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| ARCHIVE | Friday, November 29th - Cell Cycle |
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October 2024 September 2024 August 2024 July 2024 June 2024 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 December 2021 December 2020 December 2019 December 2018 | Matsuura, Y., Kaizuka, K., Inoue, Y. H. (2024). Essential Role of COPII Proteins in Maintaining the Contractile Ring Anchoring to the Plasma Membrane during Cytokinesis in Drosophila Male Meiosis. Int J Mol Sci, 25(8) PubMed ID: 38674111
Summary: Coatomer Protein Complex-II (COPII) mediates anterograde vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus. This study reports that the COPII coatomer complex is constructed dependent on a small GTPase, Sar1, in spermatocytes before and during Drosophila male meiosis. COPII-containing foci co-localized with transitional endoplasmic reticulum (tER)-Golgi units. They showed dynamic distribution along astral microtubules and accumulated around the spindle pole, but they were not localized on the cleavage furrow (CF) sites. The depletion of the four COPII coatomer subunits, Sec16, or Sar1 that regulate COPII assembly resulted in multinucleated cell production after meiosis, suggesting that cytokinesis failed in both or either of the meiotic divisions. Although contractile actomyosin and anilloseptin rings (Anillin, and septin filaments collectively) were formed once plasma membrane ingression was initiated, they were frequently removed from the plasma membrane during furrowing. This study explored the factors conveyed toward the CF sites in the membrane via COPII-mediated vesicles. DE-cadherin-containing vesicles were formed depending on Sar1 and were accumulated in the cleavage sites. Furthermore, COPII depletion inhibited de novo plasma membrane insertion. These findings suggest that COPII vesicles supply the factors essential for the anchoring and/or constriction of the contractile rings at cleavage sites during male meiosis in Drosophila. | Khodaverdian, V., Sano, T., Maggs, L., Tomarchio, G., Dias, A., Clairmont, C., Tran, M., McVey, M. (2024). REV1 Coordinates a Multi-Faceted Tolerance Response to DNA Alkylation Damage and Prevents Chromosome Shattering in Drosophila melanogaster. bioRxiv, PubMed ID: 38405884
Summary: When replication forks encounter damaged DNA, cells utilize DNA damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, this study has investigated damage tolerance responses following alkylation damage in Drosophila melanogaster. Translesion synthesis, rather than template switching, was shown to be the preferred response to alkylation-induced damage in diploid larval tissues. Furthermore, it was shown that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Drosophila larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of gamma-H2Av foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerase eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, these results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability. |
| Pazhayam, N. M., Frazier, L. K., Sekelsky, J. (2024). Centromere-proximal suppression of meiotic crossovers in Drosophila is robust to changes in centromere number, repetitive DNA content, and centromere-clustering. Genetics, 226(3) PubMed ID: 38150397 and PubMed ID
Summary: Accurate segregation of homologous chromosomes during meiosis depends on both the presence and the regulated placement of crossovers (COs). The centromere effect, or CO exclusion in pericentromeric regions of the chromosome, is a meiotic CO patterning phenomenon that helps prevent nondisjunction, thereby protecting against chromosomal disorders and other meiotic defects. Despite being identified nearly a century ago, the mechanisms behind this fundamental cellular process remain unknown, with most studies of the Drosophila centromere effect focusing on local influences of the centromere and pericentric heterochromatin. This study sought to investigate whether dosage changes in centromere number and repetitive DNA content affect the strength of the centromere effect, using phenotypic recombination mapping. Additionally, he effects of repetitive DNA function on centromere effect strength was studied using satellite DNA-binding protein mutants displaying defective centromere-clustering in meiotic nuclei. Despite what previous studies suggest, the results show that the Drosophila centromere effect is robust to changes in centromere number, repetitive DNA content, as well as repetitive DNA function. This study suggests that the centromere effect is unlikely to be spatially controlled, providing novel insight into the mechanisms behind the Drosophila centromere effect. | Sun, S., Defosse, T., Boyd, A., Sop, J., Verderose, F., Surray, D., Aziz, M., Howland, M., Wu, S., Changela, N., Jang, J., Schindler, K., Xing, J., McKim, K. S. (2024). Whole transcriptome screening for novel genes involved in meiosis and fertility in Drosophila melanogaster. Sci Rep, 14(1):3602 PubMed ID: 38351116
Summary: Reproductive success requires the development of viable oocytes and the accurate segregation of chromosomes during meiosis. Failure to segregate chromosomes properly can lead to infertility, miscarriages, or developmental disorders. A variety of factors contribute to accurate chromosome segregation and oocyte development, such as spindle assembly and sister chromatid cohesion. However, many proteins required for meiosis remain unknown. This study aimed to develop a screening pipeline for identifying novel meiotic and fertility genes using the genome of Drosophila melanogaster. To accomplish this goal, genes upregulated within meiotically active tissues were identified. More than 240 genes with no known function were silenced using RNA interference (RNAi) and the effects on meiosis and fertility were assessed. 94 genes were identified that when silenced caused infertility and/or high levels of chromosomal nondisjunction. The vast majority of these genes have human and mouse homologs that are also poorly studied. Through this screening process, novel genes were identified that are crucial for meiosis and oocyte development but have not been extensively studied in human or model organisms. Understanding the function of these genes will be an important step towards the understanding of their biological significance during reproduction. |
| Joshi, J. N., Changela, N., Mahal, L., Defosse, T., Jang, J., Wang, L. I., Das, A., Shapiro, J. G., McKim, K. (2024). Meiosis-specific functions of kinetochore protein SPC105R required for chromosome segregation in Drosophila oocytes. bioRxiv. PubMed ID: 38559067
Summary: The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. Previous work has implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and co-orientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. SPC105R's C-terminal domain (aa 1284-1960) was found to be necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, the first 473 were found most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for two activities that are critical for accurate chromosome segregation in meiosis I, lateral microtubule attachments and bi-orientation of homologs. | Wood, B. W., Shi, X., Weil, T. T. (2024). F-actin coordinates spindle morphology and function in Drosophila meiosis. PLoS Genet, 20(1):e1011111 PubMed ID: 38206959
Summary: Meiosis is a highly conserved feature of sexual reproduction that ensures germ cells have the correct number of chromosomes prior to fertilization. A subset of microtubules, known as the spindle, are essential for accurate chromosome segregation during meiosis. Building evidence in mammalian systems has recently highlighted the unexpected requirement of the actin cytoskeleton in chromosome segregation; a network of spindle actin filaments appear to regulate many aspects of this process. Thisa study shows that Drosophila oocytes also have a spindle population of actin that appears to regulate the formation of the microtubule spindle and chromosomal movements throughout meiosis. Genetic and pharmacological disruption of the actin cytoskeleton was demonstrated to have a significant impact on spindle morphology, dynamics, and chromosome alignment and segregation during maturation and the metaphase-anaphase transition. A role was revealed for calcium in maintaining the microtubule spindle and spindle actin. Together, these data highlights potential conservation of morphology and mechanism of the spindle actin during meiosis. |
Tuesday, November 26th - Metabolism and Physiology |
| Hunter-Manseau, F., Cormier, S. B., Strang, R., Pichaud, N. (2024). Fasting as a precursor to high-fat diet enhances mitochondrial resilience in Drosophila melanogaster. Insect Sci, PubMed ID: 38514255
Summary: Changes in diet type and nutrient availability can impose significant environmental stress on organisms, potentially compromising physiological functions and reproductive success. In nature, dramatic fluctuations in dietary resources are often observed and adjustments to restore cellular homeostasis are crucial to survive this type of stress. This study exposed male Drosophila melanogaster to two modulated dietary treatments: one without a fasting period before exposure to a high-fat diet and the other with a 24-h fasting period. Then mitochondrial metabolism and molecular responses to these treatments was investigated. Exposure to a high-fat diet without a preceding fasting period resulted in disrupted mitochondrial respiration, notably at the level of complex I. On the other hand, a short fasting period before the high-fat diet maintained mitochondrial respiration. Generally, transcript abundance of genes associated with mitophagy, heat-shock proteins, mitochondrial biogenesis, and nutrient sensing pathways increased either slightly or significantly following a fasting period and remained stable when flies were subsequently put on a high-fat diet, whereas a drastic decrease of almost all transcript abundances was observed for all these pathways when flies were exposed directly to a high-fat diet. Moreover, mitochondrial enzymatic activities showed less variation after the fasting period than the treatment without a fasting period. Overall, this study sheds light on the mechanistic protective effects of fasting prior to a high-fat diet and highlights the metabolic flexibility of Drosophila mitochondria in response to abrupt dietary changes and have implication for adaptation of species to their changing environment. | Aziz, R. A., Ramesh, P., Suchithra, K. V., Stothard, P., Narayana, V. K., Raghu, S. V., Shen, F. T., Young, C. C., Prasad, T. S. K., Hameed, A. (2024). Comprehensive insights into the impact of bacterial indole-3-acetic acid on sensory preferences in Drosophila melanogaster. Sci Rep, 14(1):8311 PubMed ID: 38594449
Summary: Several bacteria of environmental and clinical origins, including some human-associated strains secrete a cross-kingdom signaling molecule indole-3-acetic acid (IAA). IAA is a tryptophan (trp) derivative mainly known for regulating plant growth and development as a hormone. However, the nutritional sources that boost IAA secretion in bacteria and the impact of secreted IAA on non-plant eukaryotic hosts remained less explored. This study demonstrated significant trp-dependent IAA production in Pseudomonas juntendi NEEL19 when provided with ethanol as a carbon source in liquid cultures. IAA was further characterized to modulate the odor discrimination, motility and survivability in Drosophila melanogaster. A detailed analysis of IAA-fed fly brain proteome using high-resolution mass spectrometry showed significant alteration in the proteins governing neuromuscular features, audio-visual perception and energy metabolism as compared to IAA-unfed controls. Sex-wise variations in differentially regulated proteins were witnessed despite having similar visible changes in chemo perception and psychomotor responses in IAA-fed flies. This study not only revealed ethanol-specific enhancement in trp-dependent IAA production in P. juntendi, but also showed marked behavioral alterations in flies for which variations in an array of proteins governing odor discrimination, psychomotor responses, and energy metabolism are held responsible. This study provided novel insights into disruptive attributes of bacterial IAA that can potentially influence the eukaryotic gut-brain axis having broad environmental and clinical implications. |
| Gururaja Rao, S., Lam, A., Seeley, S., Park, J., Aruva, S., Singh, H. (2024). . The BK(Ca) (slo) channel regulates the cardiac function of Drosophila. Physiological reports, 12(7):e15996 PubMed ID: 38561252
Summary: The large conductance, calcium, and voltage-active potassium channels (BK(Ca)) were originally discovered in Drosophila melanogaster as slowpoke (slo). They are extensively characterized in fly models as ion channels for their roles in neurological and muscular function, as well as aging. BK(Ca) is known to modulate cardiac rhythm and is localized to the mitochondria. Activation of mitochondrial BK(Ca) causes cardioprotection from ischemia-reperfusion injury, possibly via modulating mitochondrial function in adult animal models. However, the role of BK(Ca) in cardiac function is not well-characterized, partially due to its localization to the plasma membrane as well as intracellular membranes and the wide array of cells present in mammalian hearts. This study demonstrates for the first time a direct role for BK(Ca) in cardiac function and cardioprotection from IR injury using the Drosophila model system. It was also discovered that the BK(Ca) channel plays a role in the functioning of aging hearts. This study establishes the presence of BK(Ca) in the fly heart and ascertains its role in aging heart function. | Jiang, N. J., Dong, X., Veit, D., Hansson, B. S., Knaden, M. (2024). Elevated ozone disrupts mating boundaries in drosophilid flies. Nat Commun, 15(1):2872 PubMed ID: 38605003
Summary: Animals employ different strategies to establish mating boundaries between closely related species, with sex pheromones often playing a crucial role in identifying conspecific mates. Many of these pheromones have carbon-carbon double bonds, making them vulnerable to oxidation by certain atmospheric oxidant pollutants, including ozone. This study investigated whether increased ozone compromises species boundaries in drosophilid flies. We show that short-term exposure to increased levels of ozone degrades pheromones of Drosophila melanogaster, D. simulans, D. mauritiana, as well as D. sechellia, and induces hybridization between some of these species. As many of the resulting hybrids are sterile, this could result in local population declines. However, hybridization between D. simulans and D. mauritiana as well as D. simulans and D. sechellia results in fertile hybrids, of which some female hybrids are even more attractive to the males of the parental species. Our experimental findings indicate that ozone pollution could potentially induce breakdown of species boundaries in insects. |
