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Mating exerts profound and multifaceted effects on the physiology of female insects, particularly influencing metabolic alterations and bolstering stress resilience. Drosophila melanogaster has emerged as an excellent model to investigate the mechanism of neurodegenerative diseases. However, interplay between mating and its impact on the Drosophila brain remains a tantalizing enigma, awaiting elucidation. Herein, we reported that mating significantly improved the climbing and jumping activity in mated females compared to the virgins in Drosophila. Mating also reduced oxidative stress in the brain. Based on the results, we found that, mated females exhibited better behavioral performance and fewer loss of dopaminergic (DA) neurons than unmated females in PINK1 RNAi flies, a well-established Parkinson's disease (PD) model. Further study demonstrated that mating led to decreased iron content in the brain, a process associated with decreased Transferrin 1 (Tsf1) and Malvolio (Mvl) and increased ferritin. Additionally, mating inhibited expression of Duox and Nox, two NADPH oxidases in Drosophila. Furthermore, Kr-h1, a transcription factor of JH, acted downstream of mating to regulate genes involved in iron metabolism and NADPH oxidases. Collectively, the findings suggested a pivotal role of mating in regulating iron metabolism and NADPH oxidases in the brain of Drosophila. Consequently, considering mating status is imperative in scientific research, particularly in the context of neurological disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cell motility is crucial for many biological processes including morphogenesis, wound healing, and cancer invasion. The WAVE regulatory complex (WRC) is a central Arp2/3 regulator driving cell motility downstream of activation by Rac GTPase. CYFIP-related Rac1 interactor (CYRI) proteins are thought to compete with WRC for interaction with Rac1 in a feedback loop regulating lamellipodia dynamics. However, the physiological role of CYRI proteins in vivo in healthy tissues is unclear. Here, we used Drosophila as a model system to study CYRI function at the cellular and organismal levels. We found that CYRI is not only a potent WRC regulator in single macrophages that controls lamellipodial spreading but also identified CYRI as a molecular brake on the Rac-WRC-Arp2/3 pathway to slow down epidermal wound healing. In addition, we found that CYRI limits invasive border cell migration by controlling cluster cohesion and migration. Thus, our data highlight CYRI as an important regulator of cellular and epithelial tissue dynamics conserved across species., (© 2024 Rötte et al.)

Two protocadherins, Dachsous and Fat, regulate organ growth in Drosophila via the Hippo pathway. Dachsous and Fat bind heterotypically to regulate the abundance and subcellular localization of a "core complex" consisting of Dachs, Dlish, and Approximated. This complex localizes to the junctional cortex where it represses Warts. Dachsous is believed to promote growth by recruiting and stabilizing this complex, while Fat represses growth by promoting its degradation. Here, we examine the functional relationships between the intracellular domains of Dachsous and Fat and the core complex. While Dachsous promotes the accumulation of core complex proteins in puncta, it is not required for their assembly. Indeed, the core complex accumulates maximally in the absence of both Dachsous and Fat. Furthermore, Dachsous represses growth in the absence of Fat by removing the core complex from the junctional cortex. Fat similarly recruits core complex components but promotes their degradation. Our findings reveal that Dachsous and Fat coordinately constrain tissue growth by repressing the core complex., (© 2024 Matakatsu and Fehon.)

Smoking, a major behavioral health burden, causes preventable and premature deaths globally. Nicotine, the addictive component present in tobacco products and Electronic cigarettes (E-cigarettes, vape), can bind to nicotinic acetylcholine receptors in the brain to trigger a dopamine release that reinforces smoking. Despite the widespread usage of nicotine, its mechanisms of toxicity, particularly in e-cigarettes, are poorly understood. Using Drosophila melanogaster as a model organism, this study aims to investigate the mechanism of the toxicity of nicotine and vape. Behavioral parameters, oxidative stress indicators, mRNA expression levels of Dopamine 1- receptor 1 (Dop1R1), Acetyl-coenzyme A synthetase (AcCoAs), and apoptotic proteins were assessed in the flies after a 5-day exposure to varying concentrations of nicotine (0.15, 0.25, and 0.35 mg/mL diet) and vape (0.06, 0.08, and 0.12 mg/mL diet). Furthermore, Gas Chromatography-Mass Spectrometry (GC/MS) and Gas Chromatography-Flame Ionization Detection (GC/FID) analyzes were conducted to gain more insight on the composition of the vape used in study. Findings indicate that both nicotine and vape exposure significantly reduced lifespan, impaired locomotor activity, and disrupted sleep patterns. Notably, nicotine exposure stimulated Dop1R1 transcription and altered Acetyl-CoA gene expression, impacting the viability and behavior of the flies. Elevated levels of reactive oxygen biomarkers were observed, contributing to cellular damage through oxidative stress and apoptotic mechanisms mediated by the Reaper and DIAP1 proteins. Additionally, the composition analysis of vape liquid revealed the presence of propylene glycol, nicotine, methyl esters, and an unidentified compound. This study highlights the complex interplay between nicotine, gene expression, and physiological responses in Drosophila., (© 2024 Wiley Periodicals LLC.)

Palm oil is the world's second most consumed vegetable oil, sourced from the tropical palm tree Elaeis guineensis. Its consumption has been associated with a higher incidence of cardiovascular disease, largely due to its elevated palmitic acid content, however those studies are contradictory and inconclusive. Wishing to contribute to this issue, the present study aims to investigate the molecular and toxicological effects of this oil and the involvement of oxidative stress, given its role in metabolic dysfunctions using Drosophila melanogaster. This study examines survival rates, and locomotor performance, oxidative status by analysis of lipid peroxidation, ROS formation, thiol levels and antioxidant enzyme activity, and metabolic parameters such as cholesterol and triglycerides, glucose, trehalose and glycogen levels. Exposure to palm oil concentrations of 10% and 30% resulted in a shortened lifespan, reduced locomotor performance, and increased lipid peroxidation, with lower thiol levels and antioxidant enzyme modulation. Cholesterol levels was increased whereas energetic fuels as glucose and glycogen and trehalose were decreased mainly after 10 days of exposure. These findings underscore the detrimental effects of high-fat diets containing palm oil on lifespan, antioxidant defenses, and metabolism in Drosophila melanogaster. This data highlights the potential risk associated with the habitual consumption of palm oil in the daily diet by population, particularly concerning cardiovascular health and metabolic function., (© 2024 John Wiley & Sons Ltd.)

