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Monday, July 31th, 2023 - Protein Expression, Evolution, Structure, and Function

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Wong, K. C., Jayapalan, J. J., Subramanian, P., Ismail, M. N. and Abdul-Rahman, P. S. (2023). Label-free quantitative mass spectrometry analysis of the circadian proteome of Drosophila melanogaster lethal giant larvae mutants reveals potential therapeutic effects of melatonin. Arch Insect Biochem Physiol: e22008. PubMed ID: 36915983
Mutation in the Drosophila melanogaster lethal giant larvae (lgl), a tumor suppressor gene with a well-established role in cellular polarity, is known to results in massive cellular proliferation and neoplastic outgrowths. Although the tumorigenic properties of lgl mutant have been previously studied, little is known about its consequences on the proteome. In this study, mass spectrometry-based label-free quantitative proteomics was employed to investigate the changes in the head and intestinal tissues proteins of Drosophila melanogaster, due to lgl mutation and following treatment with melatonin. Additionally, to uncover the time-influenced variations in the proteome during tumorigenesis and melatonin treatment, the rhythmic expression of proteins was also investigated at 6-h intervals within 24-h clock. Together, the present study has identified 434 proteins of altered expressions (p > 0.05 and fold change ±1.5) in the tissues of flies in response to lgl mutation as well as posttreatment with melatonin. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed proteins revealed that lgl mutation had significantly affected the biological functions, including metabolism, and protein synthesis and degradation, in flies' tissues. Besides, melatonin had beneficially mitigated the deleterious effects of lgl mutation by reversing the alterations in protein expression closer to baseline levels. Further, changes in protein expression in the tissues due to lgl mutation and melatonin treatment were found rhythmically orchestrated. Together, these findings provide novel insight into the pathways involved in lgl-induced tumorigenesis as well as demonstrated the efficacy of melatonin as a potential anticancer agent. Data are available via ProteomeXchange with identifier PXD033191.
Komori, Y., Takayama, K., Okamoto, N., Kamiya, M., Koizumi, W., Ihara, M., Misawa, D., Kamiya, K., Yoshinari, Y., Seike, K., Kondo, S., Tanimoto, H., Niwa, R., Sattelle, D. B. and Matsuda, K. (2023). Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids. PLoS Genet 19(2): e1010522. PubMed ID: 36795653
Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. It was recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. This study show that the Dα3 subunit co-exists with Dα1, Dα2, Dβ1, and Dβ2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dβ3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dβ3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.
Franco, G., Taillebourg, E., Delfino, E., Homolka, D., Gueguen, N., Brasset, E., Pandey, R. R., Pillai, R. S. and Fauvarque, M. O. (2023). The catalytic-dead Pcif1 regulates gene expression and fertility in Drosophila. Rna 29(5): 609-619. PubMed ID: 36754578
Eukaryotic mRNAs are modified at the 5' end with a methylated guanosine (m(7)G) that is attached to the transcription start site (TSS) nucleotide. The TSS nucleotide is 2'-O-methylated (Nm) by CMTR1 in organisms ranging from insects to human. In mammals, the TSS adenosine can be further N (6) -methylated by RNA polymerase II phosphorylated CTD-interacting factor 1 (PCIF1) to create m(6)Am. Curiously, the fly ortholog of mammalian PCIF1 is demonstrated to be catalytic-dead, and its functions are not known. This study shows that Pcif1 mutant flies display a reduced fertility which is particularly marked in females. Deep sequencing analysis of Pcif1 mutant ovaries revealed transcriptome changes with a notable increase in expression of genes belonging to the mitochondrial ATP synthetase complex. Furthermore, the Pcif1 protein is distributed along euchromatic regions of polytene chromosomes, and the Pcif1 mutation behaved as a modifier of position-effect-variegation (PEV) suppressing the heterochromatin-dependent silencing of the white gene. Similar or stronger changes in the transcriptome and PEV phenotype were observed in flies that expressed a cytosolic version of Pcif1. These results point to a nuclear cotranscriptional gene regulatory role for the catalytic-dead fly Pcif1 that is probably based on its conserved ability to interact with the RNA polymerase II carboxy-terminal domain.

Walker, D. R., Jara, K. A., Rolland, A. D., Brooks, C., Hare, W., Swansiger, A. K., Reardon, P. N., Prell, J. S. and Barbar, E. J. (2023). Linker Length Drives Heterogeneity of Multivalent Complexes of Hub Protein LC8 and Transcription Factor ASCIZ. Biomolecules 13(3). PubMed ID: 36979339
LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2-4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2-4 variants, with methodically deactivated motifs, this study shows that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2-4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2-4, with long and short linkers, forms heterogeneous complexes, whereas QT4-6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed.
Gude, F., Froese, J., Manikowski, D., Di Iorio, D., Grad, J. N., Wegner, S., Hoffmann, D., Kennedy, M., Richter, R. P., Steffes, G. and Grobe, K. (2023). Hedgehog is relayed through dynamic heparan sulfate interactions to shape its gradient. Nat Commun 14(1): 758. PubMed ID: 36765094
Cellular differentiation is directly determined by concentration gradients of morphogens. As a central model for gradient formation during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in Drosophila wing and eye discs. What is not known is how extracellular Hh spread is achieved and how it translates into precise gradients. This study shows that two separate binding areas located on opposite sides of the Hh molecule can interact directly and simultaneously with two heparan sulfate (HS) chains to temporarily cross-link the chains. Mutated Hh lacking one fully functional binding site still binds HS but shows reduced HS cross-linking. This, in turn, impairs Hhs ability to switch between both chains in vitro and results in striking Hh gradient hypomorphs in vivo. The speed and propensity of direct Hh switching between HS therefore shapes the Hh gradient, revealing a scalable design principle in morphogen-patterned tissues.
Foroutannejad, S., Good, L. L., Lin, C., Carter, Z. I., Tadesse, M. G., Lucius, A. L., Crane, B. R. and Maillard, R. A. (2023). The cofactor-dependent folding mechanism of Drosophila cryptochrome revealed by single-molecule pulling experiments. Nat Commun 14(1): 1057. PubMed ID: 36828841
The link between cofactor binding and protein activity is well-established. However, how cofactor interactions modulate folding of large proteins remains unknown. This study used optical tweezers, clustering and global fitting to dissect the folding mechanism of Drosophila Cryptochrome (dCRY), a 542-residue protein that binds FAD, one of the most chemically and structurally complex cofactors in nature. The first dCRY parts to fold were shown to be independent of FAD, but later steps are FAD-driven as the remaining polypeptide folds around the cofactor. FAD binds to largely unfolded intermediates, yet with association kinetics above the diffusion-limit. Interestingly, not all FAD moieties are required for folding: whereas the isoalloxazine ring linked to ribitol and one phosphate is sufficient to drive complete folding, the adenosine ring with phosphates only leads to partial folding. Lastly, a dCRY folding model is proposed where regions that undergo conformational transitions during signal transduction are the last to fold.

Friday, July 28th - Signaling

Kassel, S., Hanson, A. J., Benchabane, H., Saito-Diaz, K., Cabel, C. R., Goldsmith, L., Taha, M., Kanuganti, A., Ng, V. H., Xu, G., Ye, F., Picker, J., Port, F., Boutros, M., Weiss, V. L., Robbins, D. J., Thorne, C. A., Ahmed, Y. and Lee, E. (2023). USP47 deubiquitylates Groucho/TLE to promote Wnt-β-catenin signaling. Sci Signal 16(771): eabn8372. PubMed ID: 36749823
The Wnt-β-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator β-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, this study identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. USP47 was found to be required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the β-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. These data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of β-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.
Das, R., Pandey, P., Maurya, B., Pradhan, P., Sinha, D., Mukherjee, A. and Mutsuddi, M. (2023). Spoonbill positively regulates JNK signalling mediated apoptosis in Drosophila melanogaster. Eur J Cell Biol 102(2): 151300. PubMed ID: 36858008
Spoonbill (Spoon) is a putative A-kinase anchoring protein in Drosophila. This report has unravelled a novel function of Spoon protein in the regulation of the apoptotic pathway. The Drosophila TNFα homolog, Eiger, induces apoptosis via activation of the JNK pathway. This study shows that Spoonbill is a positive regulator of the Eiger-induced JNK signalling. Further genetic interaction studies show that the spoon interacts with components of the JNK pathway, TGF-β activated kinase 1 (Tak1 - JNKKK), hemipterous (hep - JNKK) and basket (bsk - JNK). Interestingly, Spoonbill alone can also induce ectopic activation of the JNK pathway in a context-specific manner. To understand the molecular mechanism underlying Spoonbill-mediated modulation of the JNK pathway, the interaction between Spoon and Drosophila JNK was assessed. basket encodes the only known JNK in Drosophila. This serine/threonine-protein kinase phosphorylates Jra/Kay, which transcriptionally regulate downstream targets like Matrix metalloproteinase 1 (Mmp1), puckered (puc), and proapoptotic genes hid, reaper and grim. Interestingly, it was found that Spoonbill colocalises and co-immunoprecipitates with the Basket protein in the developing photoreceptor neurons. It is proposed that Spoon plays a vital role in JNK-induced apoptosis. Furthermore, stress-induced JNK activation underlying Parkinson's Disease was also examined. In the Parkinson's Drosophila model of neurodegeneration, depletion of Spoonbill leads to a partial reduction of JNK pathway activation, along with improvement in adult motor activity. These observations suggest that the putative scaffold protein Spoonbill is a functional and physical interacting partner of the Drosophila JNK protein, Basket. Spoon protein is localised on the outer mitochondrial membrane (OMM), which may perhaps provide a suitable subcellular niche for activation of Drosophila Basket protein by its kinases which induce apoptosis.
Williams, A. M. and Horne-Badovinac, S. (2023). Fat2 polarizes Lar and Sema5c to coordinate the motility of collectively migrating epithelial cells. bioRxiv. PubMed ID: 36909523
Migrating epithelial cells globally align their migration machinery to achieve tissue-level movement. Biochemical signaling across leading-trailing cell-cell interfaces can promote this alignment by partitioning migratory behaviors like protrusion and retraction to opposite sides of the interface. However, how the necessary signaling proteins become organized at this site is poorly understood. The follicular epithelial cells of Drosophila melanogaster have two signaling modules at their leading-trailing interfaces-one composed of the atypical cadherin Fat2 and the receptor tyrosine phosphatase Lar, and one composed of Semaphorin 5c and its receptor Plexin A. These modules were shown to form one interface signaling system with Fat2 at its core. Trailing edge-enriched Fat2 concentrates both Lar and Sema5c at cells' leading edges, likely by slowing their turnover at this site. Once localized, Lar and Sema5c act in parallel to promote collective migration. These data suggest a model in which Fat2 couples and polarizes the distributions of multiple effectors that work together to align the migration machinery of neighboring cells.
Gui, J., Samuels, T. J., Grobicki, K. Z. A. and Teixeira, F. K. (2023). Simultaneous activation of Tor and suppression of ribosome biogenesis by TRIM-NHL proteins promotes terminal differentiation. Cell Rep 42(3): 112181. PubMed ID: 36870055
Tissue development and homeostasis depend on the balance between growth and terminal differentiation, but the mechanisms coordinating these processes remain elusive. Accumulating evidence indicates that ribosome biogenesis (RiBi) and protein synthesis, two cellular processes sustaining growth, are tightly regulated and yet can be uncoupled during stem cell differentiation. Using the Drosophila adult female germline stem cell and larval neuroblast systems, this study showed that Mei-P26 and Brat, two Drosophila TRIM-NHL paralogs, are responsible for uncoupling RiBi and protein synthesis during differentiation. In differentiating cells, Mei-P26 and Brat activate the target of rapamycin (Tor) kinase to promote translation, while concomitantly repressing RiBi. Depletion of Mei-P26 or Brat results in defective terminal differentiation, which can be rescued by ectopic activation of Tor together with suppression of RiBi. These results indicate that uncoupling RiBi and translation activities by TRIM-NHL activity creates the conditions required for terminal differentiation.
Manikowski, D., Steffes, G., Froese, J., Exner, S., Ehring, K., Gude, F., Di Iorio, D., Wegner, S. V. and Grobe, K. (2023). Drosophila hedgehog signaling range and robustness depend on direct and sustained heparan sulfate interactions.. Front Mol Biosci 10: 1130064. PubMed ID: 36911531
Morphogens determine cellular differentiation in many developing tissues in a concentration dependent manner. As a central model for gradient formation during animal development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in the Drosophila wing disc. Although heparan sulfate (HS) expression in the disc is essential for this process, it is not known whether HS regulates Hh signaling and spread in a direct or in an indirect manner. To answer this question, this study systematically screened two composite Hh binding areas for HS in vitro and expressed mutated proteins in the Drosophila wing disc. Selectively impaired HS binding was found of the second site, reducing Hh signaling close to the source and causing striking wing mispatterning phenotypes more distant from the source. These observations suggest that HS constrains Hh to the wing disc epithelium in a direct manner, and that interfering with this constriction converts Hh into freely diffusing forms with altered signaling ranges and impaired gradient robustness.
Nolan, R. B., Bontrager, C., Bowser, A., Corley, A., Fiedler, H., Flathers, C., Francis, L., Le, A., Mahmoudjafari, S., Nim, T., Muolo, C. E., Shores, B., Viermann, C., Waldren, A., Zatezalo, C., Fan, J. Y. and Price, J. L. (2023). Visual and circadian regulation of Drosophila BDBT and BDBT effects on DBT and PER localization. iScience 26(4): 106343. PubMed ID: 36994075
BRIDE OF DOUBLETIME (BDBT) interacts with the circadian kinase DOUBLETIME (DBT) and accumulates in eye foci during the dark of a light:dark cycle. BDBT foci are shown in this study to be broadly expressed in constant dark and low in constant light. Analysis of circadian photoreceptor cry and visual photoreceptor ninaE mutants showed that disappearance of eye BDBT foci requires both the CRYPTOCHROME and the RHODOPSIN-1 pathways. The arr1 and arr2 mutants, which affect rhodopsin quenching, eliminated BDBT foci under dark conditions. arr1 and arr2 mutants also caused increased nuclear PER protein. The changes in BDBT foci do not result from altered BDBT levels in the eye but from changes in its immunodetection. Knockdown of BDBT specifically in the eye produced constitutively nuclear PER and constitutively cytosolic DBT. The results show that BDBT is necessary for co-transport of DBT and PER into the nucleus and suggest that this process is regulated by a light-dependent mechanism.