| van Heerwaarden, B., Sgro, C., Kellermann, V. M. (2024). Threshold shifts and developmental temperature impact trade-offs between tolerance and plasticity. Proceedings Biological sciences, 291(2016):20232700 PubMed ID: 38320612
Summary: Mounting evidence suggests that ectotherms are already living close to their upper physiological thermal limits. Phenotypic plasticity has been proposed to reduce the impact of climate change in the short-term providing time for adaptation, but the tolerance-plasticity trade-off hypothesis predicts organisms with higher tolerance have lower plasticity. Empirical evidence is mixed, which may be driven by methodological issues such as statistical artefacts, nonlinear reaction norms, threshold shifts or selection. This study examined whether threshold shifts (organisms with higher tolerance require stronger treatments to induce maximum plastic responses) influence tolerance-plasticity trade-offs in hardening capacity for desiccation tolerance and critical thermal maximum (CT(MAX)) across Drosophila species with varying distributions/sensitivity to desiccation/heat stress. Evidence was found for threshold shifts in both traits; species with higher heat/desiccation tolerance required longer hardening treatments to induce maximum hardening responses. Species with higher heat tolerance also showed reductions in hardening capacity at higher developmental acclimation temperatures. Trade-off patterns differed depending on the hardening treatment used and the developmental temperature flies were exposed to. Based on these findings, studies that do not consider threshold shifts, or that estimate plasticity under a narrow set of environments, will have a limited ability to assess trade-off patterns and differences in plasticity across species/populations more broadly. | Zhu, H., Ludington, W. B., Spradling, A. C. (2024). Cellular and molecular organization of the Drosophila foregut. Proc Natl Acad Sci U S A, 121(11):e2318760121 PubMed ID: 38442150
Summary: The animal foregut is the first tissue to encounter ingested food, bacteria, and viruses. This study characterized the adult Drosophila foregut using transcriptomics to better understand how it triages consumed items for digestion or immune response and manages resources. Cell types were assigned and validated using GFP-tagged and Gal4 reporter lines. Foregut-associated neuroendocrine cells play a major integrative role by coordinating gut activity with nutrition, the microbiome, and circadian cycles; some express clock genes. Multiple epithelial cell types comprise the proventriculus, the central foregut organ that secretes the peritrophic matrix (PM) lining the gut. Analyzing cell types synthesizing individual PM layers revealed abundant mucin production close to enterocytes, similar to the mammalian intestinal mucosa. The esophagus and salivary gland express secreted proteins likely to line the esophageal surface, some of which may generate a foregut commensal niche housing specific gut microbiome species. Overall, these results imply that the foregut coordinates dietary sensing, hormonal regulation, and immunity in a manner that has been conserved during animal evolution. |
Monday, November 25th - Evolution |
| Cavigliasso, F., Savitsky, M., Koval, A., Erkosar, B., Savary, L., Gallart-Ayala, H., Ivanisevic, J., Katanaev, V. L., Kawecki, T. J. (2024). Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in Drosophila. PLoS Genet, 20(3):e1011204 PubMed ID: 38452112
Summary: This study investigated the contribution of a candidate gene, fezzik (fiz), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. This divergence in fiz expression is shown to be mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, fiz knockdown by RNAi was shown to result in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation. | Nunes, W. V. B., Oliveira, D. S., Dias, G. R., Carvalho, A. B., Caruso I, P., Biselli, J. M., Guegen, N., Akkouche, A., Burlet, N., Vieira, C., Carareto, C. M. A. (2024). A comprehensive evolutionary scenario for the origin and neofunctionalization of the Drosophila speciation gene Odysseus (OdsH). G3 (Bethesda), 14(3) PubMed ID: 38156703
Summary: Odysseus (OdsH) was the first speciation gene described in Drosophila related to hybrid sterility in offspring of mating between Drosophila mauritiana and Drosophila simulans. Its origin is attributed to the duplication of the gene unc-4 in the subgenus Sophophora. By using a much larger sample of Drosophilidae species, this study showed that contrary to what has been previously proposed, OdsH origin occurred 62 MYA. Evolutionary rates, expression, and transcription factor-binding sites of OdsH evidence that it may have rapidly experienced neofunctionalization in male sexual functions. Furthermore, the analysis of the OdsH peptide allowed the identification of mutations of D. mauritiana that could result in incompatibility in hybrids. In order to find if OdsH could be related to hybrid sterility, beyond Sophophora,the expression of OdsH was explored in Drosophila arizonae and Drosophila mojavensis, a pair of sister species with incomplete reproductive isolation. The data indicated that OdsH expression is not atypical in their male-sterile hybrids. In conclusion, it is proposed that the origin of OdsH occurred earlier than previously proposed, followed by neofunctionalization. These results also suggested that its role as a speciation gene might be restricted to D. mauritiana and D. simulans. |
| Mishra, P., Rundle, H. D., Agrawal, A. F. (2024). The evolution of sexual dimorphism in gene expression in response to a manipulation of mate competition.. Evolution, 78(4):746-757 PubMed ID: 38270064
Summary: Many genes are differentially expressed between males and females and patterns of sex-biased gene expression (SBGE) vary among species. Some of this variation is thought to have evolved in response to differences in mate competition among species that cause varying patterns of sex-specific selection.This study used experimental evolution to test this by quantifying SBGE and sex-specific splicing in 15 Drosophila melanogaster populations that evolved for 104 generations in mating treatments that removed mate competition via enforced monogamy, or allowed mate competition in either small, simple, or larger, structurally more complex mating environments. Consistent with sex-specific selection affecting SBGE, initially sex-biased genes diverged in expression more among treatments than unbiased genes, and there was greater expression divergence for male- than female-biased genes. It has been suggested the transcriptome should be "feminized" under monogamy because of the removal of sexual selection on males; this was not observe, likely because selection differs in additional ways between monogamy vs. polygamy. Significant divergence in average expression dimorphism between treatments was observed and, in some treatment comparisons, the direction of the divergence differed across different sex-bias categories. There was not a generalized reduction in expression dimorphism under enforced monogamy. | Pennell, T. M., Sharma, M. D., Sutter, A., Wilson, D. T., House, C. M., Hosken, D. J. (2024). The condition-dependence of male genital size and shape. Ecology and evolution 14(3):e11180 PubMed ID: 38495435
Summary: The male genitals of internal fertilisers evolve rapidly and divergently, and sexual selection is generally responsible for this. Many sexually selected traits are condition-dependent-with their expression dependent upon the resources available to be allocated to them-as revealed by genetic or environmental manipulations of condition. However, it is not clear whether male genitals are also condition-dependent. This study manipulated conditions in two ways (via inbreeding and diet) to test the condition-dependence of the genital arch of Drosophila simulans. Genital size but not genital shape was found to suffer from inbreeding depression, whereas genital size and shape were affected by dietary manipulation of condition. The differential effects of these treatments likely reflect underlying genetic architecture that has been shaped by past selection: inbreeding depression is only expected when traits have a history of directional selection, while diet impacts traits regardless of historical selection. Nonetheless, these results suggest genitals can be condition-dependent like other sexually selected traits. |
| Liu, G. Y., Jouandin, P., Bahng, R. E., Perrimon, N., Sabatini, D. M. (2024). An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway. Nat Commun, 15(1):2517 PubMed ID: 38514639
Summary: Animals sense and respond to nutrient availability in their environments, a task coordinated in part by the mTOR complex 1 (mTORC1) pathway. mTORC1 regulates growth in response to nutrients and, in mammals, senses specific amino acids through specialized sensors that bind the GATOR1/2 signaling hub. Given that animals can occupy diverse niches, it was hypothesized that the pathway might evolve distinct sensors in different metazoan phyla. Whether such customization occurs, and how the mTORC1 pathway might capture new inputs, is unknown. This study identified the Drosophila melanogaster protein Unmet expectations (CG11596) as a species-restricted methionine sensor that directly binds the fly GATOR2 complex in a fashion antagonized by S-adenosylmethionine (SAM). Tn Dipterans GATOR2 was found to rapidly evolve the capacity to bind Unmet and to thereby repurpose a previously independent methyltransferase as a SAM sensor. Thus, the modular architecture of the mTORC1 pathway allows it to co-opt preexisting enzymes to expand its nutrient sensing capabilities, revealing a mechanism for conferring evolvability on an otherwise conserved system. | Ertl, H. A., Bayala, E. X., Siddiq, M. A., Wittkopp, P. J. (2024). Divergence of Grainy head affects chromatin accessibility, gene expression, and embryonic viability in Drosophila melanogaster. bioRxiv, PubMed ID: 38645200
Summary: Pioneer factors are critical for gene regulation and development because they bind chromatin and make DNA more accessible for binding by other transcription factors. The pioneer factor Grainy head (Grh) is present across metazoans and has been shown to retain a role in epithelium development in fruit flies, nematodes, and mice despite extensive divergence in both amino acid sequence and length. This study investigated the evolution of Grh function by comparing the effects of the fly (Drosophila melanogaster) and worm (Caenorhabditis elegans) Grh orthologs on chromatin accessibility, gene expression, embryonic development, and viability in transgenic D. melanogaster.The Caenorhabditis elegans ortholog rescued cuticle development but not full embryonic viability in Drosophila melanogaster grh null mutants. At the molecular level, the C. elegans ortholog only partially rescued chromatin accessibility and gene expression. Divergence in the disordered N-terminus of the Grh protein contributes to these differences in embryonic viability and molecular phenotypes. These data show how pioneer factors can diverge in sequence and function at the molecular level while retaining conserved developmental functions at the organismal level. |
Friday, November 22nd - Gonads |
| Rinehart, L., Stewart, W. E., Luffman, N., Wawersik, M., Kerscher, O. (2024). Chigno/CG11180 and SUMO are Chinmo-interacting proteins with a role in Drosophila testes somatic support cells. PeerJ, 12:e16971 PubMed ID: 38495765
Summary: Stem cells are critical for replenishment of cells lost to death, damage or differentiation. Drosophila testes are a key model system for elucidating mechanisms regulating stem cell maintenance and differentiation. An intriguing gene identified through such studies is the transcription factor, chronologically inappropriate morphogenesis (Chinmo). Chinmo is a downstream effector of the Jak-STAT signaling pathway that acts in testis somatic stem cells to ensure maintenance of male stem cell fate and sexual identity. Defects in these processes can lead to infertility and the formation of germ cell tumors. While Chinmo's effect on testis stem cell behavior has been investigated in detail, there is still much to be learned about its structure, function, and interactions with other proteins. Using a two-hybrid screen, this study found that Chinmo interacts with itself, the small ubiquitin-like modifier SUMO, the novel protein CG11180, and four other proteins (CG4318, Ova (ovaries absent), Taf3 (TBP-associated factor 3), and CG18269). Since both Chinmo and CG11180 contain sumoylation sites and SUMO-interacting motifs (SIMs), their interaction was studied in more detail. Using site-directed mutagenesis of a unique SIM in CG11180, this study demonstrate that Chinmo's interaction with CG11180 is SUMO-dependent. Furthermore, to assess the functional relevance of both SUMO and CG11180, RNAi-mediated knockdown of both proteins was perfomed in somatic cells of the Drosophila testis. Using this approach, it was fond that CG11180 and SUMO are required in somatic cells of adult testes, and that reduction of either protein causes formation of germ cell tumors. Overall, this work suggests that SUMO may be involved in the interaction of Chinmo and CG11180 and that these genes are required in somatic cells of the adult Drosophila testis. Consistent with the CG11180 knockdown phenotype in male testes, and to underscore its connection to Chinmo, the name Chigno (Childless Gambino) for CG11180 is proposed. | Chen, J., Li, C., Sheng, Y., Zhang, J., Pang, L., Dong, Z., Wu, Z., Lu, Y., Liu, Z., Zhang, Q., Guan, X., Chen, X., Huang, J. (2024). Communication between the stem cell niche and an adjacent differentiation niche through miRNA and EGFR signaling orchestrates exit from the stem cell state in the Drosophila ovary. PLoS Biol, 22(3):e3002515 PubMed ID: 38512963
