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| ARCHIVE | Monday July 15th - Signaling |
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May 2024
April 2024 March 2024 February 2024 January 2024 December 2023 November 2023 October 2023 September 2023 August 2023 July 2023 June 2023 May 2023 April 2023 March 2023 February 2023 January 2023 December 2022 November 2022 October 2022 September 2022 August 2022 December 2021 December 2020 December 2019 December 2018 | Troost, T., Seib, E., Airich, A., Vullings, N., Necakov, A., De Renzis, S., Klein, T. (2023). The meaning of ubiquitylation of the DSL ligand Delta for the development of Drosophila. BMC Biol, 21(1):260 PubMed ID: 37974242
Summary: Ubiquitylation (ubi) of the intracellular domain of the Notch ligand Delta (Dl) by the E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) on lysines (Ks) is thought to be essential for the its signalling activity. Nevertheless, it has been previously shown that DlK2R-HA, a Dl variant where all Ks in its intracellular domain (ICD) are replaced by the structurally similar arginine (R), still possess weak activity if over-expressed. This suggests that ubi is not absolutely required for Dl signalling. However, it is not known whether the residual activity of DlK2R-HA is an effect of over-expression and, if not, whether DlK2R can provide sufficient activity for the whole development of Drosophila. To clarify these issues, this study generated and analysed Dl(attP)-DlK2R-HA, a knock-in allele into the Dl locus. This allele reveals that the sole presence of one copy of Dl(attP)-DlK2R-HA can provide sufficient activity for completion of development. It further indicates that while ubi is required for the full activity of Dl in Mib1-dependent processes, it is not essential for Neur-controlled neural development. Three modes of Dl signalling were identified that are either dependent or independent of ubi. Importantly, all modes depend on the presence of the endocytic adapter Epsin. During activation of Dl, direct binding of Epsin appears not to be an essential requirement. In addition, this analysis further reveals that the Ks are required to tune down the cis-inhibitory interaction of Dl with Notch. These results indicate that Dl can activate the Notch pathway without ubi of its ICD. It signals via three modes. Ubi is specifically required for the Mib1-dependent processes and the adjustment of cis-inhibition. In contrast to Mib1, Neur can efficiently activate Dl without ubi. Neur probably acts as an endocytic co-adapter in addition to its role as E3 ligase. Endocytosis, regulated in a ubi-dependent or ubi-independent manner is required for signalling and also suppression of cis-inhibition. The findings clarify the role of ubi of the ligands during Notch signalling. | Gutorov, R., Katz, B., Peters, M., Minke, B. (2023). Membrane lipid modulations by methyl-β-cyclodextrin uncouple the Drosophila light-activated phospholipase C from TRP and TRPL channel gating. J Biol Chem, 300(1):105484 PubMed ID: 37992804
Summary: Sterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-β-cyclodextrin (MβCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein-coupled phospholipase Cβ (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings. This study examined the effects of MβCD on Drosophila phototransduction using electrophysiology while fluorescently monitoring PIP2 hydrolysis, aiming to examine the effects of sterol modulation on PIP2 hydrolysis and the ensuing light-response in the native system. Incubation of photoreceptor cells with MβCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light response. MβCD also suppressed PLC-dependent TRP/TRPL constitutive channel activity in the dark induced by mitochondrial uncouplers, but PLC-independent activation of the channels by linoleic acid was not affected. Furthermore, MβCD suppressed a constitutively active TRP mutant-channel, trpP365, suggesting that TRP channel activity is a target of MβCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptors and simultaneously monitored PIP2-hydrolysis by translocation of fluorescently tagged Tubby protein domain, from the plasma membrane to the cytosol, revealed that MβCD virtually abolished the light response when having little effect on the light-activated PLC. Together, MβCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggesting the involvement of distinct nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel gating. |
| Strutt, H., Warrington, S., Madathil, A. C. K., Langenhan, T., Strutt, D. (2023). Molecular symmetry breaking in the Frizzled-dependent planar polarity pathway. Curr Biol, 33(24):5340-5354.e5346 PubMed ID: 37995695
Summary: The core planar polarity pathway consists of six proteins that form asymmetric intercellular complexes that segregate to opposite cell ends in developing tissues and specify polarized cell structures or behaviors. Within these complexes, the atypical cadherin Flamingo localizes on both sides of intercellular junctions, where it interacts homophilically in trans via its cadherin repeats, whereas the transmembrane proteins Frizzled and Strabismus localize to the opposite sides of apposing junctions. However, the molecular mechanisms underlying the formation of such asymmetric complexes are poorly understood. Using a novel tissue culture system, the minimum requirements were determined for asymmetric complex assembly in the absence of confounding feedback mechanisms. Complexes were shown to be intrinsically asymmetric and that an interaction of Frizzled and Flamingo in one cell with Flamingo in the neighboring cell is the key symmetry-breaking step. In contrast, Strabismus is unable to promote homophilic Flamingo trans binding and is only recruited into complexes once Frizzled has entered on the opposite side. This interaction with Strabismus requires intact intracellular loops of the seven-pass transmembrane domain of Flamingo. Once recruited, Strabismus stabilizes the intercellular complexes together with the three cytoplasmic core proteins. A model is proposed whereby Flamingo exists in a closed conformation and binding of Frizzled in one cell results in a conformational change that allows its cadherin repeats to interact with a Flamingo molecule in the neighboring cell. Flamingo in the adjacent cell then undergoes a further change in the seven-pass transmembrane region that promotes the recruitment of Strabismus. | Song, S., Cho, B., Weiner, A. T., Nissen, S. B., Ojeda Naharros, I., Sanchez Bosch, P., Suyama, K., Hu, Y., He, L., Svinkina, T., Udeshi, N. D., Carr, S. A., Perrimon, N., Axelrod, J. D. (2023). Protein phosphatase 1 regulates core PCP signaling. EMBO reports, 24(12):e56997 PubMed ID: 37975164
Summary: Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. This study used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. The catalytic subunit of protein phosphatase1, Pp1-87B, was identified, and it was shown to regulate core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, these data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, this screen serves as a resource for identifying additional regulators of PCP signaling. |
| Wani, A. R., Chowdhury, B., Luong, J., Chaya, G. M., Patel, K., Isaacman-Beck, J., Shafer, O., Kayser, M. S., Syed, M. H. (2023). Stem cell-specific ecdysone signaling regulates the development and function of a Drosophila sleep homeostat. bioRxiv, PubMed ID: 37873323
Summary: Complex behaviors arise from neural circuits that are assembled from diverse cell types. Sleep is a conserved and essential behavior, yet little is known regarding how the nervous system generates neuron types of the sleep-wake circuit. This study focused on the specification of Drosophila sleep-promoting neurons-long-field tangential input neurons that project to the dorsal layers of the fan-shaped body neuropil in the central complex (CX). Lineage analysis and genetic birth dating were used to identify two bilateral Type II neural stem cells that generate these dorsal fan-shaped body (dFB) neurons. Adult dFB neurons express Ecdysone-induced protein E93, and loss of Ecdysone signaling or E93 in Type II NSCs results in the misspecification of the adult dFB neurons. Finally, it was shown that E93 knockdown in Type II NSCs affects adult sleep behavior. These results provide insight into how extrinsic hormonal signaling acts on NSCs to generate neuronal diversity required for adult sleep behavior. These findings suggest that some adult sleep disorders might derive from defects in stem cell-specific temporal neurodevelopmental programs. | Sanchez-Martinez, A., Martinez, A. and Whitworth, A. J. (2023). FBXO7/ntc and USP30 antagonistically set the ubiquitination threshold for basal mitophagy and provide a target for Pink1 phosphorylation in vivo. PLoS Biol 21(8): e3002244. PubMed ID: 37535686
Summary: Functional analyses of genes linked to heritable forms of Parkinson's disease (PD) have revealed fundamental insights into the biological processes underpinning pathogenic mechanisms. Mutations in PARK15/FBXO7 cause autosomal recessive PD and FBXO7 has been shown to regulate mitochondrial homeostasis. This study investigated the extent to which FBXO7 and its Drosophila orthologue, ntc, share functional homology and explored its role in mitophagy in vivo. ntc mutants partially phenocopy Pink1 and parkin mutants and ntc overexpression supresses parkin phenotypes. Furthermore, ntc can modulate basal mitophagy in a Pink1- and parkin-independent manner by promoting the ubiquitination of mitochondrial proteins, a mechanism that is opposed by the deubiquitinase USP30. This basal ubiquitination serves as the substrate for Pink1-mediated phosphorylation that triggers stress-induced mitophagy. It is proposed that FBXO7/ntc works in equilibrium with USP30 to provide a checkpoint for mitochondrial quality control in basal conditions in vivo and presents a new avenue for therapeutic approaches. |
Friday July 12th - Adult Physiology and Metabolism |
| Arch, M., Vidal, M., Fuentes, E., Abat, A. S., Cardona, P. J. (2023). The reproductive status determines tolerance and resistance to Mycobacterium marinum in Drosophila melanogaster. Evolution, medicine, and public health, 11(1):332-347 PubMed ID: 37868078
Summary: Sex and reproductive status of the host have a major impact on the immune response against infection. The aim of this study was to understand their impact on host tolerance or resistance in the systemic Mycobacterium marinum infection of Drosophila melanogaster.Host survival and bacillary load were measured at time of death, as well as expression by quantitative real-time polymerase chain reaction of immune genes (diptericin and drosomycin). This study also assessed the impact of metabolic and hormonal regulation in the protection against infection by measuring expression of upd3, impl2 and ecR. The data showed increased resistance in actively mating flies and in mated females, while reducing their tolerance to infection. Data suggests that Toll and immune deficiency (Imd) pathways determine tolerance and resistance, respectively, while higher basal levels of ecR favours the stimulation of the Imd pathway. A dual role has been found for upd3 expression, linked to increased/decreased mycobacterial load at the beginning and later in infection, respectively. Finally, impl2 expression has been related to increased resistance in non-actively mating males. These results allow further assessment on the differences between sexes and highlights the role of the reproductive status in D. melanogaster to face infections, demonstrating their importance to determine resistance and tolerance against M. marinum infection. | Hou, W. Q., Wen, D. T., Zhong, Q., Mo, L., Wang, S., Yin, X. Y., Ma, X. F. (2023). Physical exercise ameliorates age-related deterioration of skeletal muscle and mortality by activating Pten-related pathways in Drosophila on a high-salt diet. Faseb j, 37(12):e23304 PubMed ID: 37971426
Summary: The phosphatase and tensin congeners (Pten) gene affects cell growth, cell proliferation, and rearrangement of connections, and it is closely related to cellular senescence, but it remains unclear the role of muscle-Pten gene in exercise against age-related deterioration in skeletal muscle and mortality induced by a high-salt diet (HSD). In here, overexpression and knockdown of muscle Pten gene were constructed by building Mhc(GAL4) /Pten(UAS-overexpression) and Mhc(GAL4) /Pten(UAS-RNAi) system in flies, and flies were given exercise training and a HSD for 2 weeks. The results showed that muscle Pten knockdown significantly reduced the climbing speed, climbing endurance, GP(X) activity, and the expression of Pten, Sirt1, PGC-1α genes, and it significantly increased the expression of Akt and ROS level, and impaired myofibril and mitochondria of aged skeletal muscle. Pten knockdown prevented exercise from countering the HSD-induced age-related deterioration of skeletal muscle. Pten overexpression has the opposite effect on skeletal muscle aging when compared to it knockdown, and it promoted exercise against HSD-induced age-related deterioration of skeletal muscle. Pten overexpression significantly increased lifespan, but its knockdown significantly decreased lifespan of flies. Thus, current results confirmed that differential expression of muscle Pten gene played an important role in regulating skeletal muscle aging and lifespan, and it also affected the adaptability of aging skeletal muscle to physical exercise since it determined the activity of muscle Pten/Akt pathway and Pten/Sirt1/PGC-1α pathway. |
| Yan, H., Ding, M., Peng, T., Zhang, P., Tian, R., Zheng, L. (2023). Regular Exercise Modulates the dfoxo/dsrebp Pathway to Alleviate High-Fat-Diet-Induced Obesity and Cardiac Dysfunction in Drosophila. Int J Mol Sci, 24(21) PubMed ID: 37958546
Summary: Obesity is a prevalent metabolic disorder associated with various diseases, including cardiovascular conditions. While exercise is recognized as an effective approach for preventing and treating obesity, its underlying molecular mechanisms remain unclear. This study aimed to explore the impact of regular exercise on high-fat-diet-induced obesity and cardiac dysfunction in Drosophila, shedding light on its molecular mechanisms by identifying its regulation of the dfoxo and dsrebp signaling pathways. The findings demonstrated that a high-fat diet leads to weight gain, fat accumulation, reduced climbing performance, and elevated triglyceride levels in Drosophila. Additionally, cardiac microfilaments in these flies exhibited irregularities, breakages, and shortening. M-mode analysis revealed that high-fat-diet-fed Drosophila displayed increased heart rates, shortened cardiac cycles, decreased systolic intervals, heightened arrhythmia indices, reduced diastolic diameters, and diminished fractional shortening. Remarkably, regular exercise effectively ameliorated these adverse outcomes. Further analysis showed that regular exercise reduced fat synthesis, promoted lipolysis, and mitigated high-fat-diet-induced cardiac dysfunction in Drosophila. These results suggest that regular exercise may mitigate high-fat-diet-induced obesity and cardiac dysfunction in Drosophila by regulating the dfoxo and dsrebp signaling pathways, offering valuable insights into the mechanisms underlying the beneficial effects of exercise on obesity and cardiac dysfunction induced by a high-fat diet. | Bamgbose, G., Tulin, A. (2024). PARP-1 is a transcriptional rheostat of metabolic and bivalent genes during development. Life science alliance, 7(2) PubMed ID: 38012002
Summary: PARP-1 participates in various cellular processes, including gene regulation. In Drosophila, PARP-1 mutants undergo developmental arrest during larval-to-pupal transition. This study investigated PARP-1 binding and its transcriptional regulatory role at this stage. The findings revealed that PARP-1 binds and represses active metabolic genes, including glycolytic genes, whereas activating low-expression developmental genes, including a subset of "bivalent" genes in third-instar larvae. These bivalent promoters, characterized by dual enrichment of low H3K4me3 and high H3K27me3, a unimodal H3K4me1 enrichment at the transcription start site (conserved in C. elegans and zebrafish), H2Av depletion, and high accessibility, may persist throughout development. In PARP-1 mutant third-instar larvae, metabolic genes typically down-regulated during the larval-to-pupal transition in response to reduced energy needs were repressed by PARP-1. Simultaneously, developmental and bivalent genes typically active at this stage were activated by PARP-1. In addition, glucose and ATP levels were significantly reduced in PARP-1 mutants, suggesting an imbalance in metabolic regulation. It is proposed that PARP-1 is essential for maintaining the delicate balance between metabolic and developmental gene expression programs to ensure proper developmental progression. |
| Kang, P., Liu, P., Kim, J., Bolton, M., Kumar, A., Miao, T., Shimell, M., O'Connor, M. B., Powell-Coffman, J., Bai, H. (2023). Ptth regulates lifespan through innate immunity pathway in Drosophila. bioRxiv, PubMed ID: 37873203
Summary: The prothoracicotropic hormone (Ptth) is well-known for its important role in controlling insect developmental timing and body size by promoting the biosynthesis and release of ecdysone. However, the role of Ptth in adult physiology is largely unexplored. Thia study showws that Ptth null mutants (both males and females) show extended lifespan and healthspan, and exhibit increased resistance to oxidative stress. Transcriptomic analysis reveals that age-dependent upregulation of innate immunity pathway is attenuated by Ptth mutants. Intriguingly, it was found that Ptth regulates the innate immunity pathway, specifically in fly oenocytes, the homology of mammalian hepatocytes. It was further shown that oenocyte-specific overexpression of Relish shortens the lifespan, while oenocyte-specific downregulation of ecdysone signaling extends lifespan. Consistently, knocking down torso, the receptor of Ptth in the prothoracic gland also promotes longevity of the flies. Thus, these data reveal a novel function of the insect hormone Ptth in longevity regulation and innate immunity in adult Drosophila. | Yu, G., Liu, S., Yang, K., Wu, Q. (2023). Reproductive-dependent effects of B vitamin deficiency on lifespan and physiology. Frontiers in nutrition,. 10:1277715 PubMed ID: 37941770
Summary: B vitamins constitute essential micronutrients in animal organisms, executing crucial roles in numerous biological processes. B vitamin deficiency can result in severe health consequences, including the impairment of reproductive functions and increased susceptibility to age-related diseases. However, the understanding of how reproduction alters the requirements of each individual B vitamins for healthy aging and lifespan remains limited. In this study, utilizing Drosophila as a model organism, the substantial impacts of deficiencies in specific B vitamins on lifespan and diverse physiological functions were revealed, with the effects being significantly shaped by reproductive status. Notably, the dietary absence of VB(1), VB(3), VB(5), VB(6), or VB(7) significantly decreased the lifespan of wild-type females, yet demonstrated relatively little effect on ovoD1 infertile mutant females' lifespan. B vitamin deficiencies also resulted in distinct impacts on the reproduction, starvation tolerance and fat metabolism of wild-type females, though no apparent effects were observed in the infertile mutant females. Moreover, a deficiency in VB(1) reshaped the impacts of macronutrient intervention on the physiology and lifespan of fertile females in a reproductive-dependent manner. Overall, this study unravels that the reproductive status of females serves as a critical modulator of the lifespan and physiological alterations elicited by B-vitamin deficiencies. |
Thursday July 11th - Genes, Enzymes and Protein Expression. Evolution, Structure and Function |
| Varland, S., Silva, R. D., Kjosas, I., Faustino, A., Bogaert, A., Billmann, M., Boukhatmi, H., Kellen, B., Costanzo, M., Drazic, A., Osberg, C., Chan, K., Zhang, X., Tong, A. H. Y., Andreazza, S., Lee, J. J., Nedyalkova, L., Usaj, M., Whitworth, A. J., Andrews, B. J., Moffat, J., Myers, C. L., Gevaert, K., Boone, C., Martinho, R. G., Arnesen, T. (2023). N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity. Nat Commun, 14(1):6774 PubMed ID: 37891180
Summary: Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, this study revealed a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. Loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility. | Peebles, K. E., LaFever, K. S., Page-McCaw, P. S., Colon, S., Wang, D., Stricker, A. M., Ferrell, N., Bhave, G., Page-McCaw, A. (2023). Peroxidasin is required for full viability in development and for maintenance of tissue mechanics in adults. Matrix biology : journal of the International Society for Matrix Biology, PubMed ID: 38000777
Summary: Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membranes that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved throughout the animal kingdom, indicating they are important, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, mutants in Drosophila, including a new CRISPR-generated catalytic null were analyzed, and homozygotes were found to be mostly lethal with 13% viable escapers. Mouse mutants also show semi-lethality, with Mendelian analysis demonstrating ∼50% lethality and ∼50% escapers. Despite the strong mutations, the homozygous fly and mouse escapers had low but detectable levels of Col4 crosslinking, indicating the existence of inefficient alternative crosslinking mechanisms, probably responsible for the viable escapers. Fly mutant phenotypes are consistent with decreased basement membrane stiffness. Interestingly, it was found that even after basement membranes are assembled and crosslinked in wild-type animals, continuing Peroxidasin activity is required in adults to maintain tissue stiffness over time. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated. |
| Markus, D., Pelletier, A., Boube, M., Port, F., Boutros, M., Payre, F., Obermayer, B., Zanet, J. (2023). The pleiotropic functions of Pri smORF peptides synchronize leg development regulators. PLoS Genet, 19(10):e1011004 PubMed ID: 37903161
Summary: The last decade witnesses the emergence of the abundant family of smORF peptides, encoded by small ORF (<100 codons), whose biological functions remain largely unexplored. Bioinformatic analyses here identify hundreds of putative smORF peptides expressed in Drosophila imaginal leg discs. Thanks to a functional screen in leg, we found smORF peptides involved in morphogenesis, including the pioneer smORF peptides Pri. Since we identified its target Ubr3 in the epidermis and pri was known to control leg development through poorly understood mechanisms, we investigated the role of Ubr3 in mediating pri function in leg. We found that pri plays several roles during leg development both in patterning and in cell survival. During larval stage, pri activates independently of Ubr3 tarsal transcriptional programs and Notch and EGFR signaling pathways, whereas at larval pupal transition, Pri peptides cooperate with Ubr3 to insure cell survival and leg morphogenesis. Our results highlight Ubr3 dependent and independent functions of Pri peptides and their pleiotropy. Moreover, we reveal that the smORF peptide family is a reservoir of overlooked developmental regulators, displaying distinct molecular functions and orchestrating leg development. | Bosch, J. A., Keith, N., Escobedo, F., Fisher, W. W., LaGraff, J. T., Rabasco, J., Wan, K. H., Weiszmann, R., Hu, Y., Kondo, S., Brown, J. B., Perrimon, N., Celniker, S. E. (2023). Molecular and functional characterization of the Drosophila melanogaster conserved smORFome. Cell Rep, 42(11):113311 PubMed ID: 37889754
Summary: Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. This study focused on a subset of 298 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, and ∼182 are conserved in plants. This study observe remarkably heterogeneous spatial and temporal expression patterns of smORF transcripts-indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains reveals a predicted enrichment of smORF polypeptides localizing to mitochondria. An embryonic ribosome profiling experiment was conducted and support was found for translation of 137 of these smORFs during embryogenesis. This study further embarked on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity. |
| Guntur, A. R., Smith, J. E., Brahmandam, A., DeBauche, P., Cronmiller, C., Lundell, M. J. (2023). ZFH-2 is required for Drosophila ovarian follicle development and is expressed at the band/interband boundaries of polytene chromosomes. Dev Biol, 504:1-11 PubMed ID: 37666353
Summary: The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. This study provides the first evidence that ZFH-2 has a role in oogenesis. ZFH-2 is expressed in the wild-type ovary and a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. This study also showed that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. It was also shown that ZFH-2 is located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190. | Zhang, Q., Deng, K., Liu, M., Yang, S., Xu, W., Feng, T., Jie, M., Liu, Z., Sheng, X., Chen, H., Jiang, H. (2023). Phase separation of BuGZ regulates gut regeneration and aging through interaction with m(6)A regulators. Nat Commun, 14(1):6700 PubMed ID: 37872148
Summary: Exploring the role of phase separation in intracellular compartment formation is an active area of research. However, the associations of phase separation with intestinal stem cell (ISC)-dependent regeneration and aging remain unclear. This study demonstrates that BuGZ, a coacervating mitotic effector, shows age- and injury-associated condensation in Drosophila ISC nuclei during interphase. BuGZ condensation promotes ISC proliferation, affecting Drosophila gut repair and longevity. Moreover, m(6)A reader YT521-B acts as the transcriptional and functional downstream of BuGZ. The binding of YT521-B promotor or m(6)A writer Ime4/ Mettl14 to BuGZ controls its coacervation, indicating that the promotor may accelerate the phase transition of its binding transcription factor. Hence, we propose that phase separation and m(6)A regulators may be critical for ameliorating ISC-dependent gut regeneration and aging and requires further study. Summary: |
Wednesday, July 10th - Gonads |
| Burghardt, E., Rakijas, J., Tyagi, A., Majumder, P., Olson, B., McDonald, J. A. (2023). Transcriptome analysis reveals temporally regulated genetic networks during Drosophila border cell collective migration. BMC Genomics, 24(1):728 PubMed ID: 38041052
Summary: Collective cell migration underlies many essential processes, including sculpting organs during embryogenesis, wound healing in the adult, and metastasis of cancer cells. At mid-oogenesis, Drosophila border cells undergo collective migration. Border cells round up into a small group at the pre-migration stage, detach from the epithelium and undergo a dynamic and highly regulated migration at the mid-migration stage, and stop at the oocyte, their final destination, at the post-migration stage. While specific genes that promote cell signaling, polarization of the cluster, formation of protrusions, and cell-cell adhesion are known to regulate border cell migration, there may be additional genes that promote these distinct active phases of border cell migration. Therefore, this study sought to identify genes whose expression patterns changed during border cell migration. RNA-sequencing was performed on border cells isolated at pre-, mid-, and post-migration stages. It is reported that 1,729 transcripts, in nine co-expression gene clusters, are temporally and differentially expressed across the three migration stages. Gene ontology analyses and constructed protein-protein interaction networks identified genes expected to function in collective migration, such as regulators of the cytoskeleton, adhesion, and tissue morphogenesis, but also uncovered a notable enrichment of genes involved in immune signaling, ribosome biogenesis, and stress responses. Finally, the in vivo expression and function of a subset of identified genes in border cells was validated. Overall, these results identified differentially and temporally expressed genetic networks that may facilitate the efficient development and migration of border cells. The genes identified in this study represent a wealth of new candidates to investigate the molecular nature of dynamic collective cell migrations in developing tissues. | Chen, X., Qi, Y., Huang, Q., Sun, C., Zheng, Y., Ji, L., Shi, Y., Cheng, X., Li, Z., Zheng, S., Cao, Y., Gu, Z., Yu, J. (2023). Single-cell transcriptome characteristics of testicular terminal epithelium lineages during aging in the Drosophila. Aging Cell:e14057 PubMed ID: 38044573
Summary: Aging is a complex biological process leading to impaired functions, with a variety of hallmarks. In the testis of Drosophila, the terminal epithelium region is involved in spermatid release and maturation, while its functional diversity and regulatory mechanism remain poorly understood. Single-cell RNA-sequencing analysis (scRNA-seq) was performed to characterize the transcriptomes of terminal epithelium in Drosophila testes at 2-, 10 and 40-Days. Terminal epithelium populations were defined with Metallothionein A (MtnA) and subdivided into six novel sub-cell clusters (EP0-EP5), and a series of marker genes were identified based on their expressions. The data revealed the functional characteristics of terminal epithelium populations, such as tight junction, focal adhesion, bacterial invasion, oxidative stress, mitochondrial function, proteasome, apoptosis and metabolism. Interestingly, it was also found that disrupting genes for several relevant pathways in terminal epithelium led to male fertility disorders. Moreover, a series of age-biased genes and pseudotime trajectory mediated state-biased genes were also discovered during terminal epithelium aging. Differentially expressed genes during terminal epithelium aging were mainly participated in the regulation of several common signatures, e.g. mitochondria-related events, protein synthesis and degradation, and metabolic processes. The Drosophila divergence and selection in the functional constraints of age-biased genes during aging was also explored, revealing that age-biased genes in epithelial cells of 2 Days group evolved rapidly and were endowed with greater evolutionary advantages. scRNA-seq analysis revealed the diversity of testicular terminal epithelium populations, providing a gene target resource for further systematic research of their functions during aging. |
| He, Z., Fang, Y., Zhang, F., Liu, Y., Cheng, X., Wang, J., Li, D., Chen, D., Wu, F. (2023). Adenine nucleotide translocase 2 (Ant2) is required for individualization of spermatogenesis of Drosophila melanogaster. Insect Sci, PubMed ID: 38112480
Summary: Successful completion of spermatogenesis is crucial for the perpetuation of the species. In Drosophila, spermatid individualization, a process involving changes in mitochondrial structure and function is critical to produce functional mature sperm. Ant2, encoding a mitochondrial adenine nucleotide translocase, is highly expressed in male testes and plays a role in energy metabolism in the mitochondria. However, its molecular function remains unclear. This study identified an important role of Ant2 in spermatid individualization. In Ant2 knockdown testes, spermatid individualization complexes composed of F-actin cones exhibited a diffuse distribution, and mature sperms were absent in the seminal vesicle, thus leading to male sterility. The most striking effects in Ant2-knockdown spermatids were decrease in tubulin polyglycylation and disruption of proper mitochondria derivatives function. Excessive apoptotic cells were also observed in Ant2-knockdown testes. To further investigate the phenotype of Ant2 knockdown in testes at the molecular level, complementary transcriptome and proteome analyses were performed. At the mRNA level, 868 differentially expressed genes were identified, of which 229 genes were upregulated and 639 were downregulated induced via Ant2 knockdown. iTRAQ-labeling proteome analysis revealed 350 differentially expressed proteins, of which 117 proteins were upregulated and 233 were downregulated. The expression of glutathione transferase (GstD5, GstE5, GstE8, and GstD3), proteins involved in reproduction were significantly regulated at both the mRNA and protein levels. These results indicate that Ant2 is crucial for spermatid maturation by affecting mitochondrial morphogenesis. | Herriage, H. C., Calvi, B. R. (2024). Premature endocycling of Drosophila follicle cells causes pleiotropic defects in oogenesis. bioRxiv, PubMed ID: 37873193
Summary: Endocycling cells grow and repeatedly duplicate their genome without dividing. Cells switch from mitotic cycles to endocycles in response to developmental signals during the growth of specific tissues in a wide range of organisms. The purpose of switching to endocycles, however, remains unclear in many tissues. Additionally, cells can switch to endocycles in response to conditional signals, which can have beneficial or pathological effects on tissues. However, the impact of these unscheduled endocycles on development is underexplored. This study uses Drosophila ovarian somatic follicle cells as a model to examine the impact of unscheduled endocycles on tissue growth and function. Follicle cells normally switch to endocycles at mid-oogenesis. Inducing follicle cells to prematurely switch to endocycles resulted in lethality of the resulting embryos. Analysis of ovaries with premature follicle cell endocycles revealed aberrant follicular epithelial structure and pleiotropic defects in oocyte growth, developmental gene amplification, and the migration of a special set of follicle cells known as border cells. Overall, these findings reveal how unscheduled endocycles can disrupt tissue growth and function to cause aberrant development. A premature switch to polyploid endocycles in Drosophila ovarian follicle cells caused pleiotropic defects in oogenesis and compromised female fertility, revealing new ways in which unscheduled endocycles cause developmental defects. |
| Kotb, N. M., Ulukaya, G., Chavan, A., Nguyen, S. C., Proskauer, L., Joyce, E., Hasson, D., Jagannathan, M., Rangan, P. (2023). Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis. bioRxiv, PubMed ID: 38014330
Summary: Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ-cell genes during differentiation and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, this study found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ-cell genes into a silenced state and activating a group of oocyte genes and Nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, crosstalk between genome architecture and NPCs is essential for successful cell fate transitions. | Wenzel, M., Aquadro, C. F. (2023). Wolbachia infection at least partially rescues the fertility and ovary defects of several new Drosophila melanogaster bag of marbles protein-coding mutants. bioRxiv, PubMed ID: 37645949
Summary: The D. melanogaster protein coding gene bag of marbles (bam) plays a key role in early male and female reproduction by forming complexes with partner proteins to promote differentiation in gametogenesis. Like another germline gene, Sex lethal, bam genetically interacts with the endosymbiont Wolbachia, as Wolbachia rescues the reduced fertility of a bam hypomorphic mutant. This study explored the specificity of the bam-Wolbachia interaction by generating 22 new bam mutants, with ten mutants displaying fertility defects. Nine of these mutants trend towards rescue by the w Mel Wolbachia variant, with eight statistically significant at the fertility and/or cytological level. In some cases, fertility was increased a striking 20-fold. There is no specificity between the rescue and the known binding regions of bam, suggesting w Mel does not interact with one singular bam partner to rescue the reproductive phenotype. Whether w Mel interacts with bam in a non-specific way was tested by increasing bam transcript levels or acting upstream in germline stem cells. A fertility assessment of a bam RNAi knockdown mutant reveals that w Mel rescue is specific to functionally mutant bam alleles and no obvious evidence was found of w Mel interaction with germline stem cells in bam mutants. |
Tuesday, July 9th - Behavior |
| Abhilash, L., Shafer, O. T. (2023). A two-process model of Drosophila sleep reveals an inter-dependence between circadian clock speed and the rate of sleep pressure decay. Sleep, PubMed ID: 37930351
Summary: Sleep is controlled by two processes - a circadian clock that regulates its timing and a homeostat that regulates the drive to sleep. Drosophila has been an insightful model for understanding both processes. For four decades, Borbely and Daan's two-process model has provided a powerful framework for understanding sleep regulation. However, the field of fly sleep has not employed such a model as a framework for the investigation of sleep. To this end, this study has adapted the two-process model to the fly and establish its utility by showing that it can provide empirically testable predictions regarding the circadian and homeostatic control of fly sleep. The ultradian rhythms previously reported for loss-of-function clock mutants in the fly are shown to be robustly detectable and a predictable consequence of a functional sleep homeostat in the absence of a functioning circadian system. We find that a model in which the circadian clock speed and homeostatic rates act without influencing each other provides imprecise predictions regarding how clock speed influences the strength of sleep rhythms and the amount of daily sleep. This study also found that quantitatively good fits between empirical values and model predictions were achieved only when clock speeds were positively correlated with rates of decay of sleep pressure. The results indicate that longer sleep bouts better reflect the homeostatic process than the current definition of sleep as any inactivity lasting five minutes or more. This two-process model represents a powerful framework for work on the molecular and physiological regulation of fly sleep. | O'Hara, M. K., Saul, C., Handa, A., Sehgal, A., Williams, J. A. (2023). The NFkappaB Dif is required for behavioral and molecular correlates of sleep homeostasis in Drosophila. bioRxiv, PubMed ID: 37905096
Summary: The nuclear factor binding the κ light chain in B-cells (NFκB) is involved in a wide range of cellular processes including development, growth, innate immunity, and sleep. However, efforts have been limited toward understanding how specific NFκB transcription factors function in sleep. Drosophila fruit flies carry three genes encoding NFκB transcription factors, Dorsal, Dorsal Immunity Factor (Dif), and Relish. Previous work found that loss of the Relish gene from fat body suppressed daily nighttime sleep, and abolished infection-induced sleep. This study shows that Dif regulates daily sleep and recovery sleep following prolonged wakefulness. Mutants of Dif showed reduced daily sleep and suppressed recovery in response to sleep deprivation. Pan-neuronal knockdown of Dif strongly suppressed daily sleep, indicating that in contrast to Relish, Dif functions from the central nervous system to regulate sleep. Based on the distribution of a Dif-associated GAL4 driver, it was hypothesized that its effects on sleep were mediated by the pars intercerebralis (PI). While RNAi knock-down of Dif in the PI reduced daily sleep, it had no effect on the recovery response to sleep deprivation. However, recovery sleep was suppressed when RNAi knock-down of Dif was distributed across a wider range of neurons. Induction of the nemuri (nur) antimicrobial peptide by sleep deprivation was suppressed in Dif mutants and pan-neuronal over-expression of nur also suppressed the Dif mutant phenotype. Together, these findings indicate that Dif functions from brain to target nemuri and to promote sleep. |
| Sten, T. H., Li, R., Hollunder, F., Eleazer, S., Ruta, V. (2023). Male-male interactions shape mate selection in Drosophila. bioRxiv, PubMed ID: 37961193
Summary: Males of many species have evolved behavioral traits to both attract females and repel rivals. Here, we explore mate selection in Drosophila from both the male and female perspective to shed light on how these key components of sexual selection - female choice and male-male competition - work in concert to guide reproductive strategies. Male flies were found to fend off competing suitors by interleaving their courtship of a female with aggressive wing flicks, which both repel competitors and generate a 'song' that obscures the female's auditory perception of other potential mates. Two higher-order circuit nodes - P1a and pC1x neurons - are coordinately recruited to allow males to flexibly interleave these agonistic actions with courtship displays, assuring they persistently pursue females until their rival falters. Together, these results suggest that female mating decisions are shaped by male-male interactions, underscoring how a male's ability to subvert his rivals is central to his reproductive success. | Safdar, M., Wessells, R. J. (2023). Octopamine Rescues Endurance and Climbing Speed in Drosophila Clk(out) Mutants with Circadian Rhythm Disruption. Cells, 12(21) PubMed ID: 37947593
Summary: Circadian rhythm disturbances are associated with various negative health outcomes, including an increasing incidence of chronic diseases with high societal costs. While exercise can protect against the negative effects of rhythm disruption, it is not available to all those impacted by sleep disruptions, in part because sleep disruption itself reduces exercise capacity. Thus, there is a need for therapeutics that bring the benefits of exercise to this population. This study investigated the relationship between exercise and circadian disturbances using a well-established Drosophila model of circadian rhythm loss, the Clkout mutant. Clkout was found to cause reduced exercise capacity, measured as post-training endurance, flight performance, and climbing speed, and these phenotypes are not rescued by chronic exercise training. However, exogenous administration of a molecule known to mediate the effects of chronic exercise, octopamine (OA), was able to effectively rescue mutant exercise performance, including the upregulation of other known exercise-mediating transcripts, without restoring the circadian rhythms of mutants. This work points the way toward the discovery of novel therapeutics that can restore exercise capacity in patients with rhythm disruption. |
| Gornostaev, N. G., Ruchin, A. B., Esin, M. N., Lazebny, O. E., Kulikov, A. M. (2023). Vertical Distribution of Fruit Flies (Diptera: Drosophilidae) in Deciduous Forests in the Center of European Russia. Insects, 14(10) PubMed ID: 37887834
Summary: Research of Diptera in temperate forests has demonstrated uneven vertical distributions of insects. In this study, we examined the vertical distribution, seasonal fluctuations, and species diversity of Drosophilidae species in the Mordovia State Reserve. This research marks the first exploration of drosophilid vertical stratification in the European part of Russia. Using traps, we collected flies in four deciduous forest sites between early June and mid-September in 2020. A total of 27,151 individuals from 10 genera and 34 drosophilid species were identified, with 6 species from 4 genera being new to the Republic of Mordovia. Drosophila obscura Fll. and Scaptodrosophila rufifrons Lw. were the most abundant species in traps. The total highest number of drosophilid flies (10,429 individuals) was captured at a height of 1.5 m, while the lowest number (5086 individuals) was recorded at 12 m. The average number of flies was 6240 and 5387 individuals at heights of 7.5 m and 3.5 m, respectively. However, the prevalence of drosophilid numbers at the 1.5-m height was not constant during the season. We found that in the second part of July the total fly counts at heights of 7.5 m and 12 m exceeded those at 1.5 m. We have described five different types of vertical distribution of drosophilids throughout the season, which differs markedly in mycetobionts and xylosaprobionts ecological groups. Species diversity demonstrated variations across different sites and tiers during the season, with peak diversity observed in June and September. | Yoshikawa, S., Tang, P., Simpson, J. H. (2023). Mechanosensory and command contributions to the Drosophila grooming sequence. bioRxiv, PubMed ID: 38045358
Summary: Flies groom in response to competing mechanosensory cues in an anterior to posterior order using specific legs. From behavior screens, this study identified a pair of cholinergic command-like neurons, Mago-no-Te (MGT), whose optogenetic activation elicits thoracic grooming by hind legs. Thoracic grooming is typically composed of body sweeps and leg rubs in alternation, but clonal analysis coupled with amputation experiments revealed that MGT activation only commands the body sweeps: initiation of leg rubbing requires contact between leg and thorax. With new electron microscopy (EM) connectome data for the ventral nerve cord (VNC), a circuit-based explanation was uncovered for why stimulation of posterior thoracic mechanosensory bristles initiates cleaning by the hind legs. Previous work showed that flies weigh mechanosensory inputs across the body to select which part to groom, but it was not known why the thorax was always cleaned last. The connectome for the VNC enabled identification if a pair of GABAergic inhibitory neurons, UMGT1, that receive diverse sensory inputs and synapse onto both MGT and components of its downstream pre-motor circuits. Optogenetic activation of UMGT1 suppresses thoracic cleaning, representing a mechanism by which mechanosensory stimuli on other body parts could take precedence in the grooming hierarchy. The pre-motor circuit downstream of MGT was mapped, including inhibitory feedback connections that may enable rhythmicity and coordination of limb movement during thoracic grooming. |
Monday, July 8th - Cancer, Tumors, and Growth |
| Martinez-Abarca Millan, A., Martin-Bermudo, M. D. (2023). Integrins Can Act as Suppressors of Ras-Mediated Oncogenesis in the Drosophila Wing Disc Epithelium. Cancers, 15(22) PubMed ID: 38001693
Summary: Cancer is the second leading cause of death worldwide. Key to cancer initiation and progression is the crosstalk between cancer cells and their microenvironment. The extracellular matrix (ECM) is a major component of the tumour microenvironment and integrins, main cell-ECM adhesion receptors, are involved in every step of cancer progression. However, accumulating evidence has shown that integrins can act as tumour promoters but also as tumour suppressor factors, revealing that the biological roles of integrins in cancer are complex. This incites a better understating of integrin function in cancer progression. To achieve this goal, simple model organisms, such as Drosophila, offer great potential to unravel underlying conceptual principles. This study found that in the Drosophila wing disc epithelium the βPS integrins act as suppressors of tumours induced by a gain of function of the oncogenic form of Ras, Ras(V)(12). βPS integrin depletion enhances the growth, delamination and invasive behaviour of Ras(V)(12) tumour cells, as well as their ability to affect the tumour microenvironment. These results strongly suggest that integrin function as tumour suppressors might be evolutionarily conserved. Drosophila can be used to understand the complex tumour modulating activities conferred by integrins, thus facilitating drug development. | Sanz, F. J., Martinez-Carrion, G., Solana-Manrique, C. and Paricio, N. (2023). Evaluation of type 1 diabetes mellitus as a risk factor of Parkinson's disease in a Drosophila model. J Exp Zool A Ecol Integr Physiol. PubMed ID: 37381093
Summary: Diabetes mellitus (DM) is a chronic metabolic disease characterized by high blood glucose levels, resulting from insulin dysregulation. Parkinson's disease (PD) is the most common neurodegenerative motor disorder caused by the selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. DM and PD are both age-associated diseases that are turning into epidemics worldwide. Previous studies have indicated that type 2 DM might be a risk factor of developing PD. However, scarce information about the link between type 1 DM (T1DM) and PD does exist. This study generated a Drosophila model of T1DM based on insulin deficiency to evaluate if T1DM could be a risk factor to trigger PD onset. As expected, model flies exhibited T1DM-related phenotypes such as insulin deficiency, increased content of carbohydrates and glycogen, and reduced activity of insulin signaling. Interestingly, the results also demonstrated that T1DM model flies presented locomotor defects as well as reduced levels of tyrosine hydroxylase (a marker of DA neurons) in brains, which are typical PD-related phenotypes. In addition, T1DM model flies showed elevated oxidative stress levels, which could be causative of DA neurodegeneration. Therefore, these results indicate that T1DM might be a risk factor of developing PD, and encourage further studies to shed light into the exact link between both diseases. |
| Guo, T., Miao, C., Liu, Z., Duan, J., Ma, Y., Zhang, X., Yang, W., Xue, M., Deng, Q., Guo, P., Xi, Y., Yang, X., Huang, X., Ge, W. (2023). Impaired dNKAP function drives genome instability and tumorigenic growth in Drosophila epithelia. J Mol Cell Biol, PubMed ID: 38059855
Summary: Mutations or dysregulated expression of NF-kappaB activating protein (NKAP) family genes have been found in human cancers. How NKAP family gene mutations promote tumor initiation and progression remains to be determined. This study characterized dNKAP, the Drosophila homolog of NKAP, and showed that impaired dNKAP function causes genome instability and tumorigenic growth in a Drosophila epithelial tumor model. dNKAP-knockdown wing imaginal discs exhibit tumorigenic characteristics, including tissue overgrowth, cell invasive behavior, abnormal cell polarity, and cell adhesion defects. dNKAP knockdown causes both R-loop accumulation and DNA damage, indicating the disruption of genome integrity. Further analysis showed that dNKAP knockdown induces c-Jun N-terminal kinase (JNK)-dependent apoptosis and causes changes in cell proliferation in distinct cell populations. Activation of the Notch and JAK/STAT signaling pathways contributes to the tumorigenic growth of dNKAP-knockdown tissues. Furthermore, JNK signaling is essential for dNKAP depletion-mediated cell invasion. Transcriptome analysis of dNKAP-knockdown tissues confirmed the misregulation of signaling pathways involved in promoting tumorigenesis and revealed abnormal regulation of metabolic pathways. dNKAP knockdown and oncogenic Ras, Notch, or Yki mutations show synergies in driving tumorigenesis, further supporting the tumor-suppressive role of dNKAP. In summary, this study demonstrates that dNKAP plays a tumor-suppressive role by preventing genome instability in Drosophila epithelia and thus provides novel insights into the roles of human NKAP family genes in tumor initiation and progression. | Pfefferkorn, R. M., Mortzfeld, B. M., Fink, C., Frieling, J. V., Bossen, J., Esser, D., Kaleta, C., Rosenstiel, P., Heine, H., Roeder, T. (2024). Recurrent Phases of Strict Protein Limitation Inhibit Tumor Growth and Restore Lifespan in A Drosophila Intestinal Cancer Model. Aging and disease, 15(1):226-244 PubMed ID: 37962464
Summary: Diets that restrict caloric or protein intake offer a variety of benefits, including decreasing the incidence of cancer. However, whether such diets pose a substantial therapeutic benefit as auxiliary cancer treatments remains unclear. This study determined the effects of severe protein depletion on tumorigenesis in a Drosophila melanogaster intestinal tumor model, using a human RAF gain-of-function allele. Severe and continuous protein restriction significantly reduced tumor growth but resulted in premature death. Therefore, a diet was developed in which short periods of severe protein restriction alternated cyclically with periods of complete feeding. This nutritional regime reduced tumor mass, restored gut functionality, and rescued the lifespan of oncogene-expressing flies to the levels observed in healthy flies on a continuous, fully nutritious diet. Furthermore, this diet reduced the chemotherapy-induced stem cell activity associated with tumor recurrence. Transcriptome analysis revealed long-lasting changes in the expression of key genes involved in multiple major developmental signaling pathways. Overall, the data suggest that recurrent severe protein depletion effectively mimics the health benefits of continuous protein restriction, without undesired nutritional shortcomings. This provides seminal insights into the mechanisms of the memory effect required to maintain the positive effects of protein restriction throughout the phases of a full diet. Finally, the repetitive form of strict protein restriction is an ideal strategy for adjuvant cancer therapy that is useful in many tumor contexts. Summary: |
