(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



The Gr64 cluster of gustatory receptors promotes survival and proteostasis of epithelial cells in Drosophila [1]

['Michael E. Baumgartner', 'School Of Cellular', 'Molecular Medicine', 'University Of Bristol', 'Bristol', 'United Kingdom', 'Alex Mastrogiannopoulos', 'Iwo Kucinski', 'The Wellcome Trust Cancer Research Uk Gurdon Institute', 'Zoology Department']

Date: 2022-07

Gustatory Receptor 64 (Gr64) genes are a cluster of 6 neuronally expressed receptors involved in sweet taste sensation in Drosophila melanogaster. Gr64s modulate calcium signalling and excitatory responses to several different sugars. Here, we discover an unexpected nonneuronal function of Gr64 receptors and show that they promote proteostasis in epithelial cells affected by proteotoxic stress. Using heterozygous mutations in ribosome proteins (Rp), which have recently been shown to induce proteotoxic stress and protein aggregates in cells, we show that Rp/+ cells in Drosophila imaginal discs up-regulate expression of the entire Gr64 cluster and depend on these receptors for survival. We further show that loss of Gr64 in Rp/+ cells exacerbates stress pathway activation and proteotoxic stress by negatively affecting autophagy and proteasome function. This work identifies a noncanonical role in proteostasis maintenance for a family of gustatory receptors known for their function in neuronal sensation.

By characterising the biology of Rp/+ cells, we identify a new function for Gr64 genes in maintaining proteostasis in epithelial cells. We find that Gr64 genes, whose expression is normally observed in neuronal cells, become up-regulated in wing disc epithelial cells upon Rp/+ mutation. Loss of Gr64 drives substantial apoptosis in noncompeting and competing Rp/+ cells and exacerbates stress pathway activation. Furthermore, loss of Gr64 exacerbates proteotoxic stress in Rp/+ cells, by reducing proteasome and autophagy function. Calcium imaging reveals reduced calcium activity in Rp/+ cells upon Gr64 down-regulation (as measured by the frequency of calcium flashes, [ 22 ]), suggesting that Gr64’s proteostasis promoting effects might be mediated by calcium signalling.

Rp/+ cells have recently been shown to suffer from chronic proteotoxic stress. They exhibit proteasome and autophagy defects, activation of the integrated stress response (ISR), a stoichiometric imbalance of large and small ribosomal subunit proteins and an accumulation of intracellular protein aggregates [ 18 – 21 ]. Importantly, boosting proteostasis rescues stress pathway activation, cell autonomous apoptosis, and competitive elimination [ 19 , 20 ]. These findings point to proteotoxic stress as a potential driver of the pathologies associated with ribosomopathies.

Mutations in ribosomal proteins or ribosome biogenesis factors can result in a class of disorders known as ribosomopathies [ 6 – 8 ]. While roles have been established for nucleolar stress, p53 activation, and translational defects in disease progression, ribosomopathy etiology remains poorly understood [ 8 ]. Ribosome protein (Rp) mutations are well studied in Drosophila, wherein the majority of cytosolic Rp encoding genes yield the so-called “Minute” phenotype when heterozygous mutant [ 9 ]. Rp/+ flies are viable and fertile but exhibit a developmental delay [ 10 ]. Epithelial cells in Rp/+ larvae exhibit reduced translation rates, increased cell-autonomous apoptosis, and stress pathway activation, including, JNK, JAK/STAT, Toll/IMD signalling, and the oxidative stress response [ 11 – 15 ]. Rp/+ cells also undergo cell competition and are eliminated from the tissue when confronted with wild-type cells in mosaic tissues [ 16 , 17 ]. Rp/+ cells are therefore said to behave as “losers” relative to wild-type “winners.”

Gustatory Receptors 64 (Gr64s) are a group of 6 tandem gustatory receptor genes (a through f) involved in mediating sensation of sugars, fatty acids, and glycerol in the adult nervous system [ 1 – 4 ]. Gr64s are thought to sense distinct ligands via distinct mechanisms: Gr64e, for example, is reported to act as a ligand gated ion channel in response to glycerol binding, whereas it acts downstream of phospholipase C in fatty acid sensation [ 4 ]. Gr64s are thought to function as heterodimers with each other and alternate gustatory receptors, as, for example, both Gr64a and Gr64f are required to mediate responses to certain sugars [ 5 ].

