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Friday, July 29th, 2022 - Immune Response

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Hegde, S., Sreejan, A., Gadgil, C. J. and Ratnaparkhi, G. S. (2022). SUMOylation of Dorsal attenuates Toll/NF-kappaB signaling.. Genetics 221(3). PubMed ID: 35567478
Summary:
In Drosophila, Toll/NF-kappaB signaling plays key roles in both animal development and in host defense. The activation, intensity, and kinetics of Toll signaling are regulated by posttranslational modifications such as phosphorylation, SUMOylation, or ubiquitination that target multiple proteins in the Toll/NF-kappaB cascade. This study has generated a CRISPR-Cas9 edited Dorsal (DL) variant that is SUMO conjugation resistant. Intriguingly, embryos laid by dlSCR mothers overcome dl haploinsufficiency and complete the developmental program. This ability appears to be a result of higher transcriptional activation by DLSCR. In contrast, SUMOylation dampens DL transcriptional activation, ultimately conferring robustness to the dorso-ventral program. In the larval immune response, dlSCR animals show an increase in crystal cell numbers, stronger activation of humoral defense genes, and high cactus levels. A mathematical model that evaluates the contribution of the small fraction of SUMOylated DL (1-5%) suggests that it acts to block transcriptional activation, which is driven primarily by DL that is not SUMO conjugated. These findings define SUMO conjugation as an important regulator of the Toll signaling cascade, in both development and host defense. These results broadly suggest that SUMO attenuates DL at the level of transcriptional activation. Furthermore, it is hypothesized that SUMO conjugation of DL may be part of a Ubc9-dependent mechanism that restrains Toll/NF-kappaB signaling.
Deshpande, R., Lee, B. and Grewal, S. S. (2022). Enteric bacterial infection in Drosophila induces whole-body alterations in metabolic gene expression independently of the Immune Deficiency (Imd) signalling pathway. G3 (Bethesda). PubMed ID: 35781508
Summary:
When infected by intestinal pathogenic bacteria, animals initiate both local and systemic defence responses. These responses are required to reduce pathogen burden and also to alter host physiology and behaviour to promote infection tolerance, and they are often mediated through alterations in host gene expression. This study has used transcriptome profiling to examine gene expression changes induced by enteric infection with the gram-negative bacteria Pseudomonas entomophila (P.e) in adult female Drosophila. Infection was found to induce a strong upregulation of metabolic gene expression, including gut and fat body-enriched genes involved in lipid transport, lipolysis, and beta-oxidation, as well as glucose and amino acid metabolism genes. Furthermore, the classic innate immune deficiency (Imd)/Relish/NF-KappaB pathway was found not to be required for, and in some cases limits, these infection-mediated increases in metabolic gene expression. Interic infection with P.e. down regulates the expression of many transcription factors and cell-cell signaling molecules, particularly those previously shown to be involved in gut-to-brain and neuronal signaling. Moreover, as with the metabolic genes, these changes occurred largely independent of the Imd pathway. Together, this study identifies many metabolic, signaling and transcription factor gene expression changes that may contribute to organismal physiological and behavioural responses to enteric pathogen infection.
Hanson, M. A., Kondo, S. and Lemaitre, B. (2022). Drosophila immunity: the Drosocin gene encodes two host defence peptides with pathogen-specific roles. Proc Biol Sci 289(1977): 20220773. PubMed ID: 35730150
Summary:
iAntimicrobial peptides (AMPs) are key to defence against infection in plants and animals. Use of AMP mutations in Drosophila has now revealed that AMPs can additively or synergistically contribute to defence in vivo. However, these studies also revealed high specificity, wherein just one AMP contributes an outsized role in combatting a specific pathogen. This study shows the Drosocin locus (CG10816) is more complex than previously described. In addition to its namesake peptide 'Drosocin', it encodes a second mature peptide from a precursor via furin cleavage. This peptide corresponds to the previously uncharacterized 'Immune-induced Molecule 7'. A polymorphism (Thr52Ala) in the Drosocin precursor protein previously masked the identification of this peptide, which this paper names 'Buletin'. Using mutations differently affecting Drosocin and Buletin, this studybshow that only Drosocin contributes to Drosocin gene-mediated defence against Enterobacter cloacae. Strikingly, it was observed that Buletin, but not Drosocin, contributes to the Drosocin gene-mediated defence against Providencia burhodogranariea, including an importance of the Thr52Ala polymorphism for survival. This study reveals that the Drosocin gene encodes two prominent host defence peptides with different specificity against distinct pathogens. This finding emphasizes the complexity of the Drosophila humoral response and demonstrates how natural polymorphisms can affect host susceptibility.
Yoo, T. J., Shim, M. S., Bang, J., Kim, J. H. and Lee, B. J. (2022). SPS1 deficiency-triggered PGRP-LC and Toll expression controls innate immunity in Drosophila S2 cells. Biol Open. PubMed ID: 35723425
Summary:
Selenophosphate synthetase 1 (SPS1) is an essential gene for the cell growth and embryogenesis in Drosophila melanogaster. A previous study reported that SPS1 deficiency stimulates the expression of genes responsible for the innate immune system, including antimicrobial peptides (AMPs), in Drosophila S2 cells. However, the underlying mechanism has not been elucidated. This study investigated the immune pathways that control the SPS1-deficiency-induced expression of AMPs in S2 cells. It was found that the activation of AMP expression is regulated by both immune deficiency (IMD) and the Toll pathway. Double knockdown of the upstream genes of each pathway with SPS1 showed that the peptidoglycan recognition protein-LC (PGRP-LC) and Toll genes are targeted by SPS1 for regulating these pathways, respectively. It was also found that the IMD and Toll pathway regulate AMP expression by cross-talking. The levels of PGRP-LC and Toll mRNAs were upregulated upon Sps1 knockdown (6.46±0.36 and 3.2±0.45-fold, respectively n=3). Overexpression of each protein also upregulated AMPs. Interestingly, PGRP-LC overexpression upregulated AMP more than Toll overexpression. These data strongly suggest that SPS1 controls the innate immune system of D. melanogaster through regulating PGRP-LC and Toll expression.
Segrist, E., Dittmar, M., Gold, B. and Cherry, S. (2021). Orally acquired cyclic dinucleotides drive dSTING-dependent antiviral immunity in enterocytes. Cell Rep 37(13): 110150. PubMed ID: 34965418
Summary:
Enteric pathogens overcome barrier immunity within the intestinal environment that includes the endogenous flora. The microbiota produces diverse ligands, and the full spectrum of microbial products that are sensed by the epithelium and prime protective immunity is unknown. Using Drosophila, this study found that the gut presents a high barrier to infection, which is partially due to signals from the microbiota, as loss of the microbiota enhances oral viral infection. Cclic dinucleotide (CDN) feeding is sufficient to protect microbiota-deficient flies from enhanced oral infection, suggesting that bacterial-derived CDNs induce immunity. Mechanistically, this study found CDN protection is dSTING- and dTBK1-dependent, leading to NF-kappaB-dependent gene expression. Furthermore, this study identified the apical nucleoside transporter, CNT2, as required for oral CDN protection. Altogether, these studies define a role for bacterial products in priming immune defenses in the gut.
Chifiriuc, M. C., Bologa, A. M., Ratiu, A. C., Ionascu, A. and Ecovoiu, A. A. (2022). Mutations of gammaCOP Gene Disturb Drosophila melanogaster Innate Immune Response to Pseudomonas aeruginosa. Int J Mol Sci 23(12). PubMed ID: 35742941
Summary:
Drosophila melanogaster (the fruit fly) is a valuable experimental platform for modeling host-pathogen interactions. It is also commonly used to define innate immunity pathways and to understand the mechanisms of both host tolerance to commensal microbiota and response to pathogenic agents. This study investigated how the host response to bacterial infection is mirrored in the expression of genes of Imd and Toll pathways when D. melanogaster strains with different γCOP genetic backgrounds are infected with Pseudomonas aeruginosa ATCC 27853. Using microarray technology, this study interrogated the whole-body transcriptome of infected versus uninfected fruit fly males with three specific genotypes, namely wild-type Oregon, γCOP(S057302)/TM6B and γCOP(14a)/γCOP(14a). While the expression of genes pertaining to Imd and Toll is not significantly modulated by P. aeruginosa infection in Oregon males, many of the components of these cascades are up- or downregulated in both infected and uninfected γCOP(S057302)/TM6B and γCOP(14a)/γCOP(14a) males. Thus, these results suggest that a γCOP genetic background modulates the gene expression profiles of Imd and Toll cascades involved in the innate immune response of D. melanogaster, inducing the occurrence of immunological dysfunctions in γCOP mutants.

Thursday July 28th - Larval and adult neural development and function

Varte, V., Kairamkonda, S., Gupta, U., Manjila, S. B., Mishra, A., Salzberg, A. and Nongthomba, U. (2022). Neuronal role of taxi is imperative for flight in Drosophila melanogaster. Gene 833: 146593. PubMed ID: 35597528
Summary:
Extensive studies in Drosophila have led to the elucidation of the roles of many molecular players involved in the sensorimotor coordination of flight. However, the identification and characterisation of new players can add novel perspectives to the process. This paper shows that the extant mutant, jumper, is a hypermorphic allele of the taxi/delilah gene, which encodes a transcription factor. The defective flight of jumper flies results from the insertion of an I-element in the 5'-UTR of taxi gene, leading to an over-expression of the taxi. The molecular lesion responsible for the taxi1 allele results from a 25 bp deletion leading to a shift in the reading frame at the C-terminus of the taxi coding sequence. Thus, the last 20 residues are replaced by 32 disparate residues in taxi1. Both taxi1, a hypomorphic allele, and the CRISPR-Cas9 knock-out (taxiKO) null allele, show a defective flight phenotype. Electrophysiological studies show taxi hypermorphs, hypomorphs, and knock out flies show abnormal neuronal firing. Neuronal-specific knock-down or over-expression of taxi cause a defect in the brain's inputs to the flight muscles, leading to reduced flight ability. Through transcriptomic analysis of the taxiKO fly head, this study identified several putative targets of Taxi that may play important roles in flight. In conclusion, from molecularly characterising jumper to establishing Taxi's role during Drosophila flight, this work shows that the forward genetics approach still can lead to the identification of novel molecular players required for neuronal transmission.
Chen, W., Gu, X., Yang, Y. T., Batterham, P. and Perry, T. (2022). Dual nicotinic acetylcholine receptor subunit gene knockouts reveal limits to functional redundancy. Pestic Biochem Physiol 184: 105118. PubMed ID: 35715057
Summary:
The nicotinic acetylcholine receptor (nAChR) subunit gene family consists of ten members in Drosophila melanogaster. The mature nAChR is a pentamer assembled from these subunits. Despite recent advances in the in vitro expression of some receptor subunit combinations (nAChR subtypes), the in vivo combinations and stoichiometry of these subtypes remains poorly defined. In addition, there are many potential nAChR signalling roles for different subtypes in insect behaviour, development and physiology. Prior work has shown that nAChR subunit mutants can display altered sleep and mating behaviour, disrupted hormone signalling and reduced locomotion, climbing ability and longevity. Teasing out the specific receptor subunits that are involved in these different functions is potentially made more difficult given that the structural similarity between members of gene families often means that there is a degree of functional redundancy. In order to circumvent this, this study created a dual knockout strain for the D&alpha1 and Dβ2 nAChR subunit genes and examined four traits including insecticide resistance. These subunits had been previously implicated in the response to a neonicotinoid insecticide, imidacloprid. The use of the dual knockout revealed that Dα1 and Dβ2 subunits are involved in signalling that leads to the inflation of wings following adult emergence from the pupal case. The Dβ1 subunit had previously been implicated as a contributor to this function. The lack of a phenotype or low penetrance of the phenotype in the Dα1 and Dβ2 single mutants compared to the dual knockout suggests that these subunits are, to some extent, functionally redundant. Stronger reductions in climbing ability and longevity in the dual knockout. These findings demonstrate that a dual knockout approach to examining members of the nAChR subunit gene family may increase the power of genetic approaches linking individual subunits and combinations thereof to particular biological functions. This approach will be valuable as the nAChRs are so widely expressed in the insect brain that they are likely to have many functions that hereto remain undetected.
Vafidis, P., Owald, D., D'Albis, T. and Kempter, R. (2022). Learning accurate path integration in ring attractor models of the head direction system. Elife 11. PubMed ID: 35723252
Summary:
Ring attractor models for angular path integration have received strong experimental support. To function as integrators, head direction circuits require precisely tuned connectivity, but it is currently unknown how such tuning could be achieved. This study proposes a network model in which a local, biologically plausible learning rule adjusts synaptic efficacies during development, guided by supervisory allothetic cues. Applied to the Drosophila head direction system, the model learns to path-integrate accurately and develops a connectivity strikingly similar to the one reported in experiments. The mature network is a quasi-continuous attractor and reproduces key experiments in which optogenetic stimulation controls the internal representation of heading, and where the network remaps to integrate with different gains in rodents. This model predicts that path integration requires self-supervised learning during a developmental phase, and proposes a general framework to learn to path-integrate with gain-1 even in architectures that lack the physical topography of a ring.
Xiao, S., Baik, L. S., Shang, X. and Carlson, J. R. (2022). Meeting a threat of the Anthropocene: Taste avoidance of metal ions by Drosophila. Proc Natl Acad Sci U S A 119(25): e2204238119. PubMed ID: 35700364
Summary:
The Anthropocene Epoch poses a critical challenge for organisms: they must cope with new threats at a rapid rate. These threats include toxic chemical compounds released into the environment by human activities. This study examined elevated concentrations of heavy metal ions as an example of anthropogenic stressors. The fruit fly Drosophila avoids nine metal ions when present at elevated concentrations that the flies experienced rarely, if ever, until the Anthropocene. This study characterized the avoidance of feeding and egg laying on metal ions, and this study identified receptors, neurons, and taste organs that contribute to this avoidance. Different subsets of taste receptors, including members of both Ir (Ionotropic receptor) and Gr (Gustatory receptor) families contribute to the avoidance of different metal ions. Metal ions activate certain bitter-sensing neurons and inhibit sugar-sensing neurons. Some behavioral responses are mediated largely through neurons of the pharynx. Feeding avoidance remains stable over 10 generations of exposure to copper and zinc ions. Some responses to metal ions are conserved across diverse dipteran species, including the mosquito Aedes albopictus. These results suggest mechanisms that may be essential to insects as they face challenges from environmental changes in the Anthropocene.
Bourouliti, A. and Skoulakis, E. M. C. (2022). Cold Shock Disrupts Massed Training-Elicited Memory in Drosophila. Int J Mol Sci 23(12). PubMed ID: 35742852
Summary:
Memory consolidation is a time-dependent process occurring over hours, days, or longer in different species and requires protein synthesis. An apparent exception is a memory type in Drosophila elicited by a single olfactory conditioning episode, which ostensibly consolidates quickly, rendering it resistant to disruption by cold anesthesia a few hours post-training. This anesthesia-resistant memory (ARM), is independent of protein synthesis. Protein synthesis independent memory can also be elicited in Drosophila by multiple massed cycles of olfactory conditioning, and this led to the prevailing notion that both of these operationally distinct training regimes yield ARM. Significantly, this study shows that, unlike bona fide ARM, massed conditioning-elicited memory remains sensitive to the amnestic treatment two hours post-training and hence it is not ARM. Therefore, there are two protein synthesis-independent memory types in Drosophila.
Chen, J., Zhu, H., Wang, R., Su, X., Ruan, Z., Pan, Y. and Peng, Q. (2022). Functional Dissection of Protein Kinases in Sexual Development and Female Receptivity of Drosophila. Front Cell Dev Biol 10: 923171. PubMed ID: 35757001
Summary:
Protein phosphorylation is crucial for a variety of biological functions, but how it is involved in sexual development and behavior is rarely known. This study= performed a screen of RNA interference targeting 177 protein kinases in Drosophila and identified 13 kinases involved in sexual development in one or both sexes. It was further identified that PKA and CASK promote female sexual behavior while not affecting female differentiation. Knocking down PKA or CASK in about five pairs of pC1 neurons in the central brain affects the fine projection but not cell number of these pC1 neurons and reduces virgin female receptivity. PKA and CASK signaling is required acutely during adulthood to promote female sexual behavior. These results reveal candidate kinases required for sexual development and behaviors and provide insights into how kinases would regulate neuronal development and physiology to fine tune the robustness of sexual behaviors.

Wednesday, July 27th - Enhancers and Transcriptonal Regulation

Bateman, J. R. and Johnson, J. E. (2022). Altering enhancer-promoter linear distance impacts promoter competition in cis and in trans. Genetics. PubMed ID: 35748724
Summary:
In Drosophila, pairing of maternal and paternal homologs can permit trans-interactions between enhancers on one homolog and promoters on another, an example of a phenomenon called transvection. When chromosomes are paired, promoters in cis and in trans to an enhancer can compete for the enhancer's activity, but the parameters that govern this competition are as yet poorly understood. To assess how the linear spacing between an enhancer and promoter can influence promoter competition in Drosophila, this study employed transgenic constructs wherein the eye-specific enhancer GMR is placed at varying distances from a heterologous hsp70 promoter driving a fluorescent reporter. While GMR activates the reporter to a high degree when the enhancer and promoter are spaced by a few hundred base pairs, activation is strongly attenuated when the enhancer is moved 3 kilobases away. By examining transcription of endogenous genes near the point of transgene insertion, this study showed that linear spacing of 3 kb between GMR and the hsp70 promoter results in elevated transcription of neighboring promoters, suggesting a loss of specificity between the enhancer and its intended transgenic target promoter. Furthermore, increasing spacing between GMR and hsp70 by just 100 bp can enhance transvection, resulting in increased activation of a promoter on a paired homolog at the expense of a promoter in cis to the enhancer. Finally, cis-/trans-promoter competition assays in which one promoter carries mutations to key core promoter elements show that GMR will skew its activity toward a wild type promoter, suggesting that an enhancer is in a balanced competition between its potential target promoters in cis and in trans.
Feng, S. and Mann, R. S. (2022). SpyChIP identifies cell type-specific transcription factor occupancy from complex tissues. Proc Natl Acad Sci U S A 119(25): e2122900119. PubMed ID: 35696584
Summary:
Chromatin immunoprecipitation (ChIP) is an important technique for characterizing protein-DNA binding in vivo. One drawback of ChIP-based techniques is the lack of cell type-specificity when profiling complex tissues. To overcome this limitation, this study developed SpyChIP to identify cell type-specific transcription factor (TF) binding sites in native physiological contexts without tissue dissociation or nuclei sorting. SpyChIP takes advantage of a specific covalent isopeptide bond that rapidly forms between the 15-amino acid SpyTag and the 17-kDa protein SpyCatcher. In SpyChIP, the target TF is fused with SpyTag by genome engineering, and an epitope tagged SpyCatcher is expressed in cell populations of interest, where it covalently binds to SpyTag-TF. Cell type-specific ChIP is obtained by immunoprecipitating chromatin prepared from whole tissues using antibodies directed against the epitope-tagged SpyCatcher. Using SpyChIP, the genome-wide binding profiles of the Hox protein Ultrabithorax (Ubx) were obtained in two distinct cell types of the Drosophila haltere imaginal disc. These results revealed extensive region-specific Ubx-DNA binding events, highlighting the significance of cell type-specific ChIP and the limitations of whole-tissue ChIP approaches. Analysis of Ubx::SpyChIP results provided insights into the relationship between chromatin accessibility and Ubx-DNA binding, as well as different mechanisms Ubx employs to regulate its downstream cis-regulatory modules. In addition to SpyChIP, it is suggested that SpyTag-SpyCatcher technology, as well as other protein pairs that form covalent isopeptide bonds, will facilitate many additional in vivo applications that were previously impractical.
Feng, S., Rastogi, C., Loker, R., Glassford, W. J., Tomas Rube, H., Bussemaker, H. J. and Mann, R. S. (2022). Transcription factor paralogs orchestrate alternative gene regulatory networks by context-dependent cooperation with multiple cofactors. Nat Commun 13(1): 3808. PubMed ID: 35778382
Summary:
In eukaryotes, members of transcription factor families often exhibit similar DNA binding properties in vitro, yet orchestrate paralog-specific gene regulatory networks in vivo. The serially homologous first (T1) and third (T3) thoracic legs of Drosophila, which are specified by the Hox proteins Scr and Ubx, respectively, offer a unique opportunity to address this paradox in vivo. Genome-wide analyses using epitope-tagged alleles of both Hox loci in the T1 and T3 leg imaginal discs, the precursors to the adult legs and ventral body regions, show that ~8% of Hox binding is paralog-specific. Binding specificity is mediated by interactions with distinct cofactors in different domains: the Hox cofactor Exd acts in the proximal domain and is necessary for Scr to bind many of its paralog-specific targets, while in the distal leg domain, the homeodomain protein Distal-less (Dll) enhances Scr binding to a different subset of loci. These findings reveal how Hox paralogs, and perhaps paralogs of other transcription factor families, orchestrate alternative downstream gene regulatory networks with the help of multiple, context-specific cofactors.
Tikhonova, E., Mariasina, S., Efimov, S., Polshakov, V., Maksimenko, O., Georgiev, P. and Bonchuk, A. (2022). Structural basis for interaction between CLAMP and MSL2 proteins involved in the specific recruitment of the dosage compensation complex in Drosophila. Nucleic Acids Res 50(11): 6521-6531. PubMed ID: 35648444
Summary:
Transcriptional regulators select their targets from a large pool of similar genomic sites. The binding of the Drosophila dosage compensation complex (DCC) exclusively to the male X chromosome provides insight into binding site selectivity rules. Previous studies showed that the male-specific organizer of the complex, MSL2, and ubiquitous DNA-binding protein CLAMP directly interact and play an important role in the specificity of X chromosome binding. The highly specific interaction between the intrinsically disordered region of MSL2 and the N-terminal zinc-finger C2H2-type (C2H2) domain of CLAMP was examined in this study. The NMR structure was obtainted of the CLAMP N-terminal C2H2 zinc finger, which has a classic C2H2 zinc-finger fold with a rather unusual distribution of residues typically used in DNA recognition. Substitutions of residues in this C2H2 domain had the same effect on the viability of males and females, suggesting that it plays a general role in CLAMP activity. The N-terminal C2H2 domain of CLAMP is highly conserved in insects. However, the MSL2 region involved in the interaction is conserved only within the Drosophila genus, suggesting that this interaction emerged during the evolution of a mechanism for the specific recruitment of the DCC on the male X chromosome in Drosophilidae.
Liang, G., Mi, D., Chang, J., On Yau, T., Xu, G., Ruan, J., Bu, W. and Gao, S. (2022). Precise annotation of Drosophila mitochondrial genomes leads to insights into AT-rich regions. Mitochondrion 65: 145-149. PubMed ID: 35779797
Summary:
In this study precise annotation of Drosophila melanogaster, D. simulans, D. grimshawi, Bactrocera oleae mitochondrial (mt) genomes were performed using pan RNA-seq analysis. Using PacBio cDNA-seq data from D. simulans, the Transcription Initiation Sites (TISs) of the mt Heavy and Light strands in Drosophila mt genomes were precisely annotated; the polyA(+) and polyA(-) motifs in the CRs are associated with TISs. The discovery of the conserved polyA(+) and polyA(-) motifs provides insights into many polyA and polyT sequences in CRs of insect mt genomes, leading to reveal the mt transcription and its regulation in invertebrates. Notably, it is proposed that: (1) polyA/polyT motifs in CRs function as signals to initiate mtDNA transcription; (2) the duplication, recombination or mutation of these polyA/polyT sequences formed the AT-rich regions during evolution; and (3) since CRs of many invertebrate species still contain many polyA/polyT sequences, there is a high probability that several TISs and TTSs exist in invertebrate mt genomes.
Dunipace, L., Newcomb, S. and Stathopoulos, A. (2022). brinker levels regulated by a promoter proximal element support germ cell homeostasis. Development 149(3). PubMed ID: 35037688
Summary:
A limited BMP signaling range in the stem cell niche of the ovary protects against germ cell tumors and promotes germ cell homeostasis. The canonical repressor of BMP signaling in both the Drosophila embryo and wing disc is the transcription factor Brinker (Brk), yet the expression and potential role of Brk in the germarium has not previously been described. This study found that brk expression requires a promoter-proximal element (PPE) to support long-distance enhancer action as well as to drive expression in the germarium. Furthermore, PPE subdomains have different activities; in particular, the proximal portion acts as a damper to regulate brk levels precisely. Using PPE mutants as well as tissue-specific RNA interference and overexpression, this study showed that altering brk expression within either the soma or the germline affects germ cell homeostasis. Remarkably, it was found that Decapentaplegic (Dpp), the main BMP ligand and canonical antagonist of Brk, is upregulated by Brk in the escort cells of the germarium, demonstrating that Brk can positively regulate this pathway.