| Li, Y., Pan, L., Li, P., Gao, F., Wang, L., Chen, J., Li, Z., Gao, Y., Gong, Y., Jin, F. (2023). Isolation of Enterococcus faecium and determination of its mechanism for promoting the growth and development of Drosophila. Sci Rep, 13(1):18726 PubMed ID: 37907538
Summary: Intestinal symbiotic microorganisms have a strong capacity to regulate the physiological functions of their host, and Drosophila serves as a useful model. Enterococcus faecium (E. faecium) is a member of the normal intestinal flora of animals. Lactic acid bacteria (LAB) such as E. faecium can promote the growth and development of Drosophila, but the mechanism of regulation of Drosophila is poorly understood. This study found that E. faecium used a carbon source to produce probiotic acids. E. faecium is a symbiotic bacterium for Drosophila, and adult flies passed on parental flora to offspring. E. faecium promoted the growth and development of Drosophila, especially under poor nutritional conditions. E. faecium shortened the developmental process for Drosophila and accelerated the transformation from larva to pupa. Finally, E. faecium promoted the growth and development of Drosophila through TOR and insulin signalling pathways. | Hofstetter, J., Ogunleye, A., Kutschke, A., Buchholz, L. M., Wolf, E., Raabe, T., Gallant, P. (2024). Spt5 interacts genetically with Myc and is limiting for brain tumor growth in Drosophila. Life science alliance, 7(1) PubMed ID: 37935464
Summary: The transcription factor SPT5 physically interacts with MYC oncoproteins and is essential for efficient transcriptional activation of MYC targets in cultured cells. This study used Drosophila to address the relevance of this interaction in a living organism. Spt5 displays moderate synergy with Myc in fast proliferating young imaginal disc cells. During later development, Spt5-knockdown has no detectable consequences on its own, but strongly enhances eye defects caused by Myc overexpression. Similarly, Spt5-knockdown in larval type 2 neuroblasts has only mild effects on brain development and survival of control flies, but dramatically shrinks the volumes of experimentally induced neuroblast tumors and significantly extends the lifespan of tumor-bearing animals. This beneficial effect is still observed when Spt5 is knocked down systemically and after tumor initiation, highlighting SPT5 as a potential drug target in human oncology. |
Friday, July 5th - Disease Models |
| Escobedo, S. E., McGovern, S. E., Jauregui-Lozano, J. P., Stanhope, S. C., Anik, P., Singhal, K., DeBernardis, R., Weake, V. M. (2023). Targeted RNAi screen identifies transcriptional mechanisms that prevent premature degeneration of adult photoreceptors. Frontiers in epigenetics and epigenomics, 1 PubMed ID: 37901602
Summary: Aging is associated with a decline in visual function and increased prevalence of ocular disease, correlating with changes in the transcriptome and epigenome of cells in the eye. This study sought to identify the transcriptional mechanisms that are necessary to maintain photoreceptor viability and function during aging. To do this, A targeted photoreceptor-specific RNAi screen was performed in Drosophila to identify transcriptional regulators whose knockdown results in premature, age-dependent retinal degeneration. From an initial set of 155 RNAi lines each targeting a unique gene and spanning a diverse set of transcription factors, chromatin remodelers, and histone modifiers, 18 high-confidence target genes were identified whose decreased expression in adult photoreceptors leads to premature and progressive retinal degeneration. These 18 target genes were enriched for factors involved in the regulation of transcription initiation, pausing, and elongation, suggesting that these processes are essential for maintaining the health of aging photoreceptors. To identify the genes regulated by these factors, the photoreceptor transcriptome was profiled in a subset of lines. Strikingly, two of the 18 target genes, Spt5 and domino, show similar changes in gene expression to those observed in photoreceptors with advanced age. Together, these data suggest that dysregulation of factors involved in transcription initiation and elongation plays a key role in shaping the transcriptome of aging photoreceptors. Further, these findings indicate that the age-dependent changes in gene expression not only correlate but might also contribute to an increased risk of retinal degeneration | Dondi, C., Vogler, G., Gupta, A., Walls, S. M., Kervadec, A., Romero, M. R., Diop, S. B., Goode, J., Thomas, J. B., Colas, A. R., Bodmer, R., Montminy, M., Ocorr, K. (2023). The nutrient sensor CRTC & Sarcalumenin / Thinman represent a new pathway in cardiac hypertrophy. bioRxiv, PubMed ID: 37873259
Summary: Obesity and type 2 diabetes are at epidemic levels and a significant proportion of these patients are diagnosed with left ventricular hypertrophy. CREB R egulated T ranscription C o-activator (CRTC) is a key regulator of metabolism in mammalian hepatocytes, where it is activated by calcineurin (CaN) to increase expression of gluconeogenic genes. CaN is known its role in pathological cardiac hypertrophy, however, a role for CRTC in the heart has not been identified. In Drosophila, CRTC null mutants have little body fat and exhibit severe cardiac restriction, myofibrillar disorganization, cardiac fibrosis and tachycardia, all hallmarks of heart disease. Cardiac-specific knockdown of CRTC, or its coactivator CREBb, mimicked the reduced body fat and heart defects of CRTC null mutants. Comparative gene expression in CRTC loss- or gain-of-function fly hearts revealed contra-regulation of genes involved in glucose, fatty acid, and amino acid metabolism, suggesting that CRTC also acts as a metabolic switch in the heart. Among the contra-regulated genes with conserved CREB binding sites, the fly ortholog of Sarcalumenin, which is a Ca (2+) -binding protein in the sarcoplasmic reticulum, was identified. Cardiac knockdown recapitulated the loss of CRTC cardiac restriction and fibrotic phenotypes, suggesting it is a downstream effector of CRTC we named thinman (tmn). Importantly, cardiac overexpression of either CaN or CRTC in flies caused hypertrophy that was reversed in a CRTC mutant background, suggesting CRTC mediates hypertrophy downstream of CaN, perhaps as an alternative to NFAT. CRTC novel role in the heart is likely conserved in vertebrates as knockdown in zebrafish also caused cardiac restriction, as in flies. These data suggest that CRTC is involved in myocardial cell maintenance and that CaN-CRTC- Sarcalumenin/ tmn signaling represents a novel and conserved pathway underlying cardiac hypertrophy. |
| Buck, S. A., Rubin, S. A., Kunkhyen, T., Treiber, C. D., Xue, X., Fenno, L. E., Mabry, S. J., Sundar, V. R., Yang, Z., Shah, D., S., Awatramani, R., Watson, A. M., Waddell, S., Cheetham, C. E. J., Logan, R. W., Freyberg, Z. (2023). Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration. bioRxiv, PubMed ID: 37873436
Summary: Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. This study discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT(+) DA neurons in mice and flies identified candidate genes that were functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. Transcription factors modulating dVGLUT-dependent DA neuroprotection were discovered and dj-1β was identified as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health. | Cuddapah, V. A., Hsu, C. T., Li, Y., Shah, H. M., Saul, C., Killiany, S., Shon, J., Yue, Z., Gionet, G., Putt, M. E., Sehgal, A. (2023). Sleepiness, not total sleep amount, increases seizure risk. bioRxiv. PubMed ID: 37873373
Summary: Sleep loss has been associated with increased seizure risk since antiquity. Despite this observation standing the test of time, how poor sleep drives susceptibility to seizures remains unclear. To identify underlying mechanisms, sleep was restricted in Drosophila epilepsy models and a method was developed to identify spontaneous seizures using quantitative video tracking. This study found that sleep loss exacerbates seizures but only when flies experience increased sleep need, or sleepiness, and not necessarily with reduced sleep quantity. This is supported by the paradoxical finding that acute activation of sleep-promoting circuits worsens seizures, because it increases sleep need without changing sleep amount. Sleep-promoting circuits become hyperactive after sleep loss and are associated with increased whole-brain activity. During sleep restriction, optogenetic inhibition of sleep-promoting circuits to reduce sleepiness protects against seizures. Downregulation of the 5HT1A serotonin receptor in sleep-promoting cells mediates the effect of sleep need on seizures, and an FDA-approved 5HT1A agonist was identified to mitigate seizures. These findings demonstrate that while homeostatic sleep is needed to recoup lost sleep, it comes at the cost of increasing seizure susceptibility. This study provides an unexpected perspective on interactions between sleep and seizures, and surprisingly implicate sleep- promoting circuits as a therapeutic target for seizure control. |
| Ahn, J. S., Mahbub, N. U., Kim, S., Kim, H. B., Choi, J. S., Chung, H. J., Hong, S. T. (2023). Nectandrin B significantly increases the lifespan of Drosophila - Nectandrin B for longevity. Aging, 15(22):12749-12762 PubMed ID: 37983180
Summary: Phytochemicals are increasingly recognized in the field of healthy aging as potential therapeutics against various aging-related diseases. Nutmeg, derived from the Myristica fragrans tree, is an example. Nutmeg has been extensively studied and proven to possess antioxidant properties that protect against aging and alleviate serious diseases such as cancer, heart disease, and liver disease. However, the specific active ingredient in nutmeg responsible for these health benefits has not been identified thus far. In this study presents evidence that Nectandrin B (NecB), a bioactive lignan compound isolated from nutmeg, significantly extended the lifespan of the fruit fly Drosophila melanogaster by as much as 42.6% compared to the control group. NecB also improved age-related symptoms including locomotive deterioration, body weight gain, eye degeneration, and neurodegeneration in aging D. melanogaster. This result represents the most substantial improvement in lifespan observed in animal experiments to date, suggesting that NecB may hold promise as a potential therapeutic agent for promoting longevity and addressing age-related degeneration. | Lenzi, C., Piat, A., Schlich, P., Ducau, J., Bregliano, J. C., Aguilaniu, H., Laurencon, A. (2023). Parental age effect on the longevity and healthspan in Drosophila melanogaster and Caenorhabditis elegans. Aging, 15(21):11720-11739 PubMed ID: 37917003
Summary: Several studies have investigated the effect of parental age on biological parameters such as reproduction, lifespan, and health; however, the results have been inconclusive, largely due to inter-species variation and/or modest effect sizes. This studsy examined the effect of parental age on the lifespan, reproductive capacity, and locomotor activity of genetic isogenic lines of the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. The progeny of successive generations of old parents had significantly shorter lifespans than the progeny of young parents in both species. Moreover, this study investigated the fertility, fecundity, and locomotor activity of C. elegans. Interestingly, both the shorter lifespan and deteriorated healthspan of the progeny were significantly improved by switching to only one generation of younger parents. Collectively, these data demonstrate that the detrimental effect of older parental age on the longevity of the progeny can be reversed, suggesting the existence of a beneficial non-genetic mechanism. |
Wednesday, July 3rd - Adult Neural Structure, Development, and Function |
| Martin, M., Gutierrez-Avino, F., Shaikh, M. N., Tejedor, F. J. (2023). A novel proneural function of Asense is integrated with the sequential actions of Delta-Notch, L'sc and Su(H) to promote the neuroepithelial to neuroblast transition. PLoS Genet, 19(10):e1010991 PubMed ID: 37871020
Summary: In order for neural progenitors (NPs) to generate distinct populations of neurons at the right time and place during CNS development, they must switch from undergoing purely proliferative, self-renewing divisions to neurogenic, asymmetric divisions in a tightly regulated manner. In the developing Drosophila optic lobe, neuroepithelial (NE) cells of the outer proliferation center (OPC) are progressively transformed into neurogenic NPs called neuroblasts (NBs) in a medial to lateral proneural wave. The cells undergoing this transition express Lethal of Scute (L'sc), a proneural transcription factor (TF) of the Acheate Scute Complex (AS-C). There is also a peak of expression of Asense (Ase), another AS-C TF, in the cells neighboring those with transient L'sc expression. These peak of Ase cells help to identify a new transitional stage as they have lost NE markers and L'sc, they receive a strong Notch signal and barely exhibit NB markers. This expression of Ase is necessary and sufficient to promote the NE to NB transition in a more robust and rapid manner than that of l'sc gain of function or Notch loss of function. Thus, these data provide the first direct evidence of a proneural role for Ase in CNS neurogenesis. Strikingly, it was found that strong Delta-Notch signaling at the lateral border of the NE triggers l'sc expression, which in turn induces ase expression in the adjacent cells through the activation of Delta-Notch signaling. These results reveal two novel non-conventional actions of Notch signaling in driving the expression of proneural factors, in contrast to the repression that Notch signaling exerts on them during classical lateral inhibition. Finally, Suppressor of Hairless (Su(H)), which seems to be upregulated late in the transitioning cells and in NBs, represses l'sc and ase, ensuring their expression is transient. Thus, these data identify a key proneural role of Ase that is integrated with the sequential activities of Delta-Notch signaling, L'sc, and Su(H), driving the progressive transformation of NE cells into NBs. | Peng, Y. X., Liu, Z. Y., Lin, P. X., Su, S. C., Gao, C. F., Wu, S. F. (2023). Reverse genetic study reveals the molecular targets of chordotonal organ TRPV channel modulators. Pesticide biochemistry and physiology, 196:105584 PubMed ID: 37945222
Summary: Insecticides have been widely used for the control of insect pests that have a significant impact on agriculture and human health. A better understanding of insecticide targets is needed for effective insecticide design and resistance management. Pymetrozine, afidopyropen and flonicamid are reported to target on proteins that located on insect chordotonal organs, resulting in the disruption of insect coordination and the inhibition of feeding. This study systematically examined the susceptibility of six Drosophila melanogaster mutants (five transient receptor potential channels and one mechanoreceptor) to three commercially used insecticides, in order to identify the receptor subunits critical to the insect's response to insecticides. The results showed that iav1, nan36a and wtrw1/i< mutants exhibited significantly reduced susceptibility to pymetrozine and afidopyropen, but not to flonicamid. The number of eggs produced by the three mutant females were significantly less than that of the w(1118) strain. Meanwhile, the longevity of all male mutants and females of nan36a and wtrw1 mutants was significantly shorter than that of the w(1118) strain as the control. However, we observed no gravitaxis defects in wtrw1 mutants and the anti-gravitaxis ofwtrw1 mutants was abolished by pymetrozine. Behavioral assays using thermogenetic tools further confirmed the bioassay results and supported the idea that Nan as a TRPV subfamily member located in Drosophila chordotonal neurons, acting as a target of pymetrozine, which interferes with Drosophila and causes motor deficits with gravitaxis defects. Taken together, this study elucidates the interactions of pymetrozine and afidopyropen with TRPV channels, Nan and Iav, and TRPA channel, Wtrw. This research provides another evidence that pymetrozine and afidopyropen might target on nan, iav and wtrw channels and provides insights into the development of sustainable pest management strategies. |
| Gonzalez, Y. R., Kamkar, F., Jafar-Nejad, P., Wang, S., Qu, D., Alvarez, L. S., Hawari, D., Sonnenfeld, M., Slack, R. S., Albert, P. R., Park, D. S., Joselin, A. (2023). PFTK1 kinase regulates axogenesis during development via RhoA activation. BMC Biol, 21(1):240 PubMed ID: 37907898
Summary: BAPFTK1/Eip63E is a member of the cyclin-dependent kinases (CDKs) family and plays an important role in normal cell cycle progression. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. This study sought to understand the function of Eip63E in the CNS by studying the fly ventral nerve cord during development. The results demonstrate that Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. Functional interaction studies performed using the same system identify an interaction between Eip63E and the small GTPase Rho1. Furthermore, deficiency of Eip63E homolog in mice, PFTK1, in a newly generated PFTK1 knockout mice results in increased axonal outgrowth confirming that the developmental defects observed in the fly model are due to defects in axogenesis. Importantly, RhoA phosphorylation and activity are affected by PFTK1 in primary neuronal cultures. GDP-bound inactive RhoA is a substrate of PFTK1 and PFTK1 phosphorylation is required for RhoA activity. In conclusion, this work establishes an unreported neuronal role of PFTK1 in axon development mediated by phosphorylation and activation of GDP-bound RhoA. The results presented add to understanding of the role of Cdks in the maintenance of RhoA-mediated axon growth and its impact on CNS development and axonal regeneration. | Ahn, J. E., Amrein, H. (2023). Opposing chemosensory functions of closely related gustatory receptors. bioRxiv, PubMed ID: 37905057
Summary: Most animals have functionally distinct populations of taste cells, expressing receptors that are tuned to compounds of different valence. In D. melanogaster, primary sensory neurons express taste receptors that are tuned to distinct groups of chemicals, thereby activating neural ensembles that elicit either feeding or avoidance behavior. Members of a family of ligand gated receptor channels, the Gustatory receptors (Grs), play a central role in these behaviors. In general, closely related, evolutionarily conserved Gr proteins are co-expressed in the same type of taste neurons, tuned to chemically related compounds, and therefore triggering the same behavioral response. This study reports that members of the Gr28 subfamily are expressed in largely non-overlapping sets of taste neurons in Drosophila larvae, detect chemicals of different valence and trigger opposing feeding behaviors. The intrinsic properties of Gr28 neurons by were determined by expressing the mammalian Vanilloid Receptor (VR1), which is activated by capsaicin, a chemical to which wildtype Drosophila larvae do not respond. When VR1 is expressed in Gr28a neurons, larvae become attracted to capsaicin, consistent with reports showing that Gr28a itself encodes a receptor for nutritious RNA. In contrast, expression of VR1 in two pairs of Gr28b.c neurons triggers avoidance to capsaicin. Moreover, neuronal inactivation experiments show that the Gr28b.c neurons are necessary for avoidance of several bitter compounds. Lastly, behavioral experiments of Gr28 deficient larvae and live Ca (2+) imaging studies of Gr28b.c neurons revealed that denatonium benzoate, a synthetic bitter compound that shares structural similarities with natural bitter chemicals, is a ligand for a receptor complex containing a Gr28b.c or Gr28b.a subunit. Thus, the Gr28 proteins, which have been evolutionarily conserved over 260 million years in insects, represent the first taste receptor subfamily in which specific members mediate behavior with opposite valence. |
| Dweck, H. K. M., Carlson, J. R. (2023). Diverse mechanisms of taste coding in Drosophila. Sci Adv, 9(46):eadj7032 PubMed ID: 37976361
Summary: Taste systems encode chemical cues that drive vital behaviors. This study has elucidated noncanonical features of taste coding using an unconventional kind of electrophysiological analysis. Taste neurons of Drosophila are much more sensitive than previously thought. They have a low spontaneous firing frequency that depends on taste receptors. Taste neurons have a dual function as olfactory neurons: They are activated by most tested odorants, including N,N-diethyl-meta-toluamide (DEET), at a distance. DEET can also inhibit certain taste neurons, revealing that there are two modes of taste response: activation and inhibition. Electrophysiological OFF responses were characterized and it was found that the tastants that elicit them are related in structure. OFF responses link tastant identity to behavior: the magnitude of the OFF response elicited by a tastant correlated with the egg laying behavior it elicited. In summary, the sensitivity and coding capacity of the taste system are much greater than previously known. | Jouandet, G. C., Alpert, M. H., Simoes, J. M., Suhendra, R., Frank, D. D., Levy, J. I., Para, A., Kath, W. L., Gallio, M. (2023). Rapid threat assessment in the Drosophila thermosensory system. Nat Commun, 14(1):7067 PubMed ID: 37923719
Summary: Neurons that participate in sensory processing often display "ON" responses, i.e., fire transiently at the onset of a stimulus. ON transients are widespread, perhaps universal to sensory coding, yet their function is not always well-understood. This study shows that ON responses in the Drosophila thermosensory system extrapolate the trajectory of temperature change, priming escape behavior if unsafe thermal conditions are imminent. First, it was shown that second-order thermosensory projection neurons (TPN-IIIs) and their Lateral Horn targets (TLHONs), display ON responses to thermal stimuli, independent of direction of change (heating or cooling) and of absolute temperature. Instead, they track the rate of temperature change, with TLHONs firing exclusively to rapid changes (>0.2 °C/s). Next, connectomics were used to track TLHONs' output to descending neurons that control walking and escape, and modeling and genetic silencing to demonstrate how ON transients can flexibly amplify aversive responses to small thermal change. These results suggest that, across sensory systems, ON transients may represent a general mechanism to systematically anticipate and respond to salient or dangerous conditions. |
Tuesday, July 2nd - Adult Physiology and Metabolism |
| Malik, D. M., Sengupta, A., Sehgal, A., Weljie, A. M. (2023). Altered Metabolism During the Dark Period in Drosophila Short Sleep Mutants. bioRxiv, PubMed ID: 37961245
Summary: Sleep is an almost universally required state in biology. Disrupted sleep has been associated with adverse health risks including metabolic perturbations. Sleep is in part regulated via circadian mechanisms, however, metabolic dysfunction at different times of day arising from sleep disruption is unclear. This study used targeted liquid chromatography-mass spectrometry to probe metabolic alterations using high-resolution temporal sampling of two Drosophila short sleep mutants, fumin and sleepless, across a circadian day. Discriminant analyses revealed overall distinct metabolic profiles for mutants when compared to a wild type dataset. Altered levels of metabolites involved in nicotinate/nicotinamide, alanine, aspartate, and glutamate, glyoxylate and dicarboxylate metabolism, and the TCA cycle were observed in mutants suggesting increased energetic demands. Furthermore, rhythmicity analyses revealed fewer 24 hr rhythmic metabolites in both mutants. Interestingly, mutants displayed two major peaks in phases while wild type displayed phases that were less concerted. In contrast to 24 hr rhythmic metabolites, an increase in the number of 12 hr rhythmic metabolites was observed in fumin while sleepless displayed a decrease. These results support that decreased sleep alters the overall metabolic profile with short sleep mutants displaying altered metabolite levels associated with a number of pathways in addition to altered neurotransmitter levels. | Tang, C., Li, Q., Wang, X., Yu, Z., Ping, X., Qin, Y., Liu, Y., Zheng, L. (2024). Cardiac Timeless Trans-Organically Regulated by miR-276 in Adipose Tissue Modulates Cardiac Function. Function (Oxford, England), 5(1):zqad064 PubMed ID: 38058384
Summary: The interconnection between cardiac function and circadian rhythms is of great importance. While the role of the biological clock gene Timeless (Tim) in circadian rhythm has been extensively studied, its impact on cardiac function remains largely been unexplored. Previous research has provided experimental evidence for the regulation of the heart by adipose tissue and the targeting of miR-276a/b on Timeless. However, the extent to which adipose tissue regulates cardiac Timeless genes trans-organically through miR-276a/b, and subsequently affects cardiac function, remains uncertain. Therefore, the objective of this study was to investigate the potential trans-organ modulation of the Timeless gene in the heart by adipose tissue through miR-276a/b. Cardiac-specific Timeless knockdown and overexpression was shown to result in a significant increase in heart rate (HR) and a significant decrease in Heart period (HP), diastolic intervals (DI), systolic intervals (SI), diastolic diameter (DD), and systolic diameter (SD). miR-276b systemic knockdown resulted in a significant increase in DI, arrhythmia index (AI), and fractional shortening (FS) significantly increased and SI, DD and SD significantly decreased. Adipose tissue-specific miR-276a/b knockdown and miR-276a overexpression resulted in a significant increase in HR and a significant decrease in DI and SI, which were improved by exercise intervention. This study presents a novel finding that highlights the significance of the heart circadian clock gene Timeless in heart function. Additionally, it demonstrates that adipose tissue exerts trans-organ modulation on the expression of the heart Timeless gene via miR-276a/b. |
| Li, X., Karpac, J. (2023). A distinct Acyl-CoA binding protein (ACBP6) shapes tissue plasticity during nutrient adaptation in Drosophila. Nat Commun, 14(1):7599 PubMed ID: 37989752
Summary: Nutrient availability is a major selective force in the evolution of metazoa, and thus plasticity in tissue function and morphology is shaped by adaptive responses to nutrient changes. Utilizing Drosophila, this study revealed that distinct calibration of acyl-CoA metabolism, mediated by Acbp6 (Acyl-CoA binding-protein 6), is critical for nutrient-dependent tissue plasticity. Drosophila Acbp6, which arose by evolutionary duplication and binds acyl-CoA to tune acetyl-CoA metabolism, is required for intestinal resizing after nutrient deprivation through activating intestinal stem cell proliferation from quiescence. Disruption of acyl-CoA metabolism by Acbp6 attenuation drives aberrant 'switching' of metabolic networks in intestinal enterocytes during nutrient adaptation, impairing acetyl-CoA metabolism and acetylation amid intestinal resizing. We also identified STAT92e, whose function is influenced by acetyl-CoA levels, as a key regulator of acyl-CoA and nutrient-dependent changes in stem cell activation. These findings define a regulatory mechanism, shaped by acyl-CoA metabolism, that adjusts proliferative homeostasis to coordinately regulate tissue plasticity during nutrient adaptation. | Mappin, F., Bellantuono, A. J., Ebrahimi, B., DeGennaro, M. (2023). Odor-evoked transcriptomics of Aedes aegypti mosquitoes. PLoS One, 18(10):e0293018 PubMed ID: 37874813
Summary: Modulation of odorant receptors mRNA induced by prolonged odor exposure is highly correlated with ligand-receptor interactions in Drosophila as well as mammals of the Muridae family. If this response feature is conserved in other organisms, this presents an intriguing initial screening tool when searching for novel receptor-ligand interactions in species with predominantly orphan olfactory receptors. This study demonstrates that mRNA modulation in response to 1-octen-3-ol odor exposure occurs in a time- and concentration-dependent manner in Aedes aegypti mosquitoes. To investigate gene expression patterns at a global level, an odor-evoked transcriptome was generated associated with 1-octen-3-ol odor exposure. Transcriptomic data revealed that ORs and OBPs were transcriptionally responsive whereas other chemosensory gene families showed little to no differential expression. Alongside chemosensory gene expression changes, transcriptomic analysis found that prolonged exposure to 1-octen-3-ol modulated xenobiotic response genes, primarily members of the cytochrome P450, insect cuticle proteins, and glucuronosyltransferases families. Together, these findings suggest that mRNA transcriptional modulation of olfactory receptors caused by prolonged odor exposure is pervasive across taxa and can be accompanied by the activation of xenobiotic responses. |
| Malik, D. M., Rhoades, S. D., Zhang, S. L., Sengupta, A., Barber, A., Haynes, P., Arnadottir, E. S., Pack, A., Kibbey, R. G., Sehgal, A., Weljie, A. M. (2023). Glucose Challenge Uncovers Temporal Fungibility of Metabolic Homeostasis Throughout the Day. bioRxiv, PubMed ID: 37961230
Summary: Rhythmicity is a central feature of behavioral and biological processes including metabolism, however, the mechanisms of metabolite cycling are poorly understood. A robust oscillation in a network of key metabolite pathways downstream of glucose is described in humans, then these pathways mechanistically probed through purpose-built (13)C(6)-glucose isotope tracing in Drosophila every 4h. A temporal peak in biosynthesis was noted by broad labelling of pathways downstream of glucose in wild-type flies shortly following lights on. Krebs cycle labelling was generally increased in a hyperactive mutant (fumin) along with glycolysis labelling primarily observed at dawn. Surprisingly, neither underlying feeding rhythms nor the presence of food explains the rhythmicity of glucose processing across genotypes. These results are consistent with clinical data demonstrating detrimental effects of mis-timed energy intake. This approach provides a window into the dynamic range of metabolic processing ability through the day and mechanistic basis for exploring circadian metabolic homeostasis in disease states. | Qiao, S., Bernasek, S., Gallagher, K. D., Yamada, S., Bagheri, N., Amaral, L. A. N., Carthew, R. W. (2023). Energy metabolism modulates the regulatory impact of activators on gene expression. bioRxiv, PubMed ID: 37961620
Summary: Gene expression is a regulated process fueled by ATP consumption. Therefore, regulation must be coupled to constraints imposed by the level of energy metabolism. This study explored this relationship both theoretically and experimentally. A stylized mathematical model predicts that activators of gene expression have variable impact depending on metabolic rate. Activators become less essential when metabolic rate is reduced and more essential when metabolic rate is enhanced. In the Drosophila eye, expression dynamics of the yan gene are less affected by loss of EGFR-mediated activation when metabolism is reduced, and the opposite effect is seen when metabolism is enhanced. The effects are also seen at the level of pattern regularity in the adult eye, where loss of EGFR-mediated activation is mitigated by lower metabolism. It is proposed that gene activation is tuned by energy metabolism to allow for faithful expression dynamics in the face of variable metabolic conditions. |
Monday, July 1st - Monday - Chromatin, DNA, and Chromosome Dynamics |
| Gemeinhardt, T. M., Regy, R. M., Mendiola, A. J., Ledterman, H. J., Henrickson, A., Phan, T. M., Kim, Y. C., Demeler, B., Kim, C. A., Mittal, J., Francis, N. J. (2023). How a disordered linker in the Polycomb protein Polyhomeotic tunes phase separation and oligomerization. bioRxiv, PubMed ID: 37961422
Summary: The Polycomb Group (PcG) complex PRC1 represses transcription, forms condensates in cells, and modifies chromatin architecture. These processes are connected through the essential, polymerizing Sterile Alpha Motif (SAM) present in the PRC1 subunit Polyhomeotic (Ph). In vitro, Ph SAM drives formation of short oligomers and phase separation with DNA or chromatin in the context of a Ph truncation ("mini-Ph"). Oligomer length is controlled by the long disordered linker (L) that connects the SAM to the rest of Ph--replacing Drosophila Ph linker (PhL) with the evolutionarily diverged human PHC3L strongly increases oligomerization. How the linker controls SAM polymerization, and how polymerization and the linker affect condensate formation are not know. This analyzed PhL and PHC3L using biochemical assays and molecular dynamics (MD) simulations. PHC3L promotes mini-Ph phase separation and makes it relatively independent of DNA. In MD simulations, basic amino acids in PHC3L form contacts with acidic amino acids in the SAM. Engineering the SAM to make analogous charge-based contacts with PhL increased polymerization and phase separation, partially recapitulating the effects of the PHC3L. Ph to PHC3 linker swaps and SAM surface mutations alter Ph condensate formation in cells, and Ph function in Drosophila imaginal discs. Thus, SAM-driven phase separation and polymerization are conserved between flies and mammals, but the underlying mechanisms have diverged through changes to the disordered linker. | Shaukat, A., Bakhtiari, M. H., Chaudhry, D. S., Khan, M. H. F., Akhtar, J., Abro, A. H., Haseeb, M. A., Sarwar, A., Mazhar, K., Umer, Z., Tariq, M. (2024). Mask exhibits trxG-like behavior and associates with H3K27ac marked chromatin. Dev Biol, 505:130-140 PubMed ID: 37981061
Summary: The Trithorax group (trxG) proteins counteract the repressive effect of Polycomb group (PcG) complexes and maintain transcriptional memory of active states of key developmental genes. Although chromatin structure and modifications appear to play a fundamental role in this process, it is not clear how trxG prevents PcG-silencing and heritably maintains an active gene expression state. This study reports a hitherto unknown role of Drosophila Multiple ankyrin repeats single KH domain (Mask), which emerged as one of the candidate trxG genes in our reverse genetic screen. The genome-wide binding profile of Mask correlates with known trxG binding sites across the Drosophila genome. In particular, the association of Mask at chromatin overlaps with CBP and H3K27ac, which are known hallmarks of actively transcribed genes by trxG. Importantly, Mask predominantly associates with actively transcribed genes in Drosophila. Depletion of Mask not only results in the downregulation of trxG targets but also correlates with diminished levels of H3K27ac. The fact that Mask positively regulates H3K27ac levels in flies was also found to be conserved in human cells. Strong suppression of Pc mutant phenotype by mutation in mask provides physiological relevance that Mask contributes to the anti-silencing effect of trxG, maintaining expression of key developmental genes. Since Mask is a downstream effector of multiple cell signaling pathways, it is proposed that Mask may connect cell signaling with chromatin mediated epigenetic cell memory governed by trxG. |
| Godneeva, B., Fejes Toth, K., Quan, B., Chou, T. F., Aravin, A. A. (2023). Impact of Germline Depletion of Bonus on Chromatin State in Drosophila Ovaries. Cells, 12(22) PubMed ID: 37998364
Summary: Gene expression is controlled via complex regulatory mechanisms involving transcription factors, chromatin modifications, and chromatin regulatory factors. Histone modifications, such as H3K27me3, H3K9ac, and H3K27ac, play an important role in controlling chromatin accessibility and transcriptional output. In vertebrates, the Transcriptional Intermediary Factor 1 (TIF1) family of proteins play essential roles in transcription, cell differentiation, DNA repair, and mitosis. This study focused on Bonus, the sole member of the TIF1 family in Drosophila, to investigate its role in organizing epigenetic modifications. The findings demonstrated that depleting Bonus in ovaries leads to a mild reduction in the H3K27me3 level over transposon regions and alters the distribution of active H3K9ac marks on specific protein-coding genes. Additionally, through mass spectrometry analysis, we identified novel interacting partners of Bonus in ovaries, such as PolQ, providing a comprehensive understanding of the associated molecular pathways. Furthermore, our research revealed Bonus's interactions with the Polycomb Repressive Complex 2 and its co-purification with select histone acetyltransferases, shedding light on the underlying mechanisms behind these changes in chromatin modifications. | Golovnin, A., Melnikova, L., Babosha, V., Pokholkova, G. V., Slovohotov, I., Umnova, A., Maksimenko, O., Zhimulev, I. F., Georgiev, P. (2023). The N-Terminal Part of Drosophila CP190 Is a Platform for Interaction with Multiple Architectural Proteins. Int J Mol Sci, 24(21) PubMed ID: 37958900
Summary: CP190 is a co-factor in many Drosophila architectural proteins, being involved in the formation of active promoters and insulators. CP190 contains the N-terminal BTB/POZ (Broad-Complex, Tramtrack and Bric a brac/POxvirus and Zinc finger) domain and adjacent conserved regions involved in protein interactions. This study examined the functional roles of these domains of CP190 in vivo. The best-characterized architectural proteins with insulator functions, Pita, Su(Hw), and M1BP, Opbp, and ZIPIC, interacted with one or both of the highly conserved regions in the N-terminal part of CP190. Transgenic lines of D. melanogaster expressing CP190 mutants with a deletion of each of these domains were obtained. The results showed that these mutant proteins only partially compensated for the functions of CP190, weakly binding to selective chromatin sites. Further analysis confirmed the essential role of these domains in recruitment to regulatory regions associated with architectural proteins. The N-terminal of CP190 was found to be sufficient for recruiting Z4 and Chromator proteins and successfully achieving chromatin opening. Taken together, our results and the results of previous studies showed that the N-terminal region of CP190 is a platform for simultaneous interaction with various DNA-binding architectural proteins and transcription complexes. |
| Al Zouabi, L., Stefanutti, M., Roumeliotis, S., Le Meur, G., Boumard, B., Riddiford, N., Rubanova, N., Bohec, M., Gervais, L., Servant, N., Bardin, A. J. (2023). Chromatin state transitions in the Drosophila intestinal lineage identify principles of cell-type specification. Dev Cell, 58(24):3048-3063 PubMed ID: 38056452
Summary: Tissue homeostasis relies on rewiring of stem cell transcriptional programs into those of differentiated cells. This study investigate changes in chromatin occurring in a bipotent adult stem cells. Combining mapping of chromatin-associated factors with statistical modeling, genome-wide transitions during differentiation were identified in the adult Drosophila intestinal stem cell (ISC) lineage. Active, stem-cell-enriched genes transition to a repressive heterochromatin protein-1-enriched state more prominently in enteroendocrine cells (EEs) than in enterocytes (ECs), in which the histone H1-enriched Black state is preeminent. In contrast, terminal differentiation genes associated with metabolic functions follow a common path from a repressive, primed, histone H1-enriched Black state in ISCs to active chromatin states in EE and EC cells. Furthermore, Lineage priming was found to have an important function in adult ISCs, and histone H1 as a mediator of this process. These data define underlying principles of chromatin changes during adult multipotent stem cell differentiation. | Al Zouabi, L., Stefanutti, M., Roumeliotis, S., Le Meur, G., Boumard, B., Riddiford, N., Rubanova, N., Bohec, M., Gervais, L., Servant, N., Bardin, A. J. (2023). Molecular underpinnings and environmental drivers of loss of heterozygosity in Drosophila intestinal stem cells . Cell Rep 38032794
Summary: During development and aging, genome mutation leading to loss of heterozygosity (LOH) can uncover recessive phenotypes within tissue compartments. This phenomenon occurs in normal human tissues and is prevalent in pathological genetic conditions and cancers. While studies in yeast have defined DNA repair mechanisms that can promote LOH, the predominant pathways and environmental triggers in somatic tissues of multicellular organisms are not well understood. This study investigated mechanisms underlying LOH in intestinal stem cell in Drosophila. Infection with the pathogenic bacteria, Erwinia carotovora carotovora 15, but not Pseudomonas entomophila, increases LOH frequency. Using whole genome sequencing of somatic LOH events, it was demonstrated that they arise primarily via mitotic recombination. Molecular features and genetic evidence argue against a break-induced replication mechanism and instead support cross-over via double Holliday junction-based repair. This study provides a mechanistic understanding of mitotic recombination, an important mediator of LOH, and its effects on stem cells in vivo. |
Friday, June 28th - Larval and Adult Neural Development, Structure, and Function |
| Rathore, S., Stahl, A., Benoit, J. B., Buschbeck, E. K. (2023). Exploring the molecular makeup of support cells in insect camera eyes. BMC Genomics, 24(1):702 PubMed ID: 37993800
Summary: Animals typically have either compound eyes, or camera-type eyes, both of which have evolved repeatedly in the animal kingdom. Both eye types include two important kinds of cells: photoreceptor cells, which can be excited by light, and non-neuronal support cells (SupCs), which provide essential support to photoreceptors. At the molecular level deeply conserved genes that relate to the differentiation of photoreceptor cells have fueled a discussion on whether or not a shared evolutionary origin might be considered for this cell type. In contrast, only a handful of studies, primarily on the compound eyes of Drosophila melanogaster, have demonstrated molecular similarities in SupCs. D. melanogaster SupCs (Semper cells and primary pigment cells) are specialized eye glia that share several molecular similarities with certain vertebrate eye glia, including Muller glia. This led the authors to ask if there could be conserved molecular signatures of SupCs, even in functionally different eyes such as the image-forming larval camera eyes of the sunburst diving beetle Thermonectus marmoratus. To investigate this possibility, an in-depth comparative whole-tissue transcriptomics approach was used. Specifically, the larval principal camera eyes were dissected into SupC- and retina-containing regions, and the respective transcriptomes were generated. This analysis revealed several common features of SupCs including enrichment of genes that are important for glial function (e.g. gap junction proteins such as innexin 3), glycogen production (glycogenin), and energy metabolism (glutamine synthetase 1 and 2). To evaluate similarities, these transcriptomes with those of fly (Semper cells) and vertebrate (Muller glia) eye glia as well as respective retinas. T. marmoratus SupCs were found to have distinct genetic overlap with both fly and vertebrate eye glia. These results suggest that T. marmoratus SupCs are a form of glia, and like photoreceptors, may be deeply conserved. | Fuse, N., Hashiba, H., Ishibashi, K., Suzuki, T., Nguyen, Q. D., Fujii, K., Ikeda-Ohtsubo, W., Kitazawa, H., Tanimoto, H., Kurata, S. (2023). Neural control of redox response and microbiota-triggered inflammation in Drosophila gut. Frontiers in immunology. 14:1268611 PubMed ID: 37965334
Summary: The neural system plays a critical role in controlling gut immunity, and the gut microbiota contributes to this process. However, the roles and mechanisms of gut-brain-microbiota interactions remain unclear. To address this issue, Drosophila was employed as a model organism. Previous work has shown that NP3253 neurons, which are connected to the brain and gut, are essential for resistance to oral bacterial infections. This study aimed to investigate the role of NP3253 neurons, enteric neurons innervating the anterior midgut, in the regulation of gut immunity. RNA-seq analysis of the adult Drosophila gut was performed after genetically inactivating the NP3253 neurons. Flies were reared under oral bacterial infection and normal feeding conditions. In addition, samples were prepared under germ-free conditions to evaluate the role of the microbiota in gut gene expression. The genes regulated by NP3253 neurons were knocked down, and their susceptibility to oral bacterial infections was examined. Immune-related gene expression was found to be upregulated in NP3253 neuron-inactivated flies compared to the control. However, this upregulation was abolished in axenic flies, suggesting that the immune response was abnormally activated by the microbiota in NP3253 neuron-inactivated flies. In addition, redox-related gene expression was downregulated in NP3253 neuron-inactivated flies, and this downregulation was also observed in axenic flies. Certain redox-related genes were required for resistance to oral bacterial infections, suggesting that NP3253 neurons regulate the redox responses for gut immunity in a microbiota-independent manner. These results show that NP3253 neurons regulate the appropriate gene expression patterns in the gut and contribute to maintain homeostasis during oral infections. |
| Barth-Maron, A., D'Alessandro, I., Wilson, R. I. (2023). Interactions between specialized gain control mechanisms in olfactory processing. Curr Biol, 33(23):5109-5120.e5107 PubMed ID: 37967554
Summary: Gain control is a process that adjusts a system's sensitivity when input levels change. Neural systems contain multiple mechanisms of gain control, but it is not understood why so many mechanisms are needed or how they interact. This study investigated these questions in the Drosophila antennal lobe, where several types of inhibitory interneurons were identified with specialized gain control functions. Some interneurons are nonspiking, with compartmentalized calcium signals, and they specialize in intra-glomerular gain control. Conversely, other interneurons were found to be recruited by strong and widespread network input; they specialize in global presynaptic gain control. Using computational modeling and optogenetic perturbations, this study shows how these mechanisms can work together to improve stimulus discrimination while also minimizing temporal distortions in network activity. These results demonstrate how the robustness of neural network function can be increased by interactions among diverse and specialized mechanisms of gain control. | Gazso-Gerhat, G., Gombos, R., Toth, K., Kaltenecker, P., Szikora, S., Bíro, J., Csapo, E., Asztalos, Z., Mihaly, J. (2023). FRL and DAAM are required for lateral adhesion of interommatidial cells and patterning of the retinal floor. Development, 150(22) PubMed ID: 37997920
Summary: Optical insulation of the unit eyes (ommatidia) is an important prerequisite of precise sight with compound eyes. Separation of the ommatidia is ensured by pigment cells that organize into a hexagonal lattice in the Drosophila eye, forming thin walls between the facets. Cell adhesion, mediated by apically and latero-basally located junctional complexes, is crucial for stable attachment of these cells to each other and the basal lamina. Whereas former studies have focused on the formation and remodelling of the cellular connections at the apical region, this study reports a specific alteration of the lateral adhesion of the lattice cells, leaving the apical junctions largely unaffected. We found that DAAM and FRL, two formin-type cytoskeleton regulatory proteins, play redundant roles in lateral adhesion of the interommatidial cells and patterning of the retinal floor. We show that formin-dependent cortical actin assembly is crucial for latero-basal sealing of the ommatidial lattice. It is expected that the investigation of these previously unreported eye phenotypes will pave the way toward a better understanding of the three-dimensional aspects of compound eye development. |
| Dweck, H. K. M., Carlson, J. R. (2023). Diverse mechanisms of taste coding in Drosophila. Sci Adv, 9(46):eadj7032 PubMed ID: 37976361
Summary: Taste systems encode chemical cues that drive vital behaviors. This study has elucidated noncanonical features of taste coding using an unconventional kind of electrophysiological analysis. Taste neurons of Drosophila are much more sensitive than previously thought. They have a low spontaneous firing frequency that depends on taste receptors. Taste neurons have a dual function as olfactory neurons: They are activated by most tested odorants, including N,N-diethyl-meta-toluamide (DEET), at a distance. DEET can also inhibit certain taste neurons, revealing that there are two modes of taste response: activation and inhibition. Electrophysiological OFF responses were characterized and it was found that the tastants that elicit them are related in structure. OFF responses link tastant identity to behavior: the magnitude of the OFF response elicited by a tastant correlated with the egg laying behavior it elicited. In summary, the sensitivity and coding capacity of the taste system are much greater than previously known. | Togashi, H., Davis, S. R., Sato, M. (2024). From soap bubbles to multicellular organisms: Unraveling the role of cell adhesion and physical constraints in tile pattern formation and tissue morphogenesis. Dev Biol, 506:1-6 PubMed ID: 37995916
Summary: Tile patterns, in which numerous cells are arranged in a regular pattern, are found in a variety of multicellular organisms and play important functional roles. Such regular arrangements of cells are regulated by various cell adhesion molecules. On the other hand, cell shape is also known to be regulated by physical constraints similar to those of soap bubbles. In particular, circumference minimization plays an important role, and cell adhesion negatively affects this process, thereby regulating tissue morphogenesis based on physical properties. This study focused on the Drosophila compound eye and the mouse auditory epithelium, and summarizes the mechanisms of tile pattern formation by cell adhesion molecules such as cadherins, Irre Cell Recognition Modules (IRMs), and nectins. Phenomena that cannot be explained by physical stability based on cortical tension alone have been reported in the tile pattern formation in the compound eye, suggesting that previously unexplored forces such as cellular concentric expansion force may play an important role. |
Thursday, June 27th - Cytoskeleton and Junctions |
| Ly, M., Schimmer, C., Hawkins, R., K, E. R., Fernandez-Gonzalez, R. (2024). Integrin-based adhesions promote cell-cell junction and cytoskeletal remodelling to drive embryonic wound healing. J Cell Sci, 137(5) PubMed ID: 37970744
Summary: Embryos repair wounds rapidly, with no inflammation or scarring. Embryonic wound healing is driven by the collective movement of the cells around the lesion. The cells adjacent to the wound polarize the cytoskeletal protein actin and the molecular motor non-muscle myosin II, which accumulate at the wound edge forming a supracellular cable around the wound. Adherens junction proteins, including E-cadherin, are internalized from the wound edge and localize to former tricellular junctions at the wound margin, in a process necessary for cytoskeletal polarity. The cells adjacent to wounds in the Drosophila embryonic epidermis polarized Talin, a core component of cell-extracellular matrix (ECM) adhesions, which preferentially accumulated at the wound edge. Integrin knockdown and inhibition of integrin binding delayed wound closure and reduced actin polarization and dynamics around the wound. Additionally, disrupting integrins caused a defect in E-cadherin reinforcement at tricellular junctions along the wound edge, suggesting crosstalk between integrin-based and cadherin-based adhesions. The results show that cell-ECM adhesion contributes to embryonic wound repair and reveal an interplay between cell-cell and cell-ECM adhesion in the collective cell movements that drive rapid wound healing. | Xu, Y., Wang, B., Bush, I., Saunders, H. A., Wildonger, J., Han, C. (2023). Light-induced trapping of endogenous proteins reveals spatiotemporal roles of microtubule and kinesin-1 in dendrite patterning of Drosophila sensory neurons. bioRxiv, PubMed ID: 37873262
