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The evolutionarily conserved immune deficiency (IMD) signaling pathway shields Drosophila against bacterial infections. It regulates the expression of antimicrobial peptides encoding genes through the activation of the NF-κB transcription factor Relish. Tight regulation of the signaling cascade ensures a balanced immune response, which is otherwise highly harmful. Several phosphorylation events mediate intracellular progression of the IMD pathway. However, signal termination by dephosphorylation remains largely elusive. Here, we identify the highly conserved protein phosphatase 4 (PP4) complex as a bona fide negative regulator of the IMD pathway. RNA interference–mediated gene silencing of PP4-19c, PP4R2, and Falafel, which encode the catalytic and regulatory subunits of the phosphatase complex, respectively, caused a marked upregulation of bacterial-induced antimicrobial peptide gene expression in both Drosophila melanogaster S2 cells and adult flies. Deregulated IMD signaling is associated with reduced lifespan of PP4-deficient flies in the absence of any infection. In contrast, flies overexpressing this phosphatase are highly sensitive to bacterial infections. Altogether, our results highlight an evolutionarily conserved function of PP4c in the regulation of NF-κB signaling from Drosophila to mammals.

Drosophila model is intensively studied for the development of cancer. The diminutive (dMyc), a homolog of the human MYC gene, is responsible for cell- apoptosis and its upregulation is responsible for determining the fate of cancerous growth in humans and Drosophila model. This work implores the requirement of dMyc and its expression as one of the major regulator of cancer with other proteins and repression of dMyc mRNA in Drosophila S2 cells. Here we report protein complex of Argonaute 1 (AGO1), Bag of marbles (Bam), and Brain tumor (Brat) proteins and not the individual factor of this complex repression of dMyc mRNA in Drosophila Schneider 2 cells and promote differentiation in cystoblast of Drosophila ovary. These results exhibit the significant role of this complex, including master differentiation factor Bam with other various differentiation factor Brat and microRNA pathway component AGO1, which may negatively regulate dMyc mRNA and so the dMyc protein.

Background/aim KT2 is a lysine/tryptophan-rich peptide modified from Crocodylus siamensis Leucrocin I. In this study, we examined the cell toxicity, cellular uptake, anti-migration and anti-invasion activities of KT2 in A375.S2 human melanoma cells. Materials and methods A375.S2 cells were treated with KT2 peptide and then we performed MTT assay, study of cellular uptake by a confocal microscope, wound healing assay, transwell migration/invasion assay, and evaluation of the expression of metastasis-associated proteins. Results KT2 can be internalized through the plasma membrane and can slightly alter cell morphology, decrease the percentage of viable cells and inhibit cell migration and invasion of A375.S2 cells in a dose-dependent manner. This peptide suppressed MMP-2 activity, as measured by gelatine zymography assay. The protein level of MMP-2 was decreased by KT2. KT2 also down-regulated metastasis pathway-related molecules, including FAK, RhoA, ROCK1, GRB2, SOS-1, p-JNK, p-c-Jun, PI3K, p-AKT (Thr308), p-AKT (Ser473), p-p38, MMP-9, NF-kB, and uPA. Conclusion These results indicate that KT2 inhibits the migration and invasion of human melanoma cells by decreasing MMP-2 and MMP-9 expression through inhibition of FAK, uPA, MAPK, PI3K/AKT NF-kB, and RhoA-ROCK signalling pathways. These findings suggest that KT2 deserves further investigation as an anti-metastatic agent for human melanoma.

Apoptosis and compensatory proliferation, two intertwined cellular processes essential for both development and adult homeostasis, are often initiated by the mis-regulation of centrosomal proteins, damaged DNA, and defects in mitosis. Fly Anastral spindle 3 (Ana3) is a member of the pericentriolar matrix proteins and known as a key component of centriolar cohesion and basal body formation. We report here that ana3m19 is a suppressor of lethality induced by the overexpression of Sol narae (Sona), a metalloprotease in a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family. ana3m19 has a nonsense mutation that truncates the highly conserved carboxyl terminal region containing multiple Armadillo repeats. Lethality induced by Sona overexpression was completely rescued by knockdown of Ana3, and the small and malformed wing and hinge phenotype induced by the knockdown of Ana3 was also normalized by Sona overexpression, establishing a mutually positive genetic interaction between ana3 and sona. p35 inhibited apoptosis and rescued the small wing and hinge phenotype induced by knockdown of ana3. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies and reduced the number of dying cells, demonstrating that Ana3 actively promotes cell survival. Knockdown of Ana3 decreased the levels of both intra- and extracellular Sona in wing discs, while overexpression of Ana3 in S2 cells dramatically increased the levels of both cytoplasmic and exosomal Sona due to the stabilization of Sona in the lysosomal degradation pathway. We propose that one of the main functions of Ana3 is to stabilize Sona for cell survival and proliferation.

The membrane-bound, long form of MGAT4D, termed MGAT4D-L, inhibits MGAT1 activity in transfected cells and reduces the generation of complex N-glycans. MGAT1 is the GlcNAc-transferase that initiates complex and hybrid N-glycan synthesis. We show here that Drosophila MGAT1 was also inhibited by MGAT4D-L in S2 cells. In mammalian cells, expression of MGAT4D-L causes the substrate of MGAT1 (Man5GlcNAc2Asn) to accumulate on glycoproteins, a change that is detected by the lectin Galanthus nivalis agglutinin (GNA). Using GNA binding as an assay for the inhibition of MGAT1 in MGAT4D-L transfectants, we performed site-directed mutagenesis to determine requirements for MGAT1 inhibition. Deletion of 25 amino acids (aa) from the C terminus inactivated MGAT4D-L, but deletion of 20 aa did not. Conversion of the five key amino acids (PSLFQ) to Ala, or deletion of PSLFQ in the context of full-length MGAT4D-L, also inactivated MGAT1 inhibitory activity. Nevertheless, mutant, inactive MGAT4D-L interacted with MGAT1 in co-immuno-precipitation experiments. The PSLFQ sequence also occurs in MGAT4A and MGAT4B GlcNAc-transferases. However, neither inhibited MGAT1 in transfected CHO cells. MGAT4D-L inhibitory activity could be partially transferred by attaching PSLFQ or the 25-aa C terminus of MGAT4D-L to the C terminus of MGAT1. Mutation of each amino acid in PSLFQ to Ala identified both Leu and Phe as independently essential for MGAT4D-L activity. Thus, replacement of either Leu-395 or Phe-396 with Ala led to inactivation of MGAT4D-L inhibitory activity. These findings provide new insights into the mechanism of inhibition of MGAT1 by MGAT4D-L, and for the development of small molecule inhibitors of MGAT1.