Results and discussion

Mining the list of genes differentially expressed in cells heterozygous mutant for the ribosomal protein RpS3, we observed up-regulation of all 6 Gr64 gustatory receptors relative to wild-type cells (Fig 1A and [11]). Gr64s were also up-regulated in cells mutant in mahjong (mahj), an E3 ubiquitin ligase, whose mutation also leads to proteotoxic stress and cell competition [11,19,23,24]. This was conspicuous, as Gr64s have no known nonneuronal nor larval function. In order to explore the role of Gr64s in cells affected by proteotoxic stress, we tested the effect of removing one copy of the Gr64 locus on RpS3+/- larvae, using a deficiency spanning the Gr64 locus, along with rescuing constructs of other affected genes (ΔGr64) [1]. ΔGr64/ΔGr64 flies present with no known phenotypes other than deficient gustatory responses [1]. RpS3+/-, ΔGr64+/- wing discs, however, exhibited a marked increase in apoptosis over levels seen in wing discs carrying either mutation alone (S1A–S1D Fig), indicating that RpS3+/- cells are acutely reliant on Gr64s for their survival. We confirmed this result using a precise CRISPR/Cas-9 deletion of the Gr64 cluster, Gr64af [4] (Fig 1B and 1C). Gr64 did not contribute to survival in the non-Minute context, as non-Minute wing discs homozygous null for Gr64 exhibited no increase in apoptosis relative to the wild type (S1E and S1F Fig). This is consistent with the fact that Gr64s appear to be only minimally expressed in wing discs (Flybase and [11]). Importantly, Gr64 mutations also led to increased apoptosis in wing discs heterozygous mutant for RpS17 or RpS23, 2 other Rp genes (S1G–S1J Fig), indicating a general requirement for Gr64 for multiple Rp mutants. RpS23 discs homozygous mutant for Gr64 further showed morphological defects with a loss of the characteristic pouch and hinge folds observed in wing discs (S1I and S1J Fig). To confirm that the effect observed was specifically caused by the Gr64 mutations, we sought to rescue the effect by overexpressing Gr64 in RpS3+/-, Gr64+/- mutants using a UAS construct driving expression of 5 of the 6 Gr64 genes (Gr64abcd-GFP-f) [1]. Overexpression of Gr64 in the P compartment resulted in toxicity and wiped out the compartment (Fig 1D), probably due to unphysiologically high expression levels. However, this specific UAS-Gr64 transgene has been shown to provide rescuing activity in the absence of Gal4 drivers [1]. Accordingly, we tested whether we could similarly observe rescue of Gr64+/- induced death by using the rescuing UAS-Gr64 transgene in the absence of Gal4. Indeed, UAS-Gr64 substantially rescued Gr64+/- induced death in RpS3+/- cells, confirming that the observed effect is due to the Gr64 mutation (Fig 1E and 1F).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Noncompeting RpS3+/- cells depend on Gr64 for their survival. (A) Fold change differences in Gr64 transcript expression relative to WT in wing discs heterozygous mutant for RpS3 (as detected in 2 separate mutant alleles: RpS3{Plac92} or RpS3*) or homozygous mutant for mahjong (mahj). Numbers and p-values are derived from [11]. (B) Wing discs heterozygous for a precise deletion of the Gr64 cluster (Gr64af) (left panel), heterozygous mutant for RpS3 (middle panel), or heterozygous mutant for both (right panel), assessed for cell death via immunostaining for cleaved-Dcp1 (red). (C) Quantification of cell death from wing discs of the same genotypes as in (B) (n Gr64 = 9, n RpS3 = 10, n RpS3,Gr64 = 9, 2-sided Mann–Whitney U test). (D) Representative image of a wing disc heterozygous mutant for RpS3 and Gr64 expressing the UAS-Gr64abcd-GFP-f construct driven by hhGal4 and stained with anti-Ci (cyan) to label the anterior compartment, and with anti-cleaved-Dcp1 (red). (E) Wing discs heterozygous mutant for RpS3 (left panel), heterozygous for both RpS3 and Gr64 (middle panel), or heterozygous for both and expressing the UAS-Gr64abcd-GFP-f construct, without Gal4 driver (right panel), assessed for cell death by immunostaining for cleaved-Dcp1 (red). (F) Quantification of cell death from wing discs of the same genotypes as in (E) (n RpS3 = 10, n RpS3,Gr64 = 10, n RpS3,Gr64,UAS-Gr64 = 15, 2-sided Mann–Whitney U test). (G) WT (left panel) or RpS3+/- wing discs (right panel) expressing Gr64f-RNAi in the posterior compartment driven by hh-Gal4 and assessed for cell death with a staining for cleaved caspase3 (red). (H) Quantification of cell death from wing discs of the same genotype as in (G, right panel) (n = 12, 2-sided Wilcoxon signed rank test). Horizontal lines in C and F indicates the median. For this and all other figures, scale bars correspond to 50 μm, and white dashed lines denote compartment boundaries, where the anterior compartment is shown on the left side of the image and ventral is up. Numerical data can be found in the “Fig 1” sheet of S1 Data. Gr64, Gustatory Receptor 64; RNAi, RNA interference; Rp, ribosome protein; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001710.g001