Summary: Animal development involves numerous molecular events, whose spatiotemporal properties largely determine the biological outcomes. Conventional methods for studying gene function lack the necessary spatiotemporal resolution for precise dissection of developmental mechanisms. Optogenetic approaches are powerful alternatives, but most existing tools rely on exogenous designer proteins that produce narrow outputs and cannot be applied to diverse or endogenous proteins. To address this limitation, OptoTrap, a light-inducible protein trapping system was developed that allows manipulation of endogenous proteins tagged with GFP or split GFP. This system turns on fast and is reversible in minutes or hours. This study generated OptoTrap variants optimized for neurons and epithelial cells and demonstrate effective trapping of endogenous proteins of diverse sizes, subcellular locations, and functions. Furthermore, OptoTrap allowed instant disruption of microtubules and inhibits the kinesin-1 motor in specific dendritic branches of Drosophila sensory neurons. Using OptoTrap, direct evidence was obtained that microtubules support the growth of highly dynamic dendrites. Similarly, targeted manipulation of Kinesin heavy chain revealed differential spatiotemporal requirements of kinesin-1 in the patterning of low- and high-order dendritic branches, suggesting that different cargos are needed for the growth of these branches. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds great promise for studying developmental mechanisms in a wide range of cell types and developmental stages. |
| Christophers, B., Leahy, S. N., Soffar, D. B., von Saucken, V. E., Broadie, K., Baylies, M. K. (2023). Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission. bioRxiv, PubMed ID: 38045306
Summary: Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Previous work has shown Drosophila cofilin (DmCFL) knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. It was hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, an RNA sequencing analysis was conducted which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. DmCFL is enriched in the muscle postsynaptic compartment and DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, no significant changes were founs in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand our understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology. | Sato, Y., Yoshimura, K., Matsuda, K., Haraguchi, T., Marumo, A., Yamagishi, M., Sato, S., Ito, K., Yajima, J. (2023). Membrane-bound myosin IC drives the chiral rotation of the gliding actin filament around its longitudinal axis. Sci Rep, 13(1):19908 PubMed ID: 37963943
Summary: Myosin IC, a single-headed member of the myosin I family, specifically interacts with anionic phosphatidylinositol 4,5-bisphosphate (PI[4,5]P(2)) in the cell membrane via the pleckstrin homology domain located in the myosin IC tail. Myosin IC is widely expressed and physically links the cell membrane to the actin cytoskeleton; it plays various roles in membrane-associated physiological processes, including establishing cellular chirality, lipid transportation, and mechanosensing. This study evaluated the motility of full-length myosin IC of Drosophila melanogaster via the three-dimensional tracking of quantum dots bound to actin filaments that glided over a membrane-bound myosin IC-coated surface. The results revealed that myosin IC drove a left-handed rotational motion in the gliding actin filament around its longitudinal axis, indicating that myosin IC generated a torque perpendicular to the gliding direction of the actin filament. The quantification of the rotational motion of actin filaments on fluid membranes containing different PI(4,5)P(2) concentrations revealed that the rotational pitch was longer at lower PI(4,5)P(2) concentrations. These results suggest that the torque generated by membrane-bound myosin IC molecules can be modulated based on the phospholipid composition of the cell membrane. |
| Kroeger, B., Manning, S. A., Fonseka, Y., Oorschot, V., Crawford, S. A., Ramm, G., Harvey, K. F. (2023). Basal spot junctions of Drosophila epithelial tissues respond to morphogenetic forces and regulate Hippo signaling.. Dev Cell, PubMed ID: 38134928
Summary: Organ size is controlled by numerous factors including mechanical forces, which are mediated in part by the Hippo pathway. In growing Drosophila epithelial tissues, cytoskeletal tension influences Hippo signaling by modulating the localization of key pathway proteins to different apical domains. This study discovered a Hippo signaling hub at basal spot junctions, which form at the basal-most point of the lateral membranes and resemble adherens junctions in protein composition. Basal spot junctions recruit the central kinase Warts via Ajuba and E-cadherin, which prevent Warts activation by segregating it from upstream Hippo pathway proteins. Basal spot junctions are prominent when tissues undergo morphogenesis and are highly sensitive to fluctuations in cytoskeletal tension. They are distinct from focal adhesions, but the latter profoundly influences basal spot junction abundance by modulating the basal-medial actomyosin network and tension experienced by spot junctions. Thus, basal spot junctions couple morphogenetic forces to Hippo pathway activity and organ growth. | Ikawa, K., Hiro, S., Kondo, S., Ohsawa, S., Sugimura, K. (2023). Coronin-1 promotes directional cell rearrangement in Drosophila wing epithelium. Cell structure and function 48(2):251-257 PubMed ID: 38030242
Summary: Directional cell rearrangement is a critical process underlying correct tissue deformation during morphogenesis. Although the involvement of F-actin regulation in cell rearrangement has been established, the role and regulation of actin binding proteins (ABPs) in this process are not well understood. This study investigated the function of Coronin-1, a WD-repeat actin-binding protein, in controlling directional cell rearrangement in the Drosophila pupal wing. Transgenic flies expressing Coronin-1-EGFP were generated using CRISPR-Cas9. Coronin-1 was obserced to localize at the reconnecting junction during cell rearrangement, which is dependent on actin interacting protein 1 (AIP1) and cofilin, actin disassemblers and known regulators of wing cell rearrangement. Loss of Coronin-1 function reduces cell rearrangement directionality and hexagonal cell fraction. These results suggest that Coronin-1 promotes directional cell rearrangement via its interaction with AIP1 and cofilin, highlighting the role of ABPs in the complex process of morphogenesis. |
Wednesday, June 26th - Disease Models |
| Kang, J., Zhang, C., Wang, Y., Peng, J., Berger, B., Perrimon, N., Shen, J. (2024). Lipophorin receptors genetically modulate neurodegeneration caused by reduction of Psn expression in the aging Drosophila brain. Genetics, 226(1) PubMed ID: 37996068
Summary: Mutations in the Presenilin (PSEN) genes are the most common cause of early-onset familial Alzheimer's disease (FAD). Studies in cell culture, in vitro biochemical systems, and knockin mice showed that PSEN mutations are loss-of-function mutations, impairing γ-secretase activity. Mouse genetic analysis highlighted the importance of Presenilin (PS) in learning and memory, synaptic plasticity and neurotransmitter release, and neuronal survival, and Drosophila studies further demonstrated an evolutionarily conserved role of PS in neuronal survival during aging. However, molecular pathways that interact with PS in neuronal survival remain unclear. To identify genetic modifiers that modulate PS-dependent neuronal survival, this study developed a new Drosophila Psn model that exhibits age-dependent neurodegeneration and increases of apoptosis. Following a bioinformatic analysis, top ranked candidate genes were tested by selective knockdown (KD) of each gene in neurons using two independent RNAi lines in Psn KD models. Interestingly, 4 of the 9 genes enhancing neurodegeneration in Psn KD flies are involved in lipid transport and metabolism. Specifically, neuron-specific KD of lipophorin receptors, lpr1 and lpr2, dramatically worsens neurodegeneration in Psn KD flies, and overexpression of lpr1 or lpr2 does not alleviate Psn KD-induced neurodegeneration. Furthermore, lpr1 or lpr2 KD alone also leads to neurodegeneration, increased apoptosis, climbing defects, and shortened lifespan. Lastly, heterozygotic deletions of lpr1 and lpr2 or homozygotic deletions of lpr1 or lpr2 similarly lead to age-dependent neurodegeneration and further exacerbate neurodegeneration in Psn KD flies. These findings show that LpRs modulate Psn-dependent neuronal survival and are critically important for neuronal integrity in the aging brain. | Li, L., Wei, Z., Tang, Y., Jin, M., Yao, H., Li, X., Li, Q., Tan, J., Xiao, B. (2023). Icaritin greatly attenuates β-amyloid-induced toxicity in vivo. CNS neuroscience & therapeutics. PubMed ID: 37990437
Summary: The accumulation and deposition of β-amyloid (Aβ; see Drosophila Appl) has always been considered a major pathological feature of Alzheimer's disease (AD). The latest and mainstream amyloid cascade hypothesis indicates that all the main pathological changes in AD are attributed to the accumulation of soluble Aβ. However, the exploration of therapeutic drugs for Aβ toxicity has progressed slowly. This study aims to investigate the protective effects of Icaritin on the Aβ-induced Drosophila AD model and its possible mechanism. To identify the effects of Icaritin on AD, an excellent Drosophila AD model named Aβ(arc) (arctic mutant Aβ(42)) Drosophila. Climbing ability, flight ability, and longevity were used to evaluate the effects of Icaritin on AD phenotypes. Aβarc was determined by immunostaining and ELISA. To identify the effects of Icaritin on oxidative stress, the detection of ROS, hydrogen peroxide, MDA, SOD, catalase, GST, and Caspase-3 was performed. To identify the effects of Icaritin on energy metabolism, the detection of ATP and lactate was performed. Transcriptome analysis and qRT-PCR verifications were used to detect the genes directly involved in oxidative stress and energy metabolism. Mitochondrial structure and function were detected by an electron microscopy assay, a mitochondrial membrane potential assay, and a mitochondrial respiration assay. It was discovered that Icaritin almost completely rescues the climbing ability, flight ability, and longevity of Aβarc Drosophila. Aβarc was dramatically reduced by Icaritin treatment. It was also found that Icaritin significantly reduces oxidative stress and greatly improves impaired energy metabolism. Importantly, transcriptome analysis and qRT-PCR verifications showed that many key genes, directly involved in oxidative stress and energy metabolism, are restored by Icaritin. Next, it was found that Icaritin perfectly restores the integrity of mitochondrial structure and function damaged by Aβarc toxicity. This study suggested that Icaritin is a potential drug to deal with the toxicity of Aβarc, at least partially realized by restoring the mitochondria/oxidative stress/energy metabolism axis, and holds potential for translation to human AD. |
| Thorpe, H. J., Owings, K. G., Aziz, M. C., Haller, M., Coelho, E., Chow, C. Y. (2023). Drosophila models of PIGA-CDG mirror patient phenotypes. bioRxiv, PubMed ID: 37961693
Summary: Mutations in the phosphatidylinositol glycan biosynthesis class A (PIGA) gene cause a rare, X-linked recessive congenital disorder of glycosylation (CDG). PIGA-CDG is characterized by seizures, intellectual and developmental delay, and congenital malformations. The PIGA gene encodes an enzyme involved in the first step of GPI anchor biosynthesis. There are over 100 GPI anchored proteins that attach to the cell surface and are involved in cell signaling, immunity, and adhesion. Little is known about the pathophysiology of PIGA-CDG. This study describes the first Drosophila model of PIGA-CDG and demonstrate that loss of PIG-A function in Drosophila accurately models the human disease. As expected, complete loss of PIG-A function is larval lethal. Heterozygous null animals appear healthy, but when challenged, have a seizure phenotype similar to what is observed in patients. To identify the cell-type specific contributions to disease, neuron- and glia-specific knockdown of PIG-A were generated. Neuron-specific knockdown resulted in reduced lifespan and a number of neurological phenotypes, but no seizure phenotype. Glia-knockdown also reduced lifespan and, notably, resulted in a very strong seizure phenotype. RNAseq analyses demonstrated that there are fundamentally different molecular processes that are disrupted when PIG-A function is eliminated in different cell types. In particular, loss of PIG-A in neurons resulted in upregulation of glycolysis, but loss of PIG-A in glia resulted in upregulation of protein translation machinery. This study demonstrates that Drosophila is a good model of PIGA-CDG and provide new data resources for future study of PIGA-CDG and other GPI anchor disorders. | Khan, M. R., Yin, X., Kang, S. U., Mitra, J., Wang, H., Ryu, T., Brahmachari, S., Karuppagounder, S. S., Kimura, Y., Jhaldiyal, A., Kim, H. H., Gu, H., Chen, R., Redding-Ochoa, J., Troncoso, J., Na, C. H., Ha, T., Dawson, V. L., Dawson, T. M. (2023). Enhanced mTORC1 signaling and protein synthesis in pathologic α-synuclein cellular and animal models of Parkinson's disease. Science translational medicine, 15(724):eadd0499 PubMed ID: 38019930
Summary: Pathologic α-synuclein plays an important role in the pathogenesis of &alpha-synucleinopathies such as Parkinson's disease (PD). Disruption of proteostasis is thought to be central to pathologic α-synuclein toxicity; however, the molecular mechanism of this deregulation is poorly understood. Complementary proteomic approaches in cellular and animal models of PD were used to identify and characterize the pathologic α-synuclein interactome. This study reports that the highest biological processes that interacted with pathologic α-synuclein in mice included RNA processing and translation initiation. Regulation of catabolic processes that include autophagy were also identified. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein 2 (TSC2) and to trigger the activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which augmented mRNA translation and protein synthesis, leading to neurodegeneration. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescued the dopamine neuron loss, behavioral deficits, and aberrant biochemical signaling in the α-synuclein preformed fibril mouse model and Drosophila transgenic models of pathologic α-synuclein-induced degeneration. Pathologic α-synuclein furthermore led to a destabilization of the TSC1-TSC2 complex, which plays an important role in mTORC1 activity. Constitutive overexpression of TSC2 rescued motor deficits and neuropathology in α-synuclein flies. Biochemical examination of PD postmortem brain tissues also suggested deregulated mTORC1 signaling. These findings establish a connection between mRNA translation deregulation and mTORC1 pathway activation that is induced by pathologic α-synuclein in cellular and animal models of PD. |
| Na, D., Lim, D. H., Hong, J. S., Lee, H. M., Cho, D., Yu, M. S., Shaker, B., Ren, J., Lee, B., Song, J. G., Oh, Y., Lee, K., Oh, K. S., Lee, M. Y., Choi, M. S., Choi, H. S., Kim, Y. H., Bui, J. M., Lee, K., Kim, H. W., Lee, Y. S., Gsponer, J. (2023). A multi-layered network model identifies Akt1 as a common modulator of neurodegeneration. Molecular systems biology 19(12):e11801 PubMed ID: 37984409
Summary: The accumulation of misfolded and aggregated proteins is a hallmark of neurodegenerative proteinopathies. Although multiple genetic loci have been associated with specific neurodegenerative diseases (NDs), molecular mechanisms that may have a broader relevance for most or all proteinopathies remain poorly resolved. This study developed a multi-layered network expansion (MLnet) model to predict protein modifiers that are common to a group of diseases and, therefore, may have broader pathophysiological relevance for that group. When applied to the four NDs Alzheimer's disease (AD), Huntington's disease, and spinocerebellar ataxia types 1 and 3, multiple members of the insulin pathway, including PDK1, Akt1, InR, and sgg (GSK-3β), were predicted as common modifiers. These modifiers were validated with the help of four Drosophila ND models. Further evaluation of Akt1 in human cell-based ND models revealed that activation of Akt1 signaling by the small molecule SC79 increased cell viability in all models. Moreover, treatment of AD model mice with SC79 enhanced their long-term memory and ameliorated dysregulated anxiety levels, which are commonly affected in AD patients. These findings validate MLnet as a valuable tool to uncover molecular pathways and proteins involved in the pathophysiology of entire disease groups and identify potential therapeutic targets that have relevance across disease boundaries. MLnet can be used for any group of diseases and is available as a web tool. | Johnstone, J. N., Mirth, C. K., Johnson, T. K., Schittenhelm, R. B., Piper, M. D. W. (2023). GCN2 mediates access to stored amino acids for somatic maintenance during Drosophila ageing. bioRxiv, PubMed ID: 38014136
Summary: Many mechanistic theories of ageing argue that a progressive failure of somatic maintenance, the use of energy and resources to prevent and repair damage to the cell, underpins ageing. To sustain somatic maintenance an organism must acquire dozens of essential nutrients from the diet, including essential amino acids (EAAs), which are physiologically limiting for many animals. In Drosophila, adulthood deprivation of each individual EAA yields vastly different lifespan trajectories, and adulthood deprivation of one EAA, phenylalanine (Phe), has no associated lifespan cost; this is despite each EAA being strictly required for growth and reproduction. Moreover, survival under any EAA deprivation depends entirely on the conserved AA sensor GCN2, a component of the integrated stress response (ISR), suggesting that a novel ISR-mediated mechanism sustains lifelong somatic maintenance during EAA deprivation. Flies chronically deprived of dietary Phe continue to incorporate Phe into new proteins, and challenging flies to increase the somatic requirement for Phe shortens lifespan under Phe deprivation. Further, this study showed that autophagy is required for full lifespan under Phe deprivation, and that activation of the ISR can partially rescue the shortened lifespan of GCN2-nulls under Phe deprivation. A mechanism is proposed by which GCN2, via the ISR, activates autophagy during EAA deprivation, breaking down a larvally-acquired store of EAAs to support somatic maintenance. These data refine understanding of the strategies by which flies sustain lifelong somatic maintenance, which determines length of life in response to changes in the nutritional environment. |
Tuesday, June 25th - Enhancers and Transcriptional Regulation |
| Fischer, M. D., Graham, P., Pick, L. (2024). The ftz upstream element drives late ftz stripes but is not required for regulation of Ftz target genes. Dev Biol, 505:141-147 PubMed ID: 37977522
Summary: The regulation of gene expression in precise, rapidly changing spatial patterns is essential for embryonic development. Multiple enhancers have been identified for the evolving expression patterns of the cascade of Drosophila segmentation genes that establish the basic body plan of the fly. Classic reporter transgene experiments identified multiple cis-regulatory elements (CREs) that are sufficient to direct various aspects of the evolving expression pattern of the pair-rule gene fushi tarazu (ftz). These include enhancers that coordinately activate expression in all seven stripes and stripe-specific elements that activate expression in one or more ftz stripes. Of the two 7-stripe enhancers, analysis of reporter transgenes demonstrated that the upstream element (UPS) is autoregulatory, requiring direct binding of Ftz protein to direct striped expression. This study asked about the endogenous role of the UPS by precisely deleting this 7-stripe enhancer. In ftzΔUPS(7S) homozygotes, ftz stripes appear in the same order as wildtype, and all but stripe 4 are expressed at wildtype levels by the end of the cellular blastoderm stage. This suggests that the zebra element and UPS harbor information to direct stripe 4 expression, although previous deletion analyses failed to identify a stripe-specific CRE within these two 7-stripe enhancers. However, the UPS is necessary for late ftz stripe expression, with all 7 stripes decaying earlier than wildtype in ftzΔUPS(7S) homozygotes. Despite this premature loss of ftz expression, downstream target gene regulation proceeds as in wildtype, and segmentation is unperturbed in the overwhelming majority of animals. It is proposed that this late-acting enhancer provides a buffer against perturbations in gene expression but is not required for establishment of Ftz cell fates. Overall, these results demonstrate that multiple enhancers, each directing distinct aspects of an overall gene expression pattern, contribute to fine-tuning the complex patterns necessary for embryonic development. | Silver, B. D., Willett, C. G., Maher, K. A., Wang, D., Deal, R. B. (2023). Differences in transcription initiation directionality underlie distinctions between plants and animals in chromatin modification patterns at genes and cis-regulatory elements. bioRxiv, PubMed ID: 37961418
Summary: Transcriptional initiation is among the first regulated steps controlling eukaryotic gene expression. High-throughput profiling of fungal and animal genomes has revealed that RNA Polymerase II (Pol II) often initiates transcription in both directions at the promoter transcription start site (TSS), but generally only elongates productively into the gene body. Additionally, Pol II can initiate transcription in both directions at cis-regulatory elements (CREs) such as enhancers. These bidirectional Pol II initiation events can be observed directly with methods that capture nascent transcripts, and they are also revealed indirectly by the presence of transcription-associated histone modifications on both sides of the TSS or CRE. Previous studies have shown that nascent RNAs and transcription-associated histone modifications in the model plant Arabidopsis thaliana accumulate mainly in the gene body, suggesting that transcription does not initiate widely in the upstream direction from genes in this plant. Transcription-associated histone modifications and nascent transcripts at both TSSs and CREs were compared in Arabidopsis thaliana, Drosophila melanogaster, and Homo sapiens. The results provide evidence for mostly unidirectional Pol II initiation at both promoters and gene-proximal CREs of Arabidopsis thaliana, whereas bidirectional transcription initiation is observed widely at promoters in both Drosophila melanogaster and Homo sapiens, as well as CREs in Drosophila. Furthermore, the distribution of transcription-associated histone modifications around TSSs in the Oryza sativa (rice) and Glycine max (soybean) genomes suggests that unidirectional transcription initiation is the norm in these genomes as well. These results suggest that there are fundamental differences in transcriptional initiation directionality between flowering plant and metazoan genomes, which are manifested as distinct patterns of chromatin modifications around RNA polymerase initiation sites. |
| Taskiran, II, Spanier, K. I., Dickmanken, H., Kempynck, N., Pancikova, A., Eksi, E. C., Hulselmans, G., Ismail, J. N., Theunis, K., Vandepoel, R., Christiaens, V., Mauduit, D., Aerts, S. (2023). Cell-type-directed design of synthetic enhancers. Nature, PubMed ID: 38086419
Summary: Transcriptional enhancers act as docking stations for combinations of transcription factors and thereby regulate spatiotemporal activation of their target genes. It has been a long-standing goal in the field to decode the regulatory logic of an enhancer and to understand the details of how spatiotemporal gene expression is encoded in an enhancer sequence. This study shows that deep learning models(2-6), can be used to efficiently design synthetic, cell-type-specific enhancers, starting from random sequences, and that this optimization process allows detailed tracing of enhancer features at single-nucleotide resolution. The function of fully synthetic enhancers was evaluatednto specifically target Kenyon cells or glial cells in the fruit fly brain using transgenic animals. Enhancer design was further exploited to create 'dual-code' enhancers that target two cell types and minimal enhancers smaller than 50 base pairs that are fully functional. By examining the state space searches towards local optima, enhancer codes were characterized through the strength, combination and arrangement of transcription factor activator and transcription factor repressor motifs. Finally, the same strategies were applied to successfully design human enhancers, which adhere to enhancer rules similar to those of Drosophila enhancers. Enhancer design guided by deep learning leads to better understanding of how enhancers work and shows that their code can be exploited to manipulate cell states. | Cheng, Y., Chan, F., Kassis, J. A. (2023). The activity of engrailed imaginal disc enhancers is modulated epigenetically by chromatin and autoregulation. PLoS Genet, 19(11):e1010826 PubMed ID: 37967127
Summary: engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal discs; in contrast, small IDE-reporter transgenes are expressed mainly in the anterior compartment. En was found to bind to the IDEs, and suggesting that it may directly repress IDE function and modulate En expression levels. Two en IDEs, O and S were discrovered. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from an endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether these data suggest that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic memory set by the embryonic enhancers. This epigenetic regulation is similar to that of the Ultrabithorax IDEs and it is suggested that the activity of late-acting enhancers in other genes may be similarly regulated. |
| Wang, Y., Salazar, R., Simonetta, L., Sorrentino, V., Gatton, T. J., Wu, B., Vecsey, C. G., Carrillo, R. A. (2023). hkb is required for DIP-alpha expression and target recognition in the Drosophila neuromuscular circuit. bioRxiv, PubMed ID: 37905128
Summary: The human nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins (CSPs). In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth and cell survival. However, the upstream regulation and downstream signaling mechanisms of Dprs and DIPs are not clear. In the Drosophila larval neuromuscular system, DIP-α is expressed in the dorsal and ventral type-Is motor neurons (MNs). F1 dominant modifier genetic screen was conducted to identify regulators of Dprs and DIPs. The transcription factor, huckebein (hkb), was found to genetically interact with DIP-α and is important for target recognition specifically in the dorsal Is MN, but not the ventral Is MN. Loss of hkb led to complete removal of DIP-α expression. We then confirmed that this specificity is through the dorsal Is MN specific transcription factor, even-skipped (eve), which acts downstream of hkb. Genetic interaction between hkb and eve revealed that they act in the same pathway to regulate dorsal Is MN connectivity. This study provides insight into the transcriptional regulation of DIP-α and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons. | Syed, S., Duan, Y., Lim, B. (2023). Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos. Elife, 12 PubMed ID: 37934571
Summary: It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. This study utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, a mutation in any one of these binding sites was found to cause a drastic reduction in transcriptional amplitude, resulting in a reduction in mRNA production of the target gene. Evidence is provided of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. It is proposed that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates. |
Monday, June 24th - Signaling |
| Tian, A., Wang, X. F., Xu, Y., Morejon, V., Huang, Y. C., Nwapuda, C., Deng, W. M. (2023). EGFR signaling controls directionality of epithelial multilayer formation upon loss of cell polarity. The EMBO journal, 42(24):e113856 PubMed ID: 37953688
Summary: Apical-basal polarity is maintained by distinct protein complexes that reside in membrane junctions, and polarity loss in monolayered epithelial cells can lead to formation of multilayers, cell extrusion, and/or malignant overgrowth. Yet, how polarity loss cooperates with intrinsic signals to control directional invasion toward neighboring epithelial cells remains elusive. Using the Drosophila ovarian follicular epithelium as a model, this study found that posterior follicle cells with loss of lethal giant larvae (lgl) or Discs large (Dlg) accumulate apically toward germline cells, whereas cells with loss of Bazooka (Baz) or atypical protein kinase C (aPKC) expand toward the basal side of wildtype neighbors. Further studies revealed that these distinct multilayering patterns in the follicular epithelium were determined by epidermal growth factor receptor (EGFR) signaling and its downstream target Pointed, a zinc-finger transcription factor. Additionally, Rho kinase was identified as a Pointed target that regulates formation of distinct multilayering patterns. These findings provide insight into how cell polarity genes and receptor tyrosine kinase signaling interact to govern epithelial cell organization and directional growth that contribute to epithelial tumor formation. | Shi, L., Ma, H., Wang, J., Ma, M., Zhao, H., Li, Z., Wang, J. H., Wu, S., Zhou, Z., Dong, M. Q., Li, Z. (2023). An EMC-Hpo-Yki axis maintains intestinal homeostasis under physiological and pathological conditions. Development, 150(24) PubMed ID: 38031990
Summary: alanced control of stem cell proliferation and differentiation underlines tissue homeostasis. Disruption of tissue homeostasis often results in many diseases. However, how endogenous factors influence the proliferation and differentiation of intestinal stem cells (ISCs) under physiological and pathological conditions remains poorly understood. This study found that the evolutionarily conserved endoplasmic reticulum membrane protein complex (EMC) negatively regulates ISC proliferation and intestinal homeostasis. Compromising EMC function in progenitors leads to excessive ISC proliferation and intestinal homeostasis disruption. Mechanistically, the EMC associates with and stabilizes Hippo (Hpo) protein, the key component of the Hpo signaling pathway. In the absence of EMC, Yorkie (Yki) is activated to promote ISC proliferation due to Hpo destruction. The EMC-Hpo-Yki axis also functions in enterocytes to maintain intestinal homeostasis. Importantly, the levels of the EMC are dramatically diminished in tunicamycin-treated animals, leading to Hpo destruction, thereby resulting in intestinal homeostasis disruption due to Yki activation. Thus, this study uncovers the molecular mechanism underlying the action of the EMC in intestinal homeostasis maintenance under physiological and pathological conditions and provides new insight into the pathogenesis of tunicamycin-induced tumorigenesis. |
| Jackson, J. A., Denk-Lobnig, M., Kitzinger, K. A., Martin, A. C. (2023). Change in RhoGAP and RhoGEF availability drives transitions in cortical patterning and excitability in Drosophila. bioRxiv, PubMed ID: 37986763
Summary: Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. While mechanisms have been established for individual cells' dynamic behaviors, mechanisms and specific molecules that result in developmental transitions in vivo are still poorly understood. This study took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. A RhoGEF (Pebble) and RhoGAP (RhoGAP15B) pair was identified whose relocalization from nucleus to cortex results in actomyosin waves in egg chambers. Furthermore, Overexpression of a different RhoGEF and RhoGAP pair resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly and RhoGAP recruitment by ~4 seconds. Overall, this study showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types. | Chen, J., Stork, T., Kang, Y., Nardone, K. A. M., Auer, F., Farrell, R. J., Jay, T. R., Heo, D., Sheehan, A., Paton, C., Nagel, K. I., Schoppik, D., Monk, K. R., Freeman, M. R. (2024). Astrocyte growth is driven by the Tre1/S1pr1 phospholipid-binding G protein-coupled receptor. Neuron, 112(1):93-112.e110 PubMed ID: 38096817
Summary: Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways regulating astrocyte morphogenesis remain poorly defined. This study shows the Drosophila lipid-binding G protein-coupled receptor (GPCR) Tre1 is required for astrocytes to establish their intricate morphology in vivo. The lipid phosphate phosphatases Wunen/Wunen2 also regulate astrocyte morphology and, via Tre1, mediate astrocyte-astrocyte competition for growth-promoting lipids. Loss of s1pr1, the functional analog of Tre1 in zebrafish, disrupts astrocyte process elaboration, and live imaging and pharmacology demonstrate that S1pr1 balances proper astrocyte process extension/retraction dynamics during growth. Loss of Tre1 in flies or S1pr1 in zebrafish results in defects in simple assays of motor behavior. Tre1 and S1pr1 are thus potent evolutionarily conserved regulators of the elaboration of astrocyte morphological complexity and, ultimately, astrocyte control of behavior. |
| Bakopoulos, D., Golenkina, S., Dark, C., Christie, E. L., Sanchez-Sanchez, B. J., Stramer, B. M., Cheng, L. Y. (2023). Convergent insulin and TGF-beta signalling drives cancer cachexia by promoting aberrant fat body ECM accumulation in a Drosophila tumour model. EMBO reports, 24(12):e57695 PubMed ID: 38014610
Summary: This study found that in the adipose tissue of wildtype animals, insulin and TGF-β signalling converge via a BMP antagonist short gastrulation (sog) to regulate ECM remodelling. In tumour bearing animals, Sog also modulates TGF-β signalling to regulate ECM accumulation in the fat body. TGF-β signalling causes ECM retention in the fat body and subsequently depletes muscles of fat body-derived ECM proteins. Activation of insulin signalling, inhibition of TGF-β signalling, or modulation of ECM levels via SPARC, Rab10 or Collagen IV in the fat body, is able to rescue tissue wasting in the presence of tumour. Together, our study highlights the importance of adipose ECM remodelling in the context of cancer cachexia. | Nakamura, M., Parkhurst, S. M. (2023). Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds. bioRxiv, PubMed ID: 38105960
Summary: To survive daily damage, the formation of actomyosin ring at the wound periphery is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. This study found that rapid recruitment of all three Drosophila calcium responding and phospholipid binding Annexin proteins (AnxB9, AnxB10, AnxB11) to distinct regions around the wound are regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, it was found that reduced extracellular calcium and depletion of intracellular calcium affect cell wound repair differently, despite these two conditions exhibiting similar GCaMP signals. Thus, the results suggest that, in addition to initiating repair events, both the quantity and sources of calcium influx are important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair. |
Friday, June 21st - Adult Neural Development, Structure, and Function |
| Munroe, J. A., Doe, C. Q. (2023). Imp is expressed in INPs and newborn neurons where it regulates neuropil targeting in the central complex. Neural Dev, 18(1):9 PubMed ID: 38031099
Summary: The generation of neuronal diversity remains incompletely understood. In Drosophila, the central brain is populated by neural stem cells derived from progenitors called neuroblasts (NBs). There are two types of NBs, type 1 and 2. T1NBs have a relatively simple lineage, whereas T2NBs expand and diversify the neural population with the generation of intermediate neural progenitors (INPs), contributing many neurons to the adult central complex, a brain region essential for navigation. However, it is not fully understood how neural diversity is created in T2NB and INP lineages. Imp, an RNA-binding protein, is expressed in T2NBs in a high-to-low temporal gradient, while the RNA-binding protein Syncrip forms an opposing gradient. It remains unknown if Imp expression is carried into INPs; whether it forms a gradient similar to NBs; and whether INP expression of Imp is required for generating neuronal identity or morphology. This study shows that Imp/Syp are both present in INPs, but not always in opposing gradients. Newborn INPs adopt their Imp/Syp levels from their parental T2NBs; that Imp and Syp are expressed in stage-specific high-to-low gradients in INPs. In addition, there is a late INP pulse of Imp. Neurons born from old INPs (E-PG and PF-R neurons) have altered morphology following both Imp knock-down and Imp overexpression. It is concluded that Imp functions in INPs and newborn neurons to determine proper neuronal morphology and central complex neuropil organization. | Rabah, Y., Frances, R., Minatchy, J., Guedon, L., Desnous, C., Placais, P. Y., Preat, T. (2023). Glycolysis-derived alanine from glia fuels neuronal mitochondria for memory in Drosophila. Nature metabolism, 5(11):2002-2019 PubMed ID: 37932430
Summary: Glucose is the primary source of energy for the brain; however, it remains controversial whether, upon neuronal activation, glucose is primarily used by neurons for ATP production or if it is partially oxidized in astrocytes, as proposed by the astrocyte-neuron lactate shuttle model for glutamatergic neurons. Thus, an in vivo picture of glucose metabolism during cognitive processes is missing. This study uncovered in Drosophila melanogaster a glia-to-neuron alanine transfer involving alanine aminotransferase that sustains memory formation. Following associative conditioning, glycolysis in glial cells produces alanine, which is back-converted into pyruvate in cholinergic neurons of the olfactory memory center to uphold their increased mitochondrial needs. Alanine, as a mediator of glia-neuron coupling, could be an alternative to lactate in cholinergic systems. In parallel, a dedicated glial glucose transporter imports glucose specifically for long-term memory, by directly transferring it to neurons for use by the pentose phosphate pathway. These results demonstrate in vivo the compartmentalization of glucose metabolism between neurons and glial cells during memory formation. |
| Tanaka, R., Zhou, B., Agrochao, M., Badwan, B. A., Au, B., Matos, N. C. B., Clark, D. A. (2023). Neural mechanisms to incorporate visual counterevidence in self-movement estimation. Curr Biol, 33(22):4960-4979. PubMed ID: 37918398
Summary: In selecting appropriate behaviors, animals should weigh sensory evidence both for and against specific beliefs about the world. For instance, animals measure optic flow to estimate and control their own rotation. However, existing models of flow detection can be spuriously triggered by visual motion created by objects moving in the world. This study shows that stationary patterns on the retina, which constitute evidence against observer rotation, suppress inappropriate stabilizing rotational behavior in the fruit fly Drosophila. In silico experiments show that artificial neural networks (ANNs) that are optimized to distinguish observer movement from external object motion similarly detect stationarity and incorporate negative evidence. Employing neural measurements and genetic manipulations, this study identified components of the circuitry for stationary pattern detection, which runs parallel to the fly's local motion and optic-flow detectors. The results show how the fly brain incorporates negative evidence to improve heading stability, exemplifying how a compact brain exploits geometrical constraints of the visual world. | Longden, K. D., Rogers, E. M., Nern, A., Dionne, H., Reiser, M. B. (2023). Different spectral sensitivities of ON- and OFF-motion pathways enhance the detection of approaching color objects in Drosophila. Nat Commun, 14(1):7693 PubMed ID: 38001097
Summary: Color and motion are used by many species to identify salient objects. They are processed largely independently, but color contributes to motion processing in humans, for example, enabling moving colored objects to be detected when their luminance matches the background. This study demonstrates an unexpected, additional contribution of color to motion vision in Drosophila. Behavioral ON-motion responses are more sensitive to UV than for OFF-motion, and cellular pathways were identified connecting UV-sensitive R7 photoreceptors to ON and OFF-motion-sensitive T4 and T5 cells, using neurogenetics and calcium imaging. Remarkably, this contribution of color circuitry to motion vision enhances the detection of approaching UV discs, but not green discs with the same chromatic contrast, and it was shown how this could generalize for systems with ON- and OFF-motion pathways. The results provide a computational and circuit basis for how color enhances motion vision to favor the detection of saliently colored objects. |
| Klose, M. K., Kim, J., Levitan, E. S. (2023). Activity-dependent capture of neuropeptide vesicles prepares clock neuron synapses for daily release. bioRxiv, PubMed ID: 38106047
Summary: Drosophila brain sLNv clock neurons release the neuropeptide PDF to control circadian rhythms. Strikingly, PDF content in sLNv terminals is rhythmic with a peak in the morning. Peak content drops because of activity-dependent release from dense-core vesicles (DCVs), but the mechanism for the daily increase in presynaptic PDF in the hours prior to release is unknown. Although transport from the soma was proposed to drive the daily increase in presynaptic PDF, live imaging in sLNv neurons shows that anterograde axonal DCV transport is constant throughout the day. Instead, capture of circulating DCVs, indicated by decreased retrograde axonal transport, rhythmically boosts presynaptic neuropeptide content. Genetic manipulations demonstrate that the late night increase in capture requires electrical activity but is independent of daily morphological changes. These results suggest that each day, during the hours of ongoing electrical activity, a toggle switches from inducing vesicle capture to triggering exocytosis, thereby maximizing daily rhythmic bursts of synaptic neuropeptide release by clock neurons. | Sanfilippo, P., Kim, A. J., Bhukel, A., Yoo, J., Mirshahidi, P. S., Pandey, V., Bevir, H., Yuen, A., Mirshahidi, P. S., Guo, P., Li, H. S., Wohlschlegel, J. A., Aso, Y., Zipursky, S. L. (2023). Mapping of multiple neurotransmitter receptor subtypes and distinct protein complexes to the connectome. bioRxiv, PubMed ID: 37873314
Summary: Neurons express different combinations of neurotransmitter receptor (NR) subunits and receive inputs from multiple neuron types expressing different neurotransmitters. Localizing NR subunits to specific synaptic inputs has been challenging. This study used epitope tagged endogenous NR subunits, expansion light-sheet microscopy, and EM connectomics to molecularly characterize synapses in Drosophila. In directionally selective motion sensitive neurons, different multiple NRs elaborated a highly stereotyped molecular topography with NR localized to specific domains receiving cell-type specific inputs. Developmental studies suggested that NRs or complexes of them with other membrane proteins determines patterns of synaptic inputs. In support of this model, a transmembrane protein associated selectively with a subset of spatially restricted synapses was identified, and through genetic analysis its requirement for synapse formation was demonstrate. It is proposed that mechanisms which regulate the precise spatial distribution of NRs provide a molecular cartography specifying the patterns of synaptic connections onto dendrites. |
Thursday, June 20th - Behavior |
| Puppato, S., Fiorenza, G., Carraretto, D., Gomulski, L. M., Gasperi, G., Caceres, C., Grassi, A., Mancini, M. V., De Cristofaro, A., Ioriatti, C., Guilhot, R., Malacrida, A. R. (2023). High promiscuity among females of the invasive pest species Drosophila suzukii. Mol Ecol, 32(22):6018-6026 PubMed ID: 37804145
Summary: Drosophila suzukii, the spotted-wing drosophila, is a highly invasive fruit fly that spread from Southern Asia across most regions of Asia and, in the last 15 years, has invaded Europe and the Americas. It is an economically important pest of small fruits such as berries and stone fruits. Drosophila suzukii speciated by adapting to cooler, mountainous, and forest environments. In temperate regions, it evolved seasonal polyphenism traits which enhanced its survival during stressful winter population bottlenecks. Consequently, in these temperate regions, the populations undergo seasonal reproductive dynamics. Despite its economic importance, no data are available on the behavioural reproductive strategies of this fly. The presence of polyandry, for example, has not been determined despite the important role it might play in the reproductive dynamics of populations. This study explored the presence of polyandry in an established population in Trentino, a region in northern Italy. In this area, D. suzukii overcomes the winter bottleneck and undergoes a seasonal reproductive fluctuation. A high remating frequency was observed in females during the late spring demographic explosion that led to the abundant summer population. The presence of a high degree of polyandry and shared paternity associated with the post-winter population increase raises the question of the possible evolutionary adaptive role of this reproductive behaviour in D. suzukii. | Mathejczyk, T. F., Babo E, J., Schonlein, E., Grinda, N. V., Greiner, A., Okroznik, N., Belusic, G., Wernet, M. F. (2023). Behavioral responses of free-flying Drosophila melanogaster to shiny, reflecting surfaces. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 209(6):929-941 PubMed ID: 37796303
Summary: Active locomotion plays an important role in the life of many animals, permitting them to explore the environment, find vital resources, and escape predators. Most insect species rely on a combination of visual cues such as celestial bodies, landmarks, or linearly polarized light to navigate or orient themselves in their surroundings. In nature, linearly polarized light can arise either from atmospheric scattering or from reflections off shiny non-metallic surfaces like water. Multiple reports have described different behavioral responses of various insects to such shiny surfaces. The goal of this study was to test whether free-flying Drosophila melanogaster, a molecular genetic model organism and behavioral generalist, also manifests specific behavioral responses when confronted with such polarized reflections. Fruit flies were placed in a custom-built arena with controlled environmental parameters (temperature, humidity, and light intensity). Flight detections and landings were quantified for three different stimuli: a diffusely reflecting matt plate, a small patch of shiny acetate film, and real water. Hydrated and dehydrated fly populations were compared, since the state of hydration may change the motivation of flies to seek or avoid water. This analysis reveals for the first time that flying fruit flies indeed use vision to avoid flying over shiny surfaces. |
| Berne, A., Zhang, T., Shomar, J., Ferrer, A. J., Valdes, A., Ohyama, T., Klein, M. (2023). Mechanical vibration patterns elicit behavioral transitions and habituation in crawling Drosophila larvae. Elife, 12 PubMed ID: 37855833
Summary: How animals respond to repeatedly applied stimuli, and how animals respond to mechanical stimuli in particular, are important questions in behavioral neuroscience. Adaptation to repeated mechanical agitation was studied using the Drosophila larva. Vertical vibration stimuli elicit a discrete set of responses in crawling larvae: continuation, pause, turn, and reversal. Through high-throughput larva tracking, this study characterize how the likelihood of each response depends on vibration intensity and on the timing of repeated vibration pulses. By examining transitions between behavioral states at the population and individual levels, how the animals habituate to the stimulus patterns was studied. Time constants associated with desensitization to prolonged vibration, with re-sensitization during removal of a stimulus, and additional layers of habituation were identified that operate in the overall response. Known memory-deficient mutants exhibit distinct behavior profiles and habituation time constants. An analogous simple electrical circuit suggests possible neural and molecular processes behind adaptive behavior. | Chowdhury, B., Abhilash, L., Ortega, A., Liu, S., Shafer, O. (2023). Homeostatic control of deep sleep and molecular correlates of sleep pressure in Drosophila. Elife, 12 PubMed ID: 37906092
Summary: Homeostatic control of sleep is typically addressed through mechanical stimulation-induced forced wakefulness and the measurement of subsequent increases in sleep. A major confound attends this approach: biological responses to deprivation may reflect a direct response to the mechanical insult rather than to the loss of sleep. Similar confounds accompany all forms of sleep deprivation and represent a major challenge to the field. This study describes a new paradigm for sleep deprivation in Drosophila that fully accounts for sleep-independent effects. The results reveal that deep sleep states are the primary target of homeostatic control and establish the presence of multi-cycle sleep rebound following deprivation. Furthermore, this study established that specific deprivation of deep sleep states results in state-specific homeostatic rebound. Finally, by accounting for the molecular effects of mechanical stimulation during deprivation experiments, it was shown that serotonin levels track sleep pressure in the fly's central brain. The results illustrate the critical need to control for sleep-independent effects of deprivation when examining the molecular correlates of sleep pressure and call for a critical reassessment of work that has not accounted for such non-specific effects. |
| Maruko, A., Iijima, K. M., Ando, K. (2023). Dissecting the daily feeding pattern: Peripheral CLOCK/CYCLE generate the feeding/fasting episodes and neuronal molecular clocks synchronize them. iScience, 26(11):108164 PubMed ID: 37915609
Summary: A 24-h rhythm of feeding behavior, or synchronized feeding/fasting episodes during the day, is crucial for survival. Internal clocks and light input regulate rhythmic behaviors, but how they generate feeding rhythms is not fully understood. This study aimed to dissect the molecular pathways that generate daily feeding patterns. By measuring the semidiurnal amount of food ingested by single flies, this study demonstrated that the generation of feeding rhythms under light:dark conditions requires quasimodo (qsm) but not molecular clocks. Under constant darkness, rhythmic feeding patterns consist of two components: CLOCK (CLK) in digestive/metabolic tissues generating feeding/fasting episodes, and the molecular clock in neurons synchronizing them to subjective daytime. Although CLK is a part of the molecular clock, the generation of feeding/fasting episodes by CLK in metabolic tissues was independent of molecular clock machinery. These results revealed novel functions of qsm and CLK in feeding rhythms in Drosophila. | Keles, M. F., Sapci, A., Brody, C., Palmer, I., Le, C., Tastan, O., Keles, S., Wu, M. N. (2023). Deep Phenotyping of Sleep in Drosophila. bioRxiv, PubMed ID: 37961473
Summary: Sleep is an evolutionarily conserved behavior, whose function is unknown. This paper presents a method for deep phenotyping of sleep in Drosophila, consisting of a high-resolution video imaging system, coupled with closed-loop laser perturbation to measure arousal threshold. To quantify sleep-associated microbehaviors, a deep-learning network was trained to annotate body parts in freely moving flies, and a semi-supervised computational pipeline was developed to classify behaviors. Quiescent flies exhibit a rich repertoire of microbehaviors, including proboscis pumping (PP) and haltere switches, which vary dynamically across the night. Using this system, the effects were characterized of optogenetically activating two putative sleep circuits. These data reveal that activating dFB neurons produces micromovements, inconsistent with sleep, while activating R5 neurons triggers PP followed by behavioral quiescence. These findings suggest that sleep in Drosophila is polyphasic with different stages and set the stage for a rigorous analysis of sleep and other behaviors in this species. |