Many studies have revealed the advantages of using three-dimensional (3D) culture over traditional two-dimensional (2D) monolayer techniques. The 3D cell culture models represent biologically relevant approaches to better mimic the tumor organization observed in vivo. These models have high potential for anticancer drug development and screening, for which challenges include tumor cell resistance to treatment and the risk of toxicity for patients. Many anticancer drug candidates do not reach clinical trials as they fail preclinical tests in vivo, although they were promising in 2D cultures. Models from 3D cell cultures are proposed as intermediate screening filters between 2D and in vivo assays. It has been suggested that ruthenium complexes have great potential for breast cancer treatment. Here, we tested the trans-[Ru(PPh3)2(N,N-dimethyl-N-thiophenylthioureato-k2O,S)(bipy)]PF6 complex in breast cancer cell lines using different 3D culture techniques, including the embedded, ‘on top’ and disease-on-a-chip (DOC) models that reproduce physical aspects of the tumor microenvironment. The complex had pronounced but distinct cytotoxic effects on tumors cultured in collagen I of appropriate stiffness for the disease stage; the extent of induced apoptosis depends on the preinvasive (S2 cells) and invasive (T4-2 and MDA-MB-231 cells) nature of triple-negative breast cancer models. Remarkably, in the DOC model, which simulates breast ductal architecture, the complex was cytotoxic for T4-2 tumors but had no remarkable effect on the differentiated luminal epithelial S1 cells, demonstrating selectivity against cancer cells. In addition, a lower concentration of the complex abrogated the malignant phenotype of the T4-2 cells with reduction of EGFR, p50 NFκB, and β1-integrin expression. To the best of our knowledge, this work uniquely demonstrates the effects of a ruthenium complex on the induction of apoptosis and on the phenotypic reversion of tumor cells in 3D cultures. These results warrant moving to in vivo evaluation of this ruthenium complex.

Material complementario: https://www.frontiersin.org/articles/10.3389/fviro.2021.756559/full#supplementary-material Bovine leukemia virus (BLV) is an oncogenic deltaretrovirus that infects cattle worldwide. In Uruguay, it is estimated that more than 70% of dairy cattle are infected, causing serious economic losses due to decreased milk production, increased calving interval, and livestock losses due to lymphosarcoma. Several attempts to develop vaccine candidates that activate protective immune responses against BLV were performed, but up to date, there is no vaccine that ensures efficient protection and/or decreased viral transmission. The development and application of new vaccines that effectively control BLV infection represent amajor challenge for countries with a high prevalence of infection. In this study, we generated two Drosophila melanogaster S2 stable cell lines capable of producing BLV virus-like particles (BLV-VLPs). One of them, BLV-VLP1, expressed both Gag and Env wild-type (Envwt) full-length proteins, whereas BLV-VLP2 contain Gag together with a mutant form of Env non-susceptible to proteolytic maturation by cellular furin type enzymes (EnvFm).We showed that Envwt is properly cleaved by cellular furin, whereas EnvFm is produced as a full-length gp72 precursor, which undergoes some partial cleavage. We observed that said mutation does not drastically affect its expression or its entry into the secretory pathway of S2 insect cells. In addition, it is expressed on the membrane and retains significant structural motifs when expressed in S2 insect cells. Morphology and size of purified BLV-VLPs were analyzed by transmission electron microscopy and dynamic light scattering, showing numerous non-aggregated and approximately spherical particles of variable diameter (70–200 nm) as previously reported for retroviral VLPs produced using different expression systems. Furthermore, we identified two N-glycosylation patterns rich in mannose in EnvFm protein displayed on VLP2. Our results suggest that the VLPs produced in Drosophila S2 cells could be a potential immunogen to be used in the development of BLV vaccines that might contribute, in conjunction with other control strategies, to reduce the transmission of the virus. CSIC I+D 2014 ANII: ALI_1_2016_2_129851; POS_NAC_2015_1_109471 PEDECIBA-FOCEM: COF 03/11 CAP: BFPD_2020_1#28143834

Enhancer sequences control gene expression and comprise binding sites (motifs) for different transcription factors (TFs). Despite extensive genetic and computational studies, the relationship between DNA sequence and regulatory activity is poorly understood and enhancerde novodesign is considered impossible. Here we built a deep learning model, DeepSTARR, to quantitatively predict the activities of thousands of developmental and housekeeping enhancers directly from DNA sequence inDrosophila melanogasterS2 cells. The model learned relevant TF motifs and higher-order syntax rules, including functionally non-equivalent instances of the same TF motif that are determined by motif-flanking sequence and inter-motif distances. We validated these rules experimentally and demonstrated their conservation in human by testing more than 40,000 wildtype and mutantDrosophilaand human enhancers. Finally, we designed and functionally validated synthetic enhancers with desired activitiesde novo.

The Polycomb group (PcG) complex PRC1 localizes in the nucleus in the form of condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph), forms phase separated condensates with DNA or chromatin in vitro, suggesting PcG bodies may form by SAM-driven phase separation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. We therefore hypothesize that sequences outside of mini-Ph, which are predicted to be intrinsically disordered, are required for proper condensate formation. We identified three distinct low complexity regions in Ph based on sequence composition. We systematically tested the role of each of these sequences in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affects Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occur when the central, glutamine rich IDR is removed, which results in large Ph condensates. Like mini-Ph condensates, these condensates exclude chromatin. Chromatin fractionation experiments indicate that removal of the glutamine rich IDR reduces chromatin binding, while removal of either of the other IDRs increases chromatin binding. Our data suggest all three IDRs, and functional interactions among them, regulate Ph condensate size and number. Our results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM driven phase separation and highlight the complexity of regulation of biological condensates housed in single proteins.

In recent years, the convergence of multiple technologies and experimental approaches has led to the expanded use of cultured Drosophila cells as a model system. Their ease of culture and maintenance, susceptibility to RNA interference, and imaging characteristics have led to extensive use in both traditional experimental approaches as well as high-throughput RNAi screens. Here we describe Drosophila S2 cell culture and preparation for live-cell and fixed-cell fluorescence microscopy and scanning electron microscopy.

