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Maintenance of proteostasis by Drosophila Rer1 is essential for competitive cell survival and Myc-driven overgrowth [1]

['Pranab Kumar Paul', 'Cell', 'Developmental Signaling Laboratory', 'Department Of Biological Sciences', 'Indian Institute Of Science Education', 'Research Bhopal', 'Bhopal', 'Madhya Pradesh', 'Shruti Umarvaish', 'Shivani Bajaj']

Date: 2024-04

Defects in protein homeostasis can induce proteotoxic stress, affecting cellular fitness and, consequently, overall tissue health. In various growing tissues, cell competition based mechanisms facilitate detection and elimination of these compromised, often referred to as ‘loser’, cells by the healthier neighbors. The precise connection between proteotoxic stress and competitive cell survival remains largely elusive. Here, we reveal the function of an endoplasmic reticulum (ER) and Golgi localized protein Rer1 in the regulation of protein homeostasis in the developing Drosophila wing epithelium. Our results show that loss of Rer1 leads to proteotoxic stress and PERK-mediated phosphorylation of eukaryotic initiation factor 2α. Clonal analysis showed that rer1 mutant cells are identified as losers and eliminated through cell competition. Interestingly, we find that Rer1 levels are upregulated upon Myc-overexpression that causes overgrowth, albeit under high proteotoxic stress. Our results suggest that increased levels of Rer1 provide cytoprotection to Myc-overexpressing cells by alleviating the proteotoxic stress and thereby supporting Myc-driven overgrowth. In summary, these observations demonstrate that Rer1 acts as a novel regulator of proteostasis in Drosophila and reveal its role in competitive cell survival.

In developing tissues, cells can stochastically acquire defects that can reduce their fitness. To maintain the overall health of tissues, these unfit cells are identified by the healthier neighboring cells and eliminated via a juxtacrine-acting cellular fitness sensing mechanism called cell competition. An example of such physiological regulation of cellular fitness is the maintenance of proteostasis. Defects in maintaining proteostasis cause proteotoxic stress. Interestingly, proteotoxic stress is observed not only in the unfit loser cells but also in the overgrowing super-competitor cells, for instance, cells with higher levels of Myc. How cell competition is linked to the maintenance of proteostasis is poorly understood. In this study, we have characterized for the first time the function of Drosophila Rer1 protein in development. We demonstrate that Rer1 is essential for maintaining protein homeostasis and loss of Rer1 activates stress-induced unfolded protein responses. Cells lacking Rer1 are identified as unfit cells and become losers when juxtaposed to the normal neighboring cells. Moreover, we show that Myc-overexpressing cells upregulate Rer1 levels, which allows them to maintain a higher demand for stress regulation, caused by increased protein translation. In this work, we propose that Rer1 functions as a stress regulator and that modulating its levels could provide cytoprotection under stress conditions.

Funding: This work was supported by the Science and Engineering Research Board (SERB), Department of Science & Technology, Government of India (grant number: CRG/2021/004686 to VC). The laboratory of V.C. is also supported by intramural funds from IISER Bhopal and the Department of Biotechnology-EMR (grant number: BT/PR34467/BRB/10/1831/2019 to VC). P.K.P. received fellowship from the Council of Scientific & Industrial Research (09/1020/(0127)/2017-EMR-I). W.A. acknowledges the financial support of the Vlaams Instituut voor Biotechnologie (VIB), KU Leuven (grant number: C14/21/095 and KA.20/085 to WA), the Fonds Wetenschappelijk Onderzoek (FWO) (grant number: I001322N to WA), and the Stichting Alzheimer Onderzoek België (grant number: #2020/0030 to WA). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Paul 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.

By creating a rer1 loss-of-function mutant, we show that rer1 is an essential gene in Drosophila. Furthermore, we found that loss of Rer1 creates proteotoxic stress in the developing wing epithelium, and when surrounded by wild-type cells, the clonal population of rer1 mutant cells attained the loser fate and were eliminated specifically via the process of cell competition. We have also analyzed the role of Rer1 in Myc-induced overgrowth and Rer1 levels were found to be upregulated upon Myc-overexpression. More importantly, we found that loss of Rer1 is sufficient to suppress Myc-induced overgrowth. In summary, our results demonstrate that Rer1 is an essential protein for proper maintenance of protein homeostasis and competitive cell survival in a developing tissue.