To determine whether the survival function of Gr64 reflects a systemic or cell autonomous role, we knocked down Gr64 specifically in the posterior compartment, using the hedgehog-Gal4 driver. We used a RNA interference (RNAi) line against Gr64f, which, given that the Gr64 cluster locus is polycistronic [1], is likely to silence multiple Gr64s. Gr64f-RNAi expression in wild-type discs yielded no appreciable change in levels of apoptosis (Fig 1G), whereas expression of Gr64f-RNAi in RpS3+/- wing discs caused a strong increase in apoptosis, specifically in the RNAi expressing cells (Fig 1G and 1H). A modest level of cell death was also observed in the non-RNAi compartment, perhaps due to apoptosis-induced apoptosis or to a systemic effect of Gr64 silencing. Expression of Gr64f-RNAi in clones in RpS3+/- discs yielded a similar result (S2A–S2C Fig). These data argue that RpS3+/- cells are cell autonomously dependent on Gr64 for their survival, although an additional non-cell autonomous contribution cannot be ruled out.

Next, we asked whether the requirement of Gr64 in Rp/+ cells is also observed in other imaginal discs. Both haltere (S2D–S2F Fig) and leg discs (S2G–S2I Fig) showed increased death when Gr64 was knocked down. This effect, however, was less pronounced than in wing discs. Eye discs instead did not appear to be affected by single copy removal of Gr64 (S2J–S2L Fig). Thus, Gr64 is required for RpS3+/- survival across many but not all imaginal discs.

Having established a prosurvival role for Gr64 in noncompeting RpS3+/- cells, we then tested whether Gr64s contribute to the survival of competing RpS3+/- losers. We therefore generated RpS3+/- losers competing against wild-type winners in wing discs carrying heterozygous mutations in one of any of the 6 Gr64 genes [25] (Fig 2). Strikingly, heterozygosity for any Gr64 yielded a substantial increase in RpS3+/- loser cell death at the winner/loser interface (Fig 2A–2H). Furthermore, loser cell clones were smaller in wing discs carrying mutations in Gr64b, Gr64c, Gr64d, Gr64e, or Gr64f, but not Gr64a (Fig 2I). RpS3+/- loser cells are therefore dependent on Gr64s in both noncompetitive and competitive conditions. While these results might suggest that each specific Gr64 protein contributes to RpS3+/- survival and these constructs have been used successfully in screens of Gr64 gustatory function [2], because of the polycistronic nature of the locus, it is difficult to draw conclusions on the requirements of individual Gr64 isoforms here, as mutations in one coding sequence might affect the regulation and behaviour of the others.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Heterozygosity at Gr64 loci exacerbates competitive RpS3+/- loser cell elimination. (A–G) Representative images of wing discs containing RpS3+/- loser cells (green) competing against wild-type winners (unlabelled) and stained for cleaved-Dcp1 (red). RpS3+/- clones were generated in a wild-type background (A) or in wing discs heterozygous for any one of the Gr64 genes a through f (mutations used were Gr64aGAL4, Gr64bLEXA, Gr64cLEXA, Gr64d1, Gr64eLEXA, and Gr64fLEXA) (B–G). (H) Quantification of the percentage of cells undergoing apoptosis at loser clone borders in wing discs of genotypes as shown in (A–G). Statistics reflect multiple logistic regression across 3 replicates (details provided in Materials and methods). (I) Quantification of loser cell growth in wing discs of the genotypes shown in (A–G), as measured by the percent loser clone coverage of the pouch. Statistics reflect Student t test with FDR p-correction. n control = 16, n Gr64aGAL4 = 15, n Gr64bLEXA = 15, n Gr64cLEXA = 11, n Gr64d[ 1 ] = 12, n Gr64eLEXA = 9, n Gr64fLEXA = 8. For all quantifications, the horizontal line indicates the mean. Scale bars correspond to 50 μm. Numerical data can be found in the “Fig 2” sheet of S1 Data. FDR, false discovery rate; Gr64, Gustatory Receptor 64; Rp, ribosome protein. https://doi.org/10.1371/journal.pbio.3001710.g002