Thursday, July 27th - Disease Models

Jangam, S., Briere, L. C., Jay, K., Andrews, J. C., Walker, M. A., Rodan, L. H., High, F. A., Yamamoto, S., Sweetser, D. A. and Wangler, M. (2023). A de novo missense variant in EZH1 associated with developmental delay exhibits functional deficits in Drosophila melanogaster. medRxiv. PubMed ID: 36778246
EZH1 (Enhancer of Zeste, homolog 1), a Polycomb Repressive Complex-2 (PRC2) component, is involved in a myriad of cellular processes through modifying histone 3 lysine27 (H3K27) residues. EZH1 represses transcription of downstream target genes through H3K27 trimethylation (H3K27me3). Genetic mutations in histone modifiers have been associated with developmental disorders, while EZH1 has not yet been linked to any human disease. However, the paralog EZH2 is associated with Weaver syndrome. This study reports a previously undiagnosed individual with a novel neurodevelopmental phenotype identified to have a de novo variant in EZH1, p.Ala678Gly, through exome sequencing. The individual presented in infancy with neurodevelopmental delay and hypotonia and was later noted to have proximal muscle weakness. The variant, p.A678G, is in the SET domain, known for its methyltransferase activity, and was the best candidate variant found in the exome. Human EZH1 / 2 are homologous to fly Enhancer of zeste E(z), an essential gene in flies, and the residue (A678 in humans, A691 in Drosophila) is conserved. To further study this variant, Drosophila null alleles were obtained and transgenic flies expressing wild-type (E(z) (WT)) and the variant (E(z) (A691G)) were generated. The E(z) (A691G) variant led to hyper H3K27me3 while the E(z) (WT) did not, suggesting this is as a gain-of-function allele. When expressed under the tubulin promotor in vivo the variant rescued null-lethality similar to wild-type but the E(z) (A691G) flies exhibit bang sensitivity and shortened lifespan. In conclusion, this study presents a novel EZH1 de novo variant associated with a neurodevelopmental disorder. Furthermore, it was found that this variant has a functional impact in Drosophila. Biochemically this allele leads to increased H3K27me3 suggesting gain-of-function, but when expressed in adult flies the E(z) (A691G) has some characteristics of partial loss-of-function which may suggest it is a more complex allele in vivo.
Alvarez-Rendon, J. P. and Riesgo-Escovar, J. R. (2023). Activation of the Cap'n'collar C pathway (Nrf2 pathway in vertebrates) signaling in insulin pathway compromised Drosophila melanogaster flies ameliorates the diabetic state upon pro-oxidant conditions. Gen Comp Endocrinol 335: 114229. PubMed ID: 36781022
The insulin pathway is a crucial central system for metabolism and growth. The Nrf2 signaling pathway functions to counteract oxidative stress. This study examined the consequences of an oxidative stress challenge to insulin compromised and control adult flies of different ages, varying the activation state of the Nrf2 pathway in flies, the Cap'n'collar C pathway. For this, two different pro-oxidative conditions were employed: 3 % hydrogen peroxide or 20 mM paraquat laced in the food. In both cases, wild type (control) flies die within a few days, yet there are significant differences between males and females, and also within flies of different ages (seven versus thirty days old flies). The same conditions were repeated with young (seven days old) flies that were heterozygous for a loss-of-function mutation in Keap1. There were no significant differences. Two hypomorphic viable conditions of the insulin pathway were tested (heteroallelic combination for the insulin receptor and the S6 Kinase), challenged in the same way: Whereas they also die in the pro-oxidant conditions, they fare significantly better when heterozygous for Keap1, in contrast to controls. Locomotion was also monitored in all of these conditions, and, in general, significant differences were foundbetween flies without and with a mutant allele (heterozygous) for Keap1. The results point to altered oxidative stress conditions in diabetic flies. These findings suggest that modest activation of the Cap'n'collar C pathway may be a treatment for diabetic symptoms.
Ferguson, J. M., Gonzalez-Gonzalez, A., Kaiser, J. A., Winzer, S. M., Anast, J. M., Ridenhour, B., Miura, T. A. and Parent, C. E. (2023). Hidden variable models reveal the effects of infection from changes in host survival. PLoS Comput Biol 19(2): e1010910. PubMed ID: 36812266
The impacts of disease on host vital rates can be demonstrated using longitudinal studies, but these studies can be expensive and logistically challenging. This study examined the utility of hidden variable models to infer the individual effects of infectious disease from population-level measurements of survival when longitudinal studies are not possible. This approach seeks to explain temporal deviations in population-level survival after introducing a disease causative agent when disease prevalence cannot be directly measured by coupling survival and epidemiological models. This approach was tested using an experimental host system (Drosophila melanogaster) with multiple distinct pathogens to validate the ability of the hidden variable model to infer per-capita disease rates. This approach was then applied to a disease outbreak in harbor seals (Phoca vituline) that had data on observed strandings but no epidemiological data. The hidden variable modeling approach successfully detected the per-capita effects of disease from monitored survival rates in both the experimental and wild populations. This approach may prove useful for detecting epidemics from public health data in regions where standard surveillance techniques are not available and in the study of epidemics in wildlife populations, where longitudinal studies can be especially difficult to implement.
Di Leva, F., Filosi, M., Oyston, L., Silvestri, E., Picard, A., Lavdas, A. A., Lobbestael, E., Baekelandt, V., Neely, G. G., Pramstaller, P. P., Hicks, A. A. and Corti, C. (2023). Increased Levels of the Parkinson's Disease-Associated Gene ITPKB Correlate with Higher Expression Levels of alpha-Synuclein, Independent of Mutation Status. Int J Mol Sci 24(3). PubMed ID: 36768321
Autosomal dominant mutations in the gene encoding α-synuclein (SNCA) were the first to be linked with hereditary Parkinson's disease (PD). Duplication and triplication of SNCA has been observed in PD patients, together with mutations at the N-terminal of the protein, among which A30P and A53T influence the formation of fibrils. By overexpressing human α-synuclein in the neuronal system of Drosophila, this study functionally validated the ability of IP3K2, an ortholog of the GWAS identified risk gene, Inositol-trisphosphate 3-kinase B (ITPKB), to modulate α-synuclein toxicity in vivo. ITPKB mRNA and protein levels were also increased in SK-N-SH cells overexpressing wild-type α-synuclein, A53T or A30P mutants. Kinase overexpression was detected in the cytoplasmic and in the nuclear compartments in all α-synuclein cell types. By quantifying mRNAs in the cortex of PD patients, higher levels of ITPKB mRNA were observed when SNCA was expressed more (p < 0.05), compared to controls. A positive correlation was also observed between SNCA and ITPKB expression in the cortex of patients, which was not seen in the controls. This observation was replicated in a public dataset. These data, generated in SK-N-SH cells and in cortex from PD patients, show that the expression of α-synuclein and ITPKB is correlated in pathological situations.
Huang, Y., Wen, D., Yuan, Y. and Chen, W. (2023). Gene Set Enrichment Analysis and Genetic Experiment Reveal Changes in Cell Signaling Pathways Induced by alpha-Synuclein Overexpression. Biomedicines 11(2). PubMed ID: 36830800
Abnormal accumulation of alpha synuclein (α-Syn) in sporadic and familial Parkinson's disease (PD) may be a key step in its pathogenesis. In this study, the expression matrix of the GSE95427 dataset after α-Syn overexpression in human glioma cell line H4 was obtained from the GEO database. The Gene Set Enrichment Analysis (GSEA) method was used to reanalyze this dataset to evaluate the possible functions of åalpha;-Syn. The results showed that the tumor necrosis factor alpha (TNF-α) signal was significantly activated in α-Syn-overexpressing cells, and oxidative phosphorylation signal, extracellular matrix signal, cell cycle related signal and fatty acid metabolism signal were significantly inhibited. Moreover, the alpha;-Syn-expressing transgenic Drosophila model of Parkinson's disease and knocked-down eiger, a TNF superfamily ligand homologue, indicated that the TNF-α pathway plays a role in the common pathogenesis of synucleinopathies. This analysis based on GSEA data provides more clues for a better understanding of α-Syn function.
Jacquemyn, J., Kuenen, S., Swerts, J., Pavie, B., Vijayan, V., Kilic, A., Chabot, D., Wang, Y. C., Schoovaerts, N., Corthout, N. and Verstreken, P. (2023). Parkinsonism mutations in DNAJC6 cause lipid defects and neurodegeneration that are rescued by Synj1. NPJ Parkinsons Dis 9(1): 19. PubMed ID: 36739293
Recent evidence links dysfunctional lipid metabolism to the pathogenesis of Parkinson's disease, but the mechanisms are not resolved. This study generated a new Drosophila knock-in model of DNAJC6/Auxilin and found that the pathogenic mutation causes synaptic dysfunction, neurological defects and neurodegeneration, as well as specific lipid metabolism alterations. In these mutants, membrane lipids containing long-chain polyunsaturated fatty acids, including phosphatidylinositol lipid species that are key for synaptic vesicle recycling and organelle function, are reduced. Overexpression of another protein mutated in Parkinson's disease, Synaptojanin-1, known to bind and metabolize specific phosphoinositides, rescues the DNAJC6/Auxilin lipid alterations, the neuronal function defects and neurodegeneration. This work reveals a functional relation between two proteins mutated in Parkinsonism and implicates deregulated phosphoinositide metabolism in the maintenance of neuronal integrity and neuronal survival.

Wednesday, July 26th - Adult Physiology

Shianiou, G., Teloni, S. and Apidianakis, Y. (2023). Intestinal Immune Deficiency and Juvenile Hormone Signaling Mediate a Metabolic Trade-off in Adult Drosophila Females. Metabolites 13(3). PubMed ID: 36984780
A trade-off hypothesis pertains to the biased allocation of limited resources between two of the most important fitness traits, reproduction and survival to infection. This quid pro quo manifests itself within animals prioritizing their energetic needs according to genetic circuits balancing metabolism, germline activity and immune response. Key evidence supporting this hypothesis includes dipteran fecundity being compromised by systemic immunity, and female systemic immunity being compromised by mating. This study revealed a local trade-off taking place in the female Drosophila midgut upon immune challenge. Genetic manipulation of intestinal motility, permeability, regeneration and three key midgut immune pathways provides evidence of an antagonism between specific aspects of intestinal defense and fecundity. That is, juvenile hormone (JH)-controlled egg laying, lipid droplet utilization and insulin receptor expression are specifically compromised by the immune deficiency (Imd) and the dual oxidase (Duox) signaling in the midgut epithelium. Moreover, antimicrobial peptide (AMP) expression under the control of the Imd pathway is inhibited upon mating and JH signaling in the midgut. Local JH signaling is further implicated in midgut dysplasia, inducing stem cell-like clusters and gut permeability. Thus, midgut JH signaling compromises host defense to infection by reducing Imd-controlled AMP expression and by inducing dysplasia, while midgut signaling through the Imd and Duox pathways compromises JH-guided metabolism and fecundity.
Pant, A., Melkani, Y. and Melkani, G. (2023). Automated evaluation of cardiac contractile dynamics and aging prediction using machine learning in a Drosophila model. Res Sq. PubMed ID: 36993511:
The Drosophila model has proven tremendously powerful for understanding pathophysiological bases of several human disorders including aging and cardiovascular disease. Relevant high-speed imaging and high-throughput lab assays generate large volumes of high-resolution videos, necessitating next-generation methods for rapid analysis. This study presents a platform for deep learning-assisted segmentation applied to optical microscopy of Drosophila hearts and the first to quantify cardiac physiological parameters during aging. An experimental test dataset is used to validate a Drosophila aging model. Then two novel methods were used to predict fly aging: deep-learning video classification and machine-learning classification via cardiac parameters. Both models suggest excellent performance, with an accuracy of 83.3% (AUC 0.90) and 77.1% (AUC 0.85), respectively. Furthermore, beat-level dynamics are reported for predicting the prevalence of cardiac arrhythmia. The presented approaches can expedite future cardiac assays for modeling human diseases in Drosophila and can be extended to numerous animal/human cardiac assays under multiple conditions. Significance Current analysis of Drosophila cardiac recordings is capable of limited cardiac physiological parameters and are error-prone and time-consuming. This study presents the first deep-learning pipeline for high-fidelity automatic modeling of Drosophila contractile dynamics. Methods are presented for automatically calculating all relevant parameters for diagnosing cardiac performance in aging model. Using the machine and deep learning age-classification approach, aging hearts can be predicted with an accuracy of 83.3% (AUC 0.90) and 77.1% (AUC 0.85), respectively.
Diaz, A. V., Matheny, T., Stephenson, D., Nemkov, T., D'Alessandro, A. and Reis, T. (2023). Spenito-dependent metabolic sexual dimorphism intrinsic to fat storage cells. bioRxiv. PubMed ID: 36824729
Metabolism in males and females is distinct. Differences are usually linked to sexual reproduction, with circulating signals (e.g. hormones) playing major roles. By contrast, sex differences prior to sexual maturity and intrinsic to individual metabolic tissues are less understood. 'This study analyzed Drosophila melanogaster larvae and find that males store more fat than females, the opposite of the sexual dimorphism in adults. Metabolic differences are intrinsic to the major fat storage tissue, including many differences in the expression of metabolic genes. Previous work identified fat storage roles for Spenito (Nito), a conserved RNA-binding protein and regulator of sex determination. Nito knockdown specifically in the fat storage tissue abolished fat differences between males and females. Nito is required for sex-specific expression of the master regulator of sex determination, Sex-lethal (Sxl). "Feminization" of fat storage cells via tissue-specific overexpression of a Sxl target gene made larvae lean, reduced the fat differences between males and females, and induced female-like metabolic gene expression. Altogether, this study supports a model in which Nito autonomously controls sexual dimorphisms and differential expression of metabolic genes in fat cells in part through its regulation of the sex determination pathway.
Padavannil, A., Murari, A., Rhooms, S. K., Owusu-Ansah, E. and Letts, J. A. (2023). Resting mitochondrial complex I from Drosophila melanogaster adopts a helix-locked state. Elife 12. PubMed ID: 36952377
Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway 'deactive' state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. This study reports the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. Although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. A helix-locked state was identified in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.
Freoa, L., Chevin, L. M., Christol, P., Méléard, S., Rera, M., Veber, A. and Gibert, J. M. (2023). Drosophilids with darker cuticle have higher body temperature under light. Sci Rep 13(1): 3513. PubMed ID: 36864153
Cuticle pigmentation was shown to be associated with body temperature for several relatively large species of insects, but it was questioned for small insects. This study used a thermal camera to assess the association between drosophilid cuticle pigmentation and body temperature increase when individuals are exposed to light. Mutants were compared of large effects within species (Drosophila melanogaster ebony and yellow mutants). Then the impact of naturally occurring pigmentation variation within species complexes (Drosophila americana/Drosophila novamexicana and Drosophila yakuba/Drosophila santomea) was examined. Finally lines of D. melanogaster with moderate differences in pigmentation were examined. Significant differences were found in temperatures for each of the four pairs analyzed. The temperature differences appeared to be proportional to the differently pigmented area: between Drosophila melanogaster ebony and yellow mutants or between Drosophila americana and Drosophila novamexicana, for which the whole body is differently pigmented, the temperature difference was around 0.6 &frh;C ± 0.2 °C. By contrast, between D. yakuba and D. santomea or between Drosophila melanogaster Dark and Pale lines, for which only the posterior abdomen is differentially pigmented, a temperature difference of about 0.14 °C ± 0.10 °C was detected. This strongly suggests that cuticle pigmentation has ecological implications in drosophilids regarding adaptation to environmental temperature.
Gomez Ortega, J., Raubenheimer, D., Tyagi, S., Mirth, C. K. and Piper, M. D. W. (2023). Biosynthetic constraints on amino acid synthesis at the base of the food chain may determine their use in higher-order consumer genomes. PLoS Genet 19(2): e1010635. PubMed ID: 36780875
Dietary nutrient composition is essential for shaping important fitness traits and behaviours. Many organisms are protein limited, and for Drosophila melanogaster this limitation manifests at the level of the single most limiting essential Amino Acid (AA) in the diet. The identity of this AA and its effects on female fecundity is readily predictable by a procedure called exome matching in which the sum of AAs encoded by a consumer's exome is used to predict the relative proportion of AAs required in its diet. However, the exome matching calculation does not weight AA contributions to the overall profile by protein size or expression. This study updates the exome matching calculation to include these weightings. Surprisingly, although nearly half of the transcriptome is differentially expressed when comparing male and female flies, this study found that creating transcriptome-weighted exome matched diets for each sex did not enhance their fecundity over that supported by exome matching alone. These data indicate that while organisms may require different amounts of dietary protein across conditions, the relative proportion of the constituent AAs remains constant. Interestingly, this study also found that exome matched AA profiles are generally conserved across taxa and that the composition of these profiles might be explained by energetic and elemental limitations on microbial AA synthesis. Thus, it appears that ecological constraints amongst autotrophs shape the relative proportion of AAs that are available across trophic levels and that this constrains biomass composition.

Tuesday July 25th - Immune Response

Hanson, M. A. and Lemaitre, B. (2023). Antimicrobial peptides do not directly contribute to aging in Drosophila, but improve lifespan by preventing dysbiosis. Dis Model Mech. PubMed ID: 36847474
Antimicrobial peptides (AMPs) are innate immune effectors first studied for their role in host defense. Recent studies have implicated these peptides in the clearance of aberrant cells and in neurodegenerative syndromes. In Drosophila, many AMPs are produced downstream of Toll and Imd NF-κB pathways upon infection. Upon aging, AMPs are upregulated, drawing attention to these molecules as possible causes of age-associated inflammatory diseases. However, functional studies overexpressing or silencing these genes have been inconclusive. Using an isogenic set of AMP gene deletions, this study investigated the net impact of AMPs on aging. Overall, no major effect of individual AMPs on lifespan was found, with the possible exception of Defensin. However, ΔAMP14 flies lacking seven AMP gene families display reduced lifespan. Increased bacterial load in the food of aged ΔAMP14 flies suggests their lifespan reduction is due to microbiome dysbiosis, consistent with a previous study. Moreover, germ-free conditions extends the lifespan of ΔAMP14 flies. Overall, these results do not point to an overt role of individual AMPs in lifespan. Instead, this study found that AMPs collectively impact lifespan by preventing dysbiosis during aging.
Kato, D., Miura, K. and Yokoi, K. (2023). Analysis of the Toll and Spaetzle Genes Involved in Toll Pathway-Dependent Antimicrobial Gene Induction in the Red Flour Beetle, Tribolium castaneum (Coleoptera; Tenebrionidae) Int J Mol Sci 24(2). PubMed ID: 36675034

Insects rely only on their innate immune system to protect themselves from pathogens. Antimicrobial peptide (AMP) production is the main immune reaction in insects. In Drosophila melanogaster, the reaction is regulated mainly by the Toll and immune deficiency (IMD) pathways. Spaetzle proteins, activated by immune signals from upstream components, bind to Toll proteins, thus, activating the Toll pathway, which in turn, induces AMP genes. Previous studies have shown the difference in immune systems related to Toll and IMD pathways between D. melanogaster and Tribolium castaneum. In T. castaneum, nine Toll and seven spaetzle (spz) genes were identified. To extend understanding of AMP production by T. castaneum, functional assays of Toll and spaetzle genes related to Toll-pathway-dependent AMP gene expression were conducted in T. castaneum under challenge with bacteria or budding yeast. The results revealed that Toll3 and Toll4 double-knockdown and spz7 knockdown strongly and moderately reduced the Toll-pathway-dependent expression of AMP genes, respectively. Moreover, Toll3 and Toll4 double-knockdown pupae more rapidly succumbed to entomopathogenic bacteria than the control pupae, but spz7 knockdown pupae did not. The results suggest that Toll3 and Toll4 play a large role in Toll-pathway-dependent immune reactions, whereas spz7 plays a small part (Kato, 2023).

Parks, S. C., Okakpu, O. K., Azizpor, P., Nguyen, S., Martinez-Beltran, S., Claudio, I., Anesko, K., Bhatia, A., Dhillon, H. S. and Dillman, A. R. (2023). Parasitic nematode secreted phospholipase A(2) suppresses cellular and humoral immunity by targeting hemocytes in Drosophila melanogaster. Front Immunol 14: 1122451. PubMed ID: 37006283

A key aspect of parasitic nematode infection is the nematodes' ability to evade and/or suppress host immunity. This immunomodulatory ability is likely driven by the release of hundreds of excretory/secretory proteins (ESPs) during infection. While ESPs have been shown to display immunosuppressive effects on various hosts, understanding of the molecular interactions between individual proteins released and host immunity requires further study. A recently discovered secreted phospholipase A2 (sPLA(2)) released from the entomopathogenic nematode (EPN) Steinernema carpocapsae was named Sc-sPLA(2). This study reports that Sc-sPLA(2) increased mortality of Drosophila melanogaster infected with Streptococcus pneumoniae and promoted increased bacterial growth. Furthermore, the data showed that Sc-sPLA(2) was able to downregulate both Toll and Imd pathway-associated antimicrobial peptides (AMPs) including drosomycin and defensin, in addition to suppressing phagocytosis in the hemolymph. Sc-sPLA(2) was also found to be toxic to D. melanogaster with the severity being both dose- and time-dependent. Collectively, these data highlighted that Sc-sPLA(2) possessed both toxic and immunosuppressive capabilities (Parks, 2023).

Elguero, J. E., Liu, G., Tiemeyer, K., Gandevia, H., Duro, L. and McCall, K. (2023). Defective phagocytosis leads to neurodegeneration through systemic increased innate immune signaling. bioRxiv. PubMed ID: 36711924

In nervous system development, disease and injury, neurons undergo programmed cell death, leaving behind cell corpses that are removed by phagocytic glia. Altered glial phagocytosis has been implicated in several neurological diseases including Alzheimer's disease, Parkinson's disease, and traumatic brain injury. To untangle the links between glial phagocytosis and neurodegeneration, this study investigated Drosophila mutants lacking the phagocytic receptor Draper. Loss of Draper leads to persistent neuronal cell corpses and age-dependent neurodegeneration. This study investigated whether the phagocytic defects observed in draper mutants lead to chronic increased immune activation that promotes neurodegeneration. A major immune response in Drosophila is the activation of two NFκB signaling pathways that produce antimicrobial peptides, primarily in the fat body. This study found that the antimicrobial peptide Attacin-A is highly upregulated in the fat body of aged draper mutants and that inhibition of the Immune deficiency (Imd) pathway in the glia and fat body of draper mutants led to reduced neurodegeneration, indicating that immune activation promotes neurodegeneration in draper mutants. Taken together, these findings indicate that phagocytic defects lead to neurodegeneration via increased immune signaling, both systemically and locally in the brain (Elguero, 2023).