Microtubule (MT) regulation is essential for oocyte development. In Drosophila , MT stability, polarity, abundance, and orientation undergo dynamic changes across developmental stages. In our effort to identify novel microtubule-associated proteins that regulate MTs in the Drosophila ovary, we identified a previously uncharacterized gene, CG18190, which encodes a novel MT end-binding (EB) protein, which we propose to name EB-SUN. We show that EB-SUN colocalizes with EB1 at growing MT plus-ends in Drosophila S2 cells. Tissue-specific and developmental expression profiles from Paralog Explorer reveal that EB-SUN is predominantly expressed in the ovary and early embryos, while EB1 is ubiquitously expressed. Furthermore, as early as oocyte determination, EB-SUN comets are highly concentrated in oocytes during oogenesis. EB-SUN knockout (KO) results in decreased MT density at the onset of mid-oogenesis (stage 7) and delays oocyte growth during late mid-oogenesis (stage 9). Combining EB-SUN KO with EB1 knockdown (KD) in germ cells significantly further reduces MT density at stage 7. Hatching assays of single protein depletion reveal distinct roles for EB-SUN and EB1 in early embryogenesis, likely due to differences in their expression and binding partners. Notably, all eggs from EB-SUN KO/EB1 KD females fail to hatch, suggesting partial redundancy between these proteins., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

The drugs mifepristone and rapamycin were compared for their relative ability to increase the life span of mated female Drosophila melanogaster . Titration of rapamycin indicated an optimal concentration of approximately 50 μM, which increased median life span here by average +81%. Meta-analysis of previous mifepristone titrations indicated an optimal concentration of approximately 466 μM, which increased median life span here by average +114%. Combining mifepristone with various concentrations of rapamycin did not produce further increases in life span, and instead reduced life span relative to either drug alone. Assay of maximum midgut diameter indicated that rapamycin was equally efficacious as mifepristone in reducing mating-induced midgut hypertrophy. The mito-QC mitophagy reporter is a previously described green fluorescent protein (GFP)-mCherry fusion protein targeted to the outer mitochondrial membrane. Inhibition of GFP fluorescence by the acidic environment of the autophagolysosome yields an increased red/green fluorescence ratio indicative of increased mitophagy. Creation of a multi-copy mito-QC reporter strain facilitated assay in live adult flies, as well as in dissected midgut tissue. Mifepristone was equally efficacious as rapamycin in activating the mito-QC mitophagy reporter in the adult female fat-body and midgut. The data suggest that mifepristone and rapamycin act through a common pathway to increase mated female Drosophila life span, and implicate increased mitophagy and decreased midgut hypertrophy in that pathway.

In this study, we raised the following question: "Does metamorphosis, being a "reboot" of all systems of the organism, erase the changes that occurred at earlier stages of insect development?" To answer this question, we investigated several behavioral, metabolic and neuroendocrine parameters in Drosophila melanogaster imago that had undergone heat stress at the 3rd larval instar (32 °C, 48 h). We discovered that larval stress negatively affected feeding and locomotor behavior, as well as total lipid content in adult flies. At the same time, these flies demonstrated a considerable increase in carbohydrate content and expression level of insulin/insulin-like growth factor signaling (IIS) pathway genes, dfoxo, dilp6 and dInR. The data obtained allow us to conclude that metamorphosis does not erase the effect of stress exposure at early developmental stages and causes dramatic changes in carbohydrate and lipid metabolism as well as locomotor activity of adult insects, which is at least in part due to changes in IIS activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Acyl-CoA binding domain-containing proteins (ACBDs) perform diverse but often uncharacterised functions linked to cellular lipid metabolism. Human ACBD4 and ACBD5 are closely related peroxisomal membrane proteins, involved in tethering of peroxisomes to the ER and capturing fatty acids for peroxisomal β-oxidation. ACBD5 deficiency causes neurological abnormalities including ataxia and white matter disease. Peroxisome-ER contacts depend on an ACBD4/5-FFAT motif, which interacts with ER-resident VAP proteins. As ACBD4/5-like proteins are present in most fungi and all animals, we combined phylogenetic analyses with experimental approaches to improve understanding of their evolution and functions. Notably, all vertebrates exhibit gene sequences for both ACBD4 and ACBD5, while invertebrates and fungi possess only a single ACBD4/5-like protein. Our analyses revealed alterations in domain structure and FFAT sequences, which help understanding functional diversification of ACBD4/5-like proteins. We show that the Drosophila melanogaster ACBD4/5-like protein possesses a functional FFAT motif to tether peroxisomes to the ER via Dm_Vap33. Depletion of Dm_Acbd4/5 caused peroxisome redistribution in wing neurons and reduced life expectancy. In contrast, the ACBD4/5-like protein of the filamentous fungus Ustilago maydis lacks a FFAT motif and does not interact with Um_Vap33. Loss of Um_Acbd4/5 resulted in an accumulation of peroxisomes and early endosomes at the hyphal tip. Moreover, lipid droplet numbers increased, and mitochondrial membrane potential declined, implying altered lipid homeostasis. Our findings reveal differences between tethering and metabolic functions of ACBD4/5-like proteins across evolution, improving our understanding of ACBD4/5 function in health and disease. The need for a unifying nomenclature for ACBD proteins is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Zinc excretion is crucial for zinc homeostasis. However, the mechanism of zinc excretion has not been well characterized. Zinc homeostasis in Drosophila seems well conserved to mammals. In this study, we screened all members of the zinc transporters ZnT (SLC30) and Zip (SLC39) for their potential roles in Drosophila hindgut, an insect organ that belongs to the excretory system. The results indicated that Catecholamines up (Catsup, CG10449), a ZIP member localized to the Golgi, is responsible for zinc homeostasis in the hindgut of Drosophila hindgut-specific knockdown of Catsup leads to a developmental arrest in the larval stage, which could be rescued well by human ZIP7. Further study suggested that Catsup RNAi in the hindgut reduced zinc levels in the excretory system (containing the Malpighian tubule and hindgut) but exhibited systemic zinc overload. Besides, more calculi were observed in the Malpighian tubules of Catsup RNAi flies. The developmental arrest and calculi in the Malpighian tubules of hindgut-specific Catsup RNAi flies could be rescued by dietary zinc restriction but hypersensitivity to zinc. These results will help us understand the fundamental process of zinc excretion in higher eukaryotes., (© 2024 Royal Entomological Society.)

In recent years, there has been a remarkable surge in the approval of therapeutic protein drugs, particularly recombinant glycoproteins. Drosophila melanogaster S2 cells have become an appealing platform for the production of recombinant proteins due to their simplicity and low cost in cell culture. However, a significant limitation associated with using the S2 cell expression system is its propensity to introduce simple paucimannosidic glycosylation structures, which differs from that in the mammalian expression system. It is well established that the glycosylation patterns of glycoproteins have a profound impact on the physicochemical properties, bioactivity, and immunogenicity. Therefore, understanding the mechanisms behind these glycosylation modifications and implementing measures to address it has become a subject of considerable interest. This review aims to comprehensively summarize recent advancements in glycosylation modification in S2 cells, with a particular focus on comparing the glycosylation patterns among S2, other insect cells, and mammalian cells, as well as developing strategies for altering the glycosylation patterns of recombinant glycoproteins., (© 2024 Wiley Periodicals LLC.)