Rp/+ mutations cause stress pathway activation and cellular malfunctions, many of which are linked with the loser status [11,12,18–21,26]. To evaluate how Gr64 loss affects cellular stress responses, we identified milder expression conditions for Gr64f-RNAi, using the posterior compartment-specific engrailed-Gal4 driver. With these conditions, Gr64f knockdown in noncompeting RpS3+/- cells exhibited a comparatively mild increase in apoptosis (Fig 3A and 3B), allowing us to investigate Gr64 function without widespread cell death. Gr64f-RNAi yielded increased activation of the oxidative stress response, as measured by GstD1-GFP reporter expression [27] (Fig 3C and 3D). Gr64f-RNAi did not yield an increase in GstD1-GFP signal in wild-type wing discs (S3A and S3B Fig), demonstrating that this result is specific to the RpS3+/- context. Consistent with these results, an increase in GstD1-GFP expression was also observed in RpS3+/- wing discs heterozygous for ΔGr64 (S3C and S3D Fig). Furthermore, Gr64f-RNAi expression in RpS3+/- discs resulted in a mild increase in JNK pathway and in the ISR, as measured by immunostaining for phosphorylated JNK (Fig 3E and 3F) and phosphorylated eIF2α (Fig 3G and 3H), respectively. No differences in phospho-JNK (S4A and S4B Fig) or phospho-eIF2α (S4C–S4D Fig) levels were seen in the posterior compartments of otherwise genetically identical wing discs lacking the Gr64f-RNAi, confirming that this result is due to RNAi expression. These data indicate that loss of Gr64 exacerbates stress responses seen in Rp/+ cells.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Loss of Gr64 worsens stress pathway activation in RpS3+/-. (A, B) RpS3+/- wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment stained for cell death, as detected by cleaved Dcp1 (green) (A) along with quantification in (B) (n = 7, 2-sided Wilcoxon signed rank test). (C, D) GstD1-GFP reporter expression (green) in RpS3+/- wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment (C) along with quantification in (D) (n = 6, 2-sided paired t test). (E, F) RpS3+/- wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment and stained for phosphorylated JNK (green) (E) along with quantification in (F) (n = 7, 2-sided paired t test). (G, H) RpS3+/- wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment and stained for phosphorylated eIF2α (green) (G) along with quantification in (H) (n = 7, 2-sided paired t test). Scale bars correspond to 50 μm. Numerical data can be found in the “Fig 3” sheet of S1 Data. Gr64, Gustatory Receptor 64; RNAi, RNA interference; Rp, ribosome protein. https://doi.org/10.1371/journal.pbio.3001710.g003

We next investigated how Gr64 alleviates stress signalling and improves viability of Rp/+ cells. We previously reported that the ISR and oxidative stress response in RpS3+/- cells are driven by accumulation of protein aggregates, accompanied by reduced degradation of proteins by the proteasome and by autophagy, leading to sustained proteotoxic stress [19]. Thus, we tested the effect of Gr64 reduction on proteostasis pathways using ProteoFlux and ReFlux, genetically encoded reporters of proteasome and autophagy function, respectively, which we previously developed [19]. In these reporters, GFP is tagged for degradation through the proteasome or the autophagosome by fusion to CL1 or ref(2)P, respectively. These GFP fusions are expressed under the control of a heat shock promoter, which enables heat-shock induced pulse-chase experiments, whereby decline in GFP signal is a quantitative measure of proteasomal or autophagosomal degradation rates [19]. Using these tools, we found that Gr64f-RNAi causes a further reduction in proteasomal (Fig 4A–4C) and autophagosomal (Fig 4D–4F) degradation rates, beyond the defects already present in RpS3+/-. These data indicate that loss of Gr64 yields a substantial impairment in cellular proteolytic pathways. Furthermore, Gr64f-RNAi yields an increase in ref(2)P-positive foci (Fig 4G), structures that we have previously shown to colocalise with poly-ubiquitinylated aggregates in RpS3+/- cells [19]. We conclude that Gr64 loss causes increased stress signalling and cell death by exacerbating proteotoxic stress.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Loss of Gr64 exacerbates proteostasis defects in RpS3+/-. (A–C) RpS3+/- wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment, marked with RFP (red) and expressing the CL1-GFP/ProteoFlux construct (green), 0 hours (A) or 4 hours (B) after heat shock along with quantification in (C) (n 0hr = 3, n 2hr = 14, n 4hr = 5, 2-sided paired t test, the horizontal line indicates the mean and the whiskers reflect the 95% confidence interval). (D–F) RpS3+/- wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment, marked with RFP (red) and expressing the ref(2)P-GFP/ReFlux construct (green), 0 hours (E) or 6 hours (F) after heat shock along with quantification in (D) (n 0hr = 4, n 3hr = 3, n 6hr = 6, 2-sided paired t test, the horizontal line indicates the mean, and the whiskers reflect the 95% confidence interval). (G) RpS3+/- wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment stained for ref(2)P (green). (H, I) RpS3+/- wing discs expressing Gr64f-RNAi driven by enGal4 in the posterior compartment and assessed for translation through an OPP translation reporter assay (grey) (H) along with quantification in (I) (n = 8, 2-sided paired t test). (J) Quantification of the number of calcium flashes in anterior and posterior compartments of wing discs heterozygous for RpS3 that also express Gr64f-RNAi in the posterior compartment with enGal4 (n Ant = 30, n Post = 30, 2-sided Wilcoxon signed rank test; the horizontal line indicates the median and the whiskers the interquartile range). (K) Proposed model: heterozygous mutation in a ribosomal protein mutant gene triggers proteotoxic stress, making Rp/+ cells highly dependent upon the autophagosome and proteasome for proteostasis. Gr64 gene cluster is up-regulated in Rp/+ cells, where it supports cell survival and inhibits proteotoxic stress by promoting autophagy and proteasome functions, possibly via calcium signalling. Scale bars correspond to 50 μm. Numerical data can be found in the “Fig 4” sheet of S1 Data. Gr64, Gustatory Receptor 64; RNAi, RNA interference; Rp, ribosome protein. https://doi.org/10.1371/journal.pbio.3001710.g004

