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Wednesday, February 28th - Oncogenesis and Growth

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Yin, H., Wang, Z., Wang, D., Nuer, M., Han, M., Ren, P., Ma, S., Lin, C., Chen, J., Xian, H., Ai, D., Li, X., Ma, S., Lin, Z. and Pan, Y. (2023). TIMELESS promotes the proliferation and migration of lung adenocarcinoma cells by activating EGFR through AMPK and SPHK1 regulation. Eur J Pharmacol 955: 175883. PubMed ID: 37433364
Summary:
Lung adenocarcinoma (LUAD) has high morbidity and is prone to recurrence. TIMELESS (TIM), which regulates circadian rhythms in Drosophila, is highly expressed in various tumors. Tumor samples from patients with LUAD patient data from public databases were used to confirm the relationship of TIM expression with lung cancer. LUAD cell lines were used and siRNA of TIM was adopted to knock down TIM expression in LUAD cells, and further cell proliferation, migration and colony formation were analyzed. By using Western blot and qPCR, the influence was detected of TIM on epidermal growth factor receptor (EGFR), sphingosine kinase 1 (SPHK1) and AMP-activated protein kinase (AMPK). With proteomics analysis, this study comprehensively inspected the different changed proteins influenced by TIM, and global bioinformatic analysis was performed. TIM expression was found to be elevated in LUAD and that this high expression was positively correlated with more advanced tumor pathological stages and shorter overall and disease-free survival. TIM knockdown inhibited EGFR activation and also AKT/mTOR phosphorylation. This study also clarified that TIM regulated the activation of SPHK1 in LUAD cells. And with SPHK1 siRNA to knock down the expression level of SPHK1, it was found that EGFR activation were inhibited greatly too. Quantitative proteomics techniques combined with bioinformatics analysis clarified the global molecular mechanisms regulated by TIM in LUAD. The results of proteomics suggested that mitochondrial translation elongation and termination were altered, which were closely related to the process of mitochondrial oxidative phosphorylation. It was further confirmed that TIM knockdown reduced ATP content and promoted AMPK activation in LUAD cells. This study revealed that siTIM could inhibit EGFR activation through activating AMPK and inhibiting SPHK1 expression, as well as influencing mitochondrial function and altering the ATP level; TIM's high expression in LUAD is an important factor and a potential key target in LUAD.
Bertrand, M., Szeremeta, F., Hervouet-Coste, N., Sarou-Kanian, V., Landon, C., Morisset-Lopez, S., Decoville, M. (2023). An adult Drosophila glioma model to highlight metabolic dysfunctions and evaluate the role of the serotonin 5-HT(7) receptor as a potential therapeutic target. Faseb j, 37(11):e23230 PubMed ID: 37781977
Summary:
Gliomas account for 50% of brain cancers and are therefore the most common brain tumors. Molecular alterations involved in adult gliomas have been identified and mainly affect tyrosine kinase receptors with amplification and/or mutation of the epidermal growth factor receptor (EGFR) and its associated signaling pathways. Several targeted therapies have been developed, but current treatments remain ineffective for glioblastomas, the most severe forms. Thus, it is a priority to identify new pharmacological targets. Drosophila glioma models established in larvae and adults are useful to identify new genes and signaling pathways involved in glioma progression. This study used a Drosophila glioma model in adults, to characterize metabolic disturbances associated with glioma and assess the consequences of 5-HT(7) R expression on glioma development. First, by using in vivo magnetic resonance imaging, it was shown that expression of the constitutively active forms of EGFR and PI3K in adult glial cells induces brain enlargement. Then, altered cellular metabolism was exploited by using high-resolution magic angle spinning NMR and (1) H-(13) C heteronuclear single quantum coherence solution states. Discriminant metabolites identified highlight the rewiring of metabolic pathways in glioma and associated cachexia phenotypes. Finally, the expression of 5-HT(7) R in this adult model attenuates phenotypes associated with glioma development. Collectively, this whole-animal approach in Drosophila provides several rapid and robust phenotype readouts, such as enlarged brain volume and glioma-associated cachexia, as well as to determine the metabolic pathways involved in glioma genesis and finally to confirm the interest of the 5-HT(7) R in the treatment of glioma.
Kinoshita, Y., Shiratsuchi, N., Araki, M., Inoue, Y. H. (2023). Anti-Tumor Effect of Turandot Proteins Induced via the JAK/STAT Pathway in the mxc Hematopoietic Tumor Mutant in Drosophila. Cells, 12(16) PubMed ID: 37626857
Summary:
Several antimicrobial peptides suppress the growth of lymph gland (LG) tumors in Drosophila multi sex comb (mxc) mutant larvae. The activity of another family of polypeptides, called Turandots, is also induced via the JAK/STAT pathway after bacterial infection; however, their influence on Drosophila tumors remains unclear. The JAK/STAT pathway was activated in LG tumors, fat body, and circulating hemocytes of mutant larvae. The mRNA levels of Turandot (Tot) genes increased markedly in the mutant fat body and declined upon silencing Stat92E in the fat body, indicating the involvement of the JAK/STAT pathway. Furthermore, significantly enhanced tumor growth upon a fat-body-specific silencing of the mRNAs demonstrated the antitumor effects of these proteins. The proteins were found to be incorporated into small vesicles in mutant circulating hemocytes (as previously reported for several antimicrobial peptides) but not normal cells. In addition, more hemocytes containing these proteins were found to be associated with tumors. The mutant LGs contained activated effector caspases, and a fat-body-specific silencing of Tots inhibited apoptosis and increased the number of mitotic cells in the LG, thereby suggesting that the proteins inhibited tumor cell proliferation. Thus, Tot proteins possibly exhibit antitumor effects via the induction of apoptosis and inhibition of cell proliferation.
Voutyraki, C., Choromidis, A., Meligkounaki, A., Vlachopoulos, N. A., Theodorou, V., Grammenoudi, S., Athanasiadis, E., Monticelli, S., Giangrande, A., Delidakis, C. and Zacharioudaki, E. (2023). Growth deregulation and interaction with host hemocytes contribute to tumor progression in a Drosophila brain tumor model. Proc Natl Acad Sci U S A 120(33): e2221601120. PubMed ID: 37549261
Summary:
Tumors constantly interact with their microenvironment. This study presented data on a Notch-induced neural stem cell (NSC) tumor in Drosophila, which can be immortalized by serial transplantation in adult hosts. This tumor arises in the larva by virtue of the ability of Notch to suppress early differentiation-promoting factors in NSC progeny. Guided by transcriptome data, this study has addressed both tumor-intrinsic and microenvironment-specific factors and how they contribute to tumor growth and host demise. The growth promoting factors Myc, Imp, and Insulin receptor in the tumor cells are important for tumor expansion and killing of the host. From the host's side, hemocytes, professional phagocytic blood cells, are found associated with tumor cells. Phagocytic receptors, like NimC1, are needed in hemocytes to enable them to capture and engulf tumor cells, restricting their growth. In addition to their protective role, hemocytes may also increase the host's morbidity by their propensity to produce damaging extracellular reactive oxygen species.
Bosso, G., Cipressa, F., Tullo, L., Cenci, G. (2023). Co-amplification of CBX3 with EGFR or RAC1 in human cancers corroborated by a conserved genetic interaction among the genes. Cell Death Discov, 9(1):317 PubMed ID: 37633946
Summary:
Chromobox Protein 3 (CBX3; Drosophila homlog - HP1b) overexpression is a common event occurring in cancer, promotes cancer cell proliferation and represents a poor prognosis marker in a plethora of human cancers. This study describes that a wide spectrum of human cancers harbors a co-amplification of CBX3 gene with either EGFR or RAC1, which yields a statistically significant increase of both mRNA and protein levels of CBX3, EGFR and RAC1. It was also revealed that the simultaneous overexpression of CBX3, RAC1 and EGFR gene products correlates with a worse prognosis compared to the condition when CBX3, RAC1 and EGFR are singularly upregulated. Furthermore, this study also showed that a co-occurrence of low-grade amplification, in addition to high-grade amplification, between CBX3 and EGFR or RAC1 is associated with a reduced patient lifespan. Finally, CBX3 and RAC1/EGFR genetically interact in the model organism Drosophila melanogaster, suggesting that the simultaneous overexpression as well as well the co-occurrence of high- or low-grade copy number alterations in these genes is not accidental and could reflect evolutionarily conserved functional relationships.
Johannessen, J. A., Formica, M., Haukeland, A. L. C., Brathen, N. R., Al Outa, A., Aarsund, M., Therrien, M., Enserink, J. M., Knaevelsrud, H. (2023). The human leukemic oncogene MLL-AF4 promotes hyperplastic growth of hematopoietic tissues in Drosophila larvae. iScience, 26(10):107726 PubMed ID: 37720104
Summary:
MLL-rearranged (MLL-r) leukemias are among the leukemic subtypes with poorest survival, and treatment options have barely improved over the last decades. Despite increasing molecular understanding of the mechanisms behind these hematopoietic malignancies, this knowledge has had poor translation into the clinic. This study reports a Drosophila melanogaster model system to explore the pathways affected in MLL-r leukemia. Expression of the human leukemic oncogene MLL-AF4 in the Drosophila hematopoietic system resulted in increased levels of circulating hemocytes and an enlargement of the larval hematopoietic organ, the lymph gland. Strikingly, depletion of Drosophila orthologs of known interactors of MLL-AF4, such as DOT1L, rescued the leukemic phenotype. In agreement, treatment with small-molecule inhibitors of DOT1L also prevented the MLL-AF4-induced leukemia-like phenotype. Taken together, this model provides an in vivo system to unravel the genetic interactors involved in leukemogenesis and offers a system for improved biological understanding of MLL-r leukemia.