Wednesday, June 19th - Evolution |
| Schrider, D. R. (2023). Allelic gene conversion softens selective sweeps. bioRxiv, PubMed ID: 38106127
Summary: The prominence of positive selection, in which beneficial mutations are favored by natural selection and rapidly increase in frequency, is a subject of intense debate. Positive selection can result in selective sweeps, in which the haplotype(s) bearing the adaptive allele "sweep" through the population, thereby removing much of the genetic diversity from the region surrounding the target of selection. Two models of selective sweeps have been proposed: classical sweeps, or "hard sweeps", in which a single copy of the adaptive allele sweeps to fixation, and "soft sweeps", in which multiple distinct copies of the adaptive allele leave descendants after the sweep. Soft sweeps can be the outcome of recurrent mutation to the adaptive allele, or the presence of standing genetic variation consisting of multiple copies of the adaptive allele prior to the onset of selection. Importantly, soft sweeps will be common when populations can rapidly adapt to novel selective pressures, either because of a high mutation rate or because adaptive alleles are already present. The prevalence of soft sweeps is especially controversial, and it has been noted that selection on standing variation or recurrent mutations may not always produce soft sweeps. This study shows that the inverse is true: selection on single-origin de novo mutations may often result in an outcome that is indistinguishable from a soft sweep. This is made possible by allelic gene conversion, which "softens" hard sweeps by copying the adaptive allele onto multiple genetic backgrounds, a process referred to as a "pseudo-soft" sweep. We carried out a simulation study examining the impact of gene conversion on sweeps from a single de novo variant in models of human, Drosophila, and Arabidopsis populations. The fraction of simulations in which gene conversion had produced multiple haplotypes with the adaptive allele upon fixation was appreciable. Indeed, under realistic demographic histories and gene conversion rates, even if selection always acts on a single-origin mutation, sweeps involving multiple haplotypes are more likely than hard sweeps in large populations, especially when selection is not extremely strong. Thus, even when the mutation rate is low or there is no standing variation, hard sweeps are expected to be the exception rather than the rule in large populations. These results also imply that the presence of signatures of soft sweeps does not necessarily mean that adaptation has been especially rapid or is not mutation limited. | Stromberg, K. A., Spain, T., Tomlin, S. A., Powell, J., Amarillo, K. D., Schroeder, C. M. (2023). Evolutionary diversification reveals distinct somatic versus germline cytoskeletal functions of the Arp2 branched actin nucleator protein. Curr Biol, 33(24):5326-5339.e5327 PubMed ID: 37977138
Summary: Branched actin networks are critical in many cellular processes, including cell motility and division. Arp2, a protein within the seven-membered Arp2/3 complex, is responsible for generating branched actin. Given its essential roles, Arp2 evolves under stringent sequence conservation throughout eukaryotic evolution. Recurrent evolutionary diversification of Arp2 in Drosophila was unexpectedly discovered, 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 somatic tissue, suggesting they have preserved the ability to polymerize branched actin even in a non-native species. However, it was found that Arp2D- and Arp2D2-expressing males display defects throughout sperm development, with Arp2D resulting in more pronounced deficiencies and subfertility, suggesting the Arp2 paralogs are cross-species incompatible in the testis. Focus was placed on Arp2D, and two highly diverged structural regions-the D-loop and C terminus were pinpointed, and it was found that they contribute to germline defects in D. melanogaster sperm development. However, while the Arp2D C terminus is suboptimal in the D. melanogaster testis, it is essential for Arp2D somatic function. Testis cytology of the paralogs' native species revealed striking differences in germline actin structures, indicating unique cytoskeletal requirements. These findings suggest canonical Arp2 function differs between somatic versus germline contexts, and Arp2 paralogs may have recurrently evolved for species-specialized actin branching in the testis. |
| Bitter, M. C., Berardi, S., Oken, H., Huynh, A., Schmidt, P., Petrov, D. A. (2023). Continuously fluctuating selection reveals extreme granularity and parallelism of adaptive tracking. bioRxiv, PubMed ID: 37904939
Summary: Temporally fluctuating environmental conditions are a ubiquitous feature of natural habitats. Yet, how finely natural populations adaptively track fluctuating selection pressures via shifts in standing genetic variation is unknown. This study generated high-frequency, genome-wide allele frequency data from a genetically diverse population of Drosophila melanogaster in extensively replicated field mesocosms from late June to mid-December, a period of ~12 generations. Adaptation throughout the fundamental ecological phases of population expansion, peak density, and collapse was underpinned by extremely rapid, parallel changes in genomic variation across replicates. Yet, the dominant direction of selection fluctuated repeatedly, even within each of these ecological phases. Comparing patterns of allele frequency change to an independent dataset procured from the same experimental system demonstrated that the targets of selection are predictable across years. In concert, the results reveal fitness-relevance of standing variation that is likely to be masked by inference approaches based on static population sampling, or insufficiently resolved time-series data. Such fine-scaled temporally fluctuating selection may be an important force maintaining functional genetic variation in natural populations and an important stochastic force affecting levels of standing genetic variation genome-wide. | Collet, J. M., Nidelet, S., Fellous, S. (2023). Genetic independence between traits separated by metamorphosis is widespread but varies with biological function. Proceedings Biological sciences, 290(2010):20231784 PubMed ID: 37935368
Summary: Why is metamorphosis so pervasive? Does it facilitate the independent (micro)evolution of quantitative traits in distinct life stages, similarly to how it enables some limbs and organs to develop at specific life stages? This hypothesis was tested by measuring the expression of 6400 genes in 41 Drosophila melanogaster inbred lines at larval and adult stages. Only 30% of the genes showed significant genetic correlations between larval and adult expression. By contrast, 46% of the traits showed some level of genetic independence between stages. Gene ontology terms enrichment revealed that across stages correlated traits were often involved in proteins synthesis, insecticide resistance and innate immunity, while a vast number of genes expression traits associated with energy metabolism were independent between life stages. These results were compared to a similar case: genetic constraints between males and females in gonochoric species (i.e. sexual antagonism). It was expected that selection for the separation between males and females to be higher than between juvenile and adult functions, as gonochorism is a more common strategy in the animal kingdom than metamorphosis. Surprisingly, it was found that inter-stage constraints were lower than inter-sexual genetic constraints. Overall, these results show that metamorphosis enables a large part of the transcriptome to evolve independently at different life stages. |
| Li, M., Chen, D. S., Junker, I. P., Szorenyi, F., Chen, G. H., Berger, A. J., Comeault, A. A., Matute, D. R., Ding, Y. (2023). Ancestral neural circuits potentiate the origin of a female sexual behavior. bioRxiv, PubMed ID: 38106147
Summary: Courtship interactions are remarkably diverse in form and complexity among species. How neural circuits evolve to encode new behaviors that are functionally integrated into these dynamic social interactions is unknown. This study reports a recently originated female sexual behavior in the island endemic Drosophila species D. santomea, where females signal receptivity to male courtship songs by spreading their wings, which in turn promotes prolonged songs in courting males. Copulation success depends on this female signal and correlates with males' ability to adjust his singing in such a social feedback loop. Functional comparison of sexual circuitry across species suggests that a pair of descending neurons, which integrates male song stimuli and female internal state to control a conserved female abdominal behavior, drives wing spreading in D. santomea. This co-option occurred through the refinement of a pre-existing, plastic circuit that can be optogenetically activated in an outgroup species. Combined, these results show that the ancestral potential of a socially-tuned key circuit node to engage the wing motor program facilitates the expression of a new female behavior in appropriate sensory and motivational contexts. More broadly, this work provides insights into the evolution of social behaviors, particularly female behaviors, and the underlying neural mechanisms. | Clark, J. M., Gibbs, A. G. (2023). Starvation selection reduces and delays larval ecdysone production and signaling. The Journal of experimental biology, 226(18) PubMed ID: 37671530
Summary: Previous studies have shown that selection for starvation resistance in Drosophila melanogaster results in delayed eclosion and increased adult fat stores. It is assumed that these traits are caused by the starvation selection pressure, but its mechanism is unknown. This study found that starvation-selected (SS) population stores more fat during larval development and has extended larval development and pupal development time. Developmental checkpoints in the third instar associated with ecdysteroid hormone pulses are increasingly delayed. The delay in the late larval period seen in the SS population is indicative of reduced and delayed ecdysone signaling. An enzyme immunoassay for ecdysteroids (with greatest affinity to the metabolically active 20-hydroxyecdysone and the α-ecdysone precursor) confirmed that the SS population had reduced and delayed hormone production compared with that of fed control (FC) flies. Feeding third instar larvae on food supplemented with α-ecdysone partially rescued the developmental delay and reduced subsequent adult starvation resistance. This work suggests that starvation selection causes reduced and delayed production of ecdysteroids in the larval stage and affects the developmental delay phenotype that contributes to subsequent adult fat storage and starvation resistance. |
Tuesday, July 18th - Chromatin |
| Khodursky, S., Zheng, E. B., Svetec, N., Durkin, S. M., Benjamin, S., Gadau, A., Wu, X., Zhao, L. (2023). The evolution and mutational robustness of chromatin accessibility in Drosophila. Genome Biol, 24(1):232 PubMed ID: 37845780
Summary: The evolution of genomic regulatory regions plays a critical role in shaping the diversity of life. While this process is primarily sequence-dependent, the enormous complexity of biological systems complicates the understanding of the factors underlying regulation and its evolution. This study applied deep neural networks as a tool to investigate the sequence determinants underlying chromatin accessibility in different species and tissues of Drosophila. Hybrid convolution-attention neural networks were trained to accurately predict ATAC-seq peaks using only local DNA sequences as input. These models generalize well across substantially evolutionarily diverged species of insects, implying that the sequence determinants of accessibility are highly conserved. Using this model to examine species-specific gains in accessibility, evidence was found suggesting that these regions may be ancestrally poised for evolution. Using in silico mutagenesis, it was shown that accessibility can be accurately predicted from short subsequences in each example. However, in silico knock-out of these sequences does not qualitatively impair classification, implying that accessibility is mutationally robust. Subsequently, it was shown that accessibility is predicted to be robust to large-scale random mutation even in the absence of selection. Conversely, simulations under strong selection demonstrate that accessibility can be extremely malleable despite its robustness. Finally, motifs predictive of accessibility were identified, recovering both novel and previously known motifs. These results demonstrate the conservation of the sequence determinants of accessibility and the general robustness of chromatin accessibility, as well as the power of deep neural networks to explore fundamental questions in regulatory genomics and evolution. | Anderson, J. T., Henikoff, S., Ahmad, K. (2023). Chromosome-specific maturation of the epigenome in the Drosophila male germline. Elife, 12 PubMed ID: 38032818
Summary: Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome, and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, active chromatin features in the testes were profiled from wildtype and meiotic arrest mutants, and this was integrated with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, widespread patterns associated with regulation of the sex chromosomes were detailed. The results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome. |
| Rogers, M. F., Marshall, O. J., Secombe, J. (2023). KDM5-mediated activation of genes required for mitochondrial biology is necessary for viability in Drosophila. Development, 150(21) PubMed ID: 37800333
Summary: Histone-modifying proteins play important roles in the precise regulation of the transcriptional programs that coordinate development. KDM5 family proteins interact with chromatin through demethylation of H3K4me3 as well as demethylase-independent mechanisms that remain less understood. To gain fundamental insights into the transcriptional activities of KDM5 proteins, this study examined the essential roles of the single Drosophila Kdm5 ortholog during development. KDM5 performs crucial functions in the larval neuroendocrine prothoracic gland, providing a model to study its role in regulating key gene expression programs. Integrating genome binding and transcriptomic data, this study identified that KDM5 regulates the expression of genes required for the function and maintenance of mitochondria, and it was found that loss of KDM5 causes morphological changes to mitochondria. This is key to the developmental functions of KDM5, as expression of the mitochondrial biogenesis transcription factor Ets97D, homolog of GABPα, is able to suppress the altered mitochondrial morphology as well as the lethality of Kdm5 null animals. Together, these data establish KDM5-mediated cellular functions that are important for normal development and could contribute to KDM5-linked disorders when dysregulated. | Melnikova, L., Molodina, V., Babosha, V., Kostyuchenko, M., Georgiev, P., Golovnin, A. (2023). The MADF-BESS Protein CP60 Is Recruited to Insulators via CP190 and Has Redundant Functions in Drosophila. Int J Mol Sci, 24(19) PubMed ID: 37834476
Summary: Drosophila CP190 and CP60 are transcription factors that are associated with centrosomes during mitosis. CP190 is an essential transcription factor and preferentially binds to housekeeping gene promoters and insulators through interactions with architectural proteins, including Su(Hw) and dCTCF. CP60 belongs to a family of transcription factors that contain the N-terminal MADF domain and the C-terminal BESS domain, which is characterized by the ability to homodimerize. This study shows that the conserved CP60 region adjacent to MADF is responsible for interacting with CP190. In contrast to the well-characterized MADF-BESS transcriptional activator Adf-1, CP60 is recruited to most chromatin sites through its interaction with CP190, and the MADF domain is likely involved in protein-protein interactions but not in DNA binding. The deletion of the Map60 gene showed that CP60 is not an essential protein, despite the strong and ubiquitous expression of CP60 at all stages of Drosophila development. Although CP60 is a stable component of the Su(Hw) insulator complex, the inactivation of CP60 does not affect the enhancer-blocking activity of the Su(Hw)-dependent gypsy insulator. Overall, these results indicate that CP60 has an important but redundant function in transcriptional regulation as a partner of the CP190 protein. |
| Dubruille, R., Herbette, M., Revel, M., Horard, B., Chang, C. H., Loppin, B. (2023). Histone removal in sperm protects paternal chromosomes from premature division at fertilization. Science, 382(6671):725-731 PubMed ID: 37943933
Summary: The global replacement of histones with protamines in sperm chromatin is widespread in animals, including insects, but its actual function remains enigmatic. In the Drosophila paternal effect mutant paternal loss (pal), sperm chromatin retains germline histones H3 and H4 genome wide without impairing sperm viability. However, after fertilization, pal sperm chromosomes are targeted by the egg chromosomal passenger complex and engage into a catastrophic premature division in synchrony with female meiosis II. pal encodes a rapidly evolving transition protein specifically required for the eviction of (H3-H4)(2) tetramers from spermatid DNA after the removal of H2A-H2B dimers. This study thus reveals an unsuspected role of histone eviction from insect sperm chromatin: safeguarding the integrity of the male pronucleus during female meiosis. | Lu, F., Park, B. J., Fujiwara, R., Wilusz, J. E., Gilmour, D. S., Lehmann, R., Lionnet, T. (2024). Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription. bioRxiv, PubMed ID: 37873455
Summary: Numerous components of the transcription machinery, including RNA polymerase II (Pol II), accumulate in regions of high local concentration known as clusters, which are thought to facilitate transcription. Using the histone locus of Drosophila nurse cells as a model, this study found that Pol II forms long-lived, transcriptionally poised clusters distinct from liquid droplets, which contain unbound and paused Pol II. Depletion of the Integrator complex endonuclease module, but not its phosphatase module or Pol II pausing factors disperses these Pol II clusters. Consequently, histone transcription fails to reach peak levels during S-phase and aberrantly continues throughout the cell cycle. It is proposed that Pol II clustering is a regulatory step occurring near promoters that limits rapid gene activation to defined times. Using the Drosophila histone locus as a model, this study has shown that clustered RNA polymerase II is poised for synchronous activation. |
Monday, June 16th - Genes, Proteins and Enzymes - Evolution, Structure, and Function |
| Summers, J. A., Yarbrough, M., Liu, M., McDonald, W. H., Hudson, B. G., Pastor-Pareja, J. C., Boudko, S. P. (2023). Collagen IV of basement membranes: IV. Adaptive mechanism of collagen IV scaffold assembly in Drosophila. J Biol Chem, 299(12):105394 PubMed ID: 37890775
Summary: Collagen IV is an essential structural protein in all metazoans. It provides a scaffold for the assembly of basement membranes, a specialized form of extracellular matrix, which anchors and signals cells and provides microscale tensile strength. Defective scaffolds cause basement membrane destabilization and tissue dysfunction. Scaffolds are composed of α-chains that coassemble into triple-helical protomers of distinct chain compositions, which in turn oligomerize into supramolecular scaffolds. Chloride ions mediate the oligomerization via NC1 trimeric domains, forming an NC1 hexamer at the protomer-protomer interface. The chloride concentration-'chloride pressure'-on the outside of cells is a primordial innovation that drives the assembly and dynamic stabilization of collagen IV scaffolds. However, a Cl-independent mechanism is operative in Ctenophora, Ecdysozoa, and Rotifera, which suggests evolutionary adaptations to environmental or tissue conditions. An understanding of these exceptions, such as the example of Drosophila, could shed light on the fundamentals of how NC1 trimers direct the oligomerization of protomers into scaffolds. This study investigated the NC1 assembly of Drosophila. The crystal structure of the NC1 hexamer was solved, the chain composition of protomers was determined, and Drosophila was found to adapt an evolutionarily unique mechanism of scaffold assembly that requires divalent cations. By studying the Drosophila case this study highlighted the mechanistic role of chloride pressure for maintaining functionality of the NC1 domain in humans. Moreover, it was discovered that the NC1 trimers encode information for homing protomers to distant tissue locations, providing clues for the development of protein replacement therapy for collagen IV genetic diseases. | Holst, J. D., Murphy, L. G., Gorman, M. J., Ragan, E. J. (2023). Comparison of insect and human cytochrome b561 proteins: Insights into candidate ferric reductases in insects. PLoS One, 18(12):e0291564 PubMed ID: 38039324
Summary: Cytochrome b561 (cytb561) proteins comprise a family of transmembrane oxidoreductases that transfer single electrons across a membrane. Most eukaryotic species, including insects, possess multiple cytb561 homologs. To learn more about this protein family in insects, a bioinformatics-based investigation of cytb561 family members was carried out from nine species representing eight insect orders. A phylogenetic analysis was performed to classify insect cytb561 ortholog groups. Then sequence analyses was conducted and protein models were analyzed to predict structural elements that may impact the biological functions and localization of these proteins, with a focus on possible ferric reductase activity. The study revealed three ortholog groups, designated CG1275, Nemy, and CG8399, and a fourth group of less-conserved genes. CG1275 and Nemy proteins are similar to a human ferric reductase, duodenal cytochrome b561 (Dcytb), and have many conserved amino acid residues that function in substrate binding in Dcytb. Notably, CG1275 and Nemy proteins contain a conserved histidine and other residues that play a role in ferric ion reduction by Dcytb. Nemy proteins were distinguished by a novel cysteine-rich cytoplasmic loop sequence. CG8399 orthologs are similar to a putative ferric reductase in humans, stromal cell-derived receptor 2. Like other members of the CYBDOM class of cytb561 proteins, these proteins contain reeler, DOMON, and cytb561 domains. Drosophila melanogaster CG8399 is the only insect cytb561 with known ferric reductase activity. Investigation of the DOMON domain in CG8399 proteins revealed a probable heme-binding site and a possible site for ferric reduction. The fourth group includes a subgroup of proteins with a conserved "KXXXXKXH" non-cytoplasmic loop motif that may be a substrate binding site and is present in a potential ferric reductase, human tumor suppressor cytochrome b561. This study provides a foundation for future investigations of the biological functions of cytb561 genes in insects. |
| Marshall, A. C., Cummins, J., Kobelke, S., Zhu, T., Widagdo, J., Anggono, V., Hyman, A., Fox, A. H., Bond, C. S., Lee, M. (2023). Different Low-complexity Regions of SFPQ Play Distinct Roles in the Formation of Biomolecular Condensates. J Mol Biol, 435(24):168364 PubMed ID: 37952770
Summary: Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism underlying the complex spatiotemporal organisation of molecules within the cell. Long disordered regions of low sequence complexity (LCRs) are a common feature of proteins that form liquid-like microscopic biomolecular condensates. In particular, RNA-binding proteins with prion-like regions have emerged as key drivers of liquid demixing to form condensates such as nucleoli, paraspeckles and stress granules. Splicing factor proline- and glutamine-rich (SFPQ) is an RNA- and DNA-binding protein essential for DNA repair and paraspeckle formation. SFPQ contains two LCRs of different length and composition. This study shows that the shorter C-terminal LCR of SFPQ is the main region responsible for the condensation of SFPQ in vitro and in the cell nucleus. In contrast, this study found that the longer N-terminal prion-like LCR of SFPQ attenuates condensation of the full-length protein, suggesting a more regulatory role in preventing aberrant condensate formation in the cell. The compositions of these respective LCRs are discussed with reference to current literature. Thesee data add nuance to the emerging understanding of biomolecular condensation, by providing the first example of a common multifunctional nucleic acid-binding protein with an extensive prion-like region that serves to regulate rather than drive condensate formation. | Vedelek, V., Vedelek, B., Lorincz, P., Juhasz, G., Sinka, R. (2023). A comparative analysis of fruit fly and human glutamate dehydrogenases in Drosophila melanogaster sperm development. Frontiers in cell and developmental biology, 11:1281487 PubMed ID: 38020911
Summary: Glutamate dehydrogenases are enzymes that take part in both amino acid and energy metabolism. Their role is clear in many biological processes, from neuronal function to cancer development. The putative testis-specific Drosophila glutamate dehydrogenase, Bb8, is required for male fertility and the development of mitochondrialGdh and the testis-specific Bb8. Both human GLUD1 and GLUD2 can rescue the bb8 (ms) mutant phenotype, with superior performance by GLUD2. The role was tested of three conserved amino acids observed in both Bb8 and GLUD2 in Gdh mutants, which showed their importance in the glutamate dehydrogenase function. The findings of this study indicate that Drosophila Bb8 and human GLUD2 could be novel examples of convergent molecular evolution. Furthermore, the importance of glutamate levels in mitochondrial homeostasis was tested during spermatogenesis by ectopic expression of the mitochondrial glutamate transporter Aralar1, which caused mitochondrial abnormalities in fly spermatids. The data presented in this study offer evidence supporting the significant involvement of glutamate metabolism in sperm development. |
| Bilska, B., Damulewicz, M., Abaquita, T. A. L., Pyza, E. (2023). Changes in heme oxygenase level during development affect the adult life of Drosophila melanogaster. Frontiers in cellular neuroscience, 17:1239101 PubMed ID: 37876913
Summary: Heme oxygenase (HO) has been shown to control various cellular processes in both mammals and Drosophila melanogaster. This study investigated how changes in HO levels in neurons and glial cells during development affect adult flies, by using the TARGET Drosophila system to manipulate the expression of the ho gene. The obtained data showed differences in adult survival, maximum lifespan, climbing, locomotor activity, and sleep, which depended on the level of HO (after ho up-regulation or downregulation), the timing of expression (chronic or at specific developmental stages), cell types (neurons or glia), sex (males or females), and age of flies. In addition to ho, the effects of changing the mRNA level of the Drosophila CNC factor gene (NRF2 homolog in mammals and master regulator of HO), were also examined to compare with those observed after changing ho expression. HO levels in neurons and glia must be maintained at an appropriate physiological level during development to ensure the well-being of adults. This study also found that the downregulation of ho in either neurons or glia in the brain is compensated by ho expressed in the retina. | Krishnan, H., Ahmed, S., Hubbard, S. R., Miller, W. T. (2024). Biochemical characterization of the Drosophila insulin receptor kinase and longevity-associated mutants. Faseb j, 38(1):e23355 PubMed ID: 38071609
Summary: Drosophila melanogaster (fruit fly) insulin receptor (D-IR) is highly homologous to the human counterpart. Like the human pathway, D-IR responds to numerous insulin-like peptides to activate cellular signals that regulate growth, development, and lipid metabolism in fruit flies. Allelic mutations in the D-IR kinase domain elevate life expectancy in fruit flies. This study developed a robust heterologous expression system to express and purify wild-type and longevity-associated mutant D-IR kinase domains to investigate enzyme kinetics and substrate specificities. D-IR exhibits remarkable similarities to the human insulin receptor kinase domain but diverges in substrate preferences. Longevity-associated mutations reduce D-IR catalytic activity. Deletion of the unique kinase insert domain portion or mutations proximal to activating tyrosines do not influence kinase activity, suggesting their potential role in substrate recruitment and downstream signaling. Through biochemical investigations, this study enhances comprehension of D-IR's role in Drosophila physiology, complementing genetic studies and expanding knowledge of the catalytic functions of this conserved signaling pathway. |
Friday, June 13th - Disease Models |
| Lai, Y., Reina-Gonzalez, P., Maor, G., Miller, G. W., Sarkar, S. (2023). Biotin rescues manganese-induced Parkinson's disease phenotypes and neurotoxicity. bioRxiv, PubMed ID: 38045419
Summary: Occupational exposure to manganese (Mn) induces manganism and has been widely linked as a contributing environmental factor to Parkinson's disease (PD), featuring dramatic signature overlaps between the two in motor symptoms and clinical hallmarks. However, the molecular mechanism underlying such link remains elusive, and for combating PD, effective mechanism-based therapies are lacking. This study developed an adult Drosophila model of Mn toxicity to recapitulate key parkinsonian features, spanning behavioral deficits, neuronal loss, and dysfunctions in lysosome and mitochondria. Global metabolomics was performed on flies at an early stage of toxicity, and metabolism of the B vitamin, biotin (vitamin B (7)) was identified as a master pathway underpinning Mn toxicity with systemic, body-brain increases in Mn-treated groups compared to the controls. Using Btnd (RNAi) mutant flies, we show that biotin depletion exacerbates Mn-induced neurotoxicity, parkinsonism, and mitochondrial dysfunction; while in Mn-exposed wild-type flies, biotin feeding dramatically ameliorates these pathophenotypes. We further show in human induced stem cells (iPSCs)- differentiated midbrain dopaminergic neurons that the supplemented biotin protects against Mn-induced neuronal loss, cytotoxicity, and mitochondrial dysregulation. Finally, human data profiling biotin-related proteins show for PD cases elevated circulating levels of biotin transporters but not of metabolic enzymes compared to healthy controls, suggesting humoral biotin transport as a key event involved in PD. Taken together, these findings identified compensatory biotin pathway as a convergent, systemic driver of Mn toxicity and parkinsonian pathology, providing new basis for devising effective countermeasures against manganism and PD. | Atilano, M. L., Hull, A., Romila, C. A., Adams, M. L., Wildfire, J., Ureña, E., Dyson, M., Ivan-Castillo-Quan, J., Partridge, L., Kinghorn, K. J. (2023). Autophagic dysfunction and gut microbiota dysbiosis cause chronic immune activation in a Drosophila model of Gaucher disease. PLoS Genet, 19(12):e1011063 PubMed ID: 38127816
Summary: Mutations in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD) and are the greatest known genetic risk factors for Parkinson's disease (PD). Communication between the gut and brain and immune dysregulation are increasingly being implicated in neurodegenerative disorders such as PD. This study shows that flies lacking the Gba1b gene, the main fly orthologue of GBA1, display widespread NF-kB signalling activation, including gut inflammation, and brain glial activation. Intestinal autophagic defects, gut dysfunction, and microbiome dysbiosis were also demonstrated. Remarkably, modulating the microbiome of Gba1b knockout flies, by raising them under germ-free conditions, partially ameliorates lifespan, locomotor and immune phenotypes. Moreover, modulation of the immune deficiency (IMD) pathway is detrimental to the survival of Gba1 deficient flies. It was also revealed that direct stimulation of autophagy by rapamycin treatment achieves similar benefits to germ-free conditions independent of gut bacterial load. Consistent with this, pharmacologically blocking autophagosomal-lysosomal fusion, mimicking the autophagy defects of Gba1 depleted cells, was shown to be sufficient to stimulate intestinal immune activation. Overall, these data elucidate a mechanism whereby an altered microbiome, coupled with defects in autophagy, drive chronic activation of NF-kB signaling in a Gba1 loss-of-function model. It also highlights that elimination of the microbiota or stimulation of autophagy to remove immune mediators, rather than prolonged immunosuppression, may represent effective therapeutic avenues for GBA1-associated disorders. |
| Chvilicek, M. M., Seguin, A., Lathen, D. R., Titos, I., Cummins-Beebe, P. N., Pabon, M. A., Miscevic, M., Nickel, E. A., Merrill, C. B., Rodan, A. R., Rothenfluh, A. (2023). Large genetic analysis of alcohol resistance and tolerance reveals an inverse correlation and suggests 'true' tolerance mutants. bioRxiv, PubMed ID: 37873285
Summary: Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce the same behavioral effects. Tolerance is historically not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and between labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. This study has analyzed a large amount of data to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes. Additionally, it was shown that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on this analysis, a method is providing for using a linear regression equation to assess the residuals of potential tolerance mutants. These residuals provide predictive insight into the likelihood of a mutant being a 'true' tolerance mutant, and a framework is offered for understanding the relationship between initial resistance and tolerance. | Liang, Y., Pan, C., Yin, T., Wang, L., Gao, X., Wang, E., Quang, H., Huang, D., Tan, L., Xiang, K., Wang, Y., Alexander, P. B., Li, Q. J., Yao, T. P., Zhang, Z., Wang, X. F. (2024). Branched-Chain Amino Acid Accumulation Fuels the Senescence-Associated Secretory Phenotype. Adv Sci (Weinh), 11(2):e2303489 PubMed ID: 37964763
Summary: The essential branched-chain amino acids (BCAAs) leucine, isoleucine, and valine play critical roles in protein synthesis and energy metabolism. Despite their widespread use as nutritional supplements, BCAAs' full effects on mammalian physiology remain uncertain due to the complexities of BCAA metabolic regulation. Here a novel mechanism linking intrinsic alterations in BCAA metabolism is identified to cellular senescence and the senescence-associated secretory phenotype (SASP), both of which contribute to organismal aging and inflammation-related diseases. Altered BCAA metabolism driving the SASP is mediated by robust activation of the BCAA transporters Solute Carrier Family 6 Members 14 and 15 as well as downregulation of the catabolic enzyme BCAA transaminase 1 during onset of cellular senescence, leading to highly elevated intracellular BCAA levels in senescent cells. This, in turn, activates the mammalian target of rapamycin complex 1 (mTORC1) to establish the full SASP program. Transgenic Drosophila models further indicate that orthologous BCAA regulators are involved in the induction of cellular senescence and age-related phenotypes in flies, suggesting evolutionary conservation of this metabolic pathway during aging. Finally, experimentally blocking BCAA accumulation attenuates the inflammatory response in a mouse senescence model, highlighting the therapeutic potential of modulating BCAA metabolism for the treatment of age-related and inflammatory diseases. |
| Yamaguchi, M., Huynh, M. A., Chiyonobu, T., Yoshida, H. (2023). Knockdown of Chronophage in the nervous system mimics features of neurodevelopmental disorders caused by BCL11A/B variants. Exp Cell Res, 433(2):113827 PubMed ID: 37926342
Summary: Neurodevelopmental disorders (NDD) are a group of disorders that include intellectual disability. Although several genes have been implicated in NDD, the molecular mechanisms underlying its pathogenesis remain unclear. Therefore, it is important to develop novel models to analyze the functions of NDD-causing genes in vivo. Recently, rare pathogenic variants of the B-cell lymphoma/leukemia11A/B (BCL11A/B) gene have been identified in several patients with NDD. Drosophila carries the Chronophage (Cph) gene, which has been predicted to be a homolog of BCL11A/B based on the conservation of the amino acid sequence. This study investigated whether nervous system-specific knockdown of Cph mimics NDD phenotypes in Drosophila. Nervous system-specific knockdown of Cph induced learning and locomotor defects in larvae and epilepsy-like behaviors in adults. The number of synaptic branches was also elevated in the larval neuromuscular junction without a corresponding increase in the number of boutons. Furthermore, the expression levels of putative target genes that are Drosophila homologs of the mammalian BCL11 target genes were decreased in Cph knockdown flies. These results suggest that Cph knockdown flies are a promising model for investigating the pathology of NDD-induced BCL11A/B dysfunction. | Sun, Y., Dai, H., Dai, X., Yin, J., Cui, Y., Liu, X., Gonzalez, G., Yuan, J., Tang, F., Wang, N., Perlegos, A. E., Bonini, N. M., Yang, X. W., Gu, W., Wang, Y. (2023). m(1)A in CAG repeat RNA binds to TDP-43 and induces neurodegeneration. Nature, 623(7987):580-587 PubMed ID: 37938769
Summary: Microsatellite repeat expansions within genes contribute to a number of neurological diseases such as ALS. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology. This study revealed that the adenosine in CAG repeat RNA can be methylated to N(1)-methyladenosine (m(1)A) by TRMT61A, and that m(1)A can be demethylated by ALKBH3. It was also observed that the m(1)A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m(1)A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m(1)A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, this study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m(1)A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion. |
Thursday, June 13th - RNAs and Transposons |
| Morillo, L., Paternina, T., Alasseur, Q., Genovesio, A., Schwartz, S., Le Hir, H. (2023). Comprhensive mapping of exon junction complex binding sites reveals universal EJC deposition in Drosophila. BMC Biol, 21(1):246 PubMed ID: 37936138
Summary: The exon junction complex (EJC) is involved in most steps of the mRNA life cycle, ranging from splicing to nonsense-mediated mRNA decay (NMD). It is assembled by the splicing machinery onto mRNA in a sequence-independent manner. A fundamental open question is whether the EJC is deposited onto all exon‒exon junctions or only on a subset of them. Several previous studies have made observations supportive of the latter, yet these have been limited by methodological constraints. This study sought to overcome these limitations via the integration of two different approaches for transcriptome-wide mapping of EJCs. The results revealed that nearly all, if not all, internal exons consistently harbor an EJC in Drosophila, demonstrating that EJC presence is an inherent consequence of the splicing reaction. Furthermore,this study underscores the limitations of eCLIP methods in fully elucidating the landscape of RBP (RNA-binding protein) binding sites. These findings highlight how highly specific (low false positive) methodologies can lead to erroneous interpretations due to partial sensitivity (high false negatives). This study contributes to understanding of EJC deposition and its association with pre-mRNA splicing. The universal presence of EJC on internal exons underscores its significance in ensuring proper mRNA processing. Additionally, these observations highlight the need to consider both specificity and sensitivity in RBP mapping methodologies. | Wang, M., Liang, A. M., Zhou, Z. Z., Pang, T. L., Fan, Y. J., Xu, Y. Z. (2023). Deletions of singular U1 snRNA gene significantly interfere with transcription and 3'-end mRNA formation. PLoS Genet, 19(11):e1011021 PubMed ID: 37917726
Summary: Small nuclear RNAs (snRNAs) are structural and functional cores of the spliceosome. In metazoan genomes, each snRNA has multiple copies/variants, up to hundreds in mammals. However, the expressions and functions of each copy/variant in one organism have not been systematically studied. Focusing on U1 snRNA genes, this study investigated all five copies in Drosophila melanogaster using two series of constructed strains. Analyses of transgenic flies that each have a U1 promoter-driven gfp revealed that U1:21D is the major and ubiquitously expressed copy, and the other four copies have specificities in developmental stages and tissues. Mutant strains that each have a precisely deleted copy of U1-gene exhibited various extents of defects in fly morphology or mobility, especially deletion of U1:82Eb. Interestingly, splicing was changed at limited levels in the deletion strains, while large amounts of differentially-expressed genes and alternative polyadenylation events were identified, showing preferences in the down-regulation of genes with 1-2 introns and selection of proximal sites for 3'-end polyadenylation. In vitro assays suggested that Drosophila U1 variants pulled down fewer SmD2 proteins compared to the canonical U1. This study demonstrates that all five U1-genes in Drosophila have physiological functions in development and play regulatory roles in transcription and 3'-end formation. |
| Ghosh, S., Chakraborti, S., Devi, D., Sahu, R., Mandal, S., Mandal, L. (2024). A conserved nutrient responsive axis mediates autophagic degradation of miRNA-mRNA hybrids in blood cell progenitors. Nucleic Acids Res, 52(1):385-403 PubMed ID: 37994707
Summary: In animals, microRNAs are amongst the primary non-coding RNAs involved in regulating the gene expression of a cell. Most mRNAs in a cell are targeted by one or many miRNAs. Although several mechanisms can be attributed to the degradation of miRNA and mRNA within a cell, but the involvement of autophagy in the clearance of miRNA and its target mRNA is not known. This study discovered a leucine-responsive axis in blood cell progenitors that can mediate an autophagy-directed degradation of miRNA-bound mRNA in Drosophila melanogaster and Homo sapiens. This previously unknown miRNA clearance axis is activated upon amino acid deprivation that can traffic miRNA-mRNA-loaded Argonaute for autophagic degradation in a p62-dependent manner. Thus, this research not only reports a novel axis that can address the turnover of a catalytically active miRISC but also elucidates a slicer-independent mechanism through which autophagy can selectively initiate the clearance of target mRNA. | Zhang, Y., Duan, Y. (2023). Genome-Wide Analysis on Driver and Passenger RNA Editing Sites Suggests an Underestimation of Adaptive Signals in Insects. Genes, 14(10) PubMed ID: 37895300
Summary: Adenosine-to-inosine (A-to-I) RNA editing leads to a similar effect to A-to-G mutations. RNA editing provides a temporo-spatial flexibility for organisms. Nonsynonymous (Nonsyn) RNA editing in insects is over-represented compared with synonymous (Syn) editing, suggesting adaptive signals of positive selection on Nonsyn editing during evolution. We utilized the brain RNA editome of Drosophila melanogaster to systematically study the LD (r(2)) between editing sites and infer its impact on the adaptive signals of RNA editing. Pairs of editing sites (PESs) were identified from the transcriptome. For CDS PESs of two consecutive editing sites, their occurrence was significantly biased to type-3 PES (Syn-Nonsyn). The haplotype frequency of type-3 PES exhibited a significantly higher abundance of AG than GA, indicating that the rear Nonsyn site is the driver that promotes the editing of the front Syn site (passenger). The exclusion of passenger Syn sites dramatically amplifies the adaptive signal of Nonsyn RNA editing. This study for the first time quantitatively demonstrates that the linkage between RNA editing events comes from hitchhiking effects and leads to the underestimation of adaptive signals for Nonsyn editing. This work provides novel insights for studying the evolutionary significance of RNA editing events. |
| Luo, Y., He, P., Kanrar, N., Fejes Toth, K., Aravin, A. A. (2023). Maternally inherited siRNAs initiate piRNA cluster formation. Mol Cell, 83(21):3835-3851.e3837 PubMed ID: 37875112
Summary: PIWI-interacting RNAs (piRNAs) guide transposable element repression in animal germ lines. In Drosophila, piRNAs are produced from heterochromatic loci, called piRNA clusters, which act as information repositories about genome invaders. piRNA generation by dual-strand clusters depends on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, which is deposited on clusters guided by piRNAs, forming a positive feedback loop in which piRNAs promote their own biogenesis. However, how piRNA clusters are formed before cognate piRNAs are present remains unknown. This study reports spontaneous de novo piRNA cluster formation from repetitive transgenic sequences. Cluster formation occurs over several generations and requires continuous trans-generational maternal transmission of small RNAs. Maternally supplied small interfering RNAs (siRNAs) were found to trigger de novo cluster activation in progeny. In contrast, siRNAs are dispensable for cluster function after its establishment. These results reveal an unexpected interplay between the siRNA and piRNA pathways and suggest a mechanism for de novo piRNA cluster formation triggered by siRNAs. | Zhang, S., Wang, R., Zhu, X., Zhang, L., Liu, X., Sun, L. (2023). Characteristics and expression of lncRNA and transposable elements in Drosophila aneuploidy. iScience, 26(12):108494 PubMed ID: 38125016
Summary: Aneuploidy can globally affect the expression of the whole genome, which is detrimental to organisms. Dosage-sensitive regulators usually have multiple intermolecular interactions, and changes in their stoichiometry are responsible for the dysregulation of the regulatory network. Currently, studies on noncoding genes in aneuploidy are relatively rare. The characteristics and expression profiles of long noncoding RNAs (lncRNAs) and transposable elements (TEs) were studied in aneuploid Drosophila. It is found that lncRNAs and TEs are affected by genomic imbalance and appear to be more sensitive to an inverse dosage effect than mRNAs. Several dosage-sensitive lncRNAs and TEs were detected for their expression patterns during embryogenesis, and their biological functions in the ovary and testes were investigated using tissue-specific RNAi. This study advances understanding of the noncoding sequences in imbalanced genomes and provides a novel perspective for the study of aneuploidy-related human diseases such as cancer. |
Wednesday, June 12th - Junctions and Vesicles |
| Shaheen, A., Richter Gorey, C. L., Sghaier, A., Dason, J. S. (2023). Cholesterol is required for activity-dependent synaptic growth. J Cell Sci, 136(22) PubMed ID: 37902091
Summary: Changes in cholesterol content of neuronal membranes occur during development and brain aging. Little is known about whether synaptic activity regulates cholesterol levels in neuronal membranes and whether these changes affect neuronal development and function. This study generated transgenic flies that express the cholesterol-binding D4H domain of perfringolysin O toxin and found increased levels of cholesterol in presynaptic terminals of Drosophila larval neuromuscular junctions following increased synaptic activity. Reduced cholesterol impaired synaptic growth and largely prevented activity-dependent synaptic growth. Presynaptic knockdown of adenylyl cyclase phenocopied the impaired synaptic growth caused by reducing cholesterol. Furthermore, the effects of knocking down adenylyl cyclase and reducing cholesterol were not additive, suggesting that they function in the same pathway. Increasing cAMP levels using a dunce mutant with reduced phosphodiesterase activity failed to rescue this impaired synaptic growth, suggesting that cholesterol functions downstream of cAMP. A protein kinase A (PKA) sensor was used to show that reducing cholesterol levels reduced presynaptic PKA activity. Collectively, these results demonstrate that enhanced synaptic activity increased cholesterol levels in presynaptic terminals and that these changes likely activate the cAMP-PKA pathway during activity-dependent growth. | Guangming, G., Mei, C., Qinfeng, Y., Xiang, G., Chenchen, Z., Qingyuan, S., Wei, X., Junhua, G. (2023). Neurexin and neuroligins jointly regulate synaptic degeneration at the Drosophila neuromuscular junction based on TEM studies. Frontiers in cellular neuroscience, 17:1257347 PubMed ID: 38026694
Summary: The Drosophila larval neuromuscular junction (NMJ) is a well-known model system and is often used to study synapse development. This study shows synaptic degeneration at NMJ boutons, primarily based on transmission electron microscopy (TEM) studies. When degeneration starts, the subsynaptic reticulum (SSR) swells, retracts and folds inward, and the residual SSR then degenerates into a disordered, thin or linear membrane. The axon terminal begins to degenerate from the central region, and the T-bar detaches from the presynaptic membrane with clustered synaptic vesicles to accelerate large-scale degeneration. There are two degeneration modes for clear synaptic vesicles. In the first mode, synaptic vesicles without actin filaments degenerate on the membrane with ultrafine spots and collapse and disperse to form an irregular profile with dark ultrafine particles. In the second mode, clear synaptic vesicles with actin filaments degenerate into dense synaptic vesicles, form irregular dark clumps without a membrane, and collapse and disperse to form an irregular profile with dark ultrafine particles. Last, all residual membranes in NMJ boutons degenerate into a linear shape, and all the residual elements in axon terminals degenerate and eventually form a cluster of dark ultrafine particles. Swelling and retraction of the SSR occurs prior to degradation of the axon terminal, which degenerates faster and with more intensity than the SSR. NMJ bouton degeneration occurs under normal physiological conditions but is accelerated in Drosophila neurexin (dnrx) dnrx273, Drosophila neuroligin (dnlg) dnlg1 and dnlg4 mutants and dnrx83;dnlg3 and dnlg2;dnlg3 double mutants, which suggests that both neurexin and neuroligins play a vital role in preventing synaptic degeneration. |
| Chen, X., Perry, S., Wang, B., Wang, S., Hu, J., Loxterkamp, E., Dickman, D., Han, C. (2023). Tissue-specific knockout in Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles. bioRxiv, PubMed ID: 37808853
Summary: Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions in animal development. However, this approach has been successfully applied in only a small number of Drosophila tissues. The Drosophila motor nervous system is an excellent model system for studying the biology of neuromuscular junction (NMJ). To expand tissue-specific CRISPR to the Drosophila motor system, this study presents a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. The efficacy of this toolkit was validated by knocking out known genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. Using these tools, an essential role for SNARE pathways in NMJ maintenance was demonstrated. Furthermore, it was demonstrated that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating the retrograde Gbb signaling. Lastly, axon termini of motoneurons were found to rely on ESCRT-mediated intra-axonal membrane trafficking to lease extracellular vesicles at the NMJ. | Christophers, B., Leahy, S. N., Soffar, D. B., von Saucken, V. E., Broadie, K., Baylies, M. K. (2023). Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission. bioRxiv, PubMed ID: 38045306