The Drosophila melanogaster brain is a complex organ with various cell types, orchestrating the development, physiology, and behaviors of the fly. While each cell type in Drosophila brain is known to express a unique gene set, their complete genetic profile is still unknown. Advances in the RNA sequencing techniques at single-cell resolution facilitate identifying novel cell type markers and/or re-examining the specificity of the available ones. In this study, exploiting a single-cell RNA sequencing data of Drosophila optic lobe, we categorized the cells based on their expression pattern for known markers, then the genes with enriched expression in astrocytes were identified. CG11000 was identified as a gene with a comparable expression profile to the Eaat1 gene, an astrocyte marker, in every individual cell inside the Drosophila optic lobe and midbrain, as well as in the entire Drosophila brain throughout its development. Consistent with our bioinformatics data, immunostaining of the brains dissected from transgenic adult flies showed co-expression of CG11000 with Eaat1 in a set of single cells corresponding to the astrocytes in the Drosophila brain. Physiologically, inhibiting CG11000 through RNA interference disrupted the normal development of male D. melanogaster , while having no impact on females. Expression suppression of CG11000 in adult flies led to decreased locomotion activity and also shortened lifespan specifically in astrocytes, indicating the gene's significance in astrocytes. We designated this gene as ' deathstar ' due to its crucial role in maintaining the star-like shape of glial cells, astrocytes, throughout their development into adult stage.

Reticulophagy, which directs the endoplasmic reticulum (ER) to the phagophore for sequestration within an autophagosome and subsequent lysosomal degradation via specific receptors, is essential for ER quality control and is implicated in various diseases. This study utilizes Drosophila to establish an in vivo model for reticulophagy. Starvation-induced reticulophagy is detected across multiple tissues in Drosophila . Whole-body upregulation or downregulation of the expression of reticulophagy receptors, atl and Rtnl1 , negatively affects fly health. Notably, moderate upregulation of reticulophagy in neuronal tissues by overexpressing these receptors reduces age-related degeneration. In a Drosophila Alzheimer model expressing human APP (amyloid beta precursor protein), reticulophagy is compromised. Correcting reticulophagy by enhancing atl and Rtnl1 expression in the neurons promotes APP degradation, significantly reducing neurodegenerative symptoms. However, overexpression of mutated atl and Rtnl1 , which disrupts the interaction of the corresponding proteins with Atg8, does not alleviate these symptoms, emphasizing the importance of receptor functionality. These findings support modulating reticulophagy as a therapeutic strategy for aging and neurodegenerative diseases associated with ER protein accumulation.

Metamorphosis plays an important role in the evolutionary success of insects. Accumulating evidence indicated that microRNAs (miRNAs) are involved in the regulation of processes associated with insect metamorphosis. However, the miRNAs coordinated with juvenile hormone (JH)-regulated metamorphosis remain poorly reported. In the present study, using high-throughput miRNA sequencing combined with Drosophila genetic approaches, we demonstrated that miR-iab-8, which primarily targets homeotic genes to modulate haltere-wing transformation and sterility was up-regulated by JH and involved in JH-mediated metamorphosis. Overexpression of miR-iab-8 in the fat body resulted in delayed development and failure of larval-pupal transition. Furthermore, metabolomic analysis results revealed that overexpression of miR-iab-8 caused severe energy metabolism defects especially the lipid metabolism, resulting in significantly reduced triacylglycerol (TG) content and glycerophospholipids but enhanced accumulation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In line with this, Nile red staining demonstrated that during the third larval development, the TG content in the miR-iab-8 overexpression larvae was continuously decreased, which is opposite to the control. Additionally, the transcription levels of genes committed to TG synthesis and breakdown were found to be significantly increased and the expression of genes responsible for glycerophospholipids metabolism were also altered. Overall, we proposed that JH induced miR-iab-8 expression to perturb the lipid metabolism homeostasis especially the TG storage in the fat body, which in turn affected larval growth and metamorphosis., (© 2024 Royal Entomological Society.)

The Myocyte enhancer factor-2 (MEF2) transcription factor plays a vital role in orchestrating muscle differentiation. While MEF2 cannot effectively induce myogenesis in naïve cells, it can potently accelerate myogenesis in mesodermal cells. This includes in Drosophila melanogaster imaginal disc myoblasts, where triggering premature muscle gene expression in these adult muscle progenitors has become a paradigm for understanding the regulation of the myogenic program. Here, we investigated the global consequences of MEF2 overexpression in the imaginal wing disc myoblasts, by combining RNA-sequencing with RT-qPCR and immunofluorescence. We observed the formation of sarcomere-like structures that contained both muscle and cytoplasmic myosin, and significant upregulation of muscle gene expression, especially genes essential for myofibril formation and function. These transcripts were functional since numerous myofibrillar proteins were detected in discs using immunofluorescence. Interestingly, muscle genes whose expression is restricted to the adult stages were not activated in these adult myoblasts. These studies confirm a broad activation of the myogenic program in response to MEF2 expression and suggest that additional regulatory factors are required for promoting the adult muscle-specific program. Our findings contribute to understanding the regulatory mechanisms governing muscle development and highlight the multifaceted role of MEF2 in orchestrating this intricate process., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans -Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process., Competing Interests: SS, SP, SF, JV, ES, MM No competing interests declared, PW Reviewing editor, eLife, (© 2023, Suárez Freire et al.)