Tuesday, July 26th - Chromatin and DNA replication andchromosome dynamics

Cho, C. Y., Seller, C. A. and O'Farrell, P. H. (2022). Temporal control of late replication and coordination of origin firing by self-stabilizing Rif1-PP1 hubs in Drosophila. Proc Natl Acad Sci U S A 119(26): e2200780119. PubMed ID: 35733247
Summary:
In the metazoan S phase, coordinated firing of clusters of origins replicates different parts of the genome in a temporal program. Despite advances, neither the mechanism controlling timing nor that coordinating firing of multiple origins is fully understood. Rif1, an evolutionarily conserved inhibitor of DNA replication, recruits protein phosphatase 1 (PP1) and counteracts firing of origins by S-phase kinases. During the midblastula transition (MBT) in Drosophila embryos, Rif1 forms subnuclear hubs at each of the large blocks of satellite sequences and delays their replication. Each Rif1 hub disperses abruptly just prior to the replication of the associated satellite sequences. This study shows that the level of activity of the S-phase kinase, DDK, accelerated this dispersal program, and that the level of Rif1-recruited PP1 retarded it. Further, Rif1-recruited PP1 supported chromatin association of nearby Rif1. This influence of nearby Rif1 can create a "community effect" counteracting kinase-induced dissociation such that an entire hub of Rif1 undergoes switch-like dispersal at characteristic times that shift in response to the balance of Rif1-PP1 and DDK activities. A model is proposed in which the spatiotemporal program of late replication in the MBT embryo is controlled by self-stabilizing Rif1-PP1 hubs, whose abrupt dispersal synchronizes firing of associated late origins.
Chetverina, D., Vorobyeva, N. E., Mazina, M. Y., Fab, L. V., Lomaev, D., Golovnina, A., Mogila, V., Georgiev, P., Ziganshin, R. H. and Erokhin, M. (2022). Comparative interactome analysis of the PRE DNA-binding factors: purification of the Combgap-, Zeste-, Psq-, and Adf1-associated proteins. Cell Mol Life Sci 79(7): 353. PubMed ID: 35676368
Summary:
The Polycomb group (PcG) and Trithorax group (TrxG) proteins are key epigenetic regulators controlling the silenced and active states of genes in multicellular organisms, respectively. While precise mechanisms of the PcG/TrxG protein recruitment remain unknown, the important role is suggested to belong to sequence-specific DNA-binding factors. At the same time, it was demonstrated that the PRE DNA-binding proteins are not exclusively localized to PREs but can bind other DNA regulatory elements, including enhancers, promoters, and boundaries. To gain an insight into the PRE DNA-binding protein regulatory network, in this study, differences in abundance of the Combgap, Zeste, Psq, and Adf1 PRE DNA-binding proteins were sought. While there were no conspicuous differences in co-localization of these proteins with other functional transcription factors, it was shown that Combgap and Zeste are more tightly associated with the Polycomb repressive complex 1 (PRC1), while Psq interacts strongly with the TrxG proteins, including the BAP SWI/SNF complex. The Adf1 interactome contained Mediator subunits as the top interactors. In addition, Combgap efficiently interacted with AGO2, NELF, and TFIID. Combgap, Psq, and Adf1 have architectural proteins in their networks. This study further investigated the existence of direct interactions between different PRE DNA-binding proteins and demonstrated that Combgap-Adf1, Psq-Dsp1, and Pho-Spps can interact in the yeast two-hybrid assay.
Wright, D. and Schaeffer, S. W. (2022). The relevance of chromatin architecture to genome rearrangements in Drosophila. Philos Trans R Soc Lond B Biol Sci 377(1856): 20210206. PubMed ID: 35694744
Summary:
DNA within chromosomes in the nucleus is non-randomly organized into chromosome territories, compartments and topologically associated domains (TADs). Chromosomal rearrangements have the potential to alter chromatin organization and modify gene expression leading to selection against these structural variants. Drosophila pseudoobscura has a wealth of naturally occurring gene arrangements that were generated by overlapping inversion mutations caused by two chromosomal breaks that rejoin the central region in reverse order. Unlike humans, Drosophila inversion heterozygotes do not have negative effects associated with crossing over during meiosis because males use achiasmate mechanisms for proper segregation, and aberrant recombinant meiotic products generated in females are lost in polar bodies. As a result, Drosophila populations are found to harbour extensive inversion polymorphisms. It is not clear, however, whether chromatin architecture constrains which inversions breakpoints persist in populations. This study mapped the breakpoints of seven inversions in D. pseudoobscura to the TAD map to determine if persisting inversion breakpoints are more likely to occur at boundaries between TADs. The results show that breakpoints occur at TAD boundaries more than expected by chance. Some breakpoints may alter gene expression within TADs supporting the hypothesis that position effects contribute to inversion establishment. =
Bag, I., Chen, Y., D'Orazio, K., Lopez, P., Wenzel, S., Takagi, Y. and Lei, E. P. (2022). Isha is a su(Hw) mRNA-binding protein required for gypsy insulator function. G3 (Bethesda). PubMed ID: 35708663
Summary:
Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. This study utilized RNA-affinity pull down coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif (RRM) and RNA Polymerase II (Pol II) C-terminal domain (CTD)-interacting domain (CID). Isha was found to physically interacts with total and elongating Pol II and associates with chromatin at the 5' end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with the core gypsy insulator component CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. These results reveal a novel factor Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.
Bi, C. L., Cheng, Q., Yan, L. Y., Wu, H. Y., Wang, Q., Wang, P., Cheng, L., Wang, R., Yang, L., Li, J., Tie, F., Xie, H. and Fang, M. (2022). A prominent gene activation role for C-terminal binding protein in mediating PcG/trxG proteins through Hox gene regulation. Development 149(11). PubMed ID: 35666088
Summary:
The evolutionarily conserved C-terminal binding protein (CtBP) has been well characterized as a transcriptional co-repressor. This study reports a previously unreported function for CtBP, showing that lowering CtBP dosage genetically suppresses Polycomb group (PcG) loss-of-function phenotypes while enhancing that of trithorax group (trxG) in Drosophila, suggesting that the role of CtBP in gene activation is more pronounced in fly development than previously thought. In fly cells, this study shows that CtBP is required for the derepression of the most direct PcG target genes, which are highly enriched by homeobox transcription factors, including Hox genes. Using ChIP and co-IP assays, this study demonstrate that CtBP is directly required for the molecular switch between H3K27me3 and H3K27ac in the derepressed Hox loci. In addition, CtBP physically interacts with many proteins, such as UTX, CBP, Fs(1)h and RNA Pol II, that have activation roles, potentially assisting in their recruitment to promoters and Polycomb response elements that control Hox gene expression. Therefore, this study reveals a prominent activation function for CtBP that confers a major role for the epigenetic program of fly segmentation and development.
Roy, S., Juste, S. S., Sneider, M., Auradkar, A., Klanseck, C., Li, Z., Julio, A. H. F., Del Amo, V. L., Bier, E. and Guichard, A. (2022). . Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells. Sci Adv 8(26): eabo0721. PubMed ID: 35776792
Summary:
Repair of double-strand breaks (DSBs) in somatic cells is primarily accomplished by error-prone nonhomologous end joining and less frequently by precise homology-directed repair preferentially using the sister chromatid as a template. In this study, a Drosophila system performs efficient somatic repair of both DSBs and single-strand breaks (SSBs) using intact sequences from the homologous chromosome in a process referred to as homologous chromosome-templated repair (HTR). Unexpectedly, HTR-mediated allelic conversion at the white locus was more efficient (40 to 65%) in response to SSBs induced by Cas9-derived nickases D10A or H840A than to DSBs induced by fully active Cas9 (20 to 30%). Repair phenotypes elicited by Nickase versus Cas9 differ in both developmental timing (late versus early stages, respectively) and the production of undesired mutagenic events (rare versus frequent). Nickase-mediated HTR represents an efficient and unanticipated mechanism for allelic correction, with far-reaching potential applications in the field of gene editing.

Monday, July 25th - RNA and Transposons

Kofler, R., Nolte, V. and Schlotterer, C. (2022). The transposition rate has little influence on the plateauing level of the P-element. Mol Biol Evol. PubMed ID: 35731857
Summary:
The popular trap model assumes that the invasion of transposable elements (TEs) in mammals and invertebrates are stopped by piRNAs that emerge after insertion of the TE into a piRNA cluster. It remains, however, still unclear which factors influence the dynamics of TE invasions. The activity of the TE (i.e. transposition rate) is one frequently discussed key factor. This study took advantage of the temperature dependent activity of the P-element, a widely studied eukaryotic TE, to test how TE activity affects the dynamics of a TE invasion. P-element invasion dynamics were monitored in experimental Drosophila simulans populations at hot and cold culture conditions. Despite marked differences in transposition rates, the P-element reached very similar copy numbers at both temperatures. The reduction of the insertion rate upon approaching the copy number plateau was accompanied by similar amounts of piRNAs against the P-element at both temperatures. Nevertheless, fewer numbers of P-element insertions were observed in piRNA clusters than expected, which is not compatible with a simple trap model. The ping-pong cycle, which degrades TE transcripts, becomes typically active after the copy number plateaued. A model, with few parameters, was generated that largely captures the observed invasion dynamics. It is concluded that the transposition rate has at the most only a minor influence on TE abundance, but other factors, such as paramutations or selection against TE insertions are shaping the TE composition.
Munden, A., Benton, M. L., Capra, J. A. and Nordman, J. T. (2022). R-loop Mapping and Characterization During Drosophila Embryogenesis Reveals Developmental Plasticity in R-loop Signatures. J Mol Biol 434(13): 167645. PubMed ID: 35609632
Summary:
R-loops are involved in transcriptional regulation, DNA and histone post-translational modifications, genome replication and genome stability. To what extent R-loop abundance and genome-wide localization is actively regulated during metazoan embryogenesis is unknown. Drosophila embryogenesis provides a powerful system to address these questions due to its well-characterized developmental program, the sudden onset of zygotic transcription and available genome-wide data sets. This study measured the overall abundance and genome localization of R-loops in early and late-stage embryos relative to Drosophila cultured cells. It was demonstrated that absolute R-loop levels change during embryogenesis and that RNaseH1 catalytic activity is critical for embryonic development. R-loop mapping by strand-specific DRIP-seq reveals that R-loop localization is plastic across development, both in the genes which form R-loops and where they localize relative to gene bodies. Importantly, these changes are not driven by changes in the transcriptional program. Negative GC skew and absolute changes in AT skew are associated with R-loop formation in Drosophila. Furthermore, this study demonstrated that while some chromatin binding proteins and histone modifications such as H3K27me3 are associated with R-loops throughout development, other chromatin factors associated with R-loops in a developmental specific manner. These findings highlight the importance and developmental plasticity of R-loops during Drosophila embryogenesis.
Flanagan, K., Baradaran-Heravi, A., Yin, Q., Dao Duc, K., Spradling, A. C. and Greenblatt, E. J. (2022). FMRP-dependent production of large dosage-sensitive proteins is highly conserved. Genetics. PubMed ID: 35731217
Summary:
Mutations in FMR1 are the most common heritable cause of autism spectrum disorder. FMR1 encodes an RNA binding protein, FMRP, which binds to long, autism-relevant transcripts and is essential for normal neuronal and ovarian development. In contrast to the prevailing model that FMRP acts to block translation elongation, previous work found that FMRP activates translation initiation of large proteins in Drosophila oocytes. This study now provides evidence that FMRP-dependent translation is conserved and occurs in the mammalian brain. Comparisons of mammalian cortex and Drosophila oocyte ribosome profiling data show that translation of FMRP-bound mRNAs decreases to a similar magnitude in FMRP-deficient tissues from both species. The steady state levels of several FMRP targets were reduced in the Fmr1 KO mouse cortex, including a ∼2-fold reduction of Auts2, a gene implicated in an autosomal dominant autism spectrum disorder. In order to distinguish between effects on elongation and initiation, a novel metric was used to detect rate-limiting ribosome stalling. No evidence was found that FMRP target protein production is governed by translation elongation rates. FMRP translational activation of large proteins may be critical for normal human development, as more than 20 FMRP targets including Auts2 are dosage-sensitive and are associated with neurodevelopmental disorders caused by haploinsufficiency.
Fang, J. and Lerit, D. A. (2022). Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly. Development 149(13). PubMed ID: 35661190
Summary:
As the microtubule-organizing centers of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin-like protein (PLP) directs formation of a pericentriolar material (PCM) scaffold required for PCM organization and microtubule-organizing center function. This study investigated the post-transcriptional regulation of Plp mRNA. Conserved binding sites were identified for cytoplasmic polyadenylation element binding (CPEB) proteins within the Plp 3'-untranslated region and examine the role of the CPEB ortholog Oo18 RNA-binding protein (Orb) in Plp mRNA regulation. The data show that Orb interacts biochemically with Plp mRNA to promote polyadenylation and PLP protein expression. Loss of orb, but not orb2, diminishes PLP levels in embryonic extracts. Consequently, PLP localization to centrosomes and its function in PCM scaffolding are compromised in orb mutant embryos, resulting in genomic instability and embryonic lethality. Moreover, this study found that PLP overexpression restores centrosome scaffolding and rescues the cell division defects caused by orb depletion. These data suggest that Orb modulates PLP expression at the level of Plp mRNA polyadenylation and demonstrates that the post-transcriptional regulation of core, conserved centrosomal mRNAs is crucial for centrosome function.
Salerno-Kochan, A., Horn, A., Ghosh, P., Nithin, C., Koscielniak, A., Meindl, A., Strauss, D., Krutyholowa, R., Rossbach, O., Bujnicki, J. M., Gaik, M., Medenbach, J. and Glatt, S. (2022). Molecular insights into RNA recognition and gene regulation by the TRIM-NHL protein Mei-P26. Life Sci Alliance 5(8). PubMed ID: 35512835
Summary:
The TRIM-NHL protein Meiotic P26 (Mei-P26) acts as a regulator of cell fate in Drosophila. Its activity is critical for ovarian germline stem cell maintenance, differentiation of oocytes, and spermatogenesis. Mei-P26 functions as a post-transcriptional regulator of gene expression; however, the molecular details of how its NHL domain selectively recognizes and regulates its mRNA targets have remained elusive. This study presents the crystal structure of the Mei-P26 NHL domain at 1.6 Å resolution and identifies key amino acids that confer substrate specificity and distinguish Mei-P26 from closely related TRIM-NHL proteins. Furthermore, mRNA targets of Mei-P26 were identified in cultured Drosophila cells, and it was shown that Mei-P26 can act as either a repressor or activator of gene expression on different RNA targets. This work reveals the molecular basis of RNA recognition by Mei-P26 and the fundamental functional differences between otherwise very similar TRIM-NHL proteins.
de Faria, I. J. S., Aguiar, E., Olmo, R. P., Alves da Silva, J., Daeffler, L., Carthew, R. W., Imler, J. L. and Marques, J. T. (2022). Invading viral DNA triggers dsRNA synthesis by RNA polymerase II to activate antiviral RNA interference in Drosophila. Cell Rep 39(12): 110976. PubMed ID: 35732126
Summary:
dsRNA sensing triggers antiviral responses against RNA and DNA viruses in diverse eukaryotes. In Drosophila, Invertebrate iridescent virus 6 (IIV-6), a large DNA virus, triggers production of small interfering RNAs (siRNAs) by the dsRNA sensor Dicer-2. This study shows that host RNA polymerase II (RNAPII) bidirectionally transcribes specific AT-rich regions of the IIV-6 DNA genome to generate dsRNA. Both replicative and naked IIV-6 genomes trigger production of dsRNA in Drosophila cells, implying direct sensing of invading DNA. Loquacious-PD, a Dicer-2 co-factor essential for the biogenesis of endogenous siRNAs, is dispensable for processing of IIV-6-derived dsRNAs, which suggests that they are distinct. Consistent with this finding, inhibition of the RNAPII co-factor P-TEFb affects the synthesis of endogenous, but not virus-derived, dsRNA. Altogether, these results suggest that a non-canonical RNAPII complex recognizes invading viral DNA to synthesize virus-derived dsRNA, which activates the antiviral siRNA pathway in Drosophila.

