Drosophila Homolog Research Articles

Drosophila AHR limits tumor growth and stem cell proliferation in the intestine

Background The aryl hydrocarbon receptor (AHR) plays important roles in intestinal homeostasis, limiting tumour growth and promoting differentiation in the intestinal epithelium. Spineless, the Drosophila homolog of AHR, has only been studied in the context of development but not in the adult intestine. Methods The role of Spineless in the Drosophila midgut was studied by overexpression or knockdown of Spineless in infection and tumour models and RNA sequencing of sorted midgut progenitor cells. Results We show that spineless is upregulated in the adult intestinal epithelium after infection with Pseudomonas entomophila (P.e.). Spineless knockdown increased stem cell proliferation following infection-induced injury. Spineless overexpression limited intestinal stem cell proliferation and reduced survival after infection. In two tumour models, using either Notch RNAi or constitutively active Yorkie, Spineless suppressed tumour growth and doubled the lifespan of tumour-bearing flies. At the transcriptional level it reversed the gene expression changes induced in Yorkie tumours, counteracting cell proliferation and altered metabolism. Conclusions These findings demonstrate a new role for Spineless in the adult Drosophila midgut and highlight the evolutionarily conserved functions of AHR/Spineless in the control of proliferation and differentiation of the intestinal epithelium.

Whole organism and tissue-specific analysis of pexophagy in Drosophila.

Peroxisomes are essential organelles involved in critical metabolic processes in animals such as fatty acid oxidation, ether phospholipid production and reactive oxygen species detoxification. We have generated transgenic Drosophila melanogaster models expressing fluorescent reporters for the selective autophagy of peroxisomes, a process known as pexophagy. We show that these reporters are colocalized with a peroxisomal marker and that they can reflect pexophagy induction by iron chelation and inhibition by depletion of the core autophagy protein Atg5. Using light sheet microscopy, we have been able to obtain a global overview of pexophagy levels across the entire organism at different stages of development. Tissue-specific control of pexophagy is exemplified by areas of peroxisome abundance but minimal pexophagy, observed in clusters of oenocytes surrounded by epithelial cells where pexophagy is much more evident. Enhancement of pexophagy was achieved by feeding flies with the iron chelator deferiprone, in line with past results using mammalian cells. Specific drivers were used to visualize pexophagy in neurons, and to demonstrate that specific depletion in the larval central nervous system of Hsc70-5, the Drosophila homologue of the chaperone HSPA9/mortalin, led to a substantial elevation in pexophagy.

Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions.

The network of proteins at the interface between cell-cell adherens junctions and the actomyosin cytoskeleton provides robust yet dynamic connections that facilitate cell shape change and motility. While this was initially thought to be a simple linear connection via classic cadherins and their associated catenins, we now have come to appreciate that many more proteins are involved, providing robustness and mechanosensitivity. Defining the full set of proteins in this network remains a key objective in our field. Proximity proteomics provides a means to define these networks. Mammalian Afadin and its Drosophila homolog Canoe are key parts of this protein network, facilitating diverse cell shape changes during gastrulation and other events of embryonic morphogenesis. Here we report results of several proximity proteomics screens, defining proteins in the neighborhood of both the N- and C-termini of mammalian Afadin in the premier epithelial model, MDCK cells. We compare our results with previous screens done in other cell types, and with proximity proteomics efforts with other junctional proteins. These reveal the value of multiple screens in defining the full network of neighbors and offer interesting insights into the overlap in protein composition between different epithelial cell junctions.

