ABSTRACT
Spotted-wing drosophila, Drosophila suzukii (Matsumura), is an invasive vinegar fly that is a major threat to the small fruits industries globally. Insect capa genes encode multiple neuropeptides, including CAPA-periviscerokinin (CAPA-PVK) peptides, that are specifically known to cause diuresis or anti-diuresis in various organisms. Here we identified and characterized a corresponding G protein-coupled receptor (GPCR) of the D. suzukii CAPA-PVK peptides: CAPA receptor (CAPA-R). To better characterize the behavior of D. suzukii CAPA-R, we used insect cell-based functional expression assays to evaluate responses of CAPA-R against D. suzukii CAPA-PVKs, CAPA-PVKs from five species in Insecta, one species from Mollusca, modified CAPA-PVK peptides, and some PRXamide family peptides: pyrokinin (PK), diapause hormone (DH), and ecdysis-triggering hormone (ETH). Functional studies revealed that the D. suzukii CAPA-R is strongly activated by both of its own natural D. suzukii CAPA-PVKs, and interestingly, it was strongly activated by other CAPA-PVK peptides from Frankliniella occidentallis (Thysanoptera), Solenopsis invicta (Hymenoptera), Helicoverpa zea (Lepidoptera) and Plutella xylostella (Lepidoptera). However, D. suzukii CAPA-R was not activated by Mollusca CAPA-PVK or the other PRXamide peptides. Gene expression analyses showed that the CAPA-R was highly expressed in the Malpighian tubules and moderately in hindgut compared to other digestive organs or the rest of body, supporting diuretic/antidiuretic functionality. When compared across life stages of D. suzukii, expression of CAPA-R was approximately 1.5x greater in the third instar than the other stages and minimally detected in the eggs, 4-day old pupae and 3-day old adults. Our results functionally characterized the D. suzukii CAPA-R and a few short peptides were identified as potential biological targets to exploit the CAPA-R for D. suzukii management.
Subject(s)
Drosophila Proteins , Drosophila , Neuropeptides , Animals , Female , Amino Acid Sequence , Drosophila/metabolism , Drosophila/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Gastrointestinal Tract/metabolism , Insect Hormones/metabolism , Larva/growth & development , Larva/metabolism , Larva/genetics , Neuropeptides/metabolism , Neuropeptides/genetics , Pupa/growth & development , Pupa/metabolism , Pupa/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/geneticsABSTRACT
RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.
Subject(s)
Cytosol , Drosophila Proteins , Endocytosis , Lysosomes , RNA Interference , RNA, Double-Stranded , Animals , RNA, Double-Stranded/metabolism , Lysosomes/metabolism , Cytosol/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Chloride Channels/metabolism , Chloride Channels/genetics , Cell Line , Intracellular Membranes/metabolism , Permeability , Drosophila/metabolism , Drosophila/geneticsABSTRACT
Mitophagy is the degradation of mitochondria via the autophagy-lysosome system, disruption of which has been linked to multiple neurodegenerative diseases. As a flux process involving the identification, tagging, and degradation of subcellular components, the analysis of mitophagy benefits from the microscopy analysis of fluorescent reporters. Studying the pathogenic mechanisms of disease also benefits from analysis in animal models in order to capture the complex interplay of molecular and cell biological phenomena. Here, we describe protocols to analyze mitophagy reporters in Drosophila by light microscopy.
Subject(s)
Mitochondria , Mitophagy , Animals , Mitochondria/metabolism , Genes, Reporter , Drosophila/metabolism , Microscopy, Fluorescence/methods , Drosophila melanogaster/metabolism , Lysosomes/metabolism , Autophagy/physiology , Drosophila Proteins/metabolism , Drosophila Proteins/geneticsABSTRACT
Extracellular vesicles are known for intercellular signaling roles but can also serve to simply dispose of unwanted cargoes. In this issue, Bostelman and Broihier discuss new work from Rodal and colleagues (https://doi.org/10.1083/jcb.202405025) that refutes prior work by showing that extracellular vesicles at Drosophila neuromuscular junctions are not required for signaling and instead likely serve a proteostasis role.