Here, we investigated the role of Retention in Endoplasmic Reticulum-1 (Rer1) protein in the competitive cell proliferation in the developing Drosophila wing epithelium. Mutations in the rer1 gene were first described in yeast, where it was identified in a screen as a factor required for proper transport of Sec12p between the endoplasmic reticulum (ER) and Golgi [ 36 ]. Later studies have shown that Rer1 is also required for the assembly of multisubunit protein complexes, for example, the tetrameric γ-secretase complex, yeast iron transporter and skeletal muscle nicotinic acetylcholine receptor (nAChR) [ 37 – 42 ]. Rer1 is also known to regulate ER homeostasis, and therefore loss of Rer1 has been shown to induce ER stress in yeast and worms [ 43 ]. Despite the fact that Rer1 is evolutionarily conserved from yeast to mammals, its function in the development of organisms remains largely unknown [ 43 – 45 ].

Interestingly, some perturbations can also provide a competitive advantage to the cells over their wild-type neighbors. For instance, the overexpression of a proto-oncogene Myc, a master regulator of cell proliferation and growth, enhances the relative fitness of the cells. Thus, clonal expression of Myc generates super-competitor cells, which proliferate at the expense of the wild-type neighbors [ 10 , 26 ]. Myc drives cellular growth through its ability to upregulate the expression of a large number of genes and enhance the activity of several crucial metabolic pathways [ 27 – 29 ]. However, Myc-overexpression also leads to proteotoxic stress due to increased protein synthesis [ 30 – 32 ]. Thus, Myc-driven overgrowth is dependent on the activation of the cytoprotective unfolded protein response pathways (UPR) [ 33 ]. This includes phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) via PERK (PKR-like ER kinase) and induction of autophagy to reduce protein translation and clear misfolded proteins, respectively [ 34 , 35 ]. However, a clear understanding of how Myc and UPR cooperate to promote a proliferative cellular environment remains unclear.

Moreover, the loser fate is associated with a number of other physiological changes impacting cell fitness. These changes include, 1) reduced metabolic activity due to alteration in the mTOR pathway activity [ 18 , 19 ], 2) loss of apico-basal polarity as a consequence of mutations of the scribble, dlg, and lgl genes [ 20 ], 3) defects in endosomal trafficking caused by mutations in the rab5 gene [ 21 ], and 4) deregulation of signaling pathways such as Wnt, BMP, and Hippo [ 22 – 24 ]. Cells bearing these perturbations are eliminated through cell competition involving JNK-dependent activation of the proapoptotic factors [ 6 , 25 ].

The development of healthy tissue requires the removal of viable but suboptimal cells. In several growing tissues, this vital culling process is orchestrated through a specific cell-cell interaction called cell competition. In this intricate mechanism, unfit cells, also called “loser”, are eliminated by their surrounding fitter counterparts, the “winner” cells [ 1 , 2 ], thereby maintaining tissue health [ 3 ]. The best-known example of cell competition is described in the developing Drosophila epithelium using the heterozygous mutations in a ribosomal protein (Rp) gene (also known as Minute). The Rp +/- flies are viable, however, under mosaic condition the Rp +/- cells are eliminated from the developing epithelium when juxtaposed with the neighboring wild-type (Rp +/+ ) cells [ 4 – 6 ]. Although the Rp +/- mutation affects cellular physiology autonomously, caspase-dependent apoptosis is observed mostly at the boundary between Rp +/- cells and nearby Rp +/+ cells, which is a hallmark of cell competition [ 6 , 7 ]. The loser fate of the slow growing Rp +/- cells was suggested to be due to reduced protein translation [ 8 – 10 ]. However, recent studies have shown that Rp +/- cells exhibit high proteotoxic stress [ 11 – 14 ] and activate the expression of bZip transcription factor Xrp-1, which plays an essential role in the elimination of the Rp +/- cells [ 11 , 15 , 16 ]. Interestingly, Xrp-1 appears to be responsible for the manifestation of various defects in Rp +/- cells, including reduced global translation and proteotoxic stress, contributing to the loser status [ 17 ].

Results

Rer1 is required for Drosophila larval development We first set out to characterize the role of Rer1 during Drosophila development. To this end, we generated a rer1 knockout mutant by imprecise excision of a p-element insertion in the rer1 locus (see materials and methods). A loss-of-function mutation in rer1 containing a 1560 bp deletion in the coding region was identified (Fig 1A). Quantitative RT-PCR analysis in the homozygous mutant (rer1–/–) animals confirmed a complete loss of rer1 mRNA levels, indicating a complete loss-of-function (S1A Fig). Further analysis showed that the rer1–/–larvae failed to develop into pupae and died during the larval stages (S1B Fig). To rule out the possibility of lethality arising due to a second site mutation in another essential gene, we performed rescue experiments using a genomic-rescue construct expressing GFP-tagged Rer1 via the endogenous promoter (see materials and methods). The expression of GFP-rer1 in homozygous rer1–/–flies led to a complete rescue of lethality, confirming the specificity of the mutant (S1B Fig). These results underscore the indispensability of Rer1 in Drosophila development. PPT PowerPoint slide