LPL is essential for intravascular lipid metabolism and is of high medical relevance. Since LPL is notoriously unstable, there is an unmet need for a robust expression system producing high quantities of active and pure recombinant human LPL (hLPL). We showed previously that bovine LPL purified from milk is unstable at body temperature (Tm is 34.8°C), but in the presence of the endothelial transporter glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), LPL is stabile (Tm increases to 57.6°C). Building on this information, we now designed an expression system for hLPL using Drosophila Schneider 2 cells grown in suspension at high cell density and at an advantageous temperature of 25°C. We cotransfected Schneider 2 cells with hLPL, lipase maturation factor 1, and soluble GPIHBP1 to provide an efficient chaperoning and stabilization of LPL in all compartments during synthesis and after secretion into the conditioned medium. For LPL purification, we used heparin-Sepharose affinity chromatography, which disrupted LPL-GPIHBP1 complexes causing GPIHBP1 to elute with the flow-through of the conditioned media. This one-step purification procedure yielded high quantities of pure and active LPL (4-28 mg/l). Purification of several hLPL variants (furin cleavage-resistant mutant R297A, active-site mutant S132A, and lipid-binding-deficient mutant W390A-W393A-W394A) as well as murine LPL underscores the versatility and robustness of this protocol. Notably, we were able to produce and purify LPL containing the cognate furin cleavage site. This method provides an efficient and cost-effective approach to produce large quantities of LPL for biophysical and large-scale drug discovery studies.

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo, moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. Here, we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt. This article has an associated First Person interview with the first author of the paper., Summary: In S2 cells, the phase-separated Sec bodies form upon the combined activation of salt-inducible kinases, IRE1 and PERK.

Suppressor of Hairy wing [Su(Hw)] is an insulator protein that participates in regulating chromatin architecture and gene repression in Drosophila. In previous studies we have shown that Su(Hw) is also required for pre-replication complex (pre-RC) recruitment on Su(Hw)-bound sites (SBSs) in Drosophila S2 cells and pupa. Here, we describe the effect of Su(Hw) on developmentally regulated amplification of 66D and 7F Drosophila amplicons in follicle cells (DAFCs), widely used as models in replication studies. We show Su(Hw) binding co-localizes with all known DAFCs in Drosophila ovaries, whereas disruption of Su(Hw) binding to 66D and 7F DAFCs causes a two-fold decrease in the amplification of these loci. The complete loss of Su(Hw) binding to chromatin impairs pre-RC recruitment to all amplification regulatory regions of 66D and 7F loci at early oogenesis (prior to DAFCs amplification). These changes coincide with a considerable Su(Hw)-dependent condensation of chromatin at 66D and 7F loci. Although we observed the Brm, ISWI, Mi-2, and CHD1 chromatin remodelers at SBSs genome wide, their remodeler activity does not appear to be responsible for chromatin decondensation at the 66D and 7F amplification regulatory regions. We have discovered that, in addition to the CBP/Nejire and Chameau histone acetyltransferases, the Gcn5 acetyltransferase binds to 66D and 7F DAFCs at SBSs and this binding is dependent on Su(Hw). We propose that the main function of Su(Hw) in developmental amplification of 66D and 7F DAFCs is to establish a chromatin structure that is permissive to pre-RC recruitment.

Cells respond to stress with translational arrest, robust transcriptional changes, and transcription-independent formation of mRNP assemblies termed stress granules (SGs). Despite considerable interest in the role of SGs in oxidative, unfolded-protein, and viral stress responses, whether and how SGs contribute to stress-induced transcription has not been rigorously examined. To address this issue, we characterized transcriptional changes in Drosophila S2 cells induced by acute oxidative-stress and assessed how these were altered under conditions that disrupted SG assembly. Sodium-arsenite stress for 3 hours predominantly resulted in the induction or upregulation of stress-responsive mRNAs whose levels peaked during cell recovery after stress cessation. The stress-transcriptome is enriched in mRNAs coding for protein chaperones, including HSP70 and low molecular-weight heat shock proteins, glutathione transferases, and several non-coding RNAs. Oxidative stress also induced prominent cytoplasmic stress granules that disassembled 3-hours after stress cessation. As expected, RNAi-mediated knockdown of the conserved G3BP1/ Rasputin protein inhibited stress-granule assembly. However, this disruption had no significant effect on the stress-induced transcriptional response or stress-induced translational arrest. Thus, SG assembly and stress-induced effects on gene expression appear to be driven by distinctive signaling processes. We suggest that while SG assembly represents a fast, transient mechanism, the transcriptional response enables a slower, longer-lasting mechanism for adaptation to and recovery from cell stress.

In mammals, plant lectins have been shown to possess immunomodulatory properties, acting in both the innate and adaptive immune system to modulate the production of mediators of the immune response, ultimately improving host defences. At present, knowledge of immunomodulatory effects of plant lectins in insects is scarce. Treatment of insect cells with the Orysa sativa lectin, Orysata, was previously reported to induce cell aggregation, mimicking the immune process of encapsulation. In this project we investigated the potential immunomodulatory effects of this mannose-binding lectin using Drosophila melanogaster S2 cells. Identification of the Orysata binding partners on the surface of S2 cells through a pull-down assay and proteomic analysis revealed 221 putative interactors, several of which were immunity-related proteins. Subsequent qPCR analysis revealed the upregulation of Toll- and immune deficiency (IMD)-regulated antimicrobial peptides (Drs, Mtk, AttA and Dpt) and signal transducers (Rel and Hid) belonging to the IMD pathway. In addition, the iron-binding protein Transferrin 3 was identified as a putative interactor for Orysata, and treatment of S2 cells with Orysata was shown to reduce the intracellular iron concentration. All together, we believe these results offer a new perspective on the effects by which plant lectins influence insect cells and contribute to the study of their immunomodulatory properties. This article is protected by copyright. All rights reserved.

Summary The Drosophila circadian clock is driven by a transcriptional feedback loop in which CLOCK-CYCLE (CLK-CYC) binds E-boxes to transcribe genes encoding the PERIOD-TIMELESS (PER-TIM) repressor, which releases CLK-CYC from E-boxes to inhibit transcription. CLOCKWORK ORANGE (CWO) reinforces PER-TIM repression by binding E-boxes to maintain PER-TIM bound CLK-CYC off DNA, but also promotes CLK-CYC transcription through an unknown mechanism. To determine how CWO activates CLK-CYC transcription, we identified CWO target genes that are upregulated in the absence of CWO repression, conserved in mammals, and preferentially expressed in brain pacemaker neurons. Among the genes identified was a putative ortholog of mouse Clock Interacting Protein Circadian (Cipc), which represses CLOCK-BMAL1 transcription. Reducing or eliminating Drosophila Cipc expression shortens period, while overexpressing Cipc lengthens period, which is consistent with previous work showing that Drosophila Cipc represses CLK-CYC transcription in S2 cells. Cipc represses CLK-CYC transcription in vivo, but not uniformly, as per is strongly repressed, tim less so, and vri hardly at all. Long period rhythms in cwo mutant flies are largely rescued when Cipc expression is reduced or eliminated, indicating that increased Cipc expression mediates the period lengthening of cwo mutants. Consistent with this behavioral rescue, eliminating Cipc rescues the decreased CLK-CYC transcription in cwo mutant flies, where per is strongly rescued, tim is moderately rescued, and vri shows little rescue. These results suggest a mechanism for CWO-dependent CLK-CYC activation: CWO inhibition of CIPC repression promotes CLK-CYC transcription. This mechanism may be conserved since cwo and Cipc perform analogous roles in the mammalian circadian clock.