Subject(s)
Extracellular Vesicles , Neuromuscular Junction , Animals , Extracellular Vesicles/metabolism , Neuromuscular Junction/metabolism , Signal Transduction , Synapses/metabolism , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila/metabolism , Cell Communication , ProteostasisABSTRACT
The Drosophila Id gene extramacrochaetae (emc) is required during Drosophila eye development for proper cell fate specification within the R7 equivalence group. Without emc, R7 cells develop like R1/6 cells, and there are delays and deficits in differentiation of non-neuronal cone cells. Although emc encodes an Inhibitor of DNA-binding (Id) protein that is known to antagonize proneural bHLH protein function, no proneural gene is known for R7 or cone cell fates. These fates are also independent of daughterless (da), which encodes the ubiquitous E protein heterodimer partner of proneural bHLH proteins. We report here that the effects of emc mutations disappear in the absence of da, and are partially mimicked by forced expression of Da dimers, indicating that emc normally restrains da from interfering with R7 and cone cell specification, as occurs in emc mutants. emc, and da, regulate three known contributors to R7 fate, which are Notch signaling, Rap1, and Sevenless. R7 specification is partially restored to emc mutant cells by mutation of RapGap1, confirming that Rap1 activity, in addition to Notch activity, is a critical target of emc. These findings exemplify how mutations of an Id protein gene can affect processes that do not require any bHLH protein, by restraining Da activity within physiological bounds.
Subject(s)
Drosophila Proteins , Eye , Mutation , Receptors, Notch , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Receptors, Notch/metabolism , Eye/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Signal Transduction , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Drosophila/genetics , Drosophila/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Expression Regulation, Developmental , Telomere-Binding Proteins/metabolism , Telomere-Binding Proteins/genetics , Protein Binding , Transcription Factors/metabolism , Transcription Factors/genetics , Shelterin Complex , Repressor ProteinsABSTRACT
Ewing sarcoma (EwS) is a cancer that arises in the bones and soft tissues, typically driven by the Ewing's sarcoma breakpoint region 1-Friend leukemia virus integration 1 (EWS-FLI) oncogene. Implementation of genetically modified animal models of EwS has proved difficult largely owing to EWS-FLI's high toxicity. The EWS-FLI1FS frameshift variant that circumvents toxicity but is still able to perform key oncogenic functions provided the first study model in Drosophila. However, the quest for Drosophila lines expressing full-length, unmodified EWS-FLI remained open. Here, we show that EWS-FLI1FS's lower toxicity is owed to reduced protein levels caused by its frameshifted C-terminal peptide, and report new strategies through which we have generated Drosophila lines that express full-length, unmodified EWS-FLI. Using these lines, we have found that the upregulation of transcription from GGAA-microsatellites (GGAAµSats) presents a positive linear correlation within a wide range of EWS-FLI protein concentrations. In contrast, rather counterintuitively, GGAAµSats-independent transcriptomic dysregulation presents relatively minor differences across the same range, suggesting that GGAAµSat-dependent and -independent transcriptional upregulation present different kinetics of response with regards to changing EWS-FLI protein concentration. Our results underpin the functional relevance of varying EWS-FLI expression levels and provide experimental tools to investigate, in Drosophila, the effect of the EWS-FLI 'high' and 'low' states that have been reported and are suspected to be important for EwS in humans.
Subject(s)
Oncogene Proteins, Fusion , Proto-Oncogene Protein c-fli-1 , RNA-Binding Protein EWS , Animals , RNA-Binding Protein EWS/metabolism , RNA-Binding Protein EWS/genetics , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/metabolism , Proto-Oncogene Protein c-fli-1/genetics , Humans , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology , Animals, Genetically Modified , Drosophila/genetics , Drosophila/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolismABSTRACT
Despite the deep conservation of the DNA damage response (DDR) pathway, cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo, and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila, activates epidermal growth factor receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which, in turn, modulates the DDR in a Chk2-, p53-, and E2F1-dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with prosurvival, antiapoptotic activities, thereby facilitating developmental robustness in the face of genomic damage.