Li, Q., Zhang, C., Zhang, C., Duan, R. and Hua, Y. (2023). CG4968 positively regulates the immune deficiency pathway by targeting Imd protein in Drosophila. PeerJ 11: e14870. PubMed ID: 36778143
Drosophila melanogaster relies solely on innate immunity to defend against various microbial pathogens. Although it is well-known that the adaptor protein Imd undergoes K63-linked ubiquitination to activate the downstream signaling cascades, its involvement with K48-linked ubiquitination and what is responsible for controlling this modification remain largely unknown. This study explored the immunological function of CG4968, which encodes a typical ovarian tumour-associated protease (OTU)-type deubiquitinase (Dub) in flies. In vitro and in vivo evidence demonstrated that CG4968 plays a positive role in governing the immune deficiency (IMD), but not the Toll innate immune response in an OTU domain-dependent manner. Mechanistically, it was found that CG4968 is associated with Imd to restrict its K48-linked ubiquitination, thereby contributing to its turnover. Collectively, this study uncovered a novel regulatory mechanism involving the K48-linked ubiquitination of Imd in Drosophila innate immunity.
Zhu, Y., Liu, L., Zhang, C., Zhang, C., Han, T., Duan, R., Jin, Y., Guo, H., She, K., Xiao, Y., Goto, A., Cai, Q. and Ji, S. (2022). Endoplasmic reticulum-associated protein degradation contributes to Toll innate immune defense in Drosophila melanogaster. Front Immunol 13: 1099637. PubMed ID: 36741393

In Drosophila, the endoplasmic reticulum-associated protein degradation (ERAD) is engaged in regulating pleiotropic biological processes, with regard to retinal degeneration, intestinal homeostasis, and organismal development. The extent to which it functions in controlling the fly innate immune defense, however, remains largely unknown. This study shows that blockade of the ERAD in fat bodies antagonizes the Toll but not the IMD innate immune defense in Drosophila. Genetic approaches further suggest a functional role of Me31B in the ERAD-mediated fly innate immunity. Moreover, evidence is provided that silence of Xbp1 other than PERK or Atf6 partially rescues the immune defects by the dysregulated ERAD in fat bodies. Collectively, this study uncovers an essential function of the ERAD in mediating the Toll innate immune reaction in Drosophila (Zhu, 2022).

Monday, July 24th - Evolution

Fujii, S., Ahn, J. E., Jagge, C., Shetty, V., Janes, C., Mohanty, A., Slotman, M., Adelman, Z. N. and Amrein, H. (2023). RNA Taste Is Conserved in Dipteran Insects. J Nutr. PubMed ID: 36907444
Ribonucleosides and RNA are an underappreciated nutrient group essential during Drosophila larval development and growth. Detection of these nutrients requires at least one of the 6 closely related taste receptors encoded by the Gr28 genes, one of the most conserved insect taste receptor subfamilies. This study investigated whether blow fly larvae and mosquito larvae, which shared the last ancestor with Drosophila about 65 and 260 million years ago, respectively, can taste RNA and ribose. Whether the Gr28 homologous genes of the mosquitoes Aedes aegypti and Anopheles gambiae can sense these nutrients when expressed in transgenic Drosophila larvae was also tested. Taste preference in blow flies was examined by adapting a 2-choice preference assay that has been well-established for Drosophila larvae. For the mosquito Aedes aegypti, a new 2-choice preference assay was developed that accommodates the aquatic environment of these insect larvae. Finally, Gr28 homologs in these species were identified and expressed in Drosophila melanogaster to determine their potential function as RNA receptors. Larvae of the blow fly Cochliomyia macellaria and Lucilia cuprina are strongly attracted to RNA (0.5 mg/mL) in the 2-choice feeding assays (P <l 0.05). Similarly, the mosquito Aedes aegypti larvae showed a strong preference for RNA (2.5 mg/mL) in an aquatic 2-choice feeding assay. Moreover, when Gr28 homologs of Aedes or Anopheles mosquitoes are expressed in appetitive taste neurons of Drosophila melanogaster larvae lacking their Gr28 genes, preference for RNA (0.5 mg/mL) and ribose (0.1 M) is rescued (P < 0.05). The appetitive taste for RNA and ribonucleosides in insects emerged about 260 million years ago, the time mosquitoes and fruit flies diverged from their last common ancestor. Like sugar receptors, receptors for RNA have been highly conserved during insect evolution, suggesting that RNA is a critical nutrient for fast-growing insect larvae.
Pelletier, K., Pitchers, W. R., Mammel, A., Northrop-Albrecht, E., Marquez, E. J., Moscarella, R. A., Houle, D. and Dworkin, I. (2023). Complexities of recapitulating polygenic effects in natural populations: replication of genetic effects on wing shape in artificially selected and wild caught populations of Drosophila melanogaster. Genetics. PubMed ID: 36961731
Identifying the genetic architecture of complex traits is important to many geneticists, including those interested in human disease, plant and animal breeding, and evolutionary genetics. Advances in sequencing technology and statistical methods for genome-wide association studies (GWAS) have allowed for the identification of more variants with smaller effect sizes, however, many of these identified polymorphisms fail to be replicated in subsequent studies. In addition to sampling variation, this failure to replicate reflects the complexities introduced by factors including environmental variation, genetic background, and differences in allele frequencies among populations. Using Drosophila melanogaster wing shape, it was asked if it were possible to replicate allelic effects of polymorphisms first identified in a GWAS in three genes: dachsous (ds), extra-macrochaete (emc) and neuralized (neur), using artificial selection in the lab, and bulk segregant mapping in natural populations. It was demonstrated that multivariate wing shape changes associated with these genes are aligned with major axes of phenotypic and genetic variation in natural populations. Following seven generations of artificial selection along the ds shape change vector, genetic differentiation of variants was observed in ds and genomic regions containing other genes in the hippo signaling pathway. This suggests a shared direction of effects within a developmental network. Artificial selection was also performed with the emc shape change vector, which is not a part of the hippo signaling network, but which exhibited a largely shared direction of effects. The response to selection along the emc vector was similar to that of ds, suggesting that the available genetic diversity of a population, summarized by the genetic (co)variance matrix (G), influenced alleles captured by selection. Despite the success with artificial selection, bulk segregant analysis using natural populations did not detect these same variants, likely due to the contribution of environmental variation and low minor allele frequencies, coupled with small effect sizes of the contributing variants.
Zakharenko, L. P., Petrovskii, D. V., Bobrovskikh, M. A., Gruntenko, N. E., Yakovleva, E. Y., Markov, A. V. and Putilov, A. A. (2023). Motus Vita Est: Fruit Flies Need to Be More Active and Sleep Less to Adapt to Either a Longer or Harder Life. Clocks Sleep 5(1): 98-115. PubMed ID: 36975551
Activity plays a very important role in keeping bodies strong and healthy, slowing senescence, and decreasing morbidity and mortality. Drosophila models of evolution under various selective pressures can be used to examine whether increased activity and decreased sleep duration are associated with the adaptation of this nonhuman species to longer or harder lives. For several years, descendants of wild flies were reared in a laboratory without and with selection pressure. To maintain the "salt" and "starch" strains, flies from the wild population (called "control") were reared on two adverse food substrates. The "long-lived" strain was maintained through artificial selection for late reproduction. The 24 h patterns of locomotor activity and sleep in flies from the selected and unselected strains (902 flies in total) were studied in constant darkness for at least, 5 days. Compared to the control flies, flies from the selected strains demonstrated enhanced locomotor activity and reduced sleep duration. The most profound increase in locomotor activity was observed in flies from the starch (short-lived) strain. Additionally, the selection changed the 24 h patterns of locomotor activity and sleep. For instance, the morning and evening peaks of locomotor activity were advanced and delayed, respectively, in flies from the long-lived strain. Flies become more active and sleep less in response to various selection pressures. These beneficial changes in trait values might be relevant to trade-offs among fitness-related traits, such as body weight, fecundity, and longevity.
Walter, G. M. (2023). Experimental Evidence That Phenotypic Evolution but Not Plasticity Occurs along Genetic Lines of Least Resistance in Homogeneous Environments. Am Nat 201(4): E70-e89. PubMed ID: 36957997
Genetic correlations concentrate genetic variation in certain directions of the multivariate phenotype. Adaptation and, under some models, plasticity is expected to occur in the direction of the phenotype containing the greatest amount of genetic variation (g(max)). However, this may hinge on environmental heterogeneity, which can affect patterns of genetic variation. This study used experimental evolution to test whether plasticity and phenotypic evolution follow g(max) during adaptation to environments that varied in environmental heterogeneity. For >25 generations, Drosophila melanogaster populations were exposed to six homogeneous or spatially and temporally heterogeneous treatments involving hot (25°C) and cold (16°C) temperatures. Five wing traits were assayed in both temperatures. Wing morphology diverged between populations evolving in homogeneous hot and cold temperatures in a direction of the phenotype containing a large proportion of genetic variance and that aligned closely with g(max) at 16°C but not at 2°°C. Spatial heterogeneity produced an intermediate phenotype, which was associated with similar genetic variance across assay temperatures compared with all other treatments. Surprisingly, plasticity across assay temperatures was in a different direction to phenotypic evolution and aligned better with maternal variance than g(max). Together, these results provide experimental evidence for evolution along genetic lines of least resistance in homogeneous environments but no support for predicting plastic responses from the orientation of genetic variation. These results also suggest that spatial heterogeneity could maintain genetic variation that increases the stability of genetic variance across environments.
Williams, A. M., Ngo, T. M., Figueroa, V. E. and Tate, A. T. (2023). The Effect of Developmental Pleiotropy on the Evolution of Insect Immune Genes. Genome Biol Evol 15(3). PubMed ID: 36911982
The pressure to survive ever-changing pathogen exposure explains the frequent observation that immune genes are among the fastest evolving in the genomes of many taxa, but an intriguing proportion of immune genes also appear to be under purifying selection. Though variance in evolutionary signatures of immune genes is often attributed to differences in gene-specific interactions with microbes, this explanation neglects the possibility that immune genes participate in other biological processes that could pleiotropically constrain adaptive selection. This study, analyzed available transcriptomic and genomic data from Drosophila melanogaster and related species to test the hypothesis that there is substantial pleiotropic overlap in the developmental and immunological functions of genes involved in immune signaling and that pleiotropy would be associated with stronger signatures of evolutionary constraint. The results suggest that pleiotropic immune genes do evolve more slowly than those having no known developmental functions and that signatures of constraint are particularly strong for pleiotropic immune genes that are broadly expressed across life stages. These results support the general yet untested hypothesis that pleiotropy can constrain immune system evolution, raising new fundamental questions about the benefits of maintaining pleiotropy in systems that need to rapidly adapt to changing pathogen pressures.
Oladipupo, S. O., Carroll, J. D. and Beckmann, J. F. (2023). Convergent Aedes and Drosophila CidB interactomes suggest cytoplasmic incompatibility targets are conserved. Insect Biochem Mol Biol 155: 103931. PubMed ID: 36933571
Wolbachia-mediated cytoplasmic incompatibility (CI) is a conditional embryonic lethality induced when Wolbachia-modified sperm fertilizes an uninfected egg. The Wolbachia proteins, CidA and CidB control CI. CidA is a rescue factor that reverses lethality. CidA binds to CidB. CidB contains a deubiquitinating enzyme and induces CI. Precisely how CidB induces CI and what it targets are unknown. Likewise, how CidA prevents sterilization by CidB is not clear. To identify CidB substrates in mosquitos pull-down assays were conducted using recombinant CidA and CidB mixed with Aedes aegypti lysates to identify the protein interactomes of CidB and the CidB/CidA protein complex. These data allow cross comparison CidB interactomes across taxa for Aedes and Drosophila. The data replicate several convergent interactions, suggesting that CI targets conserved substrates across insects. The data support a hypothesis that CidA rescues CI by tethering CidB away from its substrates. Specifically, this study identified ten convergent candidate substrates including P32 (protamine-histone exchange factor), karyopherin alpha, ubiquitin-conjugating enzyme, and Bicoid stabilizing factor. Future analysis on how these candidates contribute to CI will clarify mechanisms.

Friday, July 21st - Gonads

Doyle, D. A., Burian, F. N., Aharoni, B., Klinder, A. J., Menzel, M. M., Nifras, G. C. C., Shabazz-Henry, A. L., Palma, B. U., Hidalgo, G. A., Sottolano, C. J., Ortega, B. M. and Niepielko, M. G. (2023). Evolutionary changes in germ granule mRNA content are driven by multiple mechanisms in Drosophila. bioRxiv. PubMed ID: 36865184
The co-packaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to post-transcriptionally regulate mRNAs that function in germline development and maintenance. In D. melanogaster , mRNAs accumulate in germ granules by forming homotypic clusters, aggregates that contain multiple transcripts from a specific gene. Nucleated by Oskar (Osk), homotypic clusters in D. melanogaster are generated through a stochastic seeding and self-recruitment process that requires the 3' UTR of germ granule mRNAs. Interestingly, the 3' UTR belonging to germ granule mRNAs, such as nanos ( nos ), have considerable sequence variations among Drosophila species. Thus, it was hypothesized that evolutionary changes in the 3' UTR influences germ granule development. To test this hypothesis, the homotypic clustering of nos and polar granule component (pgc) was investigated in four Drosophila species, and it was concluded that homotypic clustering is a conserved developmental process used to enrich germ granule mRNAs. Additionally, it was discovered that the number of transcripts found in nos and/or pgc clusters could vary significantly among species. By integrating biological data with computational modeling, it was determined that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels, and/or homotypic clustering efficacy. Finally, it was found that the nos 3' UTR from different species can alter the efficacy of nos homotypic clustering, resulting in germ granules with reduced nos accumulation. These findings highlight the impact that evolution has on the development of germ granules and may provide insight into processes that modify the content of other classes of biomolecular condensates.
Rincon-Ortega, L., Valencia-Exposito, A., Kabanova, A., Gonzalez-Reyes, A. and Martin-Bermudo, M. D. (2023). Integrins control epithelial stem cell proliferation in the Drosophila ovary by modulating the Notch pathway. Front Cell Dev Biol 11: 1114458. PubMed ID: 36926523
Cell proliferation and differentiation show a remarkable inverse relationship. The temporal coupling between cell cycle withdrawal and differentiation of stem cells (SCs) is crucial for epithelial tissue growth, homeostasis and regeneration. Proliferation vs. differentiation SC decisions are often controlled by the surrounding microenvironment, of which the basement membrane (BM; a specialized form of extracellular matrix surrounding cells and tissues), is one of its main constituents. Years of research have shown that integrin-mediated SC-BM interactions regulate many aspects of SC biology, including the proliferation-to-differentiation switch. However, these studies have also demonstrated that the SC responses to interactions with the BM are extremely diverse and depend on the cell type and state and on the repertoire of BM components and integrins involved. This study shows that eliminating integrins from the follicle stem cells (FSCs) of the Drosophila ovary and their undifferentiated progeny increases their proliferation capacity. This results in an excess of various differentiated follicle cell types, demonstrating that cell fate determination can occur in the absence of integrins. Because these phenotypes are similar to those found in ovaries with decreased laminin levels, the results point to a role for the integrin-mediated cell-BM interactions in the control of epithelial cell division and subsequent differentiation. Finally, it was shown that integrins regulate proliferation by restraining the activity of the Notch/Delta pathway during early oogenesis. This work increases knowledge of the effects of cell-BM interactions in different SC types and should help improve understanding of the biology of SCs and exploit their therapeutic potential.
Yu, J., Li, Z., Fu, Y., Sun, F., Chen, X., Huang, Q., He, L., Yu, H., Ji, L., Cheng, X., Shi, Y., Shen, C., Zheng, B. and Sun, F. (2023). Single-cell RNA-sequencing reveals the transcriptional landscape of ND-42 mediated spermatid elongation via mitochondrial derivative maintenance in Drosophila testes. Redox Biol 62: 102671. PubMed ID: 36933391
During spermatogenesis, mitochondria extend along the whole length of spermatid tail and offer a structural platform for microtubule reorganization and synchronized spermatid individualization, that eventually helps to generate mature sperm in Drosophila. However, the regulatory mechanism of spermatid mitochondria during elongation remains largely unknown. This study demonstrated that NADH dehydrogenase (ubiquinone) 42 kDa subunit (ND-42) was essential for male fertility and spermatid elongation in Drosophila. Moreover, ND-42 depletion led to mitochondrial disorders in Drosophila testes. Based on single-cell RNA-sequencing (scRNA-seq), this study identified 15 distinct cell clusters, including several unanticipated transitional subpopulations or differentiative stages for testicular germ cell complexity in Drosophila testes. Enrichments of the transcriptional regulatory network in the late-stage cell populations revealed key roles of ND-42 in mitochondria and its related biological processes during spermatid elongation. Notably, it was demonstrated that ND-42 depletion led to maintenance defects of the major mitochondrial derivative and the minor mitochondrial derivative by affecting mitochondrial membrane potential and mitochondrial-encoded genes. This study proposes a novel regulatory mechanism of ND-42 for spermatid mitochondrial derivative maintenance, contributing to a better understanding of spermatid elongation.
Diegmiller, R., Imran Alsous, J., Li, D., Yamashita, Y. M. and Shvartsman, S. Y. (2023). Fusome topology and inheritance during insect gametogenesis. PLoS Comput Biol 19(2): e1010875. PubMed ID: 36821548
From insects to mammals, oocytes and sperm develop within germline cysts comprising cells connected by intercellular bridges (ICBs). In numerous insects, formation of the cyst is accompanied by growth of the fusome-a membranous organelle that permeates the cyst. Fusome composition and function are best understood in Drosophila melanogaster: during oogenesis, the fusome dictates cyst topology and size and facilitates oocyte selection, while during spermatogenesis, the fusome synchronizes the cyst's response to DNA damage. Despite its distinct and sex-specific roles during insect gametogenesis, elucidating fusome growth and inheritance in females and its structure and connectivity in males has remained challenging. This study took advantage of advances in three-dimensional (3D) confocal microscopy and computational image processing tools to reconstruct the topology, growth, and distribution of the fusome in both sexes. In females, the experimental findings inform a theoretical model for fusome assembly and inheritance and suggest that oocyte selection proceeds through an 'equivalency with a bias' mechanism. In males, it was found that cell divisions can deviate from the maximally branched pattern observed in females, leading to greater topological variability. This work consolidates existing disjointed experimental observations and contributes a readily generalizable computational approach for quantitative studies of gametogenesis within and across species.
Fang, Y., Zhang, F., Zhan, Y., Lu, M., Xu, D., Wang, J., Li, Q., Zhao, L. and Su, Y. (2023). RpS3 Is Required for Spermatogenesis of Drosophila melanogaster. Cells 12(4). PubMed ID: 36831240
Ribosomal proteins (RPs) constitute the ribosome, thus participating in the protein biosynthesis process. Emerging studies have suggested that many RPs exhibit different expression levels across various tissues and function in a context-dependent manner for animal development. Drosophila melanogaster RpS3 encodes the ribosomal protein S3, one component of the 40S subunit of ribosomes. t RpS3 was found to be highly expressed in the reproductive organs of adult flies and its depletion in male germline cells led to severe defects in sperm production and male fertility. Immunofluorescence staining showed that RpS3 knockdown had little effect on early germ cell differentiation, but strongly disrupted the spermatid elongation and individualization processes. Furthermore, abnormal morphology and activity were pbserved of mitochondrial derivatives in the elongating spermatids of RpS3-knockdown testes, which could cause the failure of axoneme elongation. This study also found that RpS3 RNAi inhibited the formation of the individualization complex that takes charge of disassociating the spermatid bundle. In addition, excessive apoptotic cells were detected in the RpS3-knockdown testes, possibly to clean the defective spermatids. Together, the data demonstrated that RpS3 plays an important role in regulating spermatid elongation and individualization processes and, therefore, is required for normal Drosophila spermatogenesis.
Frantz, S. I., Small, C. M., Cresko, W. A. and Singh, N. D. (2023). Ovarian Transcriptional Response to Wolbachia Infection in D. melanogaster in the Context of Between-Genotype Variation in Gene Expression. G3 (Bethesda). PubMed ID: 36857313
Wolbachia is a maternally-transmitted endosymbiotic bacteria that infects a wide variety of arthropod and nematode hosts. The effects of Wolbachia on host biology are far-reaching and include changes in host gene expression. However, previous work on the host transcriptional response has generally been investigated in the context of a single host genotype. Thus, the relative effect of Wolbachia infection versus host genotype on gene expression is unknown. This study explicitly tested the relative roles of Wolbachia infection and host genotype on host gene expression by comparing the ovarian transcriptomes of four strains of D. melanogaster infected and uninfected with Wolbachia. The data suggest that infection explains a small amount of transcriptional variation, particularly in comparison to variation in gene expression among strains. However, infection specifically affects genes related to cell cycle, translation, and metabolism. Enrichment of cell division and recombination processes was foundmamong genes with infection-associated differential expression. Broadly, the transcriptomic changes identified in this study provide novel understanding of the relative magnitude of the effect of Wolbachia infection on gene expression in the context of host genetic variation, and also point to genes that are consistently differentially expressed in response to infection among multiple genotypes.