Friday, July 22rd - Signaling

Palermo, J., Keene, A. C. and DiAngelo, J. R. (2022). Expression of a constitutively active insulin receptor in Drosulfakinin (Dsk) neurons regulates metabolism and sleep in Drosophila. Biochem Biophys Rep 30: 101280. PubMed ID: 35600902
Summary:
The ability of organisms to sense their nutritional environment and adjust their behavior accordingly is critical for survival. Insulin-like peptides (ilps) play major roles in controlling behavior and metabolism; however, the tissues and cells that insulin acts on to regulate these processes are not fully understood. In the fruit fly, Drosophila melanogaster, insulin signaling has been shown to function in the fat body to regulate lipid storage, but whether ilps act on the fly brain to regulate nutrient storage is not known. This study manipulated insulin signaling in defined populations of neurons in Drosophila and measure glycogen and triglyceride storage. Expressing a constitutively active form of the insulin receptor (dInR) in the insulin-producing cells had no effect on glycogen or triglyceride levels. However, activating insulin signaling in the Drosulfakinin (Dsk)-producing neurons led to triglyceride accumulation and increased food consumption. The expression of ilp2, ilp3 and ilp5 was increased in flies with activated insulin signaling in the Dsk neurons, which along with the feeding phenotype, may cause the triglyceride storage phenotypes observed in these flies. In addition, expressing a constitutively active dInR in Dsk neurons resulted in decreased sleep in the fed state and less starvation-induced sleep suppression suggesting a role for insulin signaling in regulating nutrient-responsive behaviors. Together, these data support a role for insulin signaling in the Dsk-producing neurons for regulating behavior and maintaining metabolic homeostasis.
Moore, S. L., Adamini, F. C., Coopes, E. S., Godoy, D., Northington, S. J., Stewart, J. M., Tillett, R. L., Bieser, K. L. and Kagey, J. D. (2022). Patched and Costal-2 mutations lead to differences in tissue overgrowth autonomy. Fly (Austin) 16(1): 176-189. PubMed ID: 35468034
Summary:
Genetic screens are used in Drosophila melanogaster to identify genes key in the regulation of organismal development and growth. These screens have defined signalling pathways necessary for tissue and organismal development, which are evolutionarily conserved across species, including Drosophila. This study used an FLP/FRT mosaic system to screen for conditional regulators of cell growth and cell division in the Drosophila eye. The conditional nature of this screen utilizes a block in the apoptotic pathway to prohibit the mosaic mutant cells from dying via apoptosis. From this screen, two different mutants were identified that mapped to the Hedgehog signalling pathway. Previously, a novel Ptc mutation was described, and this study adds to the understanding of disrupting the Hh pathway with a novel allele of Cos2. Both of these Hh components are negative regulators of the pathway, yet they depict mutant differences in the type of overgrowth created. Ptc mutations lead to overgrowth consisting of almost entirely wild-type tissue (non-autonomous overgrowth), while the Cos2 mutation results in tissue that is overgrown in both the mutant and wild-type clones (both autonomous and non-autonomous). These differences in tissue overgrowth are consistent in the Drosophila eye and wing. The observed difference is correlated with different deregulation patterns of pMad, the downstream effector of DPP signalling. This finding provides insight into pathway-specific differences that help to better understand intricacies of developmental processes and human diseases that result from deregulated Hedgehog signalling, such as basal cell carcinoma.
Kong, D., Zhao, S., Xu, W., Dong, J. and Ma, X. (2022). Fat body-derived Spz5 remotely facilitates tumor-suppressive cell competition through Toll-6-α-Spectrin axis-mediated Hippo activation. Cell Rep 39(12): 110980. PubMed ID: 35732124
Summary:
Tumor-suppressive cell competition is an evolutionarily conserved process that selectively removes precancerous cells to maintain tissue homeostasis. Using the polarity-deficiency-induced cell competition model in Drosophila, this study identify Toll-6, a Toll-like receptor family member, as a driver of tension-mediated cell competition through α-Spectrin (α-Spec)-Yorkie (Yki) cascade. Toll-6 aggregates along the boundary between wild-type and polarity-deficient clones, where Toll-6 physically interacts with the cytoskeleton network protein α-Spec to increase mechanical tension, resulting in actomyosin-dependent Hippo pathway activation and the elimination of scrib mutant cells. Furthermore, this study show that Spz5 secreted from fat body, the key innate organ in fly, facilitates the elimination of scrib clones by binding to Toll-6. These findings uncover mechanisms by which fat bodies remotely regulate tumor-suppressive cell competition of polarity-deficient tumors through inter-organ crosstalk and identified the Toll-6-α-Spec axis as an essential guardian that prevents tumorigenesis via tension-mediated cell elimination.
Rubio-Ferrera, I., Baladron-de-Juan, P., Clarembaux-Badell, L., Truchado-Garcia, M., Jordan-Alvarez, S., Thor, S., Benito-Sipos, J. and Monedero Cobeta, I. (2022). Selective role of the DNA helicase Mcm5 in BMP retrograde signaling during Drosophila neuronal differentiation. PLoS Genet 18(6): e1010255. PubMed ID: 35737938
Summary:
The MCM2-7 complex is a highly conserved hetero-hexameric protein complex, critical for DNA unwinding at the replicative fork during DNA replication. Overexpression or mutation in MCM2-7 genes is linked to and may drive several cancer types in humans. In mice, mutations in MCM2-7 genes result in growth retardation and mortality. All six MCM2-7 genes are also expressed in the developing mouse CNS, but their role in the CNS is not clear. This study used the central nervous system (CNS) of Drosophila melanogaster to begin addressing the role of the MCM complex during development, focusing on the specification of a well-studied neuropeptide expressing neuron: the Tv4/FMRFa neuron. In a search for genes involved in the specification of the Tv4/FMRFa neuron this study identified Mcm5 and found that it plays a highly specific role in the specification of the Tv4/FMRFa neuron. Other components of the MCM2-7 complex phenocopies Mcm5, indicating that the role of Mcm5 in neuronal subtype specification involves the MCM2-7 complex. Surprisingly, no evidence was found of reduced progenitor proliferation, and instead it was found that Mcm5 is required for the expression of the type I BMP receptor Tkv, which is critical for the FMRFa expression. These results suggest that the MCM2-7 complex may play roles during CNS development outside of its well-established role during DNA replication.
Akiyama, T., Seidel, C. W. and Gibson, M. C. (2022). The feedback regulator Nord controls Dpp/BMP signaling via extracellular interaction with Dally in the Drosophila wing. Dev Biol 488: 91-103. PubMed ID: 35609633
Summary:
The Drosophila BMP 2/4 homologue Decapentaplegic (Dpp) acts as a morphogen to regulate diverse developmental processes, including wing morphogenesis. Transcriptional feedback regulation of this pathway ensures tightly controlled signaling outputs to generate the precise pattern of the adult wing. Nevertheless, few direct Dpp target genes have been explored and understanding of feedback regulation remains incomplete. This study employed transcriptional profiling following dpp conditional knockout to identify nord, a novel Dpp/BMP feedback regulator. nord mutants generated by CRISPR/Cas9 mutagenesis produce a smaller wing and display low penetrance venation defects. At the molecular level, nord encodes a secreted heparin-binding protein, and this study shows that its overexpression is sufficient to antagonize Dpp/BMP signaling. Mechanistically, it was demonstrated that Nord physically interacts with the Dpp/BMP co-receptor Dally and promotes its degradation. In sum, it is proposed that Nord fine-tunes Dpp/BMP signaling by regulating Dally availability on the cell surface, with implications for both developmental and disease models.
Brace, E. J., Essuman, K., Mao, X., Palucki, J., Sasaki, Y., Milbrandt, J. and DiAntonio, A. (2022). Distinct developmental and degenerative functions of SARM1 require NAD+ hydrolase activity. PLoS Genet 18(6): e1010246. PubMed ID: 35737728
Summary:
SARM1 is the founding member of the TIR-domain family of NAD+ hydrolases and the central executioner of pathological axon degeneration. SARM1-dependent degeneration requires NAD+ hydrolysis. Prior to the discovery that SARM1 is an enzyme, SARM1 was studied as a TIR-domain adaptor protein with non-degenerative signaling roles in innate immunity and invertebrate neurodevelopment, including at the Drosophila neuromuscular junction (NMJ). This study explored whether the NADase activity of SARM1 also contributes to developmental signaling. Transgenic Drosophila lines were established that express SARM1 variants with normal, deficient, and enhanced NADase activity, and their function were tested in NMJ development. It was found that NMJ overgrowth scales with the amount of NADase activity, suggesting an instructive role for NAD+ hydrolysis in this developmental signaling pathway. While degenerative and developmental SARM1 signaling share a requirement for NAD+ hydrolysis, this study demonstrates that these signals use distinct upstream and downstream mechanisms. These results identify SARM1-dependent NAD+ hydrolysis as a heretofore unappreciated component of developmental signaling. SARM1 now joins sirtuins and Parps as enzymes that regulate signal transduction pathways via mechanisms that involve NAD+ cleavage, greatly expanding the potential scope of SARM1 TIR NADase functions.

Thursday, July 21st - Disease Models

McAdow, J., Yang, S., Ou, T., Huang, G., Dobbs, M. B., Gurnett, C. A., Greenberg, M. J. and Johnson, A. N. (2022). A pathogenic mechanism associated with myopathies and structural birth defects involves TPM2-directed myogenesis. JCI Insight 7(12). PubMed ID: 35579956
Summary:
Nemaline myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle-specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as cap myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so this study expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found 4 variants significantly affected muscle development and muscle function. Transient overexpression of the 4 variants also disrupted the morphogenesis of mouse myotubes in vitro and negatively affected zebrafish muscle development in vivo. This study used transient overexpression assays in zebrafish to characterize two potentially novel TPM2 variants and one recurring variant that was identified in patients with DA (V129A, E139K, A155T, respectively) and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in these assays correlated with the severity of clinical phenotypes observed in patients with DA, suggesting disrupted myogenesis is a potentially novel pathomechanism of TPM2 disorders and that myogenic assays can predict the clinical severity of TPM2 variants.
O'Hanlon, M. E., Tweedy, C., Scialo, F., Bass, R., Sanz, A. and Smulders-Srinivasan, T. K. (2022). Mitochondrial electron transport chain defects modify Parkinson's disease phenotypes in a Drosophila model. Neurobiol Dis 171: 105803. PubMed ID: 35764292
Summary:
Defects in Mitochondria have been implicated in Parkinson's disease (PD). A targeted heterozygous enhancer/suppressor screen was performed using Drosophila mutations within mitochondrial electron transport chain (ETC) genes against a null PD mutation in parkin. The interactions were assessed by climbing assays at 2-5 days as an indicator of motor function. A strong enhancer mutation in COX5A was examined further for L-dopa rescue, oxygen consumption, mitochondrial content, and reactive oxygen species. A later timepoint of 16-20 days was also investigated for both COX5A and a suppressor mutation in cyclope. Mutations in individual genes for subunits within the mitochondrial respiratory complexes have interactions with parkin, while others do not, irrespective of complex. One intriguing mutation in a complex IV subunit (cyclope) shows a suppressor rescue effect at early time points, improving the gross motor defects caused by the PD mutation, providing a strong candidate for drug discovery. Most mutations, however, show varying degrees of enhancement or slight suppression of the PD phenotypes. Thus, individual mitochondrial mutations within different oxidative phosphorylation complexes have different interactions with PD with regard to degree and direction. Upon further investigation of the strongest enhancer (COX5A), the mechanism by which these interactions occur initially does not appear to be based on defects in ATP production, but rather may be related to increased levels of reactive oxygen species. This work highlights some key subunits potentially involved in mechanisms underlying PD pathogenesis, implicating ETC complexes other than complex I in PD.
Kishino, Y., Matsukawa, K., Matsumoto, T., Miyazaki, R., Wakabayashi, T., Nonaka, T., Kametani, F., Hasegawa, M., Hashimoto, T. and Iwatsubo, T. (2022). Casein kinase 1delta/epsilon phosphorylates fused in sarcoma (FUS) and ameliorates FUS-mediated neurodegeneration. J Biol Chem: 102191. PubMed ID: 35753345
Summary:
Aberrant cytoplasmic accumulation of an RNA-binding protein, Fused in sarcoma (FUS), characterizes the neuropathology of subtypes of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, although the effects of post-translational modifications of FUS, especially phosphorylation, on its neurotoxicity have not been fully characterized. This study shows that casein kinase 1δ phosphorylates FUS at 10 serine/threonine residues in vitro using mass spectrometric analyses. Phosphorylation by casein kinase 1δ or 1ε significantly increased the solubility of FUS in human embryonic kidney 293 cells. In transgenic Drosophila that overexpress wild-type or P525L ALS-mutant human FUS in the retina or in neurons it was found that coexpression of human casein kinase 1δ or its Drosophila isologue Dco in the photoreceptor neurons significantly ameliorated the observed retinal degeneration, and neuronal coexpression of human casein kinase 1δ extended fly lifespan. Taken together, these data suggest a novel regulatory mechanism of the assembly and toxicity of FUS through casein kinase 1δ/ε-mediated phosphorylation, which could represent a potential therapeutic target in FUS proteinopathies.
Knabbe, J., Protzmann, J., Schneider, N., Berger, M., Dannehl, D., Wei, S., Strahle, C., Tegtmeier, M., Jaiswal, A., Zheng, H., Kruger, M., Rohr, K., Spanagel, R., Bilbao, A., Engelhardt, M., Scholz, H. and Cambridge, S. B. (2022). Single-dose ethanol intoxication causes acute and lasting neuronal changes in the brain. Proc Natl Acad Sci U S A 119(25): e2122477119. PubMed ID: 35700362
Summary:
Alcohol intoxication at early ages is a risk factor for the development of addictive behavior. To uncover neuronal molecular correlates of acute ethanol intoxication, this study used stable-isotope-labeled mice combined with quantitative mass spectrometry to screen more than 2,000 hippocampal proteins, of which 72 changed synaptic abundance up to twofold after ethanol exposure. Among those were mitochondrial proteins and proteins important for neuronal morphology, including MAP6 and ankyrin-G. Based on these candidate proteins, acute and lasting molecular, cellular, and behavioral changes were found following a single intoxication in alcohol-naive mice. Immunofluorescence analysis revealed a shortening of axon initial segments. Longitudinal two-photon in vivo imaging showed increased synaptic dynamics and mitochondrial trafficking in axons. Knockdown of mitochondrial trafficking in dopaminergic neurons abolished conditioned alcohol preference in Drosophila flies. This study introduces mitochondrial trafficking as a process implicated in reward learning and highlights the potential of high-resolution proteomics to identify cellular mechanisms relevant for addictive behavior.
Jarabo, P., Barredo, C. G., de Pablo, C., Casas-Tinto, S. and Martin, F. A. (2022). Alignment between glioblastoma internal clock and environmental cues ameliorates survival in Drosophila. Commun Biol 5(1): 644. PubMed ID: 35773327
Summary:
Virtually every single living organism on Earth shows a circadian (i.e. "approximately a day") internal rhythm that is coordinated with planet rotation (i.e. 24 hours). External cues synchronize the central clock of the organism. Consequences of biological rhythm disruptions have been extensively studied on cancer. Still, mechanisms underlying these alterations, and how they favor tumor development remain largely unknown. This study shows that glioblastoma-induced neurodegeneration also causes circadian alterations in Drosophila. Preventing neurodegeneration in all neurons by genetic means reestablishes normal biological rhythms. Interestingly, in early stages of tumor development, the central pacemaker lengthens its period, whereas in later stages this is severely disrupted. The re-adjustment of the external light:dark period to longer glioblastoma-induced internal rhythms delays glioblastoma progression and ameliorates associated deleterious effects, even after the tumor onset.
Du, X., Wang, Y., Wang, J., Liu, X., Chen, J., Kang, J., Yang, X. and Wang, H. (2022). d-Chiro-Inositol extends the lifespan of male Drosophila melanogaster better than d-Pinitol through insulin signaling and autophagy pathways. Exp Gerontol 165: 111856. PubMed ID: 35644418
Summary:
d-Pinitol (DP) is the methylated product of d-Chiro-Inositol (DCI), which is one of the nine isomers of inositol with optical activity. Both substances possess antioxidant activity. This study was conducted to investigate and compare the antioxidant and life-prolonging effects of DCI and DP on male Drosophila melanogaster. Results showed that DCI and DP prolonged the lifespan and improved the climbing, anti-stress, and antioxidant activities. After treatment with DCI and DP, intestinal homeostasis was improved and the abnormal proliferation of intestinal stem cells (ISCs) was attenuated. Furthermore, real-time PCR revealed downregulated expression levels of PI3K and Akt and upregulated expression levels of Dilp5 and FOXO, which consequently activated Atg1, Atg5, Atg8a, and Atg8b and increased the number of lysosomes. Altogether, DCI exerts a slightly better effect than DP based on various indicators. RNAi D. melanogaster lifespan and molecular docking results further suggested that DCI and DP could prolong longevity through insulin signaling (IIS) and autophagy pathways.

Wednesday, July 20th - Signaling

Palermo, J., Keene, A. C. and DiAngelo, J. R. (2022). Expression of a constitutively active insulin receptor in Drosulfakinin (Dsk) neurons regulates metabolism and sleep in Drosophila. Biochem Biophys Rep 30: 101280. PubMed ID: 35600902
Summary:
The ability of organisms to sense their nutritional environment and adjust their behavior accordingly is critical for survival. Insulin-like peptides (ilps) play major roles in controlling behavior and metabolism; however, the tissues and cells that insulin acts on to regulate these processes are not fully understood. In the fruit fly, Drosophila melanogaster, insulin signaling has been shown to function in the fat body to regulate lipid storage, but whether ilps act on the fly brain to regulate nutrient storage is not known. This study manipulated insulin signaling in defined populations of neurons in Drosophila and measure glycogen and triglyceride storage. Expressing a constitutively active form of the insulin receptor (dInR) in the insulin-producing cells had no effect on glycogen or triglyceride levels. However, activating insulin signaling in the Drosulfakinin (Dsk)-producing neurons led to triglyceride accumulation and increased food consumption. The expression of ilp2, ilp3 and ilp5 was increased in flies with activated insulin signaling in the Dsk neurons, which along with the feeding phenotype, may cause the triglyceride storage phenotypes observed in these flies. In addition, expressing a constitutively active dInR in Dsk neurons resulted in decreased sleep in the fed state and less starvation-induced sleep suppression suggesting a role for insulin signaling in regulating nutrient-responsive behaviors. Together, these data support a role for insulin signaling in the Dsk-producing neurons for regulating behavior and maintaining metabolic homeostasis.
Moore, S. L., Adamini, F. C., Coopes, E. S., Godoy, D., Northington, S. J., Stewart, J. M., Tillett, R. L., Bieser, K. L. and Kagey, J. D. (2022). Patched and Costal-2 mutations lead to differences in tissue overgrowth autonomy. Fly (Austin) 16(1): 176-189. PubMed ID: 35468034
Summary:
Genetic screens are used in Drosophila melanogaster to identify genes key in the regulation of organismal development and growth. These screens have defined signalling pathways necessary for tissue and organismal development, which are evolutionarily conserved across species, including Drosophila. This study used an FLP/FRT mosaic system to screen for conditional regulators of cell growth and cell division in the Drosophila eye. The conditional nature of this screen utilizes a block in the apoptotic pathway to prohibit the mosaic mutant cells from dying via apoptosis. From this screen, two different mutants were identified that mapped to the Hedgehog signalling pathway. Previously, a novel Ptc mutation was described, and this study adds to the understanding of disrupting the Hh pathway with a novel allele of Cos2. Both of these Hh components are negative regulators of the pathway, yet they depict mutant differences in the type of overgrowth created. Ptc mutations lead to overgrowth consisting of almost entirely wild-type tissue (non-autonomous overgrowth), while the Cos2 mutation results in tissue that is overgrown in both the mutant and wild-type clones (both autonomous and non-autonomous). These differences in tissue overgrowth are consistent in the Drosophila eye and wing. The observed difference is correlated with different deregulation patterns of pMad, the downstream effector of DPP signalling. This finding provides insight into pathway-specific differences that help to better understand intricacies of developmental processes and human diseases that result from deregulated Hedgehog signalling, such as basal cell carcinoma.
Kong, D., Zhao, S., Xu, W., Dong, J. and Ma, X. (2022). Fat body-derived Spz5 remotely facilitates tumor-suppressive cell competition through Toll-6-α-Spectrin axis-mediated Hippo activation. Cell Rep 39(12): 110980. PubMed ID: 35732124
Summary:
Tumor-suppressive cell competition is an evolutionarily conserved process that selectively removes precancerous cells to maintain tissue homeostasis. Using the polarity-deficiency-induced cell competition model in Drosophila, this study identify Toll-6, a Toll-like receptor family member, as a driver of tension-mediated cell competition through α-Spectrin (α-Spec)-Yorkie (Yki) cascade. Toll-6 aggregates along the boundary between wild-type and polarity-deficient clones, where Toll-6 physically interacts with the cytoskeleton network protein α-Spec to increase mechanical tension, resulting in actomyosin-dependent Hippo pathway activation and the elimination of scrib mutant cells. Furthermore, this study show that Spz5 secreted from fat body, the key innate organ in fly, facilitates the elimination of scrib clones by binding to Toll-6. These findings uncover mechanisms by which fat bodies remotely regulate tumor-suppressive cell competition of polarity-deficient tumors through inter-organ crosstalk and identified the Toll-6-α-Spec axis as an essential guardian that prevents tumorigenesis via tension-mediated cell elimination.
Rubio-Ferrera, I., Baladron-de-Juan, P., Clarembaux-Badell, L., Truchado-Garcia, M., Jordan-Alvarez, S., Thor, S., Benito-Sipos, J. and Monedero Cobeta, I. (2022). Selective role of the DNA helicase Mcm5 in BMP retrograde signaling during Drosophila neuronal differentiation. PLoS Genet 18(6): e1010255. PubMed ID: 35737938
Summary:
The MCM2-7 complex is a highly conserved hetero-hexameric protein complex, critical for DNA unwinding at the replicative fork during DNA replication. Overexpression or mutation in MCM2-7 genes is linked to and may drive several cancer types in humans. In mice, mutations in MCM2-7 genes result in growth retardation and mortality. All six MCM2-7 genes are also expressed in the developing mouse CNS, but their role in the CNS is not clear. This study used the central nervous system (CNS) of Drosophila melanogaster to begin addressing the role of the MCM complex during development, focusing on the specification of a well-studied neuropeptide expressing neuron: the Tv4/FMRFa neuron. In a search for genes involved in the specification of the Tv4/FMRFa neuron this study identified Mcm5 and found that it plays a highly specific role in the specification of the Tv4/FMRFa neuron. Other components of the MCM2-7 complex phenocopies Mcm5, indicating that the role of Mcm5 in neuronal subtype specification involves the MCM2-7 complex. Surprisingly, no evidence was found of reduced progenitor proliferation, and instead it was found that Mcm5 is required for the expression of the type I BMP receptor Tkv, which is critical for the FMRFa expression. These results suggest that the MCM2-7 complex may play roles during CNS development outside of its well-established role during DNA replication.
Akiyama, T., Seidel, C. W. and Gibson, M. C. (2022). The feedback regulator Nord controls Dpp/BMP signaling via extracellular interaction with Dally in the Drosophila wing. Dev Biol 488: 91-103. PubMed ID: 35609633
Summary:
The Drosophila BMP 2/4 homologue Decapentaplegic (Dpp) acts as a morphogen to regulate diverse developmental processes, including wing morphogenesis. Transcriptional feedback regulation of this pathway ensures tightly controlled signaling outputs to generate the precise pattern of the adult wing. Nevertheless, few direct Dpp target genes have been explored and understanding of feedback regulation remains incomplete. This study employed transcriptional profiling following dpp conditional knockout to identify nord, a novel Dpp/BMP feedback regulator. nord mutants generated by CRISPR/Cas9 mutagenesis produce a smaller wing and display low penetrance venation defects. At the molecular level, nord encodes a secreted heparin-binding protein, and this study shows that its overexpression is sufficient to antagonize Dpp/BMP signaling. Mechanistically, it was demonstrated that Nord physically interacts with the Dpp/BMP co-receptor Dally and promotes its degradation. In sum, it is proposed that Nord fine-tunes Dpp/BMP signaling by regulating Dally availability on the cell surface, with implications for both developmental and disease models.
Brace, E. J., Essuman, K., Mao, X., Palucki, J., Sasaki, Y., Milbrandt, J. and DiAntonio, A. (2022). Distinct developmental and degenerative functions of SARM1 require NAD+ hydrolase activity. PLoS Genet 18(6): e1010246. PubMed ID: 35737728
Summary:
SARM1 is the founding member of the TIR-domain family of NAD+ hydrolases and the central executioner of pathological axon degeneration. SARM1-dependent degeneration requires NAD+ hydrolysis. Prior to the discovery that SARM1 is an enzyme, SARM1 was studied as a TIR-domain adaptor protein with non-degenerative signaling roles in innate immunity and invertebrate neurodevelopment, including at the Drosophila neuromuscular junction (NMJ). This study explored whether the NADase activity of SARM1 also contributes to developmental signaling. Transgenic Drosophila lines were established that express SARM1 variants with normal, deficient, and enhanced NADase activity, and their function were tested in NMJ development. It was found that NMJ overgrowth scales with the amount of NADase activity, suggesting an instructive role for NAD+ hydrolysis in this developmental signaling pathway. While degenerative and developmental SARM1 signaling share a requirement for NAD+ hydrolysis, this study demonstrates that these signals use distinct upstream and downstream mechanisms. These results identify SARM1-dependent NAD+ hydrolysis as a heretofore unappreciated component of developmental signaling. SARM1 now joins sirtuins and Parps as enzymes that regulate signal transduction pathways via mechanisms that involve NAD+ cleavage, greatly expanding the potential scope of SARM1 TIR NADase functions.

