has been shown to increase levels of XBP1s with reduced transcriptional activity due to changes in palmitoylation (Chen et al., 2020). From these studies, we hypothesized that the accumulation of the metabolites upstream of LET-767 could be reducing the transcriptional activity of XBP-1S. To this end, we tested whether an upstream metabolite of LET-767 was sufficient to reduce UPR^ER induction in huh7 cells containing a 5 x unfolded protein response element (UPRE) GFP reporter and EIF2A promoter driving mCherry. As fatty acid elongation extends fatty acids beyond the 16 carbons synthesized by fatty acid synthase, we supplemented our reporter line with the 18 carbon fatty acid metabolites upstream and downstream of the 3-ketoacyl reductase in combination with Tunicamycin to induce ER stress (Figure 6B–C). We observed that the upstream metabolite, 3-oxostearic acid, reduced the normalized 5xUPRE reporter induction. In comparison, the downstream metabolites, 3-hydroxystearic acid and stearic acid, did not reduce the normalized 5xUPRE reporter signal. Instead, stearic acid caused a slight induction of the reporter signal, in agreement with studies showing that saturated lipids induce the UPR^ER (Volmer et al., 2013). This indicated that the 5xUPRE was specifically sensitive to the 3-oxo metabolite upstream of the let-767 pathway and that this interaction between lipid metabolism and the UPR^ER is likely conserved in mammalian cells.

N2 Bristol, LIU1 (ldrIs[dhs-3p::dhs-3::GFP]), SJ4005 (zcIs4[hsp-4p::GFP]), SJ17 (xbp-1(zc12) III; zcIs4 [hsp-4p:GFP] V), SJ4100 (zcIs13[hsp-6p::GFP]), CL2070 (dvIs70[hsp-16.2p::GFP]), VS25 (hjIs[vha-6p::GFP::C34B2.10(SP12) +unc-119(+)]),EG6703 (unc-119(ed3); cxTi10816; oxEx1582[eft-3p::GFP +Cbr-unc-119]) strains were obtained from the Caenorhabditis Genetics Center (CGC). AGD2192 (uthSi60[vha-6p::ER-signal-sequence::mRuby::HDEL::unc-54 UTR, cb-unc-119(+)] I; unc-119(ed3) III) (Daniele et al., 2020). Transgenic strains created for this study were generated from EG6703 via the MosSCI method (Yanagitani et al., 2011) or through crossing strains.

Transgenic strains created:

All worms were maintained at 20 °C on NGM agar plates seeded with OP50 E. coli bacteria. Prior to experiments, worms were bleach synchronized as described in Higuchi-Sanabria et al., 2018, followed by overnight L1 arrest in M9 buffer (22 mM KH2PO4 monobasic, 42.3 mM Na2HPO4, 85.6 mM NaCl, 1 mM MgSO4) at 20 °C. For RNAi experiments, arrested L1s were plated on 1 µm IPTG, 100 µg/mL Carbenicillin NGM agar plates maintained at 20 °C and seeded with RNAi bacteria grown in LB +100 µg/mL Carbenicillin.

Transcriptional reporter strains were imaged using a Leica DFC3000 G camera mounted on a Leica M205 FA microscope. Worms were grown to day 1 of adulthood at 20 °C, hand-picked, and immobilized with 100 mM Sodium Azide M9 buffer on NGM agar plates. Raw images were cropped, and contrast matched using ImageJ software.

For the initial screen of hsp-4p::GFP reporter animals, fluorescence was scored by eye using the following criteria: 2=increased fluorescence, 1=possible increase in fluorescence, 0=no change, –0.5=small regions of dimmer fluorescence, –1=small regions of complete loss of fluorescence, –1.5=globally dimmer fluorescence and some regions of no fluorescence, –2=global loss of fluorescence and small regions of dim fluorescence, –2.5=global loss of fluorescence except for regions within spermatheca, –3=complete loss of fluorescence.

Confocal images were acquired using a Leica Stellaris 5 confocal platform with a ×63 objective. Day 1 worms were picked onto 100 mM Sodium Azide M9 buffer on slides and imaged within 30 min. Raw images were cropped and independently contrast optimized for clarity using ImageJ software.

Transcriptional reporter strains were grown to day 1 of adulthood at 20 °C. Animals were collected into a 15 mL conical tube with M9 buffer and allowed to settle at the bottom before supernatant was aspirated. Animals were resuspended in M9 buffer and analyzed using a Union Biometrica COPAS Biosorter (P/N: 350-5000-000) as described in Bar-Ziv et al., 2020. Animals which saturated the signal capacity of 65532 or were outliers in both animal size parameters (i.e., Extinction and Time Of Flight) were censored. Mann-Whitney statistical tests were performed on fluorescence normalized to animal extinction using GraphPad Prism software. Integrated fluorescence is normalized to integrated extinction (as a proxy for size) during quantification. Biosorter plots shown are of populations from a single experiment.