Thursday, July 20th - Cytoskeleton and Junctions

Schmidt, A., Finegan, T., Haring, M., Kong, D., Fletcher, A. G., Alam, Z., Grosshans, J., Wolf, F. and Peifer, M. (2023). Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins. bioRxiv. PubMed ID: 36909597
During embryonic development dramatic cell shape changes and movements re-shape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. View of this linkage have evolved, and it is now realize that linkage is mediated by a mechanosensitive multiprotein complex assembled via multivalent connections. This study combined genetic, cell biological and modeling approaches to define the mechanism of action and functions of an important player, Drosophila Polychaetoid, homolog of mammalian ZO-1. The data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. Next, structured illumination microscopy was used to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins.The implications for the protein network at the junction-cytoskeletal interface is discussed, suggesting different proteins localize and function in distinct ways but combine to produce robust connections.
Stromberg, K. A., Spain, T., Tomlin, S. A., Amarillo, K. D. and Schroeder, C. M. (2023). Evolutionary diversification reveals distinct somatic versus germline cytoskeletal functions of the Arp2 branched actin nucleator protein. bioRxiv. PubMed ID: 36909544
Branched actin networks are critical in many cellular processes, including cell motility and division. Arp2, a protein within the 7-membered Arp2/3 complex, is responsible for generating branched actin. Given its essential roles, Arp2 evolves under stringent sequence conservation throughout eukaryotic evolution. This study unexpectedly discovered recurrent evolutionary diversification of Arp2 in Drosophila, yielding independently arising paralogs Arp2D in obscura species and Arp2D2 in montium species. Both paralogs are unusually testis-enriched in expression relative to Arp2. Whether their sequence divergence from canonical Arp2 led to functional specialization was investigated by replacing Arp2 in D. melanogaster with either Arp2D or Arp2D2. Despite their divergence, it was surprisingly found that both complement Arp2's essential function in the soma, suggesting they have preserved the ability to polymerize branched actin even in a non-native species. However, it was found that Arp2D -expressing males are subfertile and display many defects throughout sperm development. Two highly diverged structural regions in Arp2D were pinpointed that contribute to these defects: subdomain 2 and the C-terminus. It was expected that germline function would be rescued by replacing Arp2D's long and charged C-terminus with Arp2's short C-terminus, yet surprisingly, the essential somatic function of Arp2D was lost. Therefore, while Arp2D's structural divergence is incompatible with D. melanogaster sperm development, its unique C-terminus has evolved a critical role in actin polymerization. These findings suggest canonical Arp2's function differs between somatic versus germline contexts, and Arp2 paralogs have recurrently evolved and specialized for actin branching in the testis.
Zhu, H. and B, O. S. (2023). Actomyosin pulsing rescues embryonic tissue folding from disruption by myosin fluctuations. bioRxiv. PubMed ID: 36993262
During early development, myosin II mechanically reshapes and folds embryo tissue. A much-studied example is ventral furrow formation in Drosophila, marking the onset of gastrulation. Furrowing is driven by contraction of actomyosin networks on apical cell surfaces, but how the myosin patterning encodes tissue shape is unclear, and elastic models failed to reproduce essential features of experimental celf contraction profiles. The myosin patterning exhibits substanatial cell-to-cell fluctuations with pulsatile time-dependence, a striking but unexplained feature of morphogenesis in many organisms. In this study, using biophysical modeling wviscous forces were found to offer the principle resistance to actomyosin-driven apical constriction. In consequence, tissue shape is encoded in the direction-dependent curvature of the myosin patterning which orients an anterior-posterior furrow. Tissue contraction is highly sensitive to cell-to-cell myosin fluctuations, explaining furrowing failure in genetically perturbed embryos whose fluctuations are temporally persistent. In wild-type embryos, this catastrophic outcome is averted by pulsatile myosin time-dependence, a time-averaging effect that rescues furrowing. This low pass filter mechanism may underlie the usage of actomyosin pulsing in diverse morphogenetic processes across many organisms.
Daeden, A., Mietke, A., Derivery, E., Seum, C., Julicher, F. and Gonzalez-Gaitan, M. (2023). Polarized branched Actin modulates cortical mechanics to produce unequal-size daughters during asymmetric division. Nat Cell Biol 25(2): 235-245. PubMed ID: 36747081
The control of cell shape during cytokinesis requires a precise regulation of mechanical properties of the cell cortex. Only few studies have addressed the mechanisms underlying the robust production of unequal-sized daughters during asymmetric cell division. This study reports that unequal daughter-cell sizes resulting from asymmetric sensory organ precursor divisions in Drosophila are controlled by the relative amount of cortical branched Actin between the two cell poles. This was demonstrated by mistargeting the machinery for branched Actin dynamics using nanobodies and optogenetics. It is thereby possible to engineer the cell shape with temporal precision and thus the daughter-cell size at different stages of cytokinesis. Most strikingly, inverting cortical Actin asymmetry causes an inversion of daughter-cell sizes. These findings uncover the physical mechanism by which the sensory organ precursor mother cell controls relative daughter-cell size: polarized cortical Actin modulates the cortical bending rigidity to set the cell surface curvature, stabilize the division and ultimately lead to unequal daughter-cell size.
Das, M., Cheng, D., Matzat, T. and Auld, V. J. (2023). Innexin-Mediated Adhesion between Glia Is Required for Axon Ensheathment in the Peripheral Nervous System. J Neurosci 43(13): 2260-2276. PubMed ID: 36801823
Glia are essential to protecting and enabling nervous system function and a key glial function is the formation of the glial sheath around peripheral axons. Each peripheral nerve in the Drosophila larva is ensheathed by three glial layers, which structurally support and insulate the peripheral axons. How peripheral glia communicate with each other and between layers is not well established, and this study investigated the role of Innexins in mediating glial function in the Drosophila periphery. Of the eight Drosophila Innexins, it was found two (Inx1 and Inx2) are important for peripheral glia development. In particular loss of Inx1 and Inx2 resulted in defects in the wrapping glia leading to disruption of the glia wrap. Of interest loss of Inx2 in the subperineurial glia also resulted in defects in the neighboring wrapping glia. Inx plaques were observed between the subperineurial glia and the wrapping glia suggesting that gap junctions link these two glial cell types. Inx2 is key to Ca(2+) pulses in the peripheral subperineurial glia but not in the wrapping glia, and no evidence was found of gap junction communication between subperineurial and wrapping glia. Rather there is clear evidence that Inx2 plays an adhesive and channel-independent role between the subperineurial and wrapping glia to ensure the integrity of the glial wrap.
Ermanoska, B., Asselbergh, B., Morant, L., Petrovic-Erfurth, M. L., Hosseinibarkooie, S., Leitao-Gonçalves, R., Almeida-Souza, L., Bervoets, S., Sun, L., Lee, L., Atkinson, D., Khanghahi, A., Tournev, I., Callaerts, P., Verstreken, P., Yang, X. L., Wirth, B., Rodal, A. A., Timmerman, V., Goode, B. L., Godenschwege, T. A. and Jordanova, A. (2023). Tyrosyl-tRNA synthetase has a noncanonical function in actin bundling. Nat Commun 14(1): 999. PubMed ID: 36890170
Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, YARS1 dysfunction was linked to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, this work shows that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration.

Wednesday, July 19th - Adult Neural Development and Function

Ozoe, Y., Matsubara, Y., Tanaka, Y., Yoshioka, Y., Ozoe, F., Shiotsuki, T., Nomura, K., Nakao, T. and Banba, S. (2023). Controlled expression of nicotinic acetylcholine receptor-encoding genes in insects uncovers distinct mechanisms of action of the neonicotinoid insecticide dinotefuran. Pestic Biochem Physiol 191: 105378. PubMed ID: 36963946
Dinotefuran, a neonicotinoid, is a unique insecticide owing to its structure and action. This study took two approaches that employed insects with controlled expression of nicotinic acetylcholine receptor (nAChR)-encoding genes to gain insight into the uniqueness of dinotefuran. First, The insecticidal activity of dinotefuran and imidacloprid was examined against brown planthoppers (Nilaparvata lugens), in which the expression of eight (of 13) individual subunit-encoding genes was specifically reduced using RNA interference. Knockdown of the tested gene, except one, resulted in a decrease in sensitivity to imidacloprid, whereas the sensitivity of N. lugens to dinotefuran decreased only when two of the eight genes were knocked down. These findings imply that a major dinotefuran-targeted nAChR subtype may contain specific subunits although imidacloprid acts on a broad range of receptor subtypes. Next, the effects were examined of knockout of Drosophila α1 subunit-encoding gene (Dα1) on the insecticidal effects of dinotefuran and imidacloprid. Dα1-deficient flies (Dα1(KO)) demonstrated the same sensitivity to dinotefuran as control flies, but a decreased sensitivity to imidacloprid. This difference was attributed to a reduction in imidacloprid-binding sites in Dα1(KO) flies, whereas the binding of dinotefuran remained unchanged. RNA sequencing analysis indicated that Dα2 expression was specifically enhanced in Dα1(KO) flies. These findings suggest that changes in Dα1 and Dα2 expression contribute to the differences in the insecticidal activity of dinotefuran and imidacloprid in Dα1(KO) flies. Overall, these findings suggest that dinotefuran acts on distinct nAChR subtypes.
Wohl, M. P., Liu, J. and Asahina, K. (2023). Drosophila tachykininergic neurons modulate the activity of two groups of receptor-expressing neurons to regulate aggressive tone. J Neurosci. PubMed ID: 36977580
Neuropeptides influence animal behaviors through complex molecular and cellular mechanisms, the physiological and behavioral effects of which are difficult to predict solely from synaptic connectivity. Many neuropeptides can activate multiple receptors, whose ligand affinity and downstream signaling cascades are often different from one another. Although it is known that the diverse pharmacological characteristics of neuropeptide receptors form the basis of unique neuromodulatory effects on distinct downstream cells, it remains unclear exactly how different receptors shape the downstream activity patterns triggered by a single neuronal neuropeptide source. This study uncovered two separate downstream targets that are differentially modulated by Tachykinin, an aggression-promoting neuropeptide in Drosophila. Tachykinin from a single male-specific neuronal type recruits two separate downstream groups of neurons. One downstream group, synaptically connected to the tachykinergic neurons, expresses the receptor TkR86C and is necessary for aggression. In this case, tachykinin supports cholinergic excitatory synaptic transmission between the tachykinergic and TkR86C downstream neurons. The other downstream group expresses the TkR99D receptor and is recruited primarily when tachykinin is over-expressed in the source neurons. Differential activity patterns in the two groups of downstream neurons correlate with levels of male aggression triggered by the tachykininergic neurons. These findings highlight how the amount of neuropeptide released from a small number of neurons can reshape the activity patterns of multiple downstream neuronal populations. The results lay the foundation for further investigations into the neurophysiological mechanism by which a neuropeptide controls complex behaviors.
Lange, A. P. and Wolf, F. W. (2023). Alcohol tolerance encoding in sleep regulatory circadian neurons in Drosophila. bioRxiv. PubMed ID: 36778487
Alcohol tolerance is a simple form of behavioral and neural plasticity that occurs with the first drink. Neural plasticity in tolerance is likely a substrate for longer term adaptations that can lead to alcohol use disorder. Drosophila develop tolerance with characteristics similar to vertebrates, and it is useful model for determining the molecular and circuit encoding mechanisms in detail. Rapid tolerance, measured after the first alcohol exposure is completely metabolized, is localized to specific brain regions that are not interconnected in an obvious way. A forward neuroanatomical screen was used to identify three new neural sites for rapid tolerance encoding. One of these was comprised of two groups of neurons, the DN1a and DN1p glutamatergic neurons, that are part of the Drosophila circadian clock. Rapid tolerance was localized to the two DN1a neurons that regulate arousal by light at night, temperature-dependent sleep timing, and night-time sleep. Two clock neurons that regulate evening activity, LNd6 and the 5th LNv, are postsynaptic to the DN1as and they promote rapid tolerance via the metabotropic glutamate receptor. Thus, rapid tolerance to alcohol overlaps with sleep regulatory neural circuitry, suggesting a mechanistic link.
Song, C. and Broadie, K. (2023). Fragile X mental retardation protein coordinates neuron-to-glia communication for clearance of developmentally transient brain neurons. Proc Natl Acad Sci U S A 120(12): e2216887120. PubMed ID: 36920921
In the developmental remodeling of brain circuits, neurons are removed by glial phagocytosis to optimize adult behavior. Fragile X mental retardation protein (FMRP) regulates neuron-to-glia signaling to drive glial phagocytosis for targeted neuron pruning. This study finds that FMRP acts in a mothers against decapentaplegic (Mad)-insulin receptor (InR)-protein kinase B (Akt) pathway to regulate pretaporter (Prtp) and amyloid precursor protein-like (APPL) signals directing this glial clearance. Neuronal RNAi of Drosophila fragile X mental retardation 1 (dfmr1) elevates mad transcript levels and increases pMad signaling. Neuronal dfmr1 and mad RNAi both elevate phospho-protein kinase B (pAkt) and delay neuron removal but cause opposite effects on InR expression. Genetically correcting pAkt levels in the mad RNAi background restores normal remodeling. Consistently, neuronal dfmr1 and mad RNAi both decrease Prtp levels, whereas neuronal InR and akt RNAi increase Prtp levels, indicating FMRP works with pMad and insulin signaling to tightly regulate Prtp signaling and thus control glial phagocytosis for correct circuit remodeling. Neuronal dfmr1 and mad and akt RNAi all decrease APPL levels, with the pathway signaling higher glial endolysosome activity for phagocytosis. These findings reveal a FMRP-dependent control pathway for neuron-to-glia communication in neuronal pruning, identifying potential molecular mechanisms for devising fragile X syndrome treatments.
Wong, K. K. L., Li, T., Fu, T. M., Liu, G., Lyu, C., Kohani, S., Xie, Q., Luginbuhl, D. J., Upadhyayula, S., Betzig, E. and Luo, L. (2023). Origin of wiring specificity in an olfactory map revealed by neuron type-specific, time-lapse imaging of dendrite targeting. Elife 12. PubMed ID: 36975203
How does wiring specificity of neural maps emerge during development? Formation of the adult Drosophila olfactory glomerular map begins with patterning of projection neuron (PN) dendrites at the early pupal stage. To better understand the origin of wiring specificity of this map, this study created genetic tools to systematically characterize dendrite patterning across development at PN type-specific resolution. PNs were found to use lineage and birth order combinatorially to build the initial dendritic map. Specifically, birth order directs dendrite targeting in rotating and binary manners for PNs of the anterodorsal and lateral lineages, respectively. Two-photon- and adaptive optical lattice light-sheet microscope-based time-lapse imaging reveals that PN dendrites initiate active targeting with direction-dependent branch stabilization on the timescale of seconds. Moreover, PNs that are used in both the larval and adult olfactory circuits prune their larval-specific dendrites and re-extend new dendrites simultaneously to facilitate timely olfactory map organization. This work highlights the power and necessity of type-specific neuronal access and time-lapse imaging in identifying wiring mechanisms that underlie complex patterns of functional neural maps.
Lapraz, F., Boutres, C., Fixary-Schuster, C., De Queiroz, B. R., Placais, P. Y., Cerezo, D., Besse, F., Preat, T. and Noselli, S. (2023). Asymmetric activity of NetrinB controls laterality of the Drosophila brain. Nat Commun 14(1): 1052. PubMed ID: 36828820
Left-Right (LR) asymmetry of the nervous system is widespread across animals and is thought to be important for cognition and behaviour. But in contrast to visceral organ asymmetry, the genetic basis and function of brain laterality remain only poorly characterized. In this study, RNAi screening was performed to identify genes controlling brain asymmetry in Drosophila. The conserved NetrinB (NetB) pathway was found to be required for a small group of bilateral neurons to project asymmetrically into a pair of neuropils (Asymmetrical Bodies, AB) in the central brain in both sexes. While neurons project unilaterally into the right AB in wild-type flies, netB mutants show a bilateral projection phenotype and hence lose asymmetry. Developmental time course analysis reveals an initially bilateral connectivity, eventually resolving into a right asymmetrical circuit during metamorphosis, with the NetB pathway being required just prior symmetry breaking. This study shows using unilateral clonal analysis that netB activity is required specifically on the right side for neurons to innervate the right AB. It was shown that loss of NetB pathway activity leads to specific alteration of long-term memory, providing a functional link between asymmetrical circuitry determined by NetB and animal cognitive functions.