Tuesday, July 19th - Gonads

Valentino, M., Ortega, B. M., Ulrich, B., Doyle, D. A., Farnum, E. D., Joiner, D. A., Gavis, E. R. and Niepielko, M. G. (2022). Computational modeling offers new insight into Drosophila germ granule development. Biophys J. PubMed ID: 35288123
Summary:
The packaging of specific mRNAs into ribonucleoprotein granules called germ granules is required for germline proliferation and maintenance. During Drosophila germ granule development, mRNAs such as nanos (nos) and polar granule component (pgc) localize to germ granules through a stochastic seeding and self-recruitment process that generates homotypic clusters: aggregates containing multiple copies of a specific transcript. Germ granules vary in mRNA composition with respect to the different transcripts that they contain and their quantity. However, what influences germ granule mRNA composition during development is unclear. To gain insight into how germ granule mRNA heterogeneity arises, a computational model was created that simulates granule development. Although the model includes known mechanisms that were converted into mathematical representations, additional unreported mechanisms proved to be essential for modeling germ granule formation. The model was validated by predicting defects caused by changes in mRNA and protein abundance. Broader application of the model was demonstrated by quantifying nos and pgc localization efficacies and the contribution that an element within the nos 3' untranslated region has on clustering. For the first time, a mathematical representation of Drosophila germ granule formation is described, offering quantitative insight into how mRNA compositions arise while providing a new tool for guiding future studies.
Kogan, G. L., Mikhaleva, E. A., Olenkina, O. M., Ryazansky, S. S., Galzitskaya, O. V., Abramov, Y. A., Leinsoo, T. A., Akulenko, N. V., Lavrov, S. A. and Gvozdev, V. A. (2022). Extended disordered regions of ribosome-associated NAC proteins paralogs belong only to the germline in Drosophila melanogaster. Sci Rep 12(1): 11191. PubMed ID: 35778515
Summary:
The nascent polypeptide-associated complex (NAC) consisting of α- and β-subunits is an essential ribosome-associated protein conserved in eukaryotes. NAC is a ubiquitously expressed co-translational regulator of nascent protein folding and sorting providing for homeostasis of cellular proteins. This study reports on discovering the germline-specific NACαβ paralogs (gNACs), whose β-subunits, non-distinguishable by ordinary immunodetection, are encoded by five highly homologous gene copies, while the α-subunit is encoded by a single αNAC gene. The gNAC expression is detected in the primordial embryonic and adult gonads via immunostaining. The germline-specific α and β subunits differ from the ubiquitously expressed paralogs by the extended intrinsically disordered regions (IDRs) acquired at the N- and C-termini of the coding regions, predicted to be phosphorylated. The presence of distinct phosphorylated isoforms of gNAC-β subunits is confirmed by comparing of their profiles by 2D-isoeletrofocusing resolution before and after phosphatase treatment of testis ribosomes. It was revealed that the predicted S/T sites of phosphorylation in the individual orthologous IDRs of gNAC-β sequences of Drosophila species are positionally conserved despite these disordered regions are drastically different. The IDR-dependent molecular crowding and specific coordination of NAC and other proteostasis regulatory factors at the ribosomes of germinal cells is proposed. These findings imply that there may be a functional crosstalk between the germinal and ubiquitous α- and β-subunits based on assessing their depletion effects on the fly viability and gonad development.
Zhang, C., Kim, A. J., Rivera-Perez, C., Noriega, F. G. and Kim, Y. J. (2022). The insect somatostatin pathway gates vitellogenesis progression during reproductive maturation and the post-mating response. Nat Commun 13(1): 969. PubMed ID: 35181671
Summary:
Vitellogenesis (yolk accumulation) begins upon eclosion and continues through the process of sexual maturation. Upon reaching sexual maturity, vitellogenesis is placed on hold until it is induced again by mating. However, the mechanisms that gate vitellogenesis in response to developmental and reproductive signals remain unclear. This study has identified the neuropeptide allatostatin-C (AstC)-producing neurons that gate both the initiation of vitellogenesis that occurs post-eclosion and its re-initiation post-mating. During sexual maturation, the AstC neurons receive excitatory inputs from Sex Peptide Abdominal Ganglion (SAG) neurons. In mature virgin females, high sustained activity of SAG neurons shuts off vitellogenesis via continuous activation of the AstC neurons. Upon mating, however, Sex Peptide inhibits SAG neurons, leading to deactivation of the AstC neurons. As a result, this permits both JH biosynthesis and the progression of vitellogenesis in mated females. This work has uncovered a central neural circuit that gates the progression of oogenesis.
Logan, G., Chou, W. C. and McCartney, B. M. (2022). A Diaphanous and Enabled-dependent asymmetric actin cable array repositions nuclei during Drosophila oogenesis. Development 149(13). PubMed ID: 35686626
Summary:
Cells reposition their nuclei for diverse specialized functions through a wide variety of cytoskeletal mechanisms. During Drosophila oogenesis, 15 nurse cells connected by ring canals to each other and the oocyte contract, 'dumping' their cytoplasm into the oocyte. Prior to dumping, actin cables initiate from the nurse cell cortex and elongate toward their nuclei, pushing them away from ring canals to prevent obstruction. How the cable arrays reposition nuclei is unknown. This study found that these arrays are asymmetric, with regional differences in actin cable growth rate dependent on the differential localization of the actin assembly factors Enabled and Diaphanous. Enabled mislocalization produces a uniform growth rate. In oocyte-contacting nurse cells with asymmetric cable arrays, nuclei move away from ring canals. With uniform arrays, these nuclei move toward the adjacent ring canal instead. This correlated with ring canal nuclear blockage and incomplete dumping. These data suggest that nuclear repositioning relies on the regulated cortical localization of Diaphanous and Enabled to produce actin cable arrays with asymmetric growth that push nuclei away from ring canals, enabling successful oogenesis.
Seong, K. H. and Kang, S. (2022). Noncanonical function of the Sex lethal gene controls the protogyny phenotype in Drosophila melanogaster. Sci Rep 12(1): 1455. PubMed ID: 35087103
Summary:
Drosophila melanogaster females eclose on average 4 h faster than males owing to sexual differences in the pupal period, referred to as the protogyny phenotype. To elucidate the mechanism underlying the protogyny phenotype, a newly developed Drosophila Individual Activity Monitoring and Detecting System (DIAMonDS) was used that detects the precise timing of both pupariation and eclosion in individual flies. Although sex transformation induced by tra-2, tra alteration, or msl-2 knockdown-mediated disruption of dosage compensation showed no effect on the protogyny phenotype, stage-specific whole-body knockdown and mutation of the Drosophila master sex switch gene, Sxl, was found to disrupt the protogyny phenotype. Thus, Sxl establishes the protogyny phenotype through a noncanonical pathway in D. melanogaster.
Miao, G., Guo, L. and Montell, D. J. (2022). Border cell polarity and collective migration require the spliceosome component Cactin. J Cell Biol 221(7). PubMed ID: 35612426
Summary:
Border cells are an in vivo model for collective cell migration. This study identify the gene cactin as essential for border cell cluster organization, delamination, and migration. In Cactin-depleted cells, the apical proteins aPKC and Crumbs (Crb) become abnormally concentrated, and overall cluster polarity is lost. Apically tethering excess aPKC is sufficient to cause delamination defects, and relocalizing apical aPKC partially rescues delamination. Cactin is conserved from yeast to humans and has been implicated in diverse processes. In border cells, Cactin's evolutionarily conserved spliceosome function is required. Whole transcriptome analysis revealed alterations in isoform expression in Cactin-depleted cells. Mutations in two affected genes, Sec23 and Sec24CD, which traffic Crb to the apical cell surface, partially rescue border cell cluster organization and migration. Overexpression of Rab5 or Rab11, which promote Crb and aPKC recycling, similarly rescues. Thus, a general splicing factor is specifically required for coordination of cluster polarity and migration, and migrating border cells are particularly sensitive to splicing and cell polarity disruptions.

Monday July 18th - Larval and Adult Physiology and Metabolism

D'Souza, L. C., Dwivedi, S., Raihan, F., Yathisha, U. G., Raghu, S. V., Mamatha, B. S. and Sharma, A. (2022). Hsp70 overexpression in Drosophila hemocytes attenuates benzene-induced immune and developmental toxicity via regulating ROS/JNK signaling pathway. Environ Toxicol 37(7): 1723-1739. PubMed ID: 35301792
Summary:
Benzene, a ubiquitous environmental chemical, is known to cause immune dysfunction and developmental defects. This study aims to investigate the relation between benzene-induced immune dysfunction and developmental toxicity in a genetically tractable animal model, Drosophila melanogaster. Further, the study explored the protective role of Heat Shock Protein 70 (Hsp70) against benzene-induced immunotoxicity and subsequent developmental impact. Drosophila larvae exposed to benzene (1.0, 10.0, and 100.0 mM) were examined for total hemocyte (immune cells) count, phagocytic activity, oxidative stress, apoptosis, and their developmental delay and reduction were analyzed. Benzene exposure for 48 h reduced the total hemocytes count and phagocytic activity, along with an increase in the Reactive Oxygen Species (ROS), and lipid peroxidation in the larval hemocytes. Subsequently, JNK-dependent activation of the apoptosis (Caspase-3 dependent) was also observed. During their development, benzene exposure to Drosophila larvae led to 3 days of delay in development, and ~40% reduced adult emergence. Hsp70-overexpression in hemocytes was found to mitigate benzene-induced oxidative stress and abrogated the JNK-mediated apoptosis in hemocytes, thus restoring total hemocyte count and improving phagocytotic activity. Further, hsp70-overexpression in hemocytes also lessened the benzene-induced developmental delay (rescue of 2.5 days) and improved adult emergence (~20%) emergence, revealing a possible control of immune cells on the organism's development and survival. Overall, this study established that hsp70-overexpression in the Drosophila hemocytes confers protection against benzene-induced immune injury via regulating the ROS/JNK signaling pathway, which helps in the organism's survival and development.
Abuhattum, S., Kotzbeck, P., Schlussler, R., Harger, A., Ariza de Schellenberger, A., Kim, K., Escolano, J. C., Muller, T., Braun, J., Wabitsch, M., Tschop, M., Sack, I., Brankatschk, M., Guck, J., Stemmer, K. and Taubenberger, A. V. (2022). Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens. Sci Rep 12(1): 10325. PubMed ID: 35725987
Summary:
Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, this study set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. Differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, this study has dissected relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. This work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders.
Krittika, S. and Yadav, P. (2022). Alterations in lifespan and sleep/wake duration under selective monochromes of visible light in Drosophila melanogaster. Biol Open. PubMed ID: 35735020
Summary:
Rapid technology development, exposure to gadgets, and artificial lights (with different monochromes) have disturbed lifestyles and the circadian clock, which otherwise confers better regulation of behavioral patterns and sleep/wake cycles in most organisms including Drosophila melanogaster. This study assayed the effect of LD12:12 hr (light: dark) monochromatic lights (violet, blue, green, yellow, orange, and red) on the lifespan, activity, and sleep of the D. melanogaster. A shortened lifespan was observed under 12h of violet, blue, green, and yellow lights, while significantly reduced activity levels under the light phase of blue and green light as compared to their dark phase is observed. Significant increase in the evening anticipation index of flies under blue and green light alongside increased and decreased sleep depth during the day and night respectively suggests the light avoidance, while there is no effect of colored light on the waking time, daily active time, and sleep time. Thus, this study shows short and long-term exposure to certain colored lights in terms of reduced lifespan and locomotor activity, which cause qualitative as well as quantitative changes in the sleep of flies; probably as a sign of aversion towards a specific light.
Dunham, K. E. and Venton, B. J. (2022). SSRI antidepressants differentially modulate serotonin reuptake and release in Drosophila. J Neurochem. PubMed ID: 35736504
Summary:
Selective serotonin reuptake inhibitor (SSRI) antidepressants are commonly prescribed treatments for depression, but their effects on serotonin reuptake and release are not well understood. Drosophila expresses the serotonin transporter (dSERT), the major target of SSRIs, but real-time serotonin changes after SSRIs have not been characterized in this model. Various doses of fluoxetine (Prozac), escitalopram (Lexapro), citalopram (Celexa), and paroxetine (Paxil), were applied to ventral nerve cord (VNC) tissue and optogenetically-stimulated serotonin release was measured with fast-scan cyclic voltammetry (FSCV). Fluoxetine increased reuptake from 1-100 μM, but serotonin concentration only increased at 100 μM. Thus, fluoxetine occupies dSERT and slows clearance, but does not affect concentration. Escitalopram and paroxetine increased serotonin concentrations at all doses, but escitalopram increased reuptake more. Citalopram showed lower concentration changes and faster reuptake profiles compared to escitalopram, so the racemic mixture of citalopram does not change reuptake as much as the S-isomer. Dose response curves were constructed to compare dSERT affinities and paroxetine showed the highest affinity and fluoxetine the lowest. These data demonstrate SSRI mechanisms are complex, with separate effects on reuptake or release.
Anderson, L., Camus, M. F., Monteith, K. M., Salminen, T. S. and Vale, P. F. (2022). Variation in mitochondrial DNA affects locomotor activity and sleep in Drosophila melanogaster. Heredity (Edinb). PubMed ID: 35764697
Summary:
Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved among many taxa. Locomotion is a trait that is highly reliant on metabolic function and expected to be greatly affected by disruptions to mitochondrial performance. To this end, this study aimed to examine how activity and sleep vary between Drosophila melanogaster strains with different geographic origins, how these patterns are affected by mitochondrial DNA (mtDNA) variation, and how breaking up co-evolved mito-nuclear gene combinations affect the studied activity traits. The results demonstrate that Drosophila strains from different locations differ in sleep and activity, and that females are generally more active than males. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, it was possible to quantify the among-line variance attributable to mitochondrial DNA, and it was established that mtDNA variation affects both activity and sleep, in a sex-specific manner. Altogether this study highlights the important role that mitochondrial genome variation plays on organismal physiology and behaviour.
Cabrita, A., Medeiros, A. M., Pereira, T., Rodrigues, A. S., Kranendonk, M. and Mendes, C. S. (2022). Motor dysfunction in Drosophila melanogaster as a biomarker for developmental neurotoxicity. iScience 25(7): 104541. PubMed ID: 35769875
Summary:
Adequate alternatives to conventional animal testing are needed to study developmental neurotoxicity (DNT). This study used kinematic analysis to assess DNT of known (toluene (TOL) and chlorpyrifos (CPS)) and putative (β-N-methylamino-L-alanine (BMAA)) neurotoxic compounds. Drosophila melanogaster was exposed to these compounds during development and evaluated for survival and adult kinematic parameters using the FlyWalker system, a kinematics evaluation method. At concentrations that do not induce general toxicity, the solvent DMSO had a significant effect on kinematic parameters. Moreover, while TOL did not significantly induce lethality or kinematic dysfunction, CPS not only induced developmental lethality but also significantly impaired coordination in comparison to DMSO. Interestingly, BMAA, which was not lethal during development, induced motor decay in young adult animals, phenotypically resembling aged flies, an effect later attenuated upon aging. Furthermore, BMAA induced abnormal development of leg motor neuron projections. These results suggest that the kinematic approach can assess potential DNT of chemical compounds.