Tuesday, February 27th - Chromatin

Koury, S. A. (2023). Female meiotic drive shapes the distribution of rare inversion polymorphisms in Drosophila melanogaster. Genetics, 225(2) PubMed ID: 37616566
Summary:
In all species, new chromosomal inversions are constantly being formed by spontaneous rearrangement and then stochastically eliminated from natural populations. In Drosophila, when new chromosomal inversions overlap with a preexisting inversion in the population, their rate of elimination becomes a function of the relative size, position, and linkage phase of the gene rearrangements. These altered dynamics result from complex meiotic behavior wherein overlapping inversions generate asymmetric dyads that cause both meiotic drive/drag and segmental aneuploidy. In this context, patterns in rare inversion polymorphisms of a natural population can be modeled from the fundamental genetic processes of forming asymmetric dyads via crossing-over in meiosis I and preferential segregation from asymmetric dyads in meiosis II. Here, a mathematical model of crossover-dependent female meiotic drive is developed and parameterized with published experimental data from Drosophila melanogaster laboratory constructs. This mechanism is demonstrated to favor smaller, distal inversions and accelerate the elimination of larger, proximal inversions. Simulated sampling experiments indicate that the paracentric inversions directly observed in natural population surveys of D. melanogaster are a biased subset that both maximizes meiotic drive and minimizes the frequency of lethal zygotes caused by this cytogenetic mechanism. Incorporating this form of selection into a population genetic model accurately predicts the shift in relative size, position, and linkage phase for rare inversions found in this species. The model and analysis presented in this study suggest that this weak form of female meiotic drive is an important process influencing the genomic distribution of rare inversion polymorphisms.
Kingsley, G., Skagia, A., Passaretti, P., Fernandez-Cuesta, C., Reynolds-Winczura, A., Koscielniak, K., Gambus, A. (2023). DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation. Nucleic Acids Res, 51(18):9748-9763 PubMed ID: 37638758
Summary:
Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. Here we show that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients.
Shu, S., Jiang, M., Deng, X., Yue, W., Cao, X., Zhang, K., Wang, Z., He, H., Cui, J., Wang, Q., Qu, K., Fang, Y. (2023). Heterochromatic silencing of immune-related genes in glia is required for BBB integrity and normal lifespan in drosophila. Aging Cell, 22(10):e13947 PubMed ID: 37594178
Summary:
Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. This study reports that adult-onset downregulation of a Drosophila gene CG32529/GLAD led to shortened lifespan and age-dependent brain degeneration. This regulation exhibited cell type and subtype-specificity, involving mainly surface glia (comprising the BBB) and cortex glia (wrapping neuronal soma) in flies. In accordance, pan-glial knockdown of GLAD disrupted BBB integrity and the glial meshwork. GLAD expression in fly heads decreased with age, and the RNA-seq analysis revealed that the most affected transcriptional changes by RNAi-GLAD were associated with upregulation of immune-related genes. Furthermore, a series of lifespan rescue experiments was conducted and the results indicated that the profound upregulation of immune and related pathways was not the consequence but cause of the degenerative phenotypes of the RNAi-GLAD flies. Finally, this study showed that GLAD encoded a heterochromatin-associating protein that bound to the promoters of an array of immune-related genes and kept them silenced during the cell cycle. Together, these findings demonstrate a previously unappreciated role of heterochromatic gene silencing in repressing immunity in fly glia, which is required for maintaining BBB and brain integrity as well as normal lifespan.
Yang, J., Tang, R., Chen, S., Chen, Y., Yuan, K., Huang, R., Wang, L. (2023). Exposure to high-sugar diet induces transgenerational changes in sweet sensitivity and feeding behavior via H3K27me3 reprogramming. Elife, 12 PubMed ID: 37698486
Summary:
Human health is facing a host of new threats linked to unbalanced diets, including high-sugar diet (HSD), which contributes to the development of both metabolic and behavioral disorders. Studies have shown that diet-induced metabolic dysfunctions can be transmitted to multiple generations of offspring and exert long-lasting health burden. Meanwhile, whether and how diet-induced behavioral abnormalities can be transmitted to the offspring remains largely unclear. This study showed that ancestral HSD exposure suppressed sweet sensitivity and feeding behavior in the offspring in Drosophila. These behavioral deficits were transmitted through the maternal germline and companied by the enhancement of H3K27me3 modifications. PCL-PRC2 complex, a major driver of H3K27 trimethylation, was upregulated by ancestral HSD exposure, and disrupting its activity eliminated the transgenerational inheritance of sweet sensitivity and feeding behavior deficits. Elevated H3K27me3 inhibited the expression of a transcriptional factor Cad and suppressed sweet sensitivity of the sweet-sensing gustatory neurons, reshaping the sweet perception and feeding behavior of the offspring. Taken together, this study uncovered a novel molecular mechanism underlying behavioral abnormalities spanning multiple generations of offspring upon ancestral HSD exposure, which would contribute to the further understanding of long-term health risk of unbalanced diet.
Sarkar, K., Kotb, N. M., Lemus, A., Martin, E. T., McCarthy, A., Camacho, J., Iqbal, A., Valm, A. M., Sammons, M. A., Rangan, P. (2023). A feedback loop between heterochromatin and the nucleopore complex controls germ-cell-to-oocyte transition during Drosophila oogenesise. Dev Cell 58(22):2580-2596 PubMed ID: 37673064
Summary:
Germ cells differentiate into oocytes that launch the next generation upon fertilization. How the highly specialized oocyte acquires this distinct cell fate is poorly understood. During Drosophila oogenesis, H3K9me3 histone methyltransferase SETDB1 translocates from the cytoplasm to the nucleus of germ cells concurrently with oocyte specification. This study discovered that nuclear SETDB1 is required for silencing a cohort of differentiation-promoting genes by mediating their heterochromatinization. Intriguingly, SETDB1 is also required for upregulating 18 of the ∼30 nucleoporins (Nups) that compose the nucleopore complex (NPC), promoting NPC formation. NPCs anchor SETDB1-dependent heterochromatin at the nuclear periphery to maintain H3K9me3 and gene silencing in the egg chambers. Aberrant gene expression due to the loss of SETDB1 or Nups results in the loss of oocyte identity, cell death, and sterility. Thus, a feedback loop between heterochromatin and NPCs promotes transcriptional reprogramming at the onset of oocyte specification, which is critical for establishing oocyte identity.
Santos, A. S., Ramos, E. S., Valente-Gaiesky, V. L. S., de Melo Sene, F., Manfrin, M. H. (2023). Evidences of differential methylation in the genome during development in the cactophilic Drosophila species. Genesis:e23554 PubMed ID: 37750176
Summary:
DNA methylation with 5-methylcytosine (5mC) has been reported in the genome of several eukaryotes, with marked differences between vertebrates and invertebrates. DNA methylation is poorly understood as its role in evolution in insects. Drosophila gouveai (cluster Drosophila buzzatii) presents larvae that develop obligatorily in necrotic tissues of cacti in nature, with the distribution of populations in South America, and plasticity of phenotypes in insect-plant interaction. Organisms were characterized at developmental stages, and variations at multiple methylation-sensitive loci were analyzed in pupae, and adult flies using methylation sensitive amplification polymorphism. 326 loci with CCGG targets were obtained in the genome of D. gouveai. Genomic regions with molecular lengths from 100 to 700 pb were most informative about methylation states. Multiple loci show differences in methylation-sensitive sites (MSL) concerning developmental stages, such as in pupae (MSL = 40), female reproductive tissue (MSL = 76), and male reproductive tissues (MSL = 58). These results are the first evidence of genome-wide methylation in D. gouveai organisms.