The activity of multiple organelles must be coordinated to ensure cellular lipid homeostasis. This includes the peroxisomes which metabolise certain lipids and lipid droplets which act as neutral lipid storage centres. Direct organellar contact between peroxisomes and lipid droplets has been observed, and interaction between proteins associated with the membranes of these organelles has been shown, but the functional role of these interactions is not clear. In Drosophila cells, we identified a novel localization of a subset of three transmembrane Peroxin proteins (Peroxin3, Peroxin13, and Peroxin14), normally required for peroxisome biogenesis, to newly formed lipid droplets. This event was not linked to significant changes in peroxisome size or number, nor was recruitment of other Peroxin proteins or mature peroxisomes observed. The presence of these Peroxin proteins at lipid droplets influences their function as changes in the relative levels of Peroxin14 associated with the lipid droplet surface directly affected the presence of regulatory perilipin and lipases with corresponding effects on triglyceride storage.Summary statementInteractions between peroxisomes and lipid droplets is thought to help coordinate management of cellular lipids. Peroxin proteins are required for peroxisome biogenesis. A spectrum of effects on triacylglyceride storage was seen when each of the 12 conserved Peroxins are knocked down in the Drosophila fat body with Peroxin14 knockdown having the largest effect. When Drosophila S2 cells were cultured in excess oleic acid, Peroxin3, Peroxin13, and Peroxin14, but not other Peroxins were localized to lipid droplets independently of other peroxisome markers. The presence of Peroxin14 at the lipid droplet surface altered recruitment of perilipin and lipase proteins.

A constellation of chromosome conformation capture methods (С-methods) are an important tool for biochemical analysis of the spatial interactions between DNA regions that are separated in the primary sequence. All these methods are based on the long sequence of basic steps of treating cells, nuclei, chromatin, and finally DNA, thus representing a significant technical challenge. Here, we present an in-depth study of the basic steps in the chromatin conformation capture procedure (3С), which was performed using Drosophila Schneider 2 cells as a model. We investigated the steps of cell lysis, nuclei washing, nucleoplasm extraction, chromatin treatment with SDS/Triton X-100, restriction enzyme digestion, chromatin ligation, reversion of cross-links, DNA extraction, treatment of a 3C library with RNases, and purification of the 3C library. Several options were studied, and optimal conditions were found. Our work contributes to the understanding of the 3C basic steps and provides a useful guide to the 3C procedure.

Stress granules are phase separated assemblies formed around mRNAs whose identities remain elusive. The techniques available to identify the RNA content of stress granules rely on their physical purification, and are therefore not suitable for single cells and tissues displaying cell heterogeneity. Here, we adapted TRIBE (Target of RNA-binding proteins Identified by Editing) to detect stress granule RNAs by fusing a stress granule RNA-binding protein (FMR1) to the catalytic domain of an RNA-editing enzyme (ADAR). RNAs colocalized with this fusion are edited, producing mutations that are detectable by sequencing. We first optimized the expression of this fusion protein so that RNA editing preferentially occurs in stress granules. We then show that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells, and in Drosophila tissues, such as 398 neuronal stress granule mRNAs encoding ATP binding, cell cycle and transcription factors. This new method opens the possibility to identify the RNA content of stress granules as well other RNA based assemblies in single cells derived from tissues.

Previous work with the classic T4 endonuclease V digestion of DNA from irradiated Drosophila cells followed by Southern hybridization led to the conclusion that Drosophila lacks transcription-coupled repair (TCR). This conclusion was reinforced by the Drosophila Genome Project, which revealed that Drosophila lacks Cockayne syndrome WD repeat protein (CSA), CSB, or UV-stimulated scaffold protein A (UVSSA) homologs, whose orthologs are present in eukaryotes ranging from Arabidopsis to humans that carry out TCR. A recently developed in vivo excision assay and the excision repair-sequencing (XR-Seq) method have enabled genome-wide analysis of nucleotide excision repair in various organisms at single-nucleotide resolution and in a strand-specific manner. Using these methods, we have discovered that Drosophila S2 cells carry out robust TCR comparable with that observed in mammalian cells. Our findings provide critical new insights into the mechanisms of TCR among various different species.

The anoxia-tolerant fruit fly, Drosophila melanogaster, has routinely been used to examine cellular mechanisms responsible for anoxic and oxidative stress resistance. Nitric oxide (NO), an important cellular signaling molecule, and its downstream activation of cGMP-dependent protein kinase G (PKG) has been implicated as a protective mechanism against ischemic injury in diverse animal models from insects to mammals. In Drosophila, increased PKG signaling results in increased survival of animals exposed to anoxic stress. To determine if activation of the NO/cGMP/PKG pathway is protective at the cellular level, the present study employed a pharmacological protocol to mimic hypoxic injury in Drosophila S2 cells. The commonly used S2 cell line was derived from a primary culture of late stage (20–24 h old) Drosophila melanogaster embryos. Hypoxic stress was induced by exposure to either sodium azide (NaN3) or cobalt chloride (CoCl2). During chemical hypoxic stress, NO/cGMP/PKG activation protected against cell death and this mechanism involved modulation of downstream mitochondrial ATP-sensitive potassium ion channels (mitoKATP). The cellular protection afforded by NO/cGMP/PKG activation during ischemia-like stress may be an adaptive cytoprotective mechanism and modulation of this signaling cascade could serve as a potential therapeutic target for protection against hypoxia or ischemia-induced cellular injury.

The peach-potato aphid, Myzus persicae, is a serious crop pest that has developed imidacloprid resistance, mainly through overexpression of CYP6CY3. Here, we established a metabolic assay using Drosophila S2 cells that stably expressed CYP6CY3. We found that CYP6CY3 showed metabolic activity against imidacloprid, as well as acetamiprid, clothianidin, and thiacloprid, but had no activity against dinotefuran. Our study suggested that stable gene expression in Drosophila S2 cells is useful for examining which insecticide is metabolized by P450 monooxygenases.

Cell density regulates many aspects of cell properties and behaviors including metabolism, growth, cytoskeletal structure and locomotion. Importantly, the responses by cultured cells to density signals also uncover key mechanisms that govern animal development and diseases in vivo. Here we characterized a density-responsive reporter system in transgenic Drosophila S2 cells. We show that the reporter genes are strongly induced in a cell density-dependent and reporter-independent fashion. The rapid and reversible induction occurs at the level of mRNA accumulation. We show that multiple DNA elements within the transgene sequences, including a metal response element from the metallothionein gene, contribute to the reporter induction. The reporter induction correlates with changes in multiple cell density and growth regulatory pathways including hypoxia, apoptosis, cell cycle and cytoskeletal organization. Potential applications of such a density-responsive reporter will be discussed.