Friday, July 15th - Stem Cells

Ariyapala, I. S., Buddika, K., Hundley, H. A., Calvi, B. R. and Sokol, N. S. (2022). The RNA binding protein Swm is critical for Drosophila melanogaster intestinal progenitor cell maintenance. Genetics. PubMed ID: 35762963
Summary:
The regulation of stem cell survival, self-renewal, and differentiation is critical for the maintenance of tissue homeostasis. Although the involvement of signaling pathways and transcriptional control mechanisms in stem cell regulation have been extensively investigated, the role of post-transcriptional control is still poorly understood. This study shows that the nuclear activity of the RNA-binding protein Second Mitotic Wave Missing (Swm) is critical for Drosophila melanogaster intestinal stem cells (ISCs) and their daughter cells, enteroblasts (EBs), to maintain their progenitor cell properties and functions. Loss of swm causes ISCs and EBs to stop dividing and instead detach from the basement membrane, resulting in severe progenitor cell loss. swm loss is further characterized by nuclear accumulation of poly(A)+ RNA in progenitor cells. Swm associates with transcripts involved in epithelial cell maintenance and adhesion, and the loss of swm, while not generally affecting the levels of these Swm-bound mRNAs, leads to elevated expression of proteins encoded by some of them, including the fly ortholog of Filamin. Taken together, this study indicates a nuclear role for Swm in adult stem cell maintenance, raising the possibility that nuclear post-transcriptional regulation of mRNAs encoding cell adhesion proteins ensures proper attachment of progenitor cells.
Wang, Y. W., Wreden, C. C., Levy, M., Meng, J. L., Marshall, Z. D., MacLean, J. and Heckscher, E. (2022). Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord. Elife 11. PubMed ID: 35723253
Summary:
How circuits self-assemble starting from neuronal stem cells is a fundamental question in developmental neurobiology. This study addressed how neurons from different stem cell lineages wire with each other to form a specific circuit motif. In Drosophila larvae, developmental genetics (Twin spot MARCM, Multi-color Flip Out, permanent labeling) was combined with circuit analysis (calcium imaging, connectomics, network science). For many lineages, neuronal progeny are organized into subunits called temporal cohorts. Temporal cohorts are subsets of neurons born within a tight time window that have shared circuit level function. Sharp transitions in patterns of input connectivity were found at temporal cohort boundaries. In addition, a feed-forward circuit was identified that encodes the onset of vibration stimuli. This feed-forward circuit is assembled by preferential connectivity between temporal cohorts from different lineages. Connectivity does not follow the often-cited early-to-early, late-to-late model. Instead, the circuit is formed by sequential addition of temporal cohorts from different lineages, with circuit output neurons born before circuit input neurons. Further, this study generated new tools for the fly community. These data raise the possibility that sequential addition of neurons (with outputs oldest and inputs youngest) could be one fundamental strategy for assembling feed-forward circuits.
Easwaran, S., Van Ligten, M., Kui, M. and Montell, D. J. (2022). Enhanced germline stem cell longevity in Drosophila diapause. Nat Commun 13(1): 711. PubMed ID: 35132083
Summary:
In many species including humans, aging reduces female fertility. Intriguingly, some animals preserve fertility longer under specific environmental conditions. For example, at low temperature and short day-length, Drosophila melanogaster enters a state called adult reproductive diapause. As in other stressful conditions, ovarian development arrests at the yolk uptake checkpoint; however, mechanisms underlying fertility preservation and post-diapause recovery are largely unknown. This study reports that diapause causes more complete arrest than other stresses yet preserves greater recovery potential. During dormancy, germline stem cells (GSCs) incur DNA damage, activate p53 and Chk2, and divide less. Despite reduced niche signaling, germline precursor cells do not differentiate. GSCs adopt an atypical, suspended state connected to their daughters. Post-diapause recovery of niche signaling and resumption of division contribute to restoring GSCs. Mimicking one feature of quiescence, reduced juvenile hormone production, enhanced GSC longevity in non-diapausing flies. Thus, diapause mechanisms provide approaches to GSC longevity enhancement.
Kim, A. A., Nguyen, A., Marchetti, M., Du, X., Montell, D. J., Pruitt, B. L. and O'Brien, L. E. (2022). Independently paced calcium oscillations in progenitor and differentiated cells in an ex vivo epithelial organ. J Cell Sci. PubMed ID: 35722729
Summary:
Cytosolic calcium is a highly dynamic, tightly regulated, and broadly conserved cellular signal. Calcium dynamics have been studied widely in cellular monocultures, yet organs in vivo comprise heterogeneous populations of stem and differentiated cells. This study examined calcium dynamics in the adult Drosophila intestine, a self-renewing epithelial organ in which stem cells continuously produce daughters that differentiate into either enteroendocrine cells or enterocytes. Live imaging of whole organs ex vivo reveals that stem cell daughters adopt strikingly distinct patterns of calcium oscillations after differentiation: Enteroendocrine cells exhibit single-cell calcium oscillations, while enterocytes exhibit rhythmic, long-range calcium waves. These multicellular waves do not propagate through immature progenitors (stem cells and enteroblasts), whose oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates calcium oscillations in all cell types--even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. These findings establish that cells adopt fate-specific modes of calcium dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently.
Zhao, H., Shi, L., Li, Z., Kong, R., Ren, X., Ma, R., Jia, L., Ma, M., Lu, S., Xu, R., Binari, R., Wang, J. H., Dong, M. Q., Perrimon, N. and Li, Z. (2022). The Yun/Prohibitin complex regulates adult Drosophila intestinal stem cell proliferation through the transcription factor E2F1. Proc Natl Acad Sci U S A 119(6). PubMed ID: 35115400
Summary:
Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. From an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), this study identified a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. It was demonstrated that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, this study found that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, this study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation.
Aggarwal, P., Liu, Z., Cheng, G. Q., Singh, S. R., Shi, C., Chen, Y., Sun, L. V. and Hou, S. X. (2022). Disruption of the lipolysis pathway results in stem cell death through a sterile immunity-like pathway in adult Drosophila. Cell Rep 39(12): 110958. PubMed ID: 35732115
Summary:
Previous work has shown that the Arf1-mediated lipolysis pathway sustains stem cells and cancer stem cells (CSCs); its ablation resulted in necrosis of stem cells and CSCs, which further triggers a systemic antitumor immune response. This study shows that knocking down Arf1 in intestinal stem cells (ISCs) causes metabolic stress, which promotes the expression and translocation of ISC-produced damage-associated molecular patterns (DAMPs; Pretaporter [Prtp] and calreticulin [Calr]). DAMPs regulate macroglobulin complement-related (Mcr) expression and secretion. The secreted Mcr influences the expression and localization of enterocyte (EC)-produced Draper (Drpr) and LRP1 receptors (pattern recognition receptors [PRRs]) to activate autophagy in ECs for ATP production. The secreted ATP possibly feeds back to kill ISCs by activating inflammasome-like pyroptosis. This study identified an evolutionarily conserved pathway that sustains stem cells and CSCs, and its ablation results in an immunogenic cascade that promotes death of stem cells and CSCs as well as antitumor immunity.

Thursday, July 14th - Cytoskeleton and Junctions

Zhao, A. J., Montes-Laing, J., Perry, W. M. G., Shiratori, M., Merfeld, E., Rogers, S. L. and Applewhite, D. A. (2022). The Drosophila spectraplakin Short stop regulates focal adhesion dynamics by cross-linking microtubules and actin. Mol Biol Cell 33(5): ar19. PubMed ID: 35235367
Summary:
The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. This study examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted an analysis of focal adhesion dynamics. The rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, this study found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell-matrix adhesion.
Krishnan, R. K., Halachmi, N., Baskar, R., Bakhrat, A., Zarivach, R., Salzberg, A. and Abdu, U. (2021). Revisiting the Role of beta-Tubulin in Drosophila Development: β-tubulin60D is not an Essential Gene, and its Novel Pin (1) Allele has a Tissue-Specific Dominant-Negative Impact. Front Cell Dev Biol 9: 787976. PubMed ID: 35111755
Summary:
Diversity in cytoskeleton organization and function may be achieved through alternative tubulin isotypes and by a variety of post-translational modifications. The Drosophila genome contains five different β-tubulin paralogs, which may play an isotype tissue-specific function in vivo. One of these genes, the β-tubulin60D gene, which is expressed in a tissue-specific manner, was found to be essential for fly viability and fertility. To further understand the role of the β-tubulin60D gene, new β-tubulin60D null alleles (β-tubulin60D M) using the CRISPR/Cas9 system and found that the homozygous flies were viable and fertile. Moreover, using a combination of genetic complementation tests, rescue experiments, and cell biology analyses, Pin 1, an unknown dominant mutant with bristle developmental defects, was identified as a dominant-negative allele of β-tubulin60D. A missense mutation in the Pin1 mutant that results in an amino acid replacement from the highly conserved glutamate at position 75 to lysine (E75K). Analyzing the β-tubulin structure suggests that this E75K alteration destabilizes the alpha-helix structure and may also alter the GTP-Mg(2+) complex binding capabilities. These results revisited the credence that β-tubulin60D is required for fly viability and revealed for the first time in Drosophila, a novel dominant-negative function of missense β-tubulin60D mutation in bristle morphogenesis.
Hunt, E. L., Rai, H. and Harris, T. J. C. (2022). SCAR/WAVE complex recruitment to a supracellular actomyosin cable by myosin activators and a junctional Arf-GEF during Drosophila dorsal closure. Mol Biol Cell 33(8): br12. PubMed ID: 35476600
Summary:
Expansive Arp2/3 actin networks and contractile actomyosin networks can be spatially and temporally segregated within the cell, but the networks also interact closely at various sites, including adherens junctions. However, molecular mechanisms coordinating these interactions remain unclear. This study found that the SCAR/WAVE complex, an Arp2/3 activator, is enriched at adherens junctions of the leading edge actomyosin cable during Drosophila dorsal closure. Myosin activators were both necessary and sufficient for SCAR/WAVE accumulation at leading edge junctions. The same myosin activators were previously shown to recruit the cytohesin Arf-GEF Steppke to these sites, and mammalian studies have linked Arf small G protein signaling to SCAR/WAVE activation. During dorsal closure, Steppke was found to be required for SCAR/WAVE enrichment at the actomyosin-linked junctions. Arp2/3 also localizes to adherens junctions of the leading edge cable. It is proposed that junctional actomyosin activity acts through Steppke to recruit SCAR/WAVE and Arp2/3 for regulation of the leading edge supracellular actomyosin cable during dorsal closure.
Zhang, N., Zhou, S. J., Ji, H. H. and Li, X. D. (2022). Effects of the IQ1 motif of Drosophila myosin-5 on the calcium interaction of calmodulin. Cell Calcium 103: 102549. PubMed ID: 35144093
Summary:
In Drosophila compound eyes, myosin-5 (DmMyo5) plays a key role in organelle transportation, including transporting pigment granules from the distal end to the proximal end of the photoreceptor cells to regulate the amount of light reaching the photosensitive membrane organelle rhabdomere. It is generally accepted that, upon exposure to light, the dark-adapted compound eyes produce a rapid rise of free Ca(2+) concentration, which in turn activates DmMyo5 to transport pigment granules. Considering the dynamic and compartmentation of Ca(2+) signaling in photoreceptor cells during light exposure, it is necessary to understand the kinetics of Ca(2+) interaction with DmMyo5. This study investigated the interaction of Ca(2+) with Drosophila calmodulin (CaM) in complex with the IQ1 of DmMyo5 using steady-state and kinetic approaches. These results show that IQ1 binding substantially increases the Ca(2+) affinity of CaM and decreases the dissociation rate of Ca(2+) from CaM. In addition, it was found that Mlc-C, the light chain associated with the IQ2 of DmMyo5, has little effect on the Ca(2+) kinetics of CaM in IQ1. It is proposed that, by decreasing the Ca(2+) dissociation rate from CaM, IQ1 delays the deactivation of DmMyo5 after Ca(2+) transition, thereby prolonging the DmMyo5-driven transportation of pigment granules.
Selvaggi, L., Ackermann, M., Pasakarnis, L., Brunner, D. and Aegerter, C. M. (2022). Force measurements of Myosin II waves at the yolk surface during Drosophila dorsal closure. Biophys J 121(3): 410-420. PubMed ID: 34971619
Summary:
The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. A patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. This study used this tool to quantify the in vivo force production of Myosin II waves that were observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing quantitative values on an active Myosin-driven force, this study elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and the mechanical properties of the dorsal yolk cell surface was characterized.
Brooks, D., Bawa, S., Bontrager, A., Stetsiv, M., Guo, Y. and Geisbrecht, E. R. (2022). Independent pathways control muscle tissue size and sarcomere remodeling. Dev Biol 490: 1-12. PubMed ID: 35760368
Summary:
Cell growth and proliferation must be balanced during development to attain a final adult size with the appropriate proportions of internal organs to maximize fitness and reproduction. While multiple signaling pathways coordinate Drosophila development, it is unclear how multi-organ communication within and between tissues converge to regulate systemic growth. One such growth pathway, mediated by insulin-like peptides that bind to and activate the insulin receptor in multiple target tissues, is a primary mediator of organismal size. This study uncover a signaling role for the NUAK serine/threonine kinase in muscle tissue that impinges upon insulin pathway activity to limit overall body size, including a reduction in the growth of individual organs. In skeletal muscle tissue, manipulation of NUAK or insulin pathway components influences sarcomere number concomitant with modulation of thin and thick filament lengths, possibly by modulating the localization of Lasp, a nebulin repeat protein known to set thin filament length. This mode of sarcomere remodeling does not occur in other mutants that also exhibit smaller muscles, suggesting that a sensing mechanism exists in muscle tissue to regulate sarcomere growth that is independent of tissue size control.

Wednesday July 13rd - Cell Cycle

Cartwright, T. N., Harris, R. J., Meyer, S. K., Mon, A. M., Watson, N. A., Tan, C., Marcelot, A., Wang, F., Zinn-Justin, S., Traktman, P. and Higgins, J. M. G. (2022). Dissecting the roles of Haspin and VRK1 in histone H3 phosphorylation during mitosis. Sci Rep 12(1): 11210. PubMed ID: 35778595
Summary:
Protein kinases that phosphorylate histones are ideally-placed to influence the behavior of chromosomes during cell division. Indeed, a number of conserved histone phosphorylation events occur prominently during mitosis and meiosis in most eukaryotes, including on histone H3 at threonine-3 (H3T3ph). At least two kinases, Haspin and VRK1 (NHK-1/ballchen in Drosophila), have been proposed to carry out this modification. Phosphorylation of H3 by Haspin has defined roles in mitosis, but the significance of VRK1 activity towards histones in dividing cells has been unclear. In this study, using in vitro kinase assays, KiPIK screening, RNA interference, and CRISPR/Cas9 approaches, a direct role for VRK1, or its paralogue VRK2, in the phosphorylation of threonine-3 or serine-10 of Histone H3 in mitosis could not be substantiated, although loss of VRK1 did slow cell proliferation. It is concluded that the role of VRKs, and their more recently identified association with neuromuscular disease and importance in cancers of the nervous system, are unlikely to involve mitotic histone kinase activity. In contrast, Haspin is required to generate H3T3ph during mitosis.
Fu, Y., Lv, Z., Kong, D., Fan, Y. and Dong, B. (2022). High abundance of CDC45 inhibits cell proliferation through elevation of HSPA6. Cell Prolif 55(7): e13257. PubMed ID: 35642733
Summary:
CDC45 is the core component of CMG (CDC45-MCMs-GINS) complex that plays important role in the initial step of DNA replication in eukaryotic cells. The expression level of cdc45 is under the critical control for the accurate cell cycle progression. A systematic analysis of the effect of high dose of CDC45 on cell physiology and behaviors is unclear. The present study aimed to investigate the effects and mechanisms of high dose of CDC45 on cell behaviors. cdc45 was overexpressed in cultured cell lines, Ciona, and Drosophila embryos, respectively. The cell cycle progression. High levels of cdc45 from different species (human, mammal, ascidian, and Drosophila) were found to inhibit cell cycle in vitro and in vivo. High dose of CDC45 blocks cells entering into S phase. However, DNA damage and cell apoptosis were not detected. hspa6 was the most upregulated gene in HeLa cells overexpressing cdc45 as detected using RNA-seq analysis and qRT-PCR validation. Overexpression of Hs-hspa6 inhibited proliferation rate and DNA replication in HeLa cells, mimicking the phenotype of cdc45 overexpression. RNAi against hspa6 partially rescued the cell proliferation defect caused by high dose of CDC45. This study suggests that high abundance of CDC45 stops cell cycle. Instead of inducing apoptosis, excessive CDC45 prevents cell entering S phase probably due to promoting hspa6 expression.
Sainz de la Maza, D., Hof-Michel, S., Phillimore, L., Bokel, C. and Amoyel, M. (2022). Cell-cycle exit and stem cell differentiation are coupled through regulation of mitochondrial activity in the Drosophila testis. Cell Rep 39(6): 110774. PubMed ID: 35545055
Summary:
Whereas stem and progenitor cells proliferate to maintain tissue homeostasis, fully differentiated cells exit the cell cycle. How cell identity and cell-cycle state are coordinated during differentiation is still poorly understood. The Drosophila testis niche supports germline stem cells and somatic cyst stem cells (CySCs). CySCs give rise to post-mitotic cyst cells, providing a tractable model to study the links between stem cell identity and proliferation. While cell-cycle progression was shown to be required for CySC self-renewal, the E2f1/Dp transcription factor is dispensable for self-renewal but instead must be silenced by the Drosophila retinoblastoma homolog, Rbf, to permit differentiation. Continued E2f1/Dp activity inhibits the expression of genes important for mitochondrial activity. Furthermore, promoting mitochondrial biogenesis rescues the differentiation of CySCs with ectopic E2f1/Dp activity but not their cell-cycle exit. In sum, E2f1/Dp coordinates cell-cycle progression with stem cell identity by regulating the metabolic state of CySCs.
Vazquez-Pianzola, P., Beuchle, D., Saro, G., Hernandez, G., Maldonado, G., Brunssen, D., Meister, P. and Suter, B. (2022). Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal-D. Development. PubMed ID: 35723263
Summary:
Bicaudal-D (BicD) is a dynein adaptor that transports different cargoes along microtubules. Reducing the activity of BicD specifically in freshly laid Drosophila eggs by acute protein degradation revealed that BicD is needed to produce normal female meiosis II products, to prevent female meiotic products from re-entering the cell cycle, and for pronuclear fusion. As BicD is required to localize the spindle assembly checkpoint (SAC) components Mad2 and BubR1 to the female meiotic products, it appears that BicD functions to localize them to control metaphase arrest of polar bodies. BicD interacts with Clathrin heavy chain (Chc), and both proteins localize to centrosomes, mitotic spindles, and the tandem spindles during female meiosis II. Furthermore, BicD is required to correctly localize clathrin and the microtubule-stabilizing factors, D-TACC and Msps, to the meiosis II spindles, suggesting that failure to localize these proteins may perturb SAC function. Furthermore, right after the establishment of the female pronucleus, D-TACC and C. elegans BicD, tacc, and Chc are also needed for pronuclear fusion, pointing to the possibility that the underlying mechanism might be more widely used.
Binh, T. D., Nguyen, Y. D. H., Pham, T. L. A., Komori, K., Nguyen, T. Q. C., Taninaka, M. and Kamei, K. (2022). Dysfunction of lipid storage droplet-2 suppresses endoreplication and induces JNK pathway-mediated apoptotic cell death in Drosophila salivary glands. Sci Rep 12(1): 4302. PubMed ID: 35277579
Summary:
The lipid storage droplet-2 (LSD-2) protein of Drosophila is a homolog of mammalian perilipin 2, which is essential for promoting lipid accumulation and lipid droplet formation. The function of LSD-2 as a regulator of lipolysis has also been demonstrated. However, other LSD-2 functions remain unclear. To investigate the role of LSD-2, tissue-specific depletion in the salivary glands of Drosophila was performed using a combination of the Gal4-upstream activating sequence system and RNA interference. LSD-2 depletion inhibited the entry of salivary gland cells into the endoreplication cycle and delayed this process by enhancing CycE expression, disrupting the development of this organ. The deficiency of LSD-2 expression enhanced reactive oxygen species production in the salivary gland and promoted JNK-dependent apoptosis by suppressing dMyc expression. This phenomenon did not result from lipolysis. Therefore, LSD-2 is vital for endoreplication cell cycle and cell death programs.
Yang, S., McAdow, J., Du, Y., Trigg, J., Taghert, P. H. and Johnson, A. N. (2022). Spatiotemporal expression of regulatory kinases directs the transition from mitosis to cellular morphogenesis in Drosophila. Nat Commun 13(1): 772. PubMed ID: 35140224
Summary:
Embryogenesis depends on a tightly regulated balance between mitosis, differentiation, and morphogenesis. Understanding how the embryo uses a relatively small number of proteins to transition between growth and morphogenesis is a central question of developmental biology, but the mechanisms controlling mitosis and differentiation are considered to be fundamentally distinct. This study shows the mitotic kinase Polo, which regulates all steps of mitosis in Drosophila, also directs cellular morphogenesis after cell cycle exit. In mitotic cells, the Aurora kinases activate Polo to control a cytoskeletal regulatory module that directs cytokinesis. In the post-mitotic mesoderm, the control of Polo activity transitions from the Aurora kinases to the uncharacterized kinase Back Seat Driver (Bsd), where Bsd and Polo cooperate to regulate muscle morphogenesis. Polo and its effectors therefore direct mitosis and cellular morphogenesis, but the transition from growth to morphogenesis is determined by the spatiotemporal expression of upstream activating kinases.