Early embryos undergo profound changes in their genomic architecture to establish the totipotent state, enabling pioneer factors to access chromatin and drive zygotic genome activation (ZGA). However, the mechanisms by which the totipotent state is established and properly interpreted by pioneer factors to allow orderly ZGA remain unknown. Here, we identify the H3.3-specific chaperone HIRA as a factor involving establishing totipotent-state chromatin in Drosophila early embryos. Through cophase separation with HIRA, the pioneer factor GAGA factor (GAF) efficiently binds to H3.3-marked nucleosomes to activate major-wave zygotic genes. Importantly, dPCIF1, a chromatin-associated protein, antagonized the GAF-HIRA interaction by competitively binding to HIRA, thereby restricting GAF on earlier chromatin and avoiding premature ZGA. Hence, the coordinated action of HIRA and dPCIF1 ensures sequential ZGA from the minor to major wave in early embryos. This study provides insights into understanding how a totipotent state is established and properly controlled during ZGA., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Centromeres depend on chromatin containing the conserved histone H3 variant CENP-A for function and inheritance, while the role of centromeric DNA repeats remains unclear. Retroelements are prevalent at centromeres across taxa and represent a potential mechanism for promoting transcription to aid in CENP-A incorporation or for generating RNA transcripts to maintain centromere integrity., Results: In this study, we probe into the transcription and RNA localization of the centromere-enriched retroelement G2/Jockey-3 (hereafter referred to as Jockey-3) in Drosophila melanogaster, currently the only in vivo model with assembled centromeres. We find that Jockey-3 is a major component of the centromeric transcriptome and produces RNAs that localize to centromeres in metaphase. Leveraging the polymorphism of Jockey-3 and a de novo centromere system, we show that these RNAs remain associated with their cognate DNA sequences in cis, suggesting they are unlikely to perform a sequence-specific function at all centromeres. We show that Jockey-3 transcription is positively correlated with the presence of CENP-A and that recent Jockey-3 transposition events have occurred preferentially at CENP-A-containing chromatin., Conclusions: We propose that Jockey-3 preferentially inserts at the centromere to ensure its own selfish propagation, while contributing to transcription across these regions. Given the conservation of retroelements as centromere components through evolution, our findings may offer a basis for understanding similar associations in other species., Competing Interests: Declarations Ethics approval and consent to participate Ethics approval is not applicable for this study. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Cell competition is an evolutionarily conserved quality control process that eliminates suboptimal or potentially dangerous cells. Although differential metabolic states act as direct drivers of competition, how these are measured across tissues is not understood. Here, we demonstrate that vesicular glutamate transporter (VGlut) and autocrine glutamate signaling are required for cell competition and Myc-driven super-competition in the Drosophila epithelia. We find that the loss of glutamate-stimulated VGlut>NMDAR>CaMKII>CrebB signaling triggers loser status and cell death under competitive settings via the autocrine induction of TNF. This in turn drives TNFR>JNK activation, triggering loser cell elimination and PDK/LDH-dependent metabolic reprogramming. Inhibiting caspases or preventing loser cells from transferring lactate to their neighbors nullifies cell competition. Further, in a Drosophila model for premalignancy, Myc-overexpressing clones co-opt this signaling circuit to acquire super-competitor status. Targeting glutamate signaling converts Myc "super-competitor" clones into "losers," highlighting new therapeutic opportunities to restrict the evolution of fitter clones., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

LC3 (microtubule-associated protein 1 light chain 3, called Atg8 in yeast and Drosophila ) is one of the most well-studied autophagy-related proteins. LC3 controls the selectivity of autophagic degradation by interacting with LIR (LC3-interacting region) motifs also known as AIM (Atg8-interacting motifs) on selective autophagy receptors that carry cargo for degradation. Although the function of Atg8 family proteins is primarily cytoplasmic, they are also enriched in the nucleus. Despite the accumulating evidence indicating the presence of Atg8 proteins in the nucleus, the mechanisms by which they are targeted to the nucleus, their interactions with nuclear components, and their nuclear role in remain poorly understood. Here, we used yeast two-hybrid screening, and we identified transcription factor Deformed wings (Dwg) as an Atg8a-interacting protein in Drosophila . Dwg-Atg8a interaction is LIR motif-dependent. We have created Dwg Y129A/I132A LIR mutant flies and shown that they exhibit elevated autophagy, improved resistance to oxidative stress, and starvation. Our results provide novel insights into the transcriptional regulation of autophagy in Drosophila .

Host-pathogen interactions are shaped by the metabolic status of both the host and pathogen. The host must regulate metabolism to fuel the immune response, while the pathogen must extract metabolic resources from the host to enable its own survival. In this study, we focus on the metabolic interactions of Mycobacterium abscessus with Drosophila melanogaster . We identify MAB_1132c as an asparagine transporter required for pathogenicity in M. abscessus . We show that this requirement is specifically associated with damage to the host: flies infected with MAB_1132c knockout bacteria, or with wild-type bacteria grown in asparagine-restricted conditions, are longer lived without showing a significant change in bacterial load. This is associated with a reduction in the host innate immune response, demonstrated by the decreased transcription of antimicrobial peptides as well as a significant reduction in the ability of the infection to disrupt systemic insulin signaling. Much of the increase in host survival during infection with asparagine-limited M. abscessus can be attributed to alterations in unpaired cytokine signaling. This demonstrates that asparagine transport in M. abscessus prior to infection is not required for replicative fitness in vivo but does significantly influence the interaction with the host immune responses., Competing Interests: Competing interests statement:The authors declare no competing interest.

Neurexin, a molecule associated with autism spectrum disorders, is thought to function mainly in neurons. Recently, it was reported that Neurexin is also present in muscle, but the role of Neurexin in muscle is still poorly understood. Here, we demonstrate that the overexpression of Neurexin in muscles effectively restored the locomotor function of Drosophila neurexin mutants, while rescuing effects are observed within the nervous. Notably, the defects in muscle structure and function caused by Neurexin deficiency were similar to those caused by mutations in dystroglycan, a gene associated with progressive muscular dystrophy. The absence of Neurexin leads to muscle attachment defects, emphasizing the essential role of Neurexin in muscle integrity. Furthermore, Neurexin deficiency reduces Dystroglycan glycosylation on the cell surface, which is crucial for maintaining proper muscle structure and function. Finally, Neurexin guides Dystroglycan to the glycosyltransferase complex through interactions with Rotated Abdomen, a homolog of mammalian POMT1. Our findings reveal that Neurexin mediates muscle development and function through Dystroglycan glycosylation, suggesting a potential association between autism spectrum disorders and muscular dystrophy., (© 2024. The Author(s).)