Transcriptional cofactors (COFs) communicate regulatory cues from enhancers to promoters and are central effectors of transcription activation and gene expression1. Although some COFs have been shown to prefer certain promoter types2–5 over others (for example, see refs 6,7), the extent to which different COFs display intrinsic specificities for distinct promoters is unclear. Here we use a high-throughput promoter-activity assay in Drosophila melanogaster S2 cells to screen 23 COFs for their ability to activate 72,000 candidate core promoters (CPs). We observe differential activation of CPs, indicating distinct regulatory preferences or ‘compatibilities’8,9 between COFs and specific types of CPs. These functionally distinct CP types are differentially enriched for known sequence elements2,4, such as the TATA box, downstream promoter element (DPE) or TCT motif, and display distinct chromatin properties at endogenous loci. Notably, the CP types differ in their relative abundance of H3K4me3 and H3K4me1 marks (see also refs 10–12), suggesting that these histone modifications might distinguish trans-regulatory factors rather than promoter- versus enhancer-type cis-regulatory elements. We confirm the existence of distinct COF–CP compatibilities in two additional Drosophila cell lines and in human cells, for which we find COFs that prefer TATA-box or CpG-island promoters, respectively. Distinct compatibilities between COFs and promoters can explain how different enhancers specifically activate distinct sets of genes9, alternative promoters within the same genes, and distinct transcription start sites within the same promoter13. Thus, COF–promoter compatibilities may underlie distinct transcriptional programs in species as divergent as flies and humans. A screen of 23 transcriptional cofactors for their ability to activate 72,000 candidate core promoters in Drosophila melanogaster identified distinct compatibility groups, providing insight into mechanisms that underlie the selective activation of transcriptional programs.

Transcription factor E93 is a steroid hormone ecdysone early response gene and plays crucial roles in both the degradation of larval tissues and the formation of adult organs during insect metamorphosis with the prepupal-pupal-adult transition. However, the molecular mechanism underlying E93 regulation is poorly understood. In this study, we found that specific knockdown of the E93 gene in the Drosophila wing disrupted wing development. Analyzing ChIP-seq signals for E93 in Drosophila wing identified that the decapentaplegic (Dpp) gene was a potential downstream target of E93. ChIP-PCR analysis and dual-luciferase reporter assay confirmed that E93 could bind to the Dpp promoter and enhanced its activity. Furthermore, the expressions of Dpp and other components in the Dpp signaling pathway were upregulated following E93 overexpression in Drosophila S2 cells but were decreased after E93 knockdown in the wing. Moreover, the impairment of the Dpp signaling pathway phenocopied the defects of E93 knockdown on wing development. Taken together, our results suggest that E93 modulates the Dpp signaling pathway to regulate wing development during Drosophila metamorphosis.

The ribonucleoprotein (RNP) spliceosome machinery triggers the precursor RNA splicing process in eukaryotes. Major spliceosome defects are implicated in male infertility; however, the underlying mechanistic links between the spliceosome and the ribosome in Drosophila testes remains largely unresolved. Small ribonucleoprotein particle protein SmD3 (SmD3) is a novel germline stem cell (GSC) regulatory gene identified in our previous screen of Drosophila testes. In the present study, using genetic manipulation in a Drosophila model, we demonstrated that SmD3 is required for the GSC niche and controls the self-renewal and differentiation of GSCs in the testis. Using in vitro assays in Schneider 2 cells, we showed that SmD3 also regulates the homeostasis of proliferation and apoptosis in Drosophila. Furthermore, using liquid chromatography-tandem mass spectrometry methods, SmD3 was identified as binding with ribosomal protein (Rp)L18, which is a key regulator of the large subunit in the ribosome. Moreover, SmD3 was observed to regulate spliceosome and ribosome subunit expression levels and controlled spliceosome and ribosome function via RpL18. Significantly, our findings revealed the genetic causes and molecular mechanisms underlying the stem cell niche and the crosstalk between the spliceosome and the ribosome.-Yu, J., Luan, X., Yan, Y., Qiao, C., Liu, Y., Zhao, D., Xie, B., Zheng, Q., Wang, M., Chen, W., Shen, C., He, Z., Hu, X., Huang, X., Li, H., Chen, B., Zheng, B., Chen, X., Fang, J. Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila.

How the nuclear lamina (NL) impacts on global chromatin architecture is poorly understood. Here, we show that NL disruption in Drosophila S2 cells leads to chromatin compaction and repositioning from the nuclear envelope. This increases the chromatin density in a fraction of topologically-associating domains (TADs) enriched in active chromatin and enhances interactions between active and inactive chromatin. Importantly, upon NL disruption the NL-associated TADs become more acetylated at histone H3 and less compact, while background transcription is derepressed. Two-colour FISH confirms that a TAD becomes less compact following its release from the NL. Finally, polymer simulations show that chromatin binding to the NL can per se compact attached TADs. Collectively, our findings demonstrate a dual function of the NL in shaping the 3D genome. Attachment of TADs to the NL makes them more condensed but decreases the overall chromatin density in the nucleus by stretching interphase chromosomes., The role of the nuclear lamina (NL) in chromatin architecture is still poorly understood. Here, the authors provide evidence that disruption of the NL in Drosophila cells leads to overall chromatin compaction and repositioning from the nuclear envelope, whereas lamina-associated regions become less compacted and transcription within them is increased.

Kinesin-8 motor proteins control chromosome alignment in a variety of species, but the specific biochemical activity responsible is unclear. Edzuka and Goshima find that Drosophila kinesin-8 (Klp67A) exhibits both microtubule plus end–stabilizing and –destabilizing activities in vitro. In cells, Klp67A, and likely human kinesin-8 (KIF18A) as well, stabilize the kinetochore–microtubule attachment during mitosis., Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, it is unclear how this motor protein family controls chromosome alignment, as multiple biochemical activities, including inconsistent ones between studies, have been identified. Here, we find that Drosophila kinesin-8 (Klp67A) possesses both microtubule (MT) plus end–stabilizing and –destabilizing activity, in addition to kinesin-8's commonly observed MT plus end–directed motility and tubulin-binding activity in vitro. We further show that Klp67A is required for stable kinetochore–MT attachment during prometaphase in S2 cells. In the absence of Klp67A, abnormally long MTs interact in an “end-on” fashion with kinetochores at normal frequency. However, the interaction is unstable, and MTs frequently become detached. This phenotype is rescued by ectopic expression of the MT plus end–stabilizing factor CLASP, but not by artificial shortening of MTs. We show that human kinesin-8 (KIF18A) is also important to ensure proper MT attachment. Overall, these results suggest that the MT-stabilizing activity of kinesin-8 is critical for stable kinetochore–MT attachment.