Tuesday, July 12 - Adult neural development and function

Wu, S. T., Chen, J. Y., Martin, V., Ng, R., Zhang, Y., Grover, D., Greenspan, R. J., Aljadeff, J. and Su, C. Y. (2022). Valence opponency in peripheral olfactory processing. Proc Natl Acad Sci U S A 119(5). PubMed ID: 35091473
Summary:
A hallmark of complex sensory systems is the organization of neurons into functionally meaningful maps, which allow for comparison and contrast of parallel inputs via lateral inhibition. However, it is unclear whether such a map exists in olfaction. This study addressed this question by determining the organizing principle underlying the stereotyped pairing of olfactory receptor neurons (ORNs) in Drosophila sensory hairs, wherein compartmentalized neurons inhibit each other via ephaptic coupling. Systematic behavioral assays reveal that most paired ORNs antagonistically regulate the same type of behavior. Such valence opponency is relevant in critical behavioral contexts including place preference, egg laying, and courtship. Odor-mixture experiments show that ephaptic inhibition provides a peripheral means for evaluating and shaping countervailing cues relayed to higher brain centers. Furthermore, computational modeling suggests that this organization likely contributes to processing ratio information in odor mixtures. This olfactory valence map may have evolved to swiftly process ethologically meaningful odor blends without involving costly synaptic computation.
Chen, D. S., Clark, A. G. and Wolfner, M. F. (2022). Octopaminergic/tyraminergic Tdc2 neurons regulate biased sperm usage in female Drosophila melanogaster. Genetics. PubMed ID: 35736370
Summary:
In polyandrous internally fertilizing species, a multiply-mated female can use stored sperm from different males in a biased manner to fertilize her eggs. The female's ability to assess sperm quality and compatibility is essential for her reproductive success, and represents an important aspect of postcopulatory sexual selection. In Drosophila melanogaster, previous studies demonstrated that the female nervous system plays an active role in influencing progeny paternity proportion, and suggested a role for octopaminergic/tyraminergic Tdc2 neurons in this process. This study reports that inhibiting Tdc2 neuronal activity causes females to produce a higher-than-normal proportion of first-male progeny. This difference is not due to differences in sperm storage or release, but instead is attributable to the suppression of second-male sperm usage bias that normally occurs in control females. It was further shown that a subset of Tdc2 neurons innervating the female reproductive tract is largely responsible for the progeny proportion phenotype that is observed when Tdc2 neurons are inhibited globally. On the contrary, overactivation of Tdc2 neurons does not further affect sperm storage and release or progeny proportion. These results suggest that octopaminergic/tyraminergic signaling allows a multiply-mated female to bias sperm usage, and identify a new role for the female nervous system in postcopulatory sexual selection.
Sood, C., Justis, V. T., Doyle, S. E. and Siegrist, S. E. (2022). Notch signaling regulates neural stem cell quiescence entry and exit in Drosophila. Development 149(4). PubMed ID: 35112131
Summary:
Stem cells enter and exit quiescence as part of normal developmental programs and to maintain tissue homeostasis in adulthood. Although it is clear that stem cell intrinsic and extrinsic cues, local and systemic, regulate quiescence, it remains unclear whether intrinsic and extrinsic cues coordinate to control quiescence and how cue coordination is achieved. This study reports that Notch signaling coordinates neuroblast intrinsic temporal programs with extrinsic nutrient cues to regulate quiescence in Drosophila. When Notch activity is reduced, quiescence is delayed or altogether bypassed, with some neuroblasts dividing continuously during the embryonic-to-larval transition. During embryogenesis before quiescence, neuroblasts express Notch and the Notch ligand Delta. After division, Delta is partitioned to adjacent GMC daughters where it transactivates Notch in neuroblasts. Over time, in response to intrinsic temporal cues and increasing numbers of Delta-expressing daughters, neuroblast Notch activity increases, leading to cell cycle exit and consequently, attenuation of Notch pathway activity. Quiescent neuroblasts have low to no active Notch, which is required for exit from quiescence in response to nutrient cues. Thus, Notch signaling coordinates proliferation versus quiescence decisions.
Suzuki, Y., Kurata, Y. and Sakai, T. (2022). Dorsal-lateral clock neurons modulate consolidation and maintenance of long-term memory in Drosophila. Genes Cells 27(4): 266-279. PubMed ID: 35094465
Summary:
A newly formed memory is initially unstable. However, if it is consolidated into the brain, the consolidated memory is stored as stable long-term memory (LTM). Despite the recent progress, the molecular and cellular mechanisms of LTM have not yet been fully elucidated. The fruitfly Drosophila melanogaster, for which various genetic tools are available, has been used to clarify the molecular mechanisms of LTM. Using the Drosophila courtship-conditioning assay as a memory paradigm, previous work identified that the circadian clock gene period (per) plays a vital role in consolidating LTM, suggesting that per-expressing clock neurons are critically involved in LTM. However, it is still incompletely understood which clock neurons are essential for LTM. This study shows that dorsal-lateral clock neurons (LNds) play a crucial role in LTM. Using an LNd-specific split-GAL4 line, this study confirmed that disruption of synaptic transmission in LNds impaired LTM maintenance. On the other hand, induction of per RNAi or the dominant-negative transgene of Per in LNds impaired LTM consolidation. These results reveal that transmitter release and Per function in LNds are involved in courtship memory processing.
Andreani, T., Rosensweig, C., Sisobhan, S., Ogunlana, E., Kath, W. and Allada, R. (2022). Circadian programming of the ellipsoid body sleep homeostat in Drosophila. Elife 11. PubMed ID: 35735904
Summary:
Homeostatic and circadian processes collaborate to appropriately time and consolidate sleep and wake. To understand how these processes are integrated, brief sleep deprivation was scheduled at different times of day in Drosophila, and elevated morning rebound was compared to evening. These effects depend on discrete morning and evening clock neurons, independent of their roles in circadian locomotor activity. In the R5 ellipsoid body sleep homeostat, this study identified elevated morning expression of activity dependent and presynaptic gene expression as well as the presynaptic protein BRUCHPILOT consistent with regulation by clock circuits. These neurons also display elevated calcium levels in response to sleep loss in the morning, but not the evening consistent with the observed time-dependent sleep rebound. These studies reveal the circuit and molecular mechanisms by which discrete circadian clock neurons program a homeostatic sleep center.
Chen, N., Zhang, Y., Adel, M., Kuklin, E. A., Reed, M. L., Mardovin, J. D., Bakthavachalu, B., VijayRaghavan, K., Ramaswami, M. and Griffith, L. C. (2022). Local translation provides the asymmetric distribution of CaMKII required for associative memory formation. Curr Biol 32(12): 2730-2738. PubMed ID: 35545085
Summary:
How compartment-specific local proteomes are generated and maintained is inadequately understood, particularly in neurons, which display extreme asymmetries. This study shows that local enrichment of Ca(2+)/calmodulin-dependent protein kinase II CaMKII) in axons of Drosophila mushroom body neurons is necessary for cellular plasticity and associative memory formation. Enrichment is achieved via enhanced axoplasmic translation of CaMKII mRNA, through a mechanism requiring the RNA-binding protein Mub and a 23-base Mub-recognition element in the CaMKII 3' UTR. Perturbation of either dramatically reduces axonal, but not somatic, CaMKII protein without altering the distribution or amount of mRNA in vivo, and both are necessary and sufficient to enhance axonal translation of reporter mRNA. Together, these data identify elevated levels of translation of an evenly distributed mRNA as a novel strategy for generating subcellular biochemical asymmetries. They further demonstrate the importance of distributional asymmetry in the computational and biological functions of neurons.

Monday, July 11th - Disease models

Haddadi, M. and Ataei, R. (2022). wde, calpA, if, dap160, and poe genes knock down Drosophila models exhibit neurofunctional deficit. Gene 829: 146499. PubMed ID: 35447243
Summary:
Intellectual disability (ID) is a heterogeneous disorder with high prevalence and remarkable social and cost burdens. Novel genetic variants of ATF7IP, CAPN9, ITGAV, ITSN1, and UBR4 genes are reported to be associated with the ID among Iranian families. However, in vivo validation is required to confirm the functional role of these variants in ID development. Drosophila melanogaster is a convenient model for such functional investigations as its genome bears ortholog of more than 75% of the disease-causing genes in human and represents numerous approaches to study defects in neuronal function. In this connection, RNAi gene silencing was applied to wde, calpA, if, dap160, and poe genes, the Drosophila ortholog of the selected human genes, and then consequent structural and functional changes in neurons were studied by means of immunohistochemistry and confocal microscopy of mushroom bodies (MBs) and validated behavioural assays including larvae and adult conditioning learning and memories, and ethanol sensitivity. Down-regulation of these genes led to neuronal loss which was evident by decline in total fluorescent signal intensity in micrographs of MBs structure. The gene silencing caused neuronal dysfunction and induction of ID-like symptoms manifested by deficits in larval preference learning, and short-term olfactory memory and courtship suppression learning in adults. Moreover, the RNAi flies showed higher sensitivity to ethanol vapour. Interestingly, the poe knock-down flies exhibited the most severe phenotypes among other genes. Altogether, this study is highly applicable to confirm pathogenecity of the selected ID gene variants in Iranian population.
Buhl, E., Kim, Y. A., Parsons, T., Zhu, B., Santa-Maria, I., Lefort, R. and Hodge, J. J. L. (2022). Effects of Eph/ephrin signalling and human Alzheimer's disease-associated EphA1 on Drosophila behaviour and neurophysiology. Neurobiol Dis 170: 105752. PubMed ID: 35569721
Summary:
Alzheimer's disease (AD) is the most prevalent neurodegenerative disease placing a great burden on people living with it, careers and society. Yet, the underlying patho-mechanisms remain unknown and treatments limited. To better understand the molecular changes associated with AD, genome-wide association studies (GWAS) have identified hundreds of candidate genes linked to the disease, like the receptor tyrosine kinase EphA1. However, demonstration of whether and how these genes cause pathology is largely lacking. Utilising fly genetics, this study generated the first Drosophila model of human wild-type and P460L mutant EphA1 and tested the effects of Eph/ephrin signalling on AD-relevant behaviour and neurophysiology. EphA1 mis-expression did not cause neurodegeneration, shorten lifespan or affect memory but flies mis-expressing the wild-type or mutant receptor were hyper-aroused, had reduced sleep, a stronger circadian rhythm and increased clock neuron activity and excitability. Over-expression of endogenous fly Eph and RNAi-mediated knock-down of Eph and its ligand ephrin affected sleep architecture and neurophysiology. Eph over-expression led to stronger circadian morning anticipation while ephrin knock-down impaired memory. A dominant negative form of the GTPase Rho1, a potential intracellular effector of Eph, led to hyper-aroused flies, memory impairment, less anticipatory behaviour and neurophysiological changes. These results demonstrate a role of Eph/ephrin signalling in a range of behaviours affected in AD. This presents a starting point for studies into the underlying mechanisms of AD including interactions with other AD-associated genes, like Rho1, Ankyrin, Tau and APP with the potential to identify new targets for treatment.
Schmidt, C. A., Min, L. Y., McVay, M. H., Giusto, J. D., Brown, J. C., Salzler, H. R. and Matera, A. G. (2022). Mutations in Drosophila tRNA processing factors cause phenotypes similar to Pontocerebellar Hypoplasia. Biol Open 11(3). PubMed ID: 35132432
Summary:
Mature transfer (t)RNAs are generated by multiple RNA processing events, which can include the excision of intervening sequences. The tRNA splicing endonuclease (TSEN) complex is responsible for cleaving these intron-containing pre-tRNA transcripts. In humans, TSEN copurifies with CLP1, an RNA kinase. Despite extensive work on CLP1, its in vivo connection to tRNA splicing remains unclear. Interestingly, mutations in CLP1 or TSEN genes cause neurological diseases in humans that are collectively termed Pontocerebellar Hypoplasia (PCH). In mice, loss of Clp1 kinase activity results in premature death, microcephaly and progressive loss of motor function. To determine if similar phenotypes are observed in Drosophila, this study characterized mutations in crowded-by-cid (cbc), the CLP1 ortholog, as well as in the fly ortholog of human TSEN54. Analyses of organismal viability, larval locomotion and brain size revealed that mutations in both cbc and Tsen54 phenocopy those in mammals in several details. In addition to an overall reduction in brain lobe size, increased cell death was found in mutant larval brains. Ubiquitous or tissue-specific knockdown of cbc in neurons and muscles reduced viability and locomotor function. These findings indicate that PCH can be successfully modeled in a genetically-tractable invertebrate.
Taylor, J. R., Wood, J. G., Mizerak, E., Hinthorn, S., Liu, J., Finn, M., Gordon, S., Zingas, L., Chang, C., Klein, M. A., Denu, J. M., Gorbunova, V., Seluanov, A., Boeke, J. D., Sedivy, J. M. and Helfand, S. L. (2022). Sirt6 regulates lifespan in Drosophila melanogaster. Proc Natl Acad Sci U S A 119(5). PubMed ID: 35091469
Summary:
Sirt6 is a multifunctional enzyme that regulates diverse cellular processes such as metabolism, DNA repair, and aging. Overexpressing Sirt6 extends lifespan in mice, but the underlying cellular mechanisms are unclear. Drosophila melanogaster are an excellent model to study genetic regulation of lifespan; however, despite extensive study in mammals, very little is known about Sirt6 function in flies. This study characterized the Drosophila ortholog of Sirt6, dSirt6, and examined its role in regulating longevity; dSirt6 is a nuclear and chromatin-associated protein with NAD(+)-dependent histone deacetylase activity. dSirt6 overexpression (OE) in flies produces robust lifespan extension in both sexes, while reducing dSirt6 levels shortens lifespan. dSirt6 OE flies have normal food consumption and fertility but increased resistance to oxidative stress and reduced protein synthesis rates. Transcriptomic analyses reveal that dSirt6 OE reduces expression of genes involved in ribosome biogenesis, including many dMyc target genes. dSirt6 OE partially rescues many effects of dMyc OE, including increased nuclear size, up-regulation of ribosome biogenesis genes, and lifespan shortening. Last, dMyc haploinsufficiency does not convey additional lifespan extension to dSirt6 OE flies, suggesting dSirt6 OE is upstream of dMyc in regulating lifespan. These results provide insight into the mechanisms by which Sirt6 OE leads to longer lifespan.
Marcogliese, P. C., Dutta, D., Ray, S. S., Dang, N. D. P., Zuo, Z., Wang, Y., Lu, D., Fazal, F., Ravenscroft, T. A., Chung, H., Kanca, O., Wan, J., Douine, E. D., Network, U. D., Pena, L. D. M., Yamamoto, S., Nelson, S. F., Might, M., Meyer, K. C., Yeo, N. C. and Bellen, H. J. (2022). Loss of IRF2BPL impairs neuronal maintenance through excess Wnt signaling. Sci Adv 8(3): eabl5613. PubMed ID: 35044823
Summary:
De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, its function was studied in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits, the Drosophila ortholog, represses Wnt transcription in flies. In contrast, neuronal depletion of Pits leads to increased wingless (wg) levels in the brain and is associated with axonal loss, whereas inhibition of Wg signaling is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects with an associated increase in wnt1 transcription and downstream signaling. WNT1 is also increased in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Last, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt transcription and signaling.
Pulianmackal, A. J., Kanakousaki, K., Flegel, K., Grushko, O. G., Gourley, E., Rozich, E. and Buttitta, L. A. (2022). Misregulation of Nucleoporins 98 and 96 leads to defects in protein synthesis that promote hallmarks of tumorigenesis. Dis Model Mech 15(3). PubMed ID: 35107131
Summary:
Nucleoporin 98KD (Nup98) is a promiscuous translocation partner in hematological malignancies. Most disease models of Nup98 translocations involve ectopic expression of the fusion protein under study, leaving the endogenous Nup98 loci unperturbed. Overlooked in these approaches is the loss of one copy of normal Nup98 in addition to the loss of Nup96 - a second Nucleoporin encoded within the same mRNA and reading frame as Nup98 - in translocations. Nup98 and Nup96 are also mutated in a number of other cancers, suggesting that their disruption is not limited to blood cancers. This study found that reducing Nup98-96 function in Drosophila melanogaster (in which the Nup98-96 shared mRNA and reading frame is conserved) de-regulates the cell cycle. Evidence was found of overproliferation in tissues with reduced Nup98-96, counteracted by elevated apoptosis and aberrant signaling associated with chronic wounding. Reducing Nup98-96 function led to defects in protein synthesis that triggered JNK signaling and contributed to hallmarks of tumorigenesis when apoptosis was inhibited. It is suggested that partial loss of Nup98-96 function in translocations could de-regulate protein synthesis, leading to signaling that cooperates with other mutations to promote tumorigenesis.

Friday, July 8th - Chromatin

Bughio, F. J. and Maggert, K. A. (2022). Live analysis of position-effect variegation in Drosophila reveals different modes of action for HP1a and Su(var)3-9. Proc Natl Acad Sci U S A 119(25): e2118796119. PubMed ID: 35704756
Summary:
Position-effect variegation (PEV) results from the juxtaposition of euchromatic and heterochromatic components of eukaryotic genomes, silencing genes near the new euchromatin/heterochromatin junctions. Silencing is itself heritable through S phase, giving rise to distinctive random patterns of cell clones expressing the genes intermixed with clones in which the genes are silenced. Much of what is known about epigenetic inheritance in the soma stems from work on PEV aimed at identifying the components of the silencing machinery and its mechanism of inheritance. The roles of two central gene activities-the Su(var)3-9-encoded histone H3-lysine-9 methyltransferase and the Su(var)205-encoded methyl-H3-lysine-9 binding protein heterochromatin protein 1 (HP1a)-have been inferred from terminal phenotypes, leaving considerable gaps in understanding of how PEV behaves through development. This study investigate the PEV phenotypes of Su(var)3-9 and Su(var)205 mutations in live developing tissues. Mutation in Su(var)205 compromises the initial establishment of PEV in early embryogenesis. Later gains of heterochromatin-induced gene silencing are possible but are unstable and lost rapidly. In contrast, a strain with mutation in Su(var)3-9 exhibits robust silencing early in development but fails to maintain it through subsequent cell divisions. This analyses show that, while the terminal phenotypes of these mutations may appear identical, they have arrived at them through different developmental trajectories. How these findings expand and clarify existing models for epigenetic inheritance of heterochromatin-induced gene silencing is discussed.
Pettie, N., Llopart, A. and Comeron, J. M. (2022). Meiotic, genomic and evolutionary properties of crossover distribution in Drosophila yakuba. PLoS Genet 18(3): e1010087. PubMed ID: 35320272
Summary:
The number and location of crossovers across genomes are highly regulated during meiosis, yet the key components controlling them are fast evolving, hindering understanding of the mechanistic causes and evolutionary consequences of changes in crossover rates. This study applied a novel and highly efficient approach to generate whole-genome high-resolution crossover maps in D. yakuba to tackle multiple questions that benefit from being addressed collectively within an appropriate phylogenetic framework, in this case the D. melanogaster species subgroup. The genotyping of more than 1,600 individual meiotic events allowed identification of several key distinct properties relative to D. melanogaster. D. yakuba, in addition to higher crossover rates than D. melanogaster, has a stronger centromere effect and crossover assurance than any Drosophila species analyzed to date. The presence of an active crossover-associated meiotic drive mechanism is reported for the X chromosome that results in the preferential inclusion in oocytes of chromatids with crossovers. This evolutionary and genomic analyses suggest that the genome-wide landscape of crossover rates in D. yakuba has been fairly stable and captures a significant signal of the ancestral crossover landscape for the whole D. melanogaster subgroup, even informative for the D. melanogaster lineage. Contemporary crossover rates in D. melanogaster, on the other hand, do not recapitulate ancestral crossovers landscapes. As a result, the temporal stability of crossover landscapes observed in D. yakuba makes this species an ideal system for applying population genetic models of selection and linkage, given that these models assume temporal constancy in linkage effects.
Batut, P. J., Bing, X. Y., Sisco, Z., Raimundo, J., Levo, M. and Levine, M. S. (2022). Genome organization controls transcriptional dynamics during development. Science 375(6580): 566-570. PubMed ID: 35113722
Summary:
Past studies offer contradictory claims for the role of genome organization in the regulation of gene activity. This study shows through high-resolution chromosome conformation analysis that the Drosophila genome is organized by two independent classes of regulatory sequences, tethering elements and insulators. Quantitative live imaging and targeted genome editing demonstrate that this two-tiered organization is critical for the precise temporal dynamics of Hox gene transcription during development. Tethering elements mediate long-range enhancer-promoter interactions and foster fast activation kinetics. Conversely, the boundaries of topologically associating domains (TADs) prevent spurious interactions with enhancers and silencers located in neighboring TADs. These two levels of genome organization operate independently of one another to ensure precision of transcriptional dynamics and the reliability of complex patterning processes.
Stow, E. C., Simmons, J. R., An, R., Schoborg, T. A., Davenport, N. M. and Labrador, M. (2022). A Drosophila insulator interacting protein suppresses enhancer-blocking function and modulates replication timing. Gene 819: 146208. PubMed ID: 35092858
Summary:
The different roles of insulator protein partners in the Drosophila genome and how they confer functional specificity remain poorly understood. The Suppressor of Hairy wing [Su(Hw)] insulator is targeted to the nuclear lamina, preferentially localizes at euchromatin/heterochromatin boundaries, and is associated with the gypsy retrotransposon. Insulator activity relies on the ability of the Su(Hw) protein to bind the DNA at specific sites and interact with Mod(mdg4)67.2 and CP190 partner proteins. HP1 and insulator partner protein 1 (HIPP1) is a partner of Su(Hw), but how HIPP1 contributes to the function of Su(Hw) insulator complexes is unclear. This study demonstrates that HIPP1 colocalizes with the Su(Hw) insulator complex in polytene chromatin and in stress-induced insulator bodies. Overexpression of either HIPP1 or Su(Hw) or mutation of the HIPP1 crotonase-like domain (CLD) causes defects in cell proliferation by limiting the progression of DNA replication. It was also shown that HIPP1 overexpression suppresses the Su(Hw) insulator enhancer-blocking function, while mutation of the HIPP1 CLD does not affect Su(Hw) enhancer blocking. These findings demonstrate a functional relationship between HIPP1 and the Su(Hw) insulator complex and suggest that the CLD, while not involved in enhancer blocking, influences cell cycle progression.
Sun, Y., Li, Z., Li, W. and Xue, L. (2022). Loss of Bicra impairs Drosophila learning and choice abilities. Neurosci Lett 769: 136432. PubMed ID: 34974109
Summary:
The Drosophila Bicra (CG11873) gene encodes the sole ortholog of mammalian GLTSCR1 and GLTSCR1L, which are components of a chromatin remodeling complex involved in neoplasia and metastasis of cancer cells. Bicra is highly expressed in Drosophila larval CNS and adult brain, yet its physiological functions in the nervous system remain elusive. This study reports that Bicra is expressed in both neurons and glia of adult brains, and is required for courtship learning and choice ability of male flies. The function of Bicra in the mushroom body, and in particular, Bicra expression in neurons but not glia, is responsible for the male courtship learning and choice performance. This study unravels a novel function of Bicra in cognition-related courtship behaviors in Drosophila, and may provide insight into the neuronal functions of its mammalian orthologs.
Tsai, A. and Crocker, J. (2022). Nuclear morphogenesis: forming a heterogeneous nucleus during embryogenesis. Development 149(4). PubMed ID: 35142344
Summary:
An embryo experiences increasingly complex spatial and temporal patterns of gene expression as it matures, guiding the morphogenesis of its body. Using super-resolution fluorescence microscopy in Drosophila melanogaster embryos, it was observed that the nuclear distributions of transcription factors and histone modifications undergo a similar transformation of increasing heterogeneity. This spatial partitioning of the nucleus could lead to distinct local regulatory environments in space and time that are tuned for specific genes. Accordingly, transcription sites driven by different cis-regulatory regions each had their own temporally and spatially varying local histone environments, which could facilitate the finer spatial and temporal regulation of genes to consistently differentiate cells into organs and tissues. Thus, 'nuclear morphogenesis' may be a microscopic counterpart of the macroscopic process that shapes the animal body.

