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Animals sense and respond to nutrient availability in their environments, a task coordinated in part by the mTOR complex 1 (mTORC1) pathway. mTORC1 regulates growth in response to nutrients and, in mammals, senses specific amino acids through specialized sensors that bind the GATOR1/2 signaling hub. Given that animals can occupy diverse niches, we hypothesized that the pathway might evolve distinct sensors in different metazoan phyla. Whether such customization occurs, and how the mTORC1 pathway might capture new inputs, is unknown. Here, we identify the Drosophila melanogaster protein Unmet expectations (CG11596) as a species-restricted methionine sensor that directly binds the fly GATOR2 complex in a fashion antagonized by S-adenosylmethionine (SAM). We find that in Dipterans GATOR2 rapidly evolved the capacity to bind Unmet and to thereby repurpose a previously independent methyltransferase as a SAM sensor. Thus, the modular architecture of the mTORC1 pathway allows it to co-opt preexisting enzymes to expand its nutrient sensing capabilities, revealing a mechanism for conferring evolvability on an otherwise conserved system. Unmet expectations, a previously uncharacterized fly protein, is a SAM sensor for the mTORC1 pathway. Tracing the evolution of Unmet reveals that the pathway uses the GATOR2 complex to capture and repurpose ancestral enzymes as nutrient sensors.

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair. In response to gut epithelial damage, Drosophila stem cells proliferate to produce large polyploid enterocytes (EC), which comprise the bulk of the epithelium. Here, the authors show that stress-dependent EGFR/MAP kinase signalling drives both endoreplication and cell growth in newborn ECs.

The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/β-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program. The liver is segregated into spatially organized areas that serve distinct functions, though how these zones are patterned remains unclear. Here they show that mTORC1 controls spatial segregation of liver metabolic functions via modulation of Wnt signaling, and find that impaired zonation is also observed in pigs given total parenteral nutrition.

Mammary and extramammary Paget’s Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways — including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.

Adenosine monophosphate-activated protein kinase (AMPK) acts as a major sensor of cellular energy status in cancers and is critically involved in cell sensitivity to anticancer agents. Here, we showed that AMPK was inactivated in lymphoma and related to the upregulation of the mammalian target of rapamycin (mTOR) pathway. AMPK activator metformin potentially inhibited the growth of B- and T-lymphoma cells. Strong antitumor effect was also observed on primary lymphoma cells while sparing normal hematopoiesis ex vivo . Metformin-induced AMPK activation was associated with the inhibition of the mTOR signaling without involving AKT. Moreover, lymphoma cell response to the chemotherapeutic agent doxorubicin and mTOR inhibitor temsirolimus was significantly enhanced when co-treated with metformin. Pharmacologic and molecular knock-down of AMPK attenuated metformin-mediated lymphoma cell growth inhibition and drug sensitization. In vivo , metformin induced AMPK activation, mTOR inhibition and remarkably blocked tumor growth in murine lymphoma xenografts. Of note, metformin was equally effective when given orally. Combined treatment of oral metformin with doxorubicin or temsirolimus triggered lymphoma cell autophagy and functioned more efficiently than either agent alone. Taken together, these data provided first evidence for the growth-inhibitory and drug-sensitizing effect of metformin on lymphoma. Selectively targeting mTOR pathway through AMPK activation may thus represent a promising new strategy to improve treatment of lymphoma patients.

Animals have a determined species-specific body size that results from the combined action of hormones and signaling pathways regulating growth rate and duration. In Drosophila , the steroid hormone ecdysone controls developmental transitions, thereby regulating the duration of the growth period. Here we show that ecdysone promotes the growth of imaginal discs in mid-third instar larvae, since imaginal discs from larvae with reduced or no ecdysone synthesis are smaller than wild type due to smaller and fewer cells. We show that insulin-like peptides are produced and secreted normally in larvae with reduced ecdysone synthesis and upstream components of insulin/insulin-like signaling are activated in their discs. Instead, ecdysone appears to regulate the growth of imaginal discs via Thor/4E-BP , a negative growth regulator downstream of the insulin/insulin-like growth factor/Tor pathways. Discs from larvae with reduced ecdysone synthesis have elevated levels of Thor , while mutations in Thor partially rescue their growth. The regulation of organ growth by ecdysone is evolutionarily conserved in hemimetabolous insects, as shown by our results obtained using Blattella germanica . In summary, our data provide new insights into the relationship between components of the insulin/insulin-like/Tor and ecdysone pathways in the control of organ growth.