Monday, February 26th - Adult Physiology and Metabolism

Dobson, A. J., Voigt, S., Kumpitsch, L., Langer, L., Voigt, E., Ibrahim, R., Dowling, D. K., Reinhardt, K. (2023). Mitonuclear interactions shape both direct and parental effects of diet on fitness and involve a SNP in mitoribosomal 16s rRNA. PLoS Biol, 21(8):e3002218 PubMed ID: 37603597
Summary:
Nutrition is a primary determinant of health, but responses to nutrition vary with genotype. Epistasis between mitochondrial and nuclear genomes may cause some of this variation, but which mitochondrial loci and nutrients participate in complex gene-by-gene-by-diet interactions? Furthermore, it remains unknown whether mitonuclear epistasis is involved only in the immediate responses to changes in diet, or whether mitonuclear genotype might modulate sensitivity to variation in parental nutrition, to shape intergenerational fitness responses. In Drosophila melanogaster this study shows that mitonuclear epistasis shapes fitness responses to variation in dietary lipids and amino acids. It was also shown that mitonuclear genotype modulates the parental effect of dietary lipid and amino acid variation on offspring fitness. Effect sizes for the interactions between diet, mitogenotype, and nucleogenotype were equal to or greater than the main effect of diet for some traits, suggesting that dietary impacts cannot be understood without first accounting for these interactions. Associating phenotype to mtDNA variation in a subset of populations implicated a C/T polymorphism in mt:lrRNA, which encodes the 16S rRNA of the mitochondrial ribosome. This association suggests that directionally different responses to dietary changes can result from variants on mtDNA that do not change protein coding sequence, dependent on epistatic interactions with variation in the nuclear genome.
Gruss, I., Twardowski, J., Samsel-Czekala, M., Beznosiuk, J., Wandzel, C., Twardowska, K., Wiglusz, R. J. (2023). The isothermal Boltzmann-Gibbs entropy reduction affects survival of the fruit fly Drosophila melanogaster. Sci Rep, 13(1):14166 PubMed ID: 37644276
Summary:
This study presents is the first experimental evidence of the effect of isothermal changes in entropy on a living organism. In greater detail, the effect of the reduction of the total Boltzmann-Gibbs entropy (S) of the aquatic environment on the survival rate and body mass of the fruit fly Drosophila melanogaster was investigated. The tests were carried out in standard thermodynamic states at room temperature of 296.15 K and ambient atmospheric pressure of 1 bar. Two variants of entropy reduction (ΔS) were tested for ΔS = 28.49 and 51.14 J K(-1) mol(-1) compared to the blind and control samples. The entropy level was experimentally changed, using the quantum system for isothermal entropy reduction. This system is based on quantum bound entanglement of phonons and the phenomenon of phonon resonance (interference of phonon modes) in condensed matter (Silicon dioxide (SiO(2)) and single crystals of Silicon (Si(0)), Aluminum (Al(0)) plates ("chips"), glass, and water). All studied organisms were of the same age (1 day). Mortality was observed daily until the natural death of the organisms. The investigations showed that changes in the Boltzmann-Gibbs entropy affected the survival and body mass of the fruit flies. On the one hand, the reduction in entropy under isothermal conditions in the aquatic environment for ΔS = 28.49 J K(-1) mol(-1) resulted in an extension of the lifespan and an increase in the body mass of female fruit flies. On the other hand, the almost twofold reduction in this entropy for ΔS = 51.14 J K(-1) mol(-1) shortened the lives of the males. Thus, the lifespan and body mass of flies turned out to be a specific reaction of metabolism related to changes in the entropy of the aquatic environment.
Kashio, S., Masuda, S., Miura, M. (2023). Involvement of neuronal tachykinin-like receptor at 86C in Drosophila disc repair via regulation of kynurenine metabolism. iScience, 26(9):107553 PubMed ID: 37636053
Summary:
Neurons contribute to the regeneration of projected tissues; however, it remains unclear whether they are involved in the non-innervated tissue regeneration. This study shows that a neuronal tachykinin-like receptor at 86C (TkR86C) is required for the repair of non-innervated wing discs in Drosophila. Using a genetic tissue repair system in Drosophila larvae, genetic screening was performed for G protein-coupled receptors to search for signal mediatory systems for remote tissue repair. An evolutionarily conserved neuroinflammatory receptor, TkR86C, was identified as the candidate receptor. Neuron-specific knockdown of TkR86C impaired disc repair without affecting normal development. The humoral metabolites of the kynurenine (Kyn) pathway regulated in the fat body were investigated because of their role as tissue repair-mediating factors. Neuronal knockdown of TkR86C hampered injury-dependent changes in the expression of vermillion in the fat body and humoral Kyn metabolites. These data indicate the involvement of TkR86C neurons upstream of Kyn metabolism in non-autonomous tissue regeneration.
Guilhot, R., Xuereb, A., Lagmairi, A., Olazcuaga, L., Fellous, S. (2023). Microbiota acquisition and transmission in Drosophila flies. iScience, 26(9):107656 PubMed ID: 37670792
Summary:
Understanding the ecological and evolutionary dynamics of host-microbiota associations notably involves exploring how members of the microbiota assemble and whether they are transmitted along host generations. This study investigated the larval acquisition of facultative bacterial and yeast symbionts of Drosophila melanogaster and Drosophila suzukii in ecologically realistic setups. Fly mothers and fruit were major sources of symbionts. Microorganisms associated with adult males also contributed to larval microbiota, mostly in D. melanogaster. Yeasts acquired at the larval stage maintained through metamorphosis, adult life, and were transmitted to offspring. All these observations varied widely among microbial strains, suggesting they have different transmission strategies among fruits and insects. This approach shows microbiota members of insects can be acquired from a diversity of sources and highlights the compound nature of microbiotas. Such microbial transmission events along generations should favor the evolution of mutualistic interactions and enable microbiota-mediated local adaptation of the insect host.
Socha, C., Pais, I. S., Lee, K. Z., Liu, J., Liegeois, S., Lestradet, M., Ferrandon, D. (2023). Fast drosophila enterocyte regrowth after infection involves a reverse metabolic flux driven by an amino acid transporter. iScience, 26(9):107490 PubMed ID: 37636057
Summary:
Upon exposure to a bacterial pore-forming toxin, enterocytes rapidly purge their apical cytoplasm into the gut lumen, resulting in a thin intestinal epithelium. The enterocytes regain their original shape and thickness within 16 h after the ingestion of the bacteria. This study shows that the regrowth of Drosophila enterocytes entails an inversion of metabolic fluxes from the organism back toward the intestine. A proton-assisted transporter, Arcus, was identified that is required for the reverse absorption of amino acids and the timely recovery of the intestinal epithelium. Arcus is required for a peak of amino acids appearing in the hemolymph shortly after infection. The regrowth of enterocytes involves the insulin signaling pathway and Myc. The purge decreases Myc mRNA levels, which subsequently remain at low levels in the arcus mutant. Interestingly, the action of arcus and Myc in the intestinal epithelium is not cell-autonomous, suggesting amino acid fluxes within the intestinal epithelium.
Mari, M., Voutyraki, C., Zacharioudaki, E., Delidakis, C., Filippidis, G. (2023). Lipid content evaluation of Drosophila tumour associated haemocytes through Third Harmonic Generation measurements. Journal of biophotonics, 16(12):e202300171 PubMed ID: 37643223
Summary:
Non-linear microscopy is a powerful imaging tool to examine structural properties and subcellular processes of various biological samples. The competence of Third Harmonic Generation (THG) includes the label free imaging with diffraction-limited resolution and three-dimensional visualization with negligible phototoxicity effects. In this study, THG records and quantifies the lipid content of Drosophila haemocytes, upon encountering normal or tumorigenic neural cells, in correlation with their shape or their state. The lipid accumulations of adult haemocytes were shown to be similar before and after encountering normal cells. In contrast, adult haemocytes prior to their interaction with cancer cells have a low lipid index, which increases while they are actively engaged in phagocytosis only to decrease again when haemocytes become exhausted. This dynamic change in the lipid accrual of haemocytes upon encountering tumour cells could potentially be a useful tool to assess the phagocytic capacity or activation state of tumour-associated haemocytes.

