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The Hedgehog (Hh) pathway plays diverse roles in cellular processes by activating the transcription factor Cubitus interruptus (Ci). Abnormal regulation of this pathway has been linked to various human diseases. While previous studies have focused on how Ci is regulated in the cytoplasm, the control of nuclear Ci remains poorly understood. In this study, we have found that the transcriptional cofactor Taiman (Tai) functions as an inhibitor of the Hh pathway. Tai interferes with the response of Hh signal, rather than Hh secretion. Our epistatic analyses reveal that Tai works in parallel with Ci to reduce its activity, thereby counteracting organ overgrowth and the activation of target genes caused by Ci overexpression. Specifically, Tai interacts with Ci to decrease its binding to target gene promoters. The Hh signal weakens the interaction between Ci and Tai, releasing the inhibition on Ci. Importantly, this regulatory mechanism is conserved from Drosophila to mammalian cells. Moreover, NCOA1-3 are the mammalian ortholog of Drosophila protein Tai, but only NCOA2 plays a similar role in inhibiting the Hh pathway. These findings reveal an additional way to modulate the transcriptional activity of nuclear Ci., Competing Interests: Competing interests statement:The authors declare no competing interest.

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.

Alzheimer's disease is a complex neurodegenerative condition characterized by the accumulation of amyloid beta plaques, leading to memory loss, cognitive decline, and impaired autonomous behavior. Despite extensive research, an effective treatment remains elusive. The buildup of amyloid beta plaques (Aβ42) in the brain causes oxidative stress and disrupts normal molecular signaling, adversely affecting neuron function. Previous research has identified factors that can either exacerbate or mitigate neurodegenerative diseases. Our study aimed to uncover new factors involved in the pathogenesis of Alzheimer's disease. Using Drosophila as a model organism, we employed the Gal4/UAS system to express human Aβ42 in the flies' retinal neurons which led to neurodegenerative changes in their compound eyes. To identify genetic modifiers, we conducted a screen by co-expressing microRNAs and found that miR-282 acts as a suppressor. Overexpressing miR-282 in the GMR > Aβ42 background reduced Aβ42-induced neurodegeneration. Further analysis using prediction tools and RNA interference experiments identified three potential downstream targets of miR-282: calpain-B, knot, and scabrous. Downregulating these genes via RNA interference in the GMR > Aβ42 background mitigated neurodegeneration. Our research highlights miR-282 as a novel molecule that may influence the progression of Alzheimer's disease, offering potential avenues for future therapeutic or diagnostic developments., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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).)

Due to their position on glomerular capillaries, podocytes are continuously counteracting biomechanical filtration forces. Most therapeutic interventions known to generally slow or prevent the progression of chronic kidney disease appear to lower these biomechanical forces on podocytes, highlighting the critical need to better understand podocyte mechano-signalling pathways. Here we investigated whether the mechanotransducer Piezo is involved in a mechanosensation pathway in Drosophila nephrocytes, the podocyte homologue in the fly. Loss of function analysis in Piezo depleted nephrocytes reveal a severe morphological and functional phenotype. Further, pharmacological activation of endogenous Piezo with Yoda1 causes a significant increase of intracellular Ca ++ upon exposure to a mechanical stimulus in nephrocytes, as well as filtration disturbances. Elevated Piezo expression levels also result in a severe nephrocyte phenotype. Interestingly, expression of Piezo which lacks mechanosensitive channel activity, does not result in a severe nephrocyte phenotype, suggesting the observed changes in Piezo wildtype overexpressing cells are caused by the mechanosensitive channel activity. Moreover, blocking Piezo activity using the tarantula toxin GsMTx4 reverses the phenotypes observed in nephrocytes overexpressing Piezo. Taken together, here we provide evidence that Piezo activity levels need to be tightly regulated to maintain normal pericardial nephrocyte morphology and function., Competing Interests: Declarations Competing interests The authors declare no conflicts of interest., (© 2024. The Author(s).)