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TIFF original image Download: Fig 1. rer1–/–clones show reduced growth and cell death at the clone boundary. (A) Schematic representation of the rer1KO line. Upon imprecise excision of a p-element inserted in the coding sequence, a 1560bp deletion in the rer1 gene was obtained. (B-D) Wing imaginal disc harboring rer1–/–clones induced by hs-FLP at 72, 96 and 120 hrs prior to dissection of third-instar larvae. RFP-negative (black) represents rer1–/–, lighter red areas represent heterozygous rer1–/RFP, and brighter red areas represent RFP/RFP (rer1+/+; twin spot). (E) The relative size of mutant (RFP-negative) versus twin spots (RFP/RFP) areas at 72 hrs (N = 10 wing discs), 96 hrs (N = 12 wing discs), and 120 hrs (N = 11 wing discs), measured within the white dotted lines. Statistical analysis was performed using the Ordinary one-way ANOVA with Tukey’s multiple comparison test (**** p<0.0001, ** p<0.0036). (F-I) Third-instar larval wing epithelium with hs-FLP-induced (96 hrs AHS) mitotic clones of (F-F’) WT (wild-type; rer1+/+), and (G-G’) rer1–/–genotypes, immuno-stained for the anti-cleaved Dcp-1. (H-H’) A magnified image of the inset (white box) in G. (I-I”) rer1–/–clones in GFP-rer1 background stained with anti-cleaved Dcp-1. I” shows the expression of GFP-Rer1 in the wing imaginal disc. (J) Quantification of the relative size of rer1–/–(RFP-negative) versus twin spots (RFP/RFP) areas in WT control (F, N = 10 wing discs); rer1–/–(G, N = 12 wing discs) and rescue in GFP-Rer1, rer1–/–(I, N = 6 wing discs). Statistical analysis was performed using the Ordinary one-way ANOVA with Tukey’s multiple comparison test (**** p<0.0001). (K) Quantification of cell death at the center and border of rer1–/–clones (two-sided Wilcoxon signed-rank test; N = 23 clones present in 12 wing discs; **** p<0.0001, SB = 20 μm. Also see S1 and S2 Figs. https://doi.org/10.1371/journal.pgen.1011171.g001

Cells lacking Rer1 show reduced survival in the developing wing epithelium We next analyzed the importance of Rer1 at the tissue level using the developing Drosophila wing imaginal discs. We first depleted Rer1 in the posterior compartment of the wing discs by expressing rer1-RNAi using the hedgehog (hh)-Gal4 driver (S1C Fig). To test the efficiency of the knockdown, we expressed rer1-RNAi in the GFP-rer1 genomic-rescue flies. Here, we observed a strong downregulation of the GFP-Rer1 levels (S1D Fig), suggesting that rer1-RNAi effectively downregulated the Rer1 levels. We assessed the impact of Rer1 depletion on cell death by analyzing the levels of cleaved Death caspase-1 (Dcp-1) and Acridine Orange (AO) as apoptosis markers. Rer1 depletion in the posterior compartment led to a strong increase in both Dcp-1 and AO positive cells as compared to the control anterior compartment (S1E–S1H Fig; quantified in S1I and S1J Fig, respectively). Intriguingly, despite the increased cell death, the adult wing of these flies appeared normal (S1K–S1N Fig; quantified in S1O Fig). To delve further, we generated rer1–/–clones using the heat-shock-inducible Flippase (FLP)-Flp recognition target (FRT)-system (see materials and methods). Clones were induced during early larval stages (48 hrs AEL) and wing discs were dissected at 72 and 96 hrs after heat-shock (AHS). Moreover, some larvae that were delayed and could reach up to 120 hrs were also dissected and analyzed. In these experiments, we observe that the rer1–/–(RFP-negative) clones area reduced over time as compared to the rer1+/+ clones (RFP/RFP; also called twin spot) (Fig 1B–1D; quantified in Fig 1E), indicating progressive removal of rer1–/–cells from the epithelium. Moreover, generation of the rer1–/–clones did not alter the overall wing size (S2A–S2D Fig; quantified in S2E Fig), indicating that the loss of rer1–/–cells was compensated by the neighboring cells.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011171

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