Drosophila aux orchestrates the phosphorylation-dependent assembly of the lysosomal V-ATPase in glia and contributes to SNCA/α-synuclein degradation

ABSTRACT Glia contribute to the neuropathology of Parkinson disease (PD), but how they react opposingly to be beneficial or detrimental under pathological conditions, like promoting or eliminating SNCA/α-syn (synuclein alpha) inclusions, remains elusive. Here we present evidence that aux (auxilin), the Drosophila homolog of the PD risk factor GAK (cyclin G associated kinase), regulates the lysosomal degradation of SNCA/α-syn in glia. Lack of glial GAK/aux increases the lysosome number and size, regulates lysosomal acidification and hydrolase activity, and ultimately blocks the degradation of substrates including SNCA/α-syn. Whereas SNCA/α-syn accumulates prominently in lysosomes devoid of glial aux, levels of injected SNCA/α-syn preformed fibrils are further enhanced in the absence of microglial GAK. Mechanistically, aux mediates phosphorylation at the serine 543 of Vha44, the V1 C subunit of the vacuolar-type H+-translocating ATPase (V-ATPase), and regulates its assembly to control proper acidification of the lysosomal milieu. Expression of Vha44, but not the Vha44 variant lacking S543 phosphorylation, restores lysosome acidity, locomotor deficits, and DA neurodegeneration upon glial aux depletion, linking this pathway to PD. Our findings identify a phosphorylation-dependent switch controlling V-ATPase assembly for lysosomal SNCA/α-syn degradation in glia. Targeting the clearance of glial SNCA/α-syn inclusions via this lysosomal pathway could potentially be a therapeutic approach to ameliorate the disease progression in PD. Abbreviation: aux: auxilin; GAK: cyclin G associated kinase; LTG: LysoTracker Green; LTR: LysoTracker Red; MR: Magic Red; PD: Parkinson disease; SNCA/a-syn: synuclein alpha; V-ATPase: vacuolar-type H+-translocating ATPase

Structural basis for the interaction between the Drosophila RTK Sevenless (dROS1) and the GPCR BOSS

Sevenless, the Drosophila homologue of ROS1 (University of Rochester Sarcoma) (herein, dROS1) is a receptor tyrosine kinase (RTK) essential for the differentiation of Drosophila R7 photoreceptor cells. Activation of dROS1 is mediated by binding to the extracellular region (ECR) of the GPCR (G protein coupled receptor) BOSS (Bride Of Sevenless) on adjacent cells. Activation of dROS1 by BOSS leads to subsequent downstream signaling pathways including SOS (Son of Sevenless). However, the physical basis for how dROS1 interacts with BOSS has long remained unknown. Here we provide a cryo-EM structure of dROS1’s extracellular region, which mediates ligand binding. We show that the extracellular region of dROS1 adopts a folded-over conformation stabilized by an N-terminal domain comprised of two disulfide stapled helical hairpins. We further narrowed down the interacting binding epitopes on both dROS1 and BOSS using hydrogen-deuterium exchange mass spectrometry (HDX-MS). This includes beta-strands in dROS1’s third Fibronectin type III (FNIII) domain and a C-terminal peptide in BOSS’ ECR. Our mutagenesis studies, coupled with AlphaFold complex predictions, support a binding interaction mediated by a hydrophobic interaction and beta-strand augmentation between these regions. Our findings provide a fundamental understanding of the regulatory function of dROS1 and further provide mechanistic insight into the human ortholog and oncogene ROS1.

SARS-CoV-2 membrane protein induces neurodegeneration via affecting Golgi-mitochondria interaction