Tuesday, July 18th - Disease Models

Yang, D., Xiao, F., Li, J., Wang, S., Fan, X., Ni, Q., Li, Y., Zhang, M., Yan, T., Yang, M. and He, Z. (2023). Age-related ceRNA networks in adult Drosophila ageing. Front Genet 14: 1096902. PubMed ID: 36926584
As Drosophila is an extensively used genetic model system, understanding of its regulatory networks has great significance in revealing the genetic mechanisms of ageing and human diseases. Competing endogenous RNA (ceRNA)-mediated regulation is an important mechanism by which circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) regulate ageing and age-related diseases. However, extensive analyses of the multiomics (circRNA/miRNA/mRNA and lncRNA/miRNA/mRNA) characteristics of adult Drosophila during ageing have not been reported. This study, differentially expressed circRNAs and microRNAs (miRNAs) between 7 and 42-day-old flies were screened and identified. Then, the differentially expressed mRNAs, circRNAs, miRNAs, and lncRNAs between the 7- and 42-day old flies were analysed to identify age-related circRNA/miRNA/mRNA and lncRNA/miRNA/mRNA networks in ageing Drosophila. Several key ceRNA networks were identified, such as the dme_circ_0009500/dme_miR-289-5p/CG31064, dme_circ_0009500/dme_miR-289-5p/frizzled, dme_circ_0009500/dme_miR-985-3p/Abl, and XLOC_027736/dme_miR-985-3p/Abl XLOC_189909/dme_miR-985-3p/Abl networks. Furthermore, real-time quantitative PCR (qPCR) was used to verify the expression level of those genes. Those results suggest that the discovery of these ceRNA networks in ageing adult Drosophila provide new information for research on human ageing and age-related diseases.
Yusuff, T., Chang, Y. C., Sang, T. K., Jackson, G. R. and Chatterjee, S. (2023). Codon-optimized TDP-43 mediates neurodegeneration in a Drosophila model of ALS/FTLD. Front Genet 14: 881638. PubMed ID: 36968586
Transactive response DNA binding protein-43 (TDP-43) is known to mediate neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The exact mechanism by which TDP-43 exerts toxicity in the brains, spinal cord, and lower motor neurons of affected patients remains unclear. In a novel Drosophila melanogaster model, this study reports gain-of-function phenotypes due to misexpression of insect codon-optimized version of human wild-type TDP-43 (CO-TDP-43) using both the binary GAL4/UAS system and direct promoter fusion constructs. The CO-TDP-43 model showed robust tissue specific phenotypes in the adult eye, wing, and bristles in the notum. Compared to non-codon optimized transgenic flies, the CO-TDP-43 flies produced increased amount of high molecular weight protein, exhibited pathogenic phenotypes, and showed cytoplasmic aggregation with both nuclear and cytoplasmic expression of TDP-43. Further characterization of the adult retina showed a disruption in the morphology and function of the photoreceptor neurons with the presence of acidic vacuoles that are characteristic of autophagy. Based on these observations, it is proposed that TDP-43 has the propensity to form toxic protein aggregates via a gain-of-function mechanism, and such toxic overload leads to activation of protein degradation pathways such as autophagy. The novel codon optimized TDP-43 model is an excellent resource that could be used in genetic screens to identify and better understand the exact disease mechanism of TDP-43 proteinopathies and find potential therapeutic targets.
Wei, J. J., Li, X. J., Liu, W., Chai, X. J., Zhu, X. Y., Sun, P. H., Liu, F., Zhao, Y. K., Huang, J. L., Liu, Y. F. and Zhao, S. T. (2023). Eucommia Polysaccharides Ameliorate Aging-Associated Gut Dysbiosis: A Potential Mechanism for Life Extension in Drosophila. Int J Mol Sci 24(6). PubMed ID: 36982954
The gut microbiota is increasingly considered to play a key role in human immunity and health. The aging process alters the microbiota composition, which is associated with inflammation, reactive oxygen species (ROS), decreased tissue function, and increased susceptibility to age-related diseases. It has been demonstrated that plant polysaccharides have beneficial effects on the gut microbiota, particularly in reducing pathogenic bacteria abundance and increasing beneficial bacteria populations. However, there is limited evidence of the effect of plant polysaccharides on age-related gut microbiota dysbiosis and ROS accumulation during the aging process. To explore the effect of Eucommiae polysaccharides (EPs) on age-related gut microbiota dysbiosis and ROS accumulation during the aging process of Drosophila, a series of behavioral and life span assays of Drosophila with the same genetic background in standard medium and a medium supplemented with EPs were performed. Next, the gut microbiota composition and protein composition of Drosophila in standard medium and the medium supplemented with EPs were detected using 16S rRNA gene sequencing analysis and quantitative proteomic analysis. This study shows that supplementation of Eucommiae polysaccharides (EPs) during development leads to the life span extension of Drosophila. Furthermore, EPs decreased age-related ROS accumulation and suppressed Gluconobacter, Providencia, and Enterobacteriaceae in aged Drosophila. Increased Gluconobacter, Providencia, and Enterobacteriaceae in the indigenous microbiota might induce age-related gut dysfunction in Drosophila and shortens their life span. This study demonstrates that EPs can be used as prebiotic agents to prevent aging-associated gut dysbiosis and reactive oxidative stress.
Tao, X., Liu, J., Diaz-Perez, Z., Foley, J. R., Stewart, T. M., Casero, R. A. and Zhai, R. G. (2023). Reduction of Spermine Synthase Suppresses Tau Accumulation Through Autophagy Modulation in Tauopathy. bioRxiv. PubMed ID: 36993333
Tauopathy, including Alzheimer Disease (AD), is characterized by Tau protein accumulation and autophagy dysregulation. Emerging evidence connects polyamine metabolism with the autophagy pathway, however the role of polyamines in Tauopathy remains unclear. The present study investigated the role of spermine synthase (SMS) in autophagy regulation and tau protein processing in Drosophila and human cellular models of Tauopathy. A previous study showed that Drosophila spermine synthase (dSms) deficiency impairs lysosomal function and blocks autophagy flux. Interestingly, partial loss-of-function of SMS in heterozygous dSms flies extends lifespan and improves the climbing performance of flies with human Tau (hTau) overexpression. Mechanistic analysis showed that heterozygous loss-of-function mutation of dSms reduces hTau protein accumulation through enhancing autophagic flux. Measurement of polyamine levels detected a mild elevation of spermidine in flies with heterozygous loss of dSms . SMS knock-down in human neuronal or glial cells also upregulates autophagic flux and reduces Tau protein accumulation. Proteomics analysis of postmortem brain tissue from AD patients showed a significant albeit modest elevation of SMS protein level in AD-relevant brain regions compared to that of control brains consistently across several datasets. Taken together, this study uncovers a correlation between SMS protein level and AD pathogenesis and reveals that SMS reduction upregulates autophagy, promotes Tau clearance, and reduces Tau protein accumulation. These findings provide a new potential therapeutic target of Tauopathy.
Praschberger, R., Kuenen, S., Schoovaerts, N., Kaempf, N., Singh, J., Janssens, J., Swerts, J., Nachman, E., Calatayud, C., Aerts, S., Poovathingal, S. and Verstreken, P. (2023). Neuronal identity defines α-synuclein and tau toxicity. Neuron. PubMed ID: 36948206
Pathogenic α-synuclein and tau are critical drivers of neurodegeneration, and their mutations cause neuronal loss in patients. Whether the underlying preferential neuronal vulnerability is a cell-type-intrinsic property or a consequence of increased expression levels remains elusive. This study explored cell-type-specific α-synuclein and tau expression in human brain datasets and use deep phenotyping as well as brain-wide single-cell RNA sequencing of >200 live neuron types in fruit flies to determine which cellular environments react most to α-synuclein or tau toxicity. Phenotypic and transcriptomic evidence was detectedof differential neuronal vulnerability independent of α-synuclein or tau expression levels. Comparing vulnerable with resilient neurons in Drosophila enabled prediction of numerous human neuron subtypes with increased intrinsic susceptibility to pathogenic α-synuclein or tau. By uncovering synapse- and Ca(2+) homeostasis-related genes as tau toxicity modifiers, this work paves the way to leverage neuronal identity to uncover modifiers of neurodegeneration-associated toxic proteins.
Popovic, R., Mukherjee, A., Leal, N. S., Morris, L., Yu, Y., Loh, S. H. Y. and Miguel Martins, L. (2023). Blocking dPerk in the intestine suppresses neurodegeneration in a Drosophila model of Parkinson's disease. Cell Death Dis 14(3): 206. PubMed ID: 36949073
Parkinson's disease (PD) is characterised by selective death of dopaminergic (DA) neurons in the midbrain and motor function impairment. Gastrointestinal issues often precede motor deficits in PD, indicating that the gut-brain axis is involved in the pathogenesis of this disease. The features of PD include both mitochondrial dysfunction and activation of the unfolded protein response (UPR) in the endoplasmic reticulum (ER). PINK1 is a mitochondrial kinase involved in the recycling of defective mitochondria, and PINK1 mutations cause early-onset PD. Like PD patients, pink1 mutant Drosophila show degeneration of DA neurons and intestinal dysfunction. These mutant flies also lack vital proteins due to sustained activation of the kinase R-like endoplasmic reticulum kinase (dPerk), a kinase that induces the UPR. This study investigated the role of dPerk in intestinal dysfunction. Intestinal expression of dPerk impairs mitochondrial function, induces cell death, and decreases lifespan. This study found that suppressing dPerk in the intestine of pink1-mutant flies rescues intestinal cell death and is neuroprotective. It is concluded that in a fly model of PD, blocking gut-brain transmission of UPR-mediated toxicity, is neuroprotective.

Monday, July 17th - Behavior

Nielsen, T. M., Baldwin, J. and Fedorka, K. M. (2023). Gene-poor Y-chromosomes substantially impact male trait heritabilities and may help shape sexually dimorphic evolution. Heredity (Edinb) 130(4): 236-241. PubMed ID: 36759734
How natural selection facilitates sexually dimorphic evolution despite a shared genome is unclear. The patrilineal inheritance of Y-chromosomes makes them an appealing solution. However, they have largely been dismissed due to their gene-poor, heterochromatic nature and because the additive genetic variation necessary for adaptive evolution is theoretically difficult to maintain. Further, previous empirical work has revealed mostly Y-linked sign epistatic variance segregating within populations, which can often impede adaptive evolution. To assess the evolutionary impact of Y-linked variation, this study established replicate populations in Drosophila simulans containing multiple Y-chromosomes (YN populations) or a single Y-chromosome variant (Y1 populations) drawn from a single population. Male and female heritabilities were assessed for several traits known to be influenced by Y-chromosomes, including the number of sternopleural bristles, abdominal bristles, sex comb teeth, and tibia length. A decrease in YN heritabilities compared with Y1 would be consistent with Y-chromosome variation being sign epistatic. A decrease in Y1 heritabilities would be consistent with Y-chromosome variation being additive, though additive-by-additive epistatic variation cannot be entirely dismissed. Female heritability estimates served as controls and were not expected to differ. It was found that male Y1 populations exhibited lower heritabilities for all traits except tibia length; consistent with Y-linked additivity (on average YN trait heritabilities were 25% greater than Y1). Female estimates showed no difference. These data suggest Y-chromosomes should play an important role in male trait evolution and may even influence sexually dimorphic evolution by shaping traits shared by both sexes.
Layh, S., Nagarajan-Radha, V., Lemos, B. and Dowling, D. K. (2023). Y chromosome-linked variation affects locomotor activity in male Drosophila melanogaster and is robust to differences in thermal environment. Heredity (Edinb). PubMed ID: 36914794
Although containing genes important for sex determination, genetic variation within the Y chromosome was traditionally predicted to contribute little to the expression of sexually dimorphic traits. This prediction was shaped by the assumption that the chromosome harbours few protein-coding genes, and that capacity for Y-linked variation to shape adaptation would be hindered by the chromosome's lack of recombination and holandric inheritance. Consequently, most studies exploring the genotypic contributions to sexually dimorphic traits have focused on the autosomes and X chromosome. Yet, several studies have now demonstrated that the Y chromosome harbours variation affecting male fitness, moderating the expression of hundreds of genes across the nuclear genome. Furthermore, emerging results have shown that expression of this Y-linked variation may be sensitive to environmental heterogeneity, leading to the prediction that Y-mediated gene-by-environment interactions will shape the expression of sexually dimorphic phenotypes. This study tested this prediction, investigating whether genetic variation across six distinct Y chromosome haplotypes affects the expression of locomotor activity, at each of two temperatures (20 and 28 °C) in male fruit flies (Drosophila melanogaster). Locomotor activity is a sexually dimorphic trait in this species, previously demonstrated to be under intralocus sexual conflict. This study demonstrated Y haplotype effects on male locomotor activity, but the rank order and magnitude of these effects were unaltered by differences in temperature. This study contributes to a growing number of studies demonstrating Y-linked effects moderating expression of traits evolving under sexually antagonistic selection, suggesting a role for the Y chromosome in shaping outcomes of sexual conflict.
Verschut, T. A., Ng, R., Doubovetzky, N. P., Le Calvez, G., Sneep, J. L., Minnaard, A. J., Su, C. Y., Carlsson, M. A., Wertheim, B. and Billeter, J. C. (2023). Aggregation pheromones have a non-linear effect on oviposition behavior in Drosophila melanogaster. Nat Commun 14(1): 1544. PubMed ID: 36941252
Female fruit flies (Drosophila melanogaster) oviposit at communal sites where the larvae may cooperate or compete for resources depending on group size. This offers a model system to determine how females assess quantitative social information. The concentration of pheromones found on a substrate was found to increase linearly with the number of adult flies that have visited that site. Females prefer oviposition sites with pheromone concentrations corresponding to an intermediate number of previous visitors, whereas sites with low or high concentrations are unattractive. This dose-dependent decision is based on a blend of 11-cis-Vaccenyl Acetate (cVA) indicating the number of previous visitors and heptanal (a novel pheromone deriving from the oxidation of 7-Tricosene), which acts as a dose-independent co-factor. This response is mediated by detection of cVA by odorant receptor neurons Or67d and Or65a, and at least five different odorant receptor neurons for heptanal. These results identify a mechanism allowing individuals to transform a linear increase of pheromones into a non-linear behavioral response.
Sato, A., Yew, J. Y. and Takahashi, A. (2023). Effect of acetic acid bacteria colonization on oviposition and feeding site choice in Drosophila suzukii and its related species. bioRxiv. PubMed ID: 36993389
Oviposition site choice has a large impact on offspring performance. Unlike other vinegar flies that colonize decaying fruits, Drosophila suzukii lay eggs into hard ripening fruits by using their enlarged and serrated ovipositors (oviscapts). This behavior has an advantage over other species by providing access to the host fruit earlier and avoiding competition. However, the larvae are not fully adapted to a low-protein diet, and the availability of intact healthy fruits is seasonally restricted. Thus, to investigate oviposition site preference for microbial growth in this species, an oviposition assay was conducted using single species of commensal Drosophila acetic acid bacteria, Acetobacter and Gluconobacter. The oviposition site preferences for media with or without bacterial growth were quantified in multiple strains of D. suzukii and its closely related species, D. subpulchrella and D. biarmipes , and a typical fermenting-fruit consumer, D. melanogaster. These comparisons demonstrated a continuous degree of preference for sites with Acetobacter growth both within and across species, suggesting that the niche separation is notable but not complete. The preference for Gluconobacter showed large variations among replicates and no clear differences between the strains. In addition, the lack of interspecific differences in feeding site preference for Acetobacter -containing media implies that the interspecific divergence in oviposition site preference occurred independently from the feeding site preference.
Limbania, D., Turner, G. L. and Wasserman, S. M. (2023). Dehydrated Drosophila melanogaster track a water plume in tethered flight. iScience 26(3): 106266. PubMed ID: 36915685
Perception of sensory stimuli can be modulated by changes in internal state to drive contextually appropriate behavior. For example, dehydration is a threat to terrestrial animals, especially to Drosophila melanogaster due to their large surface area to volume ratio, particularly under the energy demands of flight. While hydrated D. melanogaster avoid water cues, while walking, dehydration leads to water-seeking behavior. This study shows that in tethered flight, hydrated flies ignore a water stimulus, whereas dehydrated flies track a water plume. Antennal occlusions eliminate odor and water plume tracking, whereas inactivation of moist sensing neurons in the antennae disrupts water tracking only upon starvation and dehydration. Elimination of the olfactory coreceptor eradicates odor tracking while leaving water-seeking behavior intact in dehydrated flies. These results suggest that while similar hygrosensory receptors may be used for walking and in-flight hygrotaxis, the temporal dynamics of modulating the perception of water vary with behavioral state.
Wan, X., Shen, P., Shi, K., Li, J., Wu, F. and Zhou, C. (2023). A Neural Circuit Controlling Virgin Female Aggression Induced by Mating-related Cues in Drosophila. Neurosci Bull. PubMed ID: 36941515
Females increase aggression for mating opportunities and for acquiring reproductive resources. Although the close relationship between female aggression and mating status is widely appreciated, whether and how female aggression is regulated by mating-related cues remains poorly understood. This study reports an interesting observation that Drosophila virgin females initiate high-frequency attacks toward mated females. 11-cis-vaccenyl acetate (cVA), a male-derived pheromone transferred to females during mating, was shown to promote virgin female aggression. A cVA-responsive neural circuit was subsequently reveal consisting of four orders of neurons, including Or67d, DA1, aSP-g, and pC1 neurons, that mediate cVA-induced virgin female aggression. It was also determined that aSP-g neurons release acetylcholine (ACh) to excite pC1 neurons via the nicotinic ACh receptor nAChRα7. Together, beyond revealing cVA as a mating-related inducer of virgin female aggression, these results identify a neural circuit linking the chemosensory perception of mating-related cues to aggressive behavior in Drosophila females.