Friday, February 23rd - RNA and Transposons

Flynn, J. M., Ahmed-Braimah, Y. H., Long, M., Wing, R. A., Clark, A. G. (2023). High quality genome assemblies reveal evolutionary dynamics of repetitive DNA and structural rearrangements in the Drosophila virilis sub-group. bioRxiv, PubMed ID: 37645834
Summary:
High-quality genome assemblies across a range of non-traditional model organisms can accelerate the discovery of novel aspects of genome evolution. The Drosophila virilis group has several attributes that distinguish it from more highly studied species in the Drosophila genus, such as an unusual abundance of repetitive elements and extensive karyotype evolution, in addition to being an attractive model for speciation genetics. This study used long-read sequencing to assemble five genomes of three virilis group species and characterized sequence and structural divergence and repetitive DNA evolution. The contiguous genome assemblies allow characterization of chromosomal arrangements with ease and can facilitate analysis of inversion breakpoints. A small panel of resequenced strains was leveraged to explore the genomic pattern of divergence and polymorphism in this species and show that known demographic histories largely predicts the extent of genome-wide segregating polymorphism. It was further found that a neo-X chromosome in D. americana displays X-like levels of nucleotide diversity. Unusual repetitive elements were found to be responsible for much of the divergence in genome composition among species. Helitron-derived tandem repeats tripled in abundance on the Y chromosome in D. americana compared to D. novamexicana, accounting for most of the difference in repeat content between these sister species. Repeats with characteristics of both transposable elements and satellite DNAs expanded by three-fold, mostly in euchromatin, in both D. americana and D. novamexicana compared to D. virilis. These results represent a major advance in understanding of genome biology in this emerging model clade.
Meng, L. W., Yuan, G. R., Chen, M. L., Zheng, L. S., Dou, W., Peng, Y., Bai, W. J., Li, Z. Y., Vontas, J., Wang, J. J. (2023). Cuticular competing endogenous RNAs regulate insecticide penetration and resistance in a major agricultural pest. BMC Biol, 21(1):187 PubMed ID: 37667263
Summary:
The continuously developing pesticide resistance is a great threat to agriculture and human health. Understanding the mechanisms of insecticide resistance is a key step in dealing with the phenomenon. Insect cuticle is recently documented to delay xenobiotic penetration which breaks the previous stereotype that cuticle is useless in insecticide resistance, while the underlying mechanism remains scarce. This study found the integument contributes over 40.0% to insecticide resistance via different insecticide delivery strategies in oriental fruit fly. A negative relationship exists between cuticle thickening and insecticide penetration in resistant/susceptible, also in field strains of oriental fruit fly which is a reason for integument-mediated resistance. These investigations uncover a regulator of insecticide penetration that miR-994 mimic treatment causes cuticle thinning and increases susceptibility to malathion, whereas miR-994 inhibitor results in opposite phenotypes. The target of miR-994 is a most abundant cuticle protein (CPCFC) in resistant/susceptible integument expression profile, which possesses capability of chitin-binding and influences the cuticle thickness-mediated insecticide penetration. This analyses found an upstream transcriptional regulatory signal of miR-994 cascade, long noncoding RNA (lnc19419), that indirectly upregulates CPCFC in cuticle of the resistant strain by sponging miR-994. Thus, this study has elucidated the mechanism of cuticular competing endogenous RNAs for regulating insecticide penetration and demonstrate it also exists in field strain of oriental fruit fly. This study unveil a regulatory axis of lnc19419 ~ miR-994 ~ CPCFC on the cuticle thickness that leads to insecticide penetration resistance. These findings indicate that competing endogenous RNAs regulate insecticide resistance by modulating the cuticle thickness and provide insight into the resistance mechanism in insects.
Kim, C. J., Kim, H. H., Kim, H. K., Lee, S., Jang, D., Kim, C., Lim, D. H. (2023). MicroRNA miR-263b-5p Regulates Developmental Growth and Cell Association by Suppressing Laminin A in Drosophila. Biology, 12(8) PubMed ID: 37626982
Summary:
Basement membranes (BMs) play important roles under various physiological conditions in animals, including ecdysozoans. During development, BMs undergo alterations through diverse intrinsic and extrinsic regulatory mechanisms; however, the full complement of pathways controlling these changes remain unclear. This study found that fat body-overexpression of Drosophila miR-263b, which is highly expressed during the larval-to-pupal transition, resulted in a decrease in the overall size of the larval fat body, and ultimately, in a severe growth defect accompanied by a reduction in cell proliferation and cell size. Interestingly, it was further observed that a large proportion of the larval fat body cells were prematurely disassociated from each other. Moreover, evidence is presented that miR-263b-5p suppresses the main component of BMs, Laminin A (LanA). Through experiments using RNA interference (RNAi) of LanA, it was found that its depletion phenocopied the effects in miR-263b-overexpressing flies. Overall, these findings suggest a potential role for miR-263b in developmental growth and cell association by suppressing LanA expression in the Drosophila fat body.
Duan, Y., Xu, Y., Song, F., Tian, L., Cai, W., Li, H. (2023). Differential adaptive RNA editing signals between insects and plants revealed by a new measurement termed haplotype diversity. Biology direct, 18(1):47 PubMed ID: 37592344
Summary:
C-to-U RNA editing in plants is believed to confer its evolutionary adaptiveness by reversing unfavorable DNA mutations. This "restorative hypothesis" has not yet been tested genome-wide. In contrast, A-to-I RNA editing in insects like Drosophila and honeybee is already known to benefit the host by increasing proteomic diversity in a spatial-temporal manner (namely "diversifying hypothesis"). This study profiled the RNA editomes of multiple tissues of Arabidopsis thaliana, Drosophila melanogaster, and Apis melifera. The haplotype diversity (HD) of RNA molecules was unprecedentedly defined based on nonsynonymous editing events (recoding sites). Signals of adaptation is confirmed in Arabidopsis by observing higher frequencies and levels at nonsynonymous editing sites over synonymous sites. Compared to A-to-I recoding sites in Drosophila, the C-to-U recoding sites in Arabidopsis show significantly lower HD, presumably due to the stronger linkage between C-to-U events. It is concluded that C-to-U RNA editing in Arabidopsis is adaptive but it is not designed for diversifying the proteome like A-to-I editing in Drosophila. Instead, C-to-U recoding sites resemble DNA mutations. These observation supports the restorative hypothesis of plant C-to-U editing which claims that editing is used for fixing unfavorable genomic sequences.
Zhang, Z., Bae, B., Cuddleston, W. H., Miura, P. (2023). Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing. Nat Commun, 14(1):5506 PubMed ID: 37679364
Summary:
Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. This study investigated the coordination of cassette exon (CE) splicing and APA in Drosophila using a targeted long-read sequencing approach called Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to quantify inclusion of alternative exons in connection with alternative 3' ends. Using PL-Seq, genes were identified that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.
Venkei, Z. G., Gainetdinov, I., Bagci, A., Starostik, M. R., Choi, C. P., Fingerhut, J. M., Chen, P., Balsara, C., Whitfield, T. W., Bell, G. W., Feng, S., Jacobsen, S. E., Aravin, A. A., Kim, J. K., Zamore, P. D., Yamashita, Y. M. (2023). A maternally programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila. Nat Cell Biol, 25(10):1495-1505 PubMed ID: 37723298
Summary:
In animals, PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In Drosophila and other species with a maternally specified germline, piRNAs deposited in the egg initiate piRNA biogenesis in the progeny. However, Y chromosome loci cannot participate in such a chain of intergenerational inheritance. How then can the biogenesis of Y-linked piRNAs be initiated? Using Suppressor of Stellate (Su(Ste)), a Y-linked Drosophila melanogaster piRNA locus as a model, this study showed that Su(Ste) piRNAs are made in the early male germline via 5'-to-3' phased piRNA biogenesis initiated by maternally deposited 1360/Hoppel transposon piRNAs. Notably, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, this study uncovers a developmentally programmed, intergenerational mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus.