It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-RTEs are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) derived from exogenous retroviruses that integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. Indeed, there is some evidence that gypsy may exhibit retroviral properties. Given that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration, this raises the question of whether gypsy replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome., Author summary The genomes of animals and plants contain a vast quantity of so called “junk DNA” that does not provide obvious function to the organism. But it is increasingly clear that “junk DNA” has more important contributions to both normal function and to dysfunction that can cause disease. A great deal of this “junk” is made up by so called “retrotransposons”, which have many similarities to viruses. Many of them are actually evolutionary relatives of retroviruses, and are able to replicate themselves and insert new copies into the host genome. But unlike retroviruses, which are infectious and replicate by moving from one cell to another, retrotransposons replicate within one cell and re-insert their new copies back into the chromosomes of the cell in which they originated. In this publication, we studied replication of gypsy, which is a well known retrotransposon in fruit flies. We found that gypsy has the ability to replicate within a cell, like a retrotransposon and also has the ability to replicate by moving to a new cell like a retrovirus.

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism.Drosophila melanogastergenome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show thatDrosophilaeIF4E-1 and eIF4E-3 occur in PBs where might play a role in mRNA storage and translational repression. We also demonstrate that the DEAD-box RNA helicase Me31B, a component of PBs, physically interacts with eIF4E-1 and eIF4E-3 both in the yeast two-hybrid system and FRET inDrosophilaS2 cells.Moreover, truncated and point mutated Me31B proteins indicate that the binding sites of Me31B for eIF4E-1 and eIF4E-3 are located in different domains. Residues Y401-L407 (at the carboxy-terminal) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminal) are critical for interaction with eIF4E-3. Thus, Me31B represents a novel type of eIF4E-interacting protein. Our observations suggest that Me31B might recognize different eIF4E isoforms in different tissues, which could be the key to silencing specific messengers. They provide further evidence that alternative forms of eIF4E and their interactions with various partners add complexity to the control of gene expression in eukaryotes.

Germline stem cells (GSCs) are the progenitor cells of the germline for the lifetime of an animal. InDrosophila, these cells reside in a cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem cell-daughter cell transition is tightly regulated by a number of processes, including an array of proteins required for genome stability. The germline stem-cell maintenance factor Stonewall (Stwl) associates with heterochromatin, but its molecular function is poorly understood. We performed RNA-Seq onstwlmutant ovaries and found significant derepression of many transposon families but not heterochromatic genes. We also discovered that testis-enriched genes, including the differentiation factorbgcnand a large testis-specific cluster on chromosome 2, are upregulated or ectopically expressed instwlmutant ovaries. Surprisingly, we also found that RNAi knockdown ofstwlin somatic S2 cells results in ectopic expression of these genes.Using parallel ChIP-Seq and RNA-Seq experiments in S2 cells, we discovered that Stwl binds upstream of transcription start sites and localizes to heterochromatic sequences. We also find that Stwl is enriched at repetitive sequences associated with telomeres. Finally, we identify Stwl binding motifs that are shared with known insulator binding proteins. We propose that Stwl affects gene regulation by binding insulators and establishing chromatin boundaries.

Interleukins (ILs) are cytokines with crucial functions in innate and adaptive immunity. IL genes are only found in vertebrates, except for IL-16, which has been cloned in some arthropod species. However, the function of this gene in invertebrates is unknown. In the present study, an IL-16–like gene (EsIL-16) was identified from the Chinese mitten crab Eriocheir sinensis. EsIL-16 was predicted to encode a precursor (proEsIL-16) that shares similarities with pro-IL-16 proteins from insects and vertebrates. We show that caspase-3 processes proEsIL-16 into an approximately 144-kDa N-terminal prodomain with nuclear import activity and an approximately 34-kDa mature peptide that might be secreted into the extracellular region. EsIL-16 mRNA could be detected in all analyzed tissues and was significantly upregulated after immune challenge both in vitro and in vivo. T7 phage display library screening suggested potential binding activity between EsIL-16 and integrin, which was confirmed by coimmunoprecipitation assay. Interestingly, EsIL-16 promoted cell proliferation via integrin β1 in primary cultured crab hemocytes and Drosophila S2 cells. Furthermore, the interaction between EsIL-16 and integrin β1 was necessary to efficiently protect the host from bacterial infection. To our knowledge, this study revealed integrin β1 as a receptor for IL-16 and the function of this interaction in hemocyte proliferation in invertebrates for the first time. These results provide new insights into the regulation of innate immune responses in invertebrates and shed the light on the evolution of ILs within the animal kingdom.

Ataxin-2 is a conserved translational control protein associated with spinocerebellar ataxia type II (SCA2) and amyotrophic lateral sclerosis (ALS) as well as an important target for ALS therapeutics under development. Despite its clinical and biological significance, Ataxin-2’s activities, mechanisms and functions are not well understood. While Drosophila Ataxin-2 (Atx2) mediates mRNP condensation via a C-terminal intrinsically disordered domain (cIDR), how Ataxin-2 IDRs work with structured (Lsm, Lsm-AD and PAM2) domains to enable positive and negative regulation of target mRNAs remains unclear. Using TRIBE (Targets of RNA-Binding Proteins Identified by Editing) technology, we identified and analysed Atx-2 target mRNAs in the Drosophila brain. We show that Atx2 preferentially interacts with AU-rich elements (AREs) in 3’UTRs and plays a broad role in stabilization of identified target mRNAs. Strikingly, Atx2 interaction with its targets is dependent on the cIDR domain required for neuronal-granule formation. In contrast, Atx2 lacking its Lsm domain not only interacts more efficiently with the target mRNA identified, but also forms larger RNP granules. Providing an extensive dataset of Atx2-interacting brain mRNAs, our results demonstrate that Atx2: (a) interacts with target mRNAs within RNP granules; (b) modulates the turnover of these target mRNAs; (c) has an additional essential role outside of mRNP granules; and (d) contains distinct protein domains that drive or oppose RNP-granule assembly. These findings increase understanding of neuronal translational control mechanisms and inform Ataxin-2-based interventions in development for SCA2 and ALS.