Ubiquitin controls many cellular processes via its posttranslational conjugation onto substrates. Its use is highly variable due to its ability to form poly-ubiquitin chains with various topologies. Among them, linear chains have emerged as important regulators of immune responses and protein degradation. Previous studies in Drosophila melanogaster found that expression of linear poly-ubiquitin that cannot be dismantled into single moieties leads to their ubiquitination and degradation or, alternatively, to their conjugation onto proteins. However, it remains largely unknown which proteins are sensitive to linear poly-ubiquitin. To address this question, here we expanded the toolkit to modulate linear chains and conducted ultra-deep coverage proteomics from flies that express noncleavable, linear chains comprising 2, 4, or 6 moieties. We found that these chains regulate shared and distinct cellular processes in Drosophila by impacting hundreds of proteins, such as the circadian factor Cryptochrome. Our results provide key insight into the proteome subsets and cellular pathways that are influenced by linear poly-ubiquitin chains with distinct lengths and suggest that the ubiquitin system is exceedingly pliable., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The biological rhythms generated by the endogenous circadian clocks across the tree of life regulate numerous behavioral, metabolic, and physiological processes. Although evidence from various studies in Drosophila melanogaster indicates the importance of the core circadian clock genes in the intricate interplay between the circadian clock and metabolism, little is known about the contribution of the circadian photoreceptor/s in this process. The deep brain circadian photoreceptor CRYPTOCHROME (CRY) is essential for resetting the clock in response to light and is also highly expressed in metabolically active tissues in Drosophila. In this study, we sought to explore the possible roles played by CRY in triglyceride (TG) metabolism. We observed that the cry mutant (cry01) flies exhibited increased starvation resistance and TG levels under both 12-hour (h) light:12-h dark cycle (LD) and under constant light compared with the control w1118 flies. We also observed that cry01 flies had significantly increased food intake, glycogen concentrations, and lifespan under LD. In addition, cryptochrome seemed to affect TG levels in adult flies in response to calorie-restricted and high-fat diets. These results suggest a role for the circadian photoreceptor CRY in TG metabolism in Drosophila., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Chemical detection is vital for animal survival, aiding in avoiding toxins and selecting nutritious foods. While Drosophila larvae exhibit appetitive feeding behavior toward ribose, an important sugar for RNA, nucleotide, and nucleoside synthesis, how adult Drosophila perceives ribose remains unclear. Through behavioral and electrophysiological investigations, we unexpectedly discovered that adult flies actively avoid ribose. Our external electrophysiological analysis revealed that ribose is detected through bitter-sensing gustatory receptor neurons in S-type sensilla, suggesting its perception as a bitter compound. Additionally, we identify painless as crucial for both ribose aversion and the neuronal response to ribose., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Defining the time of action for morphogens requires tools capable of temporally controlled perturbations. To study how the transcription factor Dorsal affects patterning of the Drosophila embryonic dorsal-ventral axis, we used two light-inducible tags that trigger either nuclear export or degradation of Dorsal under blue light. Nuclear export of Dorsal leads to loss of the high-threshold, ventrally expressed target gene snail (sna), while the low-threshold, laterally expressed target gene short-gastrulation (sog) is retained. In contrast, degradation of Dorsal results in retention of sna, loss of sog, and lower nuclear levels compared to when Dorsal is exported from the nucleus. To understand why nuclear export causes loss of sna but degradation does not, we investigated Dorsal kinetics using photobleaching and found that it rapidly re-enters the nucleus even under blue-light conditions favoring export. The associated kinetics of Dorsal being rapidly imported and exported continuously are likely responsible for loss of sna but, alternatively, can support sog. Collectively, our results indicate that this dynamic patterning process is influenced by both Dorsal concentration and nuclear retention., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Ribonucleoprotein (RNP) granules are membraneless condensates that organize the intracellular space by compartmentalization of specific RNAs and proteins. Studies have shown that RNA tunes the phase behaviour of RNA-binding proteins, but the role of intermolecular RNA-RNA interactions in RNP granules in vivo remains less explored. Here we determine the role of a sequence-specific RNA-RNA kissing-loop interaction in assembly of mesoscale oskar RNP granules in the female Drosophila germline. We show that a two-nucleotide mutation that disrupts kissing-loop-mediated oskar messenger RNA dimerization impairs condensate formation in vitro and oskar granule assembly in the developing oocyte, leading to defective posterior localization of the RNA and abrogation of oskar-associated processing bodies upon nutritional stress. This specific trans RNA-RNA interaction acts synergistically with the scaffold RNA-binding protein, Bruno, in driving condensate assembly. Our study highlights the architectural contribution of an mRNA and its specific secondary structure and tertiary interactions to the formation of an RNP granule that is essential for embryonic development., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Objectives: The mitochondrial enzyme L-2-hydroxyglutarate dehydrogenase (L2HGDH) regulates the abundance of L-2-hydroxyglutarate (L-2HG), a potent signaling metabolite capable of influencing chromatin architecture, mitochondrial metabolism, and cell fate decisions. Loss of L2hgdh activity in humans induces ectopic L-2HG accumulation, resulting in neurodevelopmental defects, altered immune cell function, and enhanced growth of clear cell renal cell carcinomas. To better understand the molecular mechanisms that underlie these disease pathologies, we used the fruit fly Drosophila melanogaster to investigate the endogenous functions of L2hgdh., Methods: L2hgdh mutant adult male flies were analyzed under normoxic and hypoxic conditions using a combination of semi-targeted metabolomics and RNA-seq. These multi-omic analyses were complemented by tissue-specific genetic studies that examined the effects of L2hgdh mutations on the Drosophila renal system (Malpighian tubules; MTs)., Results: Our studies revealed that while L2hgdh is not essential for growth or viability under standard culture conditions, L2hgdh mutants are hypersensitive to hypoxia and expire during the reoxygenation phase with severe disruptions of mitochondrial metabolism. Moreover, we find that the fly renal system is a key site of L2hgdh activity, as L2hgdh mutants that express a rescuing transgene within the MTs survive hypoxia treatment and exhibit normal levels of mitochondrial metabolites. We also demonstrate that even under normoxic conditions, L2hgdh mutant MTs experience significant metabolic stress and are sensitized to aberrant growth upon Egfr activation., Conclusions: These findings present a model in which renal L2hgdh activity limits systemic L-2HG accumulation, thus indirectly regulating the balance between glycolytic and mitochondrial metabolism, enabling successful recovery from hypoxia exposure, and ensuring renal tissue integrity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