Friday, July 14th - Apoptosis and Autophagy

Toyoshima-Sasatani, M., Imura, F., Hamatake, Y., Fukunaga, A. and Negishi, T. (2023). Mutation and apoptosis are well-coordinated for protecting against DNA damage-inducing toxicity in Drosophila. Genes Environ 45(1): 11. PubMed ID: 36949493
No reports have comprehensively explored the direct relationship between apoptosis and somatic cell mutations induced by various mutagenic factors. Mutation was examined by the wing-spot test, which is used to detect somatic cell mutations, including chromosomal recombination. Apoptosis was observed in the wing discs by acridine orange staining in situ. After treatment with chemical mutagens, ultraviolet light (UV), and X-ray, both the apoptotic frequency and mutagenic activity increased in a dose-dependent manner at non-toxic doses. When DNA repair-deficient Drosophila strains were used, the correlation coefficient of the relationship between apoptosis and mutagenicity, differed from that of the wild-type. To explore how apoptosis affects the behavior of mutated cells, the spot size was determined, i.e., the number of mutated cells in a spot. In parallel with an increase in apoptosis, the spot size increased with MNU or X-ray treatment dose-dependently; however, this increase was not seen with UV irradiation. In addition, BrdU incorporation, an indicator of cell proliferation, in the wing discs was suppressed at 6 h, with peak at 12 h post-treatment with X-ray, and that it started to increase again at 24 h; however, this was not seen with UV irradiation. It is concluded that damage-induced apoptosis and mutation might be coordinated with each other, and the frequency of apoptosis and mutagenicity are balanced depending on the type of DNA damage. From the data of the spot size and BrdU incorporation, it is possible that mutated cells replace apoptotic cells due to their high frequency of cell division, resulting in enlargement of the spot size after MNU or X-ray treatment. It is consider that the induction of mutation, apoptosis, and/or cell growth varies in multi-cellular organisms depending on the type of the mutagens, and that their balance and coordination have an important function to counter DNA damage for the survival of the organism.
Abaquita, T. A. L., Damulewicz, M., Tylko, G. and Pyza, E. (2023). The dual role of heme oxygenase in regulating apoptosis in the nervous system of Drosophila melanogaster. Front Physiol 14: 1060175. PubMed ID: 36860519
Accumulating evidence from mammalian studies suggests the dual-faced character of heme oxygenase (HO) in oxidative stress-dependent neurodegeneration. The present study aimed to investigate both neuroprotective and neurotoxic effects of heme oxygenase after the Ho gene chronic overexpression or silencing in neurons of Drosophila melanogaster. The results showed early deaths and behavioral defects after pan-neuronal Ho overexpression, while survival and climbing in a strain with pan-neuronal Ho silencing were similar over time with its parental controls. It was also found that Ho can be pro-apoptotic or anti-apoptotic under different conditions. In young (7-day-old) flies, both the cell death activator gene (hid) expression and the initiator caspase Dronc activity increased in heads of flies when ho expression was changed. In addition, various expression levels of ho produced cell-specific degeneration. Dopaminergic (DA) neurons and retina photoreceptors are particularly vulnerable to changes in Ho expression. In older (30-day-old) flies, no further increase was detected in hid expression or enhanced degeneration, however, high activity of the initiator caspase was still observed. In addition, curcumin, a biologically active polyphenolic compound found in turmeric, was used to further show the involvement of neuronal Ho in the regulation of apoptosis. Under normal conditions, curcumin induced both the expression of Ho and hid, which was reversed after exposure to high-temperature stress and when supplemented in flies with Ho silencing. These results indicate that neuronal Ho regulates apoptosis and this process depends on Ho expression level, age of flies, and cell type.
Zhao, H., Long, S., Liu, S., Yuan, D., Huang, D., Xu, J., Ma, Q., Wang, G., Wang, J., Li, S., Tian, L. and Li, K. (2023). Atg1 phosphorylation is activated by AMPK and indispensable for autophagy induction in insects. Insect Biochem Mol Biol 152: 103888. PubMed ID: 36493962
Phosphorylation is a key post-translational modification in regulating autophagy in yeast and mammalians, yet it is not fully illustrated in invertebrates such as insects. ULK1/Atg1 is a functionally conserved serine/threonine protein kinase involved in autophagosome initiation. As a result of alternative splicing, Atg1 in the silkworm, Bombyx mori, is present as three mRNA isoforms, with BmAtg1c showing the highest expression levels. This study found that BmAtg1c mRNA expression, BmAtg1c protein expression and phosphorylation, and autophagy simultaneously peaked in the fat body during larval-pupal metamorphosis. Importantly, two BmAtg1c phosphorylation sites were identified at Ser269 and Ser270, which were activated by BmAMPK, the major energy-sensing kinase, upon stimulation with 20-hydroxyecdysone and starvation; additionally, these Atg1 phosphorylation sites are evolutionarily conserved in insects. The two BmAMPK-activated phosphorylation sites in BmAtg1c were found to be required for BmAMPK-induced autophagy. Moreover, the two corresponding DmAtg1 phosphorylation sites in the fruit fly, Drosophila melanogaster, are functionally conserved for autophagy induction. In conclusion, AMPK-activated Atg1 phosphorylation is indispensable for autophagy induction and evolutionarily conserved in insects, shedding light on how various groups of organisms differentially regulate ULK1/Atg1 phosphorylation for autophagy induction.
Taniguchi, K. and Igaki, T. (2023). Sas-Ptp10D shapes germ-line stem cell niche by facilitating JNK-mediated apoptosis. PLoS Genet 19(3): e1010684. PubMed ID: 36972315
The function of the stem cell system is supported by a stereotypical shape of the niche structure. In Drosophila ovarian germarium, somatic cap cells form a dish-like niche structure that allows only two or three germ-line stem cells (GSCs) reside in the niche. Despite extensive studies on the mechanism of stem cell maintenance, the mechanisms of how the dish-like niche structure is shaped and how this structure contributes to the stem cell system have been elusive. This study shows that a transmembrane protein Stranded at second (Sas) and its receptor Protein tyrosine phosphatase 10D (Ptp10D), effectors of axon guidance and cell competition via Epidermal growth factor receptor (Egfr) inhibition, shape the dish-like niche structure by facilitating c-Jun N-terminal kinase (JNK)-mediated apoptosis. Loss of Sas or Ptp10D in gonadal apical cells, but not in GSCs or cap cells, during the pre-pupal stage results in abnormal shaping of the niche structure in the adult, which allows excessive, four to six GSCs reside in the niche. Mechanistically, loss of Sas-Ptp10D elevates Egfr signaling in the gonadal apical cells, thereby suppressing their naturally-occurring JNK-mediated apoptosis that is essential for the shaping of the dish-like niche structure by neighboring cap cells. Notably, the abnormal niche shape and resulting excessive GSCs lead to diminished egg production. These data propose a concept that the stereotypical shaping of the niche structure optimizes the stem cell system, thereby maximizing the reproductive capacity.
Li, H., Yu, Z., Niu, Z., Cheng, Y., Wei, Z., Cai, Y., Ma, F., Hu, L., Zhu, J. and Zhang, W. (2023). A neuroprotective role of Ufmylation through Atg9 in the aging brain of Drosophila. Cell Mol Life Sci 80(5): 129. PubMed ID: 37086384
Ufmylation is a recently identified small ubiquitin-like modification, whose biological function and relevant cellular targets are poorly understood. This study presents evidence of a neuroprotective role for Ufmylation involving Autophagy-related gene 9 (Atg9) during Drosophila aging. The Ufm1 system ensures the health of aged neurons via Atg9 by coordinating autophagy and mTORC1, and maintaining mitochondrial homeostasis and JNK (c-Jun N-terminal kinase) activity. Neuron-specific expression of Atg9 suppresses the age-associated movement defect and lethality caused by loss of Ufmylation. Furthermore, Atg9 is identified as a conserved target of Ufm1 conjugation mediated by Ddrgk1, a critical regulator of Ufmylation. Mammalian Ddrgk1 was shown to be indispensable for the stability of endogenous Atg9A protein in mouse embryonic fibroblast (MEF) cells. Taken together, these findings might have important implications for neurodegenerative diseases in mammals.
Ikegawa, Y., Combet, C., Groussin, M., Navratil, V., Safar-Remali, S., Shiota, T., Aouacheria, A. and Yoo, S. K. (2023). Evidence for existence of an apoptosis-inducing BH3-only protein, sayonara, in Drosophila. Embo J: e110454. PubMed ID: 36727601
Cells need to sense stresses to initiate the execution of the dormant cell death program. Since the discovery of the first BH3-only protein Bad, BH3-only proteins have been recognized as indispensable stress sensors that induce apoptosis. BH3-only proteins have so far not been identified in Drosophila despite their importance in other organisms. This study identified the first Drosophila BH3-only protein and name it Sayonara. Sayonara induces apoptosis in a BH3 motif-dependent manner and interacts genetically and biochemically with the BCL-2 homologous proteins, Buffy and Debcl. There is a positive feedback loop between Sayonara-mediated caspase activation and autophagy. The BH3 motif of sayonara phylogenetically appeared at the time of the ancestral gene duplication that led to the formation of Buffy and Debcl in the dipteran lineage. This is the first identification of a bona fide BH3-only protein in Drosophila, thus providing a unique example of how cell death mechanisms can evolve both through time and across taxa.

Thursday, July 13th - Immune response

Huang, J., Lou, Y., Liu, J., Bulet, P., Cai, C., Ma, K., Jiao, R., Hoffmann, J. A., Liegeois, S., Li, Z. and Ferrandon, D. (2023). A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium. Proc Natl Acad Sci U S A 120(12): e2205140120. PubMed ID: 36917667
The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. This study demonstrates that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.
Shianiou, G., Teloni, S. and Apidianakis, Y. (2023). Intestinal Immune Deficiency and Juvenile Hormone Signaling Mediate a Metabolic Trade-off in Adult Drosophila Females. Metabolites 13(3). PubMed ID: 36984780
A trade-off hypothesis pertains to the biased allocation of limited resources between two of the most important fitness traits, reproduction and survival to infection. This quid pro quo manifests itself within animals prioritizing their energetic needs according to genetic circuits balancing metabolism, germline activity and immune response. Key evidence supporting this hypothesis includes dipteran fecundity being compromised by systemic immunity, and female systemic immunity being compromised by mating. This study reveals a local trade-off taking place in the female Drosophila midgut upon immune challenge. Genetic manipulation of intestinal motility, permeability, regeneration and three key midgut immune pathways provides evidence of an antagonism between specific aspects of intestinal defense and fecundity. That is, juvenile hormone (JH)-controlled egg laying, lipid droplet utilization and insulin receptor expression are specifically compromised by the immune deficiency (Imd) and the dual oxidase (Duox) signaling in the midgut epithelium. Moreover, antimicrobial peptide (AMP) expression under the control of the Imd pathway is inhibited upon mating and JH signaling in the midgut. Local JH signaling is further implicated in midgut dysplasia, inducing stem cell-like clusters and gut permeability. Thus, midgut JH signaling compromises host defense to infection by reducing Imd-controlled AMP expression and by inducing dysplasia, while midgut signaling through the Imd and Duox pathways compromises JH-guided metabolism and fecundity.
Robert, A., Talagrand-Reboul, E., Figueras, M. J., Ruimy, R., Boyer, L. and Lamy, B. (2023). Drosophila melanogaster Systemic Infection Model to Study Altered Virulence during Polymicrobial Infection by Aeromonas. Pathogens 12(3). PubMed ID: 36986327
Polymicrobial infections are complex infections associated with worse outcomes compared to monomicrobial infections. Simple, fast, and cost-effective animal models are needed to assess their still poorly known pathogenesis. This study developed a Drosophila melanogaster polymicrobial infection model for opportunistic pathogens and assessed its capacity to discriminate the effects of bacterial mixtures taken from cases of human polymicrobial infections by Aeromonas strains. A systemic infection was obtained by needle pricking the dorsal thorax of the flies, and the fly survival was monitored over time. Different lineages of the flies were infected by a single strain or paired strains (strain ratio 1:1). Individual strains killed more than 80% of the flies in 20 h. The course of infection could be altered with a microbial mix. The model could distinguish between the diverse effects (synergistic, antagonistic, and no difference) that resulted in a milder, more severe, or similar infection, depending on the paired strain considered. Then the determinants of the effects were determined. The effects were maintained in deficient fly lineages for the main signaling pathways (Toll deficient and IMD deficient), which suggests an active microbe/microbe/host interaction. These results indicate that the D. melanogaster systemic infection model is consistent with the study of polymicrobial infection.
Clemente, G. D. and Weavers, H. (2023). A PI3K-calcium-Nox axis primes leukocyte Nrf2 to boost immune resilience and limit collateral damage. J Cell Biol 222(6). PubMed ID: 36995284
Phagosomal reactive oxygen species (ROS) are strategically employed by leukocytes to kill internalized pathogens and degrade cellular debris. Nevertheless, uncontrolled oxidant bursts could cause serious collateral damage to phagocytes or other host tissues, potentially accelerating aging and compromising host viability. Immune cells must, therefore, activate robust self-protective programs to mitigate these undesired effects, and yet allow crucial cellular redox signaling. This study dissected in vivo the molecular nature of these self-protective pathways, their precise mode of activation, and physiological effects. Drosophila embryonic macrophages activate the redox-sensitive transcription factor Nrf2 upon corpse engulfment during immune surveillance, downstream of calcium- and PI3K-dependent ROS release by phagosomal Nox. By transcriptionally activating the antioxidant response, Nrf2 not only curbs oxidative damage but preserves vital immune functions (including inflammatory migration) and delays the acquisition of senescence-like features. Strikingly, macrophage Nrf2 also acts non-autonomously to limit ROS-induced collateral damage to surrounding tissues. Cytoprotective strategies may thus offer powerful therapeutic opportunities for alleviating inflammatory or age-related diseases.
Shrivastava, N. K. and Shakarad, M. N. (2023). Correlated responses in basal immune function in response to selection for fast development in Drosophila melanogaster. J Evol Biol. PubMed ID: 37073855
Often, immunity is invoked in the context of infection, disease and injury. However, an ever alert and robust immune system is essential for maintaining good health, but resource investment into immunity needs to be traded off against allocation to other functions. This examines the consequences of such a trade-off with growth by ascertaining various components of baseline innate immunity in two types of Drosophila melanogaster populations selected for fast development, in combination with either a long effective lifespan (FLJs) or a short effective lifespan (FEJs). Distinct immunological parameters were found to be constitutively elevated in both, FLJs and FEJs. Their ancestral control (JB) populations, and these constitutive elevated immunological parameters were associated with reduced insulin signalling and comparable total gut microbiota. The results bring into focus the inter-relationship between egg to adult development time, ecdysone levels, larval gut microbiota, insulin signalling, adult reproductive longevity and immune function. How changes in selection pressures operating on life-history traits can modulate different components of immune system is discussed.
Rai, K. E., Yin, H., Bengo, A. L. C., Cheek, M., Courville, R., Bagheri, E., Ramezan, R., Behseta, S. and Shahrestani, P. (2023). Immune defense in Drosophila melanogaster depends on diet, sex, and mating status. PLoS One 18(4): e0268415. PubMed ID: 37053140
Immune defense is a complex trait that affects and is affected by many other host factors, including sex, mating, and dietary environment. This study used the agriculturally relevant fungal emtomopathogen, Beauveria bassiana, and the model host organism Drosophila melanogaster to examine how the impacts of sex, mating, and dietary environment on immunity are interrelated. The direction of sexual dimorphism in immune defense was shown to depend on mating status and mating frequency. It was also shown that post-infection dimorphism in immune defense changes over time and is affected by dietary condition both before and after infection. Supplementing the diet with protein-rich yeast improved post-infection survival but more so when supplementation was done after infection instead of before. The multi-directional impacts among immune defense, sex, mating, and diet are clearly complex, and while this study shines light on some of these relationships, further study is warranted. Such studies have potential downstream applications in agriculture and medicine.

Wednesday, July 11, 2023 - Gonads

Radousky, Y. A., Hague, M. T. J., Fowler, S., Paneru, E., Codina, A., Rugamas, C., Hartzog, G., Cooper, B. S. and Sullivan, W. (2023). Distinct Wolbachia localization patterns in oocytes of diverse host species reveal multiple strategies of maternal transmission. Genetics. PubMed ID: 36911919
A broad array of endosymbionts radiate through host populations via vertical transmission, yet much remains unknown concerning the cellular basis, diversity and routes underlying this transmission strategy. This study addressed these issues, by examining the cellular distributions of Wolbachia strains that diverged up to 50 million years ago in the oocytes of 18 divergent Drosophila species. This analysis revealed three Wolbachia distribution patterns: 1) a tight clustering at the posterior pole plasm (the site of germline formation); 2) a concentration at the posterior pole plasm, but with a significant bacteria population distributed throughout the oocyte; 3) and a distribution throughout the oocyte, with none or very few located at the posterior pole plasm. Examination of this latter class indicates Wolbachia accesses the posterior pole plasm during the interval between late oogenesis and the blastoderm formation. One Wolbachia strain in this class concentrates in the posterior somatic follicle cells that encompass the pole plasm of the developing oocyte. In contrast, strains in which Wolbachia concentrate at the posterior pole plasm generally exhibit no or few Wolbachia in the follicle cells associated with the pole plasm. Taken together, these studies suggest that for some Drosophila species, Wolbachia invade the germline from neighboring somatic follicle cells. Phylogenomic analysis indicates that closely related Wolbachia strains tend to exhibit similar patterns of posterior localization, suggesting that specific localization strategies are a function of Wolbachia-associated factors. Previous studies revealed that endosymbionts rely on one of two distinct routes of vertical transmission: continuous maintenance in the germline (germline-to-germline) or a more circuitous route via the soma (germline-to-soma-to-germline). This study provides compelling evidence that Wolbachia strains infecting Drosophila species maintain the diverse arrays of cellular mechanisms necessary for both of these distinct transmission routes. This characteristic may account for its ability to infect and spread globally through a vast range of host insect species.
Rincon-Ortega, L., Valencia-Exposito, A., Kabanova, A., Gonzalez-Reyes, A. and Martin-Bermudo, M. D. (2023). Integrins control epithelial stem cell proliferation in the Drosophila ovary by modulating the Notch pathway. Front Cell Dev Biol 11: 1114458. PubMed ID: 36926523
Cell proliferation and differentiation show a remarkable inverse relationship. The temporal coupling between cell cycle withdrawal and differentiation of stem cells (SCs) is crucial for epithelial tissue growth, homeostasis and regeneration. Proliferation vs. differentiation SC decisions are often controlled by the surrounding microenvironment, of which the basement membrane (BM; a specialized form of extracellular matrix surrounding cells and tissues), is one of its main constituents. Years of research have shown that integrin-mediated SC-BM interactions regulate many aspects of SC biology, including the proliferation-to-differentiation switch. However, these studies have also demonstrated that the SC responses to interactions with the BM are extremely diverse and depend on the cell type and state and on the repertoire of BM components and integrins (see Myospheroid) involved. This study shows that eliminating integrins from the follicle stem cells (FSCs) of the Drosophila ovary and their undifferentiated progeny increases their proliferation capacity. This results in an excess of various differentiated follicle cell types, demonstrating that cell fate determination can occur in the absence of integrins. Because these phenotypes are similar to those found in ovaries with decreased laminin levels, these results point to a role for the integrin-mediated cell-BM interactions in the control of epithelial cell division and subsequent differentiation. Finally, this study shows that integrins regulate proliferation by restraining the activity of the Notch/Delta pathway during early oogenesis. This work increases knowledge of the effects of cell-BM interactions in different SC types and should help improve understanding of the biology of SCs and exploit their therapeutic potential.
Lyman, R. F., Lyman, R. A., Yamamoto, A., Huang, W., Harbison, S. T., Zhou, S., Anholt, R. R. H. and Mackay, T. F. C. (2023). Natural genetic variation in a dopamine receptor is associated with variation in female fertility in Drosophila melanogaster. Proc Biol Sci 290(1996): 20230375. PubMed ID: 37040806
Fertility is a major component of fitness but its genetic architecture remains poorly understood. Using a full diallel cross of 50 Drosophila Genetic Reference Panel inbred lines with whole genome sequences, this study found substantial genetic variation in fertility largely attributable to females. Genes associated with variation in female fertility were mapped by genome-wide association analysis of common variants in the fly genome. Validation of candidate genes by RNAi knockdown confirmed the role of the dopamine 2-like receptor (Dop2R) in promoting egg laying.The Dop2R effect was replicated in an independently collected productivity dataset and showed that the effect of the Dop2R variant was mediated in part by regulatory gene expression variation. This study demonstrates the strong potential of genome-wide association analysis in this diverse panel of inbred strains and subsequent functional analyses for understanding the genetic architecture of fitness traits.
Grmai, L., Michaca, M., Lackner, E., Nampoothiri, V. P. N. and Vasudevan, D. (2023). Integrated Stress Response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation. bioRxiv. PubMed ID: 36909541
Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. This study used Drosophila melanogaster as a model to establish the Integrated Stress response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor which instructs oogenesis. Atf4 was shown to regulate the lipase Brummer to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient sensing role for Atf4. This study also discovered that Atf4 promotes secretion of a fat body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, it is posited that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction: directly, by impacting yolk lipoprotein production and follicle maturation, and systemically, by regulating ovulation.
Lu, W., Lakonishok, M. and Gelfand, V. I. (2023). Drosophila oocyte specification is maintained by the dynamic duo of microtubule polymerase Mini spindles/XMAP215 and dynein. bioRxiv. PubMed ID: 36945460
n many species, only one oocyte is specified among a group of interconnected germline sister cells. In Drosophila melanogaster, 16-cell interconnected cells form a germline cyst, where one cell differentiates into an oocyte, while the rest become nurse cells that supply the oocyte with mRNAs, proteins, and organelles through intercellular cytoplasmic bridges named ring canals via microtubule-based transport. This study found that a microtubule polymerase Mini spindles (Msps), the Drosophila homolog of XMAP215, is essential for the oocyte fate determination. mRNA encoding Msps is concentrated in the oocyte by dynein-dependent transport along microtubules. Translated Msps stimulates microtubule polymerization in the oocyte, causing more microtubule plus ends to grow from the oocyte through the ring canals into nurse cells, further enhancing nurse cell-to-oocyte transport by dynein. Knockdown of msps blocks the oocyte growth and causes gradual loss of oocyte determinants. Thus, the Msps-dynein duo creates a positive feedback loop, enhancing dynein-dependent nurse cell-to-oocyte transport and transforming a small stochastic difference in microtubule polarity among sister cells into a clear oocyte fate determination.
Romanov, S. E., Shloma, V. V., Koryakov, D. E., Belyakin, S. N. and Laktionov, P. P. (2023). Insulator Protein CP190 Regulates Expression of Spermatocyte Differentiation Genes in Drosophila melanogaster Male Germline. Mol Biol (Mosk) 57(1): 109-123. PubMed ID: 36976746
CP190 protein is one of the key components of Drosophila insulator complexes, and its study is important for understanding the mechanisms of gene regulation during cell differentiation. However, Cp190 mutants die before reaching adulthood, which significantly complicates the study of its functions in imago. To overcome this problem and to investigate the regulatory effects of CP190 in adult tissues development, a conditional rescue system was designed for Cp190 mutants. Using Cre/loxP-mediated recombination, the rescue construct containing Cp190 coding sequence is effectively eliminated specifically in spermatocytes, allowing study of the effect of the mutation in male germ cells. Using high-throughput transcriptome analysis i the function of CP190 on gene expression was determined in germline cells. Cp190 mutation was found to have opposite effects on tissue-specific genes, which expression is repressed by CP190, and housekeeping genes, that require CP190 for activation. Mutation of Cp190 also promoted expression of a set of spermatocyte differentiation genes that are regulated by tMAC transcriptional complex. These results indicate that the main function of CP190 in the process of spermatogenesis is the coordination of interactions between differentiation genes and their specific transcriptional activators.