Summary: The signaling environment, or niche, often governs the initial difference in behavior of an adult stem cell and a derivative that initiates a path towards differentiation. The transition between an instructive stem cell niche and differentiation niche must generally have single-cell resolution, suggesting that multiple mechanisms might be necessary to sharpen the transition. This study examined the Drosophila ovary and found that Cap cells, which are key constituents of the germline stem cell (GSC) niche, express a conserved microRNA (miR-124). Surprisingly, loss of miR-124 activity in Cap cells leads to a defect in differentiation of GSC derivatives. Evidence is presented that the direct functional target of miR-124 in Cap cells is the epidermal growth factor receptor (EGFR) and that failure to limit EGFR expression leads to the ectopic expression of a key anti-differentiation BMP signal in neighboring somatic escort cells (ECs), which constitute a differentiation niche. It was further found that Notch signaling connects EFGR activity in Cap cells to BMP expression in ECs. It was deduced that the stem cell niche communicates with the differentiation niche through a mechanism that begins with the selective expression of a specific microRNA and culminates in the suppression of the major anti-differentiation signal in neighboring cells, with the functionally important overall role of sharpening the spatial distinction between self-renewal and differentiation environments. |
| Hikawa, N., Kashio, S., Miura, M. (2024). Mating-induced increase of kynurenine in Drosophila ovary enhances starvation resistance of progeny. J Biol Chem, 300(3):105663 PubMed ID: 38246353
Summary: The maternal nutritional environment can impact progeny development, stress tolerance, and longevity. Such phenotypic variation of offspring resulting from the maternal environment is often referred to as the 'maternal effect' and is observed across taxa, including in humans. While some mechanisms behind maternal effects have been revealed, such as histone modification, many studies rely on drastic genetic or nutritional manipulation in describing these mechanisms. This study aimed to reveal how the maternal environment is regulated under physiological conditions to affect the progeny. Specifically, metabolic regulation in oocytes in response to mating is detailed using Drosophila melanogaster. Using liquid chromatography-mass spectrometry, it was found that upon mating, the ovary metabolites shifted, predominantly toward increasing amino acids and the tryptophan/kynurenine (Kyn) pathway. This mating-induced increase in ovary Kyn was driven by increased Kyn production in the fat body, a functional counterpart of the mammalian liver and white adipose tissue and the source of Kyn storage for the ovary after mating. Furthermore, maternal Kyn repression was shown to decrease the starvation resistance of progeny and that administering exogenous Kyn to the maternal generation enhanced the starvation resistance of female progeny. Taken together, these findings point to a previously unidentified role of fat body Kyn distribution during reproduction on progeny survival. | Uttekar, B., Verma, R. K., Tomer, D., Rikhy, R. (2024). Mitochondrial morphology dynamics and ROS regulate apical polarity and differentiation in Drosophila follicle cells. Development, 151(5) PubMed ID: 38345270
Summary: Mitochondrial morphology dynamics regulate signaling pathways during epithelial cell formation and differentiation. The mitochondrial fission protein Drp1 affects the appropriate activation of EGFR and Notch signaling-driven differentiation of posterior follicle cells in Drosophila oogenesis. The mechanisms by which Drp1 regulates epithelial polarity during differentiation are not known. In this study, we show that Drp1-depleted follicle cells are constricted in early stages and present in multiple layers at later stages with decreased levels of apical polarity protein aPKC. These defects are suppressed by additional depletion of mitochondrial fusion protein . Opa1 depletion leads to mitochondrial fragmentation and increased reactive oxygen species (ROS) in follicle cells. Increasing ROS by depleting the ROS scavengers, mitochondrial SOD2 and catalase also leads to mitochondrial fragmentation. Further, the loss of Opa1, SOD2 and catalase partially restores the defects in epithelial polarity and aPKC, along with EGFR and Notch signaling in Drp1-depleted follicle cells. These results show a crucial interaction between mitochondrial morphology, ROS generation and epithelial cell polarity formation during the differentiation of follicle epithelial cells in Drosophila oogenesis. |
| Zelinger, E., Brumfeld, V., Rechav, K., Waiger, D., Kossovsky, T., Heifetz, Y. (2024). Three-dimensional correlative microscopy of the Drosophila female reproductive tract reveals modes of communication in seminal receptacle sperm storage. Communications biology, 7(1):155 PubMed ID: 38321098
Summary: In many taxa, females store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. These specialized tubules perform the essential tasks of transporting sperm through the female reproductive tract and supporting long-term sperm survival and function. Little is known about the way in which female sperm storage organs provide an environment conducive to sperm survival. This was addressed using a combined light microscopy, micro computed tomography (microCT), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) approach for high-resolution correlative three-dimensional imaging to advance our understanding of sperm-female interactions in Drosophila melanogaster. Using this multimodal approach, it was possible to scan the lower female reproductive tract and distal portion of the seminal receptacle at low magnification, and to subsequently zoom in for further analysis on an ultrastructural level. The findings highlight aspects of the way in which the seminal receptacle keeps sperm viable in the lumen, and set the stage for further studies. The methods developed are suitable not only for Drosophila but also for other organisms with soft, delicate tissues. | Heaney, J., Zhao, J., Casagranda, F., Loveland, K. L., Siddall, N. A., Hime, G. R. (2024). Drosophila Importin Alpha 1 (Dα1) Is Required to Maintain Germline Stem Cells in the Testis Niche. Cells, 13(6) PubMed ID: 38534338
Summary: Stem cell maintenance and differentiation can be regulated via the differential activity of transcription factors within stem cells and their progeny. For these factors to be active, they need to be transported from their site of synthesis in the cytoplasm into the nucleus. A tissue-specific requirement for factors involved in nuclear importation is a potential mechanism to regulate stem cell differentiation. This study has undertaken a characterization of male sterile importin alpha 1 (Dα1) null alleles in Drosophila and found that Dα1 is required for maintaining germline stem cells (GSCs) in the testis niche. The loss of GSCs can be rescued by ectopic expression of Dα1 within the germline but the animals are still infertile, indicating a second role for Dα1 in spermatogenesis. Expression of a Dα1 dominant negative transgene in GSCs confirmed a functional requirement for Dα1 in GSC maintenance but expression of the transgene in differentiating spermatogonia did not exhibit a phenotype indicating a specific role for Dα1 within GSCs. Our data indicate that Dα1 is utilized as a regulatory protein within GSCs to facilitate nuclear importation of proteins that maintain the stem cell pool. |
Thursday, November 21st - Adult Development |
| Guo, X., Wang, C., Zhang, Y., Wei, R., Xi, R. (2024). Cell-fate conversion of intestinal cells in adult Drosophila midgut by depleting a single transcription factor. Nat Commun, 15(1):2656 PubMed ID: 38531872
Summary: The manipulation of cell identity by reprograming holds immense potential in regenerative medicine, but is often limited by the inefficient acquisition of fully functional cells. This problem can potentially be resolved by better understanding the reprogramming process using in vivo genetic models, which are currently scarce. This study reports that both enterocytes (ECs) and enteroendocrine cells (EEs) in adult Drosophila midgut show a surprising degree of cell plasticity. Depleting the transcription factor Tramtrack in the differentiated ECs can initiate Prospero-mediated cell transdifferentiation, leading to EE-like cells. On the other hand, depletion of Prospero in the differentiated EEs can lead to the loss of EE-specific transcription programs and the gain of intestinal progenitor cell identity, allowing cell cycle re-entry or differentiation into ECs. Intestinal progenitor cells, ECs, and EEs were found to have a similar chromatin accessibility profile, supporting the concept that cell plasticity is enabled by pre-existing chromatin accessibility with switchable transcription programs. Further genetic analysis with this system reveals that the NuRD chromatin remodeling complex, cell lineage conflict, and age act as barriers to EC-to-EE transdifferentiation. The establishment of this genetically tractable in vivo model should facilitate mechanistic investigation of cell plasticity at the molecular and genetic level. | Rodriguez, A., Foronda, D., Cordoba, S., Felipe-Cordero, D., Baonza, A., Miguez, D. G., Estella, C. (2024). Cell proliferation and Notch signaling coordinate the formation of epithelial folds in the Drosophila leg. Development, 151(8) PubMed ID: 38512712
Summary: The formation of complex three-dimensional organs during development requires precise coordination between patterning networks and mechanical forces. In particular, tissue folding is a crucial process that relies on a combination of local and tissue-wide mechanical forces. This study investigated the contribution of cell proliferation to epithelial morphogenesis using the Drosophila leg tarsal folds as a model. Tissue-wide compression forces generated by cell proliferation, in coordination with the Notch signaling pathway, were revealed to be essential for the formation of epithelial folds in precise locations along the proximo-distal axis of the leg. As cell numbers increase, compressive stresses arise, promoting the folding of the epithelium and reinforcing the apical constriction of invaginating cells. Additionally, the Notch target dysfusion plays a key function specifying the location of the folds, through the apical accumulation of F-actin and the apico-basal shortening of invaginating cells. These findings provide new insights into the intricate mechanisms involved in epithelial morphogenesis, highlighting the crucial role of tissue-wide forces in shaping a three-dimensional organ in a reproducible manner. |
| Parasram, K., Zuccato, A., Shin, M., Willms, R., DeVeale, B., Foley, E., Karpowicz, P. (2024). The emergence of circadian timekeeping in the intestine. Nat Commun, 15(1):1788 PubMed ID: 38413599
Summary: The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-h period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, this study tracked how and when the circadian clock emerges in specific cell types. The circadian clock was found to begin abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in Ecdysone and Hnf4 signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. These data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages. | Terry, D., Schweibenz, C., Moberg, K. (2024). Local ecdysone synthesis in a wounded epithelium sustains developmental delay and promotes regeneration in Drosophila. bioRxiv, PubMed ID: 38464192 ID
Summary: Regenerative ability often declines as animals mature past embryonic and juvenile stages, suggesting that regeneration requires redirection of growth pathways that promote developmental growth. Intriguingly, the Drosophila larval epithelia require the hormone ecdysone (Ec) for growth but require a drop in circulating Ec levels to regenerate. Examining Ec dynamics more closely, this study found that transcriptional activity of the Ec-receptor (EcR) drops in uninjured regions of wing discs, but simultaneously rises in cells around the injury-induced blastema. In parallel, blastema depletion of genes encoding Ec biosynthesis enzymes blocks EcR activity and impairs regeneration but has no effect on uninjured wings. Local Ec/EcR signaling was found to be required for injury-induced pupariation delay following injury and that key regeneration regulators upd3 and Ets21c respond to Ec levels. Collectively, these data indicate that injury induces a local source of Ec within the wing blastema that sustains a transcriptional signature necessary for developmental delay and tissue repair. |
| Pandey, A., Roy, J. K. (2024). The insc-GAL4 driver marks distinct cell types in Drosophila midgut. Exp Cell Res, 435(2):113953 PubMed ID: 38278285
Summary: Drosophila geneticists frequently employ the binary GAL4-UAS system of conditional gene expression to direct expression of the desired transgene in tissues of interest. The inscuteable -GAL4 driver (insc-GAL4) expresses in the type 1 and type 2 neuroblasts of Drosophila larval brain, a frequent target tissue in many investigations. This GAL4 line additionally displayed its expression in the midgut. This study examined the expression of the UAS-mCD8GFP reporter under the command of the insc-GAL4 driver and observed that this driver expresses exclusively to intestinal stem cells (ISCs) of the Drosophila adult midgut as well as adult midgut precursors (AMPs) of the larval midgut besides its expression in larval brain. Additionally, using the G-TRACE method, it was observed that AMPs in the larval midgut consistently expressed insc-GAL4 in real-time, and the lineage expression of this GAL4 was observed in the enterocyte cells. This study reveals for the first time that insc-GAL4 is specific to larval AMPs and adult ISCs of the midgut. | Kumar, N., Rangel Ambriz, J., Tsai, K., Mim, M. S., Flores-Flores, M., Chen, W., Zartman, J. J., Alber, M. (2024). Balancing competing effects of tissue growth and cytoskeletal regulation during Drosophila wing disc development. Nat Commun, 15(1):2477 PubMed ID: 38509115
Summary: How a developing organ robustly coordinates the cellular mechanics and growth to reach a final size and shape remains poorly understood. Through iterations between experiments and model simulations that include a mechanistic description of interkinetic nuclear migration, this study shows that the local curvature, height, and nuclear positioning of cells in the Drosophila wing imaginal disc are defined by the concurrent patterning of actomyosin contractility, cell-ECM adhesion, ECM stiffness, and interfacial membrane tension. Increasing cell proliferation via different growth-promoting pathways results in two distinct phenotypes. Triggering proliferation through increases basal curvature, but an increase in growth through Dpp signaling and Myc causes tissue flattening. These distinct phenotypic outcomes arise from differences in how each growth pathway regulates the cellular cytoskeleton, including contractility and cell-ECM adhesion. The coupled regulation of proliferation and cytoskeletal regulators is a general strategy to meet the multiple context-dependent criteria defining tissue morphogenesis. |