Thursday, July 7th - Adult and larval PhySiology and metabolism

Loya, A. K., Van Houten, S. K., Glasheen, B. M. and Swank, D. M. (2022). Shortening deactivation: quantifying a critical component of cyclical muscle contraction. Am J Physiol Cell Physiol 322(4): C653-c665. PubMed ID: 34965153
Summary:
A muscle undergoing cyclical contractions requires fast and efficient muscle activation and relaxation to generate high power with relatively low energetic cost. To enhance activation and increase force levels during shortening, some muscle types have evolved stretch activation (SA), a delayed increased in force following rapid muscle lengthening. SA's complementary phenomenon is shortening deactivation (SD), a delayed decrease in force following muscle shortening. SD increases muscle relaxation, which decreases resistance to subsequent muscle lengthening. Although it might be just as important to cyclical power output, SD has received less investigation than SA. To enable mechanistic investigations into SD and quantitatively compare it to SA, this study developed a protocol to elicit SA and SD from Drosophila and Lethocerus indirect flight muscles (IFM) and Drosophila jump muscle. When normalized to isometric tension, Drosophila IFM exhibited a 118% SD tension decrease, Lethocerus IFM dropped by 97%, and Drosophila jump muscle decreased by 37%. The same order was found for normalized SA tension: Drosophila IFM increased by 233%, Lethocerus IFM by 76%, and Drosophila jump muscle by only 11%. SD occurred slightly earlier than SA, relative to the respective length change, for both IFMs; but SD was exceedingly earlier than SA for jump muscle. These results suggest SA and SD evolved to enable highly efficient IFM cyclical power generation and may be caused by the same mechanism. However, jump muscle SA and SD mechanisms are likely different, and may have evolved for a role other than to increase the power output of cyclical contractions.
Nagle, C., Bhogal, J. K., Nagengast, A. A. and DiAngelo, J. R. (2022). Transportin-serine/arginine-rich (Tnpo-SR) proteins are necessary for proper lipid storage in the Drosophila fat body. Biochem Biophys Res Commun 596: 1-5. PubMed ID: 35104661
Summary:
After a meal, excess nutrients are stored within adipose tissue as triglycerides in structures called lipid droplets. Previous genome-wide RNAi screens have identified that mRNA splicing factor genes are required for normal lipid droplet formation in Drosophila cells. Previous work has shown that mRNA splicing factors called serine/arginine-rich (SR) proteins are important for triglyceride storage in the Drosophila fat body. SR proteins shuttle in and out of the nucleus with the help of proteins called Transportins (Tnpo-SR); however, whether this transport is important for SR protein-mediated regulation of lipid storage is unknown. The purpose of this study is to characterize the role of Tnpo-SR proteins in regulating lipid storage in the Drosophila fat body. Decreasing Tnpo-SR in the adult fat body resulted in an increase in triglyceride storage and consistent with this phenotype, Tnpo-SR-RNAi flies also have increased starvation resistance. In addition, the lipid accumulation in Tnpo-SR-RNAi flies is the result of increased triglyceride stored in each fat body cell and not due to increased food consumption. Interestingly, the splicing of CPT1, an enzyme important for the β-oxidation of fatty acids, is altered in Tnpo-SR-RNAi fat bodies. The isoform that produces the less catalytically active form of CPT1 accumulates in fat bodies where Tnpo-SR levels are decreased, suggesting a decrease in lipid breakdown, potentially causing the excess triglyceride storage observed in these flies. Together, these data suggest that the transport of splicing proteins in and out of the nucleus is important for proper triglyceride storage in the Drosophila fat body.
Kubrak, O., Koyama, T., Ahrentlov, N., Jensen, L., Malita, A., Naseem, M. T., Lassen, M., Nagy, S., Texada, M. J., Halberg, K. V. and Rewitz, K. (2022). The gut hormone Allatostatin C/Somatostatin regulates food intake and metabolic homeostasis under nutrient stress. Nat Commun 13(1): 692. PubMed ID: 35121731
Summary:
The intestine is a central regulator of metabolic homeostasis. Dietary inputs are absorbed through the gut, which senses their nutritional value and relays hormonal information to other organs to coordinate systemic energy balance. However, the gut-derived hormones affecting metabolic and behavioral responses are poorly defined. This study shows that the endocrine cells of the Drosophila gut sense nutrient stress through a mechanism that involves the TOR pathway and in response secrete the peptide hormone allatostatin C, a Drosophila somatostatin homolog. Gut-derived allatostatin C induces secretion of glucagon-like adipokinetic hormone to coordinate food intake and energy mobilization. Loss of gut Allatostatin C or its receptor in the adipokinetic-hormone-producing cells impairs lipid and sugar mobilization during fasting, leading to hypoglycemia. These findings illustrate a nutrient-responsive endocrine mechanism that maintains energy homeostasis under nutrient-stress conditions, a function that is essential to health and whose failure can lead to metabolic disorders.
Najjar, H., Al-Ashmar, S., Qush, A., Al-Asmar, J., Rashwan, S., Elgamal, A., Zeidan, A. and Kamareddine, L. (2022). Enteric Pathogens Modulate Metabolic Homeostasis in the Drosophila melanogaster host. Microbes Infect: 104946. PubMed ID: 35093552
Summary:
On daily basis, living beings work out an armistice with their microbial flora and a scuffle with invading pathogens to maintain a normal state of health. Although producing virulence factors and escaping the host's immune machinery are the paramount tools used by pathogens in their 'arm race' against the host, this study provides insight into another facet of pathogenic embitterment by presenting evidence of the ability of enteric pathogens to exhibit pathogenicity through modulating metabolic homeostasis in Drosophila melanogaster. Escherichia coli and Shigella sonnei orally infected flies exhibit lipid droplet deprivation from the fat body, irregular accumulation of lipid droplets in the midgut, and significant elevation of systemic glucose and triglyceride levels. These detected metabolic alterations in infected flies could be attributed to differential regulation of peptide hormones known to be crucial for lipid metabolism and insulin signaling. Gaining a proper understanding of infection-induced alterations succeeds in curbing the pathogenesis of enteric diseases and sets the stage for promising therapeutic approaches to pursue infection-induced metabolic disorders.
Gallo, M., Vento, J. M., Joncour, P., Quagliariello, A., Maritan, E., Silva-Soares, N. F., Battistolli, M., Beisel, C. L. and Martino, M. E. (2022). Beneficial commensal bacteria promote Drosophila growth by downregulating the expression of peptidoglycan recognition proteins. iScience 25(6): 104357. PubMed ID: 35601912
Summary:
Commensal bacteria are known to promote host growth. Such effect partly relies on the capacity of microbes to regulate the host's transcriptional response. However, these evidences mainly come from comparing the transcriptional response caused by commensal bacteria with that of axenic animals, making it difficult to identify the animal genes that are specifically regulated by beneficial microbes. This study employed Drosophila melanogaster associated with Lactiplantibacillus plantarum to understand the host genetic pathways regulated by beneficial bacteria and leading to improved host growth. Microbial benefit to the host relies on the downregulation of peptidoglycan-recognition proteins. Specifically, this paper reports that bacterial proliferation triggers the lower expression of PGRP-SC1 in larval midgut, which ultimately leads to improved host growth and development. This study helps elucidate the mechanisms underlying the beneficial effect exerted by commensal bacteria, defining the role of immune effectors in the relationship between Drosophila and its gut microbes.
Van den Bergh, B. (2022). Bugs on Drugs: A Drosophila melanogaster Gut Model to Study In Vivo Antibiotic Tolerance of E. coli. Microorganisms 10(1). PubMed ID: 35056568
Summary:
With an antibiotic crisis upon us, antibiotic development and improve antibiotics' efficacy need to be boosted. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to the most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as flexible in vivo models are lacking. This study explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition.

Wednesday July 6th - Signaling

Mok, J. W. and Choi, K. W. (2022). Modulation of Hippo signaling by Mnat9 N-acetyltransferase for normal growth and tumorigenesis in Drosophila. Cell Death Dis 13(2): 101. PubMed ID: 35110540
Summary:
Hippo signaling is a conserved mechanism for controlling organ growth. Increasing evidence suggests that Hippo signaling is modulated by various cellular factors for normal development and tumorigenesis. Hence, identification of these factors is pivotal for understanding the mechanism for the regulation of Hippo signaling. Drosophila Mnat9 is a putative N-acetyltransferase that is required for cell survival by affecting JNK signaling. This study shows that Mnat9 is involved in the negative regulation of Hippo signaling. RNAi knockdown of Mnat9 in the eye disc suppresses the rough eye phenotype of overexpressing Crumbs (Crb), an upstream factor of the Hippo pathway. Conversely, Mnat9 RNAi enhances the eye phenotype caused by overexpressing Expanded (Ex) or Warts (Wts) that acts downstream to Crb. Similar genetic interactions between Mnat9 and Hippo pathway genes are found in the wing. The reduced wing phenotype of Mnat9 RNAi is suppressed by overexpression of Yorkie (Yki), while it is suppressed by knockdown of Hippo upstream factors like Ex, Merlin, or Kibra. Mnat9 co-immunoprecipitates with Mer, implying their function in a protein complex. Furthermore, Mnat9 overexpression together with Hpo knockdown causes tumorous overgrowth in the abdomen. These data suggest that Mnat9 is required for organ growth and can induce tumorous growth by negatively regulating the Hippo signaling pathway (Mok, 2022).
Banka, S., Bennington, A., ..., Kazanietz, M. G. and Millard, T. H. (2022). Activating RAC1 variants in the switch II region cause a developmental syndrome and alter neuronal morphology. Brain. PubMed ID: 35139179
Summary:
RAC1 is a highly conserved Rho GTPase critical for several cellular and developmental processes. De novo missense RAC1 variants cause a highly variable neurodevelopmental disorder. Most previously reported patients with this disorder have either severe microcephaly or severe macrocephaly. This study describes eight patients with pathogenic missense RAC1 variants affecting residues between Q61 and R68 within the switch II region of RAC1. These patients display variable combinations of developmental delay, intellectual disability, brain anomalies such as polymicrogyria, and cardiovascular defects with normocephaly or relatively milder micro- or macrocephaly. Pulldown assays, NIH3T3 fibroblasts spreading assays and staining for activated PAK1/2/3 and WAVE2 suggest that these variants increase RAC1 activity and over-activate downstream signalling targets. Axons of neurons isolated from Drosophila embryos expressing the most common of the activating variants are significantly shorter, with an increased density of filopodial protrusions. In vivo, these embryos exhibit frequent defects in axonal organization. Class IV dendritic arborisation neurons expressing this variant exhibit a significant reduction in the total area of the dendritic arbour, increased branching and failure of self-avoidance. RNAi knock down of the WAVE regulatory complex component Cyfip significantly rescues these morphological defects. These results establish that activating substitutions affecting residues Q61-R68 within the switch II region of RAC1 cause developmental syndrome (Banka, 2022).
Wijesekera, T. P., Wu, Z., Stephens, N. P., Godula, R., Lew, L. K. and Atkinson, N. S. (2022). A non-nuclear NF-kappaB modulates alcohol sensitivity but not immunity. J Neurosci. PubMed ID: 35273084
Summary:
NF-κB proteins are well known as transcription factors important in immune system activation. In this highly conserved role, they contribute to changes in behavior in response to infection and in response to a variety of other insults and experiences. In some mammalian neurons, NF-κBs can be found at the synapse and translocate to the nucleus to alter gene expression when activated by synaptic activity. This study demonstrates that, in Drosophila melanogaster, NFκB action is important both inside and outside the nucleus and that the Dif gene has segregated nuclear and non-nuclear NFκB action into different protein isoforms. The DifA isoform is a canonical nuclear-acting NFκB protein that enters the nucleus and is important for combating infection. The DifB variant, but not the DifA variant, is found in the central nervous system (mushroom bodies and antennal lobes). DifB does not enter the nucleus and co-localizes with a synaptic protein. In males and females, a DifB mutant alters alcohol behavioral sensitivity without an obvious effect on combating infection, whereas a DifA mutant does not affect alcohol sensitivity but compromises the immune response. These data are evidence that the non-nuclear DifB variant contributes to alcohol behavioral sensitivity by a nongenomic mechanism that diverges from the NF-κB transcriptional effects used in the peripheral immune system. Enrichment of DifB in brain regions rich in synapses and biochemical enrichment of DifB in the synaptoneurosome fraction indicates that the protein may act locally at the synapse (Wijesekera, 2022).
Wu, X., Niu, K., Wang, X., Zhao, J., Wang, H., Li, D., Wang, H., Miao, T., Yang, Y., Ma, H., Zhang, Y., Pan, L., Liu, R., Bai, H. and Liu, N. (2022). microRNA-252 and FoxO repress inflammaging by a dual inhibitory mechanism on Dawdle-mediated TGF-beta pathway in Drosophila. Genetics 220(3). PubMed ID: 35100390
Summary:
Inflammaging refers to low-grade, chronically activated innate immunity that has deleterious effects on healthy lifespan. However, little is known about the intrinsic signaling pathway that elicits innate immune genes during aging. Using Drosophila melanogaster, the microRNA targetomes were profiled in young and aged animals and revealed Dawdle, an activin-like ligand of the TGF-β pathway, as a physiological target of microRNA-252. microRNA-252 cooperates with Forkhead box O, a conserved transcriptional factor implicated in aging, to repress Dawdle. Unopposed Dawdle triggers hyperactivation of innate immune genes coupled with a decline in organismal survival. Using adult muscle tissues, single-cell sequencing analysis describes that Dawdle and its downstream innate immune genes are expressed in distinct cell types, suggesting a cell nonautonomous mode of regulation. The genetic cascade was determined by which Dawdle signaling leads to increased Kenny/IKKγ protein, which in turn activates Relish/NF-κB protein and consequentially innate immune genes. Finally, transgenic increase of microRNA-252 and Forkhead box O pathway factors in wild-type Drosophila extends lifespan and mitigates the induction of innate immune genes in aging. Together, it is proposed that microRNA-252 and Forkhead box O promote healthy longevity by cooperative inhibition on Dawdle-mediated inflammaging.
Zhou, Y., Liu, J. and Liu, J. L. (2022). Connecting Ras and CTP synthase in Drosophila. Exp Cell Res: 113155. PubMed ID: 35427600
Summary:
CTP synthase (CTPS), the enzyme responsible for the last step of de novo synthesis of CTP, forms filamentous structures termed cytoophidia in all three domains of life. This study reports that oncogenic Ras regulates cytoophidium formation in Drosophila intestines. Overexpressing active Ras induces elongate and abundant cytoophidia in intestinal stem cells (ISCs) and enteroblasts (EBs). Knocking-down CTPS in ISCs/EBs suppresses the over proliferation phenotype induced by ectopic expression of active Ras. Moreover, disrupting cytoophidium formation increases the number of proliferating cells in the background of overexpressing active Ras. Therefore, these results demonstrate a link between Ras and CTPS (Zhou, 2022).
Karanja, F., Sahu, S., Weintraub, S., Bhandari, R., Jaszczak, R., Sitt, J. and Halme, A. (2022). Ecdysone exerts biphasic control of regenerative signaling, coordinating the completion of regeneration with developmental progression. Proc Natl Acad Sci U S A 119(5). PubMed ID: 35086929
Summary:
In Drosophila, loss of regenerative capacity in wing imaginal discs coincides with an increase in systemic levels of the steroid hormone ecdysone. Regenerating discs release the relaxin hormone Dilp8 ) to limit ecdysone synthesis and extend the regenerative period. This study describes how regenerating tissues produce a biphasic response to ecdysone levels: lower concentrations of ecdysone promote local and systemic regenerative signaling, whereas higher concentrations suppress regeneration through the expression of broad splice isoforms. Ecdysone also promotes the expression of wingless during both regeneration and normal development through a distinct regulatory pathway. This dual role for ecdysone explains how regeneration can still be completed successfully in dilp8(-) mutant larvae: higher ecdysone levels increase the regenerative activity of tissues, allowing regeneration to reach completion in a shorter time (Karanja. 2022).