Summary: Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Previous work has shown Drosophila cofilin (DmCFL) knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. It was hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, an RNA sequencing analysis was conducted which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. DmCFL was found to be enriched in the muscle postsynaptic compartment and that DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, no significant changes were found in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology |
| Xiao, C., M'Angale, P. G., Wang, S., Lemieux, A., Thomson, T. (2023). Identifying new players in structural synaptic plasticity through dArc1 interrogation. iScience, 26(11):108048 PubMed ID: 37876812
Summary: The formation, expansion, and pruning of synapses, known as structural synaptic plasticity, is needed for learning and memory, and perturbation of plasticity is associated with many neurological disorders and diseases. Previously, it was observed that the Drosophila homolog of Activity-regulated cytoskeleton-associated protein (dArc1), forms a capsid-like structure, associates with its own mRNA, and is transported across synapses. This transfer is needed for structural synaptic plasticity. To identify mRNAs that are modified by dArc1 in presynaptic neuron and postsynaptic muscle, the expression of dArc1 was disrupted, and genomic analysis was performed with deep sequencing. dArc1 was found to affects the expression of genes involved in metabolism, phagocytosis, and RNA-splicing. Through immunoprecipitation this study also identified potential mRNA cargos of dArc1 capsids. This study suggests that dArc1 acts as a master regulator of plasticity by affecting several distinct and highly conserved cellular processes. | Justs, K. A., Sempertegui, S., Riboul, D. V., Oliva, C. D., Durbin, R. J., Crill, S., Stawarski, M., Su, C., Renden, R. B., Fily, Y., Macleod, G. T. (2023). Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals. The Journal of physiology, 601(24):5705-5732 PubMed ID: 37942946
Summary: Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. In this study, through computer simulation and drawing on previously published empirical data, it was determined that motor neuron terminals in Drosophila larvae experience highly volatile power demands. It might not be surprising then, that the mitochondria in the motor neuron terminals of both Drosophila and mice were found to be heavily decorated with phosphagen kinases - a key element in an energy storage and buffering system well-characterized in fast-twitch muscle fibres. Knockdown of arginine kinase 1 (ArgK1) in Drosophila larval motor neurons led to several bioenergetic deficits, including mitochondrial matrix acidification and a faster decline in the cytosol ATP to ADP ratio during axon burst firing. |
Monday, June 10th - Gonads |
| Takashima, Y. A., Majane, A. C., Begun, D. J. (2023). Evolution of secondary cell number and position in the Drosophila accessory gland. PLoS One, 18(10):e0278811 PubMed ID: 37878630
Summary: In animals with internal fertilization, males transfer gametes and seminal fluid during copulation, both of which are required for successful reproduction. In Drosophila and other insects, seminal fluid is produced in the paired accessory gland (AG), the ejaculatory duct, and the ejaculatory bulb. The D. melanogaster AG has emerged as an important model system for this component of male reproductive biology. Seminal fluid proteins produced in the Drosophila AG are required for proper storage and use of sperm by the females, and are also critical for establishing and maintaining a suite of short- and long-term postcopulatory female physiological responses that promote reproductive success. The Drosophila AG is composed of two main cell types. The majority of AG cells, which are referred to as main cells, are responsible for production of many seminal fluid proteins. A minority of cells, about 4%, are referred to as secondary cells. These cells, which are restricted to the distal tip of the D. melanogaster AG, may play an especially important role in the maintenance of the long-term female post-mating response. Many studies of Drosophila AG evolution have suggested that the proteins produced in the gland evolve quickly, as does the transcriptome. This study investigated the evolution of secondary cell number and position in the AG in a collection of eight species spanning the entire history of the Drosophila genus. A heretofore underappreciated rapid evolutionary rate was documented for both number and position of these specialized AG cells, raising several questions about the developmental, functional, and evolutionary significance of this variation. | Anderson, J., Henikoff, S., Ahmad, K. (2023). Chromosome-specific maturation of the epigenome in the Drosophila male germline. bioRxiv, PubMed ID: 37873332
Summary: Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, active chromatin features were profiled in the testes from wildtype and meiotic arrest mutants, and this was integrated with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, widespread patterns associated with regulation of the sex chromosomes were detailed. The results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome. |
| Wenzel, M., Aquadro, C. F. (2023). Wolbachia infection at least partially rescues the fertility and ovary defects of several new Drosophila melanogaster bag of marbles protein-coding mutants. bioRxiv, PubMed ID: 37645949
Summary: The D. melanogaster protein coding gene bag of marbles (bam) plays a key role in early male and female reproduction by forming complexes with partner proteins to promote differentiation in gametogenesis. Like another germline gene, Sex lethal, bam genetically interacts with the endosymbiont Wolbachia, as Wolbachia rescues the reduced fertility of a bam hypomorphic mutant. This study explored the specificity of the bam-Wolbachia interaction by generating 22 new bam mutants, with ten mutants displaying fertility defects. Nine of these mutants trend towards rescue by the w Mel Wolbachia variant, with eight statistically significant at the fertility and/or cytological level. In some cases, fertility was increased a striking 20-fold. There is no specificity between the rescue and the known binding regions of bam, suggesting w Mel does not interact with one singular bam partner to rescue the reproductive phenotype. Whether Mel interacts with bam in a non-specific way, by increasing bam transcript levels or acting upstream in germline stem cells. A fertility assessment of a bam RNAi knockdown mutant reveals that w Mel rescue is specific to functionally mutant bam alleles and no obvious evidence was found of w Mel interaction with germline stem cells in bam mutants. | Francois, C. M., Pihl, T., Dunoyer de Segonzac, M., Herault, C., Hudry, B. (2023). Metabolic regulation of proteome stability via N-terminal acetylation controls male germline stem cell differentiation and reproduction. Nat Commun, 14(1):6737 PubMed ID: 37872135
Summary: The molecular mechanisms connecting cellular metabolism with differentiation remain poorly understood. This study found that metabolic signals contribute to stem cell differentiation and germline homeostasis during Drosophila melanogaster spermatogenesis. External citrate, originating outside the gonad, was found to fuel the production of Acetyl-coenzyme A by germline ATP-citrate lyase (dACLY). This pathway is essential during the final spermatogenic stages, where a high Acetyl-coenzyme A level promotes N-terminal acetyltransferase B (NatB)-dependent N-terminal protein acetylation. Using genetic and biochemical experiments, this study established that N-terminal acetylation shields key target proteins, essential for spermatid differentiation, from proteasomal degradation by the ubiquitin ligase dUBR1. This work uncovers crosstalk between metabolism and proteome stability that is mediated via protein post-translational modification. It is proposed that this system coordinates the metabolic state of the organism with gamete production. More broadly, modulation of proteome turnover by circulating metabolites may be a conserved regulatory mechanism to control cell functions. |
| Guntur, A. R., Smith, J. E., Brahmandam, A., DeBauche, P., Cronmiller, C., Lundell, M. J. (2023). ZFH-2 is required for Drosophila ovarian follicle development and is expressed at the band/interband boundaries of polytene chromosomes. Dev Biol, 504:1-11 PubMed ID: 37666353
Summary: The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. This study provides the first evidence that ZFH-2 has a role in oogenesis. ZFH-2 is expressed in the wild-type ovary, and a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. It was also shown that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. ZFH-2 was shown to be located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190. | Roach, T. V., Lenhart, K. F. (2023). Mating-induced ecdysone in the testis disrupts soma-germline contacts and stem cell cytokinesis. bioRxiv, PubMed ID: 37905121
Summary: Germline maintenance relies on adult stem cells to continually replenish lost gametes over a lifetime and respond to external cues altering the demands on the tissue. Mating worsens germline homeostasis over time, yet a negative impact on stem cell behavior has not been explored. Using extended live imaging of the Drosophila testis stem cell niche, this study found that short periods of mating in young males disrupts cytokinesis in germline stem cells (GSCs). This defect leads to failure of abscission, preventing release of differentiating cells from the niche. GSC abscission failure was found to be caused by increased ecdysone hormone signaling induced upon mating, which leads to disrupted somatic encystment of the germline. Abscission failure is rescued by isolating males from females but recurs with resumption of mating. Importantly, reiterative mating also leads to increased GSC loss, requiring increased restoration of stem cells via symmetric renewal and de-differentiation. Together, these results suggest a model whereby acute mating results in hormonal changes that negatively impact GSC cytokinesis but preserves the stem cell population. |
Friday, June 7th - Cytoskeleton and Junctions |
| Madan, V., Albacete-Albacete, L., Jin, L., Scaturro, P., Watson, J. L., Muschalik, N., Begum, F., Boulanger, J., Bauer, K., Kiebler, M. A., Derivery, E., Bullock, S. L. (2023). HEATR5B associates with dynein-dynactin and promotes motility of AP1-bound endosomal membranes. The EMBO journal, 42(23):e114473 PubMed ID: 37872872
Summary: The microtubule motor dynein mediates polarised trafficking of a wide variety of organelles, vesicles and macromolecules. These functions are dependent on the dynactin complex, which helps recruit cargoes to dynein's tail and activates motor movement. How the dynein-dynactin complex orchestrates trafficking of diverse cargoes is unclear. This study identified HEATR5B, an interactor of the adaptor protein-1 (AP1) clathrin adaptor complex, as a novel player in dynein-dynactin function. HEATR5B was recovered in a biochemical screen for proteins whose association with the dynein tail is augmented by dynactin. HEATR5B binds directly to the dynein tail and dynactin and stimulates motility of AP1-associated endosomal membranes in human cells. It was also demonstrated that the Drosophila HEATR5B homologue is an essential gene that selectively promotes dynein-based transport of AP1-bound membranes to the Golgi apparatus. As HEATR5B lacks the coiled-coil architecture typical of dynein adaptors, these data point to a non-canonical process orchestrating motor function on a specific cargo. It was additionally shown that HEATR5B promotes association of AP1 with endosomal membranes independently of dynein. Thus, HEATR5B co-ordinates multiple events in AP1-based trafficking. | McParland, E. D., Amber Butcher, T., Gurley, N. J., Johnson, R. I., Slep, K. C., Peifer, M. (2023). The Dilute domain of Canoe is not essential for Canoe's role in linking adherens junctions to the cytoskeleton but contributes to robustness of morphogenesis. bioRxiv, PubMed ID: 37905001
Summary: Robust linkage between cell-cell adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The multidomain protein Drosophila Canoe and its mammalian homolog Afadin are critical for this linkage, and in their absence many events of morphogenesis fail. To define underlying mechanisms, this study took Canoe apart, using Drosophila as a model. Canoe and Afadin share five folded protein domains, followed by a large intrinsically disordered region. The largest of these folded domains is the Dilute domain, which is found in Canoe/Afadin, their paralogs, and members of the MyosinV family. To define the roles of Canoe's Dilute domain this study combined biochemical, genetic and cell biological assays. Use of the AlphaFold tools revealed the predicted structure of the Canoe/Afadin Dilute domain, providing similarities and contrasts with that of MyosinV. The biochemical data suggest one potential shared function: the ability to dimerize. We next generated Drosophila mutants with the Dilute domain cleanly deleted. Surprisingly, these mutants are viable and fertile, and CanoeΔDIL protein localizes to adherens junctions and is enriched at junctions under tension. However, when we reduce the dose of Canoe&DeltaDIL protein in a sensitized assay, it becomes clear it does not provide full wildtype function. Further, canoe&DeltalDIL mutants have defects in pupal eye development, another process that requires orchestrated cell rearrangements. Together, these data reveal the robustness in AJ-cytoskeletal connections during multiple embryonic and postembryonic events, and the power of natural selection to maintain protein structure even in robust systems. |
| Zhu, H., O'Shaughnessy, B. (2023). Actomyosin pulsing rescues embryonic tissue folding from disruption by myosin fluctuations. Research square, PubMed ID: 37886516
Summary: 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 cell contraction profiles. The myosin patterning exhibits substantial cell-to-cell fluctuations with pulsatile time-dependence, a striking but unexplained feature of morphogenesis in many organisms. Using biophysical modeling this study found that viscous forces offer the principal 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 disastrous outcome is averted by pulsatile myosin time-dependence, which rescues furrowing by eliminating high frequencies in the fluctuation power spectrum. This low pass filter mechanism may underlie the usage of actomyosin pulsing in diverse morphogenetic processes across many organisms.
| LaFountain, J. R., Jr., Seaman, C. E., Cohan, C. S., Oldenbourg, R. (2023). Sliding of antiparallel microtubules drives bipolarization of monoastral spindles. PubMed ID: 37812128
Summary: Time-lapse imaging with liquid crystal polarized light (LC-PolScope) and fluorescent speckle microscopy (FSM) enabled this study of spindle microtubules in monoastral spindles that were produced in crane-fly spermatocytes through flattening-induced centrosome displacement. Monoastral spindles are found in several other contexts: after laser ablation of one of a cell's two centrosomes, in Drosophila "urchin" mutants, in Sciara males, and in RNAi variants of Drosophila S2 cells. In all cases, just one pole has a centrosome (the astral pole); the other lacks a centrosome (the anastral pole). Thus, the question: How is the anastral half-spindle, lacking a centrosome, constructed? It was learned that monoastral spindles are assembled in two phases: Phase I assembles the astral half-spindle composed of centrosomal microtubules, and Phase II assembles microtubules of the anastral half through extension of new microtubule polymerization outward from the spindle's equatorial mid-zone. That process uses plus ends of existing centrosomal microtubules as guiding templates to assemble anastral microtubules of opposite polarity. Anastral microtubules slide outward with their minus ends leading, thereby establishing proper bipolarity just like in normal biastral spindles that have two centrosomes. |
| Zhu, Z., Becam, I., Tovey, C. A., Elfarkouchi, A., Yen, E. C., Bernard, F., Guichet, A., Conduit, P. T. (2023). Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes. J Cell Biol, 222(10) PubMed ID: 37698931
Summary: Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. This study shows that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. These data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated. | Li, L., Zhang, N., Beati, S. A. H., De Las Heras Chanes, J., di Pietro, F., Bellaiche, Y., Muller, H. J., Grosshans, J. (2024). Kinesin-1 patterns Par-1 and Rho signaling at the cortex of syncytial embryos of Drosophila. J Cell Biol, 223(1) PubMed ID: 37955925
Summary: The cell cortex of syncytial Drosophila embryos is patterned into cap and intercap regions by centrosomes, specific sets of proteins that are restricted to their respective regions by unknown mechanisms. This study found that Kinesin-1 is required for the restriction of plus- and minus-ends of centrosomal and non-centrosomal microtubules to the cap region, marked by EB1 and Patronin/Shot, respectively. Kinesin-1 also directly or indirectly restricts proteins and Rho signaling to the intercap, including the RhoGEF Pebble, Dia, Myosin II, Capping protein-α, and the polarity protein Par-1. Furthermore, we found that Par-1 is required for cap restriction of Patronin/Shot, and vice versa Patronin, for Par-1 enrichment at the intercap. In summary, these data support a model that Kinesin-1 would mediate the restriction of centrosomal and non-centrosomal microtubules to a region close to the centrosomes and exclude Rho signaling and Par-1. In addition, mutual antagonistic interactions would refine and maintain the boundary between cap and intercap and thus generate a distinct cortical pattern. |
Thursday, June 6th - Drosophila as a Model of Human Diseases |
| Filosevic Vujnovic, A., Saftic Martinovic, L., Medija, M., Andretic Waldowski, R. (2023). Distinct and Dynamic Changes in the Temporal Profiles of Neurotransmitters in Drosophila melanogaster Brain following Volatilized Cocaine or Methamphetamine Administrations. Pharmaceuticals (Basel, Switzerland), 16(10) PubMed ID: 37895961
Summary: Due to similarities in genetics, cellular response, and behavior, Drosophila is used as a model organism in addiction research. A well-described behavioral response examined in flies is the induced increase in locomotor activity after a single dose of volatilized cocaine (vCOC) and volatilized methamphetamine (vMETH), the sensitivity, and the escalation of the locomotor response after the repeated dose, the locomotor sensitization. However, knowledge about how vCOC and vMETH affect different neurotransmitter systems over time is scarce. This study used LC-MS/MS to systematically examine changes in the concentration of neurotransmitters, metabolites and non-metabolized COC and METH in the whole head homogenates of male flies one to seven hours after single and double vCOC or vMETH administrations. vMETH leads to complex changes in the levels of examined substances over time, while vCOC strongly and briefly increases concentrations of dopamine, tyramine and octopamine followed by a delayed degradation into N-acetyl dopamine and N-acetyl tyramine. The first exposure to psychostimulants leads to significant and dynamic changes in the concentrations relative to the second administration when they are more stable over several hours. Further investigations are needed to understand neurochemical and molecular changes post-psychostimulant administration. | Min, Y., Wang, X., Is, O., Patel, T. A., Gao, J., Reddy, J. S., Quicksall, Z. S., Nguyen, T., Lin, S., Tutor-New, F. Q., Chalk, J. L., Mitchell, A. O., Crook, J. E., Nelson, P. T., Van Eldik, L. J., Golde, T. E., Carrasquillo, M. M., Dickson, D. W., Zhang, K., Allen, M., Ertekin-Taner, N. (2023). Cross species systems biology discovers glial DDR2, STOM, and KANK2 as therapeutic targets in progressive supranuclear palsy. Nat Commun, 14(1):6801 PubMed ID: 37919278
Summary: Progressive supranuclear palsy (PSP) is a neurodegenerative parkinsonian disorder characterized by cell-type-specific tau lesions in neurons and glia. Prior work uncovered transcriptome changes in human PSP brains, although their cell-specificity is unknown. Further, systematic data integration and experimental validation platforms to prioritize brain transcriptional perturbations as therapeutic targets in PSP are currently lacking. This study, combined bulk tissue (n = 408) and single nucleus RNAseq (n = 34) data from PSP and control brains with transcriptome data from a mouse tauopathy and experimental validations in Drosophila tau models for systematic discovery of high-confidence expression changes in PSP with therapeutic potential. This study discovered, replicated, and annotated thousands of differentially expressed genes in PSP, many of which reside in glia-enriched co-expression modules and cells. DDR2, STOM, and KANK2 were prioritized as promising therapeutic targets in PSP with striking cross-species validations. These findings and data are shared via interactive application tool PSP RNAseq Atlas . These findings reveal robust glial transcriptome changes in PSP, provide a cross-species systems biology approach, and a tool for therapeutic target discoveries in PSP with potential application in other neurodegenerative diseases. |
| He, M. F., Liu, C. Q., Zhang, X. X., Lin, Y. M., Mao, Y. L., Qiao, J. D. (2023). Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy. J Vis Exp, (200) PubMed ID: 37902337
Summary: Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level. | Al-Ayari, E. A., Shehata, M. G., El-Hadidi, M., Shaalan, M. G. (2023). In silico SNP prediction of selected protein orthologues in insect models for Alzheimer's, Parkinson's, and Huntington's diseases. Sci Rep, 13(1):18986 PubMed ID: 37923901
Summary: Alzheimer's, Parkinson's, and Huntington's are the most common neurodegenerative diseases that are incurable and affect the elderly population. Discovery of effective treatments for these diseases is often difficult, expensive, and serendipitous. Previous comparative studies on different model organisms have revealed that most animals share similar cellular and molecular characteristics. The meta-SNP tool includes four different integrated tools (SIFT, PANTHER, SNAP, and PhD-SNP) was used to identify non synonymous single nucleotide polymorphism (nsSNPs). Prediction of nsSNPs was conducted on three representative proteins for Alzheimer's, Parkinson's, and Huntington's diseases; APPl in Drosophila melanogaster, LRRK1 in Aedes aegypti, and VCPl in Tribolium castaneum. With the possibility of using insect models to investigate neurodegenerative diseases. It is concluded from the protein comparative analysis between different insect models and nsSNP analyses that D. melanogaster is the best model for Alzheimer's representing five nsSNPs of the 21 suggested mutations in the APPl protein. Aedes aegypti is the best model for Parkinson's representing three nsSNPs in the LRRK1 protein. Tribolium castaneum is the best model for Huntington's disease representing 13 SNPs of 37 suggested mutations in the VCPl protein. This study aimed to improve human neural health by identifying the best insect to model Alzheimer's, Parkinson's, and Huntington's. |
| Nithianandam, V., Bukhari, H., Leventhal, M. J., Battaglia, R. A., Dong, X., Fraenkel, E., Feany, M. B. (2023). Integrative analysis reveals a conserved role for the amyloid precursor protein in proteostasis during aging. Nat Commun, 14(1):7034 PubMed ID: 37923712
Summary: Aβ peptides derived from the amyloid precursor protein (APP) have been strongly implicated in the pathogenesis of Alzheimer's disease. However, the normal function of APP and the importance of that role in neurodegenerative disease is less clear. The Drosophila ortholog of APP, Appl, was uncovered in an unbiased forward genetic screen for neurodegeneration mutants. Comprehensive single cell transcriptional and proteomic studies of Appl mutant flies were performed to investigate Appl function in the aging brain. An unexpected role was found for Appl in control of multiple cellular pathways, including translation, mitochondrial function, nucleic acid and lipid metabolism, cellular signaling and proteostasis. A role for Appl in regulating autophagy through TGFβ signaling was mechanistically defined, and the broader relevance of these findings was documented using mouse genetic, human iPSC and in vivo tauopathy models. The results demonstrate a conserved role for APP in controlling age-dependent proteostasis with plausible relevance to Alzheimer's disease. | Xu, J., Deng, Z., Shang, S., Wang, C., Han, H. (2023). FUNDC1 collaborates with PINK1 in regulating mitochondrial Fission and compensating for PINK1 deficiency. Biochem Biophys Res Commun, 687:149210 PubMed ID: 37931419
Summary: Parkinson's disease is presently thought to have its molecular roots in the alteration of PINK1-mediated mitophagy and mitochondrial dynamics. Finding new suppressors of the pathway is essential for developing cutting-edge treatment approaches. This study shows that FUNDC1 suppressed PINK1 mutant phenotypes in Drosophila. The restoration of PINK1-deficient phenotypes through FUNDC1 is not reliant on its LC3-binding motif Y (18)L (21) or autophagy-related pathway. Moreover, the absence of Drp1 affects the phenotypic restoration of PINK1 mediated by FUNDC1 in flies. In summary, these findings have unveiled a fresh mechanism through which FUNDC1 compensates for the loss of PINK1, operating independently of autophagy but exerting its influence via interaction with Drp1. |
Wednesday, June 5th - Homologs of Drosophila Proteins |
| Cowell, L. M., King, M., West, H., Broadsmith, M., Genever, P., Pownall, M. E., Isaacs, H. V. (2023). Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development. PLoS One, 18(10):e0286040 PubMed ID: 37856433
Summary: Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. It is proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor.This study investigated the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. Xenopus Cic was characterized and was shown to be widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with our hypothesis, we show that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. Two of these genes were investigated as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b, which encode a leucine zipper transcription factor and a ras family GTPase, respectively. Cic consensus binding sites were identified in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. We show that expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, it was shown that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. The data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding. | Torres, H. M., Hinojosa, L., VanCleave, A. M., Rodezno, T., Westendorf, J. J., Tao, J. (2023). Hdac1 and Hdac2 positively regulate Notch1 gain-of-function pathogenic signaling in committed osteoblasts of male mice. Birth defects research, PubMed ID: 37921375
Summary: Skeletal development requires precise extrinsic and intrinsic signals to regulate processes that form and maintain bone and cartilage. Notch1 is a highly conserved signaling receptor that regulates cell fate decisions by controlling the duration of transcriptional bursts. Epigenetic molecular events reversibly modify DNA and histone tails by influencing the spatial organization of chromatin and can fine-tune the outcome of a Notch1 transcriptional response. Histone deacetylase 1 and 2 (HDAC1 and HDAC2) are chromatin modifying enzymes that mediate osteoblast differentiation. While an HDAC1-Notch interaction has been studied in vitro and in Drosophila, its role in mammalian skeletal development and disorders is unclear. Osteosclerosis is a bone disorder with an abnormal increase in the number of osteoblasts and excessive bone formation. This study, tested whether Hdac1/2 contribute to the pathogenesis of osteosclerosis in a murine model of the disease owing to conditionally cre-activated expression of the Notch1 intracellular domain in immature osteoblasts. RESULTS: Importantly, selective homozygous deletions of Hdac1/2 in osteoblasts partially alleviate osteosclerotic phenotypes (Col2.3kb-Cre; TG(RosaN1ICD/+) ; Hdac1(flox/flox) ; Hdac2(flox/flox) ) with a 40% decrease in bone volume and a 22% decrease in trabecular thickness in 4 weeks old when compared to male mice with heterozygous deletions of Hdac1/2 (Col2.3 kb-Cre; TG(RosaN1ICD/+) ; Hdac1(flox/+) ; Hdac2(flox/+) ). Osteoblast-specific deletion of Hdac1/2 in male and female mice results in no overt bone phenotype in the absence of the Notch1 gain-of-function (GOF) allele. These results provide evidence that Hdac1/2 contribute to Notch1 pathogenic signaling in the mammalian skeleton. This study on epigenetic regulation of Notch1 GOF-induced osteosclerosis may facilitate further mechanistic studies of skeletal birth defects caused by Notch-related GOF mutations in human patients, such as Adams-Oliver disease, congenital heart disease, and lateral meningocele syndrome. |
| Sauty, S. M., Yankulov, K. (2023). Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-modal active/silent state at the sub-telomeres of S. cerevisiae.Epigenetics & chromatin, 16(1):40 PubMed ID: 37858268
Summary: Classical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. This study used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the different roles of PCNA and histone chaperones in the stability and the propagation of repressed states at the sub-telomeres of S. cerevisiae. Position dependent transient repression or bi-modal expression of reporter genes is shown at the VIIL sub-telomere. Mutations in the replicative clamp POL30 (PCNA) or the deletion of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the deletion of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. The physical interaction of CAF1 and RRM3 with PCNA was analyzed, and the implications of these findings for understanding of the stability and transmission of the epigenetic state of the genes id discussed. There are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. Yhe roles of CAF1, RRM3 and ASF1 in these modes of gene repression were analyzed. It is suggestd that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing. | Duan, D., Lyu, W., Chai, P., Ma, S., Wu, K., Wu, C., Xiong, Y., Sestan, N., Zhang, K., Koleske, A. J. (2023). Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation. Curr Biol, 33(21):4582-4598.e4510 PubMed ID: 37858340
Summary: Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both in vitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. This study reports that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. Abl2 was found to promote MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ688-790. It is proposed that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue. |
| Kushwaha, A., Thakur, M. K. (2024). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Molecular neurobiology. 61(1):487-497 PubMed ID: 37626270
Summary: Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ Suv39h1 (a mammalian ortholog of the Drosophila melanogaster SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It has been reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. In the current study, an attempt was made to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. It was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia. | Liu, Q., Bell, B. J., Kim, D. W., Lee, S. S., Keles, M. F., Liu, Q., Blum, I. D., Wang, A. A., Blank, E. J., Xiong, J., Bedont, J. L., Chang, A. J., Issa, H., Cohen, J. Y., Blackshaw, S., Wu, M. N. (2023). A clock-dependent brake for rhythmic arousal in the dorsomedial hypothalamus. Nat Commun, 14(1):6381 PubMed ID: 37821426
Summary: Circadian clocks generate rhythms of arousal, but the underlying molecular and cellular mechanisms remain unclear. In Drosophila, the clock output molecule WIDE AWAKE (WAKE) labels rhythmic neural networks and cyclically regulates sleep and arousal. This study shows, in a male mouse model, that mWAKE/ANKFN1 labels a subpopulation of dorsomedial hypothalamus (DMH) neurons involved in rhythmic arousal and acts in the DMH to reduce arousal at night. In vivo Ca(2+) imaging reveals elevated DMH(mWAKE) activity during wakefulness and rapid eye movement (REM) sleep, while patch-clamp recordings show that DMH(mWAKE) neurons fire more frequently at night. Chemogenetic manipulations demonstrate that DMH(mWAKE) neurons are necessary and sufficient for arousal. Single-cell profiling coupled with optogenetic activation experiments suggest that GABAergic DMH(mWAKE) neurons promote arousal. Surprisingly, the data suggest that mWAKE acts as a clock-dependent brake on arousal during the night, when mice are normally active. mWAKE levels peak at night under clock control, and loss of mWAKE leads to hyperarousal and greater DMH(mWAKE) neuronal excitability specifically at night. These results suggest that the clock does not solely promote arousal during an animal's active period, but instead uses opposing processes to produce appropriate levels of arousal in a time-dependent manner. |
Tuesday, June 4th - Enhancers and Transcriptional Regulation |
| Chaubal, A., Waldern, J. M., Taylor, C., Laederach, A., Marzluff, W. F., Duronio, R. J. (2023). Coordinated expression of replication-dependent histone genes from multiple loci promotes histone homeostasis in Drosophila. Mol Biol Cell, 34(12):ar118 PubMed ID: 37647143
Summary: Production of large amounts of histone proteins during S phase is critical for proper chromatin formation and genome integrity. This process is achieved in part by the presence of multiple copies of replication dependent (RD) histone genes that occur in one or more clusters in metazoan genomes. In addition, RD histone gene clusters are associated with a specialized nuclear body, the histone locus body (HLB), which facilitates efficient transcription and 3' end-processing of RD histone mRNA. How all five RD histone genes within these clusters are coordinately regulated such that neither too few nor too many histones are produced, a process referred to as histone homeostasis, is not fully understood. This study explored the mechanisms of coordinate regulation between multiple RD histone loci in Drosophila melanogaster and Drosophila virilis. Evidence is provided for functional competition between endogenous and ectopic transgenic histone arrays located at different chromosomal locations in D. melanogaster that helps maintain proper histone mRNA levels. Consistent with this model, in both species it was found that individual histone gene arrays can independently assemble an HLB that results in active histone transcription. These findings suggest a role for HLB assembly in coordinating RD histone gene expression to maintain histone homeostasis. | Masuda, L. H. P., Sabino, A. U., Reinitz, J., Ramos, A. F., Machado-Lima, A., Andrioli, L. P. (2024). Global repression by tailless during segmentation. Dev Biol, 505:11-23 PubMed ID: 37879494
Summary: The early expression of tll in two gap domains in the segmentation cascade of Drosophila is unusual even for most other insects. This study investigated tll regulation on pair-rule stripes. With ectopic misexpression of tll unexpected repression was detected of almost all pair-rule stripes of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz). Examining Tll embryonic ChIP-chip data with regions mapped as Cis-Regulatory Modules (CRMs) of pair-rule stripes this study verified Tll interactions to these regions. With the ChIP-chip data Tll interactions to the CRMs of gap domains and in the misexpression assay, Tll-mediated repression on Kruppel (Kr), kni (kni) and giant (gt) according to their differential sensitivity to Tll. These results with gap genes confirmed previous data from the literature and argue against indirect repression roles of Tll in the striped pattern. Moreover, the prediction of Tll binding sites in the CRMs of eve stripes and the mathematical modeling of their removal using an experimentally validated theoretical framework shows effects on eve stripes compatible with the absence of a repressor binding to the CRMs. In addition, modeling increased tll levels in the embryo results in the differential repression of eve stripes, agreeing well with the results of the misexpression assay. In genetic assays we investigated eve 5, that is strongly repressed by the ectopic domain and representative of more central stripes not previously implied to be under direct regulation of tll. While this stripe is little affected in tll-, its posterior border is expanded in gt- but detected with even greater expansion in gt-;tll-. The paper ends with a discussion of tll with key roles in combinatorial repression mechanisms to contain the expression of medial patterns of the segmentation cascade in the extremities of the embryo. |
| Ramalingam, V., Yu, X., Slaughter, B. D., Unruh, J. R., Brennan, K. J., Onyshchenko, A., Lange, J. J., Natarajan, M., Buck, M., Zeitlinger, J. (2023). Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development. Nat Commun, 14(1):5862 PubMed ID: 37735176
Summary: While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor that is one of the more than 25 different splice isoforms from the lola locus, this study showed that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters. | Lovero, D., Porcelli, D., Giordano, L., Lo Giudice, C., Picardi, E., Pesole, G., Pignataro, E., Palazzo, A., Marsano, R. M. (2023). Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors. Biology, 12(8) PubMed ID: 37627011
Summary: Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. This work have analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. The conservation of a short sequence is highlighted in the genes encoding subunits of the V-ATPase complex, and their assembly factors were analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding knowledge on the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization. |
| Eggers, N., Gkountromichos, F., Krause, S., Campos-Sparr, A., Becker, P. B. (2023). Physical interaction between MSL2 and CLAMP assures direct cooperativity and prevents competition at composite binding sites. Nucleic Acids Res, 51(17):9039-9054 PubMed ID: 37602401
Summary: MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. This study explored the nature of the cooperative binding to these GA-rich, composite sequence elements in reconstituted naive embryonic chromatin. The cooperativity was found to requires physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. The cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in the reconstitution, such as roX2 lncRNA. | Haroush, N., Levo, M., Wieschaus, E. F., Gregor, T. (2023). Functional analysis of the Drosophila eve locus in response to non-canonical combinations of gap gene expression levels. Dev Cell, 58(23):2789-2801.e2785 PubMed ID: 37890488
Summary: Transcription factor combinations play a key role in shaping cellular identity. However, the precise relationship between specific combinations and downstream effects remains elusive. This relationship was examined within the context of the Drosophila eve locus, which is controlled by gap genes. Spatiotemporal levels of four gap genes was measured in heterozygous and homozygous gap mutant embryos, and they were correlated with the striped eve activity pattern. Although changes in gap gene expression extend beyond the manipulated gene, the spatial patterns of Eve expression closely mirror canonical activation levels in wild type. Interestingly, some combinations deviate from the wild-type repertoire but still drive eve activation. Although in homozygous mutants some Eve stripes exhibit partial penetrance, stripes consistently emerge at reproducible positions, even with varying gap gene levels. The findings suggest a robust molecular canalization of cell fates in gap mutants and provide insights into the regulatory constraints governing multi-enhancer gene loci. |
Monday, June 3rd - Larval and Adult Neural Structurel, Development and Function |
| Mitchell, J. W., Midillioglu, I., Schauer, E., Wang, B., Han, C., Wildonger, J. (2023). Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons. PLoS Genet, 19(11):e1011025 PubMed ID: 37943859
Summary: Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. This study investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. CRISPR-Cas9 genome engineering approaches were used to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, an over-active channel mutant was found to severely reduce dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. The data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on these data, it is proposed that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites. | Zhao, A., Nern, A., Koskela, S., Dreher, M., Erginkaya, M., Laughland, C. W., Ludwigh, H., Thomson, A., Hoeller, J., Parekh, R., Romani, S., Bock, D. D., Chiappe, E., Reiser, M. B. (2023). A comprehensive neuroanatomical survey of the Drosophila Lobula Plate Tangential Neurons with predictions for their optic flow sensitivity. bioRxiv, PubMed ID: 37904921
Summary: Flying insects exhibit remarkable navigational abilities controlled by their compact nervous systems. Optic flow, the pattern of changes in the visual scene induced by locomotion, is a crucial sensory cue for robust self-motion estimation, especially during rapid flight. The best-known optic-flow sensitive neurons are the large tangential cells of the dipteran lobula plate. Most of these studies have focused on the large, Horizontal and Vertical System neurons, yet the lobula plate houses a much larger set of 'optic-flow' sensitive neurons, many of which have been challenging to unambiguously identify or to reliably target for functional studies. This study reports the comprehensive reconstruction and identification of the Lobula Plate Tangential Neurons in an Electron Microscopy (EM) volume of a whole Drosophila brain. This catalog of 58 LPT neurons (per brain hemisphere) contains many neurons that are described here for the first time and provides a basis for systematic investigation of the circuitry linking self-motion to locomotion control. Leveraging computational anatomy methods, It was estimated the visual motion receptive fields of these neurons, and their tuning was compared to the visual consequence of body rotations and translational movements. These neurons were matched, in most cases on a one-for-one basis, to stochastically labeled cells in genetic driver lines, to the mirror-symmetric neurons in the same EM brain volume, and to neurons in an additional EM data set. Using cell matches across data sets, the integration of optic flow patterns by neurons downstream of the LPTs were analyzed, Most central brain neurons establish sharper selectivity for global optic flow patterns than their input neurons. Furthermore, self-motion information extracted from optic flow was found to be processed in distinct regions of the central brain, pointing to diverse foci for the generation of visual behaviors |
| Jovanoski, K. D., Duquenoy, L., Mitchell, J., Kapoor, I., Treiber, C. D., Croset, V., Dempsey, G., Parepalli, S., Cognigni, P., Otto, N., Felsenberg, J., Waddell, S. (2023). Dopaminergic systems create reward seeking despite adverse consequences. Nature, 623(7986):356-365 PubMed ID: 37880370
Summary: Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking. This study describes dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. It is proposed that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals. | Ganguly, I., Heckman, E. L., Litwin-Kumar, A., Clowney, E. J., Behnia, R. (2023). Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body. bioRxiv, PubMed ID: 37873086
Summary: The arthropod mushroom body is well-studied as an expansion layer that represents olfactory stimuli and links them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their tuning and function are poorly understood. This study used the FlyWire adult whole-brain connectome to identify inputs to visual Kenyon cells. The types of visual neurons identified are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual projection neurons presynaptic to Kenyon cells receive input from large swathes of visual space, while local visual interneurons, providing smaller fractions of input, receive more spatially restricted signals that may be tuned to specific features of the visual scene. Like olfactory Kenyon cells, visual Kenyon cells receive sparse inputs from different combinations of visual channels, including inputs from multiple optic lobe neuropils. The sets of inputs to individual visual Kenyon cells are consistent with random sampling of available inputs. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the expansion coding properties appear different, with a specific repertoire of visual inputs projecting onto a relatively small number of visual Kenyon cells. |
| Anthoney, N., Tainton-Heap, L., Luong, H., Notaras, E., Kewin, A. B., Zhao, Q., Perry, T., Batterham, P., Shaw, P. J., van Swinderen, B. (2023). Experimentally induced active and quiet sleep engage non-overlapping transcriptional programs in Drosophila. Elife, 12 PubMed ID: 37910019
Summary: Sleep in mammals can be broadly classified into two different physiological categories: rapid eye movement (REM) sleep and slow-wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. This study compared two commonly used approaches for studying sleep experimentally in Drosophila: optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, gaboxadol. These different sleep-induction methods have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions. | Yang, H. H., Brezovec, L. E., Capdevila, L. S., Vanderbeck, Q. X., Adachi, A., Mann, R. S., Wilson, R. I. (2023). Fine-grained descending control of steering in walking Drosophila. bioRxiv, PubMed ID: 37904997
Summary: . . Locomotion involves rhythmic limb movement patterns that originate in circuits outside the brain. Purposeful locomotion requires descending commands from the brain, but how these commands are structured is not understood. This study investigated this issue, focusing on the control of steering in walking Drosophila. First, different limb "gestures" are described associated with different steering maneuvers. Next, a set of descending neurons was identified whose activity predicts steering. Focusing on two descending cell types downstream from distinct brain networks, this study showed that they evoke specific limb gestures: one lengthens strides on the outside of a turn, while the other attenuates strides on the inside of a turn. Notably, a single descending neuron can have opposite effects during different locomotor rhythm phases, and networks were identified positioned to implement this phase-specific gating. Together, these results show how purposeful locomotion emerges from brain cells that drive specific, coordinated modulations of low-level patterns. |
Friday, May 31st - Physiology and Metabolism |
| Alassaf, M., Rajan, A. (2023). Diet-induced glial insulin resistance impairs the clearance of neuronal debris in Drosophila brain. PLoS Biol, 21(11):e3002359 PubMed ID: 37934726
Summary: Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Using a Drosophila model, this study shows that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, this study showed that genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, this study has established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris. | Brener, A., Lorber, D., Reuveny, A., Toledano, H., Porat-Kuperstein, L., Lebenthal, Y., Weizman, E., Olender, T., Volk, T. (2023). Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae. Cells, 12(19) PubMed ID: 37830547
Summary: The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. THE findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating these findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases. |
| Li, J., Dang, P., Li, Z., Zhao, T., Cheng, D., Pan, D., Yuan, Y., Song, W. (2023). Peroxisomal ERK mediates Akh/glucagon action and glycemic control. Cell Rep, 42(10):113200 PubMed ID: 37796662
Summary: The enhanced response of glucagon and its Drosophila homolog, adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. This study found that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. Mammalian glucagon-induced hepatic ERK peroxisomal translocation was observed in diabetic subjects. Therefore, it is concluded that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control. | Zhao, Y., Johansson, E., Duan, J., Han, Z., Alenius, M. (2023). Fat- and sugar-induced signals regulate sweet and fat taste perception in Drosophila. Cell Rep, 42(11):113387 PubMed ID: 37934669
Summary: This study investigated the interplay between taste perception and macronutrients. While sugar's and protein's self-regulation of taste perception is known, the role of fat remains unclear.In Drosophila, fat overconsumption reduces fatty acid taste in favor of sweet perception. Conversely, sugar intake increases fatty acid perception and suppresses sweet taste. Genetic investigations show that the sugar signal, gut-secreted Hedgehog, suppresses sugar taste and enhances fatty acid perception. Fat overconsumption induces Unpaired 2 (Upd2) secretion from adipose tissue to the hemolymph. This study revealed taste neurons take up Upd2, which triggers Domeless suppression of fatty acid perception. It was further shown that the downstream JAK/STAT signaling enhances sweet perception and, via Socs36E, fine-tunes Domeless activity and the fatty acid taste perception. Together, these results show that sugar regulates Hedgehog signaling and fat induces Upd2 signaling to balance nutrient intake and to regulate sweet and fat taste perception. |
| Brischigliaro, M., Cabrera-Orefice, A., Arnold, S., Viscomi, C., Zeviani, M., Fernández-Vizarra, E. (2023). Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes. Elife, 12 PubMed ID: 37823874
Summary: Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. This study shows that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, it is concluded that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis. | Bobrovskikh, M. A., Gruntenko, N. E. (2023). The Role of 20-Hydroxyecdysone in the Control of Carbohydrate Levels in Drosophila melanogaster under Short-Term Heat Stress. Doklady Biochemistry and biophysics, 511(1):162-165 PubMed ID: 37833600
Summary: It is known that 20-hydroxyecdysone is one of the most important hormonal regulators of development, reproduction and adaptation to unfavorable conditions in insects. This study shows for the first time that exogenous 20-hydroxyecdysone increases the content of two main insect carbohydrates, trehalose and glucose, in Drosophila melanogaster females both in normal conditions and under short-term heat stress. It was found that the levels of both trehalose and glucose increase after 39 min of heat exposure and return to their original levels after 1.5 h. A scheme of hormonal regulation of carbohydrate content under heat stress, involving 20-hydroxyecdysone, juvenile hormone, and dopamine, is suggested. |
Thursday, May 30th - Signaling |
| Alassaf, M., Rajan, A. (2023). Diet-induced glial insulin resistance impairs the clearance of neuronal debris in Drosophila brain. PLoS Biol, 21(11):e3002359 PubMed ID: 37934726
Summary: Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Using a Drosophila model, this study shows that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, this study showed that genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, this study has established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris. | Brener, A., Lorber, D., Reuveny, A., Toledano, H., Porat-Kuperstein, L., Lebenthal, Y., Weizman, E., Olender, T., Volk, T. (2023). Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae. Cells, 12(19) PubMed ID: 37830547
Summary: The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. THE findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating these findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases. |
| Li, J., Dang, P., Li, Z., Zhao, T., Cheng, D., Pan, D., Yuan, Y., Song, W. (2023). Peroxisomal ERK mediates Akh/glucagon action and glycemic control. Cell Rep, 42(10):113200 PubMed ID: 37796662
Summary: The enhanced response of glucagon and its Drosophila homolog, adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. This study found that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. Mammalian glucagon-induced hepatic ERK peroxisomal translocation was observed in diabetic subjects. Therefore, it is concluded that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control. | Zhao, Y., Johansson, E., Duan, J., Han, Z., Alenius, M. (2023). Fat- and sugar-induced signals regulate sweet and fat taste perception in Drosophila. Cell Rep, 42(11):113387 PubMed ID: 37934669
