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Tumor necrosis factor receptors (TNFRs) control pleiotropic pro-inflammatory functions that range from apoptosis to cell survival. The ability to trigger a particular function will depend on the upstream cues, association with regulatory complexes, and downstream pathways. In Drosophila melanogaster, two TNFRs have been identified, Wengen (Wgn) and Grindelwald (Grnd). Although several reports associate these receptors with JNK-dependent apoptosis, it has recently been found that Wgn activates a variety of other functions. We demonstrate that Wgn is required for survival by protecting cells from apoptosis. This is mediated by dTRAF1 and results in the activation of p38 MAP kinase. Remarkably, Wgn is required for apoptosis-induced regeneration and is activated by the reactive oxygen species (ROS) produced following apoptosis. This ROS activation is exclusive for Wgn, but not for Grnd, and can occur after knocking down Eiger/TNFα. The extracellular cysteine-rich domain of Grnd is much more divergent than that of Wgn, which is more similar to TNFRs from other animals, including humans. Our results show a novel TNFR function that responds to stressors by ensuring p38-dependent regeneration., (© 2024. The Author(s).)

Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine-hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity., (© 2024. The Author(s).)

Exportin5 (Exp5) is the major miRNA nuclear export factor and recognizes structural features of pre-miRNA hairpins, while it also exports other minihelix-containing RNAs. In Drosophila, Exp5 is suggested to play a major role in tRNA export because the gene encoding the canonical tRNA export factor Exportin-t is missing in its genome. To understand molecular functions of fly Exp5, we studied the Exp5/RNA interactome in the cell line S2R + using the crosslinking and immunoprecipitation (CLIP) technology. The CLIP experiment captured known substrates such as tRNAs and miRNAs and detected candidates of novel Exp5 substrates including various mRNAs and long non-coding RNAs (lncRNAs). Some mRNAs and lncRNAs enriched PAR-CLIP tags compared to their expression levels, suggesting selective binding of Exp5 to them. Intronless mRNAs tended to enrich PAR-CLIP tags; therefore, we proposed that Exp5 might play a role in the export of specific classes of mRNAs/lncRNAs. This result suggested that Drosophila Exp5 might have a wider variety of substrates than initially thought. Surprisingly, Exp5 CLIP reads often contained sequences corresponding to the flanking 5'-leaders and 3'-trailers of tRNAs, which were thought to be removed prior to nuclear export. In fact, we found pre-tRNAs before end-processing were present in the cytoplasm, supporting the idea that tRNA end-processing is a cytoplasmic event. In summary, our results provide a genome-wide list of Exp5 substrate candidates and suggest that flies may lack a mechanism to distinguish pre-tRNAs with or without the flanking sequences., Competing Interests: Conflicts of interest The authors declare that they have no conflcits of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The translocator protein TSPO is an evolutionary conserved mitochondrial protein overexpressed in various contexts of neurodegeneration. Friedreich Ataxia (FA) is a neurodegenerative disease due to GAA expansions in the FXN gene leading to decreased expression of frataxin, a mitochondrial protein involved in the biosynthesis of iron-sulfur clusters. We previously reported that Tspo was overexpressed in a Drosophila model of this disease generated by CRISPR/Cas9 insertion of approximately 200 GAA in the intron of fh, the fly frataxin gene. Here, we describe a new Drosophila model of FA with 42 GAA repeats, called fh-GAAs. The smaller expansion size allowed to obtain adults exhibiting hallmarks of the FA disease, including short lifespan, locomotory defects and hypersensitivity to oxidative stress. The reduced lifespan was fully rescued by ubiquitous expression of human FXN, confirming that both frataxins share conserved functions. We observed that Tspo was overexpressed in heads and decreased in intestines of these fh-GAAs flies. Then, we further overexpressed Tspo specifically in glial cells and observed improved survival. Finally, we investigated the effects of Tspo overexpression in healthy flies. Increased longevity was conferred by glial-specific overexpression, with opposite effects in neurons. Overall, this study highlights protective effects of glial TSPO in Drosophila both in a neurodegenerative and a healthy context., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Proper connection between the sperm head and tail is critical for sperm motility and fertilization. Head-tail linkage is mediated by the head-tail coupling apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is poorly understood. Here, we use Drosophila to investigate formation and remodeling of the HTCA throughout spermiogenesis by visualizing key components of this complex. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'centriole cap' that surrounds the centriole (or basal body) as it invaginates into the surface of the nucleus. As development progresses, the centriole is laterally displaced to the side of the nucleus while the HTCA expands under the nucleus, forming what we term the 'nuclear shelf'. We next show that the proximal centriole-like (PCL) structure is positioned under the nuclear shelf, functioning as a crucial stabilizer of centriole-nucleus attachment. Together, our data indicate that the HTCA is a complex, multi-point attachment site that simultaneously engages the PCL, the centriole and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Deubiquitinases (DUBs) are essential for the maintenance of protein homeostasis and assembly of proteins into functional complexes. Despite growing interest in DUBs biological functions, the roles of DUBs in regulating intestinal stem cells (ISCs) and gut homeostasis remain largely unknown. Here, we perform an in vivo RNAi screen through induced knock-down of DUBs expression in adult midgut ISCs and enteroblasts (EBs) to identify DUB regulators of intestinal homeostasis in Drosophila. We screen 43 DUBs and identify 8 DUBs that are required for ISCs homeostasis. Knocking-down of usp1, CG7857, usp5, rpn8, usp10 and csn5 decreases the number of ISCs/EBs, while knocking-down of CG4968 and usp8 increases the number of ISCs/EBs. Moreover, knock-down of usp1, CG4968, CG7857, or rpn8 in ISCs/EBs disrupts the intestinal barrier integrity and shortens the lifespan, indicating the requirement of these DUBs for the maintenance of gut homeostasis. Furthermore, we provide evidences that USP1 mediates ISC lineage differentiation via modulating the Notch signaling activity. Our study identifies, for the first time, the deubiquitinases required for the maintenance of intestinal homeostasis in Drosophila, and provide new insights into the functional links between the DUBs and intestinal homeostasis., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hyperglycaemia-induced oxidative stress plays significant roles in the development of type 2 diabetes and its complications. This study investigates effects of magainin-AM2 on high-sucrose diet induced redox imbalance and cognitive impairment in Drosophila melanogaster. Effects of various concentrations of sucrose, magainin-AM2 or a combination of both agents on mortality, eclosion rate, generation of reactive oxygen and nitrogen species, activities of antioxidant enzymes, thiol system, and markers of cognitive functions in control and treated flies were examined. Results showed that the exposure of flies to high sucrose (30 %-60 % w/w) diet increased mortality rate (38-67 %, P < 0.001) and levels of glucose (1.8-1.9-fold, P < 0.001), hydrogen peroxide (1.4-1.5-fold, P < 0.01) and nitrite/nitrate (1.2-fold, P < 0.01). Decreased levels of total thiol (53-59 %, P < 0.01), non-protein thiols (59-63 %, P < 0.01), catalase activities (39-47 %, P < 0.01-0.05) and glutathione-s-transferase activities (31-43 %, P < 0.01-0.05) were also observed. Magainin-AM2 (0-10 μM/kg diet) did not affect fly mortality rate, levels of hydrogen peroxide and nitrite/nitrate, and activities of catalase and glutathione-s-transferase. However, the peptide produced a dose-dependent increase in total thiol 1.2-1.6-fold, P < 0.001-0.01)and increases non-protein thiol levels at 10 μM/kg diet (2.0-fold, P < 0.01). Magainin-AM2 inhibited sucrose-induced elevation of glucose (55-70 %, P < 0.001), hydrogen peroxide (11-12 %, P < 0.01) and nitrite/nitrate (20-34 %, P < 0.01-0.05). The peptide prevented sucrose-induced reduction in total and non-protein thiols (1.9-2.0-fold, P < 0.05) levels and activities of catalase (2.3-3.1-fold, P < 0.001) and glutathione-s-transferase (1.8-2.8-fold, P < 0.001-0.05). Magainin-AM2 inhibited sucrose-induced reduction in acetylcholinesterase activities (3.6-4.0-fold, P < 0.001), eclosion rate (18 %, P < 0.001) and negative geotaxis (1.3-14-fold, P < 0.001). These results indicate that beneficial actions of magainin-AM2 may also involve the prevention of hyperglycaemia-induced oxidative damage and encourage its further development as an anti-diabetic agent., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ninein (Nin) is a microtubule (MT) anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize MTs at noncentrosomal MT-organizing centers (ncMTOCs). In humans, the NIN gene is mutated in Seckel syndrome, an inherited developmental disorder. Here, we dissect the protein domains involved in Nin's localization and interactions with dynein and ensconsin (ens/MAP7) and show that the association with ens cooperatively regulates MT assembly in Drosophila fat body cells. We define domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface, localization within the nucleus, and association with Dynein light intermediate chain (Dlic) and ens, respectively. We show that Nin's association with ens synergistically regulates MT assembly. Together, these findings reveal novel features of Nin function and its regulation of a ncMTOC.