Ligands of the TGF-β/BMP superfamily are crucially involved in the regulation of growth, patterning and organogenesis and can act as long-range morphogens. Essential for understanding TGF-β/BMP signaling dynamics and regulation are tools that allow monitoring and manipulating pathway components at physiological expression levels and endogenous spatiotemporal patterns. We used genome engineering to generate a comprehensive library of endogenously epitope- or fluorescent-tagged versions of receptors, co-receptors, transcription factors and key feedback regulators of the Drosophila BMP and Activin signaling pathways. We demonstrate that the generated alleles are biologically active and can be used for assessing tissue and subcellular distribution of the corresponding proteins. Furthermore, we show that the genomic platforms can be used for in locus structure-function and cis-regulatory analyses. Finally, we present a complementary set of protein binder-based tools, which allow visualization as well as manipulation of the stability and subcellular localization of epitope-tagged proteins, providing new tools for the analysis of BMP signaling and beyond., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Drosophila female germline stem cells (GSCs) complete asymmetric mitosis in the presence of an intact, but permeable, nuclear envelope and nuclear lamina (NL). This asymmetric division requires a modified centrosome cycle, wherein mitotic centrosomes with mature pericentriolar material (PCM) embed in the NL and interphase centrosomes with reduced PCM leave the NL. This centrosome cycle requires Emerin, an NL protein required for GSC survival and germ cell differentiation. In emerin mutants, interphase GSC centrosomes retain excess PCM, remain embedded in the NL and nucleate microtubule asters at positions of NL distortion. Here, we investigate the contributions of abnormal interphase centrosomes to GSC loss. Remarkably, reducing interphase PCM in emerin mutants rescues GSC survival and partially restores germ cell differentiation. Direct tests of the effects of abnormal centrosomes were achieved by expression of constitutively active Polo kinase to drive enlargement of interphase centrosomes in wild-type GSCs. Notably, these conditions failed to alter NL structure or decrease GSC survival. However, coupling enlarged interphase centrosomes with nuclear distortion promoted GSC loss. These studies establish that Emerin maintains centrosome structure to preserve stem cell survival., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Functional divergence of transcription factors (TFs) has driven cellular and organismal complexity throughout evolution, but its mechanistic drivers remain poorly understood. Here we test for new mechanisms using CORONA (CNA) and PHABULOSA (PHB), two functionally diverged paralogs in the CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) family of TFs. We show that virtually all genes bound by PHB ( ~ 99%) are also bound by CNA, ruling out occupation of distinct sets of genes as a mechanism of functional divergence. Further, genes bound and regulated by both paralogs are almost always regulated in the same direction, ruling out opposite regulation of shared targets as a mechanistic driver. Functional divergence of CNA and PHB instead results from differential usage of shared binding sites, with hundreds of uniquely regulated genes emerging from a commonly bound genetic network. Regulation of a given gene by CNA or PHB is thus a function of whether a bound site is considered 'responsive' versus 'non-responsive' by each paralog. Discrimination between responsive and non-responsive sites is controlled, at least in part, by their lipid binding START domain. This suggests a model in which HD-ZIPIII TFs use information integrated by their START domain to generate paralog-specific transcriptional outcomes from a shared network architecture. Taken together, our study identifies a mechanism of HD-ZIPIII TF paralog divergence and proposes the ubiquitously distributed START evolutionary module as a driver of functional divergence., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Decline in mitochondrial function is linked to decreased muscle mass and strength in conditions like sarcopenia and type 2 diabetes. Despite therapeutic opportunities, there is limited and equivocal data regarding molecular cues controlling muscle mitochondrial plasticity. Here we uncovered that the mitochondrial mRNA-stabilizing protein SLIRP, in complex with LRPPRC, is a PGC-1α target that regulates mitochondrial structure, respiration, and mtDNA-encoded-mRNA pools in skeletal muscle. Exercise training effectively counteracts mitochondrial defects caused by genetically-induced LRPPRC/SLIRP loss, despite sustained low mtDNA-encoded-mRNA pools, by increasing mitoribosome translation capacity and mitochondrial quality control. In humans, exercise training robustly increases muscle SLIRP and LRPPRC protein across exercise modalities and sexes, yet less prominently in individuals with type 2 diabetes. SLIRP muscle loss reduces Drosophila lifespan. Our data points to a mechanism of post-transcriptional mitochondrial regulation in muscle via mitochondrial mRNA stabilization, offering insights into how exercise enhances mitoribosome capacity and mitochondrial quality control to alleviate defects., Competing Interests: Competing interests Since the study concluded, Solvejg Hansen, Jens Frey Halling and Anders Krogh Lemminger have become employees and shareholders of Novo Nordisk A/S, Denmark. The remaining authors declare no competing interests., (© 2024. The Author(s).)

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.

Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA-modifying enzyme that methylates wobble uridines in a subset of tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Through in vivo studies in Drosophila , we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory. ALKBH8 null animals lack wobble uridine methylation and exhibit reduced protein synthesis in the nervous system, including a specific decrease in selenoprotein levels. Either loss of ALKBH8 or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth in ALKBH8 null animals, confirming oxidative stress as the underlying cause of dysregulation. ALKBH8 null animals also exhibit associative memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA wobble uridine modification in redox homeostasis in the developing nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability., Competing Interests: Competing interests statement:The authors declare no competing interest.