Subject(s)
Apoptosis , DNA Damage , Drosophila Proteins , ErbB Receptors , Imaginal Discs , Wings, Animal , Wnt Signaling Pathway , Wnt1 Protein , Animals , ErbB Receptors/metabolism , ErbB Receptors/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Wings, Animal/metabolism , Wings, Animal/growth & development , Imaginal Discs/metabolism , Imaginal Discs/growth & development , Wnt1 Protein/metabolism , Wnt1 Protein/genetics , Apoptosis/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/growth & development , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Checkpoint Kinase 2/metabolism , Checkpoint Kinase 2/genetics , Signal Transduction , DNA Breaks, Double-Stranded , Receptors, Invertebrate Peptide/metabolism , Receptors, Invertebrate Peptide/genetics , Drosophila/metabolism , Drosophila/genetics , Transcription FactorsABSTRACT
The gut microbiota and their metabolites are closely linked to obesity-related diseases, such as type 2 diabetes, but their causal relationship and underlying mechanisms remain largely elusive. Here, we found that dysbiosis-induced tyramine (TA) suppresses high-fat diet (HFD)-mediated insulin resistance in both Drosophila and mice. In Drosophila, HFD increases cytosolic Ca2+ signaling in enterocytes, which, in turn, suppresses intestinal lipid levels. 16 S rRNA sequencing and metabolomics revealed that HFD leads to increased prevalence of tyrosine decarboxylase (Tdc)-expressing bacteria and resulting tyramine production. Tyramine acts on the tyramine receptor, TyrR1, to promote cytosolic Ca2+ signaling and activation of the CRTC-CREB complex to transcriptionally suppress dietary lipid digestion and lipogenesis in enterocytes, while promoting mitochondrial biogenesis. Furthermore, the tyramine-induced cytosolic Ca2+ signaling is sufficient to suppress HFD-induced obesity and insulin resistance in Drosophila. In mice, tyramine intake also improves glucose tolerance and insulin sensitivity under HFD. These results indicate that dysbiosis-induced tyramine suppresses insulin resistance in both flies and mice under HFD, suggesting a potential therapeutic strategy for related metabolic disorders, such as diabetes.
Subject(s)
Calcium Signaling , Diet, High-Fat , Gastrointestinal Microbiome , Insulin Resistance , Tyramine , Animals , Tyramine/metabolism , Tyramine/pharmacology , Gastrointestinal Microbiome/drug effects , Diet, High-Fat/adverse effects , Mice , Calcium Signaling/drug effects , Obesity/metabolism , Obesity/microbiology , Obesity/etiology , Male , Drosophila/metabolism , Dysbiosis/metabolism , Dysbiosis/microbiology , Mice, Inbred C57BL , Drosophila melanogaster/microbiology , Drosophila melanogaster/metabolism , Enterocytes/metabolism , Enterocytes/drug effectsABSTRACT
The Ras family genes are proto-oncogenes that are highly conserved from Drosophila to humans. In Drosophila, RasV12 is a constitutively activated form of the Ras oncoprotein, and its function in cell-cycle progression is context dependent. However, how it influences the cell cycle of female germline stem cells (GSCs) still remains unknown. Using both wild-type GSCs and bam mutant GSC-like cells as model systems, here we determined that RasV12 overexpression promotes GSC division, not growth, opposite to that in somatic wing disc cells. Ras performs this function through activating the mitogen-activated protein kinase (MAPK) signaling. This signaling is activated specifically in the M phase of mitotic germ cells, including both wild-type GSCs and bam mutant GSC-like cells. Furthermore, RasV12 overexpression triggers polyploid nurse cells to die through inducing mitotic stress. Given the similarities between Drosophila and mammalian GSCs, we propose that the Ras/MAPK signaling also promotes mammalian GSC division.
Subject(s)
Cell Division , Drosophila Proteins , Ovary , ras Proteins , Animals , Female , Ovary/cytology , Ovary/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , ras Proteins/metabolism , ras Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , MAP Kinase Signaling System , Germ Cells/metabolism , Germ Cells/cytology , Stem Cells/metabolism , Stem Cells/cytology , Mitosis , Drosophila/metabolism , Signal TransductionABSTRACT
The polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia are both known to contain long cytoplasmic processes, while localized transcripts have only been studied extensively in neurons, not glia, especially in intact nervous systems. Here, we predict 1,740 localized Drosophila glial transcripts by extrapolating from our meta-analysis of seven existing studies characterizing the localized transcriptomes and translatomes of synaptically associated mammalian glia. We demonstrate that the localization of mRNAs in mammalian glial projections strongly predicts the localization of their high-confidence Drosophila homologs in larval motor neuron-associated glial projections and are highly statistically enriched for genes associated with neurological diseases. We further show that some of these localized glial transcripts are specifically required in glia for structural plasticity at the nearby neuromuscular junction synapses. We conclude that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important in disease.