The relationship of Amyotrophic Lateral Sclerosis, Parkinson's disease, and other age-related neurodegenerative diseases with mitochondrial dysfunction has led to our study of the mitochondrial fission gene Drp1 in Drosophila melanogaster and aspects of aging. Previously, the Drp1 protein has been demonstrated to interact with the Drosophila Bcl-2 mitochondrial proteins, and Drp1 mutations can lead to mitochondrial dysfunction and neuronal loss. In this study, the Dopa decarboxylase-Gal4 ( Ddc-Gal4 ) transgene was exploited to direct the expression of Drp1 and Drp1-RNAi transgenes in select neurons. Here, the knockdown of Drp1 seems to compromise locomotor function throughout life but does not alter longevity. The co-expression of Buffy suppresses the poor climbing induced by the knockdown of the Drp1 function. The consequences of Drp1 overexpression, which specifically reduced median lifespan and diminished climbing abilities over time, can be suppressed through the directed co-overexpression of pro-survival Bcl-2 gene Buffy or by the co-knockdown of the pro-cell death Bcl-2 homologue Debcl . Alteration of the expression of Drp1 acts to phenocopy neurodegenerative disease phenotypes in Drosophila, while overexpression of Buffy can counteract or rescue these phenotypes to improve overall health. The diminished healthy aging due to either the overexpression of Drp1 or the RNA interference of Drp1 has produced novel Drosophila models for investigating mechanisms underlying neurodegenerative disease.

During animal development, the spatiotemporal properties of molecular events largely determine the biological outcomes. Conventional gene analysis methods lack the spatiotemporal resolution for precise dissection of developmental mechanisms. Although optogenetic tools exist for manipulating designer proteins in cultured cells, few have been successfully applied to endogenous proteins in live animals. Here, we report OptoTrap, a light-inducible clustering system for manipulating endogenous proteins of diverse sizes, subcellular locations, and functions in Drosophila . This system turns on fast, is reversible in minutes or hours, and contains variants optimized for neurons and epithelial cells. By using OptoTrap to disrupt microtubules and inhibit kinesin-1 in neurons, we show that microtubules support the growth of highly dynamic dendrites and that kinesin-1 is required for patterning of low- and high-order dendritic branches in differential spatiotemporal domains. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds promise for studying developmental mechanisms in a wide range of cell types and developmental stages.

Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy ( d STORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality d STORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as "gold standard" fluorophores for d STORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in d STORM. In this study, we defined CF583R's performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future d STORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts.