Animals grown on EV or RNAi bacteria were collected at day 1 of adulthood using M9 and washed 3 x. M9 was aspirated and trizol added before 3 cycles of freeze/thaw in liquid nitrogen. Chloroform was then added at a ratio of 1:5 (chloroform:trizol). Separation of RNA was performed through centrifugation in gel phase-lock tubes. The RNA aqueous phase was transferred into new tubes containing isopropanol. RNAi was purified using the QIAGEN RNeasy Mini Kit (74106) according to the manufacturer’s instructions. cDNA was synthesized using 2 µg of RNA and the QIAGEN QuantiTect Reverse Transcriptase kit (205314) according to the manufacturer’s instructions. qPCR was performed using SYBR-green. Analysis was performed for each biological replicate using the Delta-Delta CT method with pmp-3, cdc-42, and Y45F10D.4 as housekeeping genes (Hoogewijs et al., 2008).

Crude lysate was obtained from N2 worms grown on 40 x concentrated EV bacteria at 20 °C.~120,000 day 1 adult worms were collected with M9 and washed 6 x with M9 buffer. Animals were then homogenized 20 x in 3 mL of M9 buffer using an ice-cold 15 mL Dura-Grind Stainless Steel Dounce Tissue Grinder (VWR, 62400–686). Crude lysate was transferred to 1.5 mL tubes and frozen in liquid N2.

Supplementation experiments were prepared by mixing 4 x concentrated RNAi bacteria with crude lysate at a 2:1 ratio, respectively. The lysate mixture was plated on RNAi plates and allowed to dry. Dried plates were then UV irradiated without lids for 9 min in an ultraviolet crosslinker (UVP, CL-1000) before plating L1 arrested worms on the plate.

Lipid supplementation experiments were prepared by inoculating cultures (LB +100 µg/mL Carbenicillin) with RNAi bacteria or empty vector and then adding the lipids to the specified concentration or equal volumes of ethanol. The cultures were allowed to grow overnight to saturation and then concentrated to 4 x before being plated on RNAi plates. The bacteria was allowed to dry overnight and then UV irradiated without lids for 9 min in an ultraviolet crosslinker (UVP, CL-1000) before plating L1 arrested worms on the plate.

Tunicamycin survival assays were conducted on NGM agar plates containing 25 ng/µL of tunicamycin in DMSO, or equal volume of DMSO with specified RNAi bacteria at 20 °C. Animals were moved daily for 4–7 days to new RNAi plates until progeny were no longer observed. Worms with protruding intestines, bagging phenotypes, or other forms of injury were scored as censored and not counted as part of the analysis. For combined RNAi lifespans, saturated cultures were mix 1:1 by volume.

FRAP 4D images were acquired using a 3i Marianas spinning-disc confocal platform. Photobleaching of a 10 µm x 10 µm region within a 4 µm Z-stack was performed using a 488 nm laser for 1–2ms. Raw images were processed with FIJI (Schindelin et al., 2012) into sum Z-projections and aligned using the ‘RigidBody’ setting of the StackReg ImageJ plugin (StackReg, 2021). FRAP analysis was then performed with the FRAP Profiler ImageJ plugin (FRAP profiler plugin, 2021) by selecting the 10 µm x 10 µm photobleached region as region 1 and the entire fluorescent area as region 2. Briefly, the plugin calculates percent mobile by fitting an exponential curve to the plotted normalized recovered fluorescence values to define the recovery curve and determine the fraction/percent that is able to move within the ROI.

N2 animals were grown on 40 x concentrated EV or let-767 RNAi bacteria at 20 °C, respectively. Animals were collected with M9 and frozen in liquid nitrogen to be submitted to the UC Davis West Coast Metabolomics Center (WCMC) for untargeted complex lipid analysis. Peak values of lipids identified by the WCMC were first corrected by subtracting corresponding peaks of blanks and then their abundance estimated relative to internal standards of their corresponding lipid class before calculating average fold change from 6 biological replicates.

Cells were grown in DMEM media (11995, Thermo Fisher) supplemented with 2 mM GlutaMAX (35050, Thermo Fisher), 10% FBS (VWR), Non-Essential Amino Acids (100 X, 11140, Thermo Fisher), and Penicillin-Streptomycin (100 X, 15070, Thermo Fisher) in 5% CO2 at 37 °C. Huh7 were obtained from UC Berkeley Cell Culture Facility (https://bds.berkeley.edu/facilities/cell-culture#cells) and their identity confirmed through their STR profling (CELL LINE AUTHENTICATION (ucberkeleydnasequencing.com)). Mycoplasma negative status confirmed by PCR Detection Kit.

To generate a UPRER transcriptional reporter, we designed a lentiviral vector that encodes sequence for 5 x UPR response element upstream of a minimal cFos promoter, driving sfGFP (Adamson et al., 2016). The sfGFP is fused to a PEST sequence for tighter regulation of the reporter (Corish and Tyler-Smith, 1999; Loetscher et al., 1991). The vector also allows for constitutive mCherry expression for normalization of the sfGFP signal, and neomycin resistant gene for selection. The transcriptional reporter was then transduced through lentivirus into Cas9-expressing Huh7 cells and selected for using G418 at 800 µg/mL.

Cells were plated onto 10 cm plates and allowed to grow overnight. Lipids, tunicamycin, and/or vehicle were directly added to media. Cells were harvested for flow cytometry 16–18 hours after addition of supplements.

Cells were trypsinized on ice and resuspended in cold FACS buffer (PBS with 0.1% BSA and 2 mM EDTA). Samples were filtered through a 50 µm nylon filter mesh to remove clumps prior to analysis with a five-laser LSR Fortessa (BD Bioscience). Acquired data were analyzed using FlowJo 10.7.2.