Summary: This study investigated the interplay between taste perception and macronutrients. While sugar's and protein's self-regulation of taste perception is known, the role of fat remains unclear.In Drosophila, fat overconsumption reduces fatty acid taste in favor of sweet perception. Conversely, sugar intake increases fatty acid perception and suppresses sweet taste. Genetic investigations show that the sugar signal, gut-secreted Hedgehog, suppresses sugar taste and enhances fatty acid perception. Fat overconsumption induces Unpaired 2 (Upd2) secretion from adipose tissue to the hemolymph. This study revealed taste neurons take up Upd2, which triggers Domeless suppression of fatty acid perception. It was further shown that the downstream JAK/STAT signaling enhances sweet perception and, via Socs36E, fine-tunes Domeless activity and the fatty acid taste perception. Together, these results show that sugar regulates Hedgehog signaling and fat induces Upd2 signaling to balance nutrient intake and to regulate sweet and fat taste perception. |
| Brischigliaro, M., Cabrera-Orefice, A., Arnold, S., Viscomi, C., Zeviani, M., Fernández-Vizarra, E. (2023). Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes. Elife, 12 PubMed ID: 37823874
Summary: Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. This study shows that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, it is concluded that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis. | Bobrovskikh, M. A., Gruntenko, N. E. (2023). The Role of 20-Hydroxyecdysone in the Control of Carbohydrate Levels in Drosophila melanogaster under Short-Term Heat Stress. Doklady Biochemistry and biophysics, 511(1):162-165 PubMed ID: 37833600
Summary: It is known that 20-hydroxyecdysone is one of the most important hormonal regulators of development, reproduction and adaptation to unfavorable conditions in insects. This study shows for the first time that exogenous 20-hydroxyecdysone increases the content of two main insect carbohydrates, trehalose and glucose, in Drosophila melanogaster females both in normal conditions and under short-term heat stress. It was found that the levels of both trehalose and glucose increase after 39 min of heat exposure and return to their original levels after 1.5 h. A scheme of hormonal regulation of carbohydrate content under heat stress, involving 20-hydroxyecdysone, juvenile hormone, and dopamine, is suggested. |
Thursday, May 30th - Signaling |
| Yarikipati, P., Jonusaite, S., Pleinis, J. M., Dominicci Cotto, C., Sanchez-Hernandez, D., Morrison, D. E., Goyal, S., Schellinger, J., Penalva, C., Curtiss, J., Rodan, A. R., Jenny, A. (2023). Unanticipated domain requirements for Drosophila Wnk kinase in vivo. PLoS Genet, 19(10):e1010975 PubMed ID: 37819975
Summary: WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate anonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. This study shows that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. The only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. These data thus provide novel insights into unexpected in vivo roles of specific WNK domains. | Brown, J., Su, T. T. (2024). E2F1, DIAP1, and the presence of a homologous chromosome promote while JNK inhibits radiation-induced loss of heterozygosity in Drosophila melanogaster. Genetics, 226(1) PubMed ID: 37874851
Summary: Loss of heterozygosity (LOH) can occur when a heterozygous mutant cell loses the remaining wild-type allele to become a homozygous mutant. LOH can have physiological consequences if, for example, the affected gene encodes a tumor suppressor. Fluorescent reporters were used to study the mechanisms of LOH induction by X-rays, a type of ionizing radiation (IR), in Drosophila melanogaster larval wing discs. IR is used to treat more than half of patients with cancer, so understanding its effects is of biomedical relevance. Quantitative analysis of IR-induced LOH at different positions between the telomere and the centromere on the X chromosome showed a strong sex dependence and the need for a recombination-proficient homologous chromosome, whereas, paradoxically, position along the chromosome made little difference in LOH incidence. It is proposed that published data documenting high recombination frequency within centromeric heterochromatin on the X chromosome can explain these data. Using a focused screen, E2F1 was identified as a key promotor of LOH and further testing suggests a mechanism involving its role in cell-cycle regulation. The loss of a transcriptional repressor was leveraged through LOH to express transgenes specifically in cells that have already acquired LOH. This approach identified JNK signaling and apoptosis as key determinants of LOH maintenance. These studies reveal previously unknown mechanisms for the generation and elimination of cells with chromosome aberrations after exposure to IR. |
| Zhang, J., Liu, Y., Wang, C., Vander Kooi, C. W., Jia, J. (2023). Phosphatidic acid binding to Patched contributes to the inhibition of Smoothened and Hedgehog signaling in Drosophila wing development. Science signaling, 16(807):eadd6834. PubMed ID: 37847757
Summary: Hedgehog (Hh) signaling controls growth and patterning during embryonic development and homeostasis in adult tissues. Hh binding to the receptor Patched (Ptc) elicits intracellular signaling by relieving Ptc-mediated inhibition of the transmembrane protein Smoothened (Smo). This study uncovered a role for the lipid phosphatidic acid (PA) in the regulation of the Hh pathway in Drosophila melanogaster. Deleting the Ptc C-terminal tail or mutating the predicted PA-binding sites within it prevented Ptc from inhibiting Smo in wing discs and in cultured cells. The C-terminal tail of Ptc directly interacted with PA in vitro, an association that was reduced by Hh, and increased the amount of PA at the plasma membrane in cultured cells. Smo also interacted with PA in vitro through a binding pocket located in the transmembrane region, and mutating residues in this pocket reduced Smo activity in vivo and in cells. By genetically manipulating PA amounts in vivo or treating cultured cells with PA, this study demonstrated that PA promoted Smo activation.These findings suggest that Ptc may sequester PA in the absence of Hh and release it in the presence of Hh, thereby increasing the amount of PA that is locally available to promote Smo activation. | Spencer, Z. T., Ng, V. H., Benchabane, H., Siddiqui, G. S., Duwadi, D., Maines, B., Bryant, J. M., Schwarzkopf, A., Yuan, K., Kassel, S. N., Mishra, A., Pimentel, A., Lebensohn, A. M., Rohatgi, R., Gerber, S. A., Robbins, D. J., Lee, E., Ahmed, Y. (2023). The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6. Nat Commun, 14(1):6174 PubMed ID: 37798281
Summary: The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. This study demonstrates that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/&alpha-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient. |
| Li, C., Zhu, X., Sun, X., Guo, X., Li, W., Chen, P., Shidlovskii, Y. V., Zhou, Q., Xue, L. (2023). Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis. Cell division, 18(1):16 PubMed ID: 37794497
Summary: The c-Jun N-terminal kinase (JNK) pathway is an evolutionarily conserved regulator of cell death, which is essential for coordinating tissue homeostasis. This study characterized the Drosophila Ste20-like kinase Slik as a novel modulator of JNK pathway-mediated apoptotic cell death. First, ectopic JNK signaling-triggered cell death is enhanced by slik depletion whereas suppressed by Slik overexpression. Second, loss of slik activates JNK signaling, which results in enhanced apoptosis and impaired tissue homeostasis. In addition, genetic epistasis analysis suggests that Slik acts upstream of or in parallel to Hep to regulate JNK-mediated apoptotic cell death. Moreover, Slik is necessary and sufficient for preventing physiologic JNK signaling-mediated cell death in development. Furthermore, introduction of STK10, the human ortholog of Slik, into Drosophila restores slik depletion-induced cell death and compromised tissue homeostasis. Lastly, knockdown of STK10 in human cancer cells also leads to JNK activation, which is cancelled by expression of Slik. This study has uncovered an evolutionarily conserved role of Slik/STK10 in blocking JNK signaling, which is required for cell death inhibition and tissue homeostasis maintenance in development. | Tokamov, S. A., Buiter, S., Ullyot, A., Scepanovic, G., Williams, A. M., Fernandez-Gonzalez, R., Horne-Badovinac, S., Fehon, R. G. (2024). Cortical tension promotes Kibra degradation via Par-1. Mol Biol Cell, 35(1):ar2 PubMed ID: 37903240
Summary: The Hippo pathway is an evolutionarily conserved regulator of tissue growth. Multiple Hippo signaling components are regulated via proteolytic degradation. However, how these degradation mechanisms are themselves modulated remains unexplored. Kibra is a key upstream pathway activator that promotes its own ubiquitin-mediated degradation upon assembling a Hippo signaling complex. This study demonstrates that Hippo complex-dependent Kibra degradation is modulated by cortical tension. Using classical genetic, osmotic, and pharmacological manipulations of myosin activity and cortical tension, it was shown that increasing cortical tension leads to Kibra degradation, whereas decreasing cortical tension increases Kibra abundance. This study also implicates Par-1 in regulating Kib abundance downstream of cortical tension. Par-1 promotes ubiquitin-mediated Kib degradation in a Hippo complex-dependent manner and is required for tension-induced Kib degradation. Collectively, these results reveal a previously unknown molecular mechanism by which cortical tension affects Hippo signaling and provide novel insights into the role of mechanical forces in growth control. |
Wednesday, May 29th - Apoptosis and Autophagy |
| Leung, H. H., Mansour, C., Rousseau, M., Nakhla, A., Kiselyov, K., Venkatachalam, K., Wong, C. O. (2024). Drosophila tweety facilitates autophagy to regulate mitochondrial homeostasis and bioenergetics in Glia. Glia, 72(2):433-451 PubMed ID: 37870193
Summary: Mitochondria support the energetic demands of the cells. Autophagic turnover of mitochondria serves as a critical pathway for mitochondrial homeostasis. It is unclear how bioenergetics and autophagy are functionally connected. This study identified an endolysosomal membrane protein that facilitates autophagy to regulate ATP production in glia. Drosophila tweety (tty) was determined to be highly expressed in glia and localized to endolysosomes. Diminished fusion between autophagosomes and endolysosomes in tty-deficient glia was rescued by expressing the human Tweety Homolog 1 (TTYH1). Loss of tty in glia attenuated mitochondrial turnover, elevated mitochondrial oxidative stress, and impaired locomotor functions. The cellular and organismal defects were partially reversed by antioxidant treatment. Live-cell imaging of genetically encoded metabolite sensors was performed to determine the impact of tty and autophagy deficiencies on glial bioenergetics. tty-deficient glia exhibited reduced mitochondrial pyruvate consumption accompanied by a shift toward glycolysis for ATP production. Likewise, genetic inhibition of autophagy in glia resulted in a similar glycolytic shift in bioenergetics. Furthermore, the survival of mutant flies became more sensitive to starvation, underlining the significance of tty in the crosstalk between autophagy and bioenergetics. Together, these findings uncover the role for tty in mitochondrial homeostasis via facilitating autophagy, which determines bioenergetic balance in glia. | Yoo, J., Dombrovski, M., Mirshahidi, P., Nern, A., LoCascio, S. A., Zipursky, S. L., Kurmangaliyev, Y. Z. (2023). Brain wiring determinants uncovered by integrating connectomes and transcriptomes. Curr Biol, 33(18):3998-4005. PubMed ID: 37647901
Summary: Advances in brain connectomics have demonstrated the extraordinary complexity of neural circuits. Developing neurons encounter the axons and dendrites of many different neuron types and form synapses with only a subset of them. During circuit assembly, neurons express cell-type-specific repertoires comprising many cell adhesion molecules (CAMs) that can mediate interactions between developing neurites. Many CAM families have been shown to contribute to brain wiring in different ways. It has been challenging, however, to identify receptor-ligand pairs directly matching neurons with their synaptic targets. This study integrated the synapse-level connectome of the neural circuit with the developmental expression patterns and binding specificities of CAMs on pre- and postsynaptic neurons in the Drosophila visual system. To overcome the complexity of neural circuits, focus was placed on pairs of genetically related neurons that make differential wiring choices. In the motion detection circuit, closely related subtypes of T4/T5 neurons choose between alternative synaptic targets in adjacent layers of neuropil. This choice correlates with the matching expression in synaptic partners of different receptor-ligand pairs of the Beat and Side families of CAMs. Genetic analysis demonstrated that presynaptic Side-II and postsynaptic Beat-VI restrict synaptic partners to the same layer. Removal of this receptor-ligand pair disrupts layers and leads to inappropriate targeting of presynaptic sites and postsynaptic dendrites. It is proposed that different Side/Beat receptor-ligand pairs collaborate with other recognition molecules to determine wiring specificities in the fly brain. Combining transcriptomes, connectomes, and protein interactome maps allow unbiased identification of determinants of brain wiring. |
| Willot, Q., du Toit, A., de Wet, S., Huisamen, E. J., Loos, B., Terblanche, J. S. (2023). Exploring the connection between autophagy and heat-stress tolerance in Drosophila melanogaster. Proceedings Biological sciences, 290(2006):20231305 PubMed ID: 37700658
Summary: Mechanisms aimed at recovering from heat-induced damages are closely associated with the ability of ectotherms to survive exposure to stressful temperatures. Autophagy, a ubiquitous stress-responsive catabolic process, has recently gained renewed attention as one of these mechanisms. By increasing the turnover of cellular structures as well as the clearance of long-lived protein and protein aggregates, the induction of autophagy has been linked to increased tolerance to a range of abiotic stressors in diverse ectothermic organisms. However, whether a link between autophagy and heat-tolerance exists in insect models remains unclear despite broad ecophysiological implications thereof. This study explored the putative association between autophagy and heat-tolerance using Drosophila melanogaster as a model. It was hypothesized that (1) heat-stress would cause an increase of autophagy in flies' tissues, and (2) rapamycin exposure would trigger a detectable autophagic response in adults and increase their heat-tolerance. In line with this hypothesis, it is reported that flies exposed to heat-stress present signs of protein aggregation and appear to trigger an autophagy-related homoeostatic response as a result. It was further shown that rapamycin feeding causes the systemic effect associated with target of rapamycin (TOR) inhibition, induces autophagy locally in the fly gut, and increases the heat-stress tolerance of individuals. These results argue in favour of a substantial contribution of autophagy to the heat-stress tolerance mechanisms of insects. | Cachoux, V. M. L., Balakireva, M., Gracia, M., Bosveld, F., Lopez-Gay, J. M., Maugarny, A., Gaugue, I., di Pietro, F., Rigaud, S. U., Noiret, L., Guirao, B., Bellaiche, Y. (2023). Epithelial apoptotic pattern emerges from global and local regulation by cell apical area. Curr Biol, 33(22):4807-4826.e4806 PubMed ID: 37827152
Summary: Geometry is a fundamental attribute of biological systems, and it underlies cell and tissue dynamics. Cell geometry controls cell-cycle progression and mitosis and thus modulates tissue development and homeostasis. In sharp contrast and despite the extensive characterization of the genetic mechanisms of caspase activation, little is known about whether and how cell geometry controls apoptosis commitment in developing tissues. This study combined multiscale time-lapse microscopy of developing Drosophila epithelium, quantitative characterization of cell behaviors, and genetic and mechanical perturbations to determine how apoptosis is controlled during epithelial tissue development. Early in cell lives and well before extrusion, apoptosis commitment is linked to two distinct geometric features: a small apical area compared with other cells within the tissue and a small relative apical area with respect to the immediate neighboring cells. These global and local geometric characteristics are shown to be sufficient to recapitulate the tissue-scale apoptotic pattern. Furthermore, the coupling between these two geometric features and apoptotic cells is shown to be dependent on the Hippo/YAP and Notch pathways. Overall, by exploring the links between cell geometry and apoptosis commitment, this work provides important insights into the spatial regulation of cell death in tissues and improves understanding of the mechanisms that control cell number and tissue size. |
| Yamada, T., Yoshinari, Y., Tobo, M., Habara, O., Nishimura, T. (2023). Nacα protects the larval fat body from cell death by maintaining cellular proteostasis in Drosophila. Nat Commun, 14(1):5328 PubMed ID: 37658058
Summary: Protein homeostasis (proteostasis) is crucial for the maintenance of cellular homeostasis. Impairment of proteostasis activates proteotoxic and unfolded protein response pathways to resolve cellular stress or induce apoptosis in damaged cells. However, the responses of individual tissues to proteotoxic stress and evoking cell death program have not been extensively explored in vivo. This study shows that a reduction in Nascent polypeptide-associated complex protein alpha subunit (Nacα) specifically and progressively induces cell death in Drosophila fat body cells. Nacα mutants disrupt both ER integrity and the proteasomal degradation system, resulting in caspase activation through JNK and p53. Although forced activation of the JNK and p53 pathways was insufficient to induce cell death in the fat body, the reduction of Nacα sensitized fat body cells to intrinsic and environmental stresses. Reducing overall protein synthesis by mTor inhibition or Minute mutants alleviated the cell death phenotype in Nacα mutant fat body cells. This work revealed that Nacα is crucial for protecting the fat body from cell death by maintaining cellular proteostasis, thus demonstrating the coexistence of a unique vulnerability and cell death resistance in the fat body. | Pai, Y. L., Lin, Y. J., Peng, W. H., Huang, L. T., Chou, H. Y., Wang, C. H., Chien, C. T., Chen, G. C. (2023). The deubiquitinase Leon/USP5 interacts with Atg1/ULK1 and antagonizes autophagy. Cell Death Dis, 14(8):540 PubMed ID: 37607937
Summary: Accumulating evidence has shown that the quality of proteins must be tightly monitored and controlled to maintain cellular proteostasis. Misfolded proteins and protein aggregates are targeted for degradation through the ubiquitin proteasome (UPS) and autophagy-lysosome systems. The ubiquitination and deubiquitinating enzymes (DUBs) have been reported to play pivotal roles in the regulation of the UPS system. However, the function of DUBs in the regulation of autophagy remain to be elucidated. This study found that knockdown of Leon/USP5 caused a marked increase in the formation of autophagosomes and autophagic flux under well-fed conditions. Genetic analysis revealed that overexpression of Leon suppressed Atg1-induced cell death in Drosophila. Immunoblotting assays further showed a strong interaction between Leon/USP5 and the autophagy initiating kinase Atg1/ULK1. Depletion of Leon/USP5 led to increased levels of Atg1/ULK1. These findings indicate that Leon/USP5 is an autophagic DUB that interacts with Atg1/ULK1, negatively regulating the autophagic process. |
Tuesday, May 28th - Cancer, Tumors and Growth/h3> |
| Khalili, D., Mohammed, M., Kunc, M., Sindlerova, M., Ankarklev, J., Theopold, U. (2023). Single-cell sequencing of tumor-associated macrophages in a Drosophila model. Frontiers in immunology, 14:1243797 PubMed ID: 37795097
Summary: Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized. This study used a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (Ras(V12)) to study dysplastic growth during early tumor progression. Single-cell RNA-sequencing was performed of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells. Five distinct hemocyte clusters were identified. In addition to Ras(V12) larvae, a tumor model was included where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells-they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors-antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, evidence is provided for transcript transfer between hemocytes and possibly other tissues. Taken together, these data support the usefulness of Drosophila to study the response against tumors at the organismic level. | Yu, K., Ramkumar, N., Wong, K. K. L., Tettweiler, G., Verheyen, E. M. (2023). The AMPK-like protein kinases Sik2 and Sik3 interact with Hipk and induce synergistic tumorigenesis in a Drosophila cancer model. Frontiers in cell and developmental biology, 11:1214539 PubMed ID: 37854071
Summary: Homeodomain-interacting protein kinases (Hipks) regulate cell proliferation, apoptosis, and tissue development. Overexpression of Hipk in Drosophila causes tumorigenic phenotypes in larval imaginal discs. This study found that depletion of Salt-inducible kinases Sik2 or Sik3 can suppress Hipk-induced overgrowth. Furthermore, co-expression of constitutively active forms of Sik2 or Sik3 with Hipk caused significant tissue hyperplasia and tissue distortion, indicating that both Sik2 and Sik3 can synergize with Hipk to promote tumorous phenotypes, accompanied by elevated dMyc, Armadillo/β-catenin, and the Yorkie target gene expanded. Larvae expressing these hyperplastic growths also display an extended larval phase, characteristic of other Drosophila tumour models. Examination of total protein levels from fly tissues showed that Hipk proteins were reduced when Siks were depleted through RNAi, suggesting that Siks may regulate Hipk protein stability and/or activity. Conversely, expression of constitutively active Siks with Hipk leads to increased Hipk protein levels. Furthermore, Hipk can interact with Sik2 and Sik3 by co-immunoprecipitation. Co-expression of both proteins leads to a mobility shift of Hipk protein, suggesting it is post-translationally modified. In summary, this research demonstrates a novel function of Siks in synergizing with Hipk to promote tumour growth. |
| Bosch, P. S., Cho, B., Axelrod, J. D. (2023). . Flamingo participates in multiple models of cell competition. bioRxiv, PubMed ID: 37790459
Summary: The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. This study shows that in Drosophila, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in pre-malignant cells as they grow among wildtype cells, in healthy cells to eliminate pre-malignant cells, and by supercompetitors to occupy excessive territory within wildtype tissues. "Would-be" winners that lack Fmi are unable to over-proliferate, and instead become losers. This study demonstrate that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well defined functions of Fmi. | Pranoto, I. K. A., Lee, J., Kwon, Y. V. (2023). The roles of the native cell differentiation program aberrantly recapitulated in Drosophila intestinal tumors. Cell Rep, 42(10):113245 PubMed ID: 37837622
Summary: Many tumors recapitulate the developmental and differentiation program of their tissue of origin, a basis for tumor cell heterogeneity. Although stem-cell-like tumor cells are well studied, the roles of tumor cells undergoing differentiation remain to be elucidated. This study employed Drosophila genetics to demonstrate that the differentiation program of intestinal stem cells is crucial for enabling intestinal tumors to invade and induce non-tumor-autonomous phenotypes. The differentiation program that generates absorptive cells is aberrantly recapitulated in the intestinal tumors generated by activation of the Yap1 ortholog Yorkie. Inhibiting it allows stem-cell-like tumor cells to grow but suppresses invasiveness and reshapes various phenotypes associated with cachexia-like wasting by altering the expression of tumor-derived factors. This study provides insight into how a native differentiation program determines a tumor's capacity to induce advanced cancer phenotypes and suggests that manipulating the differentiation programs co-opted in tumors might alleviate complications of cancer, including cachexia. |
| Quintero, M., Bangi, E. (2023). Disruptions in cell fate decisions and transformed enteroendocrine cells drive intestinal tumorigenesis in Drosophila. Cell Rep, 42(11):113370 PubMed ID: 37924517
Summary: Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between different cell types ensures precise regulation of stem cell behavior and cell fate decisions. These cell-cell interactions are often disrupted during tumorigenesis, but mechanisms by which they are co-opted to support tumor growth in different genetic contexts are poorly understood. This study introduces PromoterSwitch, a genetic platform established to generate large, transformed clones derived from individual adult Drosophila intestinal stem/progenitor cells. This study showed that cancer-driving genetic alterations representing common colon tumor genome landscapes disrupt cell fate decisions within transformed tissue and result in the emergence of abnormal cell fates. It was also shown that transformed enteroendocrine cells, a differentiated, hormone-secreting cell lineage, support tumor growth by regulating intestinal stem cell proliferation through multiple genotype-dependent mechanisms, which represent potential vulnerabilities that could be exploited for therapy. | Zheng, J., Guo, Y., Shi, C., Yang, S., Xu, W., Ma, X. (2023). Differential Ire1 determines loser cell fate in tumor-suppressive cell competition. Cell Rep, 42(11):113303 PubMed ID: 37924514
Summary: Tumor-suppressive cell competition (TSCC) is a conserved surveillance mechanism in which neighboring cells actively eliminate oncogenic cells. Despite overwhelming studies showing that the unfolded protein response (UPR) is dysregulated in various tumors, it remains debatable whether the UPR restrains or promotes tumorigenesis. Using Drosophila eye epithelium as a model, this study uncovered a surprising decisive role of the Ire1 branch of the UPR in regulating cell polarity gene scribble (scrib) loss-induced TSCC. Both mutation and hyperactivation of Ire1 accelerate elimination of scrib clones via inducing apoptosis and autophagy, respectively. Unexpectedly, relative Ire1 activity is also crucial for determining loser cell fate, as dysregulating Ire1 signaling in the surrounding healthy cells reversed the "loser" status of scrib clones by decreasing their apoptosis. Furthermore, it was shown that Ire1 is required for cell competition in mammalian cells. Together, these findings provide molecular insights into scrib-mediated TSCC and highlight Ire1 as a key determinant of loser cell fate. |
Friday, May 24th - Disease Models |
| Yan, L., Zhou, J., Yuan, L., Ye, J., Zhao, X., Ren, G., Chen, H. (2023). Silibinin alleviates intestinal inflammation via inhibiting JNK signaling in Drosophila. Frontiers in pharmacology, 14:1246960 PubMed ID: 37781701
Summary: Inflammatory bowel diseases (IBDs) are characterized by chronic relapsing intestinal inflammation that causes digestive system dysfunction. For years, researchers have been working to find more effective and safer therapeutic strategies to treat these diseases. Silibinin (SIL), a flavonoid compound extracted from the seeds of milk thistle plants, possesses multiple biological activities and is traditionally applied to treat liver diseases. SIL is also widely used in the treatment of a variety of inflammatory diseases attributed to its excellent antioxidant and anti-inflammatory effects. However, the efficacy of SIL against IBDs and its mechanisms remain unclear. This study, using Drosophila melanogaster as a model organism, found that SIL can effectively relieve intestinal inflammation caused by dextran sulfate sodium (DSS). The results suggested that SIL supplementation can inhibit the overproliferation of intestinal stem cells (ISCs) induced by DSS, protect intestinal barrier function, acid-base balance, and intestinal excretion function, reduce intestinal reactive oxygen species (ROS) levels and inflammatory stress, and extend the lifespan of Drosophila. Furthermore, this study demonstrated that SIL ameliorates intestinal inflammation via modulating the c-Jun N-terminal kinase (JNK) signaling pathway in Drosophila. This research aims to provide new insight into the treatment of IBDs. | Aalto, A. L., Saadabadi, A., Lindholm, F., Kietz, C., Himmelroos, E., Marimuthu, P., Salo-Ahen, O. M. H., Eklund, P., Meinander, A. (2023). Stilbenoid compounds inhibit NF-κB-mediated inflammatory responses in the Drosophila intestine. Frontiers in immunology, 14:1253805 PubMed ID: 37809071
Summary: Stilbenoid compounds have been described to have anti-inflammatory properties in animal models in vivo, and have been shown to inhibit Ca2+-influx through the transient receptor potential ankyrin 1 (TrpA1). To study how stilbenoid compounds affect inflammatory signaling in vivo, this study has utilized the fruit fly, Drosophila melanogaster, as a model system. To induce intestinal inflammation, flies were fed with the intestinal irritant dextran sodium sulphate (DSS). DSS was found to induce severe changes in the bacteriome of the Drosophila intestine, and that this dysbiosis causes activation of the NF-κB transcription factor Relish. Advantage was taken of the DSS-model to study the anti-inflammatory properties of the stilbenoid compounds pinosylvin (PS) and pinosylvin monomethyl ether (PSMME). With the help of in vivo approaches, PS and PSMME were found to be to be transient receptor ankyrin 1 (TrpA1)-dependent antagonists of NF-κB-mediated intestinal immune responses in Drosophila. This study has also computationally predicted the putative antagonist binding sites of these compounds at Drosophila TrpA1. Taken together, this study showed that the stilbenoids PS and PSMME have anti-inflammatory properties in vivo in the intestine and can be used to alleviate chemically induced intestinal inflammation in Drosophila. |
| Fan, X., Huang, T., Wang, S., Yang, Z., Song, W., Zeng, Y., Tong, Y., Cai, Y., Yang, D., Zeng, B., Zhang, M., Ni, Q., Li, Y., Li, D., Yang, M. (2023). The adaptor protein 14-3-3zeta modulates intestinal immunity and aging in Drosophila. J Biol Chem, 299(12):105414 PubMed ID: 37918806
Summary: The proteins that coordinate the complex transcriptional networks of aging have not been completely documented. Protein 14-3-3zeta is an adaptor protein that coordinates signaling and transcription factor networks, but its function in aging is not fully understood. This study showed that the protein expression of 14-3-3zeta gradually increased during aging. High levels of 14-3-3zeta led to shortened lifespan and imbalance of intestinal immune homeostasis in Drosophila, but the decrease in 14-3-3zeta protein levels by RNAi was able to significantly promote the longevity and intestinal immune homeostasis of fruit flies. Importantly, it was demonstrated that adult-onset administration of TIC10, a compound that reduces the aging-related AKT and ">extracellular signal-regulated kinase (ERK) signaling pathways, rescues the shortened lifespan of 14-3-3zeta-overexpressing flies. This finding suggests that 14-3-3zeta plays a critical role in regulating the aging process. This study elucidates the role of 14-3-3zeta in natural aging and provides the rationale for subsequent 14-3-3zeta-based antiaging research. | Diaw, S. H., Borsche, M., Streubel-Gallasch, L., Dulovic-Mahlow, M., Hermes, J., Lenz, I., Seibler, P., Klein, C., Bruggemann, N., Vos, M., Lohmann, K. (2023). Characterization of the pathogenic α-Synuclein Variant V15A in Parkinson´s disease. NPJ Parkinson's disease. 9(1):148 PubMed ID: 37903765
Summary: Despite being a major component of Lewy bodies and Lewy neurites, pathogenic variants in the gene encoding alpha-Synuclein (α-Syn) are rare. To date, only four missense variants in the SNCA gene, encoding α-Syn have unequivocally been shown to be disease-causing. This study describes a Parkinson´s disease patient with early cognitive decline carrying an as yet not fully characterized variant in SNCA. Different cellular models, including stably transfected neuroblastoma (SH-SY5Y) cell cultures, induced pluripotent stem cell (iPSC)-derived neuronal cultures, and generated a Drosophila model to elucidate the impact of the p.V15A variant on α-Syn function and aggregation properties compared to other known pathogenic variants. This study demonstrated that p.V15A increased the aggregation potential of α-Syn and the levels of apoptotic markers, and impaired the mitochondrial network. Moreover, p.V15A affects the flying ability and survival of mutant flies. Thus, this study provides supporting evidence for the pathogenicity of the p.V15A variant, suggesting its inclusion in genetic testing approaches. |
| Wang, C. W., Clemot, M., Hashimoto, T., Diaz, J. A., Goins, L. M., Goldstein, A. S., Nagaraj, R., Banerjee, U. (2023). A conserved mechanism for JNK-mediated loss of Notch function in advanced prostate cancer. Science signaling, 16(810):eabo5213 PubMed ID: 37934809
Summary: ysregulated Notch signaling is a common feature of cancer; however, its effects on tumor initiation and progression are highly variable, with Notch having either oncogenic or tumor-suppressive functions in various cancers. To better understand the mechanisms that regulate Notch function in cancer, Notch signaling was studied in a Drosophila tumor model, prostate cancer-derived cell lines, and tissue samples from patients with advanced prostate cancer. Increased activity of the Src-JNK pathway in tumors inactivated Notch signaling because of JNK pathway-mediated inhibition of the expression of the gene encoding the Notch S2 cleavage protease, Kuzbanian, which is critical for Notch activity. Consequently, inactive Notch accumulated in cells, where it was unable to transcribe genes encoding its target proteins, many of which have tumor-suppressive activities. These findings suggest that Src-JNK activity in tumors predicts Notch activity status and that suppressing Src-JNK signaling could restore Notch function in tumors, offering opportunities for diagnosis and targeted therapies for a subset of patients with advanced prostate cancer. | Zane, F., Bouzid, H., Sosa Marmol, S., Brazane, M., Besse, S., Molina, J. L., Cansell, C., Aprahamian, F., Durand, S., Ayache, J., Antoniewski, C., Todd, N., Carre, C., Rera, M. (2023). Smurfness-based two-phase model of ageing helps deconvolve the ageing transcriptional signature. Aging Cell, 22(11):e13946 PubMed ID: 37822253
Summary: Ageing is characterised at the molecular level by six transcriptional 'hallmarks of ageing', that are commonly described as progressively affected as time passes. By contrast, the 'Smurf' assay, which assesses food intake by the co-ingestion of a blue dye, which detects increased intestinal permeability. Performing whole body total RNA sequencing, it was found that Smurfness distinguishes transcriptional changes associated with chronological age from those associated with biological age. Transcriptional heterogeneity increases with chronological age in non-Smurf individuals preceding the other five hallmarks of ageing that are specifically associated with the Smurf state. Using this approach, this study devised targeted pro-longevity genetic interventions delaying entry in the Smurf state. It is anticipated that increased attention to the evolutionary conserved Smurf phenotype will bring about significant advances in understanding of the mechanisms of ageing. |
Thursday, May 23nd - Cell Cycle |
| Koury, S. A. (2023). Female meiotic drive shapes the distribution of rare inversion polymorphisms in Drosophila melanogaster. Genetics. PubMed ID: 37616566
Summary: In all species, new chromosomal inversions are constantly being formed by spontaneous rearrangement and then stochastically eliminated from natural populations. In Drosophila, when new chromosomal inversions overlap with a pre-existing inversion in the population, their rate of elimination becomes a function of the relative size, position, and linkage phase of the gene rearrangements. These altered dynamics result from complex meiotic behavior wherein overlapping inversions generate asymmetric dyads that cause both meiotic drive/drag and segmental aneuploidy. In this context, patterns in rare inversion polymorphisms of a natural population can be modeled from the fundamental genetic processes of forming asymmetric dyads via crossing-over in meiosis I and preferential segregation from asymmetric dyads in meiosis II. In this study a mathematical model of crossover-dependent female meiotic drive is developed and parameterized with published experimental data from Drosophila melanogaster laboratory constructs. This mechanism is demonstrated to favor smaller, distal inversions and accelerate the elimination of larger, proximal inversions. Simulated sampling experiments indicate that the paracentric inversions directly observed in natural population surveys of Drosophila melanogaster are a biased subset that both maximizes meiotic drive and minimizes the frequency of lethal zygotes caused by this cytogenetic mechanism. Incorporating this form of selection into a population genetic model accurately predicts the shift in relative size, position, and linkage phase for rare inversions found in this species. The model and analysis presented in this study suggest that this weak form of female meiotic drive is an important process influencing the genomic distribution of rare inversion polymorphisms. | Haseeb, M. A., Weng, K. A., Bickel, S. E. (2023). Chromatin-associated cohesin turns over extensively and forms new cohesive linkages during meiotic prophase. bioRxiv, PubMed ID: 37645916
Summary: In dividing cells, accurate chromosome segregation depends on sister chromatid cohesion, protein linkages that are established during DNA replication. Faithful chromosome segregation in oocytes requires that cohesion, first established in S phase, remain intact for days to decades, depending on the organism. Premature loss of meiotic cohesion in oocytes leads to the production of aneuploid gametes and contributes to the increased incidence of meiotic segregation errors as women age (maternal age effect). The prevailing model is that cohesive linkages do not turn over in mammalian oocytes. However, it has been previously reported that cohesion-related defects arise in Drosophila oocytes when individual cohesin subunits (see SMC1) cohesin regulators are knocked down after meiotic S phase. This study use two strategies to express a tagged cohesin subunit exclusively during mid-prophase in Drosophila oocytes and demonstrate that newly expressed cohesin is used to form de novo linkages after meiotic S phase. Moreover, nearly complete turnover of chromosome-associated cohesin occurs during meiotic prophase, with faster replacement on the arms than at the centromeres. Unlike S-phase cohesion establishment, the formation of new cohesive linkages during meiotic prophase does not require acetylation of conserved lysines within the Smc3 head. These findings indicate that maintenance of cohesion between S phase and chromosome segregation in Drosophila oocytes requires an active cohesion rejuvenation program that generates new cohesive linkages during meiotic prophase. |
| Warecki, B. and Tao, L. (2023). Centralspindlin-mediated transport of RhoGEF positions the cleavage plane for cytokinesis.. Sci Signal 16(792): eadh0601. PubMed ID: 37402224
Summary: During cytokinesis, the cell membrane furrows inward along a cleavage plane. The positioning of the cleavage plane is critical to faithful cell division and is determined by the Rho guanine nucleotide exchange factor (RhoGEF)-mediated activation of the small guanosine triphosphatase RhoA and the conserved motor protein complex centralspindlin. This study explored whether and how centralspindlin mediates the positioning of RhoGEF. In dividing neuroblasts from Drosophila melanogaster, it was observed that immediately before cleavage, first centralspindlin and then RhoGEF localized to the sites where cleavage subsequently initiated. Using in vitro assays with purified Drosophila proteins and stabilized microtubules, it was found that centralspindlin directly transported RhoGEF as cargo along single microtubules and sequestered it at microtubule plus-ends for prolonged periods of time. In addition, the binding of RhoGEF to centralspindlin appeared to stimulate centralspindlin motor activity. Thus, the motor activity and microtubule association of centralspindlin can translocate RhoGEF to areas where microtubule plus-ends are abundant, such as at overlapping astral microtubules, to locally activate RhoA and accurately position the cleavage plane during cell division. | Sperling, A. L., Fabian, D. K., Garrison, E. and Glover, D. M. (2023). A genetic basis for facultative parthenogenesis in Drosophila. Curr Biol. PubMed ID: 37516115
Summary: Facultative parthenogenesis enables sexually reproducing organisms to switch between sexual and asexual parthenogenetic reproduction. To gain insights into this phenomenon, the genomes of sexually reproducing and parthenogenetic strains of Drosophila mercatorum were sequenced, and differences were identified in the gene expression in their eggs. Then whether manipulating the expression of candidate gene homologs identified in Drosophila mercatorum could lead to facultative parthenogenesis in the non-parthenogenetic species Drosophila melanogaster was tested. This identified a polygenic system whereby increased expression of the mitotic protein kinase polo and decreased expression of a desaturase, Desat2, caused facultative parthenogenesis in the non-parthenogenetic species that was enhanced by increased expression of Myc. The genetically induced parthenogenetic Drosophila melanogaster eggs exhibit de novo centrosome formation, fusion of the meiotic products, and the onset of development to generate predominantly triploid offspring. Thus, this study demonstrated a genetic basis for sporadic facultative parthenogenesis in an animal. |
| Bakshi, A., Iturra, F. E., Alamban, A., Rosas-Salvans, M., Dumont, S., Aydogan, M. G. (2023). Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes. Cell, 186(21):4694-4709. PubMed ID: 37832525
Summary: Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. This study demonstrates in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, evidence is presented that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. It is postulated that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes. | Baker, C. C., Gallicchio, L., Matias, N. R., Porter, D. F., Parsanian, L., Taing, E., Tam, C., Fuller, M. T. (2023). Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase. Development, 150(22) PubMed ID: 37882771
Summary: During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. This study showa that the spermatocyte-specific protein Lutin (Lut) is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase. |
Wednesday, May 22nd - Immune Response |
| Jin, Q., Wang, Y., Yin, H., Jiang, H. (2023). Two clip-domain serine protease homologs, cSPH35 and cSPH242, act as a cofactor for prophenoloxidase-1 activation in Drosophila melanogaster. Frontiers in immunology, 14:1244792 PubMed ID: 37781370
Summary: Insect phenoloxidases (POs) catalyze phenol oxygenation and o-diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high M(r) cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila, PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. This study produced the recombinant cSPH35 and cSPH242 precursors, activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila PPO1 activation by MP2 (i.e., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta. In the presence of high M(r) complexes of the cSPHs, PO at a high specific activity of 260 U/μg was generated in vitro. To complement the in vitro analysis, hemolymph PO activity levels were measured in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/μl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults. | Tafesh-Edwards, G., Eleftherianos, I. (2023). The Drosophila melanogaster prophenoloxidase system participates in immunity against Zika virus infection.. Eur J Immunol, 53(12):e2350632 PubMed ID: 37793051
Summary: Drosophila melanogaster relies on an evolutionarily conserved innate immune system to protect itself from a wide range of pathogens, making it a convenient genetic model to study various human pathogenic viruses and host antiviral immunity. This study explored for the first time the contribution of the Drosophila phenoloxidase (PO) system to host survival and defenses against Zika virus (ZIKV) infection by analyzing the role of mutations in the three prophenoloxidase (PPO) genes in female and male flies. Only PPO1 and PPO2 genes contribute to host survival and appear to be upregulated following ZIKV infection in Drosophila. Also, data suggesting that a complex regulatory system exists between Drosophila PPOs, potentially allowing for a sex-dependent compensation of PPOs by one another or other immune responses such as the Toll, Imd, and JAK/STAT pathways. Furthermore, this study showed that PPO1 and PPO2 are essential for melanization in the hemolymph and the wound site in flies upon ZIKV infection. These results reveal an important role played by the melanization pathway in response to ZIKV infection, hence highlighting the importance of this pathway in insect host defense against viral pathogens and potential vector control strategies to alleviate ZIKV outbreaks. |
| Elguero, J. E., Liu, G., Tiemeyer, K., Bandyadka, S., Gandevia, H., Duro, L., Yan, Z., McCall, K. (2023). Defective phagocytosis leads to neurodegeneration through systemic increased innate immune signaling. iScience, 26(10):108052 PubMed ID: 37854687
Summary: 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. To untangle the links between glial phagocytosis and neurodegeneration, Drosophila mutants lacking the phagocytic receptor Draper were investigated. Loss of Draper leads to persistent neuronal cell corpses and age-dependent neurodegeneration. Whether the phagocytic defects observed in draper mutants lead to chronic increased immune activation that promotes neurodegeneration was investigate. It was found that the antimicrobial peptide Attacin-A is highly upregulated in the fat body of aged draper mutants and that the inhibition of the Immune deficiency (Imd) pathway in the glia and fat body of draper mutants led to reduced neurodegeneration. Taken together, these findings indicate that phagocytic defects lead to neurodegeneration via increased immune signaling, both systemically and locally in the brain. | Pan, W., Yao, X., Lin, L., Liu, X., Jin, P., Ma, F. (2023). The Relish/miR-275/Dredd mediated negative feedback loop is crucial to restoring immune homeostasis of Drosophila Imd pathway. Insect biochemistry and molecular biology, 162:104013 PubMed ID: 37804878
Summary: The NF-κB/Relish, as a core transcription factor of Drosophila immune deficiency (Imd) pathway, activates the transcriptions of antimicrobial peptides (AMPs) to combat gram-negative bacterial infections, but its role in regulating miRNA expression during immune response has less been reported. This study describes a negative feedback loop of Imd signaling mediated by Relish/miR-275/Dredd that controls Drosophila immune homeostasis after Escherichia coli (E. coli) infection. The results demonstrate that Relish may directly activate the transcription of miR-275 via binding to its promoter in vitro and vivo, particularly miR-275 further inhibits the expression of Dredd through binding to its 3'UTR to negatively control Drosophila Imd immune response. Remarkably, the ectopic expression of miR-275 significantly reduces Drosophila lifespan. More importantly, this work uncovers a new mechanism by which Relish can flexibly switch its role to maintain Drosophila immune response and homeostasis during infection. Collectively, this study not only reveals the functional duality of Relish in regulating immune response of Drosophila Imd pathway, but also provides a new insight into the maintenance of animal innate immune homeostasis. |
| Arias-Rojas, A., Frahm, D., Hurwitz, R., Brinkmann, V., Iatsenko, I. (2023). Resistance to host antimicrobial peptides mediates resilience of gut commensals during infection and aging in Drosophila. Proc Natl Acad Sci U S A, 120(36):e2305649120 PubMed ID: 37639605
Summary: Resilience to short-term perturbations, like inflammation, is a fundamental feature of microbiota, yet the underlying mechanisms of microbiota resilience are incompletely understood. This study shows that Lactiplantibacillus plantarum, a major Drosophila commensal, stably colonizes the fruit fly gut during infection and is resistant to Drosophila antimicrobial peptides (AMPs). By transposon screening, L. plantarum mutants sensitive to AMPs were identified. These mutants were impaired in peptidoglycan O-acetylation or teichoic acid D-alanylation, resulting in increased negative cell surface charge and higher affinity to cationic AMPs. AMP-sensitive mutants were cleared from the gut after infection and aging-induced gut inflammation in wild-type, but not in AMP-deficient flies, suggesting that resistance to host AMPs is essential for commensal resilience in an inflamed gut environment. Thus, this work reveals that in addition to the host immune tolerance to the microbiota, commensal-encoded resilience mechanisms are necessary to maintain the stable association between host and microbiota during inflammation. | Cai, H., Li, L., Slavik, K. M., Huang, J., Yin, T., Ai, X., Hedelin, L., Haas, G., Xiang, Z., Yang, Y., Li, X., Chen, Y., Wei, Z., Deng, H., Chen, D., Jiao, R., Martins, N., Meignin, C., Kranzusch, P. J., Imler, J. L. (2023). The virus-induced cyclic dinucleotide 2'3'-c-di-GMP mediates STING-dependent antiviral immunity in Drosophila. Immunity, 56(9):1991-2005 PubMed ID: 37659413
Summary: In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP, which triggers STING-dependent immunity. In Drosophila melanogaster, two cGAS-like receptors (cGLRs) produce 3'2'-cGAMP and 2'3'-cGAMP to activate STING. CDN-mediated immunity was explored in 14 Drosophila species covering 50 million years of evolution; 2'3'-cGAMP and 3'2'-cGAMP failed to control infection by Drosophila C virus in D. serrata and two other species. Diverse CDNs produced in a cGLR-dependent manner were descovered in response to viral infection in D. melanogaster, including 2'3'-c-di-GMP. This CDN was a more potent STING agonist than cGAMP in D. melanogaster and it also activated a strong antiviral transcriptional response in D. serrata. These results shed light on the evolution of cGLRs in flies and provide a basis for understanding the function and regulation of this emerging family of pattern recognition receptors in animal innate immunity. |
Tuesday, May 21st - Enzymes and protein expression, evolution, structure, and function |
| Yushkova, E. (2024). . Interaction effect of mutations in the genes (piwi and aub) of the Argonaute family and hobo transposons on the integral survival parameters of Drosophila melanogaster. Biogerontology, 25(1):131-146 PubMed ID: 37864608
Summary: The Argonaute family genes (piwi and aub) involved in the production of small RNAs are responsible for the regulation of many cellular processes, including the suppression of genome instability, modulation of gene activity, and transposable elements. Dysfunction of these genes and the associated activation of transposable elements adversely affect reproductive development and quality of life. The role of transposons in contrast to retrotransposons and their interaction with genes of the Argonaute family in aging processes have not been studied. This study considers a scenario in which the piwi and aub genes in the presence of functional hobo transposons can modify the effects from the level of DNA damage to lifespan. The simultaneous presence of mutation (piwi or aub) and hobo (regardless of size) in the genome has practically no effect or (less often) leads to a decrease in the level of DNA damage in ovarian cells. A high level of sterility and low ovarian reserve were noted mainly with a combination of mutations and full-sized hobo elements. The combination of these two genetic factors negatively affects the fertility of young females and embryonic survival. Isolated cases of restoration of reproductive functions with age were noted but only in females that had low fertility in the early period of life. The presence of hobo transposons contributed to an increase in the lifespan of both mutant and non-mutant females. Dysfunction of the piwi and aub genes (without hobo) can reduce the lifespan of both sexes. Together, each mutation and hobo transposons act antagonistically/additively (in females) and synergistically/antagonistically (in males) to change the lifespan. In parameters of locus-specific instability, hobo activation was more pronounced in piwi gene dysfunction. The results obtained complement data on the study of new functions of Argonaute family genes and their interactions with transposable elements in the aging process. | Krzywinska, E., Ribeca, P., Ferretti, L., Hammond, A., Krzywinski, J. (2023). A novel factor modulating X chromosome dosage compensation in Anopheles. Curr Biol, 33(21):4697-4703. PubMed ID: 37774706