Alzheimer's disease (AD), caused by amyloid beta (Aβ) plaques and Tau tangles, is a neurodegenerative disease characterized by progressive memory impairment and cognitive dysfunction. High-fat diet (HFD), which induces type 2 diabetes, exacerbates Aβ plaque deposition in the brain. To investigate the function of HFD in Tau-mediated AD, we fed an HFD to the Drosophila Tau model and found that HFD aggravates Tau-induced neurological phenotypes. Since microRNAs (miRNAs) are biomarkers for diabetes and AD, we evaluated the expression levels of common miRNAs of HFD and AD in HFD-fed Tau model fly brains. Among the common miRNAs, the expression levels of Let-7 and miR-34 were increased. We found that the inhibition of these miRNAs alleviates Tau-mediated AD phenotypes. Our research provides valuable insights into how HFD accelerates tau toxicity. Additionally, our work highlights the therapeutic potential of targeting Let-7 and miR-34 to develop innovative treatment approaches for AD., Competing Interests: Declaration of competing interest The authors have no financial interests in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The essential Drosophila RNA-binding protein Brain Tumor (Brat) represses specific genes to control embryogenesis and differentiation of stem cells. In the brain, Brat functions as a tumor suppressor that diminishes neural stem cell proliferation while promoting differentiation. Though important Brat-regulated target mRNAs have been identified in these contexts, the full impact of Brat on gene expression remains to be discovered. Here, we identify the network of Brat-regulated mRNAs by performing RNA sequencing (RNA-seq) following depletion of Brat from cultured cells. We identify 158 mRNAs, with high confidence, that are repressed by Brat. De novo motif analysis identified a functionally enriched RNA motif in the 3' untranslated regions (UTRs) of Brat-repressed mRNAs that matches the biochemically defined Brat binding site. Integrative data analysis revealed a high-confidence list of Brat-repressed and Brat-bound mRNAs containing 3'UTR Brat binding motifs. Our RNA-seq and reporter assays show that multiple 3'UTR motifs promote the strength of Brat repression, whereas motifs in the 5'UTR are not functional. Strikingly, we find that Brat regulates expression of glycolytic enzymes and the vacuolar ATPase complex, providing new insight into its role as a tumor suppressor and the coordination of metabolism and intracellular pH., (Published by Oxford University Press on behalf of Nucleic Acids Research 2024.)

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).)

Rb/E2f and DREAM complexes play vital roles in regulating cell cycle progression. To date, how they coordinate their functions to regulate cell cycle-dependent gene expression is not clear. Here, we identified a long noncoding RNA (lncRNA), which we named DREAMer , that bridges the interaction between E2f1 and the dREAM complex to regulate endoreplication specifically in Drosophila salivary gland. We show that E2f1 directly stimulates DREAMer expression, whereas DREAMer mediates the repression of e2f1 transcription by modulating the recruitment of the dREAM complex to the e2f1 promoter via a direct interaction with the dREAM component E2f2. The depletion of DREAMer impairs dREAM binding, leading to derepression of e2f1 transcription, which ultimately increases E2f1 activity and promotes the endoreplication. Furthermore, the transcriptomic analysis revealed profound changes in cell cycle-related gene expression in DREAMer KO salivary glands. Together, our findings reveal an lncRNA-mediated link between the dREAM complex and E2f1, which regulates endoreplication during development.

Notch signalling activity regulates hematopoiesis in Drosophila and vertebrates alike. Parasitoid wasp infestation of Drosophila larvae, however, requires a timely downregulation of Notch activity to allow the formation of encapsulation-active blood cells. Here, we show that the Drosophila CSL transcription factor Suppressor of Hairless [Su(H)] is phosphorylated at Serine 269 in response to parasitoid wasp infestation. As this phosphorylation interferes with the DNA binding of Su(H), it reversibly precludes its activity. Accordingly, phospho-deficient Su(H) S269A mutants are immune-compromised. A screen for kinases involved in Su(H) phosphorylation identified Pkc53E, required for normal hematopoiesis as well as for parasitoid immune response. Genetic and molecular interactions support the specificity of the Su(H)-Pkc53E relationship. Moreover, phorbol ester treatment inhibits Su(H) activity in vivo and in human cell culture. We conclude that Pkc53E targets Su(H) during parasitic wasp infestation, thereby remodelling the blood cell population required for wasp egg encapsulation., Competing Interests: SD, LF, JF, BG, MZ, HM, IP, AP, AN No competing interests declared, (© 2023, Deichsel et al.)

