Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

RNAi screening for modulators of an osmo-sensitive gene response to extracellular matrix damage reveals negative feedback and interactions with translation inhibition

  • Luke M. Chandler,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Biology and Genetics Institute, University of Florida, Gainesville, FL, United States of America

    ⨯
  • Michael Rodriguez,

    Roles Data curation, Investigation, Methodology, Validation, Writing – review & editing

    Affiliation Department of Biology and Genetics Institute, University of Florida, Gainesville, FL, United States of America

    ⨯
  • Keith P. Choe

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing

    kchoe@ufl.edu

    Affiliation Department of Biology and Genetics Institute, University of Florida, Gainesville, FL, United States of America

    ⨯

Abstract

In epidermal tissues, extracellular matrices (ECMs) function as barriers between the organism and environment. Despite being at the interface with the environment, little is known about the role of animal barrier ECMs in sensing stress and communicating with cytoprotective gene pathways in neighboring cells. We and others have identified a putative damage sensor in the C. elegans cuticle that regulates osmotic, detoxification, and innate immune response genes. This pathway is associated with circumferential collagen bands called annular furrows; mutation or loss of furrow collagens causes constitutive activation of osmotic, detoxification, and innate immune response genes. Here, we performed a genome-wide RNAi screen for modulators of osmotic stress response gene gpdh-1 in a furrow collagen mutant strain. RNAi of six genes identified in this screen were tested under other conditions and for effects on other stress responses. The functions of these genes suggest negative feedback within osmolyte accumulation pathways and interactions with ATP homeostasis and protein synthesis. Loss of these gpdh-1 modulators had distinct effects on canonical detoxification and innate immune response genes.

Citation: Chandler LM, Rodriguez M, Choe KP (2023) RNAi screening for modulators of an osmo-sensitive gene response to extracellular matrix damage reveals negative feedback and interactions with translation inhibition. PLoS ONE 18(5): e0285328. https://doi.org/10.1371/journal.pone.0285328

Editor: Myeongwoo Lee, Baylor University, UNITED STATES

Received: September 21, 2022; Accepted: April 19, 2023; Published: May 8, 2023

Copyright: © 2023 Chandler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All underlying numeric data is publicly available from the figshare repository (https://doi.org/10.6084/m9.figshare.21179404).

Funding: This study was supported by National Science Foundation grant IOS-1452948 to K.P.C; all authors received some salary from this funding source. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Extracellular matrices (ECMs) are complex networks of secreted fibrous proteins, polysaccharides, and sometimes minerals found in nearly all animal tissues [1]. Traditional functions of ECMs include structural support, tensile strength, elasticity, cell adhesion, and migration [2–4]. It is now clear that ECMs also communicate with cell signaling pathways to influence cell differentiation, tissue morphogenesis, and disease [5–7]. Proteins and growth factors in ECMs interact with cell membrane receptors to regulate migration, differentiation, and tumorigenesis [2,8–10]. Mis-regulation of ECM composition and signaling contributes to arthritis, fibrosis, cancer, and aging [11].

Barrier ECMs are the first line of defense against environmental osmotic pressures, toxins, and pathogens, and are well-positioned to sense environmental stress. Examples include a keratin and lipid-rich epidermis with a collagen foundation in mammals, a rigid chitinous exoskeleton in arthropods, and a flexible collagen-rich cuticle in nematodes [12–15]. Dermal ECM proteins have well-understood roles in transmitting acute mechanical signals to touch sensory neurons in mammals and nematodes [16,17]; alternatively, the role of animal ECMs in sensing environmental stress and regulating cytoprotective gene responses is poorly understood. In fungal cells, several transmembrane sensor proteins detect mechanical stimuli from cell wall damage or cell volume changes and regulate downstream MAPK cascades such as ‘HOG’ (High-Osmolarity Glycerol) to activate cytoprotective and cell wall repair genes [18–20]. Discovery of similar barrier ECM damage sensors in animals would provide new fundamental insights into signaling of stress responses that influence health and distributions of wild populations and degenerative diseases, aging, and longevity in humans [21–24].

The cuticle of nematodes is composed largely of collagen [12,25]. Between 14–16 years ago, genome-wide genetic screens made the surprising discovery that loss of a few cuticle collagen genes strongly activated C. elegans osmotic and innate immune response genes in the absence of stress [22,26–28]; these results suggested that the cuticle may be involved in sensing environmental stress and regulating relevant cytoprotective genes in neighboring cells. A mucin-like protein secreted by epidermal cells also negatively regulates these same cytoprotective genes further implicating the presence of an extracellular stress sensor [29,30]. To investigate the nature of this putative cuticle sensor and test if other stress responses were affected, we used RNAi to screen 40 genes that are required for diverse aspects of cuticle and epidermal integrity for activation of six core stress responses [31]. Dumpy ‘dpy’ is a class of 26 genes, most encoding cuticle collagens, that all cause a short and wide body shape when mutated or silenced. We discovered that loss of any one of a subset of six dpy genes (dpy-2, 3, 7, 8, 9, or 10) activates broad osmotic, innate immune, and detoxification stress responses without activating heat shock or organelle-specific stress responses [31]. Loss or mutation of any one of these six genes disrupts organization of circumferential bands of collagen termed ‘annular furrows’ and at least one encodes a collagen localized to furrows [31–34]. These results are consistent with furrows being part of, or interacting with, a sensor for cuticle damage that regulates three environmental stress responses related to barrier defenses.