Thursday, February 22nd - Gonads

Tu, R., Tang, X. A., Xu, R., Ping, Z., Yu, Z., Xie, T. (2023). Gap junction-transported cAMP from the niche controls stem cell progeny differentiation. Proc Natl Acad Sci U S A, 120(35):e2304168120 PubMed ID: 37603749
Summary:
The niche has been shown to control stem cell self-renewal in different tissue types and organisms. Recently, a separate niche has been proposed to control stem cell progeny differentiation, called the differentiation niche. However, it remains poorly understood whether and how the differentiation niche directly signals to stem cell progeny to control their differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells contribute to two separate niche compartments for controlling both germline stem cell (GSC) self-renewal and progeny differentiation. This study shows that IGS cells express Inx2 protein, which forms gap junctions (GJs) with germline-specific Zpg protein to control stepwise GSC lineage development, including GSC self-renewal, germline cyst formation, meiotic double-strand DNA break formation, and oocyte specification. Germline-specific Zpg and IGS-specific Inx2 knockdowns cause similar defects in stepwise GSC development. Additionally, secondary messenger cAMP is transported from IGS cells to GSCs and their progeny via GJs to activate PKA signaling for controlling stepwise GSC development. Therefore, this study demonstrates that the niche directly controls GSC progeny differentiation via the GJ-cAMP-PKA signaling axis, which provides important insights into niche control of stem cell differentiation and highlights the importance of GJ-transported cAMP in tissue regeneration. This may represent a general strategy for the niche to control adult stem cell development in various tissue types and organisms since GJs and cAMP are widely distributed.
Eichler, C. E., Li, H., Grunberg, M. E., Gavis, E. R. (2023). Localization of oskar mRNA by agglomeration in ribonucleoprotein granules. PLoS Genet, 19(8):e1010877. PubMed ID: 37624861
Summary:
Localization of oskar mRNA to the posterior of the Drosophila oocyte is essential for abdominal patterning and germline development. oskar localization is a multi-step process involving temporally and mechanistically distinct transport modes. Numerous cis-acting elements and trans-acting factors have been identified that mediate earlier motor-dependent transport steps leading to an initial accumulation of oskar at the posterior. Little is known, however, about the requirements for the later localization phase, which depends on cytoplasmic flows and results in the accumulation of large oskar ribonucleoprotein granules, called founder granules, by the end of oogenesis. Using super-resolution microscopy, this study showed that founder granules are agglomerates of smaller oskar transport particles. In contrast to the earlier kinesin-dependent oskar transport, late-phase localization depends on the sequence as well as on the structure of the spliced oskar localization element (SOLE), but not on the adjacent exon junction complex deposition. Late-phase localization also requires the oskar 3' untranslated region (3' UTR), which targets oskar to founder granules. Together, these results show that 3' UTR-mediated targeting together with SOLE-dependent agglomeration leads to accumulation of oskar in large founder granules at the posterior of the oocyte during late stages of oogenesis. In light of previous work showing that oskar transport particles are solid-like condensates, these findings indicate that founder granules form by a process distinct from that of well-characterized ribonucleoprotein granules like germ granules, P bodies, and stress granules. Additionally, they illustrate how an individual mRNA can be adapted to exploit different localization mechanisms depending on the cellular context.
Benner, L., Muron, S., Oliver, B. (2023). Female germline expression of OVO transcription factor bridges Drosophila generations. bioRxiv, PubMed ID: 37662231
Summary:
OVO is required for karyotypically female germ cell viability but has no known function in the male germline in Drosophila. ovo is autoregulated by two antagonistic isoforms, OVO-A and OVO-B. All ovo- alleles were created as partial revertants of the antimorphic ovo(D1) allele. Creation of new targeted alleles in an ovo+ background indicated that disrupting the germline-specific exon extension of ovo-B leads to an arrested egg chamber phenotype, rather than germ cell death. RNA-seq analysis, including >1K full length cDNAs, indicates that ovo utilizes a number of unannotated splice variations in the extended exon and a minor population of ovo-B transcripts utilizes an alternative splice. This indicates that classical ovo alleles such as ovoD1rv23, are not truly null for ovo, and are likely to be weak antimorphs. To generate bonafide nulls, the ovo-A and ovo-B promoters were deleted showing that only ovo-B is required for female germ cell viability and there is an early and polyphasic developmental requirement for ovo-B in the female germline. To visualize OVO expression and localization, endogenously tagged ovo was generated, and nuclear OVO was found in all differentiating female germ cells throughout oogenesis in adults. This study also found that OVO is maternally deposited into the embryo, where it showed nuclear localization in newly formed pole cells. Maternal OVO persisted in embryonic germ cells until zygotic OVO expression was detectable, suggesting that there is continuous nuclear OVO expression in the female germline in the transition from one generation to the next.
Emelyanov, A. V., Barcenilla-Merino, D., Loppin, B., Fyodorov, D. V. (2023). APOLLO, a testis-specific Drosophila ortholog of importin-4, mediates the loading of protamine-like protein Mst77F into sperm chromatin. J Biol Chem, 299(10):105212 PubMed ID: 37660905
Summary:
DNA in sperm is packed with small, charged proteins termed SNBPs (sperm nuclear basic proteins), including mammalian and Drosophila protamines. During spermiogenesis, somatic-type chromatin is taken apart and replaced with sperm chromatin in a multistep process leading to an extraordinary condensation of the genome. During fertilization, the ova face a similarly challenging task of SNBP eviction and reassembly of nucleosome-based chromatin. Despite its importance for the animal life cycle, sperm chromatin metabolism, including the biochemical machinery mediating the mutual replacement of histones and SNBPs, remains poorly studied. In Drosophila, Mst77F is one of the first SNBPs loaded into the spermatid nuclei. It persists in mature spermatozoa and is essential for sperm compaction and male fertility. By using in vitro biochemical assays this study identified chaperones that can mediate the eviction and loading of Mst77F on DNA, thus facilitating the interconversions of chromatin forms in the male gamete. Unlike NAP1 and TAP/p32 chaperones that disassemble Mst77F-DNA complexes, ARTEMIS and APOLLO, orthologs of mammalian importin-4 (IPO4), mediate the deposition of Mst77F on DNA or oligonucleosome templates, accompanied by the dissociation of histone-DNA complexes. In vivo, a mutation of testis-specific Apollo brings about a defect of Mst77F loading, abnormal sperm morphology, and male infertility. This study has identified IPO4 ortholog APOLLO as a critical component of sperm chromatin assembly apparatus in Drosophila. In addition to recognized roles in protein traffic, a nuclear transport receptor can function directly in chromatin remodeling as a dual, histone- and SNBP-specific, chaperone.
Lu, W., Lakonishok, M., Gelfand, V. I. (2023). The dynamic duo of microtubule polymerase Mini spindles/XMAP215 and cytoplasmic dynein is essential for maintaining Drosophila oocyte fate. Proc Natl Acad Sci U S A, 120(39):e2303376120 PubMed ID: 37722034
Summary:
In many species, only one oocyte is specified among a group of interconnected germline sister cells. In Drosophila melanogaster, 16 interconnected cells form a germline cyst, where one cell differentiates into an oocyte, while the rest become nurse cells that supply the oocyte with mRNAs, proteins, and organelles through intercellular cytoplasmic bridges named ring canals via microtubule-based transport. This study finds that a microtubule polymerase Mini spindles (Msps), the Drosophila homolog of XMAP215, is essential for maintenance of the oocyte specification. mRNA encoding Msps is transported and concentrated in the oocyte by dynein-dependent transport along microtubules. Translated Msps stimulates microtubule polymerization in the oocyte, causing more microtubule plus ends to grow from the oocyte through the ring canals into nurse cells, further enhancing nurse cell-to-oocyte transport by dynein. Knockdown of msps blocks the oocyte growth and causes gradual loss of oocyte determinants. Thus, the Msps-dynein duo creates a positive feedback loop, ensuring oocyte fate maintenance by promoting high microtubule polymerization activity in the oocyte, and enhancing dynein-dependent nurse cell-to-oocyte transport.
Kakino, K., Mon, H., Ebihara, T., Hino, M., Masuda, A., Lee, J. M., Kusakabe, T. (2023). Comprehensive Transcriptome Analysis in the Testis of the Silkworm, Bombyx mori. Insects, 14(8) PubMed ID: 37623394
Summary:
Spermatogenesis is an important process in reproduction and is conserved across species, but in Bombyx mori, it shows peculiarities, such as the maintenance of spermatogonia by apical cells and fertilization by dimorphic spermatozoa. This study attempted to characterize the genes expressed in the testis of B. mori, focusing on aspects of expression patterns and gene function by transcriptome comparisons between different tissues, internal testis regions, and Drosophila melanogaster. The transcriptome analysis of 12 tissues of B. mori, including those of testis, revealed the widespread gene expression of 20,962 genes and 1705 testis-specific genes. A comparative analysis of the stem region (SR) and differentiated regions (DR) of the testis revealed 4554 and 3980 specific-enriched genes, respectively. In addition, comparisons with D. melanogaster testis transcriptome revealed homologs of 1204 SR and 389 DR specific-enriched genes that were similarly expressed in equivalent regions of Drosophila testis. Moreover, gene ontology (GO) enrichment analysis was performed for SR-specific enriched genes and DR-specific enriched genes, and the GO terms of several biological processes were enriched, confirming previous findings. This study advances understanding of spermatogenesis in B. mori and provides an important basis for future research, filling a knowledge gap between fly and mammalian studies.