Worsening of proteotoxic stress in ribosome mutants could derive from an inhibition of protein catabolic processes or from an increase in protein translation, which, by producing more proteins, could cause a further burden on proteostasis. Interestingly, OPP, a global translation reporter, revealed a mild but statistically significant decrease in translation upon expression of Gr64f-RNAi (Fig 4H and 4I). This is consistent with the observed increase in eIF2α-phosphorylation (Fig 3G and 3H), which is a translation inhibitor as well as a stress pathway marker [28]. Thus, the increase in proteotoxic stress observed upon Gr64 reduction is not due to increased translation but rather to a failure to clear defective proteins.

How could a family of taste receptors play a role in proteostasis? Gr64 proteins are sweet tastant receptors that cause an increase in cytoplasmic calcium upon ligand binding [25]. Calcium is known to play a role in modulating proteostatic pathways, [29,30] suggesting that Gr64 could ameliorate proteostasis by modifying calcium signalling. We therefore investigated what effect loss of Gr64 might have on calcium signalling using a GCaMP fluorescent calcium reporter [31].Consistent with this hypothesis, we found that silencing Gr64 in the posterior compartment of RpS3+/- wing discs reduced the frequency of calcium flashes in posterior compartment cells, relative to reference RpS3+/- anterior cells, which served as internal control (Fig 4J, S1 Movie).

In this study, we have identified Gr64 taste receptors as novel players in proteostasis control and as cytoprotective regulators in epithelial cells affected by proteotoxic stress. Our data suggest that Gr64s contribute to proteostasis by promoting protein catabolism rather than by inhibiting translation (Fig 4K). This is potentially mediated by calcium signalling, as Gr64 activity typically induces calcium release [25], and calcium is involved in protein folding, the ISR, proteasome, and autophagy function [29,32–34]. Indeed, consistent with this hypothesis, our data indicate that removal of Gr64 modifies Calcium signalling in Rp/+ cells. It remains to be determined, however, whether this phenotype is causal or a consequence of the proteostasis defects observed in Gr64 knockdown conditions, and thus further work must be done to elucidate Gr64’s mechanism of action.

Taste receptors have been implicated in nontaste-related chemosensation in neuronal and neuroendocrine cells both in flies and in mammals [2,35–37]. However, a role in proteostasis and a function in epithelia have not previously been described for taste receptors. Interestingly, dysregulation of olfactory and gustatory receptors has been observed in nonolfactory human brain tissue from individuals suffering from protein-aggregate driven neurodegenerative disorders, including Alzheimer disease, Parkinson disease, and Creutzfeld–Jacob disease as well as in a mouse model of Alzheimer disease [38], and olfactory receptors are expressed near to amyloid plaques in a mouse model of Alzheimer disease [39]. It is therefore possible that gustatory and olfactory receptors play a conserved role in promoting proteostasis in both neuronal and nonneuronal cells.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001710

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.