Subject(s)
Neuroglia , Neuromuscular Junction , Neuronal Plasticity , RNA, Messenger , Animals , Neuroglia/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Neuronal Plasticity/genetics , Neuromuscular Junction/metabolism , Neuromuscular Junction/genetics , Mice , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Motor Neurons/metabolism , Transcriptome/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila/metabolism , Drosophila/geneticsABSTRACT
Stem cell loss in aging and disease is associated with nuclear deformation. Yet, how nuclear shape influences stem cell homeostasis is poorly understood. We investigated this connection using Drosophila germline stem cells, as survival of these stem cells is compromised by dysfunction of the nuclear lamina, the extensive protein network that lines the inner nuclear membrane and gives shape to the nucleus. To induce nuclear distortion in germline stem cells, we used the GAL4-UAS system to increase expression of the permanently farnesylated nuclear lamina protein, Kugelkern, a rate limiting factor for nuclear growth. We show that elevated Kugelkern levels cause severe nuclear distortion in germline stem cells, including extensive thickening and lobulation of the nuclear envelope and nuclear lamina, as well as alteration of internal nuclear compartments. Despite these changes, germline stem cell number, proliferation, and female fertility are preserved, even as females age. Collectively, these data demonstrate that disruption of nuclear architecture does not cause a failure of germline stem cell survival or homeostasis, revealing that nuclear deformation does not invariably promote stem cell loss.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Germ Cells , Homeostasis , Nuclear Lamina , Stem Cells , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Female , Germ Cells/metabolism , Drosophila melanogaster/metabolism , Stem Cells/metabolism , Nuclear Lamina/metabolism , Cell Nucleus/metabolism , Cell Proliferation , Drosophila/metabolism , Nuclear Envelope/metabolismABSTRACT
Perilipins are evolutionarily conserved from insects to mammals. Drosophila lipid storage droplet-1 (LSD-1) is a lipid storage droplet membrane surface-binding protein family member and a counterpart to mammalian perilipin 1 and is known to play a role in lipolysis. However, the function of LSD-1 during specific tissue development remains under investigation. This study demonstrated the role of LSD-1 in salivary gland development. Knockdown of Lsd-1 in the salivary gland was established using the GAL4/UAS system. The third-instar larvae of knockdown flies had small salivary glands containing cells with smaller nuclei. The null mutant Drosophila also showed the same phenotype. The depletion of LSD-1 expression induced a delay of endoreplication due to decreasing CycE expression and increasing DNA damage. Lsd-1 genetically interacted with Myc in the third-instar larvae. These results demonstrate that LSD-1 is involved in cell cycle and cell death programs in the salivary gland, providing novel insight into the effects of LSD-1 in regulating salivary gland development and the interaction between LSD-1 and Myc.
Subject(s)
Cell Death , Drosophila Proteins , Larva , Salivary Glands , Animals , Salivary Glands/metabolism , Salivary Glands/cytology , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Larva/growth & development , Larva/metabolism , Larva/genetics , Drosophila/metabolism , Drosophila/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/growth & development , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , DNA Replication , DNA-Binding Proteins , Oxidoreductases, N-Demethylating , Transcription FactorsABSTRACT
Deubiquitinases (DUBs) are essential for the maintenance of protein homeostasis and assembly of proteins into functional complexes. Despite growing interest in DUBs biological functions, the roles of DUBs in regulating intestinal stem cells (ISCs) and gut homeostasis remain largely unknown. Here, we perform an in vivo RNAi screen through induced knock-down of DUBs expression in adult midgut ISCs and enteroblasts (EBs) to identify DUB regulators of intestinal homeostasis in Drosophila. We screen 43 DUBs and identify 8 DUBs that are required for ISCs homeostasis. Knocking-down of usp1, CG7857, usp5, rpn8, usp10 and csn5 decreases the number of ISCs/EBs, while knocking-down of CG4968 and usp8 increases the number of ISCs/EBs. Moreover, knock-down of usp1, CG4968, CG7857, or rpn8 in ISCs/EBs disrupts the intestinal barrier integrity and shortens the lifespan, indicating the requirement of these DUBs for the maintenance of gut homeostasis. Furthermore, we provide evidences that USP1 mediates ISC lineage differentiation via modulating the Notch signaling activity. Our study identifies, for the first time, the deubiquitinases required for the maintenance of intestinal homeostasis in Drosophila, and provide new insights into the functional links between the DUBs and intestinal homeostasis.