To learn more about processes that influence this putative ECM damage signal, we used a dsRNA-expressing E. coli library to screen the C. elegans genome for genes that modify fluorescence of a reporter for canonical osmotic stress response gene gpdh-1; GPDH-1 is an enzyme for synthesis of glycerol [35]. Secondary assays and RT-qPCR verified gpdh-1 suppression in dpy-7 mutants by RNAi of two genes that function in ATP homeostasis; gpdh-1 expression was enhanced by RNAi of genes for tRNA ligases and organic osmolyte accumulation enzymes. RNAi of these genes had weak or opposite effects on expression of canonical detoxification and innate immune stress response genes suggesting specificity to the osmotic stress response. These results are consistent with glycerol synthesis being linked to energy balance, protein translation acting parallel to furrow disruption, and negative feedback within and between osmolyte accumulation pathways.

Materials and methods

C. elegans strains

All C. elegans strains were cultured at 20°C using standard methods [36]. The following strains were used: N2 Bristol, CB88 dpy-7(e88) X, and QV261 dpy-7(e88); kbIs24[gpdh-1p::dsRed2; myo-2p::GFP; pDP#MM051(unc-119(+))]. The gpdh-1p::dsRed2 reporter contains the same genomic DNA region as a GFP reporter used in prior studies [26]; it starts 3 kb upstream from the gpdh-1 start codon and is fused to dsRed2 at the beginning of exon 2.

RNAi screen

A genome-wide RNAi screen was performed by feeding worms Escherichia coli [HT115(DE3)] engineered to synthesize double-stranded RNA (dsRNA). RNAi clones were taken from the ORFeome RNAi feeding library (Open Biosystems, Huntsville, AL) and missing clones were supplemented from the MRC genomic library (Geneservice, Cambridge, UK). Bacteria clones were grown in 200 μl of LB broth with selective antibiotic using standard methods and washed in liquid NGM buffer [37]. Embryos from QV261 worms were collected by bleach synchronization and 20–30 were added to each well of 96-well plates containing bacteria in NGM buffer with 25 μg/ml carbenicillin and 3 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). When worms reached the young adult stage, they were screened manually for fluorescence with a Zeiss Discovery V12 Stereo microscope. Fluorescence was compared to worms fed control dsRNA clone pPD129.36 (LH4440) encoding a 202-bp dsRNA that is not homologous to any C. elegans gene; dsRNA clones were scored as candidate gpdh-1 modulators if dsRed2 fluorescence was either enhanced (greater fluorescence) or suppressed (less fluorescence) in at least 80% of worms in a well relative to wells containing worms that were fed control dsRNA clones.

RT-qPCR

RNAi agar plates contained dsRNA-expressing bacteria on NGM agar supplemented with 25 μg/ml carbenicillin and 3 mM IPTG. Worms were synchronized with bleach, placed on plates, and grown until the first day of adulthood. Some dsRNA clones were diluted with control dsRNA to avoid developmental arrest as follows: 1/2 atp-4, 1/2 pfk-1.1, 1/4 hars-1, and 1/8 rars-1. RT-qPCR assays were carried out as described previously using the delta-delta Ct method with some modifications [38]. Each replicate contained 7–15 young adult worms; lysates were treated with dsDNAse for 5 minutes at 37°C (ThermoFisher Scientific EN0771) before reverse transcription (Promega A5001). All reactions were performed in 10 μL volumes in a Realplex2 (Eppendorf). Relative expression was normalized to wild type worms on standard NGM agar using the reference gene rpl-2. Primers used for RT-qPCR are in S1 Table.

Microscopy

For fluorescence, worms were mounted on 2% agarose pads with 5 mM levamisole and imaged using an Olympus BX60 microscope with a Zeiss AxioCam MRm camera; GFP or RFP filters were used for fluorescence. Exposure settings were consistent for RFP across all conditions, and adjustments to contrast and brightness were made equally across conditions and evenly to whole images. Color was added and merged using ImageJ Version 1.53c. Furrows were imaged by differential interference contrast (DIC) on the same microscope. Body shape was measured as described previously [34].

Glycerol and motility assays

Glycerol assays were conducted on populations of worms grown on agar as described for RT-qPCR. Samples were lysed by sonication and processed as described previously using the PicoProbe Free Glycerol Fluorometric Assay Kit (Biovision K643-100) [31,39]. Values were normalized to total protein using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific 23227). For acute motility assays, worms were transferred to high NaCl agar by chunking and motility was scored as described previously [39].

Statistical analysis

Relative mRNA means were normalized to means for controls, log2 transformed, and then compared to a value of 1.0 with one-sample t-tests using Benjamini-Hochberg corrections for multiple comparisons. Glycerol levels were compared to control conditions using a one-way ANOVA with Dunnett post-hoc tests. p-values < 0.05 were taken as significant. Data was graphed with Prism 5.04 (La Jolla, CA). Gene ontology enrichment of genes was identified with DAVID functional analysis, and we report Benjamini adjusted P-values [40].