Obesity is a growing concern. 42.3% of people in the U.S were considered obese between 2017-2018. Much is still unknown about the genetic components that contribute to weight gain. In humans, the hormone glucagon is a major contributor to the body's energy regulation as it signals for the breakdown of lipids. Treatments targeting the glucagon pathway have helped patients with both weight loss and appetite suppression. Understanding the genetic modifiers of glucagon signaling and its downstream pathways could enable the development of a wider variety of effective therapeutics. In this study, we blocked the glucagon pathway in Drosophila melanogaster by reducing the expression of the fly ortholog of the glucagon receptor (AKHR). We then crossed our model to the Drosophila Genetic Reference Panel (DGRP) and looked for natural variation in fat content. We used variation in larval density to identify candidate modifier genes through a genome-wide association study. We then tested these modifier genes by increasing or decreasing their expression in the AKHR model. We screened these candidates initially with the same density assay used in the original study to narrow down to four candidate genes that substantially impacted the density of the larvae: THADA, AmyD, GluRIIC, and CG9826. We further characterized these candidates using biochemical assays to analyze stored metabolites such as triglycerides, glucose, glycogen, and protein under control, high sugar, and high fat conditions to see if the larvae are resistant to environmental changes. Our results indicate consistency between the results of the density assay and direct measurement of metabolite levels. In particular, THADA and AmyD are highlighted as interesting genes for additional study. We hope to improve our understanding of the glucagon signaling pathway, obesity, and lipid metabolism. We also aim to provide candidate genes that can be regarded as future therapeutic targets., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Nicol 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.)

Chemosensory cells across the body of Drosophila melanogaster evaluate the environment to prioritize certain behaviors. Previous mapping of gustatory receptor neurons (GRNs) on the fly labellum identified a set of neurons in L-type sensilla that express Ionotropic Receptor 94e (IR94e), but the impact of IR94e GRNs on behavior remains unclear. We used optogenetics and chemogenetics to activate IR94e neurons and found that they drive mild feeding suppression but enhance egg laying. In vivo calcium imaging revealed that IR94e GRNs respond strongly to certain amino acids, including glutamate, and that IR94e plus co-receptors IR25a and IR76b are required for amino acid detection. Furthermore, IR94e mutants show behavioral changes to solutions containing amino acids, including increased consumption and decreased egg laying. Overall, our results suggest that IR94e GRNs on the fly labellum discourage feeding and encourage egg laying as part of an important behavioral switch in response to certain chemical cues., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Reproduction is paramount to animals. For it to be successful, a coordination of social behavior, physiology, and gamete production is necessary. How are social cues perceived and how do they affect physiology and gametogenesis? While females, ranging from insects to mammals, have provided multiple insights about this coordination, its existence remains largely unknown in males. Here, by using the Drosophila male as a model, we describe a phenomenon by which the availability of potential mating partners triggers an activation state on the stem cell populations of the testis, boosting spermatogenesis. We reveal its reliance on pheromonal communication, even in the absence of mating or other interactions with females. Finally, we identify the interorgan communication signaling network responsible-muscle-secreted tumor necrosis factor alpha (TNF-α)/Eiger and neuronally secreted octopamine trigger, respectively, the Jun N-terminal kinase (JNK) pathway and a change in calcium dynamics in the cyst stem cells. As a consequence, germ line stem cells increase their proliferation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Insect developmental transitions are precisely coordinated by ecdysone and juvenile hormone (JH). We previously revealed that accumulated H3K27 trimethylation (H3K27me3) at the locus encoding JH signal transducer Hairy is involved in the larval-pupal transition in insects, but the underlying mechanism remains to be fully defined. Here, we show in Drosophila and Bombyx that Rpd3-mediated H3K27 deacetylation in the prothoracic gland during the last larval instar promotes ecdysone biosynthesis and the larval-pupal transition by enabling H3K27me3 accumulation at the Hairy locus to induce its transcriptional repression. Importantly, we find that the homeodomain transcription factor Schlank acts to switch active H3K27 acetylation (H3K27ac) to repressive H3K27me3 at the Hairy locus by directly binding to the Hairy promoter and then recruiting the histone deacetylase Rpd3 and the histone methyltransferase PRC2 component Su(z)12 through physical interactions. Moreover, Schlank inhibits Hairy transcription to facilitate the larval-pupal transition, and the Schlank signaling cascade is suppressed by JH but regulated in a positive feedback manner by ecdysone. Together, our data uncover that Schlank mediates epigenetic reprogramming of H3K27 modifications in hormone actions during insect developmental transition., Competing Interests: Competing interests statement:The authors declare no competing interest.