Friday, July 7th - Larval and Adult Development

Bossen, J., Prange, R., Kuhle, J. P., Kunzel, S., Niu, X., Hammel, J. U., Krieger, L., Knop, M., Ehrhardt, B., Uliczka, K., Krauss-Etschmann, S. and Roeder, T. (2023). Adult and Larval Tracheal Systems Exhibit Different Molecular Architectures in Drosophila. Int J Mol Sci 24(6). PubMed ID: 36982710
Knowing the molecular makeup of an organ system is required for its in-depth understanding. This analyzed the molecular repertoire of the adult tracheal system of the fruit fly Drosophila melanogaster using transcriptome studies to advance knowledge of the adult insect tracheal system. Comparing this to the larval tracheal system revealed several major differences that likely influence organ function. During the transition from larval to adult tracheal system, a shift in the expression of genes responsible for the formation of cuticular structure occurs. This change in transcript composition manifests in the physical properties of cuticular structures of the adult trachea. Enhanced tonic activation of the immune system is observed in the adult trachea, which encompasses the increased expression of antimicrobial peptides. In addition, modulatory processes are conspicuous, in this case mainly by the increased expression of G protein-coupled receptors in the adult trachea. Finally, all components of a peripheral circadian clock are present in the adult tracheal system, which is not the case in the larval tracheal system. Comparative analysis of driver lines targeting the adult tracheal system revealed that even the canonical tracheal driver line breathless (btl)-Gal4 is not able to target all parts of the adult tracheal system. This study have uncovered a specific transcriptome pattern of the adult tracheal system and provide this dataset as a basis for further analyses of the adult insect tracheal system.
Ramakrishnan, P., Joshi, A., Tulasi, M. and Yadav, P. (2022). Monochromatic visible lights modulate the timing of pre-adult developmental traits in Drosophila melanogaster. Photochem Photobiol Sci. PubMed ID: 36583814
Light exposure impacts several aspects of Drosophila development including the establishment of circadian rhythms, neuroendocrine regulation, life-history traits, etc. Introduction of artificial lights in the environment has caused almost all animals to develop ecological and physiological adaptations. White light which comprises different lights of differing wavelengths shortens the lifespan in fruit flies Drosophila melanogaster. The wavelength-specific effects of white light on Drosophila development remains poorly understood. This study shows that different wavelengths of white light differentially modulate Drosophila development in all its concomitant stages when maintained in a 12-h light: 12-h dark photoperiod. It was observed that exposure to different monochromatic lights significantly alters larval behaviours such as feeding rate and phototaxis that influence pre-adult development. Larvae grown under shorter wavelengths of light experienced an altered feeding rate. Similarly, larvae were found to avoid shorter wavelengths of light but were highly attracted to the longer wavelengths of light. Most of the developmental processes were greatly accelerated under the green light regime while in other light regimes, the effects were highly varied. Interestingly, pre-adult survivorship remained unaltered across all light regimes but light exposure was found to show its impact on sex determination. This study for the first time reveals how different wavelengths of white light modulate Drosophila development which in the future might help in developing non-invasive therapies and effective pest measures.
Friesen, S. and Hariharan, I. K. (2023). Coordinated growth of linked epithelia is mediated by the Hippo pathway. bioRxiv. PubMed ID: 36993542
An epithelium in a living organism seldom develops in isolation. Rather, most epithelia are tethered to other epithelial or non-epithelial tissues, necessitating growth coordination between layers. This study investigated how two tethered epithelial layers of the Drosophila larval wing imaginal disc, the disc proper (DP) and the peripodial epithelium (PE), coordinate their growth. DP growth is driven by the morphogens Hedgehog (Hh) and Dpp, but regulation of PE growth is poorly understood. This study found that the PE adapts to changes in growth rates of the DP, but not vice versa, suggesting a 'leader and follower' mechanism. Moreover, PE growth can occur by cell shape changes, even when proliferation is inhibited. While Hh and Dpp pattern gene expression in both layers, growth of the DP is exquisitely sensitive to Dpp levels, while growth of the PE is not; the PE can achieve an appropriate size even when Dpp signaling is inhibited. Instead, both the growth of the PE and its accompanying cell shape changes require the activity of two components of the mechanosensitive Hippo pathway, the DNA-binding protein Scalloped (Sd) and its co-activator (Yki), which could allow the PE to sense and respond to forces generated by DP growth. Thus, an increased reliance on mechanically-dependent growth mediated by the Hippo pathway, at the expense of morphogen-dependent growth, enables the PE to evade layer-intrinsic growth control mechanisms and coordinate its growth with the DP. This provides a potential paradigm for growth coordination between different components of a developing organ.
Bernasek, S. M., Hur, S. S. J., Peláez-Restrepo, N., Lachance, J. B., Bakker, R., Navarro, H. T., Sanchez-Luege, N., Amaral, L. A. N., Bagheri, N., Rebay, I. and Carthew, R. W. (2023). Ratiometric sensing of Pnt and Yan transcription factor levels confers ultrasensitivity to photoreceptor fate transitions in Drosophila. Development. PubMed ID: 36942737
Cell state transitions are often triggered by large changes in the concentrations of transcription factors and therefore large differences in their stoichiometric ratios. Whether cells can elicit transitions using modest changes in the ratios of co-expressed factors is unclear. This study investigated how cells in the Drosophila eye resolve state transitions by quantifying the expression dynamics of the ETS transcription factors Pnt and Yan. Eye progenitor cells maintain a relatively constant ratio of Pnt/Yan protein despite expressing both proteins with pulsatile dynamics. A rapid and sustained two-fold increase in the Pnt/Yan ratio accompanies transitions to photoreceptor fates. Genetic perturbations that modestly disrupt the Pnt/Yan ratio produce fate transition defects consistent with the hypothesis that transitions are normally driven by a two-fold shift in the ratio. A biophysical model based on cooperative Yan-DNA binding coupled with non-cooperative Pnt-DNA binding illustrates how two-fold ratio changes could generate ultrasensitive changes in target gene transcription to drive fate transitions. Thus, coupling cell state transitions to the Pnt/Yan ratio sensitizes the system to modest fold-changes, conferring robustness and ultrasensitivity to the developmental program.
DeSantis, D. F., Neal, S. J., Zhou, Q. and Pignoni, F. (2023). Peripodial adherens junctions regulate Ajuba-Yorkie signaling to preserve fly eye morphology. Biol Open 12(3). PubMed ID: 36912729
The Drosophila eye develops from the larval eye disc, a flattened vesicle comprised of continuous retinal and peripodial epithelia (PE). The PE is an epithelium that plays a supporting role in retinal neurogenesis, but gives rise to cuticle in the adult. This study reports that the PE is also necessary to preserve the morphology of the retinal epithelium. Depletion of the adherens junction (AJ) components β-Catenin (β-Cat), DE-Cadherin or α-Catenin from the PE leads to altered disc morphology, characterized by retinal displacement (RDis); so too does loss of the Ajuba protein Jub, an AJ-associated regulator of the transcriptional coactivator Yorkie (Yki). Restoring AJs or overexpressing Yki in β-Cat deficient PE results in suppression of RDis. Additional suppressors of AJ-dependent RDis include knockdown of Rho kinase (Rok) and Dystrophin (Dys). Furthermore, knockdown of βPS integrin (Mys) from the PE results in RDis, while overexpression of Mys can suppress RDis induced by the loss of β-Cat. It is thus proposed that AJ-Jub-Yki signaling in PE cells regulates PE cell contractile properties and/or attachment to the extracellular matrix to promote normal eye disc morphology.
Ren, M., Xu, Y., Phoon, C. K. L., Erdjument-Bromage, H., Neubert, T. A. and Schlame, M. (2023). Knockout of cardiolipin synthase disrupts postnatal cardiac development by inhibiting the maturation of mitochondrial cristae. bioRxiv. PubMed ID: 36945411
Cardiomyocyte maturation requires a massive increase in respiratory enzymes and their assembly into long-lived complexes of oxidative phosphorylation (OXPHOS). The molecular mechanisms underlying the maturation of cardiac mitochondria have not been established. To determine whether the mitochondria-specific lipid cardiolipin is involved in cardiac maturation, a cardiomyocyte-restricted knockout (KO) of cardiolipin synthase (Crls1 ) was created in mice, and the postnatal development of the heart was studied. The turnover rates were also measured of proteins and lipids in cardiolipin-deficient flight muscle from Drosophila, a tissue that has mitochondria with high OXPHOS activity like the heart. Crls1KO mice survived the prenatal period but failed to accumulate OXPHOS proteins during postnatal maturation and succumbed to heart failure at the age of 2 weeks. Turnover measurements showed that the exceptionally long half-life of OXPHOS proteins is critically dependent on cardiolipin. Cardiolipin is essential for the postnatal maturation of cardiomyocytes because it allows mitochondrial cristae to accumulate OXPHOS proteins to a high concentration and to shield them from degradation.

Thursday, July 6th - Enhancers and Transcriptional Regulation

Neidviecky, E. and Deng, H. (2023). Determination of Complex Formation between Drosophila Nrf2 and GATA4 Factors at Selective Chromatin Loci Demonstrates Transcription Coactivation. Cells 12(6). PubMed ID: 36980279
Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is highly correlated with many human diseases, but the underlying molecular mechanisms remain to be fully uncovered. GATA4 is a conserved GATA family transcription factor that is essential for cardiac and dorsal epidermal development. This study describes a novel interaction between Drosophila Nrf2 and GATA4 proteins, i.e., cap'n'collar C (CncC) and Pannier (Pnr), respectively. Using the bimolecular fluorescence complementation (BiFC) assay, a unique imaging tool for probing protein complexes in living cells, this stuy detected CncC-Pnr complexes in the nuclei of Drosophila embryonic and salivary gland cells. Visualization of CncC-Pnr BiFC signals on the polytene chromosome revealed that CncC and Pnr tend to form complexes in euchromatic regions, with a preference for loci that are not highly occupied by CncC or Pnr alone. Most genes within these loci are activated by the CncC-Pnr BiFC, but not by individually expressed CncC or Pnr fusion proteins, indicating a novel mechanism whereby CncC and Pnr interact at specific genomic loci and coactivate genes at these loci. Finally, CncC-induced early lethality can be rescued by Pnr depletion, suggesting that CncC and Pnr function in the same genetic pathway during the early development of Drosophila. Taken together, these results elucidate a novel crosstalk between the Nrf2 xenobiotic/oxidative response factor and GATA factors in the transcriptional regulation of development. This study also demonstrates that the polytene chromosome BiFC assay is a valuable tool for mapping genes that are targeted by specific transcription factor complexes.
Reiter, F., de Almeida, B. P. and Stark, A. (2023). Enhancers display constrained sequence flexibility and context-specific modulation of motif function. Genome Res 33(3): 346-358. PubMed ID: 36941077
The information about when and where each gene is to be expressed is mainly encoded in the DNA sequence of enhancers, sequence elements that comprise binding sites (motifs) for different transcription factors (TFs). Most of the research on enhancer sequences has been focused on TF motif presence, whereas the enhancer syntax, that is, the flexibility of important motif positions and how the sequence context modulates the activity of TF motifs, remains poorly understood. This study explored the rules of enhancer syntax by a two-pronged approach in Drosophila melanogaster S2 cells: (1) important TF motifs were replaced by all possible 65,536 eight-nucleotide-long sequences and (2) eight important TF motif types were pasted into 763 positions within 496 enhancers. These complementary strategies reveal that enhancers display constrained sequence flexibility and the context-specific modulation of motif function. Important motifs can be functionally replaced by hundreds of sequences constituting several distinct motif types, but these are only a fraction of all possible sequences and motif types. Moreover, TF motifs contribute with different intrinsic strengths that are strongly modulated by the enhancer sequence context (the flanking sequence, the presence and diversity of other motif types, and the distance between motifs), such that not all motif types can work in all positions. The context-specific modulation of motif function is also a hallmark of human enhancers, as was demonstrated experimentally. Overall, these two general principles of enhancer sequences are important to understand and predict enhancer function during development, evolution, and in disease.
Zappia, M. P., Kwon, Y. J., Westacott, A., Liseth, I., Lee, H. M., Islam, A., Kim, J. and Frolov, M. V. (2023). E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development. Proc Natl Acad Sci U S A 120(15): e2220770120. PubMed ID: 37011211
The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. This study used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. The impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in Pgk(ΔE2F) mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in Pgk(ΔE2F) mutants. Moreover, Pgk(ΔE2F) animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, these results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the Pgk(ΔE2F) animals underscore the importance of E2F regulation on a single E2F target, Pgk.
Bunker, J., Bashir, M., Bailey, S., Boodram, P., Perry, A., Delaney, R., Tsachaki, M., Sprecher, S. G., Nelson, E., Call, G. B. and Rister, J. (2023). Blimp-1/PRDM1 and Hr3/RORβ specify the blue-sensitive photoreceptor subtype in Drosophila by repressing the hippo pathway. Front Cell Dev Biol 11: 1058961. PubMed ID: 36960411
During terminal differentiation of the mammalian retina, transcription factors control binary cell fate decisions that generate functionally distinct subtypes of photoreceptor neurons. For instance, Otx2 and RORβ activate the expression of the transcriptional repressor Blimp-1/PRDM1 that represses bipolar interneuron fate and promotes rod photoreceptor fate. Moreover, Otx2 and Crx promote expression of the nuclear receptor Nrl that promotes rod photoreceptor fate and represses cone photoreceptor fate. Mutations in these four transcription factors cause severe eye diseases such as retinitis pigmentosa. This study shows that a post-mitotic binary fate decision in Drosophila color photoreceptor subtype specification requires ecdysone signaling and involves orthologs of these transcription factors: Drosophila Blimp-1/PRDM1 and Hr3/RORβ promote blue-sensitive (Rh5) photoreceptor fate and repress green-sensitive (Rh6) photoreceptor fate through the transcriptional repression of warts/LATS, the nexus of the phylogenetically conserved Hippo tumor suppressor pathway. Moreover, a novel interaction was identified between Blimp-1 and warts, whereby Blimp-1 represses a warts intronic enhancer in blue-sensitive photoreceptors and thereby gives rise to specific expression of warts in green-sensitive photoreceptors. Together, these results reveal that conserved transcriptional regulators play key roles in terminal cell fate decisions in both the Drosophila and the mammalian retina, and the mechanistic insights further deepen understanding of how Hippo pathway signaling is repurposed to control photoreceptor fates for Drosophila color vision.
Serebreni, L., Pleyer, L. M., Haberle, V., Hendy, O., Vlasova, A., Loubiere, V., Nemcko, F., Bergauer, K., Roitinger, E., Mechtler, K. and Stark, A. (2023). Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns. Embo j: e113519. PubMed ID: 37013908
Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Using Drosophila melanogaster S2 cells as a model, this study demonstratea that different promoter classes function via distinct PICs. Promoter DNA of developmentally regulated genes readily associates with the canonical Pol II PIC, whereas housekeeping promoters do not, and instead recruit other factors such as DREF. Consistently, TBP and DREF are differentially required by distinct promoter types. TBP and its paralog TRF2 also function at different promoter types in a partially redundant manner. In contrast, TFIIA is required at all promoters, and this study identified factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. Promoter activation by tethering these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes utilize distinct mechanisms of transcription initiation, which translate into different focused versus dispersed initiation patterns.
Sanchez, J. A., Ingaramo, M. C., Gerve, M. P., Thomas, M. G., Boccaccio, G. L. and Dekanty, A. (2023). FOXO-mediated repression of Dicer1 regulates metabolism, stress resistance, and longevity in Drosophila. Proc Natl Acad Sci U S A 120(15): e2216539120. PubMed ID: 37014862
The adipose tissue plays a crucial role in metabolism and physiology, affecting animal lifespan and susceptibility to disease. This study presents evidence that adipose Dicer1 (Dcr-1), a conserved type III endoribonuclease involved in miRNA processing, plays a crucial role in the regulation of metabolism, stress resistance, and longevity. The results indicate that the expression of Dcr-1 in murine 3T3L1 adipocytes is responsive to changes in nutrient levels and is subject to tight regulation in the Drosophila fat body, analogous to human adipose and hepatic tissues, under various stress and physiological conditions such as starvation, oxidative stress, and aging. The specific depletion of Dcr-1 in the Drosophila fat body leads to changes in lipid metabolism, enhanced resistance to oxidative and nutritional stress, and is associated with a significant increase in lifespan. Moreover, mechanistic evidence is provided showing that the JNK-activated transcription factor FOXO binds to conserved DNA-binding sites in the dcr-1 promoter, directly repressing its expression in response to nutrient deprivation. These findings emphasize the importance of FOXO in controlling nutrient responses in the fat body by suppressing Dcr-1 expression. This mechanism coupling nutrient status with miRNA biogenesis represents a novel and previously unappreciated function of the JNK-FOXO axis in physiological responses at the organismal level.