Results and conclusions

Identification of gpdh-1 modulators in a furrow collagen mutant

During hyperosmotic stress and in furrow collagen mutants, C. elegans accumulates the organic osmolyte glycerol in part by inducing gpdh-1 [26,35]; gpdh-1 encodes one of two C. elegans glycerol-3-phosphate dehydrogenase enzymes and is constitutively induced in furrow collagen mutants [26,31]. Fluorescent reporters driven by the gpdh-1 promoter correlate well with gpdh-1 mRNA levels [26,31]. In an initial genome-wide screen, 31 clones enhanced, and 242 clones suppressed gpdh-1p::dsRed2 fluorescence in dpy-7(e88) worms (S2 Table and Fig 1A). After rescreening these candidates and verifying plasmid sequences, there were eight confirmed dsRNA clones that enhanced, and seven that suppressed, gpdh-1p::dsRed2 fluorescence in at least two out of three trials (S3 Table).

thumbnail
Download:
Fig 1. Whole-genome screening identified modulators of gpdh-1 in dpy-7 worms.

(A) Schematic model of dsRNA screening and scoring of dpy-7(e88);gpdh-1p::dsRed2 worms. (B) Mean plus standard error of relative gpdh-1 mRNA levels in worms fed dsRNA expressing bacteria that changed gpdh-1p::dsRed2 fluorescence. N = 3 to 12 populations of worms, *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-sample t-test with multiple comparisons corrections versus a normalized value of 1.0.

https://doi.org/10.1371/journal.pone.0285328.g001

Using RT-qPCR in a dpy-7 background, two suppressor and three enhancer dsRNA clones were confirmed to significantly affect gpdh-1 mRNA levels in the directions expected based on reporter scoring (Fig 1B); another clone, hmit-1.2, caused a 60% increase that was near the significance threshold when adjusted for repeated measures (adjusted p = 0.098). The failure of other dsRNA clones to affect gpdh-1 mRNA in these assays could be caused by inconsistent RNAi effects or transgene array expression modification (e.g., tam-1 and add-1) [41,42]; it is also possible that some act post-transcriptionally because the gpdh-1p::dsRed2 reporter used for screening includes some coding sequence and O-GlcNAc transferase OGT-1 was recently shown to regulate GPDH-1 protein [43]. Images of gpdh-1p::dsRed2 fluorescence in dpy-7 worms fed atp-4, pfk-1.1, hmit-1.2, hars-1, pgph-3, or rars-1 dsRNA clones are shown in S1 Fig; relative gpdh-1p::dsRed2 fluorescence generally matched relative gpdh-1 mRNA changes. Loss of furrows from the cuticle can be observed with high magnification DIC [34,44]; as shown in S2 Fig, furrows were not restored by any of these dsRNA clones. Furthermore, the associated Dpy (short and wide) body shape phenotype [34] was not suppressed or enhanced by any of these dsRNA clones (S3 Fig).

The gpdh-1 modulators can be grouped into three functional categories: ATP homeostasis (atp-4 or pfk-1.1), protein synthesis (rars-1 or hars-1), and osmolyte accumulation (pgph-3 or hmit-1.2). ATP-4 is a homolog of mitochondrial coupling factor 6 and a subunit of ATP synthase, and PFK-1.1 is core glycolysis enzyme phosphofructokinase [45]. RARS-1 and HARS-1 are aminoacyl-tRNA ligases that link tRNAs to arginine and histidine, respectively, for protein translation [46]. PGPH-3 is a glycerol-3-phosphate phosphatase and catalyzes the final step of glycerol synthesis from a glycolysis intermediate. HMIT-1.2 is a homolog of H+/myo-inositol transporters that is induced by osmotic stress and promotes viability in worms cultured in high concentrations of NaCl [47].

Effects of gpdh-1 modulators vary with condition

Our RNAi screen and results in Fig 1B are from dpy-7 worms with disrupted furrows. Exposure to high osmolarity activates many of the same stress response genes as furrow disruption but also likely involves other damage signals including impaired protein translation [26,31,48,49]. We next tested silencing of gpdh-1 modulators in wild type worms under basal (51 mM NaCl) and mild hyperosmotic conditions (200 mM NaCl) (Fig 2). To make comparisons simple and to highlight the effects of RNAi within each condition, data in each panel of Fig 2 are normalized to control RNAi within each condition and results for dpy-7 worms are replotted from Fig 1B. Relative to the basal condition (wild type at 51 mM NaCl), gpdh-1 mRNA levels were induced 19.10 and 7.91-fold in dpy-7 and 200 mM NaCl worms, respectively (Fig 2B and 2C).

thumbnail
Download:
Fig 2. Effects of gpdh-1 modulators vary with condition.

Mean plus standard error of relative gpdh-1 mRNA levels in worms fed dsRNA expressing bacteria in wild type (A), dpy-7(e88) worms on 51 mM (replotted from Fig 1B) (B), and wild type worms on 200 mM NaCl agar (C). The control RNAi worms in dpy-7 (B) and 200 mM NaCl (C) had gpdh-1 mRNA levels that were elevated 19.1 and 7.9-fold relative to wild type 51 mM controls, respectively. N = 3 to 12 populations of worms, *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-sample t-test with multiple comparisons corrections versus a normalized value of 1.0.

https://doi.org/10.1371/journal.pone.0285328.g002

RNAi of atp-4 or pfk-1.1 only reduced gpdh-1 expression in dpy-7 worms with no effect under basal or hyperosmotic conditions suggesting that their effects are specific to worms with disrupted furrows (Fig 2). Hyperosmotic stress decreases protein translation [49]. Loss of aminoacyl-tRNA ligases decreases protein translation and increases gpdh-1 expression in wild type worms under basal conditions; these results are consistent with a model in which disruption of protein synthesis can act as a signal for osmotic stress [26,49]. As shown in Fig 2, RNAi of either aminoacyl-tRNA ligase gene rars-1 or hars-1 increased gpdh-1 expression under all three conditions consistent with translation inhibition being at least partially additive to furrow disruption and mild hyperosmolarity. RNAi of either osmolyte accumulation gene pgph-3 or hmit-1.2 increased gpdh-1 expression in 200 mM NaCl but had no effect on wild type worms in 51 mM NaCl (Fig 2A and 2C). These results suggest that loss of osmolyte accumulation genes reduces negative feedback onto gpdh-1 expression under conditions that normally activate hyperosmotic stress responses.