CREB-regulated transcription co-activator (CRTC) is activated by Calcineurin (CaN) to regulate gluconeogenic genes. CaN also has roles in cardiac hypertrophy. Here, we explore a cardiac-autonomous role for CRTC in cardiac hypertrophy. In Drosophila, CRTC mutants exhibit severe cardiac restriction, myofibrillar disorganization, fibrosis, and tachycardia. Cardiac-specific CRTC knockdown (KD) phenocopies mutants, and cardiac overexpression causes hypertrophy. CaN-induced hypertrophy in Drosophila is reduced in CRTC mutants, suggesting that CRTC mediates the effects. RNA sequencing (RNA-seq) of CRTC-KD and -overexpressing hearts reveals contraregulation of metabolic genes. Genes with conserved CREB sites include the fly ortholog of Sarcalumenin, a Ca 2+ -binding protein. Cardiac manipulation of this gene recapitulates the CRTC-KD and -overexpression phenotypes. CRTC KD in zebrafish also causes cardiac restriction, and CRTC KD in human induced cardiomyocytes causes a reduction in Srl expression and increased action potential duration. Our data from three model systems suggest that CaN-CRTC-Sarcalumenin signaling represents an alternate, conserved pathway underlying cardiac function and hypertrophy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The transcriptional coactivator Yorkie (Yki) regulates organ size by promoting cell proliferation. It is unclear how cells control Yki activity when exposed to harmful stimuli such as oxidative stress. In this study, we show that oxidative stress inhibits the binding of Yki to Scalloped (Sd) but promotes the interaction of Yki with another transcription factor, forkhead box O (Foxo), ultimately leading to a halt in cell proliferation. Mechanistically, Foxo normally exhibits a low binding affinity for Yki, allowing Yki to form a complex with Sd and activate proliferative genes. Under oxidative stress, Usp7 deubiquitinates Foxo to promote its interaction with Yki, thereby activating the expression of proliferation suppressors. Finally, we show that Yki is essential for Drosophila survival under oxidative stress. In summary, these findings suggest that oxidative stress reprograms Yki from a proliferation-promoting factor to a proliferation suppressor, forming a self-protective mechanism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The role of Drosophila numb in regulating Notch signaling and neurogenesis has been extensively studied, with a particular focus on its effects on the peripheral nervous system (PNS). Previous studies based on a single loss-of-function allele of numb , numb 1 , showed an antineurogenic effect on the peripheral nervous system (PNS), which revealed that the wild-type numb suppresses Notch signaling. In the current study, we examined whether this phenotype is consistently observed in loss-of-function mutations of numb. Two more numb alleles, numb EY03840 and numb EY03852 , were shown to have an antineurogenic phenotype in the PNS. We also found that introducing a wild-type numb genomic fragment into numb 1 homozygotes rescued their antineurogenic phenotype. These results demonstrated that loss-of-function mutations of numb universally induce this phenotype. Many components of Notch signaling are encoded by maternal effect genes, but no maternal effect of numb was observed in this study. The antineurogenic phenotype of numb was found to be dependent on the Enhancer of split ( E ( spl )), a downstream gene of Notch signaling. We found that the combination of E ( spl ) homozygous and numb 1 homozygous suppressed the neurogenic phenotype of the embryonic central nervous system (CNS) associated with the E ( spl ) mutation. In the E(spl) allele, genes encoding basic helix-loop-helix proteins, such as m5 , m6 , m7 , and m8, remain. Thus, in the E(spl) allele, derepression of Notch activity by numb mutation can rescue the neurogenic phenotype by increasing the expression of the remaining genes in the E(spl) complex. We also uncovered a role for numb in regulating neuronal projections. Our results further support an important role for numb in the suppression of Notch signaling during embryonic nervous system development.

In animals, mitosis involves the breakdown of the nuclear envelope and the sorting of individualized, condensed chromosomes. During mitotic exit, emerging nuclei reassemble a nuclear envelope around a single mass of interconnecting chromosomes. The molecular mechanisms of nuclear reassembly are incompletely understood. Moreover, the cellular and physiological consequences of defects in this process are largely unexplored. Here, we have characterized a mechanism essential for nuclear reassembly in Drosophila. We show that Ankle2 promotes the PP2A-dependent recruitment of BAF and Lamin at reassembling nuclei, and that failures in this mechanism result in severe nuclear defects after mitosis. We then took advantage of perturbations in this mechanism to investigate the physiological responses to nuclear reassembly defects during tissue development in vivo. Partial depletion of Ankle2, BAF, or Lamin in imaginal wing discs results in wing development defects accompanied by apoptosis. We found that blocking apoptosis strongly enhances developmental defects. Blocking p53 does not prevent apoptosis but enhances defects due to the loss of a cell cycle checkpoint. Our results suggest that apoptotic and p53-dependent responses play a crucial role in safeguarding tissue development in response to sporadic nuclear reassembly defects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li 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.)

The Drosophila corneal lens is entirely composed of chitin and other apical extracellular matrix components, and it is not known how it acquires the biconvex shape that enables it to focus light onto the retina. We show here that the zona pellucida domain-containing protein Dusky-like is essential for normal corneal lens morphogenesis. Dusky-like transiently localizes to the expanded apical surfaces of the corneal lens-secreting cells and prevents them from undergoing apical constriction and apicobasal contraction. Dusky-like also controls the arrangement of two other zona pellucida domain proteins, Dumpy and Piopio, external to the developing corneal lens. Loss of either dusky-like or dumpy delays chitin accumulation and disrupts the outer surface of the corneal lens. We find that artificially inducing apical constriction by activating myosin contraction is sufficient to similarly alter chitin deposition and corneal lens morphology. These results demonstrate the importance of cell shape in controlling the morphogenesis of overlying apical extracellular matrix structures such as the corneal lens.

Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase ( OPA1 ) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 ( hOPA1 ) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1 . This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies., Competing Interests: YN, JO, RM, SH, ES, SU, TS, AS No competing interests declared, (© 2023, Nitta, Osaka et al.)

Obesity remains one of the largest health problems in the world, arising from the excess storage of triglycerides (TAGs). However, the full complement of genes that are important for regulating TAG storage is not known. The Glut1 gene encodes a Drosophila glucose transporter that has been identified as a potential obesity gene through genetic screening. Yet, the tissue-specific metabolic functions of Glut1 are not fully understood. Here, we characterized the role of Glut1 in the fly brain by decreasing neuronal Glut1 levels with RNAi and measuring glycogen and TAGs. Glut1RNAi flies had decreased TAG and glycogen levels, suggesting a nonautonomous role of Glut1 in the fly brain to regulate nutrient storage. A group of hormones that regulate metabolism and are expressed in the fly brain are Drosophila insulin-like peptides (Ilps) 2, 3, and 5. Interestingly, we observed blunted Ilp3 and Ilp5 expression in neuronal Glut1RNAi flies, suggesting Glut1 functions in insulin-producing neurons (IPCs) to regulate whole-organism TAG and glycogen storage. Consistent with this hypothesis, we also saw fewer TAGs and glycogens and decreased expression of Ilp3 and Ilp5 in flies with IPC-specific Glut1RNAi . Together, these data suggest Glut1 functions as a nutrient sensor in IPCs, controlling TAG and glycogen storage and regulating systemic energy homeostasis.

Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick ( drm ), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis., (© 2024 Wen et al.; Published by Cold Spring Harbor Laboratory Press.)