Wednesday, February 21st - Stress

Wen, D., Xie, J., Yuan, Y., Shen, L., Yang, Y., Chen, W. (2023). The endogenous antioxidant ability of royal jelly in Drosophila is independent of Keap1/Nrf2 by activating oxidoreductase activity. Insect Sci, PubMed ID: 37632209
Summary:
Royal jelly (RJ) is a biologically active substance secreted by the hypopharyngeal and mandibular glands of worker honeybees. It is widely claimed that RJ reduces oxidative stress. However, the antioxidant activity of RJ has mostly been determined by in vitro chemical detection methods or by external administration drugs that cause oxidative stress. Whether RJ can clear the endogenous production of reactive oxygen species (ROS) in cells remains largely unknown. This study systematically investigated the antioxidant properties of RJ using several endogenous oxidative stress models of Drosophila. RJ was found to enhanced sleep quality of aging Drosophila, which is decreased due to an increase of oxidative damage with age. RJ supplementation improved survival and suppressed ROS levels in gut cells of flies upon exposure to hydrogen peroxide or to the neurotoxic agent paraquat. Moreover, RJ supplementation moderated levels of ROS in endogenous gut cells and extended lifespan after exposure of flies to heat stress. Sleep deprivation leads to accumulation of ROS in the gut cells, and RJ attenuated the consequences of oxidative stress caused by sleep loss and prolonged lifespan. Mechanistically, RJ prevented cell oxidative damage caused by heat stress or sleep deprivation, with the antioxidant activity in vivo independent of Keap1/Nrf2 signaling. RJ supplementation activated oxidoreductase activity in the guts of flies, suggesting its ability to inhibit endogenous oxidative stress and maintain health, possibly in humans.
Wang, X., Zhou, P., Zhang, Z., Huang, Q., Chen, X., Ji, L., Cheng, X., Shi, Y., Yu, S., Tang, J., Sun, C., Zhao, X., Yu, J. (2023). A Drosophila model of gestational antimony exposure uncovers growth and developmental disorders caused by disrupting oxidative stress homeostasis. Free radical biology & medicine, 208:418-429 PubMed ID: 37666440
Summary:
The toxic heavy metal antimony (Sb) is ubiquitous in our daily lives. Various models have shown that Sb induces neuronal and reproductive toxicity. However, little is known about the developmental toxicity of Sb exposure during gestation and the underlying mechanisms. To study its effects on growth and development, Drosophila stages from eggs to pupae were exposed to different Sb concentrations (0, 0.3, 0.6 and 1.2 mg/mL Sb); RNA sequencing was used to identify the underlying mechanism. The model revealed that prenatal Sb exposure significantly reduced larval body size and weight, the pupation and eclosion rates, and the number of flies at all stages. With 1.2 mg/mL Sb exposure in 3rd instar larvae, 484 genes were upregulated and 694 downregulated compared to controls. Biological analysis showed that the disrupted transcripts were related to the oxidative stress pathway, as verified by reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and glutathione (GSH) intervention experiments. Sb exposure induced oxidative stress imbalance could be rectified by chelation and antioxidant effects of NAC/GSH. The Drosophila Schneider 2 (S2) model further demonstrated that NAC and GSH greatly ameliorated cell death induced by Sb exposure. In conclusion, gestational Sb exposure disrupted oxidative stress homeostasis, thereby impairing growth and development.
Hwang, R. D., Lu, Y. N., Tang, Q., Periz, G., Park, G., Li, X., Liu, Y., Zhang, T., Wang, J. (2023). DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment. bioRxiv, PubMed ID: 37745492
Summary:
Proteotoxic stress impairs cellular homeostasis and underlies the pathogeneses of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. This study reports a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of the proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in tissues from ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.
Reyes-Ramirez, A., Belgaidi, Z., Gibert, P., Pommier, T., Siberchicot, A., Mouton, L., Desouhant, E. (2023). Larval density in the invasive Drosophila suzukii: Immediate and delayed effects on life-history traits. Ecology and evolution, 13(8):e10433 PubMed ID: 37636864
Summary:
The effects of density are key in determining population dynamics, since they can positively or negatively affect the fitness of individuals. These effects have great relevance for polyphagous insects for which immature stages develop within a single site of finite feeding resources. Drosophila suzukii is a crop pest that induces severe economic losses for agricultural production; however, little is known about the effects of density on its life-history traits. This study, (i) investigated the egg distribution resulting from females' egg-laying strategy and (ii) tested the immediate (on immatures) and delayed (on adults) effects of larval density on emergence rate, development time, potential fecundity, and adult size. The density used varied in a range between 1 and 50 larvae. 44.27% of the blueberries used for the oviposition assay contained between 1 and 11 eggs in aggregates. The high experimental density (50 larvae) has no immediate effect in the emergence rate but has effect on larval developmental time. This trait was involved in a trade-off with adult life-history traits: The time of larval development was reduced as larval density increased, but smaller and less fertile females were produced. These results clearly highlight the consequences of larval crowding on the juveniles and adults of this fly.
Shui, K., Wang, C., Zhang, X., Ma, S., Li, Q., Ning, W., Zhang, W., Chen, M., Peng, D., Hu, H., Fang, Z., Guo, A., Gao, G., Ye, M., Zhang, L. and Xue, Y. (2023). Small-sample learning reveals propionylation in determining global protein homeostasis. Nat Commun 14(1): 2813. PubMed ID: 37198164
Summary:
Proteostasis is fundamental for maintaining organismal health. However, the mechanisms underlying its dynamic regulation and how its disruptions lead to diseases are largely unclear. This study conducted in-depth propionylomic profiling in Drosophila, and develop a small-sample learning framework to prioritize the propionylation at lysine 17 of H2B (H2BK17pr) to be functionally important. Mutating H2BK17 which eliminates propionylation leads to elevated total protein level in vivo. Further analyses reveal that H2BK17pr modulates the expression of 14.7-16.3% of genes in the proteostasis network, and determines global protein level by regulating the expression of genes involved in the ubiquitin-proteasome system. In addition, H2BK17pr exhibits daily oscillation, mediating the influences of feeding/fasting cycles to drive rhythmic expression of proteasomal genes. This study not only reveals a role of lysine propionylation in regulating proteostasis, but also implements a generally applicable method which can be extended to other issues with little prior knowledge.
Xu, X., An, H., Wu, C., Sang, R., Wu, L., Lou, Y., Yang, X. and Xi, Y. (2023). HR repair pathway plays a crucial role in maintaining neural stem cell fate under irradiation stress. Life Sci Alliance 6(8). PubMed ID: 37197982
Summary:
Environmental stress can cause mutation or genomic instability in stem cells which, in some cases, leads to tumorigenesis. Mechanisms to monitor and eliminate these mutant stem cells remain elusive. Using the Drosophila larval brain as a model, this study shows that X-ray irradiation (IR) at the early larval stage leads to accumulation of nuclear Prospero (Pros), resulting in premature differentiation of neural stem cells (neuroblasts, NBs). Through NB-specific RNAi screenings, the Mre11-Rad50-Nbs1 complex and the homologous recombination (HR) repair pathway, rather than non-homologous end-joining pathway that plays, a dominant role in the maintenance of NBs under IR stress. The DNA damage sensor ATR/mei-41 is shown to act to prevent IR-induced nuclear Pros in a WRNexo-dependent manner. The accumulation of nuclear Pros in NBs under IR stress, leads to NB cell fate termination, rather than resulting in mutant cell proliferation. This study reveals an emerging mechanism for the HR repair pathway in maintaining neural stem cell fate under irradiation stress.