BackgroundNeurological complications are a significant concern of Coronavirus Disease 2019 (COVID-19). However, the pathogenic mechanism of neurological symptoms associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is poorly understood.MethodsWe used Drosophila as a model to systematically analyze SARS-CoV-2 genes encoding structural and accessory proteins and identified the membrane protein (M) that disrupted mitochondrial functions in vivo. The M protein was stereotaxically injected to further assess its effects in the brains of wild-type (WT) and 5 × FAD mice. Omics technologies, including RNA sequencing and interactome analysis, were performed to explore the mechanisms of the effects of M protein both in vitro and in vivo.ResultsSystematic analysis of SARS-CoV-2 structural and accessory proteins in Drosophila identified that the M protein induces mitochondrial fragmentation and dysfunction, leading to reduced ATP production, ROS overproduction, and eventually cell death in the indirect flight muscles. In WT mice, M caused hippocampal atrophy, neural apoptosis, glial activation, and mitochondrial damage. These changes were further aggravated in 5 × FAD mice. M was localized to the Golgi apparatus and genetically interacted with four wheel drive (FWD, a Drosophila homolog of mammalian PI4KIIIβ) to regulate Golgi functions in flies. Fwd RNAi, but not PI4KIIIα RNAi, reversed the M-induced Golgi abnormality, mitochondrial fragmentation, and ATP reduction. Inhibition of PI4KIIIβ activity suppressed the M-induced neuronal cell death. Therefore, M induced mitochondrial fragmentation and apoptosis likely through disruption of Golgi-derived PI(4)P-containing vesicles.ConclusionsM disturbs the distribution and function of Golgi, leading to mitochondrial abnormality and eventually neurodegeneration via a PI4KIIIβ-mediated mechanism. This study reveals a potential mechanism for COVID-19 neurological symptoms and opens a new avenue for development of therapeutic strategies targeting SARS-CoV-2 M or mitochondria.

Examining the potential involvement of NONO in TDP-43 proteinopathy in Drosophila.

The misfolding and aggregation of TAR DNA binding protein-43 (TDP-43), leading to the formation of cytoplasmic inclusions, emerge as a key pathological feature in a spectrum of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). TDP-43 shuttles between the nucleus and cytoplasm but forms nuclear bodies (NBs) in response to stress. These NBs partially colocalise with nuclear speckles and paraspeckles that sequester RNAs and proteins, thereby regulating many cellular functions. The laboratory of Steven Brown has recently found that the non-POU domain-containing octamer-binding protein (NONO), a component of paraspeckles, forms novel nuclear speckle-like structures in mouse cortical neurons in response to stress and sleep deprivation. These findings suggest the possibility of a functional link between NONO and TDP-43, potentially contributing to TDP-43 proteinopathy. Here, we demonstrate that pathological phenotypes caused by TDP-43 gain of function-locomotor defects and life span shortening-are exacerbated by silencing the Drosophila homolog of NONO, no on or off transient A (NonA). Additionally, NonA silencing results in an increase in nuclear TDP-43 NBs. These results provide supporting evidence for the functional link between NONO and TDP-43 and lay the foundation for dissecting underlying mechanisms.

SASH1 is a novel binding partner to disassemble Caskin1 tandem SAM homopolymer through heterogeneous SAM-SAM interaction.

Calcium/calmodulin-dependent serine protein kinase (CASK) interaction protein 1/2 (Caskin1/2) is essential neuronal synaptic scaffold protein in nervous system development. Knockouts of Caskin1/2 display severe deficits in novelty recognition and spatial memory. The tandem sterile alpha motif (SAM) domains of Caskin1/2, also conserved in their Drosophila homolog Ckn, are known to form homopolymers, yet their dynamic regulation mechanism remains unclear. In this study, SAM and SH3 domain-containing protein 1 (SASH1) was first identified as a novel binding partner of Caskin1/2 through yeast two-hybrid (Y2H) screening. The SAM-SAM interaction between SASH1 and Caskin1 was biochemically characterized by size-exclusion chromatography (SEC), isothermal titration calorimetry (ITC), and glutathione-S-transferase (GST) pull-down and co-immunoprecipitation (co-IP) assays. Structural insights from AlphaFold2-predicted models of the Caskin1-SAMs/SASH1-SAM1 complex, along with mutagenesis validations, revealed key residues at the end-helix (EH)/mid-loop (ML) interface for this interaction. More interestingly, the Caskin1-SAMs homopolymer can be disrupted by the SAM-SAM interaction, which was consistently verified by using sedimentation, transmission electron microscopy (TEM), and immunofluorescence (IF) staining in heterologous cell lines. In summary, our findings provide a solid biochemical basis for the Caskin1/SASH1 interaction and propose a potential mechanism for regulating Caskin1/2 homopolymerization via SAM-SAM interactions. More importantly, the principle governing SAM homopolymer depolymerization is generalized via suggesting two distinct types of heterogeneous SAM-SAM interactions, offering fresh insights into SAM domain-mediated homopolymerization and depolymerization.