Alternative cleavage and polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3' UTRs from the same genetic locus, potentially impacting mRNA translation, localization, and stability. Developmentally regulated APA can thus make major contributions to cell type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, ∼500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of cleavage factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knockdown of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell type-specific APA at selected genes., (© 2024 Gallicchio et al.; Published by Cold Spring Harbor Laboratory Press.)

SLC30A9 (ZnT9) is a mitochondria-resident zinc transporter. Mutations in SLC30A9 have been reported in human patients with a novel cerebro-renal syndrome. Here, we show that ZnT9 is an evolutionarily highly conserved protein, with many regions extremely preserved among evolutionarily distant organisms. In Drosophila melanogaster (the fly), ZnT9 (ZnT49B) knockdown results in acutely impaired movement and drastic mitochondrial deformation. Severe Drosophila ZnT9 (dZnT9) reduction and ZnT9-null mutant flies are pupal lethal. The phenotype of dZnT9 knockdown can be partially rescued by mouse ZnT9 expression or zinc chelator TPEN, indicating the defect of dZnT9 loss is indeed a result of zinc dyshomeostasis. Interestingly, in the mouse, germline loss of Znt9 produces even more extreme phenotypes: the mutant embryos exhibit midgestational lethality with severe development abnormalities. Targeted mutagenesis of Znt9 in the mouse brain leads to serious dwarfism and physical incapacitation, followed by death shortly. Strikingly, the GH/IGF-1 signals are almost non-existent in these tissue-specific knockout mice, consistent with the medical finding in some human patients with severe mitochondrial deficiecny. ZnT9 mutations cause mitochondrial zinc dyshomeostasis, and we demonstrate mechanistically that mitochondrial zinc elevation quickly and potently inhibits the activities of respiration complexes. These results reveal the critical role of ZnT9 and mitochondrial zinc homeostasis in mammalian development. Based on our functional analyses, we finally discussed the possible nature of the so far identified human SLC30A9 mutations., (© 2024. The Author(s).)

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Familial cases of PD are often caused by mutations of PTEN-induced kinase 1 (PINK1) and the ubiquitin ligase Parkin, both pivotal in maintaining mitochondrial quality control. CISD1, a homodimeric mitochondrial iron-sulfur-binding protein, is a major target of Parkin-mediated ubiquitination. We here discovered a heightened propensity of CISD1 to form dimers in Pink1 mutant flies and in dopaminergic neurons from PINK1 mutation patients. The dimer consists of two monomers that are covalently linked by a disulfide bridge. In this conformation CISD1 cannot coordinate the iron-sulfur cofactor. Overexpressing Cisd, the Drosophila ortholog of CISD1, and a mutant Cisd incapable of binding the iron-sulfur cluster in Drosophila reduced climbing ability and lifespan. This was more pronounced with mutant Cisd and aggravated in Pink1 mutant flies. Complete loss of Cisd, in contrast, rescued all detrimental effects of Pink1 mutation on climbing ability, wing posture, dopamine levels, lifespan, and mitochondrial ultrastructure. Our results suggest that Cisd, probably iron-depleted Cisd, operates downstream of Pink1 shedding light on PD pathophysiology and implicating CISD1 as a potential therapeutic target., Competing Interests: SB, TB, CW, MA, LR, PZ, TS, IR, VV, BM, EH, GA, RK, LZ, AM No competing interests declared, (© 2024, Bitar et al.)

The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates. In the developing Drosophila eye, differentiation of photoreceptors R1-R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We find that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologs Scratch and Scrape, as well as directly activating neuronal effector genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells., (© 2024. The Author(s).)

During development, unique combinations of transcription factors and signaling pathways carve the nascent eye-antennal disc of the fruit fly Drosophila melanogaster into several territories that will eventually develop into the compound eye, ocelli, head epidermis, bristles, antenna and maxillary palpus of the adult head. Juxtaposed patterns of Hedgehog (Hh) and Decapentaplegic (Dpp) initiate compound eye development, while reciprocal domains of Dpp and Wingless (Wg) induce formation of the antennal and maxillary palp fields. Hh and Wg signaling, but not Dpp, contribute to the patterning of the dorsal head vertex. Here, we show that combinatorial reductions of the Pax6 transcription factor Twin of Eyeless and either the Wg pathway or the Mirror (Mirr) transcription factor trigger a transformation of the ocelli into a compound eye and the neighboring head epidermis into an antenna. These changes in fate are accompanied by the ectopic expression of Dpp, which might be expected to trigger these changes in fate. However, the transformation of the field cannot be replicated by increasing Dpp levels alone despite the recreation of adjacent Hh-Dpp and Wg-Dpp domains. As such, the emergence of these ectopic organs occurs through a unique regulatory path., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here, we address the function of Drosophila Spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, the cells of which undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of Spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of Spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelope., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The Drosophila Id gene extramacrochaetae (emc) is required during Drosophila eye development for proper cell fate specification within the R7 equivalence group. Without emc, R7 cells develop like R1/6 cells, and there are delays and deficits in differentiation of non-neuronal cone cells. Although emc encodes an Inhibitor of DNA-binding (Id) protein that is known to antagonize proneural bHLH protein function, no proneural gene is known for R7 or cone cell fates. These fates are also independent of daughterless (da), which encodes the ubiquitous E protein heterodimer partner of proneural bHLH proteins. We report here that the effects of emc mutations disappear in the absence of da, and are partially mimicked by forced expression of Da dimers, indicating that emc normally restrains da from interfering with R7 and cone cell specification, as occurs in emc mutants. emc, and da, regulate three known contributors to R7 fate, which are Notch signaling, Rap1, and Sevenless. R7 specification is partially restored to emc mutant cells by mutation of RapGap1, confirming that Rap1 activity, in addition to Notch activity, is a critical target of emc. These findings exemplify how mutations of an Id protein gene can affect processes that do not require any bHLH protein, by restraining Da activity within physiological bounds., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Energy storage and endocrine functions of the Drosophila fat body make it an excellent model for elucidating mechanisms that underlie physiological and pathophysiological organismal metabolism. Combined with Drosophila's robust genetic and immunofluorescence microscopy toolkits, studies of Drosophila fat body function are ripe for cell biological analysis. Unlike the larval fat body, which is easily removed as a single, cohesive sheet of tissue, isolating intact adult fat body proves to be more challenging, thus hindering consistent immunofluorescence labeling even within a single piece of adipose tissue. Here, we describe an improved approach to handling Drosophila abdomens that ensures full access of the adult fat body to solutions generally used in immunofluorescence labeling protocols. In addition, we assess the quality of fluorescence reporter expression and antibody immunoreactivity in response to variations in fixative type, fixation incubation time, and detergent used for cellular permeabilization. Overall, we provide several recommendations for steps in a whole-mount staining protocol that results in consistent and robust immunofluorescence labeling of the adult Drosophila fat body., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