Tuesday, February 20th - Disease Models

Clabough, E. B. D., Aspili, C., Fussy, W. S., ...., Venton, B. J., Hayes, D., Sipe, C. W. (2023). Huntingtin Plays a Role in the Physiological Response to Ethanol in Drosophila. Journal of Huntington's disease, 12(3):241-252 PubMed ID: 37661891
Summary:
Huntingtin (htt) protein is an essential regulator of nervous system function through its various neuroprotective and pro-survival functions, and loss of wild-type htt function is implicated in the etiology of Huntington's disease. While its pathological role is typically understood as a toxic gain-of-function, some neuronal phenotypes also result from htt loss. Therefore, it is important to understand possible roles for htt in other physiological circumstances. To elucidate the role of htt in the context of ethanol exposure, this study investigated how loss of htt impacts behavioral and physiological responses to ethanol in Drosophila. Flies lacking htt were tested for ethanol sensitivity and tolerance, preference for ethanol using capillary feeder assays, and recovery of mobility after intoxication. Levels of dopamine neurotransmitter and numbers of dopaminergic cells in brains lacking dhtt were also measured. dhtt-null flies were found to be both less sensitive and more tolerant to ethanol exposure in adulthood. Moreover, flies lacking dhtt are more averse to alcohol than controls, and they recover mobility faster following acute ethanol intoxication. dhtt was shown to mediate these effects at least in part through the dopaminergic system, as dhtt is required to maintain normal levels of dopamine in the brain and normal numbers of dopaminergic cells in the adult protocerebrum. These results demonstrate that htt regulates the physiological response to ethanol and indicate a novel neuroprotective role for htt in the dopaminergic system, raising the possibility that it may be involved more generally in the response to toxic stimuli.
Onkar, A., Sheshadri, D., Rai, A., Gupta, A. K., Gupta, N., Ganesh, S. (2023). Increase in brain glycogen levels ameliorates Huntington's disease phenotype and rescues neurodegeneration in Drosophila. Disease models & mechanisms, 16(10) PubMed ID: 37681238
Summary:
Under normal physiological conditions, the mammalian brain contains very little glycogen, most of which is stored in astrocytes. However, the aging brain and the subareas of the brain in patients with neurodegenerative disorders tend to accumulate glycogen, the cause and significance of which remain largely unexplored. Using cellular models, a neuroprotective role for neuronal glycogen and glycogen synthase has been recently demonstrated in the context of Huntington's disease. To gain insight into the role of brain glycogen in regulating proteotoxicity, a Drosophila model of Huntington's disease was used, in which glycogen synthase is either knocked down or expressed ectopically. Enhancing glycogen synthesis in the brains of flies with Huntington's disease decreased mutant Huntingtin aggregation and reduced oxidative stress by activating auto-lysosomal functions. Further, overexpression of glycogen synthase in the brain rescues photoreceptor degeneration, improves locomotor deficits and increases fitness traits in this Huntington's disease model. This study, thus, provides in vivo evidence for the neuroprotective functions of glycogen synthase and glycogen in neurodegenerative conditions, and their role in the neuronal autophagy process.
Zhang, F., Wang, L., Jin, J., Pang, Y., Shi, H., Fang, Z., Wang, H., Du, Y., Hu, Y., Zhang, Y., Ding, X., Zhu, Z. (2023). Insights into the genetic influences of the microbiota on the life span of a host. Frontiers in microbiology, 14:1138979 PubMed ID: 37601381
Summary:
Escherichia coli (E. coli) mutant strains have been reported to extend the life span of Caenorhabditis elegans (C. elegans). However, the specific mechanisms through which the genes and pathways affect aging are not yet clear. This study fed Drosophila melanogaster (fruit fly) various E. coli single-gene knockout strains to screen mutant strains with an extended lifespan. The results showed that D. melanogaster fed with E. coli purE had the longest mean lifespan, which was verified by C. elegans. RNA-sequencing and analysis of C. elegans fed with E. coli purE (a single-gene knockout mutant) was conducted to further explore the underlying molecular mechanism. Differential gene expression (DGE) analysis, enrichment analysis, and gene set enrichment analysis (GSEA) were ised to screen vital genes and modules with significant changes in overall expression. The results suggest that E. coli mutant strains may affect the host lifespan by regulating the protein synthesis rate (cfz-2) and ATP level (catp-4). To conclude, this study could provide new insights into the genetic influences of the microbiota on the life span of a host and a basis for developing anti-aging probiotics and drugs.
Tandon, S., Sarkar, S. (2023). Glutamine stimulates the S6K/4E-BP branch of insulin signalling pathway to mitigate human poly(Q) disorders in Drosophila disease models. Nutritional neuroscience:1-12 PubMed ID: 37658796
Summary:
Since, the S6K/4E-BP sub-pathway can be stimulated by various amino acids; this study examine if oral feeding of amino acids delivers rescue against human poly(Q) toxicity in Drosophila. Drosophila models of two different poly(Q) disorders were used to test this hypothesis. Glutamine was fed to the test flies orally mixed in the food. Control and treated flies were then tested for different parameters, such as formation of poly(Q) aggregates and neurodegeneration, to evaluate glutamine's proficiency in mitigating poly(Q) neurotoxicity. This study study, for the first time, reports that glutamine feeding stimulates the growth promoting S6K/4E-BP branch of insulin signalling pathway and restricts pathogenesis of poly(Q) disorders in Drosophila disease models. It is noted that glutamine treatment restricts the formation of neurotoxic poly(Q) aggregates and minimises neuronal deaths. Further, glutamine treatment re-establishes the chromatin architecture by improving the histone acetylation which is otherwise compromised in poly(Q) expressing neuronal cells. Since, the insulin signalling pathway as well as mechanism of action of glutamine are fairly conserved between human and Drosophila, this finding strongly suggests that glutamine holds immense potential to be developed as an intervention therapy against the incurable human poly(Q) disorders.
Ciampelli, C., Galleri, G., Puggioni, S., Fais, M., Iannotta, L., Galioto, M., Becciu, M., Greggio, E., Bernardoni, R., Crosio, C., Iaccarino, C. (2023). Inhibition of the Exocyst Complex Attenuates the LRRK2 Pathological Effects. Int J Mol Sci, 24(16) PubMed ID: 37628835
Summary:
Pathological mutations in leucine-rich repeat kinase 2 (LRRK2) gene are the major genetic cause of Parkinson's disease (PD). Multiple lines of evidence link LRRK2 to the control of vesicle dynamics through phosphorylation of a subset of RAB proteins. However, the molecular mechanisms underlying these processes are not fully elucidated. Previous work demonstrated that LRRK2 increases the exocyst complex assembly by Sec8 interaction, one of the eight members of the exocyst complex, and that Sec8 over-expression mitigates the LRRK2 pathological effect in PC12 cells. This analysis was extended using LRRK2 drosophila models and shows that the LRRK2-dependent exocyst complex assembly increase is downstream of RAB phosphorylation. Moreover, exocyst complex inhibition rescues mutant LRRK2 pathogenic phenotype in cellular and drosophila models. Finally, prolonged exocyst inhibition leads to a significant reduction in the LRRK2 protein level, overall supporting the role of the exocyst complex in the LRRK2 pathway. Taken together, this study suggests that modulation of the exocyst complex may represent a novel therapeutic target for PD.
Kim, H. S., Parker, D. J., Hardiman, M. M., Munkacsy, E., Jiang, N., Rogers, A. N., Bai, Y., Brent, C., Mobley, J. A., Austad, S. N., Pickering, A. M. (2023). Early-adulthood spike in protein translation drives aging via juvenile hormone/germline signaling. Nat Commun, 14(1):5021 PubMed ID: 37596266
Summary:
Protein translation (PT) declines with age in invertebrates, rodents, and humans. It has been assumed that elevated PT at young ages is beneficial to health and PT ends up dropping as a passive byproduct of aging. In Drosophila, this study shows that a transient elevation in PT during early-adulthood exerts long-lasting negative impacts on aging trajectories and proteostasis in later-life. Blocking the early-life PT elevation robustly improves life-/health-span and prevents age-related protein aggregation, whereas transiently inducing an early-life PT surge in long-lived fly strains abolishes their longevity/proteostasis benefits. The early-life PT elevation triggers proteostatic dysfunction, silences stress responses, and drives age-related functional decline via juvenile hormone-lipid transfer protein axis and germline signaling. These findings suggest that PT is adaptively suppressed after early-adulthood, alleviating later-life proteostatic burden, slowing down age-related functional decline, and improving lifespan. This work provides a theoretical framework for understanding how lifetime PT dynamics shape future aging trajectories.