Wednesday November 20th - Transcriptional Regulation |
| Tendolkar, A., Mazo-Vargas, A., Livraghi, L., Hanly, J. J., Van Horne, K. C., Gilbert, L. E., Martin, A. (2024). Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings. Elife, 12 PubMed ID: 38261357
Summary: Hox gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila, which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia (Antp) and Ultrabithorax (Ubx). This study investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia, a Topologically Associated Domain (TAD) was shown to maintain a hindwing-enriched profile of chromatin opening around Ubx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis-regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. A series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies is described, their possible mutational basis is discussed. By leveraging the extensive wing specialization found in butterflies, this initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings. | Ko, B. S., Han, M. H., Kwon, M. J., Cha, D. G., Ji, Y., Park, E. S., Jeon, M. J., Kim, S., Lee, K., Choi, Y. H., Lee, J., Torras-Llort, M., Yoon, K. J., Lee, H., Kim, J. K., Lee, S. B. (2024). Baf-mediated transcriptional regulation of teashirt is essential for the development of neural progenitor cell lineages. Experimental & molecular medicine, 56(2):422-440 PubMed ID: 38374207
Summary: Accumulating evidence hints heterochromatin anchoring to the inner nuclear membrane as an upstream regulatory process of gene expression. Given that the formation of neural progenitor cell lineages and the subsequent maintenance of postmitotic neuronal cell identity critically rely on transcriptional regulation, it seems possible that the development of neuronal cells is influenced by cell type-specific and/or context-dependent programmed regulation of heterochromatin anchoring. This possibility was explored by genetically disrupting the evolutionarily conserved barrier-to-autointegration factor (Baf) in the Drosophila nervous system. Through single-cell RNA sequencing, it was demonstrated that Baf knockdown induces prominent transcriptomic changes, particularly in type I neuroblasts. Among the differentially expressed genes, genetic analyses identified teashirt (tsh), a transcription factor that interacts with beta-catenin, to be closely associated with Baf knockdown-induced phenotypes that were suppressed by the overexpression of tsh or beta-catenin. It was also found that Baf and tsh colocalized in a region adjacent to heterochromatin in type I NBs. Notably, the subnuclear localization pattern remained unchanged when one of these two proteins was knocked down, indicating that both proteins contribute to the anchoring of heterochromatin to the inner nuclear membrane. Overall, this study reveals that the Baf-mediated transcriptional regulation of teashirt is a novel molecular mechanism that regulates the development of neural progenitor cell lineages. |
| Tikhonova, E., Revel-Muroz, A., Georgiev, P., Maksimenko, O. (2024). Interaction of MLE with CLAMP zinc finger is involved in proper MSL proteins binding to chromosomes in Drosophila. Open biology, 14(3):230270 PubMed ID: 38471568
Summary: The Drosophila male-specific lethal (MSL) complex binds to the male X chromosome to activate transcription. It comprises five proteins (MSL1, MSL2, MSL3, Male absent on the first (MOF), and Maleless (MLE)) and two long noncoding RNAs (lncRNAs; roX1 and roX2/). The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. MSL2 is expressed only in males and interacts with the N-terminal zinc finger of the transcription factor chromatin-linked adapter for MSL proteins (CLAMP), which is important for the specific recruitment of the MSL complex to the male X chromosome. This study found that MLE's unstructured C-terminal region interacts with the sixth zinc-finger domain of CLAMP. In vitro, 4-5 zinc fingers are critical for the specific DNA-binding of CLAMP with GA repeats, which constitute the core motif at the high affinity binding sites for MSL proteins. Deleting the CLAMP binding region in MLE decreases the association of MSL proteins with the male X chromosome and increases male lethality. These results suggest that interactions of unstructured regions in MSL2 and MLE with CLAMP zinc finger domains are important for the specific recruitment of the MSL complex to the male X chromosome. | Lin, S., Lim, B. (2024). Multifaceted effects on even-skipped transcriptional dynamics upon Kruppel dosage changes. Development, 151(5) PubMed ID: 38345298
Summary: Although fluctuations in transcription factor (TF) dosage are often well tolerated, TF dosage modulation can change the target gene expression dynamics and result in significant non-lethal developmental phenotypes. Using MS2/MCP-mediated quantitative live imaging in early Drosophila embryos, this study analyzed how changing levels of the gap gene Krü,ppel (Kr) affects transcriptional dynamics of the pair-rule gene even-skipped (eve). Halving the Kr dosage leads to a transient posterior expansion of the eve stripe 2 and an anterior shift of stripe 5. Surprisingly, the most significant changes are observed in eve stripes 3 and 4, the enhancers of which do not contain Kr-binding sites. In Kr heterozygous embryos, both stripes 3 and 4 display narrower widths, anteriorly shifted boundaries and reduced mRNA production levels. Kr dosage indirectly affects stripe 3 and 4 dynamics by modulating other gap gene dynamics. Moderate body segment phenotypes of Kr heterozygotes were quantively corrolated with spatiotemporal changes in eve expression. These results indicate that nonlinear relationships between TF dosage and phenotypes underlie direct TF-DNA and indirect TF-TF interactions. |
| Gibson, T. J., Larson, E. D., Harrison, M. M. (2024). Protein-intrinsic properties and context-dependent effects regulate pioneer factor binding and function. Nat Struct Mol Biol, 31(3):548-558 PubMed ID: 38365978
Summary: Chromatin is a barrier to the binding of many transcription factors. By contrast, pioneer factors access nucleosomal targets and promote chromatin opening. Despite binding to target motifs in closed chromatin, many pioneer factors display cell-type-specific binding and activity. The mechanisms governing pioneer factor occupancy and the relationship between chromatin occupancy and opening remain unclear. Three Drosophila transcription factors with distinct DNA-binding domains and biological functions were studied: Zelda, Grainy head and Twist. The level of chromatin occupancy was demonstrated to be a key determinant of pioneering activity. Multiple factors regulate occupancy, including motif content, local chromatin and protein concentration. Regions outside the DNA-binding domain are required for binding and chromatin opening. These results show that pioneering activity is not a binary feature intrinsic to a protein but occurs on a spectrum and is regulated by a variety of protein-intrinsic and cell-type-specific features. | Pollex, T., Rabinowitz, A., Gambetta, M. C., Marco-Ferreres, R., Viales, R. R., Jankowski, A., Schaub, C., Furlong, E. E. M. (2024). Enhancer-promoter interactions become more instructive in the transition from cell-fate specification to tissue differentiation. Nat Genet, 56(4):686-696 PubMed ID: 38467791
Summary: To regulate expression, enhancers must come in proximity to their target gene. However, the relationship between the timing of enhancer-promoter (E-P) proximity and activity remains unclear, with examples of uncoupled, anticorrelated and correlated interactions. To assess this, 600 characterized enhancers or promoters were chosen with tissue-specific activity in Drosophila embryos and Capture-C was performed in FACS-purified myogenic or neurogenic cells during specification and tissue differentiation. This enabled direct comparison between E-P proximity and activity transitioning from OFF-to-ON and ON-to-OFF states across developmental conditions. This showed remarkably similar E-P topologies between specified muscle and neuronal cells, which are uncoupled from activity. During tissue differentiation, many new distal interactions emerge where changes in E-P proximity reflect changes in activity. The mode of E-P regulation therefore appears to change as embryogenesis proceeds, from largely permissive topologies during cell-fate specification to more instructive regulation during terminal tissue differentiation, when E-P proximity is coupled to activation. |
Monday November 18th - Cytoskeleton |
| Singh, A., Thale, S., Leibner, T., Lamparter, L., Ricker, A., Nusse, H., Klingauf, J., Galic, M., Ohlberger, M., Matis, M. (2024). Dynamic interplay of microtubule and actomyosin forces drive tissue extension. Nat Commun, 15(1):3198 PubMed ID: 38609383
Summary: In order to shape a tissue, individual cell-based mechanical forces have to be integrated into a global force pattern. Over the last decades, the importance of actomyosin contractile arrays, which are the key constituents of various morphogenetic processes, has been established for many tissues. Recent studies have demonstrated that the microtubule cytoskeleton mediates folding and elongation of the epithelial sheet during Drosophila morphogenesis, placing microtubule mechanics on par with actin-based processes. While these studies establish the importance of both cytoskeletal systems during cell and tissue rearrangements, a mechanistic understanding of their functional hierarchy is currently missing. This study dissected the individual roles of these two key generators of mechanical forces during epithelium elongation in the developing Drosophila wing. Wing extension, which entails columnar-to-cuboidal cell shape remodeling in a cell-autonomous manner, is driven by anisotropic cell expansion caused by the remodeling of the microtubule cytoskeleton from apico-basal to planarly polarized. Importantly, cell and tissue elongation is not associated with Myosin activity. Instead, Myosin II exhibits a homeostatic role, as actomyosin contraction balances polarized microtubule-based forces to determine the final cell shape. Using a reductionist model, this study confirmed that pairing microtubule and actomyosin-based forces is sufficient to recapitulate cell elongation and the final cell shape. These results support a hierarchical mechanism whereby microtubule-based forces in some epithelial systems prime actomyosin-generated forces. | Li, S., Liu, Z. Y., Li, H., Zhou, S., Liu, J., Sun, N., Yang, K. F., Dougados, V., Mangeat, T., Belguise, K., Feng, X. Q., Liu, Y., Wang, X. (2024). Basal actomyosin pulses expand epithelium coordinating cell flattening and tissue elongation. Nat Commun, 15(1):3000 PubMed ID: 38589403
Summary: Actomyosin networks constrict cell area and junctions to alter cell and tissue shape. However, during cell expansion under mechanical stress, actomyosin networks are strengthened and polarized to relax stress. Thus, cells face a conflicting situation between the enhanced actomyosin contractile properties and the expansion behaviour of the cell or tissue. To address this paradoxical situation, late Drosophila oogenesis was studied; an unusual epithelial expansion wave behaviour was revealed. Mechanistically, Rac1 and Rho1 integrate basal pulsatile actomyosin networks with ruffles and focal adhesions to increase and then stabilize basal area of epithelial cells allowing their flattening and elongation. This epithelial expansion behaviour bridges cell changes to oocyte growth and extension, while oocyte growth in turn deforms the epithelium to drive cell spreading. Basal pulsatile actomyosin networks exhibit non-contractile mechanics, non-linear structures and F-actin/Myosin-II spatiotemporal signal separation, implicating unreported expanding properties. Biophysical modelling incorporating these expanding properties well simulates epithelial cell expansion waves. This work thus highlights actomyosin expanding properties as a key mechanism driving tissue morphogenesis. |
| Osaka, J., Ishii, A., Wang, X., Iwanaga, R., Kawamura, H., Akino, S., Sugie, A., Hakeda-Suzuki, S., Suzuki, T. (2024). Complex formation of immunoglobulin superfamily molecules Side-IV and Beat-IIb regulates synaptic specificity. Cell Rep, 43(2):113798 PubMed ID: 38381608
Summary: Neurons establish specific synapses based on the adhesive properties of cell-surface proteins while also retaining the ability to form synapses in a relatively non-selective manner. However, comprehensive understanding of the underlying mechanism reconciling these opposing characteristics remains incomplete. This study has identified Side-IVBeat-IIb, members of the Drosophila immunoglobulin superfamily, as a combination of cell-surface recognition molecules inducing synapse formation. The Side-IV/Beat-IIb combination transduces bifurcated signaling with Side-IV's co-receptor, Kirre, and a synaptic scaffold protein, Dsyd-1. Genetic experiments and subcellular protein localization analyses showed the Side-IV/Beat-IIb/Kirre/Dsyd-1 complex to have two essential functions. First, it narrows neuronal binding specificity through Side-IV/Beat-IIb extracellular interactions. Second, it recruits synapse formation factors, Kirre and Dsyd-1, to restrict synaptic loci and inhibit miswiring. This dual function explains how the combinations of cell-surface molecules enable the ranking of preferred interactions among neuronal pairs to achieve synaptic specificity in complex circuits in vivo. | Sato, Y., Yamagishi, M., Yajima, J. (2024). Effect of temperature on actin filament corkscrewing driven by nonprocessive myosin IC. Biochem Biophys Res Commun, 703:149597 PubMed ID: 38367512