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 human dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in the pathology of Down syndrome, microcephaly, and cancer; however the exact mechanism through which it functions is unknown. Here, we have studied the role of the Drosophila ortholog of DYRK1A, Minibrain (Mnb), in brain development and organ growth. The neuroblasts (neural stem cells) that eventually give rise to differentiated neurons in the adult brain are formed from a specialized tissue in the larval optic lobe called the neuroepithelium, in a tightly regulated process. Molecular marker analysis of mnb mutants revealed alterations in the neuroepithelium and neuroblast regions of developing larval brains. Using affinity purification-mass spectrometry (AP-MS), we identified the novel Mnb binding partners Ral interacting protein (Rlip) and RALBP1 associated Eps domain containing (Reps). Rlip and Reps physically and genetically interact with Mnb, and the three proteins may form a ternary complex. Mnb phosphorylates Reps, and human DYRK1A binds to the Reps orthologs REPS1 and REPS2. Mnb also promotes re-localization of Rlip from the nucleus to the cytoplasm in cultured cells. Furthermore, Mnb engages the small GTPase Ras-like protein A (Rala) to regulate brain and wing development. This work uncovers a previously unrecognized role of Mnb in the neuroepithelium and defines the functions of the Mnb/Reps/Rlip/Rala signaling network in organ growth and neurodevelopment., 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.)

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.)

Drosophila Pins (and its mammalian homologue LGN) play a crucial role in the process of asymmetric cell division (ACD). Extensive research has established that Pins/LGN functions as a conformational switch primarily through intramolecular interactions involving the N-terminal TPR repeats and the C-terminal GoLoco (GL) motifs. The GL motifs served as binding sites for the α subunit of the trimeric G protein (Gα), which facilitates the release of the autoinhibited conformation of Pins/LGN. While LGN has been observed to specifically bind to Gα i ·GDP, Pins has been found to associate with both Drosophila Gα i ( d Gα i ) and Gα o ( d Gα o ) isoforms. Moreover, d Gα o was reported to be able to bind Pins in both the GDP- and GTP-bound forms. However, the precise mechanism underlying the influence of d Gα o on the conformational states of Pins remains unclear, despite extensive investigations into the Gα i ·GDP-mediated regulatory conformational changes in LGN/Pins. In this study, we conducted a comprehensive characterization of the interactions between Pins-GL motifs and d Gα o in both GDP- and GTP-loaded forms. Our findings reveal that Pins-GL specifically binds to GDP-loaded d Gα o . Through biochemical characterization, we determined that the intramolecular interactions of Pins primarily involve the entire TPR domain and the GL23 motifs. Additionally, we observed that Pins can simultaneously bind three molecules of d Gα o ·GDP, leading to a partial opening of the autoinhibited conformation. Furthermore, our study presents evidence contrasting with previous observations indicating the absence of binding between d Gα i and Pins-GLs, thus implying the pivotal role of d Gα o as the principal participant in the ACD pathway associated with Pins.

Drosophila nephrocytes are specialised cells that share critical functional, morphological, and molecular features with mammalian podocytes. Accordingly, nephrocytes represent a preferred invertebrate model for human glomerular disease. Here, we established a method for cell-specific isolation of the two types of Drosophila nephrocytes, garland and pericardial cells, from animals of different developmental stages and ages. Mass spectrometry-based proteomics and RNA-Seq-based transcriptomics were applied to characterise the proteome and transcriptome of the respective cells in an integrated and complementary manner. We observed characteristic changes in the proteome and transcriptome due to cellular ageing. Furthermore, functional enrichment analyses suggested that larval and adult nephrocytes, as well as garland and pericardial nephrocytes, fulfil distinct physiological functions. In addition, the pericardial nephrocytes were characterised by transcriptomic and proteomic profiles suggesting an atypical energy metabolism with very low oxidative phosphorylation rates. Moreover, the nephrocytes displayed typical signatures of extensive immune signalling and showed an active antimicrobial response to an infection. Factor-specific comparisons identified novel candidate proteins either expressed and secreted by the nephrocytes or sequestered by them. The data generated in this study represent a valuable basis for a more specific application of the Drosophila model in analysing renal cell function in health and disease., (© 2024. The Author(s).)

Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain tumor found in adult humans with a poor prognosis and average survival of 14-15 months. In order to have a comprehensive understanding of proteome and identify novel therapeutic targets, this study focused mainly on the differentially abundant proteins (DAPs) of Ras V12 -induced GBM. Ras V12 is a constitutively active Ras mutant form essential for tumor progression by continuously activating signaling pathways leading to uncontrolled tumor growth. This study used a transgenic Drosophila model with Ras V12 overexpression using the repo-GAL4 driver line, specifically in glial cells, to study GBM. The high-resolution mass spectrometry (HRMS)-based proteomic analysis of the GBM larval central nervous system identified three novel DAPs specific to mitochondria. These DAPs, probable maleylacetoacetate isomerase 2 (Q9VHD2), bifunctional methylene tetrahydrofolate dehydrogenase (Q04448), and glutamine synthetase1 (P20477), identified through HRMS were further validated by qRT-PCR. The protein-protein interaction analysis revealed interactions between Ras V12 and DAPs, with functional links to mitochondrial dynamics regulators such as Drp1, Marf, Parkin, and HtrA2. Notably, altered expressions of Q9VHD2, P20477, and Q04448 were observed during GBM progression, which offers new insights into the involvement of mitochondrial dynamic regulators in Ras V12 -induced GBM pathophysiology.

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.)

Wingless (Wg)/Wnt signaling plays a critical role in both development and adult tissue homeostasis. In the Drosophila larval wing disc epithelium, the orderly delivery of Wg/Wnt to the apical and basal cell surfaces is essential for wing development. Here, we identified Ehbp1 as the switch that dictates the direction of Wg/Wnt polarized intracellular transport: the Adaptor Protein complex 1 (AP-1) delivers Wg/Wnt to the basolateral cell surface, and its sequestration by Ehbp1 redirects Wg/Wnt for apical delivery. Genetic analyses showed that Ehbp1 specifically regulates the polarized distribution of Wg/Wnt, a process that depends on the dedicated Wg/Wnt cargo receptor Wntless. Mechanistically, Ehbp1 competes with Wntless for AP-1 binding, thereby preventing the unregulated basolateral Wg/Wnt transport. Reducing Ehbp1 expression, or removing the coiled-coil motifs within its bMERB domain, leads to basolateral Wg/Wnt accumulation. Importantly, the regulation of polarized Wnt delivery by EHBP1 is conserved in vertebrates. The generality of this switch mechanism for regulating intracellular transport remains to be determined in future studies., (© 2024. The Author(s).)

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.)

Drosophila NF-κB transcription factor Dif has been well known for its function in innate immunity, and recent study also reveals its role in neuronal cells. However, the underlying mechanisms of Dif in the brain remain elusive. In this study, we aim to investigate the function of Dif in Drosophila brain development and how Dif regulates structure and plasticity of the brain to affect aging and behaviors. Based on the analysis of differentially expressed genes, we identified key genes associated with cell division, development and aging in the brain of Dif 1 loss of function mutant. In Dif 1 larvae, we found that the metamorphosis and brain development were delayed, and cell division was decreased. In Dif 1 adults, the number of neuron cells was reduced in the brain, the lifespan and locomotor activity were decreased, protein markers associated with aging-related neurodegenerative diseases in the brain were altered in abundance or activity. Our results indicated that Dif plays a crucial role in brain plasticity and neurogenesis, dysfunction of Dif delays larval brain development and impacts proliferation of neuronal cells, resulting in aging adult brain by regulating expression of key genes in multiple signaling pathways involved in cell division, neurogenesis and aging., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lateral inhibition mediates alternative cell fate decision and produces regular cell fate patterns with fate symmetry breaking (SB) relying on the amplification of small stochastic differences in Notch activity via an intercellular negative-feedback loop. Here, we used quantitative live imaging of endogenous Scute (Sc), a proneural factor, and of a Notch activity reporter to study the emergence of sensory organ precursor cells in the pupal abdomen of Drosophila. SB was observed at low Sc levels and was not preceded by a phase of intermediate Sc expression and Notch activity. Thus, mutual inhibition may only be transient in this context. In support of the intercellular feedback loop model, cell-to-cell variations in Sc levels promoted fate divergence. The size of the apical area of competing cells did not detectably bias this fate choice. Surprisingly, cells that were in direct contact at the time of SB could adopt the sensory organ precursor cell fate, albeit at low frequency (10%). These lateral inhibition defects were corrected by cellular rearrangements, not cell fate change, highlighting the role of cell-cell intercalation in pattern refinement., 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).)