Enhancement of gpdh-1 expression by hmit-1.2 RNAi suggests crosstalk between myo-inositol and glycerol accumulation pathways. C. elegans have three hmit paralogs that are expressed in different tissues; hmit-1.1 and hmit-1.2 are recent gene duplicates with many regions of nearly identical nucleotide coding sequence [47]. As shown in Fig 3A, the hmit-1.2 dsRNA clone targets both hmit-1.1 and hmit-1.2 mRNA without affecting hmit-1.3. hmit-1.1 is expressed in the intestine with gpdh-1 and is the paralog mostly strongly induced by hyperosmotic stress and loss of furrow collagens [26,31,47]. Fig 3B shows effects of gpdh-1 modulator RNAi on hmit-1.1 expression; relative to wild type worms, hmit-1.1 mRNA levels were induced 205-fold in dpy-7 worms. RNAi of pgph-3 further enhanced hmit-1.1 mRNA 4-fold in dpy-7 worms; together with data in Fig 2B, this result suggests that crosstalk between osmolyte accumulation pathways occurs in both directions.

thumbnail
Download:
Fig 3. Glycerol and myo-inositol accumulation pathways interact via feedback.

(A) Mean plus standard error of relative hmit-1.1, hmit-1.2, and hmit-1.3 mRNA levels in dpy-7(e88) worms fed dsRNA for hmit-1.2. N = 4 populations of worms. (B) Mean plus standard error of relative hmit-1.1 mRNA levels in dpy-7(e88) worms fed dsRNA of gpdh-1 modulators. N = 6 to 7 populations of worms, *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-sample t-test with multiple comparisons corrections versus a normalized value of 1.0.

https://doi.org/10.1371/journal.pone.0285328.g003

RNAi of pfk-1.1 had no effect on hmit-1.1 mRNA (Fig 3B). Unlike their strong enhancing effect on gpdh-1 mRNA (Fig 2), neither rars-1 nor hars-1 RNAi increased hmit-1.1 mRNA suggesting that reduced protein synthesis may not activate myo-inositol accumulation mechanisms.

RNAi of rars-1 or hars-1 increases glycerol accumulation in dpy-7 worms

Given that gpdh-1 encodes a rate-limiting enzyme for glycerol synthesis and that furrow collagen mutant worms accumulate high levels of glycerol, we next tested if the gpdh-1 modulators we identified affect accumulation of glycerol in dpy-7 or wild type worms under basal conditions. In wild type worms on 51 mM NaCl, glycerol levels were not decreased significantly by RNAi of pgph-3 consistent with residual PGPH activity or other synthesis pathways being sufficient for low basal levels (Fig 4A). Alternatively, RNAi of pgph-3 decreased glycerol levels in dpy-7 worms (Fig 4B). RNAi of rars-1 or hars-1 strongly increased glycerol levels in wild type and dpy-7 worms (Fig 4). We also tested if the strong gpdh-1 effects of rars-1 or hars-1 RNAi would be reflected in motility after transfer to high motility. As shown in Fig 4C and 4D, rars-1 and hars-1 RNAi increased acute motility on high NaCl under both conditions.

thumbnail
Download:
Fig 4. gpdh-1 modulators affect glycerol accumulation.

Mean plus standard error of whole-worm glycerol levels in wild type (A) and dpy-7(e88) worms (B) fed dsRNA of gpdh-1 modulators. (A and B) N = 3 to 7 populations of worms, *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA with a Dunnett post-hoc test. (C and D) Motility of worms 20 minutes after transfer to agar containing 350 (C) or 550 mM NaCl (D); N = 6 to 10 populations of worms. (A-D) *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA with a Dunnett post-hoc test.

https://doi.org/10.1371/journal.pone.0285328.g004

RNAi of neither hmit-1.2 nor pfk-1.1 significantly changed glycerol levels consistent with small effects on gpdh-1 mRNA levels (Figs 2 and 4). There was a trend of reduced glycerol levels with RNAi of atp-4 in dpy-7 worms, but this effect did not reach the threshold for statistical significance (p = 0.0997) (Fig 4B).

gpdh-1 modulators are stress response gene specific

Lastly, we tested the effects of furrow-sensitive gpdh-1 modulators on core detoxification (gst-4) and innate immune response genes (nlp-29) [31,50–53]. Relative to wild type worms on 51 mM NaCl, gst-4 and nlp-29 mRNA levels were induced 3.1 and 33.9-fold in dpy-7;control RNAi worms, respectively (Fig 5). Compared to effects on gpdh-1 (Fig 2B), RNAi of gpdh-1 modulators had reduced or opposite effects on gst-4 and nlp-29 mRNA (Fig 5). For example, RNAi of pfk-1.1, atp-4, pgph-3, and hmit-1.2 had no effect on either gst-4 or nlp-29. RNAi of rars-1 suppressed expression of nlp-29 and RNAi of hars-1 suppressed expression of gst-4 and nlp-29. These data suggest that the effects of aminoacyl-tRNA ligase, ATP homeostasis, and osmolyte accumulation gene silencing on gpdh-1 are not shared with other stress response genes activated by furrow disruption.

thumbnail
Download:
Fig 5. gpdh-1 modulators are stress response specific.