Summary: Myosin family proteins are ATP-driven, actin filament-based motor proteins that generate force along actin filaments. In in vitro actin filament gliding assays, certain myosins generate rotation of gliding actin filaments around their long axes. This study assessed the effects of temperature on the corkscrewing motion of actin filaments, including factors like gliding and rotational velocities and corkscrewing pitch. The corkscrewing motion was driven by a nonprocessive, full-length single-headed Drosophila myosin IC attached to an antibody adsorbed onto a cover glass. An in vitro actin filament corkscrewing assay was performed at temperatures ranging from 25 °C to 35 °C. The gliding and rotational velocities and the pitch of corkscrewing actin filaments generated by myosin IC molecules was found to increase with increasing temperature. Since the pitch is determined by dividing the gliding velocity by the rotational velocity, an increase in the pitch indicates that the gliding velocity increased faster than the rotational velocity with increasing temperature. These results suggest that temperature has distinct effects on the gliding and rotational forces produced by myosin IC, with implications for interpreting the temperature effect on torque-generation mechanisms driven by myosins on actin filaments at physiological temperatures. |
| de-Carvalho, J., Tlili, S., Saunders, T. E., Telley, I. A. (2024). The positioning mechanics of microtubule asters in Drosophila embryo explants. Elife, 12 PubMed ID: 38426416
Summary: Microtubule asters are essential in localizing the action of microtubules in processes including mitosis and organelle positioning. In large cells, such as the one-cell sea urchin embryo, aster dynamics are dominated by hydrodynamic pulling forces. However, in systems with more densely positioned nuclei such as the early Drosophila embryo, which packs around 6000 nuclei within the syncytium in a crystalline-like order, it is unclear what processes dominate aster dynamics. This study take advantage of a cell cycle regulation Drosophila mutant to generate embryos with multiple asters, independent from nuclei. An ex vivo assay was used to further simplify this biological system to explore the forces generated by and between asters. Through live imaging, drug and optical perturbations, and theoretical modeling, it was demonstrate that these asters likely generate an effective pushing force over short distances. | McGory, J. M., Verma, V., Barcelos, D. M., Maresca, T. J. (2024). Multimerization of a disordered kinetochore protein promotes accurate chromosome segregation by localizing a core dynein module. J Cell Biol, 223(3) PubMed ID: 38180477
Summary: Kinetochores connect chromosomes and spindle microtubules to maintain genomic integrity through cell division. Crosstalk between the minus-end directed motor dynein and kinetochore-microtubule attachment factors promotes accurate chromosome segregation by a poorly understood pathway. This study identified a linkage between the intrinsically disordered protein Spc105 (KNL1 orthologue) and dynein using an optogenetic oligomerization assay. Core pools of the checkpoint protein BubR1 and the adaptor complex RZZ contribute to the linkage. Furthermore, a minimal segment of Spc105 with a propensity to multimerize and which contains protein binding motifs is sufficient to link Spc105 to RZZ/dynein. Deletion of the minimal region from Spc105 compromises the recruitment of its binding partners to kinetochores and elevates chromosome missegregation due to merotelic attachments. Restoration of normal chromosome segregation and localization of BubR1 and RZZ requires both protein binding motifs and oligomerization of Spc105. Together, our results reveal that higher-order multimerization of Spc105 contributes to localizing a core pool of RZZ that promotes accurate chromosome segregation. |
Friday November 15th - Adult neural development, structure, and function |
| Takagi, S., Sancer, G., Abuin, L., Stupski, S. D., Arguello, J. R., Prieto-Godino, L. L., Stern, D. L., Cruchet, S., Alvarez-Ocana, R., Wienecke, C. F. R., van Breugel, F., Jeanne, J. M., Auer, T. O., Benton, R. (2024). Sensory neuron population expansion enhances odor tracking without sensitizing projection neurons. bioRxiv, PubMed ID: 37745467
Summary: The evolutionary expansion of sensory neuron populations detecting important environmental cues is widespread, but functionally enigmatic. This study investigated this phenomenon through comparison of homologous neural pathways of Drosophila melanogaster and its close relative Drosophila sechellia, an extreme specialist for Morinda citrifolia noni fruit. D. sechellia has evolved species-specific expansions in select, noni-detecting olfactory sensory neuron (OSN) populations, through multigenic changes. Activation and inhibition of defined proportions of neurons demonstrate that OSN population increases contribute to stronger, more persistent, noni-odor tracking behavior. These sensory neuron expansions result in increased synaptic connections with their projection neuron (PN) partners, which are conserved in number between species. Surprisingly, having more OSNs does not lead to greater odor-evoked PN sensitivity or reliability. Rather, pathways with increased sensory pooling exhibit reduced PN adaptation, likely through weakened lateral inhibition. This work reveals an unexpected functional impact of sensory neuron expansions to explain ecologically-relevant, species-specific behavior. | Abubaker, M. B., Hsu, F. Y., Feng, K. L., Chu, L. A., de Belle, J. S., Chiang, A. S. (2024). Asymmetric neurons are necessary for olfactory learning in the Drosophila brain. Curr Biol, 34(5):946-957.e944 PubMed ID: 38320552
Summary: Animals have complementary parallel memory systems that process signals from various sensory modalities. In the brain of the fruit fly Drosophila melanogaster, mushroom body (MB) circuitry is the primary associative neuropil, critical for all stages of olfactory memory. These findings suggest that active signaling from specific asymmetric body (AB) neurons is also crucial for this process. These AB neurons respond to odors and electric shock separately and exhibit timing-sensitive neuronal activity in response to paired stimulation while leaving a decreased memory trace during retrieval. These experiments also show that rutabaga-encoded adenylate cyclase, which mediates coincidence detection, is required for learning and short-term memory in both AB and MB. Additive effects were observed when manipulating rutabaga co-expression in both structures. Together, these results implicate the AB in playing a critical role in associative olfactory learning and short-term memory. |
| Li, H., Li, Z., Yuan, X., Tian, Y., Ye, W., Zeng, P., Li, X. M., Guo, F. (2024). Dynamic encoding of temperature in the central circadian circuit coordinates physiological activities. Nat Commun, 15(1):2834 PubMed ID: 38565846
Summary: The circadian clock regulates animal physiological activities. How temperature reorganizes circadian-dependent physiological activities remains elusive. Using in-vivo two-photon imaging with the temperature control device, this study investigated the response of the Drosophila central circadian circuit to temperature variation and identified that DN1as serves as the most sensitive temperature-sensing neurons. The circadian clock gate DN1a's diurnal temperature response. Trans-synaptic tracing, connectome analysis, and functional imaging data reveal that DN1as bidirectionally targets two circadian neuronal subsets: activity-related E cells and sleep-promoting DN3s. Specifically, behavioral data demonstrate that the DN1a-E cell circuit modulates the evening locomotion peak in response to cold temperature, while the DN1a-DN3 circuit controls the warm temperature-induced nocturnal sleep reduction. These findings systematically and comprehensively illustrate how the central circadian circuit dynamically integrates temperature and light signals to effectively coordinate wakefulness and sleep at different times of the day, shedding light on the conserved neural mechanisms underlying temperature-regulated circadian physiology in animals. | Lillvis, J. L., Wang, K., Shiozaki, H. M., Xu, M., Stern, D. L., Dickson, B. J. (2024). Nested neural circuits generate distinct acoustic signals during Drosophila courtship. Curr Biol, 34(4):808-824.e806 PubMed ID: 38295797
Summary: Many motor control systems generate multiple movements using a common set of muscles. How are premotor circuits able to flexibly generate diverse movement patterns? This study characterizes the neuronal circuits that drive the distinct courtship songs of Drosophila melanogaster. Male flies vibrate their wings toward females to produce two different song modes-pulse and sine song-which signal species identity and male quality. Using cell-type-specific genetic reagents and the connectome, a cellular and synaptic map is provided of the circuits in the male ventral nerve cord that generate these songs and examine how activating or inhibiting each cell type within these circuits affects the song. The data reveal that the song circuit is organized into two nested feedforward pathways with extensive reciprocal and feedback connections. The larger network produces pulse song, the more complex and ancestral song form. A subset of this network produces sine song, the simpler and more recent form. Such nested organization may be a common feature of motor control circuits in which evolution has layered increasing flexibility onto a basic movement pattern. |
| Agi, E., Reifenstein, E. T., Wit, C., Schneider, T., Kauer, M., Kehribar, M., Kulkarni, A., von Kleist, M., Hiesinger, P. R. (2024). Axonal self-sorting without target guidance in Drosophila visual map formation.. Science, 383(6687):1084-1092 PubMed ID: 38452066
Summary: The idea of guidance toward a target is central to axon pathfinding and brain wiring in general. This work shows how several thousand axonal growth cones self-pattern without target-dependent guidance during neural superposition wiring in Drosophila. Ablation of all target lamina neurons or loss of target adhesion prevents the stabilization but not the development of the pattern. Intravital imaging at the spatiotemporal resolution of growth cone dynamics in intact pupae and data-driven dynamics simulations reveal a mechanism by which >30,000 filopodia do not explore potential targets, but instead simultaneously generate and navigate a dynamic filopodial meshwork that steers growth directions. Hence, a guidance mechanism can emerge from the interactions of the axons being guided, suggesting self-organization as a more general feature of brain wiring. | Tao, L., Ayembem, D., Barranca, V. J., Bhandawat, V. (2024). Neurons underlying aggressive actions that are shared by both males and females in Drosophila. bioRxiv, PubMed ID: 38464020
Summary: Aggression involves both sexually monomorphic and dimorphic actions. How the brain implements these two types of actions is poorly understood. This study found that a set of neurons, which are called CL062, previously shown to mediate male aggression also mediate female aggression. These neurons elicit aggression acutely and without the presence of a target. Although the same set of actions is elicited in males and females, the overall behavior is sexually dimorphic. The CL062 neurons do not express fruitless, a gene required for sexual dimorphism in flies, and expressed by most other neurons important for controlling fly aggression. Connectomic analysis suggests that these neurons have limited connections with fruitless expressing neurons that have been shown to be important for aggression, and signal to different descending neurons. Thus, CL062 is part of a monomorphic circuit for aggression that functions parallel to the known dimorphic circuits. |
Tuesday November 12th - Disease Models |
| Chaurasia, R., Ayajuddin, M., Ratnaparkhi, G. S., Lingadahalli, S. S., Yenisetti, S. C. (2024). A Simple Immunofluorescence Method to Characterize Neurodegeneration and Tyrosine Hydroxylase Reduction in Whole Brain of a Drosophila Model of Parkinson's Disease. Bio Protoc, 14(4):e4937 PubMed ID: 38405079
Summary: Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson's disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. This study demonstrates in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). This study presents a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories. | Imomnazarov, K., Lopez-Scarim, J., Bagheri, I., Joers, V., Tansey, M. G., Martin-Pena, A. (2024). Biochemical Fractionation of Human alpha-Synuclein in a Drosophila Model of Synucleinopathies. Int J Mol Sci, 25(7) PubMed ID: 38612454
Summary: Synucleinopathies are a group of central nervous system pathologies that are characterized by the intracellular accumulation of misfolded and aggregated α-synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of α-synuclein from its physiological to pathological form has been associated with several post-translational modifications such as phosphorylation and an increasing degree of insolubility, which also correlate with disease progression in post-mortem specimens from human patients. Neuronal expression of α-synuclein in model organisms, including Drosophila melanogaster, has been a typical approach employed to study its physiological effects. Biochemical analysis of α-synuclein solubility via high-speed ultracentrifugation with buffers of increasing detergent strength offers a potent method for identification of α-synuclein biochemical properties and the associated pathology stage. Unfortunately, the development of a robust and reproducible method for the evaluation of human α-synuclein solubility isolated from Drosophila tissues has remained elusive. This study tested different detergents for their ability to solubilize human α-synuclein carrying the pathological mutation A53T from the brains of aged flies. The effect of sonication on the solubility of human α-synuclein was assessed, and a protocol was optimized to discriminate the relative amounts of soluble/insoluble human α-synuclein from dopaminergic neurons of the Drosophila brain. The data established that, using a 5% SDS buffer, the three-step protocol separates cytosolic soluble, detergent-soluble and insoluble proteins in three sequential fractions according to their chemical properties. This protocol shows that sonication breaks down α-synuclein insoluble complexes from the fly brain, making them soluble in the SDS buffer and thus enriching the detergent-soluble fraction of the protocol. |
| Li, Y., Liu, D., Zhang, X., Rimal, S., Lu, B., Li, S. (2024). RACK1 and IRE1 participate in the translational quality control of amyloid precursor protein in Drosophila models of Alzheimer's disease. J Biol Chem, 300(3):105719 PubMed ID: 38311171