Wednesday, July 5th - Protein Expression, Evolution, Structure, and Function

Taylor, C. A. t., Cormier, K. W., Martin-Vega, A., Earnest, S., Stippec, S., Wichaidit, C. and Cobb, M. H. (2023). ERK2 Mutations Affect Interactions, Localization, and Dimerization. Biochemistry. PubMed ID: 37021821
The most frequent ERK2 (MAPK1; see Drosophila Rolled) mutation in cancers, E322K, lies in the common docking (CD) site, which binds short motifs made up of basic and hydrophobic residues present in the activators MEK1 (MAP2K1) and MEK2 (MAP2K2), in dual specificity phosphatases (DUSPs) that inactivate the kinases, and in many of their substrates. Also, part of the CD site, but mutated less often in cancers, is the preceding aspartate (D321N). These mutants were categorized as gain of function in a sensitized melanoma system. In Drosophila developmental assays, this study found that the aspartate but not the glutamate mutant caused gain-of-function phenotypes. This study catalogued additional properties of these mutants to accrue greater insight into their functions. A modest increase in nuclear retention of E322K was noted. Binding of ERK2 E322K and D321N to a small group of substrates and regulatory proteins was similar, in spite of differences in CD site integrity. Interactions with a second docking site, the F site, which should be more accessible in E322K, were modestly reduced rather than increased. The crystal structure of ERK2 E322K also indicated a disturbed dimer interface, and reduced dimerization was detected by a two-hybrid test; yet, it was detected in dimers in EGF-treated cells, although to a lesser extent than D321N or wt ERK2. These findings indicate a range of small differences in behaviors that may contribute to increased function of E322K in certain cancers.
Pueyo, J. I., Salazar, J., Grincho, C., Berni, J., Towler, B. P. and Newbury, S. F. (2023). Purriato is a conserved small open reading frame gene that interacts with the CASA pathway to regulate muscle homeostasis and epithelial tissue growth in Drosophila. Front Cell Dev Biol 11: 1117454. PubMed ID: 36968202
100s of Small Open Reading Frames (smORFs) are actively translated and display conserved sequence, structure and evolutionary signatures indicating that the translated peptides could fulfil important biological roles. Some of these smORFs display conserved functions with their mutations being associated with pathogenesis. Thus, investigating smORF roles in Drosophila will not only expand understanding of their functions but it may have an impact in human health. This study describe the function of a novel and essential Drosophila smORF gene named purriato (prto CG9034). prto belongs to an ancient gene family whose members have expanded throughout the Protostomia clade. prto encodes a transmembrane peptide which is localized in endo-lysosomes and perinuclear and plasma membranes. prto is dynamically expressed in mesodermal tissues and imaginal discs. Targeted prto knockdown (KD) in these organs results in changes in nuclear morphology and endo-lysosomal distributions correlating with the loss of sarcomeric homeostasis in muscles and reduction of mitosis in wing discs. Consequently, prto KD mutants display severe reduction of motility, and shorter wings. Finally, genetic interaction experiments show that prto function is closely associated to the CASA pathway, a conserved mechanism involved in turnover of mis-folded proteins and linked to muscle dystrophies and neurodegenerative diseases. Thus, this study shows the relevance of smORFs in regulating important cellular functions and supports the systematic characterisation of this class of genes to understand their functions and evolution.
Bauer, M., Aguilar, G., Wharton, K. A., Matsuda, S. and Affolter, M. (2023). Heterodimerization-dependent secretion of bone morphogenetic proteins in Drosophila. Dev Cell. PubMed ID: 37054707
Combinatorial signaling is key to instruct context-dependent cell behaviors. During embryonic development, adult homeostasis, and disease, bone morphogenetic proteins (BMPs) act as dimers to instruct specific cellular responses. BMP ligands can form both homodimers or heterodimers; however, obtaining direct evidence of the endogenous localization and function of each form has proven challenging. This study made use of precise genome editing and direct protein manipulation via protein binders to dissect the existence and functional relevance of BMP homodimers and heterodimers in the Drosophila wing imaginal disc. This approach identified in situ the existence of Dpp (BMP2/4)/Gbb (BMP5/6/7/8) heterodimers. Gbb was found to be secreted in a Dpp-dependent manner in the wing imaginal disc. Dpp and Gbb form a gradient of heterodimers, whereas neither Dpp nor Gbb homodimers are evident under endogenous physiological conditions. This study found that the formation of heterodimers is critical for obtaining optimal signaling and long-range BMP distribution.
Abraham, E., Rethi-Nagy, Z., Vilmos, P., Sinka, R. and Lipinszki, Z. (2023). Plk4 Is a Novel Substrate of Protein Phosphatase 5. Int J Mol Sci 24(3). PubMed ID: 36768356
The conserved Ser/Thr protein phosphatase 5 (PP5) is involved in the regulation of key cellular processes, including DNA damage repair and cell division in eukaryotes. As a co-chaperone of Hsp90, PP5 has been shown to modulate the maturation and activity of numerous oncogenic kinases. This study identified a novel substrate of PP5, the Polo-like kinase 4 (Plk4), which is the master regulator of centriole duplication in animal cells. This study shows that PP5 specifically interacts with Plk4, and is able to dephosphorylate the kinase in vitro and in vivo, which affects the interaction of Plk4 with its partner proteins. In addition, evidence is provided that PP5 and Plk4 co-localize to the centrosomes in Drosophila embryos and cultured cells. PP5 was shown not to be essential; the null mutant flies are viable without a severe mitotic phenotype; however, its loss significantly reduces the fertility of the animals. These results suggest that PP5 is a novel regulator of the Plk4 kinase in Drosophila.
Wang, X., Li, Y., Wei, H., Yang, Z., Luo, R., Gao, Y., Zhang, W., Liu, X. and Sun, L. (2023). Molecular architecture and gating mechanisms of the Drosophila TRPA1 channel. Cell Discov 9(1): 36. PubMed ID: 37015924
The transient receptor potential channel subfamily A member 1 (TRPA1) ion channel is an evolutionary conserved polymodal sensor responding to noxious temperature or chemical stimuli. Notably, the thermosensitivity of TRPA1 varies among different species or even different isoforms in the same species. However, the underlying molecular basis of its thermo-gating remains largely unknown. This study determine the structures of a heat-sensitive isoform of TRPA1 in Drosophila melanogaster in two distinct conformations with cryo-samples prepared at 8 °C. Large conformational changes are observed in the ankyrin repeat domain (ARD) and the coiled-coil domain between the two states. Remarkably, all 17 ankyrin repeats are mapped in the newly resolved conformation, forming a propeller-like architecture. Two intersubunit interfaces are identified in the amino (N)-terminal domain, and play vital roles during both heat and chemical activation as shown by electrophysiological analysis. With cryo-samples prepared at 35 °C, only one conformation is resolved, suggesting possible state transitions during heat responses. These findings provide a basis for further understanding how the ARD regulates channel functions, and insights into the gating mechanism of TRPA1.
Koller, T. O., Morici, M., Berger, M., Safdari, H. A., Lele, D. S., Beckert, B., Kaur, K. J. and Wilson, D. N. (2023). Structural basis for translation inhibition by the glycosylated drosocin peptide. Nat Chem Biol. PubMed ID: 36997646
The proline-rich antimicrobial peptide (PrAMP) Drosocin is produced by Drosophila species to combat bacterial infection. Unlike many PrAMPs, Drosocin is O-glycosylated at threonine 11, a post-translation modification that enhances its antimicrobial activity. This study demonstrates that the O-glycosylation not only influences cellular uptake of the peptide but also interacts with its intracellular target, the ribosome. Cryogenic electron microscopy structures of glycosylated Drosocin on the ribosome at 2.0-2.8-Å resolution reveal that the peptide interferes with translation termination by binding within the polypeptide exit tunnel and trapping RF1 on the ribosome, reminiscent of that reported for the PrAMP apidaecin. The glycosylation of Drosocin enables multiple interactions with U2609 of the 23S rRNA, leading to conformational changes that break the canonical base pair with A752. Collectively, this study reveals novel molecular insights into the interaction of O-glycosylated Drosocin with the ribosome, which provide a structural basis for future development of this class of antimicrobials.

Monday, July 3rd - Synapse and Vesicles

Voing, K., Michgehl, U., Mertens, N. D., Picciotto, C., Maywald, M. L., Goretzko, J., Waimann, S., Gilhaus, K., Rogg, M., Schell, C., Klingauf, J., Tsytsyura, Y., Hansen, U., van Marck, V., Edinger, A. L., Vollenbroker, B., Rescher, U., Braun, D. A., George, B., Weide, T. and Pavenstadt, H. (2023). Disruption of the Rab7-Dependent Final Common Pathway of Endosomal and Autophagic Processing Results in a Severe Podocytopathy. J Am Soc Nephrol. PubMed ID: 37022133
Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, this study focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. Mouse and Drosophila in vivo models lacking Rab7 were generated exclusively in podocytes or nephrocytes, and histologic and ultrastructural analyses was performed. To further investigate Rab7 function on lysosomal and autophagic structures, immortalized human cell lines depleted for Rab were used. RESULTS: Depletion of Rab7 in mice, Drosophila, and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro, RAB7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. It is concluded that isruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.
Stone, A., Cujic, O., Rowlett, A., Aderhold, S., Savage, E., Graham, B. and Steinert, J. R. (2023). Triose-phosphate isomerase deficiency is associated with a dysregulation of synaptic vesicle recycling in Drosophila melanogaster.Front Synaptic Neurosci 15: 1124061. PubMed ID: 36926383
Numerous neurodegenerative diseases are associated with neuronal dysfunction caused by increased redox stress, often linked to aberrant production of redox-active molecules such as nitric oxide (NO) or oxygen free radicals. One such protein affected by redox-mediated changes is the glycolytic enzyme triose-phosphate isomerase (TPI), which has been shown to undergo 3-nitrotyrosination (a NO-mediated post-translational modification) rendering it inactive. This work uses Drosophila to identify the impacts of altered TPI activity on neuronal physiology, linking aberrant TPI function and redox stress to neuronal defects. Drosophila mutants were used expressing a missense allele of the TPI protein, M81T, resulting in an inactive mutant of the TPI protein (TPIM81T, wstd1). Electrophysiological recordings of evoked and spontaneous excitatory junctional currents, alongside high frequency train stimulations and recovery protocols, were applied to the NMJ to investigate synaptic depletion and subsequent recovery. Single synaptic currents were unaltered in the presence of the wstd1 mutation, but frequencies of spontaneous events were reduced. wstd1 larvae also showed enhanced vesicle depletion rates at higher frequency stimulation, and subsequent recovery times for evoked synaptic responses were prolonged. A computational model showed that TPI mutant larvae exhibited a significant decline in activity-dependent vesicle recycling, which manifests itself as increased recovery times for the readily-releasable vesicle pool. Confocal images of NMJs showed no morphological or developmental differences between wild-type and wstd(1) but TPI mutants exhibited learning impairments as assessed by olfactory associative learning assays. These data suggests that the wstd1 phenotype is partially due to altered vesicle dynamics, involving a reduced vesicle pool replenishment, and altered endo/exocytosis processes. This may result in learning and memory impairments and neuronal dysfunction potentially also presenting a contributing factor to other reported neuronal phenotypes.
Ascencio, G., de Cruz, M. A., Abuel, J., Alvarado, S., Arriaga, Y., Conrad, E., Castro, A., Eichelberger, K., Galvan, L., Gundy, G., Inojoza Garcia, J. A., Jimenez, A., Lu, N. T., Lugar, C., Marania, R., Mendsaikhan, T., Ortega, J., Nand, N., Rodrigues, N. S., Shabazz, K., Tam, C., Valenciano, E., Hayzelden, C., Eritano, A. S. and Riggs, B. (2023). A deficiency screen of the 3rd chromosome for dominant modifiers of the Drosophila ER integral membrane protein, Jagunal. G3 (Bethesda). PubMed ID: 36932646
The mechanism surrounding chromosome inheritance during cell division has been well documented, however, organelle inheritance during mitosis is less understood. Recently, the Endoplasmic Reticulum (ER) has been shown to reorganize during mitosis, dividing asymmetrically in proneuronal cells prior to cell fate selection, indicating a programmed mechanism of inheritance. ER asymmetric partitioning in proneural cells relies on the highly conserved ER integral membrane protein, Jagunal (Jagn). Knockdown of Jagn in the compound Drosophila eye displays a pleotropic rough eye phenotype in 48% of the progeny. To identify genes involved in Jagn dependent ER partitioning pathway, a dominant modifier screen was performed of the 3rd chromosome for enhancers and suppressors of this Jagn RNAi-induced rough eye phenotype. this study screened through 181 deficiency lines covering the 3L and 3R chromosomes and identified 12 suppressors and 10 enhancers of the Jagn RNAi phenotype. Based on the functions of the genes covered by the deficiencies, genes were identified that displayed a suppression or enhancement of the Jagn RNAi phenotype. These include Division Abnormally Delayed (Dally), an heparan sulfate proteoglycan, the γ-secretase subunit Presenilin, and the ER resident protein Sec63. Based on current understanding of the function of these targets, there is a connection between Jagn and the Notch signaling pathway. Further studies will elucidate the role of Jagn and identified interactors within the mechanisms of ER partitioning during mitosis.
Grasskamp, A. T., Jusyte, M., McCarthy, A. W., Gotz, T. W. B., Ditlevsen, S. and Walter, A. M. (2023). Spontaneous neurotransmission at evocable synapses predicts their responsiveness to action potentials. Front Cell Neurosci 17: 1129417. PubMed ID: 36970416
Synaptic transmission relies on presynaptic neurotransmitter (NT) release from synaptic vesicles (SVs) and on NT detection by postsynaptic receptors. Transmission exists in two principal modes: action-potential (AP) evoked and AP-independent, "spontaneous" transmission. AP-evoked neurotransmission is considered the primary mode of inter-neuronal communication, whereas spontaneous transmission is required for neuronal development, homeostasis, and plasticity. While some synapses appear dedicated to spontaneous transmission only, all AP-responsive synapses also engage spontaneously, but whether this encodes functional information regarding their excitability is unknown. This study reports on functional interdependence of both transmission modes at individual synaptic contacts of Drosophila larval neuromuscular junctions (NMJs) which were identified by the presynaptic scaffolding protein Bruchpilot (BRP) and whose activities were quantified using the genetically encoded Ca(2+) indicator GCaMP. Consistent with the role of BRP in organizing the AP-dependent release machinery (voltage-dependent Ca(2+) channels and SV fusion machinery), most active BRP-positive synapses (>85%) responded to APs. At these synapses, the level of spontaneous activity was a predictor for their responsiveness to AP-stimulation. AP-stimulation resulted in cross-depletion of spontaneous activity and both transmission modes were affected by the non-specific Ca(2+) channel blocker cadmium and engaged overlapping postsynaptic receptors. Thus, by using overlapping machinery, spontaneous transmission is a continuous, stimulus independent predictor for the AP-responsiveness of individual synapses.
Zhou, L., Xue, X., Yang, K., Feng, Z., Liu, M. and Pastor-Pareja, J. C. (2023). Convergence of secretory, endosomal, and autophagic routes in trans-Golgi-associated lysosomes. J Cell Biol 222(1). PubMed ID: 36239631
At the trans-Golgi, complex traffic connections exist to the endolysosomal system additional to the main Golgi-to-plasma membrane secretory route. This study investigated three hits in a Drosophila screen displaying secretory cargo accumulation in autophagic vesicles: ESCRT-III component Vps20, SNARE-binding Rop, and lysosomal pump subunit VhaPPA1-1. Vps20, Rop, and lysosomal markers localize near the trans-Golgi. Furthermore, this study documents that the vicinity of the trans-Golgi is the main cellular location for lysosomes and that early, late, and recycling endosomes associate as well with a trans-Golgi-associated degradative compartment where basal microautophagy of secretory cargo and other materials occurs. Disruption of this compartment causes cargo accumulation in these hits, including Munc18 homolog Rop, required with Syx1 and Syx4 for Rab11-mediated endosomal recycling. Finally, besides basal microautophagy, this study shows that the trans-Golgi-associated degradative compartment contributes to the growth of autophagic vesicles in developmental and starvation-induced macroautophagy. These results argue that the fly trans-Golgi is the gravitational center of the whole endomembrane system.
Medeiros, A. T., Gratz, S. J., Delgado, A., Ritt, J. T. and O'Connor-Giles, K. M. (2023). Molecular and organizational diversity intersect to generate functional synaptic heterogeneity within and between excitatory neuronal subtypes. PubMed ID: bioRxiv. PubMed ID: 37034654
Synaptic heterogeneity is a hallmark of complex nervous systems that enables reliable and responsive communication in neural circuits. This study investigated the contributions of voltage-gated calcium channels (VGCCs) to synaptic heterogeneity at two closely related Drosophila glutamatergic motor neurons, one low- and one high-Pr. VGCC levels are highly predictive of heterogeneous release probability among individual active zones (AZs) of low- or high-Pr inputs, but not between neuronal subtypes. Underlying organizational differences in the AZ cytomatrix, VGCC composition, and a more compact arrangement of VGCCs alter the relationship between VGCC levels and Pr at AZs of low- vs. high-Pr inputs, explaining this apparent paradox. It was further found that the CAST/ELKS AZ scaffolding protein Bruchpilot differentially regulates VGCC levels at low- and high-Pr AZs following acute glutamate receptor inhibition, indicating that synapse-specific organization also impacts adaptive plasticity. These findings reveal intersecting levels of molecular and spatial diversity with context-specific effects on heterogeneity in synaptic strength and plasticity.
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