Subject(s)
Deubiquitinating Enzymes , Drosophila Proteins , Homeostasis , Intestines , RNA Interference , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Deubiquitinating Enzymes/metabolism , Deubiquitinating Enzymes/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Stem Cells/metabolism , Drosophila/genetics , Drosophila/metabolism , Ubiquitin-Specific Proteases/metabolism , Ubiquitin-Specific Proteases/geneticsABSTRACT
Ninein (Nin) is a microtubule (MT) anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize MTs at noncentrosomal MT-organizing centers (ncMTOCs). In humans, the NIN gene is mutated in Seckel syndrome, an inherited developmental disorder. Here, we dissect the protein domains involved in Nin's localization and interactions with dynein and ensconsin (ens/MAP7) and show that the association with ens cooperatively regulates MT assembly in Drosophila fat body cells. We define domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface, localization within the nucleus, and association with Dynein light intermediate chain (Dlic) and ens, respectively. We show that Nin's association with ens synergistically regulates MT assembly. Together, these findings reveal novel features of Nin function and its regulation of a ncMTOC.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Dyneins , Microtubule-Associated Proteins , Microtubule-Organizing Center , Microtubules , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Animals , Dyneins/metabolism , Microtubules/metabolism , Microtubule-Associated Proteins/metabolism , Microtubule-Organizing Center/metabolism , Drosophila melanogaster/metabolism , Nuclear Proteins/metabolism , Centrosome/metabolism , Protein Domains , Humans , Fat Body/metabolism , Drosophila/metabolism , Cell Nucleus/metabolism , Centrioles/metabolism , Protein Binding , Homeodomain ProteinsABSTRACT
Vitamin B6 is a water-soluble vitamin which possesses antioxidant properties. Its catalytically active form, pyridoxal 5'-phosphate (PLP), is a crucial cofactor for DNA and amino acid metabolism. The inverse correlation between vitamin B6 and cancer risk has been observed in several studies, although dietary vitamin B6 intake sometimes failed to confirm this association. However, the molecular link between vitamin B6 and cancer remains elusive. Previous work has shown that vitamin B6 deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, suggesting that genome instability may correlate the lack of this vitamin to cancer. Here we provide evidence in support of this hypothesis. Firstly, we show that PLP deficiency, induced by the PLP antagonists 4-deoxypyridoxine (4DP) or ginkgotoxin (GT), promoted tumorigenesis in eye larval discs transforming benign RasV12 tumors into aggressive forms. In contrast, PLP supplementation reduced the development of tumors. We also show that low PLP levels, induced by 4DP or by silencing the sgllPNPO gene involved in PLP biosynthesis, worsened the tumor phenotype in another Drosophila cancer model generated by concomitantly activating RasV12 and downregulating Discs-large (Dlg) gene. Moreover, we found that RasV12 eye discs from larvae reared on 4DP displayed CABs, reactive oxygen species (ROS) and low catalytic activity of serine hydroxymethyltransferase (SHMT), a PLP-dependent enzyme involved in thymidylate (dTMP) biosynthesis, in turn required for DNA replication and repair. Feeding RasV12 4DP-fed larvae with PLP or ascorbic acid (AA) plus dTMP, rescued both CABs and tumors. The same effect was produced by overexpressing catalase in RasV12 DlgRNAi 4DP-fed larvae, thus allowing to establish a relationship between PLP deficiency, CABs, and cancer. Overall, our data provide the first in vivo demonstration that PLP deficiency can impact on cancer by increasing genome instability, which is in turn mediated by ROS and reduced dTMP levels.