In nephrotic syndrome, the podocyte filtration structures are damaged in a process called foot process effacement. This is mediated by the actin cytoskeleton; however, which actins are involved and how they interact with other filtration components, like the basement membrane, remains poorly understood. Here, we used the well-established Drosophila pericardial nephrocyte-the equivalent of podocytes in flies-knockdown models (RNAi) to study the interplay of the actin cytoskeleton (Act5C, Act57B, Act42A, and Act87E), alpha- and beta-integrin (basement membrane), and the slit diaphragm (Sns and Pyd). Knockdown of an actin gene led to variations of formation of actin stress fibers, the internalization of Sns, and a disrupted slit diaphragm cortical pattern. Notably, deficiency of Act5C , which resulted in complete absence of nephrocytes, could be partially mitigated by overexpressing Act42A or Act87E , suggesting at least partial functional redundancy. Integrin localized near the actin cytoskeleton as well as slit diaphragm components, but when the nephrocyte cytoskeleton or slit diaphragm was disrupted, this switched to colocalization, both at the surface and internalized in aggregates. Altogether, the data show that the interdependence of the slit diaphragm, actin cytoskeleton, and integrins is key to the structure and function of the Drosophila nephrocyte.

RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl - /H + antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells., (© 2024. The Author(s).)

Constitutive heterochromatin, a fundamental feature of eukaryotic nucleus essential for transposon silencing and genome stability, is rebuilt on various types of repetitive DNA in the zygotic genome during early embryogenesis. However, the molecular program underlying this process remains poorly understood. Here, we show that histone H3 lysine 14 acetylation (H3K14ac) is engaged in the reinstallation of constitutive heterochromatin in Drosophila early embryos. H3K14ac partially colocalizes with H3 lysine 9 trimethylation (H3K9me3) and its methyltransferase Eggless/SetDB1 around the mid-blastula transition. Concealing H3K14ac by either antibody injection or maternal knockdown of Gcn5 diminishes Eggless/SetDB1 nuclear foci and reduces the deposition of H3K9me3. Structural analysis reveals that Eggless/SetDB1 recognizes H3K14ac via its tandem Tudor domains, and disrupting the binding interface causes defects in Eggless/SetDB1 distribution and derepression of a subset of transposons. Therefore, H3K14ac, a histone modification normally associated with active transcription, is a crucial component of the early embryonic machinery that introduces constitutive heterochromatic features to the newly formed zygotic genome., Competing Interests: Competing interests statement:The authors declare no competing interest.

Colorectal cancer remains a major global health concern. Colonoscopy, the gold-standard colorectal cancer diagnostic, relies on the visual detection of lesions and necessitates invasive biopsies for confirmation. Alternative diagnostic methods, based on nanomedicine, can facilitate early detection of malignancies. Here, we examine the uptake of surface-enhanced Raman scattering nanoparticles (SERS NPs) as a marker for intestinal tumor detection and imaging using an established Drosophila melanogaster model for gut disease. Young and old Oregon-R and w 1118 flies were orally administered SERS NPs and scanned without and upon gut lumen clearance to assess nanoparticle retention as a function of aging. Neither young nor old flies showed significant NP retention in their body after gut lumen clearance. Moreover, tumorigenic flies of the esg-Gal4/UAS-Ras V12 genotype were tested for SERS NP retention 2, 4 and 6 days after Ras V12 oncogene induction in their midgut progenitor cells. Tumorigenic flies showed a statistically significant NP retention signal at 2 days, well before midgut epithelium impairment. The signal was then visualized in scans of dissected guts revealing areas of NP uptake in the posterior midgut region of high stem cell activity.

The proper functioning of neural circuits that integrate sensory signals is essential for individual adaptation to an ever-changing environment. Many molecules can modulate neuronal activity, including neurotransmitters, receptors, and even amino acids. Here, we ask whether amino acid transporters expressed by neurons can influence neuronal activity. We found that minidiscs ( mnd ), which encodes a light chain of a heterodimeric amino acid transporter, is expressed in different cell types of the adult Drosophila brain: in mushroom body neurons (MBs) and in glial cells. Using live calcium imaging, we found that MND expressed in α/β MB neurons is essential for sensitivity to the L-amino acids: Leu, Ile, Asp, Glu, Lys, Thr, and Arg. We found that the Target Of Rapamycin (TOR) pathway but not the Glutamate Dehydrogenase (GDH) pathway is involved in the Leucine-dependent response of α/β MB neurons. This study strongly supports the key role of MND in regulating MB activity in response to amino acids.