Sex pheromones play a crucial role in species recognition and reproductive isolation. Despite being largely species-specific in drosophilids, the mechanisms underlying pheromone detection, production, and their influence on mating behavior remain poorly understood. Here, we compare the chemical profiles of Drosophila bipectinata and D. melanogaster, the mating behaviors in both species, as well as the tuning properties of Or67d receptors, which are expressed by neurons in antennal trichoid sensilla at1. Through single sensillum recordings, we demonstrate that the D. bipectinata Or67d-ortholog exhibits similar sensitivity to cis-vaccenyl acetate (cVA) as compared to D. melanogaster but in addition also responds uniquely to (Z)-11-eicosen-1-yl-acetate (Z11-20:Ac), a compound exclusively produced by D. bipectinata males. Through courtship behavior assays we found that, surprisingly, perfuming the flies with Z11-20:Ac did not reveal any aphrodisiacal or anti-aphrodisiacal effects in mating assays. The behavioral relevance of at1 neuron channels in D. bipectinata compared to D. melanogaster seems to be restricted to its formerly shown function as an aggregation pheromone. Moreover, the non-specific compound cVA affected copulation negatively in D. bipectinata and could potentially act as a premating isolation barrier. As both ligands of Or67d seem to govern different behaviors in D. bipectinata, additional neurons detecting at least one of those compounds might be involved. These results underscore the complexity of chemical signaling in species recognition and raise intriguing questions about the evolutionary implications of pheromone detection pathways in Drosophila species., (© 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.)

We have analyzed the organization of the microtubule system in photoreceptor cells and pigment cells within the adult Drosophila compound eye. Immunofluorescence localization of tubulin and of Short stop, a spectraplakin that has been reported to be involved in the anchorage of microtubule minus ends at the membrane, suggests the presence of non-centrosomal microtubule-organizing centers at the distal tip of the visual cells. Ultrastructural analyses confirm that microtubules emanate from membrane-associated plaques at the site of contact with cone cells and that all microtubules are aligned in distal-proximal direction within the photoreceptor cells. Determination of microtubule polarities demonstrated that about 95% of the microtubules in photoreceptor cells are oriented with their plus end in the direction of the synapse. Pigment cells in the eye contain only microtubules aligned in distal-proximal direction, with their plus end pointing towards the retinal floor. There, two populations of microtubules can be distinguished, single microtubules and bundled microtubules, the latter associated with actin filaments. Whereas microtubules in both photoreceptor cells and pigment cells are acetylated and mono/bi-glutamylated on α-tubulin, bundled microtubules in pigment cells are apparently also mono/bi-glutamylated on β-tubulin, providing the possibility of binding different microtubule-associated proteins., (© 2024. The Author(s).)

Neuronal cells are highly specialized cells and have a specific metabolic profile to support their function. It has been demonstrated that the metabolic profiles of different cells/tissues undergo significant reprogramming with advancing age, which has often been considered a contributing factor towards aging-related diseases including Alzheimer's (AD) and Parkinson's (PD) diseases. However, it is unclear if the metabolic changes associated with normal aging predispose neurons to disease conditions or a distinct set of metabolic alterations happen in neurons in AD or PD which might contribute to disease pathologies. To decipher the changes in neuronal metabolism with age, in AD, or in PD, we performed high-throughput steady-state metabolite profiling on heads in wildtype Drosophila and in Drosophila models relevant to AD and PD. Intriguingly, we found that the spectrum of affected metabolic pathways is dramatically different between normal aging, Tau, or Synuclein overexpressing neurons. Genetic targeting of the purine and glutamate metabolism pathways, which were dysregulated in both old age and disease conditions partially rescued the neurodegenerative phenotype associated with the overexpression of wildtype and mutant tau. Our findings support a "two-hit model" to explain the pathological manifestations associated with AD where both aging- and Tau/Synuclein- driven metabolic reprogramming events cooperate with each other, and targeting both could be a potent therapeutic strategy., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Germ cells mutant for bam or bgcn are locked in a germline stem cell (GSC)-like state, leading to tumor-like overgrowth in Drosophila ovaries. Our previous studies have demonstrated that germline overgrowth in bam mutants can be suppressed by defects in the miRNA pathway but enhanced by a null mutation in hippo. However, the genetic epistasis between the miRNA and Hippo pathways still remains unknown. Here, we determined that the miRNA pathway acts downstream of the Hippo pathway in regulating this process. Germ cells mutant for bam or bgcn and defective in both pathways divide very slowly, phenocopying those defective only in the miRNA pathway. In addition, we found that Yki, a key oncoprotein in the Hippo pathway, promotes the growth of both wild-type germ cells and bam mutant GSC-like cells. Like wild-type GSCs, bam mutant GSC-like cells predominantly stay in the G2 phase. Remarkably, many of those defective in the miRNA pathway are arrested before entering this phase. Furthermore, our studies identified bantam as a critical miRNA promoting germline overgrowth in bam or bgcn mutants. Taken together, these findings establish a genetic circuitry controlling Drosophila female germline overgrowth., (Copyright © 2024 Elsevier Inc. 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.)