FMR1 genetically interacts with DISC1 to regulate glutamatergic synaptogenesis

Synaptic development and functions have been hypothesized as crucial mechanisms of diverse neuropsychiatric disorders. Studies in past years suggest that mutations in the fragile X mental retardation 1 (FMR1) are associated with diverse mental disorders including intellectual disability, autistic spectrum disorder, and schizophrenia. In this study, we have examined genetical interactions between a select set of risk factor genes using fruit flies to find that dfmr1, the Drosophila homolog of the human FMR1 gene, exhibits functional interactions with DISC1 in synaptic development. We show that DISC1 overexpression in the dfmr1null heterozygous background causes synaptic alterations at the larval neuromuscular junctions that are distinct from those in the wild-type background. Loss of dfmr1 modifies the DISC1 overexpression phenotype in synaptic formation, suppressing the formation of synapse boutons. Interaction between the two genes was further supported molecularly by the results that dfmr1 mutations suppress the DISC1-mediated upregulations of the postsynaptic expression of a glutamate receptor and the expression of ELKS/CAST protein, Bruchpilot, in presynaptic motoneurons. Moreover, DISC1 overexpression in the dfmr1null heterozygous background causes downregulation of a MAP1 family protein, Futsch. These results thus suggest an intriguing converging mechanism controlled by FMR1 and DISC1 in the developing glutamatergic synapses.

ScRNA-seq revealed transcriptional signatures of human umbilical cord primitive stem cells and their germ lineage origin regulated by imprinted genes

A population of CD133+lin-CD45- and CD34+lin-CD45- very small embryonic-like stem cells (VSELs) has been identified in postnatal human tissues, including bone marrow (BM), mobilized peripheral blood (mPB) and umbilical cord blood (UCB). Under appropriate conditions, VSELs in vitro and in vivo differentiate into tissue-committed stem cells for all three germ layers. Molecular analysis of adult murine BM-purified VSELs revealed that these rare cells deposited during development in adult tissues (i) express a similar transcriptome as embryonic stem cells, (ii) share several markers characteristic for epiblast and migratory primordial germ cells (PGCs), (iii) highly express a polycomb group protein enhancer of zeste drosophila homolog 2 (Ezh2) and finally (iv) display a unique pattern of imprinting at crucial paternally inherited genes that promotes their quiescence. Here, by employing single-cell RNA sequencing we demonstrate for the first time that purified from UCB human VSELs defined by expression of CD34 or CD133 antigens and lack of lineage markers, including CD45 antigen express similar molecular signature as murine BM-derived VSELs. Specifically, unsupervised clustering revealed numerous subpopulations of VSELs including ones i) annotated to germline compartments, ii) regulated by parental imprinting, iii) responding to early developmental fate decisions, iv) transcription factors involved in differentiation and development, including homeobox family of genes, and v) expressing innate immunity and purinergic signaling genes.

Neuronal fatty acid-binding protein enhances autophagy and suppresses amyloid-β pathology in a Drosophila model of Alzheimer's disease.