The Ras family genes are proto-oncogenes that are highly conserved from Drosophila to humans. In Drosophila, Ras V12 is a constitutively activated form of the Ras oncoprotein, and its function in cell-cycle progression is context dependent. However, how it influences the cell cycle of female germline stem cells (GSCs) still remains unknown. Using both wild-type GSCs and bam mutant GSC-like cells as model systems, here we determined that Ras V12 overexpression promotes GSC division, not growth, opposite to that in somatic wing disc cells. Ras performs this function through activating the mitogen-activated protein kinase (MAPK) signaling. This signaling is activated specifically in the M phase of mitotic germ cells, including both wild-type GSCs and bam mutant GSC-like cells. Furthermore, Ras V12 overexpression triggers polyploid nurse cells to die through inducing mitotic stress. Given the similarities between Drosophila and mammalian GSCs, we propose that the Ras/MAPK signaling also promotes mammalian GSC division., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ribosomes are regulated by evolutionarily conserved ubiquitination/deubiquitination events. We uncover the role of the deubiquitinase OTUD6 in regulating global protein translation through deubiquitination of the RPS7/eS7 subunit on the free 40 S ribosome in vivo in Drosophila. Coimmunoprecipitation and enrichment of monoubiquitinated proteins from catalytically inactive OTUD6 flies reveal RPS7 as the ribosomal substrate. The 40 S protein RACK1 and E3 ligases CNOT4 and RNF10 function upstream of OTUD6 to regulate alkylation stress. OTUD6 interacts with RPS7 specifically on the free 40 S, and not on 43 S/48 S initiation complexes or the translating ribosome. Global protein translation levels are bidirectionally regulated by OTUD6 protein abundance. OTUD6 protein abundance is physiologically regulated in aging and in response to translational and alkylation stress. Thus, OTUD6 may promote translation initiation, the rate limiting step in protein translation, by titering the amount of 40 S ribosome that recycles., (© 2024. The Author(s).)

The strength of molecular interactions is characterized by their dissociation constants (K D ). Only high-affinity interactions (K D  ≤ 10 -8  M) are extensively investigated and support binary on/off switches. However, such analyses have discounted the presence of low-affinity binders (K D  > 10 -5  M) in the cellular environment. We assess the potential influence of low-affinity binders on high-affinity interactions. By employing Gillespie stochastic simulations and continuous methods, we demonstrate that the presence of low-affinity binders can alter the kinetics and the steady state of high-affinity interactions. We refer to this effect as 'herd regulation' and have evaluated its possible impact in two different contexts including sex determination in Drosophila melanogaster and in signalling systems that employ molecular thresholds. We have also suggested experiments to validate herd regulation in vitro. We speculate that low-affinity binders are prevalent in biological contexts where the outcomes depend on molecular thresholds impacting homoeostatic regulation., (© 2024. The Author(s).)

Despite recent studies implicating liquid-like biomolecular condensates in diverse cellular processes, many biomolecular condensates exist in a solid-like state, and their function and regulation are less understood. We show that the tumor suppressor Merlin, an upstream regulator of the Hippo pathway, localizes to both cell junctions and medial apical cortex in Drosophila epithelia, with the latter forming solid-like condensates that activate Hippo signaling. Merlin condensation required phosphatidylinositol-4-phosphate (PI4P)-mediated plasma membrane targeting and was antagonistically controlled by Pez and cytoskeletal tension through plasma membrane PI4P regulation. The solid-like material properties of Merlin condensates are essential for physiological function and protect the condensates against external perturbations. Collectively, these findings uncover an essential role for solid-like condensates in normal physiology and reveal regulatory mechanisms for their formation and disassembly.

RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

INTS11 and CPSF73 are metal-dependent endonucleases for Integrator and pre-mRNA 3'-end processing, respectively. Here, we show that the INTS11 binding partner BRAT1/CG7044, a factor important for neuronal fitness, stabilizes INTS11 in the cytoplasm and is required for Integrator function in the nucleus. Loss of BRAT1 in neural organoids leads to transcriptomic disruption and precocious expression of neurogenesis-driving transcription factors. The structures of the human INTS9-INTS11-BRAT1 and Drosophila dIntS11-CG7044 complexes reveal that the conserved C terminus of BRAT1/CG7044 is captured in the active site of INTS11, with a cysteine residue directly coordinating the metal ions. Inspired by these observations, we find that UBE3D is a binding partner for CPSF73, and UBE3D likely also uses a conserved cysteine residue to directly coordinate the active site metal ions. Our studies have revealed binding partners for INTS11 and CPSF73 that behave like cytoplasmic chaperones with a conserved impact on the nuclear functions of these enzymes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To survive daily damage, the formation of actomyosin ring at the wound edge is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that the rapid recruitment of all 3 Drosophila calcium-responding and phospholipid-binding Annexin proteins (AnxB9, AnxB10, and AnxB11) to distinct regions around the wound is regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, while the wound does not close in the absence of calcium influx, we find that reduced calcium influx can still initiate repair processes, albeit leading to severe repair phenotypes. Thus, our results suggest that, in addition to initiating repair events, the quantity of calcium influx is important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair., 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 Eph receptor, a prototypically large receptor protein tyrosine kinase, interacts with ephrin ligands, forming a bidirectional signaling system that impacts diverse brain functions. Eph receptors and ephrins mediate forward and reverse signaling, affecting neurogenesis, axon guidance, and synaptic signaling. While mammalian studies have emphasized their roles in neurogenesis and synaptic plasticity, the Drosophila counterparts are less studied, especially in glial cells, despite structural similarities. Using RNAi to modulate Eph/ephrin expression in Drosophila neurons and glia, we studied their roles in brain development and sleep and circadian behavior. Knockdown of neuronal ephrin disrupted mushroom body development, while glial knockdown had minimal impact. Surprisingly, disrupting ephrin in neurons or glial cells altered sleep and circadian rhythms, indicating a direct involvement in these behaviors independent from developmental effects. Further analysis revealed distinct sleep phenotypes between neuronal and glial knockdowns, underscoring the intricate interplay within the neural circuits that govern behavior. Glia-specific knockdowns showed altered sleep patterns and reduced circadian rhythmicity, suggesting an intricate role of glia in sleep regulation. Our findings challenge simplistic models of Eph/ephrin signaling limited to neuron-glia communication and emphasize the complexity of the regulatory networks modulating behavior. Future investigations targeting specific glial subtypes will enhance our understanding of Eph/ephrin signaling's role in sleep regulation across species., 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.)