In animals undergoing metamorphosis, the appearance of the nervous system is coincidently transformed by the morphogenesis of neurons. Such morphogenic alterations are exemplified in three types of intrinsic neurons in the Drosophila memory center. In contrast to the well-characterized remodeling of γ neurons, the morphogenesis of α/β and α'/β' neurons has not been adequately explored. Here, we show that mamo, a BTB-zinc finger transcription factor that acts as a terminal selector for α'/β' neurons, controls the formation of the correct axonal pattern of α'/β' neurons. Intriguingly, specific Mamo isoforms are preferentially expressed in α'/β' neurons to regulate the expression of axon guidance molecule Semaphorin-1a. This action directs proper axon guidance in α'/β' neurons, which is also crucial for wiring of α'/β' neurons with downstream neurons. Taken together, our results provide molecular insights into how neurons establish correct axonal patterns in circuitry assembly during adult memory center construction., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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.)

Traumatic brain injury (TBI) caused by external mechanical forces is a major health burden worldwide, but the underlying mechanism in glia remains largely unclear. We report herein that Drosophila adults exhibit a defective blood-brain barrier, elevated innate immune responses, and astrocyte swelling upon consecutive strikes with a high-impact trauma device. RNA sequencing (RNA-seq) analysis of these astrocytes revealed upregulated expression of genes encoding PDGF and VEGF receptor-related (Pvr, a receptor tyrosine kinase), adaptor protein complex 1 (AP-1 , a transcription factor complex of the c-Jun N-terminal kinase pathway) composed of Jun-related antigen (Jra) and kayak (kay), and matrix metalloproteinase 1 (Mmp1) following TBI. Interestingly, Pvr is both required and sufficient for AP-1 and Mmp1 upregulation, while knockdown of AP-1 expression in the background of Pvr overexpression in astrocytes rescued Mmp1 upregulation upon TBI, indicating that Pvr acts as the upstream receptor for the downstream AP-1-Mmp1 transduction. Moreover, dynamin-associated endocytosis was found to be an important regulatory step in downregulating Pvr signaling. Our results identify a new Pvr-AP-1-Mmp1 signaling pathway in astrocytes in response to TBI, providing potential targets for developing new therapeutic strategies for TBI., Competing Interests: TL, WS, YZ No competing interests declared, MH Reviewing Editor, eLife, (© 2023, Li et al.)

The multiprotein Target of Rapamycin (TOR) Complex 1 (TORC1) is a serine/threonine kinase that stimulates anabolic metabolism and suppresses catabolism. Deregulation of TORC1 is implicated in various human pathologies, including cancer, epilepsy, and neurodegenerative disorders. The Gap Activity Towards Rags (GATOR) complex contains two subcomplexes: GATOR1, which inhibits TORC1 activity; and GATOR2, which counteracts GATOR1s function. Structural and biochemical studies have elucidated how GATOR1 regulates TORC1 activity by acting as a GTPase activating protein for Rag GTPase. However, while cryogenic electron microscopy has determined that the structure of the multi-protein GATOR2 complex is conserved from yeast to humans, how GATOR2 inhibits GATOR1 remains unclear. Here, we describe recent whole-animal studies in Drosophila that have yielded novel insights into GATOR2 function, including identifying a novel role for the GATOR2 subunit WDR59, redefining the core proteins sufficient for GATOR2 activity, and defining a TORC1-independent role for GATOR2 in the regulation of the lysosomal autophagic endomembrane system. Additionally, the recent characterization of a novel methionine receptor in Drosophila that acts through the GATOR2 complex suggests an attractive model for the evolution of species-specific nutrient sensors. Research on GATOR2 function in Drosophila highlights how whole-animal genetic models can be used to dissect intracellular signaling pathways to identify tissue-specific functions and functional redundancies that may be missed in studies confined to rapidly proliferating cell lines.

Relevance-based selectivity and high energy cost are two distinct features of long-term memory (LTM) formation that warrant its default inhibition. Spaced repetition of learning is a highly conserved cognitive mechanism that can lift this inhibition. Here, we questioned how the spacing effect integrates experience selection and energy efficiency at the cellular and molecular levels. We showed in Drosophila that spaced training triggers LTM formation by extending over several hours an increased mitochondrial metabolic activity in neurons of the associative memory center, the mushroom bodies (MBs). We found that this effect is mediated by PKCδ, a member of the so-called 'novel PKC' family of enzymes, which uncovers the critical function of PKCδ in neurons as a regulator of mitochondrial metabolism for LTM. Additionally, PKCδ activation and translocation to mitochondria result from LTM-specific dopamine signaling on MB neurons. By bridging experience-dependent neuronal circuit activity with metabolic modulation of memory-encoding neurons, PKCδ signaling binds the cognitive and metabolic constraints underlying LTM formation into a unified gating mechanism., Competing Interests: TC, TP, AP, PP No competing interests declared, (© 2024, Comyn et al.)