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the ER exit sites under the stress of amino-acid starvation. Here we address which signaling pathways cause Sec body formation. We find that two pathways are critical. The first is a SIK dependent pathway induced by salt (NaCl) stress in a necessary and sufficient manner. The second is the activation of IRE1 (one of the key kinases mediating the Unfolded Protein Response) by absence of amino- acids, which partly leads to the depletion of intracellular ATP. However, IRE1 activation is not sufficient to induce Sec body formation and needs to be combined to salt stress. This works pioneers the role of SIK in phase transition and re-enforces the role of IRE1 as a metabolic sensor for the level of circulating amino-acids.

RNA interference (RNAi) is a posttranscriptional gene silencing method that is triggered by double-stranded RNA (dsRNA). RNAi is used to inactivate genes of interest and provides a genetic tool for loss-of-function studies in a variety of organisms.I have used this method to reveal the physiological roles of a number of endogenous proteins involved in mitochondrial DNA metabolism in Schneider cells, including the mitochondrial single-stranded DNA-binding protein. Here, I present experimental schemes of selective suppression of endogenous gene expression using RNAi in Drosophila Schneider S2 cells. With this method, the function of exogenous wild-type or mutant genes can be evaluated.

Several plant lectins, or carbohydrate-binding proteins, interact with glycan moieties on the surface of immune cells, thereby influencing the immune response of these cells. Orysata, a mannose-binding lectin from rice, has been reported to exert immunomodulatory activities on insect cells. While the natural lectin is non-glycosylated, recombinant Orysata produced in the yeast Pichia pastoris (YOry) is modified with a hyper-mannosylated N-glycan. Since it is unclear whether this glycosylation can affect the YOry activity, non-glycosylated rOrysata was produced in Escherichia coli (BOry). In a comparative analysis, both recombinant Orysata proteins were tested for their carbohydrate specificity on a glycan array, followed by the investigation of the carbohydrate-dependent agglutination of red blood cells (RBCs) and the carbohydrate-independent immune responses in Drosophila melanogaster S2 cells. Although YOry and BOry showed a similar carbohydrate-binding profiles, lower concentration of BOry were sufficient for the agglutination of RBCs and BOry induced stronger immune responses in S2 cells. The data are discussed in relation to different hypotheses explaining the weaker responses of glycosylated YOry. In conclusion, these observations contribute to the understanding how post-translational modification can affect protein function, and provide guidance in the selection of the proper expression system for the recombinant production of lectins.

Background/aim The DSL proteins, Serrate and Delta, which act as Notch receptor ligands, mediate signalling between adjacent cells, when a ligand-expressing cell binds to Notch on an adjacent receiving cell. Notch is ubiquitously expressed and DSL protein mis-expression can have devastating developmental consequences. Although transcriptional regulation of Delta and Serrate has been amply documented, we examined whether they are also regulated at the level of translation. Materials and methods We generated a series of deletions to investigate the initiation codon usage for Serrate using Drosophila S2 cells. Results Serrate mRNA contains three putative ATG initiation codons spanning a 60-codon region upstream of its signal peptide; we found that each one can act as an initiation codon, however, with a different translational efficiency. Conclusion Serrate expression is strictly regulated at the translational level.

We analyzed the whole-genome experimental maps of nucleosomes in Drosophila melanogaster and classified genes by the expression level in S2 cells (RPKM value, reads per kilobase million) as well as the number of tissues in which a gene was expressed (breadth of expression, BoE). Chromatin in 5&prime, regions of genes we classified on four states according to the hidden Markov model (4HMM). Only the Aquamarine chromatin state we considered as Active, while the rest three states we defined as Non-Active. Surprisingly, about 20/40% of genes with 5&prime, regions mapped to Active/Non-Active chromatin possessed the minimal/at least modest RPKM and BoE. We found that regardless of RPKM/BoE the genes of Active chromatin possessed the regular nucleosome arrangement in 5&prime, regions, while genes of Non-Active chromatin did not show respective specificity. Only for genes of Active chromatin the RPKM/BoE positively correlates with the number of nucleosome sites upstream/around TSS and negatively with that downstream TSS. We propose that for genes of Active chromatin, regardless of RPKM value and BoE the nucleosome arrangement in 5&prime, regions potentiates transcription, while for genes of Non-Active chromatin, the transcription machinery does not require the substantial support from nucleosome arrangement to influence gene expression.

Background Although recent studies have revealed the genome-wide distribution of R-loops, our understanding of R-loop formation is still limited. Genomes are known to have a large number of repetitive elements. Emerging evidence suggests that these sequences may play an important regulatory role. However, few studies have investigated the effect of repetitive elements on R-loop formation. Results We found different repetitive elements related to R-loop formation in various species. By controlling length and genomic distributions, we observed that satellite, long interspersed nuclear elements (LINEs), and DNA transposons were each specifically enriched for R-loops in humans, fruit flies, and Arabidopsis thaliana, respectively. R-loops also tended to arise in regions of low-complexity or simple repeats across species. We also found that the repetitive elements associated with R-loop formation differ according to developmental stage. For instance, LINEs and long terminal repeat retrotransposons (LTRs) are more likely to contain R-loops in embryos (fruit fly) and then turn out to be low-complexity and simple repeats in post-developmental S2 cells. Conclusions Our results indicate that repetitive elements may have species-specific or development-specific regulatory effects on R-loop formation. This work advances our understanding of repetitive elements and R-loop biology.

The homeostasis of the stem cell niche is regulated by both intrinsic and extrinsic factors, and the complex and ordered molecular and cellular regulatory mechanisms need to be further explored. In Drosophila testis, germline stem cells (GSCs) rely on hub cells for self-renewal and physical attachment. GSCs are also in contact with somatic cyst stem cells (CySCs). Utilizing genetic manipulation in Drosophila, we investigated the role of Wnt6 in vivo and in vitro. In Drosophila testis, we found that Wnt6 is required for GSC differentiation and CySC self-renewal. In Schneider 2 (S2) cells, we found that Wnt6 regulates cell proliferation and apoptosis. Mechanistically, we demonstrated that Wnt6 can downregulate the expression levels of Arm, Rac1 and Cdc42 in S2 cells. Notably, Rac1 and Cdc42, which act downstream of the noncanonical Wnt signalling pathway, imitated the phenotypes of Wnt6 in Drosophila testis. Thus, the newly discovered Wnt6-Rac1/Cdc42 signal axis is required for the homeostasis of the stem cell niche in the Drosophila testis.