Summary: Dosage compensation (DC), a process countering chromosomal imbalance in individuals with heteromorphic sex chromosomes, has been molecularly characterized only in mammals, Caenorhabditis elegans, and fruit flies. In Drosophila melanogaster males, it is achieved by an approximately 2-fold hypertranscription of the monosomic X chromosome mediated by the MSL complex. The complex is not assembled on female X chromosomes because production of its key protein MSL-2 is prevented due to intron retention and inhibition of translation by Sex-lethal, a female-specific protein operating at the top of the sex determination pathway.) It remains unclear how DC is mechanistically regulated in other insects. In the malaria mosquito Anopheles gambiae, an approximately 2-fold hypertranscription of the male X also occurs by a yet-unknown molecular mechanism distinct from that in D. melanogaster. This study shows that a male-specifically spliced gene call 007, which arose by a tandem duplication in the Anopheles ancestral lineage, is involved in the control of DC in males. Homozygous 007 knockouts lead to a global downregulation of the male X, phenotypically manifested by a slower development compared to wild-type mosquitoes or mutant females-however, without loss of viability or fertility. In females, a 007 intron retention promoted by the sex determination protein Femaleless, known to prevent hypertranscription from both X chromosomes introduces a premature termination codon apparently rendering the female transcripts non-productive. In addition to providing a unique perspective on DC evolution, the 007, with its conserved properties, may represent an important addition to a genetic toolbox for malaria vector control. |
| Liu, M., Xie, X. J., Li, X., Ren, X., Sun, J., Lin, Z., Hemba-Waduge, R. U., Ji, J. Y. (2023). Transcriptional coupling of telomeric retrotransposons with the cell cycle. bioRxiv, PubMed ID: 37808851
Summary: Instead of employing telomerases to safeguard chromosome ends, dipteran species maintain their telomeres by transposition of telomeric-specific retrotransposons (TRs): in Drosophila , these are HeT-A, TART, and TAHRE. Previous studies have shown how these TRs create tandem repeats at chromosome ends, but the exact mechanism controlling TR transcription has remained unclear. This study reports the identification of multiple subunits of the transcription cofactor Mediator complex and transcriptional factors Scalloped (Sd, the TEAD homolog in flies) and E2F1-Dp as novel regulators of TR transcription and telomere length in Drosophila . Depletion of multiple Mediator subunits, Dp, or Sd increased TR expression and telomere length, while over-expressing E2F1-Dp or knocking down the E2F1 regulator Rbf1 (Retinoblastoma-family protein 1) stimulated TR transcription, with Mediator and Sd affecting TR expression through E2F1-Dp. The CUT&RUN analysis revealed direct binding of CDK8, Dp, and Sd to telomeric repeats. These findings highlight the essential role of the Mediator complex in maintaining telomere homeostasis by regulating TR transcription through E2F1-Dp and Sd, revealing the intricate coupling of TR transcription with the host cell-cycle machinery, thereby ensuring chromosome end protection and genomic stability during cell division. | Kour, S., Fortuna, T., Anderson, E. N., Mawrie, D., Bilstein, J., Sivasubramanian, R., Ward, C., Roy, R., Rajasundaram, D., Sterneckert, J., Pandey, U. B. (2023). Drosha-dependent microRNAs modulate FUS-mediated neurodegeneration in vivo. Nucleic Acids Res, 51(20):11258-11276 PubMed ID: 37791873
Summary: Mutations in the Fused in Sarcoma (FUS) gene (see Drosophila Cabeza) cause the familial and progressive form of amyotrophic lateral sclerosis (ALS). FUS is a nuclear RNA-binding protein involved in RNA processing and the biogenesis of a specific set of microRNAs. This study reports that Drosha and two previously uncharacterized Drosha-dependent miRNAs are strong modulators of FUS expression and prevent the cytoplasmic segregation of insoluble mutant FUS in vivo. Depletion of Drosha mitigates FUS-mediated degeneration, survival and motor defects in Drosophila. Mutant FUS strongly interacts with Drosha and causes its cytoplasmic mis-localization into the insoluble FUS inclusions. Reduction in Drosha levels increases the solubility of mutant FUS. Interestingly, it was found that two Drosha dependent microRNAs, miR-378i and miR-6832-5p, which differentially regulate the expression, solubility and cytoplasmic aggregation of mutant FUS in iPSC neurons and mammalian cells. More importantly, this study reports different modes of action of these miRNAs against mutant FUS. Whereas miR-378i may regulate mutant FUS inclusions by preventing G3BP-mediated stress granule formation, miR-6832-5p may affect FUS expression via other proteins or pathways. Overall, this research reveals a possible association between ALS-linked FUS mutations and the Drosha-dependent miRNA regulatory circuit, as well as a useful perspective on potential ALS treatment via microRNAs. |
| Matzkin, L. M., Bono, J. M., Pigage, H. K., Allan, C. W., Diaz, F., McCoy, J. R., Green, C. C., Callan, J. B., Delahunt, S. P. (2023). Females translate male mRNA transferred during mating. bioRxiv, PubMed ID: 37790342
Summary: Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. This study develop an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. Strong evidence was found for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction. | Coronado-Zamora, M., Gonzalez, J. (2023). Transposons contribute to the functional diversification of the head, gut, and ovary transcriptomes across Drosophila natural strains. Genome research, 33(9):1541-1553 PubMed ID: 37793782
Summary: Transcriptomes are dynamic, with cells, tissues, and body parts expressing particular sets of transcripts. Transposable elements (TEs) are a known source of transcriptome diversity; however, studies often focus on a particular type of chimeric transcript, analyze single body parts or cell types, or are based on incomplete TE annotations from a single reference genome. This work has implemented a method based on de novo transcriptome assembly that minimizes the potential sources of errors while identifying a comprehensive set of gene-TE chimeras. This method was applied to the head, gut, and ovary dissected from five Drosophila melanogaster natural strains, with individual reference genomes available. ~19% of body part-specific transcripts are gene-TE chimeras. Overall, chimeric transcripts contribute a mean of 43% to the total gene expression, and they provide protein domains for DNA binding, catalytic activity, and DNA polymerase activity. This comprehensive data set is a rich resource for follow-up analysis. Moreover, because TEs are present in virtually all species sequenced to date, their role in spatially restricted transcript expression is likely not exclusive to the species analyzed in this work. |
Monday, May 20th - Enzymes and protein expression, evolution, structure, and function |
| Sayeesh, P. M., Iguchi, M., Suemoto, Y., Inoue, J., Inomata, K., Ikeya, T., Ito, Y. (2023). Interactions of the N- and C-Terminal SH3 Domains of Drosophila Drk with the Proline-Rich Peptides from Sos and Dos. Int J Mol Sci, 24(18) PubMed ID: 37762438
Summary: Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. A previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, NMR titration experiments were conducted using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. The results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. Docking simulations were performed based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, the various modes in the interactions of Drk with Sos/Dos are discussed. | Li, J., Zheng, H., Hou, J., Chen, J., Zhang, F., Yang, X., Jin, F., Xi, Y. (2023). X-linked RBBP7 mutation causes maturation arrest and testicular tumors. J Clin Invest, 133(20) PubMed ID: 37843278
Summary: Maturation arrest (MA) is a subtype of non-obstructive azoospermia, and male infertility is a known risk factor for testicular tumors. However, the genetic basis for many affected individuals remains unknown. This study identified a deleterious hemizygous variant of X-linked retinoblastoma-binding protein 7 (RBBP7) as a potential key cause of MA, which was also found to be associated with the development of Leydig cell tumors. This mutation resulted in premature protein translation termination, affecting the sixth WD40 domain of the RBBP7 and the interaction of the mutated RBBP7 with histone H4. Decreased BRCA1 and increased γH2AX were observed in the proband. In mouse spermatogonial and pachytene spermatocyte-derived cells, deprivation of rbbp7 led to cell cycle arrest and apoptosis. In Drosophila, knockdown of RBBP7/Caf1-55 in germ cells resulted in complete absence of germ cells and reduced testis size, whereas knockdown of RBBP7/Caf1-55 in cyst cells resulted in hyperproliferative testicular cells. Interestingly, male infertility caused by Caf1-55 deficiency was rescued by ectopic expression of wild-type human RBBP7 but not mutant variants, suggesting the importance of RBBP7 in spermatogenesis. This study provides insights into the mechanisms underlying the co-occurrence of MA and testicular tumors and may pave the way for innovative genetic diagnostics of these 2 diseases. |
| Yoon, H. J., Price, B. E., Parks, R. K., Ahn, S. J., Choi, M. Y. (2023). Diuretic hormone 31 activates two G protein-coupled receptors with differential second messengers for diuresis in Drosophila suzukii. Insect biochemistry and molecular biology, 162:104025 PubMed ID: 37813200
Summary: Diuretic hormones (DHs) bind to G protein-coupled receptors (GPCRs), regulating water and ion balance to maintain homeostasis in animals. Two distinct DHs are known in insects: calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. This study identified and characterized DH31 and two DH31 GPCR variants, DH31-Ra and DH31-Rb, from spotted-wing drosophila, Drosophila suzukii, a globally prevalent vinegar fly causing severe damage to small fruits. Both GPCRs are active, but DH31-Ra is the dominant receptor based on gene expression analyses and DH31 peptide binding affinities. A notable difference between the two variants lies in 1) the GPCR structures of their C-termini and 2) the utilization of second messengers, and the amino acid sequences of the two variants are identical. DH31-Ra contains 12 additional amino acids, providing different intracellular C-terminal configurations. DH31-Ra utilizes both cAMP and Ca(2+) as second messengers, whereas DH31-Rb utilizes only cAMP; this is the first time reported for an insect CT-like DH31 peptide. DH31 stimulated fluid secretion in D. suzukii adults, and secretion increased in a dose-dependent manner. However, when the fly was injected with a mixture of DH31 and CAPA, an anti-diuretic hormone, fluid secretion was suppressed. The structures are discussed of the DH31 receptors and the differential signaling pathways, including second messengers, involved in fly diuresis. These findings provide fundamental insights into the characterization of D. suzukii DH31 and DH31-Rs, and facilitate the identification of potential biological targets for D. suzukii management. | Gupta, K., Chakrabarti, S., Janardan, V., Gogia, N., Banerjee, S., Srinivas, S., Mahishi, D., Visweswariah, S. S. (2023). Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response. PLoS Genet, 19(9):e1010962 PubMed ID: 37733787
Summary: Evolutionarily conserved genes often play critical roles in organismal physiology. This study describes multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped (Metallophosphoesterase domain containing), the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. It was observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism. |
| Abbasi Yeganeh, F., Rastegarpouyani, H., Li, J., Taylor, K. A. (2023). Structure of the Drosophila melanogaster Flight Muscle Myosin Filament at 4.7 A Resolution Reveals New Details of Non-Myosin Proteins. Int J Mol Sci, 24(19) PubMed ID: 37834384
Summary: Striated muscle thick filaments are composed of myosin II and several non-myosin proteins which define the filament length and modify its function. Myosin II has a globular N-terminal motor domain comprising its catalytic and actin-binding activities and a long α-helical, coiled tail that forms the dense filament backbone. Myosin alone polymerizes into filaments of irregular length, but striated muscle thick filaments have defined lengths that, with thin filaments, define the sarcomere structure. The motor domain structure and function are well understood, but the myosin filament backbone is not. This study reports on the structure of the flight muscle thick filaments from Drosophila melanogaster at 4.7 Å resolution, which eliminates previous ambiguities in non-myosin densities. The full proximal S2 region is resolved, as are the connecting densities between the Ig domains of stretchin-klp. The proteins, flightin, and myofilin are resolved in sufficient detail to build an atomic model based on an AlphaFold prediction. The results suggest a method by which flightin and myofilin cooperate to define the structure of the thick filament and explains a key myosin mutation that affects flightin incorporation. Drosophila is a genetic model organism for which These results can define strategies for functional testing. | Keller, S. H., Deng, H., Lim, B. (2023). Regulation of the dynamic RNA Pol II elongation rate in Drosophila embryos. Cell Rep, 42(10):113225 PubMed ID: 37837623
Summary: An increasing number of studies have shown the key role that RNA polymerase II (RNA Pol II) elongation plays in gene regulation. This study systematically examine how various enhancers, promoters, and gene body composition influence the RNA Pol II elongation rate through a single-cell-resolution live imaging assay. By using reporter constructs containing 5' MS2 and 3' PP7 repeating stem loops, this study quantified the rate of RNA Pol II elongation in live Drosophila embryos. Promoters and exonic gene lengths were found to have no effect on elongation rate, while enhancers and the presence of long introns may significantly change how quickly RNA Pol II moves across a gene. Furthermore, it was observed in multiple constructs that the RNA Pol II elongation rate accelerates after the transcriptional onset of nuclear cycle 14 in Drosophila embryos. This study provides a single-cell view of various mechanisms that affect the dynamic RNA Pol II elongation rate, ultimately affecting the rate of mRNA production. |
Thursday, May 16th - Drosophila as a model for human diseases |
| Bennett, C. L., Dastidar, S., Arnold, F. J., McKinstry, S. U., Stockford, C., Freibaum, B. D., Sopher, B. L., Wu, M., Seidner, G., Joiner, W., Taylor, J. P., West, R. J. H., La Spada, A. R. (2023). Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity. Acta neuropathologica communications, 11(1):164 PubMed ID: 37845749
Summary: Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. This study considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, SETX fly lines were generated, and the effect of SETX in flies expressing either (G4C2)(58) repeats or glycine-arginine-50 [GR(50)] DPRs was evaluated. Dramatic suppression of disease phenotypes was observed in (G4C2)(58) and GR(50) Drosophila models, and a striking relocalization of GR(50) out of the nucleolus was detected in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. It is noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, the nucleolus in cells expressing GR-DPRs was directly assessed, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS. | Swinter, K., Salah, D., Rathnayake, R., Gunawardena, S. (2023). PolyQ-Expansion Causes Mitochondria Fragmentation Independent of Huntingtin and Is Distinct from Traumatic Brain Injury (TBI)/Mechanical Stress-Mediated Fragmentation Which Results from Cell Death. Cells, 12(19) PubMed ID: 37830620
Summary: Mitochondrial dysfunction has been reported in many Huntington's disease (HD) models; however, it is unclear how these defects occur. This study tested the hypothesis that excess pathogenic huntingtin (HTT) impairs mitochondrial homeostasis, using Drosophila genetics and pharmacological inhibitors in HD and polyQ-expansion disease models and in a mechanical stress-induced traumatic brain injury (TBI) model. Expression of pathogenic HTT caused fragmented mitochondria compared to normal HTT, but HTT did not co-localize with mitochondria under normal or pathogenic conditions. Expression of pathogenic polyQ (127Q) alone or in the context of Machado Joseph Disease (MJD) caused fragmented mitochondria. While mitochondrial fragmentation was not dependent on the cellular location of polyQ accumulations, the expression of a chaperone protein, excess of mitofusin (MFN), or depletion of dynamin-related protein 1 (DRP1) rescued fragmentation. Intriguingly, a higher concentration of nitric oxide (NO) was observed in polyQ-expressing larval brains and inhibiting NO production rescued polyQ-mediated fragmented mitochondria, postulating that DRP1 nitrosylation could contribute to excess fission. Furthermore, while excess PI3K, which suppresses polyQ-induced cell death, did not rescue polyQ-mediated fragmentation, it did rescue fragmentation caused by mechanical stress/TBI. Together, these observations suggest that pathogenic polyQ alone is sufficient to cause DRP1-dependent mitochondrial fragmentation upstream of cell death, uncovering distinct physiological mechanisms for mitochondrial dysfunction in polyQ disease and mechanical stress. |
| Roth, J. R., de Moraes, R. C. M., Xu, B. P., Crawley, S. R., Khan, M. A., Melkani, G. C. (2023). Rapamycin reduces neuronal mutant huntingtin aggregation and ameliorates locomotor performance in Drosophila. Frontiers in aging neuroscience, 15:1223911 PubMed ID: 37823007
Summary: Huntington's disease (HD) is a neurodegenerative disease characterized by movement and cognitive dysfunction. HD is caused by a CAG expansion in exon 1 of the HTT gene that leads to a polyglutamine (PQ) repeat in the huntingtin protein, which aggregates in the brain and periphery. Previously, Drosophila models were used to determine that Htt-PQ aggregation in the heart causes shortened lifespan and cardiac dysfunction that is ameliorated by promoting chaperonin function or reducing oxidative stress. The role of neuronal mutant huntingtin and how it affects peripheral function was further studied in this project. Normal (Htt-PQ25) or expanded mutant (Htt-PQ72) exon 1 of huntingtin was overexpressed in Drosophila neurons, and mutant huntingtin was found to cause age-dependent Htt-PQ aggregation in the brain and could cause a loss of synapsin. To determine if this neuronal dysfunction led to peripheral dysfunction,a negative geotaxis assay was performed to measure locomotor performance, and neuronal mutant huntingtin was found to cause an age-dependent decrease in locomotor performance. Next, it was found that rapamycin reduced Htt-PQ aggregation in the brain. These results demonstrate the role of neuronal Htt-PQ in dysfunction in models of HD, suggest that brain-periphery crosstalk could be important to the pathogenesis of HD, and show that rapamycin reduces mutant huntingtin aggregation in the brain. | Catterson, J. H., Minkley, L., Aspe, S., Judd-Mole, S., Moura, S., Dyson, M. C., Rajasingam, A., Woodling, N. S., Atilano, M. L., Ahmad, M., Durrant, C. S., Spires-Jones, T. L., Partridge, L. (2023). Protein retention in the endoplasmic reticulum rescues A β toxicity in Drosophila. Neurobiology of aging, 132:154-174 PubMed ID: 37837732
Summary: Amyloid β (A β; see Drosophila Appl) accumulation is a hallmark of Alzheimer's disease. In adult Drosophila brains, human A β overexpression harms climbing and lifespan. It's uncertain whether A β is intrinsically toxic or activates downstream neurodegeneration pathways. This study uncovers a novel protective role against A β toxicity: intra-endoplasmic reticulum (ER) protein accumulation with a focus on laminin and collagen subunits. Despite high A β, laminin B1 (LanB1) overexpression robustly counters toxicity, suggesting a potential A β resistance mechanism. Other laminin subunits and collagen IV also alleviate A β toxicity; combining them with LanB1 augments the effect. Imaging reveals ER retention of LanB1 without altering A β secretion. LanB1's rescue function operates independently of the IRE1α/XBP1 ER stress response. ER-targeted GFP overexpression also mitigates A β toxicity, highlighting broader ER protein retention advantages. Proof-of-principle tests in murine hippocampal slices using mouse Lamb1 demonstrate ER retention in transduced cells, indicating a conserved mechanism. Though ER protein retention generally harms, it could paradoxically counter neuronal A β toxicity, offering a new therapeutic avenue for Alzheimer's disease. |
| Han, J. E., Kang, K. H., Kim, H., Hong, Y. B., Choi, B. O., Koh, H. (2023). PINK1 and Parkin rescue motor defects and mitochondria dysfunction induced by a patient-derived HSPB3 mutant in Drosophila models. Biochem Biophys Res Commun, 682:71-76 PubMed ID: 37804589
Summary: Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with the α-crystalline domain that is critical to their chaperone activity. Within the sHSP family, three (HSPB1, HSPB3, and HSPB8) proteins are linked with inherited peripheral neuropathies, including distal hereditary motor neuropathy (dHMN) and Charco-Marie-Tooth disease (CMT). This study introduced the HSPB3 Y118H (HSPB3(Y118H)) mutant gene identified from the CMT2 family in Drosophila. With a missense mutation on its α-crystalline domain, this human HSPB3 mutant gene induced a loss of motor activity accompanied by reduced mitochondrial membrane potential in fly neuronal tissues. Moreover, mitophagy, a critical mechanism of mitochondrial quality control, is downregulated in fly motor neurons expressing HSPB3(Y118H). Surprisingly, ">PINK1 and Parkin, the core regulators of mitophagy, successfully rescued these motor and mitochondrial abnormalities in HSPB3 mutant flies. Results from the first animal model of HSPB3 mutations suggest that mitochondrial dysfunction plays a critical role in HSPB3-associated human pathology. | Nil, Z., Deshwar, A. R., Huang, Y., Barish, S., ..., Yamamoto, S., Costain, G., Bellen, H. J. (2023). Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies. American journal of human genetics, 110(11):1919-1937 PubMed ID: 37827158
Summary: Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L (homolog of Drosophila Grappa) is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. This study has identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. Thesee results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease. |
Wednesday, May 15th - Homologs of Drosophila Genes |
| Duan, D., Lyu, W., Chai, P., Ma, S., Wu, K., Wu, C., Xiong, Y., Sestan, N., Zhang, K., Koleske, A. J. (2023). Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation. Curr Biol, 33(21):4582-4598.e4510 PubMed ID: 37858340
Summary: Abl family kinases (see Drosophila Abl) are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both in vitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. This study reports that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ-790. It is proposed that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue. | Cowell, L. M., King, M., West, H., Broadsmith, M., Genever, P., Pownall, M. E., Isaacs, H. V. (2023). Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development. PLoS One, 18(10):e0286040 PubMed ID: 37856433
Summary: Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf; see Drosophila Breathless) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. It has been proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor. This study investigated the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. Xenopus Cic was characterized and it was shown to be widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with the hypothesis, it was shown that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. Two of these genes were investigated as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b (see Drosophila Ras oncogene at 85D), which encode a leucine zipper transcription factor and a ras family GTPase, respectively. Cic consensus binding sites were identified in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. Expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, it was shown that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. These data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding. |
| Sauty, S. M., Yankulov, K. (2023). Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-modal active/silent state at the sub-telomeres of S. cerevisiae. Epigenetics & chromatin, 16(1):40 PubMed ID: 37858268
Summary: Classical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. This study used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the different roles of PCNA and histone chaperones in the stability and the propagation of repressed states are shown to at the sub-telomeres of S. cerevisiae. Position dependent transient repression or bi-modal expression of reporter genes were documented at the VIIL sub-telomere. This study also showed that mutations in the replicative clamp POL30 (PCNA) or the deletion of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the deletion of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. The physical interaction of CAF1 and RRM3 with PCNA was analyzed and the implications of these findings for understanding of the stability and transmission of the epigenetic state of the genes are discussed. There are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. This study characterised the roles of CAF1, RRM3 and ASF1 in these modes of gene repression. It is suggested that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing. | Liu, Q., Bell, B. J., Kim, D. W., Lee, S. S., Keles, M. F., Liu, Q., Blum, I. D., Wang, A. A., Blank, E. J., Xiong, J., Bedont, J. L., Chang, A. J., Issa, H., Cohen, J. Y., Blackshaw, S., Wu, M. N. (2023). A clock-dependent brake for rhythmic arousal in the dorsomedial hypothalamus. Nat Commun, 14(1):6381 PubMed ID: 37821426
Summary: Circadian clocks generate rhythms of arousal, but the underlying molecular and cellular mechanisms remain unclear. In Drosophila, the clock output molecule WIDE AWAKE (WAKE) labels rhythmic neural networks and cyclically regulates sleep and arousal. This study shows, in a male mouse model, that mWAKE/ANKFN1 labels a subpopulation of dorsomedial hypothalamus (DMH) neurons involved in rhythmic arousal and acts in the DMH to reduce arousal at night. In vivo Ca(2+) imaging reveals elevated DMH(mWAKE) activity during wakefulness and rapid eye movement (REM) sleep, while patch-clamp recordings show that DMH(mWAKE) neurons fire more frequently at night. Chemogenetic manipulations demonstrate that DMH(mWAKE) neurons are necessary and sufficient for arousal. Single-cell profiling coupled with optogenetic activation experiments suggest that GABAergic DMH(mWAKE) neurons promote arousal. Surprisingly, the data suggest that mWAKE acts as a clock-dependent brake on arousal during the night, when mice are normally active. mWAKE levels peak at night under clock control, and loss of mWAKE leads to hyperarousal and greater DMH(mWAKE) neuronal excitability specifically at night. These results suggest that the clock does not solely promote arousal during an animal's active period, but instead uses opposing processes to produce appropriate levels of arousal in a time-dependent manner. |
| Adiji, O. A., McConnell, B. S., Parker, M. W. (2024). The origin recognition complex requires chromatin tethering by a hypervariable intrinsically disordered region that is functionally conserved from sponge to man. Nucleic Acids Res, 52(8):4344-4360 PubMed ID: 38381902
Summary: The first step toward eukaryotic genome duplication is loading of the replicative helicase onto chromatin. This 'licensing' step initiates with the recruitment of the origin recognition complex (ORC) to chromatin, which is thought to occur via ORC's ATP-dependent DNA binding and encirclement activity. However, previous work has shown that ATP binding is dispensable for the chromatin recruitment of fly ORC, raising the question of how metazoan ORC binds chromosomes. This study shows that the intrinsically disordered region (IDR) of fly Orc1 is both necessary and sufficient for recruitment of ORC to chromosomes in vivo and demonstrate that this is regulated by IDR phosphorylation. Consistently, it was found that the IDR confers the ORC holocomplex with ATP-independent DNA binding activity in vitro. Using phylogenetic analysis, the surprising observation was made that metazoan Orc1 IDRs have diverged so markedly that they are unrecognizable as orthologs and yet these compositionally homologous sequences are functionally conserved. Altogether, these data suggest that chromatin is recalcitrant to ORC's ATP-dependent DNA binding activity, necessitating IDR-dependent chromatin tethering, which is proposed to poise ORC to opportunistically encircle nucleosome-free regions as they become available. | Kushwaha, A., Thakur, M. K. (2024). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Molecular neurobiology 61(1):487-497 PubMed ID: 37626270
Summary: Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ Suv39h1 (a mammalian ortholog of the Drosophila melanogaster SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It has been previously reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. In the current study an attempt was made to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. This attempt was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia. |
Tuesday, May 14th - Adult neural structure, development and function |
| Banach-Latapy, A., Rincheval, V., Briand, D., Guenal, I., Speder, P. (2023). Differential adhesion during development establishes individual neural stem cell niches and shapes adult behaviour in Drosophila. PLoS Biol, 21(11):e3002352 PubMed ID: 37943883
Summary: Neural stem cells (NSCs) reside in a defined cellular microenvironment, the niche, which supports the generation and integration of newborn neurons. The mechanisms building a sophisticated niche structure around NSCs and their functional relevance for neurogenesis are yet to be understood. In the Drosophila larval brain, the cortex glia (CG) encase individual NSC lineages in membranous chambers, organising the stem cell population and newborn neurons into a stereotypic structure. CG were found to wrap around lineage-related cells regardless of their identity, showing that lineage information builds CG architecture. It was then discovered that a mechanism of temporally controlled differential adhesion using conserved complexes supports the individual encasing of NSC lineages. An intralineage adhesion through homophilic Neuroglian interactions provides strong binding between cells of a same lineage, while a weaker interaction through Neurexin-IV and Wrapper exists between NSC lineages and CG. Loss of Neuroglian results in NSC lineages clumped together and in an altered CG network, while loss of Neurexin-IV/Wrapper generates larger yet defined CG chamber grouping several lineages together. Axonal projections of newborn neurons are also altered in these conditions. Further, the loss of these 2 adhesion complexes specifically during development is linked to locomotor hyperactivity in the resulting adults. Altogether, these findings identify a belt of adhesions building a neurogenic niche at the scale of individual stem cell and provide the proof of concept that niche properties during development shape adult behaviour. | Zhao, A., Nern, A., Koskela, S., Dreher, M., Erginkaya, M., Laughland, C. W., Ludwigh, H., Thomson, A., Hoeller, J., Parekh, R., Romani, S., Bock, D. D., Chiappe, E., Reiser, M. B. (2023). A comprehensive neuroanatomical survey of the Drosophila Lobula Plate Tangential Neurons with predictions for their optic flow sensitivity. bioRxiv, PubMed ID: 37904921
Summary: Flying insects exhibit remarkable navigational abilities controlled by their compact nervous systems. Optic flow, the pattern of changes in the visual scene induced by locomotion, is a crucial sensory cue for robust self-motion estimation, especially during rapid flight. The best-known optic-flow sensitive neurons are the large tangential cells of the dipteran lobula plate. This study reports the comprehensive reconstruction and identification of the Lobula Plate Tangential Neurons in an Electron Microscopy (EM) volume of a whole Drosophila brain. This catalog of 58 LPT neurons (per brain hemisphere) contains many neurons that are described here for the first time and provides a basis for systematic investigation of the circuitry linking self-motion to locomotion control. Leveraging computational anatomy methods, it is estimated the visual motion receptive fields of these neurons and compared their tuning to the visual consequence of body rotations and translational movements. These neurons were matched, in most cases on a one-for-one basis, to stochastically labeled cells in genetic driver lines, to the mirror-symmetric neurons in the same EM brain volume, and to neurons in an additional EM data set. Using cell matches across data sets, the integration of optic flow patterns by neurons downstream of the LPTs was analyzed and most central brain neurons were found to establish sharper selectivity for global optic flow patterns than their input neurons. Furthermore, it was found that self-motion information extracted from optic flow is processed in distinct regions of the central brain, pointing to diverse foci for the generation of visual behaviors. |
| Jovanoski, K. D., Duquenoy, L., Mitchell, J., Kapoor, I., Treiber, C. D., Croset, V., Dempsey, G., Parepalli, S., Cognigni, P., Otto, N., Felsenberg, J., Waddell, S. (2023). Dopaminergic systems create reward seeking despite adverse consequences. Nature, 623(7986):356-365 PubMed ID: 37880370
Summary: Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking. Although dysfunction of the dopaminergic valuation system of the brain is known to contribute towards unconstrained reward seeking, the underlying reasons for this behaviour are unclear. This study describes dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. It is proposed that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals. | Ganguly, I., Heckman, E. L., Litwin-Kumar, A., Clowney, E. J., Behnia, R. (2023). Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body. bioRxiv, PubMed ID: 37873086
Summary: The arthropod mushroom body is well-studied as an expansion layer that represents olfactory stimuli and links them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their tuning and function are poorly understood. This study used the FlyWire adult whole-brain connectome to identify inputs to visual Kenyon cells. The types of visual neurons identified are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual projection neurons presynaptic to Kenyon cells receive input from large swathes of visual space, while local visual interneurons, providing smaller fractions of input, receive more spatially restricted signals that may be tuned to specific features of the visual scene. Like olfactory Kenyon cells, visual Kenyon cells receive sparse inputs from different combinations of visual channels, including inputs from multiple optic lobe neuropils. The sets of inputs to individual visual Kenyon cells are consistent with random sampling of available inputs. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the expansion coding properties appear different, with a specific repertoire of visual inputs projecting onto a relatively small number of visual Kenyon cells. |
| Wang, C. M., Wu, C. Y., Lin, C. E., Hsu, M. C., Lin, J. C., Huang, C. C., Lien, T. Y., Lin, H. K., Chang, T. W., Chiang, H. C. (2023). Forgotten memory storage and retrieval in Drosophila. Nat Commun, 14(1):7153 PubMed ID: 37935667
Summary: Inaccessibility of stored memory in ensemble cells through the forgetting process causes animals to be unable to respond to natural recalling cues. While accumulating evidence has demonstrated that reactivating memory-stored cells can switch cells from an inaccessible state to an accessible form and lead to recall of previously learned information, the underlying cellular and molecular mechanisms remain elusive. The current study used Drosophila as a model to demonstrate that the memory of one-trial aversive olfactory conditioning, although inaccessible within a few hours after learning, is stored in KCαβ and retrievable after mild retraining. One-trial aversive conditioning triggers protein synthesis to form a long-lasting cellular memory trace, approximately 20 days, via creb in KCαβ, and a transient cellular memory trace, approximately one day, via orb in MBON-α3. PPL1-α3 negatively regulates forgotten one-trial conditioning memory retrieval. The current study demonstrated that KCαβ, PPL1-α3, and MBON-α3 collaboratively regulate the formation of forgotten one-cycle aversive conditioning memory formation and retrieval. | Yang, H. H., Brezovec, L. E., Capdevila, L. S., Vanderbeck, Q. X., Adachi, A., Mann, R. S., Wilson, R. I. (2023). Fine-grained descending control of steering in walking Drosophila. bioRxiv, PubMed ID: 37904997
Summary: Locomotion involves rhythmic limb movement patterns that originate in circuits outside the brain. Purposeful locomotion requires descending commands from the brain, but it is not understood how these commands are structured. Focusing on the control of steering in walking Drosophila. First, different limb "gestures" associated with different steering maneuvers are described. Next, this study identified a set of descending neurons whose activity predicts steering. Focusing on two descending cell types downstream from distinct brain networks, this study shows that they evoke specific limb gestures: one lengthens strides on the outside of a turn, while the other attenuates strides on the inside of a turn. Notably, a single descending neuron can have opposite effects during different locomotor rhythm phases, and networks positioned to implement this phase-specific gating were identified. Together, these results show how purposeful locomotion emerges from brain cells that drive specific, coordinated modulations of low-level patterns. |
Monday, May 13th - Transcriptional Regulation |
| Chaubal, A., Waldern, J. M., Taylor, C., Laederach, A., Marzluff, W. F., Duronio, R. J. (2023). Coordinated expression of replication-dependent histone genes from multiple loci promotes histone homeostasis in Drosophila. Mol Biol Cell, 34(12):ar118 PubMed ID: 37647143
Summary: Production of large amounts of histone proteins during S phase is critical for proper chromatin formation and genome integrity. This process is achieved in part by the presence of multiple copies of replication dependent (RD) histone genes that occur in one or more clusters in metazoan genomes. In addition, RD histone gene clusters are associated with a specialized nuclear body, the histone locus body (HLB), which facilitates efficient transcription and 3' end-processing of RD histone mRNA. How all five RD histone genes within these clusters are coordinately regulated such that neither too few nor too many histones are produced, a process referred to as histone homeostasis, is not fully understood. This study explored the mechanisms of coordinate regulation between multiple RD histone loci in Drosophila melanogaster and Drosophila virilis. Evidence ia provided for functional competition between endogenous and ectopic transgenic histone arrays located at different chromosomal locations in D. melanogaster that helps maintain proper histone mRNA levels. Consistent with this model, in both species it was found that individual histone gene arrays can independently assemble an HLB that results in active histone transcription. These findings suggest a role for HLB assembly in coordinating RD histone gene expression to maintain histone homeostasis. | Khanbabei, A., Segura, L., Petrossian, C., Lemus, A., Cano, I., Frazier, C., Halajyan, A., Ca, D., Loza-Coll, M. (2024). Experimental validation and characterization of putative targets of Escargot and STAT, two master regulators of the intestinal stem cells in Drosophila melanogaster. Dev Biol, 505:148-163 PubMed ID: 37952851
Summary: Many organs contain adult stem cells (ASCs) to replace cells due to damage, disease, or normal tissue turnover. ASCs can divide asymmetrically, giving rise to a new copy of themselves (self-renewal) and a sister that commits to a specific cell type (differentiation). Decades of research have led to the identification of pleiotropic genes whose loss or gain of function affect diverse aspects of normal ASC biology. Genome-wide screens of these so-called genetic "master regulator" (MR) genes, have pointed to hundreds of putative targets that could serve as their downstream effectors. This study experimentally validated and characterized the regulation of several putative targets of Escargot (Esg) and the Signal Transducer and Activator of Transcription (Stat92E, a.k.a. STAT), two known MRs in Drosophila intestinal stem cells (ISCs). The results indicate that regardless of bioinformatic predictions, most experimentally validated targets show a profile of gene expression that is consistent with co-regulation by both Esg and STAT, fitting a rather limited set of co-regulatory modalities. A bioinformatic analysis of proximal regulatory sequences in specific subsets of co-regulated targets identified additional transcription factors that might cooperate with Esg and STAT in modulating their transcription. Lastly, in vivo manipulations of validated targets rarely phenocopied the effects of manipulating Esg and STAT, suggesting the existence of complex genetic interactions among downstream targets of these two MR genes during ISC homeostasis. |
| Eggers, N., Gkountromichos, F., Krause, S., Campos-Sparr, A., Becker, P. B. (2023). Physical interaction between MSL2 and CLAMP assures direct cooperativity and prevents competition at composite binding sites. Nucleic Acids Res, 51(17):9039-9054 PubMed ID: 37602401
Summary: MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. This study explored the nature of the cooperative binding to these GA-rich, composite sequence elements in reconstituted naive embryonic chromatin. The cooperativity was found to require physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. The cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in the reconstitution, such as roX2 lncRNA. | Aguilera, J., Duan, J., Lee, S. M., Ray, M., Larschan, E. (2023). The CLAMP GA-binding transcription factor regulates heat stress-induced transcriptional repression by associating with 3D loop anchors. bioRxiv, PubMed ID: 37873306
Summary: In order to survive when exposed to heat stress (HS), organisms activate stress response genes and repress constitutive gene expression to prevent the accumulation of potentially toxic RNA and protein products. Although many studies have elucidated the mechanisms that drive HS-induced activation of stress response genes across species, little is known about repression mechanisms or how genes are targeted for activation versus repression context-specifically. The mechanisms of heat stress-regulated activation have been well-studied in Drosophila, in which the GA-binding transcription factor GAF is important for activating genes upon heat stress. This study shows that a functionally distinct GA-binding transcription factor (TF) protein, CLAMP (Chromatin-linked adaptor for MSL complex proteins), is essential for repressing constitutive genes upon heat stress but not activation of the canonical heat stress pathway. HS induces loss of CLAMP-associated 3D chromatin loop anchors associated with different combinations of GA-binding TFs prior to HS if a gene becomes repressed versus activated. Overall, this study demonstrated that CLAMP promotes repression of constitutive genes upon HS, and repression and activation are associated with the loss of CLAMP-associated 3D chromatin loops bound by different combinations of GA-binding TFs. |
| Ramalingam, V., Yu, X., Slaughter, B. D., Unruh, J. R., Brennan, K. J., Onyshchenko, A., Lange, J. J., Natarajan, M., Buck, M., Zeitlinger, J. (2023). Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development. Nat Commun, 14(1):5862 PubMed ID: 37735176
Summary: While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-, a Drosophila zinc finger transcription factor that has one of the more than 25 different splice isoforms from the lolaI locus, this study shows that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters. | Lovero, D., Porcelli, D., Giordano, L., Lo Giudice, C., Picardi, E., Pesole, G., Pignataro, E., Palazzo, A., Marsano, R. M. (2023). Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors. Biology, 12(8) PubMed ID: 37627011
Summary: Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. This work analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. The conservation is highlighted of a short sequence in the genes encoding subunits of the V-ATPase complex and their assembly factors analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding knowledge of the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization. |
Friday, May 10th - Adult physiology and metabolism |
| Vujnovic, A. F., Martinovic, L., S., Medija, M., Waldowski, R. A. (2023). Distinct and Dynamic Changes in the Temporal Profiles of Neurotransmitters in Drosophila melanogaster Brain following Volatilized Cocaine or Methamphetamine Administrations. Pharmaceuticals (Basel, Switzerland), 16(10) PubMed ID: 37895961
Summary: Due to similarities in genetics, cellular response, and behavior, Drosophila is used as a model organism in addiction research. A well-described behavioral response examined in flies is the induced increase in locomotor activity after a single dose of volatilized cocaine (vCOC) and volatilized methamphetamine (vMETH), the sensitivity, and the escalation of the locomotor response after the repeated dose, the locomotor sensitization. However, knowledge about how vCOC and vMETH affect different neurotransmitter systems over time is scarce. This study used LC-MS/MS to systematically examine changes in the concentration of neurotransmitters, metabolites and non-metabolized COC and METH in the whole head homogenates of male flies one to seven hours after single and double vCOC or vMETH administrations. vMETH leads to complex changes in the levels of examined substances over time, while vCOC strongly and briefly increases concentrations of dopamine, tyramine and octopamine followed by a delayed degradation into N-acetyl dopamine and N-acetyl tyramine. The first exposure to psychostimulants leads to significant and dynamic changes in the concentrations relative to the second administration when they are more stable over several hours. Further investigations are needed to understand neurochemical and molecular changes post-psychostimulant administration. | Alassaf, M., Rajan, A. (2023). Diet-induced glial insulin resistance impairs the clearance of neuronal debris in Drosophila brain. PLoS Biol, 21(11):e3002359 PubMed ID: 37934726
Summary: Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Using a Drosophila model this study shows that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, this study has established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris. |
| Brener, A., Lorber, D., Reuveny, A., Toledano, H., Porat-Kuperstein, L., Lebenthal, Y., Weizman, E., Olender, T., Volk, T. (2023). Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae. Cells, 12(19) PubMed ID: 37830547
Summary: The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi(1)) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. The findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating these findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases. | Torre, M., Bukhari, H., Nithianandam, V., Zanella, C. A., Mata, D. A., Feany, M. B. (2023). A Drosophila model relevant to chemotherapy-related cognitive impairment. Sci Rep, 13(1):19290 PubMed ID: 37935827
Summary: Chemotherapy-related cognitive impairment (CRCI) is a common adverse effect of treatment and is characterized by deficits involving multiple cognitive domains including memory. Despite the significant morbidity of CRCI and the expected increase in cancer survivors over the coming decades, the pathophysiology of CRCI remains incompletely understood, highlighting the need for new model systems to study CRCI. Given the powerful array of genetic approaches and facile high throughput screening ability in Drosophila, the goal of this study was to validate a Drosophila model relevant to CRCI. The chemotherapeutic agents cisplatin, cyclophosphamide, and doxorubicin were administered to adult Drosophila. Neurologic deficits were observed with all tested chemotherapies, with doxorubicin and in particular cisplatin also resulting in memory deficits. Histologic and immunohistochemical analysis was performed of cisplatin-treated Drosophila tissue, demonstrating neuropathologic evidence of increased neurodegeneration, DNA damage, and oxidative stress. Thus, the Drosophila model relevant to CRCI recapitulates clinical, radiologic, and histologic alterations reported in chemotherapy patients. This new Drosophila model can be used for mechanistic dissection of pathways contributing to CRCI (and chemotherapy-induced neurotoxicity more generally) and pharmacologic screens to identify disease-modifying therapies. |
| Brischigliaro, M., Cabrera-Orefice, A., Arnold, S., Viscomi, C., Zeviani, M., Fernandez-Vizarra, E. (2023). Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes. Elife, 12 PubMed ID: 37823874
Summary: Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. This study show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, it is concluded that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis. | Li, J., Dang, P., Li, Z., Zhao, T., Cheng, D., Pan, D., Yuan, Y., Song, W. (2023). Peroxisomal ERK mediates Akh/glucagon action and glycemic control. Cell Rep, 42(10):113200 PubMed ID: 37796662
Summary: he enhanced response of glucagon and its Drosophila homolog, Adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. This study found that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. Mammalian glucagon-induced hepatic ERK peroxisomal translocation was also observed in diabetic subjects. Therefore, these results conclude that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control. |
Thursday, May 9th - Tumors, Cancer and Growth |
| Khalili, D., Mohammed, M., Kunc, M., Sindlerova, M., Ankarklev, J., Theopold, U. (2023). Single-cell sequencing of tumor-associated macrophages in a Drosophila model. Frontiers in immunology, 14:1243797 PubMed ID: 37795097
Summary: Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized. This study used a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (Ras(V12)) to study dysplastic growth during early tumor progression. Single-cell RNA-sequencing was performed of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells. Five distinct hemocyte clusters were identified. In addition to Ras(V12) larvae, a tumor model was included where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells-they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors-antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, evidence is provided for transcript transfer between hemocytes and possibly other tissues. Taken together, these data support the usefulness of Drosophila to study the response against tumors at the organismic level. | Yu, K., Ramkumar, N., Wong, K. K. L., Tettweiler, G., Verheyen, E. M. (2023). The AMPK-like protein kinases Sik2 and Sik3 interact with Hipk and induce synergistic tumorigenesis in a Drosophila cancer model. Frontiers in cell and developmental biology, 11:1214539 PubMed ID: 37854071.