Drosophila Cryptochrome (CRY) is an essential photoreceptor that mediates the resetting of the circadian clock by light. in vitro studies demonstrated a critical role of redox cycling of the FAD cofactor for CRY activation by light. However, it is unknown if CRY responds to cellular redox environment to modulate the circadian clock. We report here that the mitochondrial respiratory chain impinges on CRY activity. Inhibition of complex III and V blocks CRY-mediated degradation of TIMELESS (TIM) in response to light, and also blocks light-induced CRY degradation. On the other hand, inhibition of complex I facilitates TIM degradation even in the dark. Mutations of critical residues of the CRY C-terminus promote TIM degradation in the dark, even in the presence of complex III and V inhibitors. We propose that complex III and V activities are important for activation of CRY in response to light. Interestingly, we found that transcriptional repressor functions of Drosophila and mammalian CRY proteins are not affected by mitochondrial inhibitors. Together these data suggest that the two functions of CRY have different sensitivity to disruptions of the mitochondrial respiratory chain: one is sensitive to mitochondrial activities that enable resetting, the other is insensitive so as to sustain the molecular oscillator., (© 2024. The Author(s).)

Dual leucine zipper kinase (DLK) mediates multiple neuronal stress responses, and its expression levels are constantly suppressed to prevent excessive stress signaling. We found that Wallenda (Wnd), the Drosophila ortholog of DLK, is highly enriched in the axon terminals of Drosophila sensory neurons in vivo and that this subcellular localization is necessary for Highwire-mediated Wnd protein turnover under normal conditions. Our structure-function analysis found that Wnd palmitoylation is essential for its axon terminal localization. Palmitoylation-defective Wnd accumulated in neuronal cell bodies, exhibited dramatically increased protein expression levels, and triggered excessive neuronal stress responses. Defective intracellular transport is implicated in neurodegenerative conditions. Comprehensive dominant-negative Rab protein screening identified Rab11 as an essential factor for Wnd localization in axon terminals. Consequently, Rab11 loss-of-function increased the protein levels of Wnd and induced neuronal stress responses. Inhibiting Wnd activity significantly ameliorated neuronal loss and c-Jun N-terminal kinase signaling triggered by Rab11 loss-of-function. Taken together, these suggest that DLK proteins are constantly transported to axon terminals for protein turnover and a failure of such transport can lead to neuronal loss. Our study demonstrates how subcellular protein localization is coupled to protein turnover for neuronal stress signaling., Competing Interests: SK, YQ, MS, JK No competing interests declared, (© 2024, Kim et al.)

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.

Rabex-5 (also called RabGEF1), a protein originally characterized for its Rab5 GEF function, also has an A20-like E3 ubiquitin ligase domain. We and others reported that Rabex-5 E3 activity promotes Ras mono- and di-ubiquitination to inhibit Ras signaling in Drosophila and mammals. Subsequently, we reported that Rabex-5 inhibits Notch signaling in the Drosophila hematopoietic system. Here we report genetic interactions using Rabex-5 transgenes encoding domain-specific mutations that show that Rabex-5 requires an intact E3 domain to inhibit Notch signaling in the epithelial tissue of the developing wing. Surprisingly, we discovered that Rabex-5 with an impaired E3 domain but active Rab5 GEF domain suppresses Notch loss-of-function phenotypes and enhances both Notch duplication phenotypes and activated Ras phenotypes consistent with a model that the Rab5 GEF activity of Rabex-5 might positively regulate Ras and Notch. Positive and negative regulation of developmental signaling by its different catalytic domains could allow Rabex-5 to precisely coordinate developmental signaling to fine-tune patterning. Finally, we report that Rabex-5 also inhibits the overgrowth due to loss of PTEN or activation of PI3K but not activation of AKT. Inhibition of Ras, Notch, and PI3K signaling may explain why Rabex-5 is deleted in some cancers. Paradoxically, Rabex-5 is reported to be an oncogene in other cancers. We propose that Rabex-5 acts as a tumor suppressor via its E3 activity to inhibit Ras, Notch, and PI3K signaling and as an oncogene via its Rab5 GEF activity to enhance Ras and Notch signaling., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Reimels 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.)