Mean plus standard error of relative gst-4 (A) and nlp-29 (B) mRNA levels in dpy-7(e88) worms fed dsRNA of gpdh-1 modulators. N = 4 to 8 populations of worms, *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-sample t-test with multiple comparisons corrections versus a normalized value of 1.0.

https://doi.org/10.1371/journal.pone.0285328.g005

Discussion

Defining interactions between extracellular matrix sensors and cellular stress responses is expected to reveal novel signaling mechanisms. Unfortunately, our RNAi screen did not identify any genes with obvious signaling functions. False negative rates are high with large-scale C. elegans RNAi screens [54]; relevant signaling genes might have redundancy, could be missing from our RNAi library, or might cause lethality when silenced. From a targeted screen of 381 C. elegans predicted protein kinase genes, we recently identified a membrane kinase named DRL-1 that is required for full activation of osmotic, detoxification, and innate immune responses in dpy-7 mutant worms [39]. Although our current genome-wide screen did not identify genes likely to function directly in activation of stress responses, the genes identified reveal new insights into how osmotic stress responses are regulated by negative feedback and integrated with ATP homeostasis and protein synthesis signaling.

Disruption of ATP homeostasis genes restricts gpdh-1 activation

Glycerol metabolism is tightly linked to carbohydrate homeostasis and is central to lipid synthesis [55]. Loss of ATP-4 or PFK-1.1 would be expected to disrupt ATP homeostasis. Suppression of gpdh-1 mRNA by RNAi of atp-4 or pfk-1.1 suggests that the osmotic stress response is restricted when ATP homeostasis is disrupted in furrow collagen mutant worms. RNAi of pfk-1.1 and atp-4 had little or no effect on expression of detoxification and innate immune response genes gst-4 and nlp-29, respectively (Fig 5) suggesting that the effect of ATP homeostasis gene silencing is specific to the osmotic stress response. Surprisingly, RNAi of atp-4 or pfk-1.1 did not suppress gpdh-1 expression in worms exposed to 200 mM NaCl (Fig 2C) suggesting that interactions between gpdh-1 regulation and energy metabolism during mild hyperosmotic stress might be different than when furrows are disrupted.

A previous study conducted a similar RNAi screen for suppressors of gpdh-1 in osm-8 mutants [30]; OSM-8 encodes a protein secreted by epidermal cells with homology to mucins and is predicted to function downstream from furrow collagens [30,56]. osm-8 mutants activate the same stress responses as furrow collagen mutants with no obvious defects in cuticle organization [29,30,44]. Analysis of genes required for gpdh-1 expression in osm-8 worms identifies oxidative phosphorylation as the most enriched functional category [30,40] (S4 Fig). Therefore, ATP homeostasis may be a common requirement for full gpdh-1 activation in osm-8 and furrow collagen mutant worms. Another gene required for gpdh-1 activation in osm-8 worms is ptr-23, which encodes an uncharacterized protein with predicted transmembrane domains [30]. Like our results for pfk-1.1 and atp-4, ptr-23 was not required for gpdh-1 activation by high salt [30]. Future studies are needed to determine if ptr-23 functions in furrow collagen mutants.

Depletion of aminoacyl-tRNA ligases and furrow disruption initiate distinct responses

Protein misfolding and aggregation occur rapidly and broadly during hyperosmotic stress in C. elegans [22,48,57,58] and translation of new protein is suppressed to attenuate this damage [49,59]. Inhibition of translation pharmacologically or by depletion of aminoacyl-tRNA ligases activates gpdh-1 expression supporting a model in which uncharged tRNAs can act as a signal for hyperosmotic stress [26,49]. Depletion of aminoacyl-tRNA ligases activated gpdh-1 and glycerol accumulation additively with furrow collagen gene mutation. This result could be explained by furrow disruption only partially reducing aminoacyl-tRNA ligation or by furrow loss and uncharged tRNAs initiating different signaling pathways that converge on gpdh-1. Disruption of furrows activates canonical detoxification (gst-4) and innate immune response (nlp-29) genes together with gpdh-1 and hmit-1.1 [29,31]. Depletion of aminoacyl-tRNA ligases either had no strong effect, or suppressed, hmit-1.1, gst-4, and nlp-29 (Figs 3 and 5) consistent with distinct cytoprotective responses to uncharged tRNAs and furrow disruption. Alternatively, we and others have demonstrated striking correlation between genome-wide transcriptome changes caused by hyperosmolarity and mutation of furrow collagens [29,31].

Negative feedback within and between osmolyte accumulation pathways

Glycerol is synthesized from glycolysis intermediate metabolite dihydroxyacetone phosphate in two steps, the first reaction is catalyzed by GPDH and the second by PGPH. RNAi of pgph-3 lowered glycerol levels and enhanced gpdh-1 expression under conditions that normally induce the osmotic stress response. These results are consistent with a negative feedback loop where glycerol accumulates to an adjustable set-point and represses further gpdh-1 expression. Strong induction of hmit-1.1 by high osmolarity and furrow loss suggests that myo-inositol also has a role [31,34,47]. Under conditions that induce osmotic stress responses, RNAi of hmit-1.1/1.2 enhanced gpdh-1 expression and RNAi of pgph-3 enhanced hmit-1.1 expression suggesting that a feedback mechanism is sensitive to total internal osmolarity contributed by both glycerol and myo-inositol and represses expression of genes that promote accumulation of both osmolytes. RNAi of pgph-3 or hmit-1.1/1.2 had no effect on canonical detoxification (gst-4) and innate immune response genes (nlp-29) consistent with a feedback mechanism that is specific to osmotic responses.