Summary: Homeodomain-interacting protein kinases (Hipks) regulate cell proliferation, apoptosis, and tissue development. Overexpression of Hipk in Drosophila causes tumorigenic phenotypes in larval imaginal discs. This study found that depletion of Salt-inducible kinases Sik2 or Sik3 can suppress Hipk-induced overgrowth. Furthermore, co-expression of constitutively active forms of Sik2 or Sik3 with Hipk caused significant tissue hyperplasia and tissue distortion, indicating that both Sik2 and Sik3 can synergize with Hipk to promote tumorous phenotypes, accompanied by elevated dMyc, Armadillo/β-catenin, and the Yorkie target gene expanded. Larvae expressing these hyperplastic growths also display an extended larval phase, characteristic of other Drosophila tumour models. Examination of total protein levels from fly tissues showed that Hipk proteins were reduced when Siks were depleted through RNAi, suggesting that Siks may regulate Hipk protein stability and/or activity. Conversely, expression of constitutively active Siks with Hipk leads to increased Hipk protein levels. Furthermore, Hipk can interact with Sik2 and Sik3 by co-immunoprecipitation. Co-expression of both proteins leads to a mobility shift of Hipk protein, suggesting it is post-translationally modified. In summary, this research demonstrates a novel function of Siks in synergizing with Hipk to promote tumour growth. |
| Bosch, P. S., Cho, B., Axelrod, J. D. (2023). Flamingo participates in multiple models of cell competition. bioRxiv, PubMed ID: 37790459
Summary: The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. This study shows that in Drosophila, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in pre-malignant cells as they grow among wildtype cells, in healthy cells to eliminate pre-malignant cells, and by supercompetitors to occupy excessive territory within wildtype tissues. "Would-be" winners that lack Fmi are unable to over-proliferate, and instead become losers. This study demonstrates that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well defined functions of Fmi. | Pranoto, I. K. A., Lee, J., Kwon, Y. V. (2023). The roles of the native cell differentiation program aberrantly recapitulated in Drosophila intestinal tumors. Cell Rep, 42(10):113245 PubMed ID: 37837622ID:
Summary: Many tumors recapitulate the developmental and differentiation program of their tissue of origin, a basis for tumor cell heterogeneity. Although stem-cell-like tumor cells are well studied, the roles of tumor cells undergoing differentiation remain to be elucidated. This study employ Drosophila genetics to demonstrate that the differentiation program of intestinal stem cells is crucial for enabling intestinal tumors to invade and induce non-tumor-autonomous phenotypes. The differentiation program that generates absorptive cells is aberrantly recapitulated in the intestinal tumors generated by activation of the Yap1 ortholog Yorkie. Inhibiting it allows stem-cell-like tumor cells to grow but suppresses invasiveness and reshapes various phenotypes associated with cachexia-like wasting by altering the expression of tumor-derived factors. This study provides insight into how a native differentiation program determines a tumor's capacity to induce advanced cancer phenotypes and suggests that manipulating the differentiation programs co-opted in tumors might alleviate complications of cancer, including cachexia. |
| Zheng, J., Guo, Y., Shi, C., Yang, S., Xu, W., Ma, X. (2023). Differential Ire1 determines loser cell fate in tumor-suppressive cell competition. Cell Rep, 42(11):113303 PubMed ID: 37924514
Summary: Tumor-suppressive cell competition (TSCC) is a conserved surveillance mechanism in which neighboring cells actively eliminate oncogenic cells. Despite overwhelming studies showing that the unfolded protein response (UPR) is dysregulated in various tumors, it remains debatable whether the UPR restrains or promotes tumorigenesis. Using Drosophila eye epithelium as a model, this study uncovered a surprising decisive role of the Ire1 branch of the UPR in regulating cell polarity gene scribble (scrib) loss-induced TSCC. Both mutation and hyperactivation of Ire1 accelerate elimination of scrib clones via inducing apoptosis and autophagy, respectively. Unexpectedly, relative Ire1 activity is also crucial for determining loser cell fate, as dysregulating Ire1 signaling in the surrounding healthy cells reversed the "loser" status of scrib clones by decreasing their apoptosis. Furthermore, it was shown that Ire1 is required for cell competition in mammalian cells. Together, these findings provide molecular insights into scrib-mediated TSCC and highlight Ire1 as a key determinant of loser cell fate. | Quintero, M., Bangi, E. (2023). Disruptions in cell fate decisions and transformed enteroendocrine cells drive intestinal tumorigenesis in Drosophila. Cell Rep, 42(11):113370 PubMed ID: 37924517
Summary: Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between different cell types ensures precise regulation of stem cell behavior and cell fate decisions. These cell-cell interactions are often disrupted during tumorigenesis, but mechanisms by which they are co-opted to support tumor growth in different genetic contexts are poorly understood. This study introduced PromoterSwitch, a genetic platform established to generate large, transformed clones derived from individual adult Drosophila intestinal stem/progenitor cells. Cancer-driving genetic alterations representing common colon tumor genome landscapes were shown to disrupt cell fate decisions within transformed tissue and result in the emergence of abnormal cell fates. It was also shown that transformed enteroendocrine cells, a differentiated, hormone-secreting cell lineage, support tumor growth by regulating intestinal stem cell proliferation through multiple genotype-dependent mechanisms, which represent potential vulnerabilities that could be exploited for therapy. |
Wednesday, May 8th - Drosophila as a model for human diseases |
| Min, Y., Wang, X., Is, O., Patel, T. A., Gao, J., Reddy, J. S., Quicksall, Z. S., Nguyen, T., Lin, S., Tutor-New, F. Q., Chalk, J. L., Mitchell, A. O., Crook, J. E., Nelson, P. T., Van Eldik, L. J., Golde, T. E., Carrasquillo, M. M., Dickson, D. W., Zhang, K., Allen, M., Ertekin-Taner, N. (2023). Cross species systems biology discovers glial DDR2, STOM, and KANK2 as therapeutic targets in progressive supranuclear palsy. Nat Commun, 14(1):6801 PubMed ID: 37919278
Summary: Progressive supranuclear palsy (PSP) is a neurodegenerative parkinsonian disorder characterized by cell-type-specific tau lesions in neurons and glia. Prior work uncovered transcriptome changes in human PSP brains, although their cell-specificity is unknown. Further, systematic data integration and experimental validation platforms to prioritize brain transcriptional perturbations as therapeutic targets in PSP are currently lacking. This study combined bulk tissue (n = 408) and single nucleus RNAseq (n = 34) data from PSP and control brains with transcriptome data from a mouse tauopathy and experimental validations in Drosophila tau models for systematic discovery of high-confidence expression changes in PSP with therapeutic potential. Thousands of differentially expressed genes were discovered, replicated, and annotated in PSP, many of which reside in glia-enriched co-expression modules and cells. DDR2, STOM, and KANK2 were were prioritized as promising therapeutic targets in PSP with striking cross-species validations. These findings and data are shared via an interactive application tool PSP RNAseq Atlas. The findings reveal robust glial transcriptome changes in PSP, provide a cross-species systems biology approach, and a tool for therapeutic target discoveries in PSP with potential application in other neurodegenerative diseases. | Yan, L., Zhou, J., Yuan, L., Ye, J., Zhao, X., Ren, G., Chen, H. (2023). Silibinin alleviates intestinal inflammation via inhibiting JNK signaling in Drosophila. Frontiers in pharmacology, 14:1246960 PubMed ID: 37781701
Summary: Inflammatory bowel diseases (IBDs) are characterized by chronic relapsing intestinal inflammation that causes digestive system dysfunction. For years, researchers have been working to find more effective and safer therapeutic strategies to treat these diseases. Silibinin (SIL), a flavonoid compound extracted from the seeds of milk thistle plants, possesses multiple biological activities and is traditionally applied to treat liver diseases. SIL is also widely used in the treatment of a variety of inflammatory diseases attributed to its excellent antioxidant and anti-inflammatory effects. However, the efficacy of SIL against IBDs and its mechanisms remain unclear. This study, using Drosophila melanogaster as a model organism, found that SIL can effectively relieve intestinal inflammation caused by dextran sulfate sodium (DSS). The results suggested that SIL supplementation can inhibit the overproliferation of intestinal stem cells (ISCs) induced by DSS, protect intestinal barrier function, acid-base balance, and intestinal excretion function, reduce intestinal reactive oxygen species (ROS) levels and inflammatory stress, and extend the lifespan of Drosophila. Furthermore, this study demonstrated that SIL ameliorates intestinal inflammation via modulating the c-Jun N-terminal kinase (JNK) signaling pathway in Drosophila. This research aims to provide new insight into the treatment of IBDs. |
| Aalto, A. L., Saadabadi, A., Lindholm, F., Kietz, C., Himmelroos, E., Marimuthu, P., Salo-Ahen, O. M. H., Eklund, P., Meinander, A. (2023). Stilbenoid compounds inhibit NF-κB-mediated inflammatory responses in the Drosophila intestine. Frontiers in immunology, 14:1253805 PubMed ID: 37809071
Summary: Stilbenoid compounds have been described to have anti-inflammatory properties in animal models in vivo, and have been shown to inhibit Ca2+-influx through the transient receptor potential ankyrin 1 (TrpA1). To study how stilbenoid compounds affect inflammatory signaling in vivo, the fruit fly, Drosophila melanogaster, was used as a model system. To induce intestinal inflammation in the fly, flies were fed with the intestinal irritant dextran sodium sulphate (DSS). DSS was found to induce severe changes in the bacteriome of the Drosophila intestine, and that this dysbiosis causes activation of the NF-κB transcription factor Relish. The DSS-model was used to study the anti-inflammatory properties of the stilbenoid compounds pinosylvin (PS) and pinosylvin monomethyl ether (PSMME). With the help of in vivo approaches, PS and PSMME were identified as transient receptor ankyrin 1 (TrpA1)-dependent antagonists of NF-κB-mediated intestinal immune responses in Drosophila were computationally predicted. The putative antagonist binding sites of these compounds at Drosophila TrpA1. Taken together, this study showed that the stilbenoids PS and PSMME have anti-inflammatory properties in vivo in the intestine and can be used to alleviate chemically induced intestinal inflammation in Drosophila. | Wang, C. W., Clemot, M., Hashimoto, T., Diaz, J. A., Goins, L. M., Goldstein, A. S., Nagaraj, R., Banerjee, U. (2023). A conserved mechanism for JNK-mediated loss of Notch function in advanced prostate cancer. Science signaling, 16(810):eabo5213 PubMed ID: 37934809
Summary: Dysregulated Notch signaling is a common feature of cancer; however, its effects on tumor initiation and progression are highly variable, with Notch having either oncogenic or tumor-suppressive functions in various cancers. To better understand the mechanisms that regulate Notch function in cancer, Notch signaling was studied in a Drosophila tumor model, prostate cancer-derived cell lines, and tissue samples from patients with advanced prostate cancer. It was demonstrated that increased activity of the Src-JNK pathway in tumors inactivated Notch signaling because of JNK pathway-mediated inhibition of the expression of the gene encoding the Notch S2 cleavage protease, Kuzbanian, which is critical for Notch activity. Consequently, inactive Notch accumulated in cells, where it was unable to transcribe genes encoding its target proteins, many of which have tumor-suppressive activities. These findings suggest that Src-JNK activity in tumors predicts Notch activity status and that suppressing Src-JNK signaling could restore Notch function in tumors, offering opportunities for diagnosis and targeted therapies for a subset of patients with advanced prostate cancer. |
| Diaw, S. H., Borsche, M., Streubel-Gallasch, L., Dulovic-Mahlow, M., Hermes, J., Lenz, I., Seibler, P., Klein, C., Bruggemann, N., Vos, M., Lohmann, K. (2023). Characterization of the pathogenic alpha-Synuclein Variant V15A in Parkinson´s disease. NPJ Parkinson's disease, 9(1):148 PubMed ID: 37903765
Summary: Despite being a major component of Lewy bodies and Lewy neurites, pathogenic variants in the gene encoding alpha-Synuclein (α-Syn) are rare. To date, only four missense variants in the SNCA gene, encoding α-Syn have unequivocally been shown to be disease-causing. This study describes a Parkinson´s disease patient with early cognitive decline carrying an as yet not fully characterized variant in SNCA (NM_001146055: c.44T > C, p.V15A). Different cellular models, including stably transfected neuroblastoma (SH-SY5Y) cell cultures, induced pluripotent stem cell (iPSC)-derived neuronal cultures, and a Drosophila model was generated to elucidate the impact of the p.V15A variant on α-Syn function and aggregation properties compared to other known pathogenic variants. This study demonstrated that p.V15A increased the aggregation potential of α-Syn and the levels of apoptotic markers, and impaired the mitochondrial network. Moreover, p.V15A affects the flying ability and survival of mutant flies. Thus, this study provides supporting evidence for the pathogenicity of the p.V15A variant, suggesting its inclusion in genetic testing approaches. | Zane, F., Bouzid, H., Sosa Marmol, S., Brazane, M., Besse, S., Molina, J. L., Cansell, C., Aprahamian, F., Durand, S., Ayache, J., Antoniewski, C., Todd, N., Carre, C., Rera, M. (2023). Smurfness-based two-phase model of ageing helps deconvolve the ageing transcriptional signature. Aging Cell, 22(11):e13946 PubMed ID: 37822253
Summary: Ageing is characterised at the molecular level by six transcriptional 'hallmarks of ageing', that are commonly described as progressively affected as time passes. By contrast, the 'Smurf' assay, which assesses food intake by the co-ingestion of a blue dye, which is not absorbed by the digestive tract, separates high-and-constant-mortality risk individuals from healthy, zero-mortality risk individuals, based on increased intestinal permeability. Performing whole body total RNA sequencing, it was found that Smurfness distinguishes transcriptional changes associated with chronological age from those associated with biological age. Transcriptional heterogeneity is shown to increase with chronological age in non-Smurf individuals preceding the other five hallmarks of ageing that are specifically associated with the Smurf state. Using this approach, targeted pro-longevity genetic interventions delaying entry in the Smurf state were devised. It is anticipated that increased attention to the evolutionary conserved Smurf phenotype will bring about significant advances in understanding of the mechanisms of ageing. |
Tuesday, May 7th - Embryonic Neural Development |
| Mitchell, J. W., Midillioglu, I., Schauer, E., Wang, B., Han, C., Wildonger, J. (2023). Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons. PLoS Genet, 19(11):e1011025 PubMed ID: 37943859
Summary: Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. This study investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. CRISPR-Cas9 genome engineering approaches were used to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, it was found that an over-active channel mutant severely reduces dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. These data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on these data, it is proposed that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites. | Hsiao, Y. L., Chen, H. W., Chen, K. H., Tan, B. C., Chen, C. H. and Pi, H. (2023). Actin-related protein 6 facilitates proneural protein-induced gene activation for rapid neural differentiation. Development 150(5). PubMed ID: 36897355
Summary: Neurogenesis is initiated by basic helix-loop-helix proneural proteins. This study showed that Actin-related protein 6 (Arp6), a core component of the H2A.Z exchange complex SWR1, interacts with proneural proteins and is crucial for efficient onset of proneural protein target gene expression. Arp6 mutants exhibit reduced transcription in sensory organ precursors (SOPs) downstream of the proneural protein patterning event. This leads to retarded differentiation and division of SOPs and smaller sensory organs. These phenotypes are also observed in proneural gene hypomorphic mutants. Hypomorphic ac sc mutant recapitulates Arp6 mutant phenotypes. Arp6 also interacted with Sc and Atonal (Ato) but failed to interact with the proneural protein heterodimeric partner Daughterless. Proneural protein expression is not reduced in Arp6 mutants. Enhanced proneural gene expression fails to rescue retarded differentiation in Arp6 mutants, suggesting that Arp6 acts downstream of or in parallel with proneural proteins. H2A.Z mutants display Arp6-like retardation in SOPs. Transcriptomic analyses demonstrate that loss of Arp6 and H2A.Z preferentially decreases expression of proneural protein-activated genes. H2A.Z enrichment in nucleosomes around the transcription start site before neurogenesis correlates highly with greater activation of proneural protein target genes by H2A.Z. It is proposed that upon proneural protein binding to E-box sites, H2A.Z incorporation around the transcription start site allows rapid and efficient activation of target genes, promoting rapid neural differentiation. |
| Singh, B. N., Tran, H., Kramer, J., Kirishenko, E., Changela, N., Wang, F., Feng, Y., Kumar, D., Tu, M., Lan, J., Bizet, M., Fuks, F. and Steward, R. (2023). Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates the axon guidance genes robo2 and slit in Drosophila. Res Sq. PubMed ID: 36824980
Summary: Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases enzymes catalyzing the transition of 5mC to 5hmC in DNA and have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila because Tet knock-out animals do not reach adulthood. The identification is described of Tet-target genes in the embryo and larval brain by determining Tet DNA-binding sites throughout the genome and by mapping the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC-modified sites can be found along the entire transcript and are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are frequently involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and are sensitized to reduced levels of slit. Both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. These results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs, primarily in developing nerve cells. | Sullivan, K. G., Bashaw, G. J. (2023). Commissureless acts as a substrate adapter in a conserved Nedd4 E3 ubiquitin ligase pathway to promote axon growth across the midline. bioRxiv. PubMed ID: 37905056
Summary: In both vertebrates and invertebrates, commissural neurons prevent premature responsiveness to the midline repellant Slit by downregulating surface levels of its receptor Roundabout1 (Robo1). In Drosophila, Commissureless (Comm) plays a critical role in this process; however, there is conflicting data on the underlying molecular mechanism. This study demonstrated that the conserved PY motifs in the cytoplasmic domain of Comm are required allow the ubiquitination and lysosomal degradation of Robo1. Disruption of these motifs prevents Comm from localizing to Lamp1 positive late endosomes and to promote axon growth across the midline in vivo. In addition, a role for Nedd4 in midline crossing was identified. Genetic analysis shows that nedd4 mutations result in midline crossing defects in the Drosophila embryonic nerve cord, which can be rescued by introduction of exogenous Nedd4. Biochemical evidence shows that Nedd4 incorporates into a three-member complex with Comm and Robo in a PY motif-dependent manner. Finally, genetic evidence is presented that Nedd4 acts with Comm in the embryonic nerve cord to downregulate Robo1 levels. Taken together, these findings demonstrate that Comm promotes midline crossing in the nerve cord by facilitating Robo ubiquitination by Nedd4, ultimately leading to its degradation. |
| Carranza, A., Howard, L. J., Brown, H. E., Ametepe, A. S. and Evans, T. A. (2023). Slit-independent guidance of longitudinal axons by Drosophila Robo3. bioRxiv. PubMed ID: 37214810
Summary: Drosophila Robo3 is a member of the evolutionarily conserved Roundabout (Robo) receptor family and one of three Drosophila Robo paralogs. During embryonic ventral nerve cord development, Robo3 does not participate in canonical Slit-dependent midline repulsion, but instead regulates the formation of longitudinal axon pathways at specific positions along the medial-lateral axis. Longitudinal axon guidance by Robo3 is hypothesized to be Slit dependent, but this has not been directly tested. In this study a series of Robo3 variants was created in which the N-terminal Ig1 domain is deleted or modified, in order to characterize the functional importance of Ig1 and Slit binding for Robo3's axon guidance activity. Robo3 is shown to require its Ig1 domain for interaction with Slit and for proper axonal localization in embryonic neurons, but deleting Ig1 from Robo3 only partially disrupts longitudinal pathway formation. Robo3 variants with modified Ig1 domains that cannot bind Slit retain proper localization and fully rescue longitudinal axon guidance. These results indicate that Robo3 guides longitudinal axons independently of Slit, and that sequences both within and outside of Ig1 contribute to this Slit-independent activity. | Karkali, K., Saunders, T. E., Panayotou, G. and Martín-Blanco, E. (2023). JNK signaling in pioneer neurons organizes ventral nerve cord architecture in Drosophila embryos. Nat Commun 14(1): 675. PubMed ID: 36750572
Summary: Morphogenesis of the Central Nervous System (CNS) is a complex process that obeys precise architectural rules. Yet, the mechanisms dictating these rules remain unknown. Analyzing morphogenesis of the Drosophila embryo Ventral Nerve Cord (VNC), this study observe that a tight control of JNK signaling is essential for attaining the final VNC architecture. JNK signaling in a specific subset of pioneer neurons autonomously regulates the expression of Fasciclin 2 (Fas 2) and Neurexin IV (Nrx IV) adhesion molecules, probably via the transcription factor zfh1. Interfering at any step in this cascade affects fasciculation along pioneer axons, leading to secondary cumulative scaffolding defects during the structural organization of the axonal network. The global disorder of architectural landmarks ultimately influences nervous system condensation. In summary, these data point to JNK signaling in a subset of pioneer neurons as a key element underpinning VNC architecture, revealing critical milestones on the mechanism of control of its structural organization. |
Monday, May 6th - Cell Cycle |
| Haseeb, M. A., Weng, K. A., Bickel, S. E. (2023). Chromatin-associated cohesin turns over extensively and forms new cohesive linkages during meiotic prophase. bioRxiv, PubMed ID: 37645916
Summary: In dividing cells, accurate chromosome segregation depends on sister chromatid cohesion, protein linkages that are established during DNA replication. Faithful chromosome segregation in oocytes requires that cohesion, first established in S phase, remain intact for days to decades, depending on the organism. Premature loss of meiotic cohesion in oocytes leads to the production of aneuploid gametes and contributes to the increased incidence of meiotic segregation errors as women age (maternal age effect). The prevailing model is that cohesive linkages do not turn over in mammalian oocytes. However, it has been reported that cohesion-related defects arise in Drosophila oocytes when individual cohesin subunits (see Verthandi) or cohesin regulators are knocked down after meiotic S phase. This study used two strategies to express a tagged cohesin subunit exclusively during mid-prophase in Drosophila oocytes and demonstrate that newly expressed cohesin is used to form de novo linkages after meiotic S phase. Moreover, nearly complete turnover of chromosome-associated cohesin occurs during meiotic prophase, with faster replacement on the arms than at the centromeres. Unlike S-phase cohesion establishment, the formation of new cohesive linkages during meiotic prophase does not require acetylation of conserved lysines within the Smc3 head. These findings indicate that maintenance of cohesion between S phase and chromosome segregation in Drosophila oocytes requires an active cohesion rejuvenation program that generates new cohesive linkages during meiotic prophase. | Koury, S. A. (2023). Female meiotic drive shapes the distribution of rare inversion polymorphisms in Drosophila melanogaster. Genetics. PubMed ID: 37616566
Summary: In all species, new chromosomal inversions are constantly being formed by spontaneous rearrangement and then stochastically eliminated from natural populations. In Drosophila, when new chromosomal inversions overlap with a pre-existing inversion in the population, their rate of elimination becomes a function of the relative size, position, and linkage phase of the gene rearrangements. These altered dynamics result from complex meiotic behavior wherein overlapping inversions generate asymmetric dyads that cause both meiotic drive/drag and segmental aneuploidy. In this context, patterns in rare inversion polymorphisms of a natural population can be modeled from the fundamental genetic processes of forming asymmetric dyads via crossing-over in meiosis I and preferential segregation from asymmetric dyads in meiosis II. A mathematical model was developed of crossover-dependent female meiotic drive and was parameterized with published experimental data from Drosophila melanogaster laboratory constructs. This mechanism is demonstrated to favor smaller, distal inversions and accelerate the elimination of larger, proximal inversions. Simulated sampling experiments indicate that the paracentric inversions directly observed in natural population surveys of Drosophila melanogaster are a biased subset that both maximizes meiotic drive and minimizes the frequency of lethal zygotes caused by this cytogenetic mechanism. Incorporating this form of selection into a population genetic model accurately predicts the shift in relative size, position, and linkage phase for rare inversions found in this species. The model and analysis presented in this study suggest that this weak form of female meiotic drive is an important process influencing the genomic distribution of rare inversion polymorphisms. |
| Warecki, B. and Tao, L. (2023). Centralspindlin-mediated transport of RhoGEF positions the cleavage plane for cytokinesis. Sci Signal 16(792): eadh0601. PubMed ID: 37402224
Summary: During cytokinesis, the cell membrane furrows inward along a cleavage plane. The positioning of the cleavage plane is critical to faithful cell division and is determined by the Rho guanine nucleotide exchange factor (RhoGEF)-mediated activation of the small guanosine triphosphatase RhoA and the conserved motor protein complex centralspindlin. This study explored whether and how centralspindlin mediates the positioning of RhoGEF. In dividing neuroblasts from Drosophila melanogaster, immediately before cleavage, first centralspindlin and then RhoGEF localized to the sites where cleavage subsequently initiated. Using in vitro assays with purified Drosophila proteins and stabilized microtubules, it was found that centralspindlin directly transported RhoGEF as cargo along single microtubules and sequestered it at microtubule plus-ends for prolonged periods of time. In addition, the binding of RhoGEF to centralspindlin appeared to stimulate centralspindlin motor activity. Thus, the motor activity and microtubule association of centralspindlin can translocate RhoGEF to areas where microtubule plus-ends are abundant, such as at overlapping astral microtubules, to locally activate RhoA and accurately position the cleavage plane during cell division. | Baker, C. C., Gallicchio, L., Matias, N. R., Porter, D. F., Parsanian, L., Taing, E., Tam, C., Fuller, M. T. (2023). Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase. Development, 150(22) PubMed ID: 37882771
Summary: During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. This study shows that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase. |
| Sperling, A. L., Fabian, D. K., Garrison, E. and Glover, D. M. (2023). A genetic basis for facultative parthenogenesis in Drosophila. Curr Biol. PubMed ID: 37516115
Summary: Facultative parthenogenesis enables sexually reproducing organisms to switch between sexual and asexual parthenogenetic reproduction. To gain insights into this phenomenon, the genomes of sexually reproducing and parthenogenetic strains of Drosophila mercatorum were sequenced, and differences were identified in the gene expression in their eggs. Then whether manipulating the expression of candidate gene homologs identified in Drosophila mercatorum could lead to facultative parthenogenesis in the non-parthenogenetic species Drosophila melanogaster was tested. This identified a polygenic system whereby increased expression of the mitotic protein kinase polo and decreased expression of a desaturase, Desat2, caused facultative parthenogenesis in the non-parthenogenetic species that was enhanced by increased expression of Myc. The genetically induced parthenogenetic Drosophila melanogaster eggs exhibit de novo centrosome formation, fusion of the meiotic products, and the onset of development to generate predominantly triploid offspring. Thus, this study demonstrated a genetic basis for sporadic facultative parthenogenesis in an animal. | Bakshi, A., Iturra, F. E., Alamban, A., Rosas-Salvans, M., Dumont, S., Aydogan, M. G. (2023). Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes. Cell, 186(21):4694-4709. PubMed ID: 37832525
Summary: Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. This study demonstrates in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, evidence is presented that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. It is postulated that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes. |
Friday, May 3rd - RNAs and Transposons |
| Krzywinska, E., Ribeca, P., Ferretti, L., Hammond, A., Krzywinski, J. (2023). A novel factor modulating X chromosome dosage compensation in Anopheles. Curr Biol, 33(21):4697-4703. PubMed ID: 37774706
Summary: Dosage compensation (DC), a process countering chromosomal imbalance in individuals with heteromorphic sex chromosomes, has been molecularly characterized only in mammals, Caenorhabditis elegans, and fruit flies. In Drosophila melanogaster males, it is achieved by an approximately 2-fold hypertranscription of the monosomic X chromosome mediated by the MSL complex. The complex is not assembled on female X chromosomes because production of its key protein MSL-2 is prevented due to intron retention and inhibition of translation by Sex-lethal, a female-specific protein operating at the top of the sex determination pathway. It remains unclear how DC is mechanistically regulated in other insects. In the malaria mosquito Anopheles gambiae, an approximately 2-fold hypertranscription of the male X also occurs by a yet-unknown molecular mechanism distinct from that in D. melanogaster. This study shows that a male-specifically spliced gene called 007, which arose by a tandem duplication in the Anopheles ancestral lineage, is involved in the control of DC in males. Homozygous 007 knockouts lead to a global downregulation of the male X, phenotypically manifested by a slower development compared to wild-type mosquitoes or mutant females-however, without loss of viability or fertility. In females, a 007 intron retention promoted by the sex determination protein Femaleless, known to prevent hypertranscription from both X chromosomes, introduces a premature termination codon apparently rendering the female transcripts non-productive. In addition to providing a unique perspective on DC evolution, the 007, with its conserved properties, may represent an important addition to a genetic toolbox for malaria vector control. | Yushkova, E. (2024). Interaction effect of mutations in the genes (piwi and aub) of the Argonaute family and hobo transposons on the integral survival parameters of Drosophila melanogaster. Biogerontology, 25(1):131-146 PubMed ID: 37864608
Summary: The Argonaute family genes (piwi and aub) involved in the production of small RNAs are responsible for the regulation of many cellular processes, including the suppression of genome instability, modulation of gene activity, and transposable elements. Dysfunction of these genes and the associated activation of transposable elements adversely affect reproductive development and quality of life. The role of transposons in contrast to retrotransposons and their interaction with genes of the Argonaute family in aging processes have not been studied. This study considers a scenario in which the piwi and aub genes in the presence of functional hobo transposons can modify the effects from the level of DNA damage to lifespan. The simultaneous presence of mutation (piwi or aub) and hobo (regardless of size) in the genome has practically no effect or (less often) leads to a decrease in the level of DNA damage in ovarian cells. A high level of sterility and low ovarian reserve were noted mainly with a combination of mutations and full-sized hobo elements. The combination of these two genetic factors negatively affects the fertility of young females and embryonic survival. Isolated cases of restoration of reproductive functions with age were noted but only in females that had low fertility in the early period of life. The presence of hobo transposons contributed to an increase in the lifespan of both mutant and non-mutant females. Dysfunction of the piwi and aub genes (without hobo) can reduce the lifespan of both sexes. Together, each mutation and hobo transposons act antagonistically/additively (in females) and synergistically/antagonistically (in males) to change the lifespan. In parameters of locus-specific instability, hobo activation was more pronounced in piwi gene dysfunction. The results obtained complement data on the study of new functions of Argonaute family genes and their interactions with transposable elements in the aging process. |
| Kour, S., Fortuna, T., Anderson, E. N., Mawrie, D., Bilstein, J., Sivasubramanian, R., Ward, C., Roy, R., Rajasundaram, D., Sterneckert, J., Pandey, U. B. (2023). Drosha-dependent microRNAs modulate FUS-mediated neurodegeneration in vivo. Nucleic Acids Res, 51(20):11258-11276 PubMed ID: 37791873
Summary: Mutations in the Fused in Sarcoma (FUS) gene cause the familial and progressive form of amyotrophic lateral sclerosis (ALS). FUS is a nuclear RNA-binding protein involved in RNA processing and the biogenesis of a specific set of microRNAs. This study reports that Drosha and two previously uncharacterized Drosha-dependent miRNAs are strong modulators of FUS expression and prevent the cytoplasmic segregation of insoluble mutant FUS in vivo. Depletion of Drosha mitigates FUS-mediated degeneration, survival and motor defects in Drosophila. Mutant FUS strongly interacts with Drosha and causes its cytoplasmic mis-localization into the insoluble FUS inclusions. Reduction in Drosha levels increases the solubility of mutant FUS. Interestingly, two Drosha dependent microRNAs, miR-378i and miR-6832-5p, which differentially regulate the expression, solubility and cytoplasmic aggregation of mutant FUS in iPSC neurons and mammalian cells. More importantly, different modes of action are reported of these miRNAs against mutant FUS. Whereas miR-378i may regulate mutant FUS inclusions by preventing G3BP-mediated stress granule formation, miR-6832-5p may affect FUS expression via other proteins or pathways. Overall, this research reveals a possible association between ALS-linked FUS mutations and the Drosha-dependent miRNA regulatory circuit, as well as a useful perspective on potential ALS treatment via microRNAs. | Liu, M., Xie, X. J., Li, X., Ren, X., Sun, J., Lin, Z., Hemba-Waduge, R. U., Ji, J. Y. (2023). Transcriptional coupling of telomeric retrotransposons with the cell cycle. bioRxiv, PubMed ID: 37808851
Summary: Instead of employing telomerases to safeguard chromosome ends, dipteran species maintain their telomeres by transposition of telomeric-specific retrotransposons (TRs): in Drosophila , these are HeT-A, TART, and TAHRE. Previous studies have shown how these TRs create tandem repeats at chromosome ends, but the exact mechanism controlling TR transcription has remained unclear. This study reports the identification of multiple subunits of the transcription cofactor Mediator complex and transcriptional factors Scalloped (Sd, the TEAD homolog in flies) and E2F1-Dp as novel regulators of TR transcription and telomere length in Drosophila. Depletion of multiple Mediator subunits, Dp, or Sd increased TR expression and telomere length, while over-expressing E2F1-Dp or knocking down the E2F1 regulator Rbf1 (Retinoblastoma-family protein 1) stimulated TR transcription, with Mediator and Sd affecting TR expression through E2F1-Dp. The CUT&RUN analysis revealed direct binding of CDK8, Dp, and Sd to telomeric repeats. These findings highlight the essential role of the Mediator complex in maintaining telomere homeostasis by regulating TR transcription through E2F1-Dp and Sd, revealing the intricate coupling of TR transcription with the host cell-cycle machinery, thereby ensuring chromosome end protection and genomic stability during cell division. |
| Matzkin, L. M., Bono, J. M., Pigage, H. K., Allan, C. W., Diaz, F., McCoy, J. R., Green, C. C., Callan, J. B., Delahunt, S. P. (2023). Females translate male mRNA transferred during mating. bioRxiv, PubMed ID: 37790342
Summary: Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. This study developed an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. Strong evidence is found for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction. | Coronado-Zamora, M., Gonzalez, J. (2023). Transposons contribute to the functional diversification of the head, gut, and ovary transcriptomes across Drosophila natural strains. Genome research, 33(9):1541-1553 PubMed ID: 37793782
Summary: Transcriptomes are dynamic, with cells, tissues, and body parts expressing particular sets of transcripts. Transposable elements (TEs) are a known source of transcriptome diversity; however, studies often focus on a particular type of chimeric transcript, analyze single body parts or cell types, or are based on incomplete TE annotations from a single reference genome. This work has implemented a method based on de novo transcriptome assembly that minimizes the potential sources of errors while identifying a comprehensive set of gene-TE chimeras. This method was applied to the head, gut, and ovary dissected from five Drosophila melanogaster natural strains, with individual reference genomes available. ~19% of body part-specific transcripts were found to be gene-TE chimeras. Overall, chimeric transcripts contribute a mean of 43% to the total gene expression, and they provide protein domains for DNA binding, catalytic activity, and DNA polymerase activity. This comprehensive data set is a rich resource for follow-up analysis. Moreover, because TEs are present in virtually all species sequenced to date, their role in spatially restricted transcript expression is likely not exclusive to the species analyzed in this work. |
Thursday, May 2nd - Enzyme and protein expression, evolution, structure, and function |
| Yoon, H. J., Price, B. E., Parks, R. K., Ahn, S. J., Choi, M. Y. (2023). Diuretic hormone 31 activates two G protein-coupled receptors with differential second messengers for diuresis in Drosophila suzukii. Insect biochemistry and molecular biology, 162:104025 PubMed ID: 37813200
Summary: Diuretic hormones (DHs) bind to G protein-coupled receptors (GPCRs), regulating water and ion balance to maintain homeostasis in animals. Two distinct DHs are known in insects: calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. This study identified and characterized DH31 and two DH31 GPCR variants, DH31-Ra and DH31-Rb, from spotted-wing drosophila, Drosophila suzukii, a globally prevalent vinegar fly causing severe damage to small fruits. Both GPCRs are active, but DH31-Ra is the dominant receptor based on gene expression analyses and DH31 peptide binding affinities. A notable difference between the two variants lies in 1) the GPCR structures of their C-termini and 2) the utilization of second messengers, and the amino acid sequences of the two variants are identical. DH31-Ra contains 12 additional amino acids, providing different intracellular C-terminal configurations. DH31-Ra utilizes both cAMP and Ca(2+) as second messengers, whereas DH31-Rb utilizes only cAMP; this is the first time reported for an insect CT-like DH31 peptide. DH31 stimulated fluid secretion in D. suzukii adults, and secretion increased in a dose-dependent manner. However, when the fly was injected with a mixture of DH31 and CAPA, an anti-diuretic hormone, fluid secretion was suppressed. The structures of the DH31 receptors and the differential signaling pathways, including second messengers, involved in fly diuresis, are discussed. These findings provide fundamental insights into the characterization of D. suzukii DH31 and DH31-Rs, and facilitate the identification of potential biological targets for D. suzukii management. | Yarikipati, P., Jonusaite, S., Pleinis, J. M., Dominicci Cotto, C., Sanchez-Hernandez, D., Morrison, D. E., Goyal, S., Schellinger, J., Penalva, C., Curtiss, J., Rodan, A. R., Jenny, A. (2023). Unanticipated domain requirements for Drosophila Wnk kinase in vivo. PLoS Genet, 19(10):e1010975 PubMed ID: 37819975
Summary: WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate canonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. This study shows that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. The only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. These data thus provide novel insights into unexpected in vivo roles of specific WNK domains. |
| Sayeesh, P. M., Iguchi, M., Suemoto, Y., Inoue, J., Inomata, K., Ikeya, T., Ito, Y. (2023). Interactions of the N- and C-Terminal SH3 Domains of Drosophila Drk with the Proline-Rich Peptides from Sos and Dos. Int J Mol Sci, 24(18) PubMed ID: 37762438
Summary: Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. A previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, NMR titration experiments were conducted using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. The results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. Docking simulations were performed based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, the various modes in the interactions of Drk with Sos/Dos are discussed. | Gupta, K., Chakrabarti, S., Janardan, V., Gogia, N., Banerjee, S., Srinivas, S., Mahishi, D., Visweswariah, S. S. (2023). Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response. PLoS Genet, 19(9):e1010962 PubMed ID: 37733787
Summary: Evolutionarily conserved genes often play critical roles in organismal physiology. This study describes multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. It was observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism. |
| Keller, S. H., Deng, H., Lim, B. (2023). Regulation of the dynamic RNA Pol II elongation rate in Drosophila embryos. Cell Rep, 42(10):113225 PubMed ID: 37837623
Summary: An increasing number of studies have shown the key role that RNA polymerase II (RNA Pol II) elongation plays in gene regulation. This study systematically examine how various enhancers, promoters, and gene body composition influence the RNA Pol II elongation rate through a single-cell-resolution live imaging assay. By using reporter constructs containing 5' MS2 and 3' PP7 repeating stem loops, the rate of RNA Pol II elongation in live Drosophila embryos was quantified. Promoters and exonic gene lengths have no effect on elongation rate, while enhancers and the presence of long introns may significantly change how quickly RNA Pol II moves across a gene. Furthermore, it was observed in multiple constructs that the RNA Pol II elongation rate accelerates after the transcriptional onset of nuclear cycle 14 in Drosophila embryos. This study provides a single-cell view of various mechanisms that affect the dynamic RNA Pol II elongation rate, ultimately affecting the rate of mRNA production. | Viola, C. M., Frittmann, O., Jenkins, H. T., Shafi, T., De Meyts, P., Brzozowski, A. M. (2023). Structural conservation of insulin/IGF signalling axis at the insulin receptors level in Drosophila and humans. Nat Commun, 14(1):6271 PubMed ID: 37805602
Summary: The insulin-related hormones regulate key life processes in Metazoa, from metabolism to growth, lifespan and aging, through an evolutionarily conserved insulin signalling axis (IIS). In humans the IIS axis is controlled by insulin, two insulin-like growth factors, two isoforms of the insulin receptor (hIR-A and -B), and its homologous IGF-1R. In Drosophila, this signalling engages seven insulin-like hormones (DILP1-7) and a single receptor (dmIR). This report describes the cryoEM structure of the dmIR ectodomain:DILP5 complex, revealing high structural homology between dmIR and hIR. The excess of DILP5 yields dmIR complex in an asymmetric 'T' conformation, similar to that observed in some complexes of human IRs. However, dmIR binds three DILP5 molecules in a distinct arrangement, showing also dmIR-specific features. This work adds structural support to evolutionary conservation of the IIS axis at the IR level, and also underpins a better understanding of an important model organism. |
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