Tuesday, July 5th - Behavior

Wang, J., Fan, J. Y., Zhao, Z., Dissel, S. and Price, J. (2022). DBT affects sleep in both circadian and non-circadian neurons. PLoS Genet 18(2): e1010035. PubMed ID: 35139068
Summary:
Sleep is a very important behavior observed in almost all animals. Importantly, sleep is subject to both circadian and homeostatic regulation. The circadian rhythm determines the daily alternation of the sleep-wake cycle, while homeostasis mediates the rise and dissipation of sleep pressure during the wake and sleep period. As an important kinase, Dbt plays a central role in both circadian rhythms and development. This study investigated the sleep patterns of several ethyl methanesulfonate-induced dbt mutants and discusses the possible reasons why different sleep phenotypes were shown in these mutants. In order to reduce DBT in all neurons in which it is expressed, CRISPR-Cas9 was used to produce flies that expressed GAL4 in frame with the dbt gene at its endogenous locus, and knock-down of DBT with this construct produced elevated sleep during the day and reduced sleep at night. Loss of sleep at night is mediated by dbt loss during the sleep/wake cycle in the adult, while the increased sleep during the day is produced by reductions in dbt during development and not by reductions in the adult. Additionally, using targeted RNA interference, this study uncovered the contribution of dbt on sleep in different subsets of neurons in which dbt is normally expressed. Reduction of dbt in circadian neurons produced less sleep at night, while lower expression of dbt in noncircadian neurons produced increased sleep during the day. Importantly, independently of the types of neurons where dbt affects sleep, this study demonstrated that the PER protein is involved in DBT mediated sleep regulation.
Dollish, H. K., Kaladchibachi, S., Negelspach, D. C. and Fernandez, F. X. (2022). The Drosophila circadian phase response curve to light: Conservation across seasonally relevant photoperiods and anchorage to sunset. Physiol Behav 245: 113691. PubMed ID: 34958825
Summary:
Photic history, including the relative duration of day versus night in a 24-hour cycle, is known to influence subsequent circadian responses to light in mammals. Whether such modulation is present in Drosophila is currently unknown. This study constructed the first high-resolution Drosophila seasonal atlas for light-induced circadian phase-resetting. Testing the light responses of over 4,000 Drosophila at 120 timepoints across 5 seasonally-relevant rectangular photoperiods (i.e., LD 8:16, 10:14, 12:12, 14:10, and 16:8; 24 hourly intervals surveyed in each), it was determined that many aspects of the fly circadian PRC waveform are conserved with increasing daylength. Surprisingly though, irrespective of LD schedule, the start of the PRCs always remained anchored to the timing of subjective sunset, creating a differential overlap of the advance zone with the morning hours after subjective sunrise that was maximized under summer photoperiods and minimized under winter photoperiods. These data suggest that there may be differences in flies versus mammals as to how the photoperiod modulates the waveform and amplitude of the circadian PRC to light. On the other hand, they support the possibility that the lights-off transition determines the phase-positioning of photic PRCs across seasons and across species. More work is necessary to test this claim and whether it might factor into the timing of seasonal light responses in humans.
Scott, A. M., Yan, J. L., Baxter, C. M., Dworkin, I. and Dukas, R. (2022). The genetic basis of variation in sexual aggression: Evolution versus social plasticity. Mol Ecol 31(10): 2865-2881. PubMed ID: 35313034
Summary:
Male sexual aggression towards females is a form of sexual conflict that can result in increased fitness for males through forced copulations (FCs) or coercive matings at the cost of female lifetime fitness. This study used male fruit flies (Drosophila melanogaster) as a model system to uncover the genomic contributions to variation in FC, both due to standing variation in a wild population, and due to plastic changes associated with variation in social experience. RNAseq was used to analyse whole-transcriptome differential expression (DE) in male head tissue associated with evolved changes in FC from lineages previously selected for high and low FC rate and in male flies with varying FC rates due to social experience. Hundreds of genes were identified associated with evolved and plastic variation in FC, however only a small proportion (27 genes) showed consistent DE due to both modes of variation. This trend of low concordance in gene expression effects across broader sets of genes was confirmed to be significant in either the evolved or plastic analyses using multivariate approaches. The gene ontology terms neuropeptide hormone activity and serotonin receptor activity were significantly enriched in the set of significant genes. Of seven genes chosen for RNAi knockdown validation tests, knockdown of four genes showed the expected effect on FC behaviours. Taken together, these results provide important information about the apparently independent genetic architectures that underlie natural variation in sexual aggression due to evolution and plasticity.
Mueller, J. M., Zhang, N., Carlson, J. M. and Simpson, J. H. (2021). Variation and Variability in Drosophila Grooming Behavior. Front Behav Neurosci 15: 769372. PubMed ID: 35087385
Summary:
Behavioral differences can be observed between species or populations (variation) or between individuals in a genetically similar population (variability). This study investigated genetic differences as a possible source of variation and variability in Drosophila grooming. Grooming confers survival and social benefits. Grooming features of five Drosophila species exposed to a dust irritant were analyzed. Aspects of grooming behavior, such as anterior to posterior progression, were conserved between and within species. However, significant differences in activity levels, proportion of time spent in different cleaning movements, and grooming syntax were identified between species. All species tested showed individual variability in the order and duration of action sequences. Genetic diversity was not found to correlate with grooming variability within a species: melanogaster flies bred to increase or decrease genetic heterogeneity exhibited similar variability in grooming syntax. Individual flies observed on consecutive days also showed grooming sequence variability. Standardization of sensory input using optogenetics reduced but did not eliminate this variability. In aggregate, these data suggest that sequence variability may be a conserved feature of grooming behavior itself. These results also demonstrate that large genetic differences result in distinguishable grooming phenotypes (variation), but that genetic heterogeneity within a population does not necessarily correspond to an increase in the range of grooming behavior (variability).
Smith, M. A., Honegger, K. S., Turner, G. and de Bivort, B. (2022). Idiosyncratic learning performance in flies. Biol Lett 18(2): 20210424. PubMed ID: 35104427
Summary:
Individuals vary in their innate behaviours, even when they have the same genome and have been reared in the same environment. The extent of individuality in plastic behaviours, like learning, is less well characterized. Also unknown is the extent to which intragenotypic differences in learning generalize: if an individual performs well in one assay, will it perform well in other assays? This was investigated using the fruit fly Drosophila melanogaster, an organism long-used to study the mechanistic basis of learning and memory. Isogenic flies, reared in identical laboratory conditions and subject to classical conditioning that associated odorants with electric shock, exhibit clear individuality in their learning responses. Flies that performed well when an odour was paired with shock tended to perform well when the odour was paired with bitter taste or when other odours were paired with shock. Thus, individuality in learning performance appears to be prominent in isogenic animals reared identically, and individual differences in learning performance generalize across some aversive sensory modalities. Establishing these results in flies opens up the possibility of studying the genetic and neural circuit basis of individual differences in learning in a highly suitable model organism.
McDonough-Goldstein, C. E., Pitnick, S. and Dorus, S. (2022). Drosophila female reproductive glands contribute to mating plug composition and the timing of sperm ejection. Proc Biol Sci 289(1968): 20212213. PubMed ID: 35105240
Summary:
Reproductive traits that influence female remating and competitive fertilization rapidly evolve in response to sexual selection and sexual conflict. One such trait, observed across diverse animal taxa, is the formation of a structural plug inside the female reproductive tract (FRT), either during or shortly after mating. In Drosophila melanogaster, male seminal fluid forms a mating plug inside the female bursa, which has been demonstrated to influence sperm entry into storage and latency of female remating. Processing of the plug, including its eventual ejection from the female's reproductive tract, influences the competitive fertilization success of her mates and is mediated by female x male genotypic interactions. However, female contributions to plug formation and processing have received limited attention. Using developmental mutants that lack glandular FRT tissues, this study revealed that these tissues are essential for mating plug ejection. Proteomics was used to demonstrate that female glandular proteins, and especially proteolytic enzymes, contribute to mating plug composition and have a widespread impact on plug formation and composition. Together, these phenotypic and molecular data identify female contributions to intersexual interactions that are a potential mechanism of post-copulatory sexual selection.

Thursday, June 30th - Characterization of Genes and Proteins

Zafar, Z., Fatima, S., Bhatti, M. F., Shah, F. A., Saud, Z. and Butt, T. M. (2022). Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights. PLoS One 17(3): e0265896. PubMed ID: 35316281
Summary:
Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi.
Pegoraro, M., Sayegh Rezek, E., Fishman, B. and Tauber, E. (2022). Nucleotide Variation in Drosophila cryptochrome Is Linked to Circadian Clock Function: An Association Analysis. Front Physiol 13: 781380. PubMed ID: 35250608
Summary:
Cryptochrome (CRY) is a conserved protein associated with the circadian clock in a broad range of organisms, including plants, insects, and mammals. In Drosophila, cry is a pleiotropic gene that encodes a blue light-dedicated circadian photoreceptor, as well as an electromagnetic field sensor and a geotaxis behavior regulator. This study has generated a panel of nearly-isogenic strains that originated from various wild populations and which carry different natural alleles of cry. Sequencing of these alleles revealed substantial polymorphism, the functional role of which was elusive. To link this natural molecular diversity to gene function, association mapping was used. Such analysis revealed two major haplogroups consisting of six linked nucleotides associated with circadian phase (haplotypes All1/All2). A maximum-likelihood gene-tree that uncovered an additional pair of haplogroups (B1/B2). Behavioral analysis of the different haplotypes indicated significant effect on circadian phase and period, as well on the amount of activity and sleep. The data also suggested substantial epistasis between the All and B haplogroups. Intriguingly, circadian photosensitivity, assessed by light-pulse experiments, did not differ between the genotypes. Using CRISPR-mediated transgenic flies, the effect of B1/B2 polymorphism on circadian phase was verified. The transgenic flies also exhibited substantially different levels of cry transcription.The geographical distribution of the B1/B2 haplotypes was analyzed, focusing on a 12 bp insertion/deletion polymorphism that differentiates the two haplotypes. Analysis of cry sequences in wild populations across Europe revealed a geographical cline of B1/B2 indel frequency, which correlated with seasonal bioclimatic variables. This spatial distribution of cry polymorphism reinforces the functional importance of these haplotypes in the circadian system and local adaptation.
Yang, S. and Zhang, W. (2022). Systematic analysis of olfactory protein-protein interactions network of fruitfly, Drosophila melanogaster. Arch Insect Biochem Physiol: e21882. PubMed ID: 35249240
Summary:
Olfaction is one of the physiological traits of insect behavior. Insects have evolved a sophisticated olfactory system and use a combined coding strategy to process general odor. Drosophila melanogaster is a powerful model to reveal the molecular and cellular mechanisms of odor detection. Identifying new olfactory targets through complex interactions will contribute to a better understanding of the functions, interactions, and signaling pathways of olfactory proteins. However, the mechanism of D. melanogaster olfaction is still unclear, and more olfactory proteins are required to be discovered. This study tried to explore essential proteins in the olfactory system of D. melanogaster and conduct protein-protein interactions (PPIs) analysis. The PPIs network of the olfactory system of D. melanogaster was constructed, consisting of 863 proteins and 18,959 interactions. Various methods were used to perform functional enrichment analysis, topological analysis and cluster analysis. The results confirmed that Class B scavenger receptors (SR-Bs), glutathione S-transferases (GSTs), and UDP-glycosyltransferases (UGTs) play an essential role in olfaction of D. melanogaster. The proteins obtained in this study can be used for subsequent functional identification in D. melanogaster olfactory system.
Denha, S. A., Atang, A. E., Hays, T. S. and Avery, A. W. (2022). beta-III-spectrin N-terminus is required for high-affinity actin binding and SCA5 neurotoxicity. Sci Rep 12(1): 1726. PubMed ID: 35110634
Summary:
Recent structural studies of β-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). This study investigated in Drosophila the significance of the β-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). Pan-neuronal expression of an N-terminally truncated β-spectrin fails to rescue lethality resulting from a β-spectrin loss-of-function allele, indicating that the N-terminus is essential to β-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P substitution. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 β-spectrin and spectrin-related disease proteins.
Taylor, J. R., Wood, J. G., Mizerak, E., Hinthorn, S., Liu, J., Finn, M., Gordon, S., Zingas, L., Chang, C., Klein, M. A., Denu, J. M., Gorbunova, V., Seluanov, A., Boeke, J. D., Sedivy, J. M. and Helfand, S. L. (2022). Sirt6 regulates lifespan in Drosophila melanogaster. Proc Natl Acad Sci U S A 119(5). PubMed ID: 35091469
Summary:
Sirt6 is a multifunctional enzyme that regulates diverse cellular processes such as metabolism, DNA repair, and aging. Overexpressing Sirt6 extends lifespan in mice, but the underlying cellular mechanisms are unclear. Drosophila melanogaster are an excellent model to study genetic regulation of lifespan; however, despite extensive study in mammals, very little is known about Sirt6 function in flies. This study characterized the Drosophila ortholog of Sirt6, dSirt6, and examined its role in regulating longevity; dSirt6 is a nuclear and chromatin-associated protein with NAD(+)-dependent histone deacetylase activity. dSirt6 overexpression (OE) in flies produces robust lifespan extension in both sexes, while reducing dSirt6 levels shortens lifespan. dSirt6 OE flies have normal food consumption and fertility but increased resistance to oxidative stress and reduced protein synthesis rates. Transcriptomic analyses reveal that dSirt6 OE reduces expression of genes involved in ribosome biogenesis, including many dMyc target genes. dSirt6 OE partially rescues many effects of dMyc OE, including increased nuclear size, up-regulation of ribosome biogenesis genes, and lifespan shortening. Last, dMyc haploinsufficiency does not convey additional lifespan extension to dSirt6 OE flies, suggesting dSirt6 OE is upstream of dMyc in regulating lifespan. These results provide insight into the mechanisms by which Sirt6 OE leads to longer lifespan.
Zheng, L., Liu, J., Niu, L., Kamran, M., Yang, A. W. H., Jolma, A., Dai, Q., Hughes, T. R., Patel, D. J., Zhang, L., Prasanth, S. G., Yu, Y., Ren, A. and Lai, E. C. (2022). Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins. Genes Dev 36(3-4): 225-240. PubMed ID: 35144965
Summary:
The BEN domain (see Drosophila Insensitive) is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. This study characterized several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. Detailed functional, genomic, and structural studies of BEND3 were conducted. BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. A high-resolution structure was obtained of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

Wednesday, June 29th - Larval and Adult Neural Development

Taylor, J. R., Wood, J. G., Mizerak, E., Hinthorn, S., Liu, J., Finn, M., Gordon, S., Zingas, L., Chang, C., Klein, M. A., Denu, J. M., Gorbunova, V., Seluanov, A., Boeke, J. D., Sedivy, J. M. and Helfand, S. L. (2022). Sirt6 regulates lifespan in Drosophila melanogaster. Proc Natl Acad Sci U S A 119(5). PubMed ID: 35091469
Summary:
Sirt6 is a multifunctional enzyme that regulates diverse cellular processes such as metabolism, DNA repair, and aging. Overexpressing Sirt6 extends aging in mice, but the underlying cellular mechanisms are unclear. Drosophila melanogaster are an excellent model to study genetic regulation of lifespan; however, despite extensive study in mammals, very little is known about Sirt6 function in flies. This study characterized the Drosophila ortholog of Sirt6, dSirt6, and examined its role in regulating longevity; dSirt6 is a nuclear and chromatin-associated protein with NAD(+)-dependent histone deacetylase activity. dSirt6 overexpression (OE) in flies produces robust lifespan extension in both sexes, while reducing dSirt6 levels shortens lifespan. dSirt6 OE flies have normal food consumption and fertility but increased resistance to oxidative stress and reduced protein synthesis rates. Transcriptomic analyses reveal that dSirt6 OE reduces expression of genes involved in ribosome biogenesis, including many dMyc target genes. dSirt6 OE partially rescues many effects of dMyc OE, including increased nuclear size, up-regulation of ribosome biogenesis genes, and lifespan shortening. Last, dMyc haploinsufficiency does not convey additional lifespan extension to dSirt6 OE flies, suggesting dSirt6 OE is upstream of dMyc in regulating lifespan. These results provide insight into the mechanisms by which Sirt6 OE leads to longer lifespan.
Ko, S., Yeom, E., Chun, Y. L., Mun, H., Howard-McGuire, M., Millison, N. T., Jung, J., Lee, K. P., Lee, C., Lee, K. S., Delaney, J. R. and Yoon, J. H. (2022). Profiling of RNA-binding Proteins Interacting With Glucagon and Adipokinetic Hormone mRNAs. J Lipid Atheroscler 11(1): 55-72. PubMed ID: 35118022
Summary:
Glucagon in mammals and its homolog (adipokinetic hormone [AKH] in Drosophila melanogaster) are peptide hormones which regulate lipid metabolism by breaking down triglycerides. Although regulatory mechanisms of glucagon and AKH expression have been widely studied, post-transcriptional gene expression of glucagon has not been investigated thoroughly. This study aimed to profile proteins binding with Gcg messenger RNA (mRNA) in mouse and Akh mRNA in Drosophila. Drosophila Schneider 2 (S2) and mouse 3T3-L1 cell lysates were utilized for affinity pull down of Akh and Gcg mRNA respectively using biotinylated anti-sense DNA oligoes against target mRNAs. Mass spectrometry and computational network analysis revealed mRNA-interacting proteins residing in functional proximity. This study observed that 1) 91 proteins interact with Akh mRNA from S2 cell lysates, 2) 34 proteins interact with Gcg mRNA from 3T3-L1 cell lysates. 3) Akh mRNA interactome revealed clusters of ribosomes and known RNA-binding proteins (RBPs). 4) Gcg mRNA interactome revealed mRNA-binding proteins including Plekha7, zinc finger protein, carboxylase, lipase, histone proteins and a cytochrome, Cyp2c44. 5) Levels of Gcg mRNA and its interacting proteins are elevated in skeletal muscles isolated from old mice compared to ones from young mice.Akh mRNA in S2 cells are under active translation in a complex of RBPs and ribosomes. Gcg mRNA in mouse precursor adipocyte is in a condition distinct from Akh mRNA due to biochemical interactions with a subset of RBPs and histones. It is anticipatet that thiw study contributes to investigating regulatory mechanisms of Gcg and Akh mRNA decay, translation, and localization.
Kamemura, K., Moriya, H., Ukita, Y., Okumura, M., Miura, M. and Chihara, T. (2022). Endoplasmic reticulum proteins Meigo and Gp93 govern dendrite targeting by regulating Toll-6 localization. Dev Biol 484: 30-39. PubMed ID: 35134382
Summary:
Neuronal target recognition is performed by numerous cell-surface transmembrane proteins. Correct folding of these proteins occurs in the endoplasmic reticulum (ER) lumen of the neuronal cells before being transported to the plasma membrane of axons or dendrites. Disturbance in this protein folding process in the ER leads to dysfunction of neuronal cell surface molecules, resulting in abnormal neuronal targeting. This study reports that the ER-resident protein Meigo in Drosophila, governs the dendrite targeting of olfactory projection neurons (PNs) along the mediolateral axis of the antennal lobe by regulating Toll-6 localization. Loss of Meigo causes Toll-6 mislocalization in the PNs and mediolateral dendrite targeting defects, which are suppressed by Toll-6 overexpression. Furthermore, it was found that the ER-chaperone protein, Gp93, also regulates the mediolateral targeting of PN dendrites by localization of the Toll-6 protein. Gp93 overexpression in the PN homozygous for the meigo mutation, partially rescued the dendrite targeting defect, while meigo knockdown decreased Gp93 expression levels in cultured cells. These results indicate that the ER-proteins Meigo and Gp93 regulate dendrite targeting by attenuating the amount and localization of cell surface receptors, including Toll-6, implying the unexpected but active involvement of ER proteins in neural wiring.
Srinivasan, A. R., Tran, T. T. and Bonini, N. M. (2022). Loss of miR-34 in Drosophila dysregulates protein translation and protein turnover in the aging brain. Aging Cell 21(3): e13559. PubMed ID: 35166006
Summary:
Aging is a risk factor for neurodegenerative disease, but precise mechanisms that influence this relationship are still under investigation. Work in Drosophila melanogaster identified the microRNA miR-34 as a modifier of aging and neurodegeneration in the brain. MiR-34 mutants present aspects of early aging, including reduced lifespan, neurodegeneration, and a buildup of the repressive histone mark H3K27me3. To better understand how miR-34 regulated pathways contribute to age-associated phenotypes in the brain, this study transcriptionally profiled the miR-34 mutant brain. This identified that genes associated with translation are dysregulated in the miR-34 mutant. The brains of these animals show increased translation activity, accumulation of protein aggregation markers, and altered autophagy activity. To determine if altered H3K27me3 was responsible for this proteostasis dysregulation, the effects of increased H3K27me3 was studied by mutating the histone demethylase Utx. Reduced Utx activity enhanced neurodegeneration and mimicked the protein accumulation seen in miR-34 mutant brains. However, unlike the miR-34 mutant, Utx mutant brains did not show similar altered autophagy or translation activity, suggesting that additional miR-34-targeted pathways are involved. Transcriptional analysis of predicted miR-34 targets identified Lst8, a subunit of Tor Complex 1 (TORC1), as a potential target. It was confirmed that miR-34 regulates the 3' UTR of Lst8 and identified several additional predicted miR-34 targets that may be critical for maintaining proteostasis and brain health. Together, these results present novel understanding of the brain and the role of the conserved miRNA miR-34 in impacting proteostasis in the brain with age.
Schell, B., Legrand, P. and Fribourg, S. (2022). Crystal structure of SFPQ-NONO heterodimer. Biochimie 198: 1-7. PubMed ID: 35245601
Summary:
The Drosophila behavior/human splicing (DBHS) protein family is composed of the three members SFPQ, NONO and PSPC1. These proteins share a strong sequence and structural homology within the core-structured domains forming obligate homo- and heterodimers. This feature may lead to the simultaneous existence of six different dimeric complexes that sustain their function in many cellular processes such as pre-mRNA splicing, innate immunity, transcriptional regulation. In order to perform a complete structural analysis of all possible DBHS dimers,this study has solved the crystal structure of the missing DBHS heterodimer SFPQ-NONO at 3.0 Å resolution. Subtle changes were identfied in amino acid composition and local secondary structure of the NOPS region orientation that may modulate affinity between complexes. Interestingly this area is found mutated in aggressive skin cancers and adenocarcinomas.
Yang, N., Srivastav, S. P., Rahman, R., Ma, Q., Dayama, G., Li, S., Chinen, M., Lei, E. P., Rosbash, M. and Lau, N. C. (2022). Transposable element landscapes in aging Drosophila. PLoS Genet 18(3): e1010024. PubMed ID: 35239675
Summary:
Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression in aged animals. Does increased TE expression during aging lead to more genomic TE copies in older animals? To address this question, this study quantified TE Landscapes (TLs) via whole genome sequencing of young and aged Drosophila strains of wild-type and mutant backgrounds. TLs were quantified in whole flies and dissected brains and the feasibility of this approach in detecting new TE insertions in aging Drosophila genomes when small RNA and RNA interference (RNAi) pathways are compromised. This study also describes improved sequencing methods to quantify extra-chromosomal DNA circles (eccDNAs) in Drosophila as an additional source of TE copies that accumulate during aging. Lastly, to combat the natural progression of aging-associated TE expression, it was show that knocking down PAF1, a conserved transcription elongation factor that antagonizes RNAi pathways, may bolster suppression of TEs during aging and extend lifespan. This study suggests that in addition to a possible influence by different genetic backgrounds, small RNA and RNAi mechanisms may mitigate genomic TL expansion despite the increase in TE transcripts during aging.

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