The oncoprotein GOLPH3 (Golgi phosphoprotein 3) is an evolutionarily conserved phosphatidylinositol 4-phosphate effector, mainly localized to the Golgi apparatus, where it supports organelle architecture and vesicular trafficking. Overexpression of human GOLPH3 correlates with poor prognosis in several cancer types and is associated with enhanced signaling downstream of mTOR (mechanistic target of rapamycin). However, the molecular link between GOLPH3 and mTOR remains elusive. Studies in Drosophila melanogaster have shown that Translationally controlled tumor protein (Tctp) and 14-3-3 proteins are required for organ growth by supporting the function of the small GTPase Ras homolog enriched in the brain (Rheb) during mTORC1 (mTOR complex 1) signaling. Here we demonstrate that Drosophila GOLPH3 (dGOLPH3) physically interacts with Tctp and 14-3-3ζ. RNAi-mediated knockdown of dGOLPH3 reduces wing and eye size and enhances the phenotypes of Tctp RNAi. This phenotype is partially rescued by overexpression of Tctp, 14-3-3ζ, or Rheb. We also show that the Golgi localization of Rheb in Drosophila cells depends on dGOLPH3. Consistent with dGOLPH3 involvement in Rheb-mediated mTORC1 activation, depletion of dGOLPH3 also reduces levels of phosphorylated ribosomal S6 kinase, a downstream target of mTORC1. Finally, the autophagy flux and the expression of autophagic transcription factors of the TFEB family, which anti correlates with mTOR signaling, are compromised upon reduction of dGOLPH3. Overall, our data provide the first in vivo demonstration that GOLPH3 regulates organ growth by directly associating with mTOR signaling proteins.

Organ size is regulated by intercellular signaling for cell growth and proliferation. The TOR pathway mediates a key signaling mechanism for controlling cell size and number in organ growth. Chaperonin containing TCP-1 (CCT) is a complex that assists protein folding and function, but its role in animal development is largely unknown. Here we show that the CCT complex is required for organ growth by interacting with the TOR pathway in Drosophila . Reduction of CCT4 results in growth defects by affecting both cell size and proliferation. Loss of CCT4 causes preferential cell death anterior to the morphogenetic furrow in the eye disc and within wing pouch in the wing disc. Depletion of any CCT subunit in the eye disc results in headless phenotype. Overgrowth by active TOR signaling is suppressed by CCT RNAi. The CCT complex physically interacts with TOR signaling components including TOR, Rheb, and S6K. Loss of CCT leads to decreased phosphorylation of S6K and S6 while increasing phosphorylation of Akt. Insulin/TOR signaling is also necessary and sufficient for promoting CCT complex transcription. Our data provide evidence that the CCT complex regulates organ growth by directly interacting with the TOR signaling pathway.

Primordial follicles, consisting of granulosa cell (GC)-enveloped oocytes are maintained in a state of developmental arrest until activated to grow. The mechanism that operates to maintain this arrested state in GCs is currently unknown. Here, we show the TGFβ-activated transcription factor SMAD3 is expressed in primordial GC nuclei alongside the cell cycle proteins, cyclin D2 (CCND2) and P27. Using neonatal C57/Bl6 mouse ovaries densely populated with primordial follicles, CCND2 protein co-localised and was detected in complex with P27 by immunofluorescence and co-immunoprecipitation, respectively. In the same tissue, SMAD3 co-precipitated with DNA sequences upstream of Ccnd2 and Myc transcription start sites implicating both as direct SMAD3 targets. In older ovaries follicle growth was associated with nuclear exclusion of SMAD3 and reduced P27 and CCND2 in GCs, alongside elevated Myc expression. Brief (2 H) exposure of neonatal ovaries to TGFβ1 (10 ng/ml) in vitro led to immediate dissociation of SMAD3 from the Ccnd2 and Myc promoters. This coincided with elevated Myc and phospho-S6, an indicator of mTOR signalling, followed by a small increase in mean primordial GC number after 48 H. These findings highlight a concentration-dependent role for TGFβ signalling in the maintenance and activation of primordial follicles, through SMAD-dependent and independent signalling pathways, respectively.

The manner by which genetic diversity within a population generates individual phenotypes is a fundamental question of biology. To advance the understanding of the genotype–phenotype relationships towards the level of biochemical processes, we perform a proteome-wide association study (PWAS) of a complex quantitative phenotype. We quantify the variation of wing imaginal disc proteomes in Drosophila genetic reference panel (DGRP) lines using SWATH mass spectrometry. In spite of the very large genetic variation (1/36 bp) between the lines, proteome variability is surprisingly small, indicating strong molecular resilience of protein expression patterns. Proteins associated with adult wing size form tight co-variation clusters that are enriched in fundamental biochemical processes. Wing size correlates with some basic metabolic functions, positively with glucose metabolism but negatively with mitochondrial respiration and not with ribosome biogenesis. Our study highlights the power of PWAS to filter functional variants from the large genetic variability in natural populations. How genetic diversity generates complex phenotypes along a continuum remains a fundamental question of biology. Here—applying the emerging SWATH proteomics technology—the authors describe a proteome wide association study (PWAS) of Drosophila wing size and identify functional protein clusters associated with this trait.