Fatty acid-binding proteins (FABPs) are small cytoplasmic proteins involved in intracellular lipid transport and bind free fatty acids, cholesterol, and retinoids. FABP3, the major neuronal FABP in the adult brain, is upregulated in the CSF of patients with Alzheimer's disease (AD). However, the precise role of neuronal FABPs in AD pathogenesis remains unclear. This study investigates the contribution of fabp, the Drosophila homolog of FABP3 and FABP7, to amyloid β (Aβ) pathology using a Drosophila model. Neuronal knockdown of fabp shortened the lifespan of flies and increased age-related protein aggregates in the brain. In an AD model, fabp knockdown in neurons increased Aβ accumulation and Aβ-induced neurodegeneration, whereas fabp overexpression ameliorated Aβ pathology. Notably, fabp overexpression stimulated autophagy, which was inhibited by the knockdown of Eip75B, the Drosophila homolog of the peroxisome proliferator-activated receptor (PPAR). The PPAR activator rosiglitazone restored autophagy impaired by fabp knockdown and reduced fabp knockdown-induced increased Aβ aggregation and cell death. Furthermore, knockdown of either fabp or Eip75B in the wing imaginal disc or adult fly brain reduced the expression of Atg6 and Atg8a. Additionally, treatment of the fabp knockdown AD model flies with polyunsaturated fatty acids, such as docosahexaenoic acid or linoleic acid, partially alleviated cell death in the developing eye, restored impaired autophagy flux, reduced Aβ aggregation, and attenuated Aβ-induced cell death. Our results suggest that Drosophila fabp plays an important role in maintaining protein homeostasis during aging and protects neurons from Aβ-induced cell death by enhancing autophagy through the PPAR pathway. These findings highlight the potential importance of neuronal FABP function in AD pathogenesis.

Endurance exercise attenuates Gαq-RNAi induced hereditary obesity and skeletal muscle dysfunction via improving skeletal muscle Srl/MRCC-I pathway in Drosophila

G protein alpha q subunit (Gαq) can binds to the G protein-coupled receptor (GPCR) for signaling and is closely related to lipid metabolism. Endurance exercise is an effective means of combating acquired obesity and its complications, but the mechanisms by which endurance exercise modulates hereditary obesity and its complications are unknown. In this study, we achieved knockdown of Gαq in drosophila adipose tissue and skeletal muscle by constructing the Gαq-UAS-RNAi/Ppl-Gal4 and Gαq-UAS-RNAi/Mef2-GAl4 systems. Drosophila were subjected a three-week endurance exercise intervention, and changes in relevant indicators were detected and observed by RT-PCR, ELISA, oil red staining, immunofluorescence staining, and transmission electron microscopy. The results showed that knockdown of Gαq in both adipose tissue and skeletal muscle induced a significant increase in triglycerides accompanied by a decrease in rapid climbing ability, a decrease in Superoxide Dismutase (SOD) activity level, and a decrease in Mitochondrial respiratory chain complexI (MRCC I) content in Drosophila whole body and skeletal muscle, and down-regulated the expression of the G protein alpha q subunit (Gαq), the skeletal muscle myosin heavy chain expression gene (Mhc), mitochondrial biogenesis gene Spargal(the PGC-1alpha homologue in Drosophila). Endurance exercise significantly improved the triglyceride levels in the whole body and skeletal muscle of drosophila with Gαq knockdown in adipose tissue and skeletal muscle, as well as their ability to climb, increased SOD activity level and MRCCI content level, and up-regulated the expression of Gαq, Mhc, and Spargal(Srl). Thus, the present findings suggest that genetic defects in the Gαq gene in adipose and skeletal muscle tissues induce hereditary obesity and skeletal muscle dysfunction, and that endurance exercise attenuates this hereditary obesity and concomitant skeletal muscle dysfunction in drosophila by improving skeletal muscle fiber contractile proteins, mitochondrial function and function, and antioxidant capacity via mediating the Gαq/Mhc, Gαq/Srl/MRCC-I, and Gαq/SOD pathways.

Dynamic protein assembly and architecture of the large solitary membraneless organelle during germline development in the wasp Nasonia vitripennis.