Adhesion between epithelial cells enables the remarkable mechanical behavior of epithelial tissues during morphogenesis. However, it remains unclear how cell-cell adhesion influences mechanics in both static and dynamically flowing confluent epithelial tissues. Here, we systematically modulate E-cadherin-mediated adhesion in the Drosophila embryo and study the effects on the mechanical behavior of the germband epithelium before and during dramatic tissue remodeling and flow associated with body axis elongation. Before axis elongation, we find that increasing E-cadherin levels produces tissue comprising more elongated cells and predicted to be more fluid-like, providing reduced resistance to tissue flow. During axis elongation, we find that the dominant effect of E-cadherin is tuning the speed at which cells proceed through rearrangement events. Before and during axis elongation, E-cadherin levels influence patterns of actomyosin-dependent forces, supporting the notion that E-cadherin tunes tissue mechanics in part through effects on actomyosin. Notably, the effects of ∼4-fold changes in E-cadherin levels on overall tissue structure and flow are relatively weak, suggesting that the system is tolerant to changes in absolute E-cadherin levels over this range where an intact tissue is formed. Taken together, these findings reveal dual-and sometimes opposing-roles for E-cadherin-mediated adhesion in controlling tissue structure and dynamics in vivo, which result in unexpected relationships between adhesion and flow in confluent tissues., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Organisms experience constant nutritional flux. Mechanisms at the interface of opposing nutritional states-scarcity and surplus-enable organismal energy homeostasis. Contingent on nutritional stores, adipocytes secrete adipokines, such as the fat hormone leptin, to signal nutrient status to the central brain. Increased leptin secretion underlies metabolic dysregulation during common obesity, but the molecular mechanisms regulating leptin secretion from human adipocytes are poorly understood. Here, we report that Atg8/LC3 family proteins, best known for their role in autophagy during nutrient scarcity, play an evolutionarily conserved role during nutrient surplus by promoting adipokine secretion. We show that in a well-fed state, Atg8/LC3 promotes the secretion of the Drosophila functional leptin ortholog unpaired 2 (Upd2) and leptin from human adipocytes. Proteomic analyses reveal that LC3 directs leptin to a secretory pathway in human cells. We identified LC3-dependent extracellular vesicle (EV) loading and secretion (LDELS) as a required step for leptin release, highlighting a unique secretory route adopted by leptin in human adipocytes. In Drosophila, mutations to Upd2's Atg8 interaction motif (AIM) result in constitutive adipokine retention. Atg8-mediated Upd2 retention alters lipid storage and hunger response and rewires the bulk organismal transcriptome in a manner conducive to starvation survival. Thus, Atg8/LC3's bidirectional role in nutrient sensing-conveying nutrient surplus and responding to nutrient deprivation-enables organisms to manage nutrient flux effectively. We posit that decoding how bidirectional molecular switches-such as Atg8/LC3-operate at the nexus of nutritional scarcity and surplus will inform therapeutic strategies to tackle chronic metabolic disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Coordinated cell shape changes are a major driver of tissue morphogenesis, with apical constriction of epithelial cells leading to tissue bending. We previously identified that interplay between the apical-medial actomyosin, which drives apical constriction, and the underlying longitudinal microtubule array has a key role during tube budding of salivary glands in the Drosophila embryo. At this microtubule-actomyosin interface, a hub of proteins accumulates, and we have shown before that this hub includes the microtubule-actin crosslinker Shot and the microtubule minus-end-binding protein Patronin. Here, we identify two actin-crosslinkers, β-heavy (H)-Spectrin (also known as Karst) and Filamin (also known as Cheerio), and the multi-PDZ-domain protein Big bang as components of the protein hub. We show that tissue-specific degradation of β-H-Spectrin leads to reduction of apical-medial F-actin, Shot, Patronin and Big bang, as well as concomitant defects in apical constriction, but that residual Patronin is still sufficient to assist microtubule reorganisation. We find that, unlike Patronin and Shot, neither β-H-Spectrin nor Big bang require microtubules for their localisation. β-H-Spectrin is instead recruited via binding to apical-medial phosphoinositides, and overexpression of the C-terminal pleckstrin homology domain-containing region of β-H-Spectrin (β-H-33) displaces endogenous β-H-Spectrin and leads to strong morphogenetic defects. This protein hub therefore requires the synergy and coincidence of membrane- and microtubule-associated components for its assembly and function in sustaining apical constriction during tubulogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The gut microbiota and their metabolites are closely linked to obesity-related diseases, such as type 2 diabetes, but their causal relationship and underlying mechanisms remain largely elusive. Here, we found that dysbiosis-induced tyramine (TA) suppresses high-fat diet (HFD)-mediated insulin resistance in both Drosophila and mice. In Drosophila, HFD increases cytosolic Ca 2+ signaling in enterocytes, which, in turn, suppresses intestinal lipid levels. 16 S rRNA sequencing and metabolomics revealed that HFD leads to increased prevalence of tyrosine decarboxylase (Tdc)-expressing bacteria and resulting tyramine production. Tyramine acts on the tyramine receptor, TyrR1, to promote cytosolic Ca 2+ signaling and activation of the CRTC-CREB complex to transcriptionally suppress dietary lipid digestion and lipogenesis in enterocytes, while promoting mitochondrial biogenesis. Furthermore, the tyramine-induced cytosolic Ca 2+ signaling is sufficient to suppress HFD-induced obesity and insulin resistance in Drosophila. In mice, tyramine intake also improves glucose tolerance and insulin sensitivity under HFD. These results indicate that dysbiosis-induced tyramine suppresses insulin resistance in both flies and mice under HFD, suggesting a potential therapeutic strategy for related metabolic disorders, such as diabetes., (© 2024. The Author(s).)