ɣ-aminobutyric acid (GABA) is a four‑carbon amino acid acting as the main inhibitory transmitter in the invertebrate and vertebrate nervous systems. The metabolism of GABA is well compartmentalized in the cell and the uptake of cytosolic GABA into the mitochondrial matrix is required for its degradation. A previous study carried out in the fruit fly Drosophila melanogaster indicated that the mitochondrial aspartate/glutamate carrier (AGC) is responsible for mitochondrial GABA accumulation. Here, we investigated the transport of GABA catalysed by the human and D. melanogaster AGC proteins through a well-established method for the study of the substrate specificity and the kinetic parameters of the mitochondrial carriers. In this experimental system, the D. melanogaster spliced AGC isoforms (Aralar1-PA and Aralar1-PE) and the human AGC isoforms (AGC1/aralar1 and AGC2/citrin) are unable to transport GABA both in homo- and in hetero-exchange with either glutamate or aspartate, i.e. the canonical substrates of AGC. Moreover, GABA has no inhibitory effect on the exchange activities catalysed by the investigated AGCs. Our data demonstrate that AGC does not transport GABA and the molecular identity of the GABA transporter in human and D. melanogaster mitochondria remains unknown., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Maternal nutrition, including the availability of micronutrients such as zinc, influences the health of the offspring. Using Drosophila melanogaster, we studied the impact of zinc deficiency on development and reproduction, as well as the effects of maternal zinc status on the offspring's expression of zinc transporters across F1 to F3 generations. Zinc deficiency was induced by adding N,N,N',N'-Tetrakis (2-pyridylmethyl)-ethylenediamine (TPEN) to the diet on which the eggs representing the F0 generation flies were laid. Then, virgin F0 females were mated with control males to produce F1, and subsequently thereafter to generate F2 and F3. Offspring from F1 to F3 were analyzed for body zinc status and zinc transporter mRNA levels. We found that zinc deficiency significantly (p < 0.05) impaired the development of flies, as evidenced by a reduced eclosion rate of zinc-deficient flies. Similarly, zinc deficiency significantly (p < 0.05) reduced the egg-laying rate in F0 flies, highlighting its impact on reproductive functions. Also, zinc levels were consistently lower in the F0 and persisted in subsequent generations for both male and female offspring, indicating transgenerational alterations in zinc status. Furthermore, gene expression analysis revealed significant (p < 0.05) variations in the mRNA levels of dZip42C.1, dZnT63C, dZip71B, and dZnT35C genes across different generations and between male and female offspring. These findings indicate gender-specific dynamics of gene expression in response to zinc deficiency, suggesting potential regulatory mechanisms involved in maintaining zinc homeostasis. Our study emphasizes the detrimental effects of zinc deficiency on development and reproduction in Drosophila and highlights potential implications for offspring and human health., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Insect prophenoloxidases (PPOs) are important immunity proteins for defending against the invading pathogens and parasites. As a Type-Ⅲ copper-containing proteins, unlike Homo sapiens tyrosinases, the insect PPOs and most bacterial tyrosinases contain no signal peptides for unknown reason, however they can still be released. To this end, we fused different signal peptides to Drosophila melanogaster PPOs for in vitro and in vivo expression, respectively. We demonstrate that an artificial signal peptide can help PPO secretion in vitro. The secreted PPO appeared larger than wild-type PPO on molecular weight sizes due to glycosylation when expressed in S2 cells. Two asparagine residues for potential glycosylation in PPO1 were identified when a signal peptide was fused. After purification, the glycosylated PPO1 lost zymogen activity. When PPO1 containing a signal peptide was over-expressed in Drosophila larvae, the glycosylation and secretion of PPO1 was detected in vivo. Unlike insect PPO, human tyrosinase needs a signal peptide for protein expression and maintaining enzyme activity. An artificial signal peptide fused to bacterial tyrosinase had no influence on the protein expression and enzyme activity. These Type-Ⅲ copper-containing proteins from different organisms may evolve to perform their specific functions. Intriguingly, our study revealed that the addition of calcium inhibits PPO secretion from the transiently cultured larval hindguts in vitro, indicating that the calcium concentration may regulate PPO secretion. Taken together, insect PPOs can maintain enzyme activities without any signal peptide., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The Snail superfamily of transcription factors plays a crucial role in metazoan development; one of the most important vertebrate members of this family is Snai1 which is orthologous to the Drosophila melanogaster esg gene. This review offers a comprehensive examination of the roles of the esg gene in Drosophila development, covering its expression pattern and downstream targets, and draws parallels between the vertebrate Snai1 family proteins on controlling the epithelial-to-mesenchymal transition and esg. This gene regulates stemness, ploidy, and pluripontency. esg is expressed in various tissues during development, including the gut, imaginal discs, and neuroblasts. The functions of the esg include the suppression of differentiation in intestinal stem cells and the preservation of diploidy in imaginal cells. In the nervous system development, esg expression also inhibits neuroblast differentiation, thus regulating the number of neurons and the moment in development of neuronal differentiation. Loss of esg function results in diverse developmental defects, including defects in intestinal stem cell maintenance and differentiation, and alters imaginal disc and nervous system development. Expression levels of esg also play a role in regulating longevity and metabolism in adult stages. This review provides an overview of the current understanding of esg's developmental role, emphasizing cellular and tissue effects that arise from its loss of function. The insights gained may contribute to a better understanding of evolutionary conserved developmental mechanisms and certain metabolic diseases., (© 2024 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Switching from mitotic spermatogonia to meiotic spermatocytes is critical to producing haploid sperms during male germ cell differentiation. However, the underlying mechanisms of this switch remain largely unexplored. In Drosophila melanogaster, the gene RpL38 encodes the ribosomal protein L38, one component of the 60S subunit of ribosomes. We found that its depletion in spermatogonia severely diminished the production of mature sperms and thus led to the infertility of male flies. By examining the germ cell differentiation in testes, we found that RpL38-knockdown blocked the transition from spermatogonia to spermatocytes and accumulated spermatogonia in the testis. To understand the intrinsic reason for this blockage, we conducted proteomic analysis for these spermatogonia populations. Differing from the control spermatogonia, the accumulated spermatogonia in RpL38-knockdown testes already expressed many spermatocyte markers but lacked many meiosis-related proteins, suggesting that spermatogonia need to prepare some important proteins for meiosis to complete their switch into spermatocytes. Mechanistically, we found that the expression of bag of marbles (bam), a crucial determinant in the transition from spermatogonia to spermatocytes, was inhibited at both the mRNA and protein levels upon RpL38 depletion. We also confirmed that the bam loss phenocopied RpL38 RNAi in the testis phenotype and transcriptomic profiling. Strikingly, overexpressing bam was able to fully rescue the testis abnormality and infertility of RpL38-knockdown flies, indicating that bam is the key effector downstream of RpL38 to regulate spermatogonia differentiation. Overall, our data suggested that germ cells start to prepare meiosis-related proteins as early as the spermatogonial stage, and RpL38 in spermatogonia is required to regulate their transition toward spermatocytes in a bam-dependent manner, providing new knowledge for our understanding of the transition process from spermatogonia to spermatocytes in Drosophila spermatogenesis., (© 2024. Science China Press.)

Phosducin-like proteins (PhLP) are thioredoxin domain-containing proteins that are highly conserved across unicellular and multicellular organisms. PhLP family proteins are hypothesized to function as co-chaperones in the folding of cytoskeletal proteins. Here, we present the initial molecular, biochemical, and functional characterization of CG4511 as Drosophila melanogaster PhLP3. We cloned the gene into a bacterial expression vector and produced enzymatically active recombinant PhLP3, which showed similar kinetics to previously characterized orthologues. A fly strain homozygous for a P-element insertion in the 5' UTR of the PhLP3 gene exhibited significant downregulation of PhLP3 expression. We found these male flies to be sterile. Microscopic analysis revealed altered testes morphology and impairment of spermiogenesis, leading to a lack of mature sperm. Among the most significant observations was the lack of actin cones during sperm maturation. Excision of the P-element insertion in PhLP3 restored male fertility, spermiogenesis, and seminal vesicle size. Given the high level of conservation of PhLP3, our data suggests PhLP3 may be an important regulator of sperm development across species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Petit 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.)

Advances in the study of mRNAs have yielded major new insights into post-transcriptional control of gene expression. Focus on the spatial regulation of mRNAs in highly polarized cells has demonstrated that mRNAs translocate through cells as mRNA:protein granules (mRNPs). These complex self-assemblies containing nuclear and cytoplasmic proteins are fundamental to the coordinated translation throughout cellular development. Initial studies on translational control necessitated fixed tissue, but the last 30 years have sparked innovative live-cell studies in several cell types to deliver a far more nuanced picture of how mRNA-protein dynamics exert translational control. In this review, we weave together the events that underpin mRNA processes and showcase the pivotal studies that revealed how a multitude of protein factors engage with a transcript. We highlight a mRNA's ability to act as a 'super scaffold' to facilitate molecular condensate formation and further moderate translational control. We focus on the Drosophila melanogaster germline due to the extensive post-transcriptional regulation occurring during early oogenesis. The complexity of the spatio-temporal expression of maternal transcripts in egg chambers allows for the exploration of a wide range of mechanisms that are crucial to the life cycle of mRNAs., (© 2024 The Author(s).)