Throughout metazoa, germ cells assemble RNA-protein organelles (germ granules). In Drosophila ovaries, perinuclear nuage forms in the nurse cells, while compositionally similar polar granules form in the oocyte. A similar system appears to exist in the distantly related (∼350 million years) wasp Nasonia, with some surprising divergences. Nuage is similarly formed in Nasonia, except that anterior nurse cells accumulate significantly more nuage, in association with high levels of DNA double-strand breaks, suggesting that increased transposon activity anteriorly is silenced by high nuage levels. In the oocyte, the germ plasm forms a single granule that is 40 times larger than a homologous Drosophila polar granule. While conserved germ granule proteins are recruited to the oosome, they show unusual localization: Tudor protein forms a shell encapsulating the embryonic oosome, while small Oskar/Vasa/Aubergine granules coalesce interiorly. Wasp Vasa itself is unusual since it has an alternative splice form that includes a previously unreported nucleoporin-like phenylalanine-glycine repeat domain. Our work is consistent with the high degree of evolutionary plasticity of membraneless organelles, and describes a new experimental model and resources for studying biomolecular condensates.

Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions.

The network of proteins at the interface between cell-cell adherens junctions and the actomyosin cytoskeleton provides robust yet dynamic connections that facilitate cell shape change and motility. While this was initially thought to be a simple linear connection via classic cadherins and their associated catenins, we now have come to appreciate that many more proteins are involved, providing robustness and mechanosensitivity. Defining the full network of proteins in this network remains a key objective in our field. Proximity proteomics provides a means to define these networks. Mammalian Afadin and its Drosophila homolog Canoe are key parts of this protein network, facilitating diverse cell shape changes during gastrulation and other events of embryonic morphogenesis. Here we report results of several proximity proteomics screens, defining proteins in the neighborhood of both the N- and C-termini of mammalian Afadin in the premier epithelial model, MDCK cells. We compare our results with previous screens done in other cell types, and with proximity proteomics efforts with other junctional proteins. These reveal the value of multiple screens in defining the full network of neighbors and offer interesting insights into the overlap in protein composition between different epithelial cell junctions.

Cross-species evidence for a developmental origin of adult hypersomnia with loss of synaptic adhesion molecules beat-Ia/CADM2.

Idiopathic hypersomnia (IH) is a poorly-understood sleep disorder characterized by excessive daytime sleepiness despite normal nighttime sleep. Combining human genomics with behavioral and mechanistic studies in fish and flies, we uncover a role for beat-Ia/CADM2 , synaptic adhesion molecules of the immunoglobulin superfamily, in excessive sleepiness. Neuronal knockdown of Drosophila beat-Ia results in sleepy flies and loss of the vertebrate ortholog of beat-Ia , CADM2 , results in sleepy fish. We delineate a developmental function for beat-Ia in synaptic elaboration of neuropeptide F (NPF) neurites projecting to the suboesophageal zone (SEZ) of the fly brain. Brain connectome and experimental evidence demonstrate these NPF outputs synapse onto a subpopulation of SEZ GABAergic neurons to stabilize arousal. NPF is the Drosophila homolog of vertebrate neuropeptide Y (NPY), and an NPY receptor agonist restores sleep to normal levels in zebrafish lacking CADM2 . These findings point towards NPY modulation as a treatment target for human hypersomnia.

Destruction as a creative process: CAD-induced DNA strand breaks promote macrophage differentiation

In a recent issue of Cell Reports, Maurya etal.1 demonstrated that Drep4, the Drosophila homolog of caspase-activated DNase (CAD), drives DNA strand breaks during myeloid-like/macrophage cell differentiation and that this Drep4/CAD activity is essential for the differentiation process.