Nematodes maintain a positive internal turgor pressure against the cuticle that functions as a hydrostatic skeleton; in large species that allow direct measurements, this turgor pressure was found to be 10–70 mmHg [60–63]. Maintenance of turgor pressure is required for motility and would be expected to be under tight regulation. Glycerol and myo-inositol are organic osmolytes compatible with protein folding that are expected to contribute to turgor pressure and the hydrostatic skeleton. Mechanical strain between the cuticle and underlying tissues is one possible mechanism for sensing turgor pressure. Exposure to high osmolarity environments causes osmotic water loss and reduced turgor pressure that could initiate a mechanical signal for stress response activation [22,63–65]. Mutation or loss of furrow collagens may mimic mechanical changes caused by hyperosmotic water loss by relaxing mechanical strain on turgor pressure sensors [66–68].

Supporting information

S1 Fig. Paired bright field and merged fluorescent images of QV261 worms fed dsRNA of gpdh-1 modulators.

Note that exposure settings are the same for all fluorescent images resulting in saturation of the rars-4 RNAi worms. Scale bar is 200 microns.

https://doi.org/10.1371/journal.pone.0285328.s001

(TIF)

S2 Fig. Representative DIC images of cuticles in dpy-7 worms fed dsRNA of gpdh-1 modulators.

Furrows are lost in dpy-7 adults fed control or gpdh-1 modulator dsRNA clones. Scale bar is 25 microns.

https://doi.org/10.1371/journal.pone.0285328.s002

(TIF)

S3 Fig. RNAi of gpdh-1 modulators does not change body shape.

N = 15 individual worms per condition.

https://doi.org/10.1371/journal.pone.0285328.s003

(TIF)

S4 Fig. ATP homeostasis genes are required for gpdh-1 activation in osm-8 mutants.

Genes identified in a prior RNAi screen for regulation of gpdh-1 in osm-8 mutants [30] were analyzed for gene ontology enrichment with DAVID [40].

https://doi.org/10.1371/journal.pone.0285328.s004

(TIF)

S1 Table. Primer sequences.

https://doi.org/10.1371/journal.pone.0285328.s005

(XLSX)

S2 Table. dsRNA gene hits.

https://doi.org/10.1371/journal.pone.0285328.s006

(XLSX)

S3 Table. Confirmed dsRNA genes.

https://doi.org/10.1371/journal.pone.0285328.s007

(XLSX)

Acknowledgments

Some C. elegans strains were provided by the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN).