Dietary restriction (DR) is a potent method to enhance lifespan and healthspan, but individual responses are influenced by genetic variations. Understanding how metabolism-related genetic differences impact longevity and healthspan are unclear. To investigate this, we used metabolites as markers to reveal how different genotypes respond to diet to influence longevity and healthspan traits. We analyzed data from Drosophila Genetic Reference Panel (DGRP) strains raised under AL and DR conditions, combining metabolomic, phenotypic, and genome-wide information. We employed two computational and complementary methods across species-random forest modeling within the DGRP as our primary analysis and Mendelian randomization in human cohorts as a secondary analysis. We pinpointed key traits with cross-species relevance as well as underlying heterogeneity and pleiotropy that influence lifespan and healthspan. Notably, orotate was linked to parental age at death in humans and blocked the DR lifespan extension in flies, while threonine supplementation extended lifespan, in a strain- and sex-specific manner. Thus, utilizing natural genetic variation data from flies and humans, we employed a systems biology approach to elucidate potential therapeutic pathways and metabolomic targets for diet-dependent changes in lifespan and healthspan., (© 2024. The Author(s).)

Sexual reproduction requires the choreographed interaction of female cells and molecules with sperm and seminal fluid. In internally fertilizing animals, these interactions are managed by specialized tissues within the female reproductive tract (FRT), such as a uterus, glands, and sperm storage organs. However, female somatic reproductive tissues remain understudied, hindering insight into the molecular interactions that support fertility. Here, we report the identification, molecular characterization, and analysis of cell types throughout the somatic FRT in the premier Drosophila melanogaster model system. We find that the uterine epithelia is composed of 11 distinct cell types with well-delineated spatial domains, likely corresponding to functionally specialized surfaces that interact with gametes and reproductive fluids. Polyploidy is pervasive: More than half of lower reproductive tract cells are ≥4C. While seminal fluid proteins (SFPs) are typically thought of as male products that are transferred to females, we find that specialized cell types in the sperm storage organs heavily invest in expressing SFP genes. Rates of amino acid divergence between closely related species indicate heterogeneous evolutionary processes acting on male-limited versus female-expressed seminal fluid genes. Together, our results emphasize that more than 40% of annotated seminal fluid genes are better described as shared components of reproductive transcriptomes, which may function cooperatively to support spermatozoa. More broadly, our work provides the molecular foundation for improved technologies to catalyze the functional characterization of the FRT., Competing Interests: Competing interests statement:The authors declare no competing interest.

Alzheimer's disease (AD) is a progressive neurodegenerative condition and the primary form of dementia among elderly people. The amyloidogenic hypothesis is the main theory that explains this phenomenon and describes the extracellular accumulation of amyloid beta (Aβ) peptides. Model organisms such as Drosophila melanogaster have been utilized to improve the understanding of this disease and its treatment. This study evaluated the effects of peptide and metabolic fractions of Brazilian kefir on a strain of D. melanogaster that expresses human Aβ peptide 1-42 in the eye. The parameters assessed included ommatidial organization, vacuole area, retinal thickness, and Aβ peptide quantification. The present study revealed that the fractions, particularly the peptidic fraction, significantly reduced the vacuole area and increased the retina thickness in treated flies, indicating an improvement in neurodegeneration phenotype. The peptidic fraction was also found to alter Aβ aggregation dynamics, inhibiting Aβ fibril formation, as revealed by dynamic light scattering. This study demonstrated that kefir fractions, particularly the peptidic fraction < 10 kDa, have the potential to regulate Aβ aggregation and alleviate neurodegeneration in a Drosophila melanogaster AD-like model. These findings suggest that kefir fractions could be viable for the bioprospection of novel drug prototypes for AD treatment, providing valuable insights into strategies targeting Aβ aggregation and neurodegeneration in AD., (© 2024. The Author(s).)

The actin cytoskeleton is a key determinant of cell structure and homeostasis. However, possible tissue-specific changes to actin dynamics during aging, notably brain aging, are not understood. Here, we show that there is an age-related increase in filamentous actin (F-actin) in Drosophila brains, which is counteracted by prolongevity interventions. Critically, decreasing F-actin levels in aging neurons prevents age-onset cognitive decline and extends organismal healthspan. Mechanistically, we show that autophagy, a recycling process required for neuronal homeostasis, is disabled upon actin dysregulation in the aged brain. Remarkably, disrupting actin polymerization in aged animals with cytoskeletal drugs restores brain autophagy to youthful levels and reverses cellular hallmarks of brain aging. Finally, reducing F-actin levels in aging neurons slows brain aging and promotes healthspan in an autophagy-dependent manner. Our data identify excess actin polymerization as a hallmark of brain aging, which can be targeted to reverse brain aging phenotypes and prolong healthspan., (© 2024. The Author(s).)

Transcriptional regulation, orchestrated by the interplay between transcription factors (TFs) and enhancers, governs gene expression dynamics crucial for cellular processes. While gross qualitative fluctuations in transcription factor-dependent gene expression patterning have a long history of characterization, the roles of these factors in the nuclei retaining expression in the presence or absence of these factors are now observable using modern techniques. Our study investigates the impact of Suppressor of Hairless (Su(H)), a broadly expressed transcription factor, on enhancer-driven transcriptional modulation using Drosophila early embryos as a model system. Building upon previous findings, we employ super-resolution microscopy to dissect Su(H)'s influence on sog-Distal ( sogD ) enhancer activity specifically in nuclei with preserved sogD -driven expression in the absence of Su(H) binding. We demonstrate that Su(H) occupancy perturbations alter expression levels and bursting dynamics. Notably, Su(H) absence during embryonic development exhibits region-specific effects, inhibiting expression dorsally and stabilizing expression ventrally, implying a nuanced role in enhancer regulation. Our findings shed light on the intricate mechanisms that govern transcriptional dynamics and suggest a critical patterning role for Notch/Hairless signaling in sog expression as embryos transition to gastrulation.