Summary: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by dysregulation of the expression and processing of the amyloid precursor protein (APP). Protein quality control systems are dedicated to remove faulty and deleterious proteins to maintain cellular protein homeostasis (proteostasis). Identifying mechanisms underlying APP protein regulation is crucial for understanding AD pathogenesis. However, the factors and associated molecular mechanisms regulating APP protein quality control remain poorly defined. This study shows that mutant APP with its mitochondrial-targeting sequence ablated exhibited predominant endoplasmic reticulum (ER) distribution and led to aberrant ER morphology, deficits in locomotor activity, and shortened lifespan. Regulators were sought that could counteract the toxicity caused by the ectopic expression of this mutant APP. Genetic removal of the ribosome-associated quality control (RQC) factor RACK1 resulted in reduced levels of ectopically expressed mutant APP. By contrast, gain of RACK1 function increased mutant APP level. Additionally, overexpression of the ER stress regulator (IRE1) resulted in reduced levels of ectopically expressed mutant APP. Mechanistically, the RQC related ATPase VCP/p97 and the E3 ubiquitin ligase Hrd1 were required for the reduction of mutant APP level by IRE1. These factors also regulated the expression and toxicity of ectopically expressed wild type APP, supporting their relevance to APP biology. THESE results reveal functions of RACK1 and IRE1 in regulating the quality control of APP homeostasis and mitigating its pathogenic effects, with implications for the understanding and treatment of AD. | Loreto, J. S., Ferreira, S. A., de Almeida, P., da Rocha, J. B. T., Barbosa, N. V. (2024). Screening for Differentially Expressed Memory Genes on a Diabetes Model Induced by High-Sugar Diet in Drosophila melanogaster: Potential Markers for Memory Deficits. Molecular neurobiology, 61(3):1225-1236 PubMed ID: 37698834
Summary: Type 2 diabetes mellitus (T2DM) has been shown to affect a series of cognitive processes including memory, increasing the risk for dementia, particularly Alzheimer's disease (AD). Although increasing evidence has supported that both diseases share common features, the pathophysiological mechanisms connecting these two disorders remain to be fully elucidated. This study used Drosophila melanogaster fed on a high-sugar diet (HSD) to mimic T2DM, and investigate its effects on memory as well as identify potential molecular players associated with the memory deficits induced by HSD. Flies hatched from and reared on HSD for 7 days had a substantial decrease in short-term memory (STM). The screening for memory-related genes using transcriptome data revealed that HSD altered the expression of 33% of memory genes in relation to the control. Among the differentially expressed genes (DEGs) with a fold change (FC) higher than two, were five genes, related to synapse and memory trace formation, that could be considered strong candidates to underlie the STM deficits in HSD flies: Abl tyrosine kinase (Abl), bruchpilot (Brp), minibrain (Mnb), shaker (Sh), and gilgamesh (Gish). We also analyzed genes from the dopamine system, one of the most relevant signaling pathways for olfactory memory. Interestingly, the flies fed on HSD presented a decreased expression of the Tyrosine hydroxylase (Ple) and Dopa decarboxylase (Ddc) genes, signals of a possible dopamine deficiency. This work presents promising biomarkers to investigate molecular networks shared between T2DM and AD. |
| Liao, J. Z., Chung, H. L., Shih, C., Wong, K. K. L., Dutta, D., Nil, Z., Burns, C. G., Kanca, O., Park, Y. J., Zuo, Z., Marcogliese, P. C., Sew, K., Bellen, H. J., Verheyen, E. M. (2024). Cdk8/CDK19 promotes mitochondrial fission through Drp1 phosphorylation and can phenotypically suppress pink1 deficiency in Drosophila. Nat Commun, 15(1):3326 PubMed ID: 38637532
Summary: Cdk8 in Drosophila is the orthologue of vertebrate CDK8 and CDK19. These proteins have been shown to modulate transcriptional control by RNA polymerase II. This study found that neuronal loss of Cdk8 severely reduces fly lifespan and causes bang sensitivity. Remarkably, these defects can be rescued by expression of human CDK19, found in the cytoplasm of neurons, suggesting a non-nuclear function of CDK19/Cdk8. This study showed that Cdk8 plays a critical role in the cytoplasm, with its loss causing elongated mitochondria in both muscles and neurons. Endogenous GFP-tagged Cdk8 can be found in both the cytoplasm and nucleus. Cdk8 was shown to promote the phosphorylation of Drp1 at S616, a protein required for mitochondrial fission. Interestingly, Pink1, a mitochondrial kinase implicated in Parkinson's disease, also phosphorylates Drp1 at the same residue. Indeed, overexpression of Cdk8 significantly suppresses the phenotypes observed in flies with low levels of Pink1, including elevated levels of ROS, mitochondrial dysmorphology, and behavioral defects. In summary, it is proposed that Pink1 and Cdk8 perform similar functions to promote Drp1-mediated fission. | Chang, Y. J., Lin, K. T., Shih, O., Yang, C. H., Chuang, C. Y., Fang, M. H., Lai, W. B., Lee, Y. C., Kuo, H. C., Hung, S. C., Yao, C. K., Jeng, U. S., Chen, Y. R. (2024). Sulfated disaccharide protects membrane and DNA damages from arginine-rich dipeptide repeats in ALS. Sci Adv, 10(8):eadj0347 PubMed ID: 38394210
Summary: Hexanucleotide repeat expansion in C9ORF72 (C9) is the most prevalent mutation among amyotrophic lateral sclerosis (ALS) patients. The patients carry over ~30 to hundreds or thousands of repeats translated to dipeptide repeats (DPRs) where poly-glycine-arginine (GR) and poly-proline-arginine (PR) are most toxic. The structure-function relationship is still unknown. This study examined the minimal neurotoxic repeat number of poly-GR and found that extension of the repeat number led to a loose helical structure disrupting plasma and nuclear membrane. Poly-GR/PR bound to nucleotides and interfered with transcription. A sulfated disaccharide was screened and identified that bound to poly-GR/PR and rescued poly-GR/PR-induced toxicity in neuroblastoma and C9-ALS-iPSC-derived motor neurons. The compound rescued the shortened life span and defective locomotion in poly-GR/PR expressing Drosophila model and improved motor behavior in poly-GR-injected mouse model. Overall, these results reveal structural and toxicity mechanisms for poly-GR/PR and facilitate therapeutic development for C9-ALS. |
Monday November 11th - Behavior |
| Schwarz, J. E., Sengupta, A., Guevara, C., Barber, A. F., Hsu, C. T., Zhang, S. L., Weljie, A., Sehgal, A. (2024). Age-regulated cycling metabolites are relevant for behavior. Aging Cell, 23(4):e14082 PubMed ID: 38204362
Summary: Circadian cycles of sleep:wake and gene expression change with age in all organisms examined. Metabolism is also under robust circadian regulation, but little is known about how metabolic cycles change with age and whether these contribute to the regulation of behavioral cycles. To address this gap, cycling of metabolites in young and old Drosophila was compared and major age-related variations were found. A significant model separated the young metabolic profiles by circadian timepoint, but could not be defined for the old metabolic profiles due to the greater variation in this dataset. Of the 159 metabolites measured in fly heads, 17 were found that cycle by JTK analysis in young flies and 17 in aged. Only four metabolites overlapped in the two groups, suggesting that cycling metabolites are distinct in young and old animals. Among our top cyclers exclusive to young flies were components of the pentose phosphate pathway (PPP). As the PPP is important for buffering reactive oxygen species, and overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key component of the PPP, was previously shown to extend lifespan in Drosophila, it was asked if this manipulation also affects sleep:wake cycles. Overexpression in circadian clock neurons was found to decrease sleep in association with an increase in cellular calcium and mitochondrial oxidation, suggesting that altering PPP activity affects neuronal activity. These findings elucidate the importance of metabolic regulation in maintaining patterns of neural activity, and thereby sleep:wake cycles. | Thiem, J., Viskadourou, M., Gaitanidis, A., Stravopodis, D. J., Strauß, R., Duch, C., Consoulas, C. (2024). Biological aging of two innate behaviors of Drosophila melanogaster: Escape climbing versus courtship learning and memory. PLoS One, 19(4):e0293252 PubMed ID: 38593121
Summary: Motor and cognitive aging can severely affect life quality of elderly people and burden health care systems. In search for diagnostic behavioral biomarkers, it has been suggested that walking speed can predict forms of cognitive decline, but in humans, it remains challenging to separate the effects of biological aging and lifestyle. This study examined a possible association of motor and cognitive decline in Drosophila, a genetic model organism of healthy aging. Long term courtship memory is present in young male flies but absent already during mid life (4-8 weeks). By contrast, courtship learning index and short term memory (STM) are surprisingly robust and remain stable through mid (4-8 weeks) and healthy late life (>8 weeks), until courtship performance collapses suddenly at ~4.5 days prior to death. By contrast, climbing speed declines gradually during late life (>8 weeks). The collapse of courtship performance and short term memory close to the end of life occur later and progress with a different time course than the gradual late life decline in climbing speed. Thus, during healthy aging in male Drosophila, climbing and courtship motor behaviors decline differentially. Moreover, cognitive and motor performances decline at different time courses. Differential behavioral decline during aging may indicate different underlying causes, or alternatively, a common cause but different thresholds for defects in different behaviors. |
| Jiang, X., Sun, M., Chen, J., Pan, Y. (2024). Sex-Specific and State-Dependent Neuromodulation Regulates Male and Female Locomotion and Sexual Behaviors. Research (Washington, DC), 7:0321 PubMed ID: 38390306
Summary: Males and females display dimorphic behaviors that often involve sex-specific locomotor patterns. How the sexually dimorphic locomotion is mediated is poorly understood. This study identify a neuropeptide that oppositely regulates locomotion for efficient sexual behaviors in Drosophila males and females. Males are less active than females if isolated. However, when sexually aroused through activating homologous but sexually dimorphic pC1 neurons, males exhibit higher activity levels than females. Diuretic hormone 44 (DH44), that functions in pC1 neurons in a sex-specific way, was found to inhibit male locomotion and promote female locomotion. Surprisingly, DH44 exerts opposite effects in sexually aroused flies to promote male locomotion and suppress female locomotion, which is crucial for successful male courtship and female receptivity. These findings demonstrate sexually dimorphic and state-dependent control of locomotor activity by pC1 neuronal activity and DH44 modulation. | Zhao, H., Jiang, X., Ma, M., Xing, L., Ji, X., Pan, Y. (2024). A neural pathway for social modulation of spontaneous locomotor activity (SoMo-SLA) in Drosophila. Proc Natl Acad Sci U S A, 121(9):e2314393121 PubMed ID: 38394240
Summary: Social enrichment or social isolation affects a range of innate behaviors, such as sex, aggression, and sleep, but whether there is a shared mechanism is not clear. This study reports a neural mechanism underlying social modulation of spontaneous locomotor activity (SoMo-SLA), an internal-driven behavior indicative of internal states. Social enrichment specifically reduces spontaneous locomotor activity in male flies. We identify neuropeptides Diuretic hormone 44 (DH44) and Tachykinin (TK) to be up- and down-regulated by social enrichment and necessary for SoMo-SLA. A sexually dimorphic neural circuit was further demonstrate, in which the male-specific P1 neurons encoding internal states form positive feedback with interneurons coexpressing doublesex (dsx) and Tk to promote locomotion, while P1 neurons also form negative feedback with interneurons coexpressing dsx and DH44 to inhibit locomotion. These two opposing neuromodulatory recurrent circuits represent a potentially common mechanism that underlies the social regulation of multiple innate behaviors. |
| Jagannathan, S. R., Jeans, T., Van De Poll, M. N., van Swinderen, B. (2024). Multivariate classification of multichannel long-term electrophysiology data identifies different sleep stages in fruit flies. Sci Adv, 10(8):eadj4399 PubMed ID: 38381836
Summary: Identifying different sleep stages in humans and other mammals has traditionally relied on electroencephalograms. Such an approach is not feasible in certain animals such as invertebrates, although these animals could also be sleeping in stages. In this study long-term multichannel local field potential recordings were performed in the brains of behaving flies undergoing spontaneous sleep bouts. Aonsistent spatial recordings were acquired of local field potentials across multiple flies, allowing comparison of brain activity across awake and sleep periods. Using machine learning, distinct temporal stages of sleep were uncover and the associated spatial and spectral features across the fly brain were explored. Further, the electrophysiological correlates were analyzed of microbehaviors associated with certain sleep stages. The existence was confirmed of a distinct sleep stage associated with rhythmic proboscis extensions, and spectral features of this sleep-related behavior was shown to differ significantly from those associated with the same behavior during wakefulness, indicating a dissociation between behavior and the brain states wherein these behaviors reside. | Panos, P., Chan, T., Gowda, N., Hong, J., Kansal, R., Shen, K., McLean, E. (2024). Sex Differences in Sociability for Drosophila melanogaster with Altered Gut Microbiomes. microPublication biology, 2024 PubMed ID: 38481554
Summary: In Drosophila melanogaster the gut microbiome has been shown to influence multiple behaviors, including aggressive social behavior. This study investigated the effect of the Drosophila microbiome on pro-social behavior. It was predicted that reducing the microbiome would lead to a decrease in pro-social behavior in adult flies. After altering the flies' microbiomes, virgin male flies with reduced microbiomes were significantly less social than virgin male control flies (t=3.09, p=0.006). This difference was not observed in virgin female flies (t=0.344, p=0.73), or mated flies of either sex (males: t=0.456, p=0.66; females: t=0.271, p=0.79). These results suggest that the role of the Drosophila microbiome in pro-social behavior is dependent on both sex and previous social experience. |
Saturday November 9th - Evolution |