References

  1. 1. Karamanos NK, Theocharis AD, Piperigkou Z, Manou D, Passi A, et al. (2021) A guide to the composition and functions of the extracellular matrix. FEBS 288: 6850–6912. pmid:33605520
  2. 2. Frantz C, Stewart KM, Weaver VM (2010) The extracellular matrix at a glance. J Cell Sci 123: 4195–4200. pmid:21123617
  3. 3. LeBlue VS, MacDonald B, Kalluri R (2007) Structure and Function of Basement Membranes. Experiemental Biology and Medicine 232: 1121–1129.
  4. 4. Schmidt S, Friedl P (2010) Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms. Cell Tissue Res 399: 83–92. pmid:19921267
  5. 5. Adams JC, Watt FM (1993) Regulation of development and differentiation by the extracellular matrix. Development 117: 1183–1198. pmid:8404525
  6. 6. Padhi A, Nain AS (2019) ECM in Differentiation: A Review of Matrix Structure, Composition and Mechanical Properties. Annals of Biomedical Engineering: 1071–1089. pmid:31485876
  7. 7. Lu P, Weaver VM, Werb Z (2012) The extracellular matrix: a dynamic niche in cancer progression. J Cell Biol 196: 395–406. pmid:22351925
  8. 8. Lukashev ME, Werb Z (1998) ECM signalling: orchestrating cell behaviour and misbehaviour. Trends Cell Biol 8: 437–441. pmid:9854310
  9. 9. Berk BC, Fujiwara K, Lehoux S (2007) ECM remodeling in hypertensive heart disease. J Clin Invest 117: 568–575. pmid:17332884
  10. 10. Bateman JF, Boot-Handford RP, Lamandé SR (2009) Genetic diseases of connective tissues: cellular and extracellular effects of ECM mutations. Nat Rev Genet 10: 173–183. pmid:19204719
  11. 11. Bonnans C, Chou J, Werb Z (2014) Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 15: 786–801. pmid:25415508
  12. 12. Page AP, Johnstone IL (2007) The cuticle. WormBook: 1–15.
  13. 13. Tajiri R (2016) Cuticle itself as a central and dynamic player in shaping cuticle. 19: 30–35. pmid:28521940
  14. 14. Harris-Tyron TA, Grice EA (2022) Microbiota and maintenance of skin barrier function. Science 376: 940–945. pmid:35617415
  15. 15. Shamaprasad P, Frame CO, Moore TC, Yang A, Iacovella CR, et al. (2022) Using molecular simulation to understand the skin barrier. Prog Lipid Res: 101184. pmid:35988796
  16. 16. Emtage L, Gu G, Hartwieg E, Chalfie M (2004) Extracellular Proteins Organize the Mechanosensory Channel Complex in C. elegans Touch Receptor Neurons. Neuron 44: 795–807.
  17. 17. Hu J, Chiang L-Y, Koch M, Lewin GR (2010) Evidence for a protein tether involved in somatic touch. EMBO 29: 855–867. pmid:20075867
  18. 18. Rodríguez-Peña JM, García R, Nombela C, Arroyo J (2010) The high-osmolarity glycerol (HOG) and cell wall integrity (CWI) signalling pathways interplay: a yeast dialogue between MAPK routes. Yeast 27: 495–502. pmid:20641030
  19. 19. Rodicio R, Heinisch JJ (2010) Together we are strong—cell wall integrity sensors in yeasts. Yeast 27: 531–540. pmid:20641024
  20. 20. Kock C, Dufrene YF, Heinisch JJ (2015) Up against the wall: is yeast cell wall integrity ensured by mechanosensing in plasma membrane microdomains? Appl Environ Microbiol 81: 806–811. pmid:25398859
  21. 21. Sykiotis GP, Bohamann D (2010) Stress-activated cap’n’collar transcription factors in aging and human disease. Protein Science 3: re3. pmid:20215646
  22. 22. Choe KP (2013) Physiological and molecular mechanisms of salt and water homeostasis in the nematode Caenorhabditis elegans. Am J Physiol Regul Integr Comp Physiol 305: R175–186.
  23. 23. Tang L, Choe KP (2015) Characterization of skn-1/wdr-23 phenotypes in Caenorhabditis elegans; pleiotrophy, aging, glutathione, and interactions with other longevity pathways. Mech Ageing Dev 149: 88–98.
  24. 24. Blackwell TK, Steinbaugh MJ, Hourihan JM, Ewald CY, Isik M (2015) SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans. Free Radic Biol Med 88: 290–301. 291.
  25. 25. Johnstone IL (2000) Cuticle collagen genes: expression in Caenorhabditis elegans. Trends in Genetics 16: 21–27.
  26. 26. Lamitina T, Huang CG, Strange K (2006) Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression. Proceedings of the National Academy of Sciences of the United States of America 103: 12173–12178. pmid:16880390
  27. 27. Wheeler JM, Thomas JH (2006) Identification of a novel gene family involved in osmotic stress response in Caenorhabditis elegans. Genetics 174: 1327–1336.
  28. 28. Pujol N, Zugasti O, Wong D, Couillault C, Kurz CL, et al. (2008) Anti-Fungal Innate Immunity in C. elegans is Enhanced by Evolutionary Diversification of Antimicrobial Peptides. PLOS Pathogens 4: e1000105.
  29. 29. Rohlfing AK, Miteva Y, Hannenhalli S, Lamitina T (2010) Genetic and physiological activation of osmosensitive gene expression mimics transcriptional signatures of pathogen infection in C. elegans. PLoS One 5: e9010.
  30. 30. Rohlfing AK, Miteva Y, Moronetti L, He L, Lamitina T (2011) The Caenorhabditis elegans mucin-like protein OSM-8 negatively regulates osmosensitive physiology via the transmembrane protein PTR-23. PLoS Genet 7: e1001267.
  31. 31. Dodd W, Tang L, Lone JC, Wimberly K, Wu CW, et al. (2018) A Damage Sensor Associated with the Cuticle Coordinates Three Core Environmental Stress Responses in Caenorhabditis elegans. Genetics 208: 1467–1482.
  32. 32. McMahon L, Muriel JM, Roberts B, Quinn M, Johnstone IL (2003) Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix. Mol Biol Cell 14: 1366–1378.
  33. 33. Miao R, Li M, Zhang Q, Yang C, Wang X (2020) An ECM-to-Nucleus Signaling Pathway Activates Lysosomes for C. elegans Larval Development. Dev Cell 52: 21–37.
  34. 34. Chandler LM, Choe KP (2022) Extracellular matrix regulation of stress response genes during larval development in Caenorhabditis elegans. G3: jkac221.
  35. 35. Lamitina ST, Morrison R, Moeckel GW, Strange K (2004) Adaptation of the nematode Caenorhabditis elegans to extreme osmotic stress. Am J Physiol Cell Physiol 286: C785–791.