Hippo effector, Yorkie, is a tumor suppressor in select Drosophila squamous epithelia

Mammalian Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) and Drosophila Yorkie (Yki) are transcription cofactors of the highly conserved Hippo signaling pathway. It has been long assumed that the YAP/TAZ/Yki signaling drives cell proliferation during organ growth. However, its instructive role in regulating developmentally programmed organ growth, if any, remains elusive. Out-of-context gain of YAP/TAZ/Yki signaling often turns oncogenic. Paradoxically, mechanically strained, and differentiated squamous epithelia display developmentally programmed constitutive nuclear YAP/TAZ/Yki signaling. The unknown, therefore, is how a growth-promoting YAP/TAZ/Yki signaling restricts proliferation in differentiated squamous epithelia. Here, we show that reminiscent of a tumor suppressor, Yki negatively regulates the cell growth-promoting PI3K/Akt/TOR signaling in the squamous epithelia of Drosophila tubular organs. Thus, downregulation of Yki signaling in the squamous epithelium of the adult male accessory gland (MAG) up-regulates PI3K/Akt/TOR signaling, inducing cell hypertrophy, exit from their cell cycle arrest, and, finally, culminating in squamous cell carcinoma (SCC). Thus, blocking PI3K/Akt/TOR signaling arrests Yki loss-induced MAG-SCC. Further, MAG-SCCs, like other lethal carcinomas, secrete a cachectin, Impl2-the Drosophila homolog of mammalian IGFBP7-inducing cachexia and shortening the lifespan of adult males. Moreover, in the squamous epithelium of other tubular organs, like the dorsal trunk of larval tracheal airways or adult Malpighian tubules, downregulation of Yki signaling triggers PI3K/Akt/TOR-induced cell hypertrophy. Our results reveal that Yki signaling plays an instructive, antiproliferative role in the squamous epithelia of tubular organs.

Transcriptomic analysis of meiotic genes during the mitosis-to-meiosis transition in Drosophila females.

Germline cells produce gametes, which are specialized cells essential for sexual reproduction. Germline cells first amplify through several rounds of mitosis before switching to the meiotic program, which requires specific sets of proteins for DNA recombination, chromosome pairing, and segregation. Surprisingly, we previously found that some proteins of the synaptonemal complex, a prophase I meiotic structure, are already expressed and required in the mitotic region of Drosophila females. Here, to assess if additional meiotic genes were expressed earlier than expected, we isolated mitotic and meiotic cell populations to compare their RNA content. Our transcriptomic analysis reveals that all known meiosis I genes are already expressed in the mitotic region; however, only some of them are translated. As a case study, we focused on mei-W68, the Drosophila homolog of Spo11, to assess its expression at both the mRNA and protein levels and used different mutant alleles to assay for a premeiotic function. We could not detect any functional role for Mei-W68 during homologous chromosome pairing in dividing germ cells. Our study paves the way for further functional analysis of meiotic genes expressed in the mitotic region.

Murine glial protrusion transcripts predict localized Drosophila glial mRNAs involved in plasticity.

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.

ER-GUARD: an evolutionarily conserved antioxidant defense system at ER membranes.

Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species (ROS). The ER-transmembrane protein that provides reducing equivalents to ER and guards the cytosol for antioxidant defense remains unidentified. Here we combine AlphaFold2-based and functional reporter screens in C. elegans to identify a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. Deleting C. elegans ERGU-1 causes excessive H2O2 and transcriptional gene up-regulation through SKN-1, homolog of mammalian antioxidant master regulator NRF2. ERGU-1 deficiency also impairs organismal reproduction and behaviors. Both C. elegans and human ERGU-1 proteins localize to ER membranes and form network reticulum structures. We name this system ER-GUARD, Endoplasmic Reticulum Guardian Aegis of Redox Defense. Human and Drosophila homologs of ERGU-1 can rescue C. elegans mutant phenotypes, demonstrating evolutionarily ancient and conserved functions. Together, our results reveal an ER-membrane-specific protein machinery and defense-net system ER-GUARD for peroxide detoxification and suggest a previously unknown but conserved pathway for antioxidant defense in animal cells.