Subject(s)
Vitamin B 6 Deficiency , Animals , Vitamin B 6 Deficiency/metabolism , Vitamin B 6 Deficiency/complications , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Vitamin B 6/metabolism , Vitamin B 6/pharmacology , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Drosophila/metabolism , Pyridoxal Phosphate/metabolism , Reactive Oxygen Species/metabolism , Carcinogenesis/genetics , Carcinogenesis/pathology , Carcinogenesis/metabolism , Carcinogenesis/drug effects , ras Proteins/metabolism , Neoplasms/pathology , Neoplasms/metabolism , Neoplasms/genetics , Larva/metabolism , HumansABSTRACT
Aggregation of aberrant proteins is a common pathological hallmark in neurodegeneration such as polyglutamine (polyQ) and other repeat-expansion diseases. Here through overexpression of ataxin3 C-terminal polyQ expansion in Drosophila gut enterocytes, we generated an intestinal obstruction model of spinocerebellar ataxia type3 (SCA3) and reported a new role of nuclear-associated endosomes (NAEs)-the delivery of polyQ to the nucleoplasm. In this model, accompanied by the prominently increased RAB5-positive NAEs are abundant nucleoplasmic reticulum enriched with polyQ, abnormal nuclear envelope invagination, significantly reduced endoplasmic reticulum, indicating dysfunctional nucleocytoplasmic trafficking and impaired endomembrane organization. Consistently, Rab5 but not Rab7 RNAi further decreased polyQ-related NAEs, inhibited endomembrane disorganization, and alleviated disease model. Interestingly, autophagic proteins were enriched in polyQ-related NAEs and played non-canonical autophagic roles as genetic manipulation of autophagic molecules exhibited differential impacts on NAEs and SCA3 toxicity. Namely, the down-regulation of Atg1 or Atg12 mitigated while Atg5 RNAi aggravated the disease phenotypes both in Drosophila intestines and compound eyes. Our findings, therefore, provide new mechanistic insights and underscore the fundamental roles of endosome-centered nucleocytoplasmic trafficking and homeostatic endomembrane allocation in the pathogenesis of polyQ diseases.
Subject(s)
Autophagy , Endosomes , Peptides , Animals , Peptides/metabolism , Endosomes/metabolism , Cell Nucleus/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Active Transport, Cell Nucleus , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Machado-Joseph Disease/metabolism , Machado-Joseph Disease/genetics , Machado-Joseph Disease/pathology , Enterocytes/metabolism , Disease Models, Animal , Ataxin-3/metabolism , Ataxin-3/genetics , Drosophila/metabolismABSTRACT
Adenosine-to-inosine (A-to-I) RNA editing recodes the genome and confers flexibility for the organisms to adapt to the environment. It is believed that RNA recoding sites are well suited for facilitating adaptive evolution by increasing the proteomic diversity in a temporal-spatial manner. The function and essentiality of a few conserved recoding sites are recognized. However, the experimentally discovered functional sites only make up a small corner of the total sites, and there is still the need to expand the repertoire of such functional sites with bioinformatic approaches. In this study, we define a new category of RNA editing sites termed 'conserved editing with non-conserved recoding' and systematically identify such sites in Drosophila editomes, figuring out their selection pressure and signals of adaptation at inter-species and intra-species levels. Surprisingly, conserved editing sites with non-conserved recoding are not suppressed and are even slightly overrepresented in Drosophila. DNA mutations leading to such cases are also favoured during evolution, suggesting that the function of those recoding events in different species might be diverged, specialized, and maintained. Finally, structural prediction suggests that such recoding in potassium channel Shab might increase ion permeability and compensate the effect of low temperature. In conclusion, conserved editing with non-conserved recoding might be functional as well. Our study provides novel aspects in considering the adaptive evolution of RNA editing sites and meanwhile expands the candidates of functional recoding sites for future validation.
Subject(s)
Adenosine , Drosophila , Inosine , RNA Editing , Animals , Inosine/metabolism , Inosine/genetics , Drosophila/genetics , Drosophila/metabolism , Adenosine/metabolism , Adenosine/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Evolution, Molecular , Drosophila Proteins/genetics , Drosophila Proteins/metabolismABSTRACT
Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.
Subject(s)
Anaplastic Lymphoma Kinase , Central Nervous System , Drosophila Proteins , Animals , Central Nervous System/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Anaplastic Lymphoma Kinase/metabolism , Anaplastic Lymphoma Kinase/genetics , Larva/metabolism , Larva/genetics , Larva/growth & development , Neuropeptides/metabolism , Neuropeptides/genetics , Signal Transduction , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Drosophila/genetics , Drosophila/metabolism , Gene Expression Profiling , Gene Expression RegulationABSTRACT
The MS2-PP7 two-color live-imaging system provides insights into the spatiotemporal dynamics of nascent transcripts at tagged loci. Here, we present a protocol to quantitatively measure the rate of RNA polymerase II elongation for each actively transcribing nucleus in living Drosophila embryos. The elongation rate is calculated by measuring the effective distance and the time elapsed between MS2 and PP7 trajectories. We describe steps for preparing embryo samples, performing live imaging, and measuring the elongation rate. For complete details on the use and execution of this protocol, please refer to Keller et al.1.