Robertsonian (centric-fusion) translocation is the form of chromosomal translocation in which two long arms of acrocentric chromosomes are fused to form one metacentric. These translocations reduce the number of chromosomes while preserving existing genes and are considered to contribute to speciation. We asked whether hypomorphic mutations in genes that disrupt the formation of pericentromeric regions could lead to centric fusion. TBP-related factor 2 (Trf2) encodes an alternative general transcription factor. A decrease of TRF2 expression disrupts the structure of the pericentromeric regions and prevents their association into chromocenter. We revealed several centric fusions in two lines of Drosophila melanogaster with weak Trf2 alleles in genetic experiments. We performed an RNAi-mediated knock-down of Trf2 in Drosophila and S2 cells and demonstrated that Trf2 upregulates expression of D1&mdash, one of the major genes responsible for chromocenter formation and nuclear integrity in Drosophila. Our data, for the first time, indicate that Trf2 may be involved in transcription program responsible for structuring of pericentromeric regions and may contribute to new karyotypes formation in particular by promoting centric fusion. Insight into the molecular mechanisms of Trf2 function and its new targets in different tissues will contribute to our understanding of its phenomenon.

Cell polarity is an evolutionarily conserved process of asymmetric spatial organization within cells and is essential to tissue structure, signal transduction, cell migration, and cell division. The establishment and maintenance of polarity typically involves extensive protein-protein interactions that can be made further intricate by cell cycle-dependent regulation. These aspects can make interpreting phenotypes within traditional in vivo genetic systems challenging due to pleiotropic effects in loss-of-function experiments. Minimal reconstitution methods offer investigators the advantage of stricter control of otherwise complex systems and allow for more direct assessment of the role of individual components to the process of interest. Here I provide a detailed protocol for a cell adhesion-based method of inducing cell polarity within non-polarized Drosophila S2 cells. This technique is simple, cost effective, moderate throughput, and amenable to RNAi-based loss-of-function studies. The ability to “plug-and-play” genes of interest allows investigators to easily assess the contribution of individual protein domains and post-translational modifications to their function. The system is ideally suited to test not only the requirement of individual components but also their sufficiency, and can provide important insight into the epistatic relationship among multiple components in a protein complex. Although designed for use within Drosophila cells, the general premise and protocol should be easily adapted to mammalian cell culture or other systems that may better suit the interests of potential users.

The role of miRNAs in mediating insecticide resistance remains largely unknown, even for the model species Drosophila melanogaster. Building on prior research, this study used microinjection of synthetic miR-310s mimics into DDT-resistant 91-R flies and observed both a significant transcriptional repression of computationally-predicted endogenous target P450 detoxification genes, Cyp6g1 and Cyp6g2, and also a concomitant increase in DDT susceptibility. Additionally, co-transfection of D. melanogaster S2 cells with dual luciferase reporter constructs validated predictions that miR-310s bind to target binding sites in the 3ʹ untranslated regions (3ʹ-UTR) of both Cyp6g1 and Cyp6g2 in vitro. Findings in the current study provide empirical evidence for a link between reduced miRNA expression and an insecticidal resistance phenotype through reduced targeted post-transcriptional suppression of transcripts encoding proteins involved in xenobiotic detoxification. These insights are important for understanding the breadth of adaptive molecular changes that have contributed to the evolution of DDT resistance in D. melanogaster.

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

Research using the fruit fly Drosophila melanogaster has traditionally focused on understanding how mutations affecting gene regulation or function affect processes linked to animal development. Accordingly, flies have become an essential foundation of modern medical research through repeated contributions to our fundamental understanding of how their homologs of human genes function. Peroxisomes are organelles that metabolize lipids and reactive oxygen species like peroxides. However, despite clear linkage of mutations in human genes affecting peroxisomes to developmental defects, for many years fly models were conspicuously absent from the study of peroxisomes. Now, the few early studies linking the Rosy eye color phenotype to peroxisomes in flies have been joined by a growing body of research establishing novel roles for peroxisomes during the development or function of specific tissues or cell types. Similarly, unique properties of cultured fly Schneider 2 cells have advanced our understanding of how peroxisomes move on the cytoskeleton. Here, we profile how those past and more recent Drosophila studies started to link specific effects of peroxisome dysfunction to organ development and highlight the utility of flies as a model for human peroxisomal diseases. We also identify key differences in the function and proliferation of fly peroxisomes compared to yeast or mammals. Finally, we discuss the future of the fly model system for peroxisome research including new techniques that should support identification of additional tissue specific regulation of and roles for peroxisomes.

Objectives Stem cell niche regulated the renewal and differentiation of germline stem cells (GSCs) in Drosophila. Previously, we and others identified a series of genes encoding ribosomal proteins that may contribute to the self‐renewal and differentiation of GSCs. However, the mechanisms that maintain and differentiate GSCs in their niches were not well understood. Materials and Methods Flies were used to generate tissue‐specific gene knockdown. Small interfering RNAs were used to knockdown genes in S2 cells. qRT‐PCR was used to examine the relative mRNA expression level. TUNEL staining or flow cytometry assays were used to detect cell survival. Immunofluorescence was used to determine protein localization and expression pattern. Results Herein, using a genetic manipulation approach, we investigated the role of ribosomal protein S13 (RpS13) in testes and S2 cells. We reported that RpS13 was required for the self‐renewal and differentiation of GSCs. We also demonstrated that RpS13 regulated cell proliferation and apoptosis. Mechanistically, we showed that RpS13 regulated the expression of ribosome subunits and could moderate the expression of the Rho1, DE‐cad and Arm proteins. Notably, Rho1 imitated the phenotype of RpS13 in both Drosophila testes and S2 cells, and recruited cell adhesions, which was mediated by the DE‐cad and Arm proteins. Conclusion These findings uncover a novel mechanism of RpS13 that mediates Rho1 signals in the stem cell niche of the Drosophila testis., Objectives: Stem cell niche regulated the renewal and differentiation of germline stem cells (GSCs) in Drosophila. Previously, we and others identified a series of genes encoding ribosomal proteins that may contribute to the self‐renewal and differentiation of GSCs. However, the mechanisms that maintain and differentiate GSCs in their niches were not well understood.Materials and Methods: Flies were used to generate tissue‐specific gene knockdown. Small interfering RNAs were used to knockdown genes in S2 cells. qRT‐PCR was used to examine the relative mRNA expression level. TUNEL staining or flow cytometry assays were used to detect cell survival. Immunofluorescence was used to determine protein localization and expression pattern.Results: Herein, using a genetic manipulation approach, we investigated the role of ribosomal protein S13 (RpS13) in testes and S2 cells. We reported that RpS13 was required for the self‐renewal and differentiation of GSCs. We also demonstrated that RpS13 regulated cell proliferation and apoptosis. Mechanistically, we showed that RpS13 regulated the expression of ribosome subunits and could moderate the expression of the Rho1, DE‐cad and Arm proteins. Notably, Rho1 imitated the phenotype of RpS13 in both Drosophila testes and S2 cells, and recruited cell adhesions, which was mediated by the DE‐cad and Arm proteins.Conclusions: These findings uncover a novel mechanism of RpS13 that mediates Rho1 signals in the stem cell niche of the Drosophila testis.