  36. 36. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94.
  37. 37. Kamath RS, Ahringer J (2003) Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30: 313–321.
  38. 38. Scolaro G, Bridges K, Curry S, Jacobson S, LoPresti M, et al. (2019) Increased expression of pgph-1, T23F2.4, and cyp-14A5 in C. elegans dpy-7 mutants and by high salt. MicroPubl Bio: 10.17912.
  39. 39. Wimberly K, Choe K (2022) An extracellular matrix damage sensor signals through membrane-associated kinase DRL-1 to mediate cytoprotective responses in Caenorhabditis elegans. Genetics 220.
  40. 40. Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44–57. pmid:19131956
  41. 41. Robert VJ, Sijen T, van Wolfswinkel J, Plasterk RH (2005) Chromatin and RNAi factors protect the C. elegans germline against repetitive sequences. Genes Dev 19: 782–787.
  42. 42. Hsieh J, Liu J, Kostas SA, Chang C, Sternberg PW, et al. (1999) The RING finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans. Genes Dev 13: 2958–2970.
  43. 43. Urso SJ, Comly M, Hanover JA, Lamitina T (2020) The O-GlcNAc transferase OGT is a conserved and essential regulator of the cellular and organismal response to hypertonic stress. PLoS Genet 16: e1008821. pmid:33006972
  44. 44. Rodriguez Mendoza V, Chandler L, Liu Z, Buddendorff L, Al-Rajhi A, et al. (2022) Extracellular proteins OSM-7 and OSM-8 are required for stress response gene regulation at all post-embryonic stages. MicroPubl Biol 2022. pmid:36606078
  45. 45. Jang S, Xuan Z, Lagoy RC, Jawerth LM, Gonzalez IJ, et al. (2021) Phosphofructokinase relocalizes into subcellular compartments with liquid-like properties in vivo. Biophys J 120: 1170–1187.
  46. 46. Anderson LL, Mao X, Scott BA, Crowder CM (2009) Survival from Hypoxia in C. elegans by Inactivation of Aminoacyl-tRNA Synthetases. Science 323: 630–633.
  47. 47. Kage-Nakadai E, Uehara T, Mitani S (2011) H+/myo-inositol transporter genes, hmit-1.1 and hmit-1.2, have roles in the osmoprotective response in Caenorhabditis elegans. Biochem Biophys Res Commun 410: 471–477.
  48. 48. Choe KP, Strange K (2008) Genome-wide RNAi screen and in vivo protein aggregation reporters identify degradation of damaged proteins as an essential hypertonic stress response. Am J Physiol Cell Physiol 295: C1488–1498. pmid:18829898
  49. 49. Lee EC-H, Strange K (2012) GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling. AJP Cell Physiology 303: 1269–1277. pmid:23076791
  50. 50. Couillault C, Pujol N, Reboul J, Sabatier L, Guichou J-F, et al. (2004) TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM. Nat Immunol 5: 488–494.
  51. 51. Pujol N, Cypowyj S, Ziegler K, Millet A, Astrain A, et al. (2008) Distinct innate immune responses to infection and wounding in the C. elegans epidermis. Curr Biol 18: 481–489.
  52. 52. Choe KP, Przybysz AJ, Strange K (2009) The WD40 repeat protein WDR-23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in Caenorhabditis elegans. Mol Cell Biol 29: 2704–2715.
  53. 53. Wu CW, Deonarine A, Przybysz A, Strange K, Choe KP (2016) The Skp1 Homologs SKR-1/2 are Required for the Caenorhabditis elegans SKN-1 Antioxidant/Detoxification Response Independently of p38 MAPK. PLoS Genet 12: e1006361.
  54. 54. Simmer F, Moorman C, van der Linden AM, Kuijk E, van den Berghe PVE, et al. (2003) Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. Plos Biol 1: E12.
  55. 55. Xue LL, Chen HH, Jiang JG (2017) Implications of glycerol metabolism for lipid production. Prog Lipid Res 68: 12–25. pmid:28778473
  56. 56. Hollingsworth MA, Swanson BJ (2004) Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer 4: 45–60. pmid:14681689
  57. 57. Burkewitz K, Choe K, Strange K (2011) Hypertonic stress induces rapid and widespread protein damage in C. elegans. Am J Physiol Cell Physiol 301: C566–576.
  58. 58. Burkewitz K, Choe KP, Lee EC, Deonarine A, Strange K (2012) Characterization of the proteostasis roles of glycerol accumulation, protein degradation and protein synthesis during osmotic stress in C. elegans. PLoS One 7: e34153.
  59. 59. Kim H, Strange K (2013) Changes in translation rate modulate stress-induced damage of diverse proteins. Am J Physiol Cell Physiol 305: C 1257–1264. pmid:24153430
  60. 60. Harris J, Crofton H (1957) Structure and Function in the Nematodes: Internal Pressure and Cuticular Structure in Ascaris. Journal of Experimental Biology 34: 116–130.
  61. 61. Chapman G, Newell GE (1947) The role of the body fluid in relation to movement in soft-bodied invertebrates: the burrowing of Arenicola. Proc R Soc Med 134: 431–455.
  62. 62. Fusé M, Davey KG, Sommerville RI (1993) Osmoregulation in the parasitic nematode Pseaduoterranova decipiens. J Exp Biol 175: 127–142.
  63. 63. Choe KP, Strange K (2007) Molecular and genetic characterization of osmosensing and signal transduction in the nematode Caenorhabditis elegans. FEBS J 274: 5782–5789.
  64. 64. Choe KP, Strange K (2007) Evolutionarily conserved WNK and Ste20 kinases are essential for acute volume recovery and survival after hypertonic shrinkage in Caenorhabditis elegans. Am J Physiol Cell Physiol 293: C915–927.
  65. 65. Urso SJ, Lamitina T (2021) The C. elegans Hypertonic Stress Response: Big Insights from Shrinking Worms. Cell Physiol Biochem 55: 89–105.
  66. 66. Muralidhar A, Shabala L, Broady P, Shabala S, Garrill A (2015) Mechanisms underlying turgor regulation in the estuarine alga Vaucheria erythrospora (Xanthophyceae) exposed to hyperosmotic shock. Plant Cell Environ 38: 1514–1527. pmid:25546818
  67. 67. Rojas ER, Huang KC (2018) Regulation of microbial growth by turgor pressure. Current Opinion in Microbiology 42: 62–70. pmid:29125939
  68. 68. Mishra R, Minc N, Peter M (2022) Cells under pressure: how yeast cells respond to mechanical forces. Trends in Microbiology 30: 495–510. pmid:35000797