| Jarvis, W. M. C., Arthur, N. J., Rundle, H. D., Dyer, K. A. (2024). An experimental test of the evolutionary consequences of sympatry in Drosophila subquinaria. Evolution, 78(3):555-565 PubMed ID: 38153840
Summary: Prezygotic isolation is often stronger between sympatric as opposed to allopatric taxa, but the underlying cause can be difficult to infer from comparative studies alone. Experimental evolution, where evolutionary responses to treatments manipulating the presence/absence of heterospecific individuals are tracked, can provide a powerful complementary approach. This study used experimental evolution to investigate a naturally occurring pattern of reproductive character displacement in the mushroom-feeding fly, Drosophila subquinaria. In nature, female D. subquinaria from populations sympatric with the closely related Drosophila recens discriminate more strongly against heterospecific males than do females from allopatric populations. Starting with 16 replicate allopatric populations of D. subquinaria, the presence/absence of D. recens during mating (experimental sympatry vs. control) was manipulated and, when present, hybrids were allowed to live or they were killed each generation. Across 12 generations, heterospecific offspring production from no-choice mating trials between D. subquinaria females and D. recens males declined in both experimental sympatry treatments relative to the control, suggesting increased sexual isolation. Male cuticular hydrocarbon profiles also evolved, but only in the hybrids killed treatment. These results strongly imply that the existing reproductive character displacement in wild D. subquinaria populations was an evolutionary response to selection arising from secondary contact with D. recens. | Urum, A., Rice, G., Glassford, W., Yanku, Y., Shklyar, B., Rebeiz, M., Preger-Ben Noon, E. (2024). A developmental atlas of male terminalia across twelve species of Drosophila. Frontiers in cell and developmental biology, 12:1349275 PubMed ID: 38487271
Summary: How complex morphologies evolve is one of the central questions in evolutionary biology. Observing the morphogenetic events that occur during development provides a unique perspective on the origins and diversification of morphological novelty. One can trace the tissue of origin, emergence, and even regression of structures to resolve murky homology relationships between species. This study traced the developmental events that shape some of the most diverse organs in the animal kingdom-the male terminalia (genitalia and analia) of Drosophilids. Male genitalia are known for their rapid evolution with closely related species of the Drosophila genus demonstrating vast variation in their reproductive morphology. Confocal microscopy was used to monitor terminalia development during metamorphosis in twelve related species of Drosophila. From this comprehensive dataset, a new staging scheme is proposed for pupal terminalia development based on shared developmental landmarks, which allows one to align developmental time points between species. It was possible to trace the origin of different substructures, find new morphologies and suggest possible homology of certain substructures. Additionally, it was demonstrated that posterior lobe is likely originated prior to the split between the Drosophila melanogaster and the Drosophila yakuba clade. This dataset opens up many new directions of research and provides an entry point for future studies of the Drosophila male terminalia evolution and development. |
| Padilla Perez, D. J. (2024). Geographic and seasonal variation of the for gene reveal signatures of local adaptation in Drosophila melanogaster. J Evol Biol, 37(2):201-211 PubMed ID: 38301664
Summary: In the early 1980s, the observation that Drosophila melanogaster larvae differed in their foraging behaviour laid the foundation for the work that would later lead to the discovery of the foraging gene (for) and its associated foraging phenotypes, rover and sitter. Since then, the molecular characterization of the for gene and understanding of the mechanisms that maintain its phenotypic variants in the laboratory have progressed enormously. However, the significance and dynamics of such variation are yet to be investigated in nature. With the advent of next-generation sequencing, it is now possible to identify loci underlying the adaptation of populations in response to environmental variation. This study presents the results of a genotype-environment association analysis that quantifies variation at the for gene among samples of D. melanogaster structured across space and time. These samples consist of published genomes of adult flies collected worldwide, and at least twice per site of collection (during spring and fall). Both an analysis of genetic differentiation based on Fst values and an analysis of population structure revealed an east-west gradient in allele frequency. This gradient may be the result of spatially varying selection driven by the seasonality of precipitation. These results support the hypothesis that different patterns of gene flow as expected under models of isolation by distance and potentially isolation by environment are driving genetic differentiation among populations. Overall, this study is essential for understanding the mechanisms underlying the evolution of foraging behaviour in D. melanogaster. | Janzen, A., Pothula, R., Sychla, A., Feltman, N. R., Smanski, M. J. (2024). Predicting thresholds for population replacement gene drives. BMC Biol, 22(1):40 PubMed ID: 38369493
Summary: Threshold-dependent gene drives (TDGDs) could be used to spread desirable traits through a population, and are likely to be less invasive and easier to control than threshold-independent gene drives. Engineered Genetic Incompatibility (EGI) is an extreme underdominance system previously demonstrated in Drosophila melanogaster that can function as a TDGD when EGI agents of both sexes are released into a wild-type population. This study used a single generation fitness assay to compare the fecundity, mating preferences, and temperature-dependent relative fitness to wild-type of two distinct genotypes of EGI agents. Significant differences were found in the behavior/performance of these EGI agents that would not be predicted a priori based on their genetic design. A surprising temperature-dependent change is reported in the predicted threshold for population replacement in an EGI agent that drives ectopic expression of the developmental morphogen pyramus. The single-generation fitness assay presented here could reduce the amount of time required to estimate the threshold for TDGD strategies for which hybrid genotypes are inviable. Additionally, this work underscores the importance of empirical characterization of multiple engineered lines, as behavioral differences can arise in unique genotypes for unknown reasons. |
| Clifton, B. D., Hariyani, I., Kimura, A., Luo, F., Nguyen, A., Ranz, J. M. (2023). Paralog transcriptional differentiation in the D. melanogaster-specific gene family Sdic across populations and spermatogenesis stages. Communications biology, 6(1):1069 PubMed ID: 37864070
Summary: How recently originated gene copies become stable genomic components remains uncertain as high sequence similarity of young duplicates precludes their functional characterization. The tandem multigene family Sdic is specific to Drosophila melanogaster and has been annotated across multiple reference-quality genome assemblies. This study shows the existence of a positive correlation between Sdic copy number and total expression, plus vast intrastrain differences in mRNA abundance among paralogs, using RNA-sequencing from testis of four strains with variable paralog composition. Single cell and nucleus RNA-sequencing data expose paralog expression differentiation in meiotic cell types within testis from third instar larva and adults. Additional RNA-sequencing across synthetic strains only differing in their Y chromosomes reveal a tissue-dependent trans-regulatory effect on Sdic: upregulation in testis and downregulation in male accessory gland. By leveraging paralog-specific expression information from tissue- and cell-specific data, these results elucidate the intraspecific functional diversification of a recently expanded tandem gene family. | Harris, M., Kim, B. Y., Garud, N. (2024). Enrichment of hard sweeps on the X chromosome compared to autosomes in six Drosophila species. Genetics, 226(4) PubMed ID: 38366786
Summary: The X chromosome, being hemizygous in males, is exposed one-third of the time increasing the visibility of new mutations to natural selection, potentially leading to different evolutionary dynamics than autosomes. Recently, an enrichment was found of hard selective sweeps over soft selective sweeps on the X chromosome relative to the autosomes in a North American population of Drosophila melanogaster. To understand whether this enrichment is a universal feature of evolution on the X chromosome, diversity patterns were analyzed across 6 commonly studied Drosophila species. An increased proportion was found of regions with steep reductions in diversity and elevated homozygosity on the X chromosome compared to autosomes. To assess if these signatures are consistent with positive selection, a wide variety of evolutionary scenarios was simulated spanning variations in demography, mutation rate, recombination rate, background selection, hard sweeps, and soft sweeps; the diversity patterns observed on the X are most consistent with hard sweeps. These findings highlight the importance of sex chromosomes in driving evolutionary processes and suggest that hard sweeps have played a significant role in shaping diversity patterns on the X chromosome across multiple Drosophila species. |
Wednesday November 6th - Synapse and Vesicles |
| Son, S., Jeong, H., Lee, G., Park, J. H., Yoo, S. (2024). Biogenesis of circular RNAs in vitro and in vivo from the Drosophila Nk2.1 / scarecrow gene. bioRxiv, PubMed ID: 38463984
Summary: scarecrow (scro) encodes a fly homolog of mammalian Nkx2.1 that is vital for early fly development as well as for optic lobe development. Interestingly, scro was reported to produce a circular RNA (circRNA). This study identified 12 different scro circRNAs, which are either mono- or multi-exonic forms. The most abundant forms are circE2 carrying the second exon only and bi-exonic circE3-E4. Levels of circE2 show an age-dependent increase in adult heads, supporting a general trend of high accumulation of circRNAs in aged fly brains. Aligning sequences of introns flanking exons uncovered two pairs of intronic complementary sequences (ICSs); one pair residing in introns 1 and 2 and the other in introns 2 and 4. The first pair was demonstrated to be essential for the circE2 production in cell-based assays; furthermore, deletion of the region including potential ICS components in the intron-2 reduced in vivo production of circE2 and circE3-E4 by 80%, indicating them to be essential for the biogenesis of these isoforms. Besides the ICS, the intron regions immediately abutting exons seemed to be responsible for a basal level of circRNA formation. Moreover, the replacement of scro-ICS with those derived from laccase2 was comparably effective in scro-circRNA production, buttressing the importance of the hairpin-loop structure formed by ICS for the biogenesis of circRNA. Lastly, overexpressed scro affected outcomes of both linear and circular RNAs from the endogenous scro locus, suggesting that Scro plays a direct or indirect role in regulating expression levels of either or both forms. | Bernard, E. I. M., Towler, B. P., Rogoyski, O. M., Newbury, S. F. (2024). Characterisation of the in-vivo miRNA landscape in Drosophila ribonuclease mutants reveals Pacman-mediated regulation of the highly conserved let-7 cluster during apoptotic processes. Front Genet, 15:1272689 PubMed ID: 38444757
Summary: The control of gene expression is a fundamental process essential for correct development and to maintain homeostasis. Many post-transcriptional mechanisms exist to maintain the correct levels of each RNA transcript within the cell. Controlled and targeted cytoplasmic RNA degradation is one such mechanism with the 5'-3' exoribonuclease Pacman (XRN1) and the 3'-5' exoribonuclease Dis3L2 playing crucial roles. Loss of function mutations in either Pacman or Dis3L2 have been demonstrated to result in distinct phenotypes, and both have been implicated in human disease. One mechanism by which gene expression is controlled is through the function of miRNAs which have been shown to be crucial for the control of almost all cellular processes. Although the biogenesis and mechanisms of action of miRNAs have been comprehensively studied, the mechanisms regulating their own turnover are not well understood. This study characterised the miRNA landscape in a natural developing tissue, the Drosophila melanogaster wing imaginal disc, and assess the importance of Pacman and Dis3L2 on the abundance of miRNAs. A complex landscape of miRNA expression was revealed, and it whilst a null mutation in dis3L2 has a minimal effect on the miRNA expression profile, loss of Pacman has a profound effect with a third of all detected miRNAs demonstrating Pacman sensitivity. A role was revealed for Pacman in regulating the highly conserved let-7 cluster (containing miR-100, let-7 and miR-125), and a genetic model is presented outlining a positive feedback loop regulated by Pacman which enhances understanding of the apoptotic phenotype observed in Pacman mutants. |
| Ilık, I, A., Jaglarzar, P., Tse, K., Brandl, B., Meierhofer, D., Muller, F. J., Smith, Z. D., Aktas, T. (2024). Autonomous transposons tune their sequences to ensure somatic suppression. Nature, 626(8001):1116-1124 PubMed ID: 38355802