Serotonin, a monoamine neurotransmitter, is important in both the central nervous system (CNS) and the peripheral nervous system. Malfunction of serotonin signaling leads to various disorders. We studied serotonin signaling from serotonergic neurons inside the ventral nerve cord of Drosophila melanogaster. Serotonergic neurons and stimulated release were visualized and achieved with mCherry and channelrhodopsin-2 (an optogenetically transfected ion channel), respectively, and two electrochemical techniques quantified serotonin release and vesicular content. Mean vesicular serotonin content released during exocytosis from these neurons was 84 %, considerably higher than reported in previous studies regarding octopamine (4.5 %) and glutamate release (31 %). Serotonin content within all vesicles is uniformly changed when serotonin concentration is inhibited or enhanced. However, serotonin release exhibits two Gaussian distributions: higher frequency of small release events, and similar or slightly higher frequency of large events, resulting in differential release fractions ranging from partial (13-18 %) to full (100 %) release after treatment with agents to either enhance or diminish release. This is the first example of consistent full exocytotic release events we have observed in any system. We suggest one pool of vesicles can release significantly diverse fractions of transmitter load during exocytosis, a potentially novel pathway to regulate exocytosis and neuronal signaling., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Fat and Dachsous are evolutionarily conserved atypical cadherins that regulate polarized cell behaviors. In the Drosophila wing, they interact heterophilically between neighboring cells, localize asymmetrically to opposite cell ends, and control wing shape by regulating oriented cell rearrangements and divisions. Fat and Dachsous have 34 and 27 cadherin repeats, respectively, and previous work has identified trans interactions between their first four cadherin repeats. Here, we identify a second heterophilic binding site in their C-terminal cadherin repeats and show the conservation of this binding site in human Fat4 and Dachsous1. We provide evidence that both N- and C-terminal binding sites regulate the stability of Fat-Dachsous binding interactions and show that the N-terminal binding sites are partly dispensable for Fat-Dachsous function in vivo. Finally, we provide in vivo confirmation that the N-terminal repeats interact in an anti-parallel manner. We propose that multiple binding sites promote the clustering of Fat and Dachsous into a lattice-like array., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Invertebrate striated muscle myosin filaments are highly variable in structure. The best characterized myosin filaments are those found in insect indirect flight muscle (IFM) in which the flight-powering muscles are not attached directly to the wings. Four insect orders, Hemiptera, Diptera, Hymenoptera, and Coleoptera, have evolved IFM. IFM thick filaments from the first three orders have highly similar myosin arrangements but differ significantly among their non-myosin proteins. The cryo-electron microscopy of isolated IFM myosin filaments from the Dipteran Drosophila melanogaster described here revealed the coexistence of two distinct filament types, one presenting a tubular backbone like in previous work and the other a solid backbone. Inside an annulus of myosin tails, tubular filaments show no noticeable densities; solid filaments show four paired paramyosin densities. Both myosin heads of the tubular filaments are disordered; solid filaments have one completely and one partially immobilized head. Tubular filaments have the protein stretchin-klp on their surface; solid filaments do not. Two proteins, flightin and myofilin, are identifiable in all the IFM filaments previously determined. In Drosophila , flightin assumes two conformations, being compact in solid filaments and extended in tubular filaments. Nearly identical solid filaments occur in the large water bug Lethocerus indicus , which flies infrequently. The Drosophila tubular filaments occur in younger flies, and the solid filaments appear in older flies, which fly less frequently if at all, suggesting that the solid filament form is correlated with infrequent muscle use. We suggest that the solid form is designed to conserve ATP when the muscle is not in active use.

DNA double-strand breaks (DSBs) must be properly repaired within diverse chromatin domains to maintain genome stability. Whereas euchromatin has an open structure and is associated with transcription, facultative heterochromatin is essential to silence developmental genes and forms compact nuclear condensates, called polycomb bodies. Whether the specific chromatin properties of facultative heterochromatin require distinct DSB repair mechanisms remains unknown. Here, we integrate single DSB systems in euchromatin and facultative heterochromatin in Drosophila melanogaster and find that heterochromatic DSBs rapidly move outside polycomb bodies. These DSB movements coincide with a break-proximal reduction in the canonical heterochromatin mark histone H3 Lysine 27 trimethylation (H3K27me3). We demonstrate that DSB movement and loss of H3K27me3 at heterochromatic DSBs depend on the histone demethylase dUtx. Moreover, loss of dUtx specifically disrupts completion of homologous recombination at heterochromatic DSBs. We conclude that DSBs in facultative heterochromatin require dUtx-mediated loss of H3K27me3 to promote DSB movement and repair., (© 2024. The Author(s).)

Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Removal of cells is coordinated with the increase in number of newly dividing cells to maintain barrier function of the tissue. In Drosophila metamorphosis, larval epidermal cells (LECs) are replaced by adult precursor cells called histoblasts. Removal of LECs must counterbalance the exponentially increasing adult histoblasts. Previous work showed that the LEC removal accelerates as endocytic activity decreases throughout all LECs. Here, we show that the acceleration is accompanied by a mode switching from isolated single-cell apoptosis to clustered ones induced by the endocytic activity reduction. We identify the epidermal growth factor receptor (EGFR) pathway via extracellular-signal regulated kinase (ERK) activity as the main components downstream of endocytic activity in LECs. The reduced ERK activity, caused by the decrease in endocytic activity, is responsible for the apoptotic mode switching. Initially, ERK is transiently activated in normal LECs surrounding a single apoptotic LEC in a ligand-dependent manner, preventing clustered cell death. Following the reduction of endocytic activity, LEC apoptosis events do not provoke these transient ERK up-regulations, resulting in the acceleration of the cell elimination rate by frequent clustered apoptosis. These findings contrasted with the common perspective that clustered apoptosis is disadvantageous. Instead, switching to clustered apoptosis is required to accommodate the growth of neighboring tissues., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yuswan 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.)

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerization-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MASs). Super-resolution microscopy reveals that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking shows that, during MAS development, integrins become immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerization-driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis., (© 2024. The Author(s).)

Formation of functional organs requires cell-cell communication between different cell lineages and failure in this communication can result in severe developmental defects. Hundreds of possible interacting pairs of proteins are known, but identifying the interacting partners that ensure a specific interaction between 2 given cell types remains challenging. Here, we use the Drosophila leg model and our cell type-specific transcriptomic data sets to uncover the molecular mediators of cell-cell communication between tendon and muscle precursors. Through the analysis of gene expression signatures of appendicular muscle and tendon precursor cells, we identify 2 candidates for early interactions between these 2 cell populations: Amalgam (Ama) encoding a secreted protein and Neurotactin (Nrt) known to encode a membrane-bound protein. Developmental expression and function analyses reveal that: (i) Ama is expressed in the leg myoblasts, whereas Nrt is expressed in adjacent tendon precursors; and (ii) in Ama and Nrt mutants, myoblast-tendon cell-cell association is lost, leading to tendon developmental defects. Furthermore, we demonstrate that Ama acts downstream of the FGFR pathway to maintain the myoblast population by promoting cell survival and proliferation in an Nrt-independent manner. Together, our data pinpoint Ama and Nrt as molecular actors ensuring early reciprocal communication between leg muscle and tendon precursors, a prerequisite for the coordinated development of the appendicular musculoskeletal system., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Moucaud 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.)

Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly., (© 2024 Tam and Harris.)

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties., (© 2024 MRC Laboratory of Molecular Biology.)