Summary: Transposable elements (TEs) are a major constituent of human genes, occupying approximately half of the intronic space. During pre-messenger RNA synthesis, intronic TEs are transcribed along with their host genes but rarely contribute to the final mRNA product because they are spliced out together with the intron and rapidly degraded. Paradoxically, TEs are an abundant source of RNA-processing signals through which they can create new introns, and also functional or non-functional chimeric transcripts. The rarity of these events implies the existence of a resilient splicing code that is able to suppress TE exonization without compromising host pre-mRNA processing. This study shows that SAFB proteins protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity, via prevention of the exonization of previously integrated TEs. This unique dual role is possible because of L1's conserved adenosine-rich coding sequences that are bound by SAFB proteins. The suppressive activity of SAFB extends to tissue-specific, giant protein-coding cassette exons, nested genes and Tigger DNA transposons. Moreover, SAFB also suppresses LTR/ERV elements in species in which they are still active, such as mice and flies. A significant subset of splicing events suppressed by SAFB in somatic cells are activated in the testis, coinciding with low SAFB expression in postmeiotic spermatids. Reminiscent of the division of labour between innate and adaptive immune systems that fight external pathogens, our results uncover SAFB proteins as an RNA-based, pattern-guided, non-adaptive defence system against TEs in the soma, complementing the RNA-based, adaptive Piwi-interacting RNA pathway of the germline. | Saedi, H., Waro, G., Giacchetta, L., Tsunoda, S. (2024). miR-137 regulates PTP61F, affecting insulin signaling, metabolic homeostasis, and starvation resistance in Drosophila. Proc Natl Acad Sci U S A, 121(5):e2319475121 PubMed ID: 38252824
Summary: miR-137 is a highly conserved brain-enriched microRNA (miRNA) that has been associated with neuronal function and proliferation. This study shows that Drosophila miR-137 null mutants display increased body weight with enhanced triglyceride content and decreased locomotor activity. In addition, when challenged by nutrient deprivation, miR-137 mutants exhibit reduced motivation to feed and prolonged survival. Genetic epistasis and rescue experiments show that this starvation resistance is due to a disruption in insulin signaling. These studies further show that miR-137 null mutants exhibit a drastic reduction in levels of the phosphorylated/activated Insulin receptor, InR (InR-P). Whether this is due to the predicted miR-137 target, Protein Tyrosine Phosphatase 61F (PTP61F), ortholog of mammalian TC-PTP/PTP1B, which are known to dephosphorylate InR-P. Indeed, levels of an endogenously tagged GFP-PTP61F are significantly elevated in miR-137 null mutants, and overexpression of PTP61F alone was shown to be sufficient to mimic many of the metabolic phenotypes of miR-137 mutants. Finally, elevated levels of PTP61F in the miR-137 null mutant background were knocked-down and this rescues levels of InR-P, restores normal body weight and triglyceride content, starvation sensitivity, as well as attenuates locomotor and starvation-induced feeding defects. This study supports a model in which miR-137 is critical for dampening levels of PTP61F, thereby maintaining normal insulin signaling and energy homeostasis. |
| Crawford, B. I., Talley, M. J., Russman, J., Riddle, J., Torres, S., Williams, T., Longworth, M. S. (2024). Condensin-mediated restriction of retrotransposable elements facilitates brain development in Drosophila melanogaster. Nat Commun, 15(1):2716 PubMed ID: 38548759
Summary: Neural stem and progenitor cell (NSPC) maintenance is essential for ensuring that organisms are born with proper brain volumes and head sizes. Microcephaly is a disorder in which babies are born with significantly smaller head sizes and cortical volumes. Mutations in subunits of the DNA organizing complex condensin have been identified in microcephaly patients. However, the molecular mechanisms by which condensin insufficiency causes microcephaly remain elusive. Previous work has identified conserved roles for condensins in repression of retrotransposable elements (RTEs). This study shows that condensin subunit Cap-d3 knockdown in NSPCs of the Drosophila larval central brain increases RTE expression and mobility which causes cell death, and significantly decreases adult head sizes and brain volumes. These findings suggest that unrestricted RTE expression and activity may lead to improper brain development in condensin insufficient organisms, and lay the foundation for future exploration of causative roles for RTEs in other microcephaly models. | Rajeev, R., Mishra, R. K., Khosla, S. (2024). DNMT3L interacts with Piwi and modulates the expression of piRNAs in transgenic Drosophila. Epigenomics, 16(6):375-388 PubMed ID: 38440884
Summary: This study explored the role of Piwi protein and piRNAs in DNMT3L-mediated epigenetic inheritance. Transgenic Drosophila were used to examine the effect of ectopically expressed DNMT3L on the profile of piRNAs by sequencing of small RNAs. Previous work showed accumulation and inheritance of epimutations across multiple generations in transgenic DNMT3L Drosophila. This study shows interaction of DNMT3L with Piwi and a significant alteration in the piRNA profile across multiple generations in transgenic Drosophila. In the light of its interaction with histone H1, it is proposed that in addition to its role of modulating core histone modifications, DNMT3L allows for inheritance of epigenetic information through its collaboration with Piwi, piRNAs and histone H1. |
Tuesday November 5th - Synapse and Vesicles |
| Beckers, C. J., Mrestani, A., Komma, F., Dannhauser, S. (2024). Versatile Endogenous Editing of GluRIIA in Drosophila melanogaster. Cells, 13(4) PubMed ID: 38391936
Summary: Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the GluRIIA gene have been utilized. Furthermore, mutated and tagged versions of GluRIIA have been examined in the past, but most of these constructs were not expressed under endogenous regulatory control or involved the mentioned imprecise GluRIIA knockouts. This study performed CRISPR/Cas9-assisted gene editing at the endogenous locus of GluRIIA. This enabled the investigation of the endogenous expression pattern of GluRIIA using tagged constructs with an EGFP and an ALFA tag for super-resolution immunofluorescence imaging, including structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). All GluRIIA constructs exhibited full functionality and PHP could be induced by philanthotoxin at control levels. By applying hierarchical clustering algorithms to analyze the dSTORM data, postsynaptic receptor cluster areas of ~0.15 μm(2) were detected. Consequently, these constructs are suitable for ultrastructural analyses of GluRIIA. | Delgado, M. G., Delgado, R. (2024). Transient Synaptic Enhancement Triggered by Exogenously Supplied Monocarboxylate in Drosophila Motoneuron Synapse. Neuroscience, 539:66-75 PubMed ID: 38220128
Summary: Current evidence suggests that glial cells provide C3 carbon sources to fuel neuronal activity; however, this notion has become challenged by biosensor studies carried out in acute brain slices or in vivo, showing that neuronal activity does not rely on the import of astrocyte-produced L-lactate. Rather, stimulated neurons become net lactate exporters, as it was also shown in Drosophila neurons, in which astrocyte-provided lactate returns as lipid droplets to be stored in glial cells. In this view, this study investigated whether exogenously supplied monocarboxylates can support Drosophila motoneuron neurotransmitter release (NTR). By assessing the excitatory post-synaptic current (EPSC) amplitude under voltage-clamp as NTR indicative, both pyruvate and L-lactate (as the only carbon sources in the synapses bathing-solution) cause a large transient NTR enhancement, which declines to reach a synaptic depression state, from which the synapses do not recover. The FM1-43 (a fluorescent styryl dye that stains nerve terminals in an activity-dependent fashion) pre-synaptic loading ability , however, is maintained under monocarboxylate, suggesting that SV cycling should not contribute to the synaptic depression state. The NTR recovery was reached by supplementing the monocarboxylate medium with sucrose. However, monocarboxylate addition to sucrose medium does not enhance NTR, but it does when the disaccharide concentration becomes too reduced. Thus, when pyruvate concentrations become too reduced, exogenously supplied L-lactate could be converted to pyruvate and metabolized by the neural mitochondria, triggering the NTR enhancement. |
| Dutta, D., Kanca, O., Shridharan, R. V., Marcogliese, P. C., Steger, B., Morimoto, M., Frost, F. G., Macnamara, E., Wangler, M. F., Yamamoto, S., Jenny, A., Adams, D., Malicdan, M. C., Bellen, H. J. (2024). Loss of the endoplasmic reticulum protein Tmem208 affects cell polarity, development, and viability. Proc Natl Acad Sci U S A, 121(9):e2322582121 PubMed ID: 38381787
Summary: Nascent proteins destined for the cell membrane and the secretory pathway are targeted to the endoplasmic reticulum (ER) either posttranslationally or cotranslationally. The signal-independent pathway, containing the protein TMEM208, is one of three pathways that facilitates the translocation of nascent proteins into the ER. The in vivo function of this protein is ill characterized in multicellular organisms. This study generated a CRISPR-induced null allele of the fruit fly ortholog CG8320/Tmem208 by replacing the gene with the Kozak-GAL4 sequence. Tmem208 was shown to be broadly expressed in flies and that its loss causes lethality, although a few short-lived flies eclose. These animals exhibit wing and eye developmental defects consistent with impaired cell polarity and display mild ER stress. Tmem208 physically interacts with Frizzled (Fz), a planar cell polarity (PCP) receptor, and is required to maintain proper levels of Fz. Moreover, this study identified a child with compound heterozygous variants in TMEM208 who presents with developmental delay, skeletal abnormalities, multiple hair whorls, cardiac, and neurological issues, symptoms that are associated with PCP defects in mice and humans. Additionally, fibroblasts of the proband display mild ER stress. Expression of the reference human TMEM208 in flies fully rescues the loss of Tmem208, and the two proband-specific variants fail to rescue, suggesting that they are loss-of-function alleles. In summary, this study uncovers a role of TMEM208 in development, shedding light on its significance in ER homeostasis and cell polarity. | Szenci, G., Glatz, G., Takaes, S., Juhasz, G. (2024). The Ykt6-Snap29-Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles. Sci Rep, 14(1):3200 PubMed ID: 38331993
Summary: n the Drosophila larval salivary gland, developmentally programmed fusions between lysosomes and secretory granules (SGs) and their subsequent acidification promote the maturation of SGs that are secreted shortly before puparium formation. Subsequently, ongoing fusions between non-secreted SGs and lysosomes give rise to degradative crinosomes, where the superfluous secretory material is degraded. Lysosomal fusions control both the quality and quantity of SGs, however, its molecular mechanism is incompletely characterized. This study identified the R-SNARE Ykt6 as a novel regulator of crinosome formation, but not the acidification of maturing SGs. We show that Ykt6 localizes to Lamp1+carrier vesicles, and forms a SNARE complex with Syntaxin 13 and Snap29 to mediate fusion with SGs. These Lamp1 carriers represent a distinct vesicle population that are functionally different from canonical Arl8+, Cathepsin L+ lysosomes, which also fuse with maturing SGs but are controlled by another SNARE complex composed of Syntaxin 13, Snap29 and Vamp7. Ykt6- and Vamp7-mediated vesicle fusions also determine the fate of SGs, as loss of either of these SNAREs prevents crinosomes from acquiring endosomal PI3P. These results highlight that fusion events between SGs and different lysosome-related vesicle populations are critical for fine regulation of the maturation and crinophagic degradation of SGs. |
| Drogalin, A., Monteiro, L. S., Alves, M. J., Castro, T. G. (2024). Golgi α-mannosidase: opposing structures of Drosophila melanogaster and novel human model using molecular dynamics simulations and docking at different pHs. Journal of biomolecular structure & dynamics, 42(5):2714-2725 PubMed ID: 37158092
Summary: The search for Golgi αmannosidase II (GMII) potent and specific inhibitors has been a focus of many studies for the past three decades since this enzyme is a key target for cancer treatment. α-Mannosidases, such as those from Drosophila melanogaster or Jack bean, have been used as functional models of the human Golgi α-mannosidase II (hGMII) because mammalian mannosidases are difficult to purify and characterize experimentally. Meanwhile, computational studies have been seen as privileged tools able to explore assertive solutions to specific enzymes, providing molecular details of these macromolecules, their protonation states and their interactions. Thus, modelling techniques can successfully predict hGMII 3D structure with high confidence, speeding up the development of new hits. In this study, Drosophila melanogaster Golgi mannosidase II (dGMII) and a novel human model, developed in silico and equilibrated via molecular dynamics simulations, were both opposed for docking. These findings highlight that the design of novel inhibitors should be carried out considering the human model's characteristics and the enzyme operating pH. A reliable model is evidenced, showing a good correlation between K(i)/IC(50) experimental data and theoretical ΔG(binding) estimations in GMII, opening the possibility of optimizing the rational drug design of new derivatives. | Gao, Z., Huang, E., Wang, W., Xu, L., Xu, W., Zheng, T., Rui, M. (2024). Patronin regulates presynaptic microtubule organization and neuromuscular junction development in Drosophila. iScience, 27(2):108944 PubMed ID: 38318379
Summary: Synapses are fundamental components of the animal nervous system. Synaptic cytoskeleton is essential for maintaining proper neuronal development and wiring. Perturbations in neuronal microtubules (MTs) are correlated with numerous neuropsychiatric disorders. Despite discovering multiple synaptic MT regulators, the importance of MT stability, and particularly the polarity of MT in synaptic function, is still under investigation. This study identified Patronin, an MT minus-end-binding protein, for its essential role in presynaptic regulation of MT organization and neuromuscular junction (NMJ) development. Analyses indicate that Patronin regulates synaptic development independent of Klp10A. Subsequent research elucidates that it is Short stop (Shot), a member of the Spectraplakin family of large cytoskeletal linker molecules, works synergistically with Patronin to govern NMJ development. This study further raise the possibility that normal synaptic MT polarity contributes to proper NMJ morphology. Overall, this study demonstrates an unprecedented role of Patronin, and a potential involvement of MT polarity in synaptic development. |
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