Friday, February 16th - Evolution

Davies, N., Janicke, T., Morrow, E. H. (2023). Evidence for stronger sexual selection in males than in females using an adapted method of Bateman's classic study of Drosophila melanogaster. Evolution, 77(11):2420-2430 PubMed ID: 37624087
Summary:
Bateman's principles, originally a test of Darwin's theoretical ideas, have since become fundamental to sexual selection theory and vital to contextualizing the role of anisogamy in sex differences of precopulatory sexual selection. Despite this, Bateman's principles have received substantial criticism, and researchers have highlighted both statistical and methodological errors, suggesting that Bateman's original experiment contains too much sampling bias for there to be any evidence of sexual selection. This study uses Bateman's original method as a template, accounting for two fundamental flaws in his original experiments, (a) viability effects and (b) a lack of mating behavior observation. Experimental populations of Drosophila melanogaster consisted of wild-type focal individuals and nonfocal individuals established by backcrossing the brown eye (bw-) eye-color marker-thereby avoiding viability effects. Mating assays included direct observation of mating behavior and total number of offspring, to obtain measures of mating success, reproductive success, and standardized variance measures based on Bateman's principles. The results provide observational support for Bateman's principles, particularly that (a) males had significantly more variation in number of mates compared with females and (b) males had significantly more individual variation in total number of offspring. A significantly steeper Bateman gradient was found for males compared to females, suggesting that sexual selection is operating more intensely in males. However, female remating was limited, providing the opportunity for future study to further explore female reproductive success in correlation with higher levels of remating.
Lankinen, P., Kastally, C., Hoikkala, A. (2023). Clinal variation in the temperature and photoperiodic control of reproductive diapause in Drosophila montana females. J Insect Physiol, 150:104556 PubMed ID: 37598869
Summary:
Insect adaptation to climatic conditions at different latitudes has required changes in life-history traits linked with survival and reproduction. Several species, including Drosophila montana, show robust latitudinal variation in the critical day length (CDL), below which more than half of the emerging females enter reproductive diapause at a given temperature. This study used a novel approach to find out whether D. montana also shows latitudinal variation in the critical temperature (CTemp), above which the photoperiodic regulation of diapause is disturbed so that the females develop ovaries in daylengths that are far below their CDL. CTemp was estimated for 53 strains from different latitudes on 3 continents after measuring their diapause proportions at a range of temperatures in 12 h daylength (for 29 of the strains also in continuous darkness). In 12 h daylength, CTemp increased towards high latitudes alongside an increase in CDL, and in 3 high-latitude strains diapause proportion exceeded 50% in all temperatures. In continuous darkness, the diapause proportion was above 50% in the lowest temperature(s) in only 9 strains, all of which came from high latitudes. In the second part of the study, changes were measured in CTemp and CDL in a selection experiment favouring reproduction in short daylength (photoperiodic selection) and by exercising selection for females that reproduce in LD12:12 at low temperature (photoperiodic and temperature selection). In both experiments selection induced parallel changes in CDL and CTemp, confirming correlations seen between these traits along latitudinal clines. Overall, these findings suggest that selection towards strong photoperiodic diapause and long CDL at high latitudes has decreased the dependency of D. montana diapause on environmental temperature. Accordingly, the prevalence and timing of the diapause of D. montana is likely to be less vulnerable to climate warming in high- than low-latitude populations.
Ma, L., Zheng, C., Xu, S., Xu, Y., Song, F., Tian, L., Cai, W., Li, H., Duan, Y. (2023). A full repertoire of Hemiptera genomes reveals a multi-step evolutionary trajectory of auto-RNA editing site in insect Adar gene. RNA Biol, 20(1):703-714 PubMed ID: 37676051
Summary:
Adenosine-to-inosine (A-to-I) RNA editing, mediated by metazoan ADAR enzymes, is a prevalent post-transcriptional modification that diversifies the proteome and promotes adaptive evolution of organisms. The Drosophila Adar gene has an auto-recoding site (termed S>G site) that forms a negative-feedback loop and stabilizes the global editing activity. However, the evolutionary trajectory of Adar S>G site in many other insects remains largely unknown, preventing a deeper understanding on the significance of this auto-editing mechanism. This study retrieved the well-annotated genomes of 375 arthropod species including the five major insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera and Hemiptera) and several outgroup species. Comparative genomic analysis was performed on the Adar auto-recoding S>G site. The ancestral state of insect S>G site was found to be an uneditable serine codon (unSer); this state was largely maintained in Hymenoptera. The editable serine codon (edSer) appeared in the common ancestor of Lepidoptera, Diptera and Coleoptera and was almost fixed in the three orders. Interestingly, Hemiptera species possessed comparable numbers of unSer and edSer codons, and a few 'intermediate codons', demonstrating a multi-step evolutionary trace from unSer-to-edSer with non-synchronized mutations at three codon positions. It is argued that the evolution of Adar S>G site is the best genomic evidence supporting the 'proteomic diversifying hypothesis' of RNA editing. This work deepens our understanding on the evolutionary significance of Adar auto-recoding site which stabilizes the global editing activity and controls transcriptomic diversity.
Blunk, S., Garcia-Verdugo, H., O'Sullivan, S., Camp, J., Haines, M., Coalter, T., Williams, T. A., Nagy, L. M. (2023). Functional Divergence of the Tribolium castaneum engrailed and invected Paralogs. Insects, 14(8) PubMed ID: 37623401
Summary:
engrailed (en) and invected (inv) encode paralogous transcription factors found as a closely linked tandem duplication within holometabolous insects. Drosophila en mutants segment normally, then fail to maintain their segments. Loss of Drosophila inv is viable, while loss of both genes results in asegmental larvae. Surprisingly, the knockdown of Oncopeltus inv can result in the loss or fusion of the entire abdomen and en knockdowns in Tribolium show variable degrees of segmental loss. The consequence of losing or knocking down both paralogs on embryogenesis has not been studied beyond Drosophila. To further investigate the relative functions of each paralog and the mechanism behind the segmental loss, Tribolium double and single knockdowns of en and inv were analyzed. The most common cuticular phenotype of the double knockdowns was small, limbless, and open dorsally, with all but a single, segmentally iterated row of bristles. Less severe knockdowns had fused segments and reduced appendages. The Tribolium paralogs appear to act synergistically: the knockdown of either Tribolium gene alone was typically less severe, with all limbs present, whereas the most extreme single knockdowns mimic the most severe double knockdown phenotype. Morphological abnormalities unique to either single gene knockdown were not found. inv expression was not affected in the Tribolium en knockdowns, but hh expression was unexpectedly increased midway through development. Thus, while the segmental expression of en/inv is broadly conserved within insects, the functions of en and inv are evolving independently in different lineages.
Sproul, J. S., Hotaling, S., Heckenhauer, J. ..., Pauls, S. U., Frandsen, P. B. (2023). Analyses of 600+ insect genomes reveal repetitive element dynamics and highlight biodiversity-scale repeat annotation challenges. Genome research, 33(10):1708-1717 PubMed ID: 37739812
Summary:
Repetitive elements (REs) are integral to the composition, structure, and function of eukaryotic genomes, yet remain understudied in most taxonomic groups. This study investigated REs across 601 insect species and report wide variation in RE dynamics across groups. Analysis of associations between REs and protein-coding genes revealed dynamic evolution at the interface between REs and coding regions across insects, including notably elevated RE-gene associations in lineages with abundant long interspersed nuclear elements (LINEs). This study leveraged this large, empirical data set to quantify impacts of long-read technology on RE detection and investigate fundamental challenges to RE annotation in diverse groups. In long-read assemblies, ∼36% more REs than in short-read assemblies, with long terminal repeats (LTRs) showing 162% increased detection, whereas DNA transposons and LINEs showed less respective technology-related bias. In most insect lineages, 25%-85% of repetitive sequences were "unclassified" following automated annotation, compared with only ∼13% in Drosophila species. Although the diversity of available insect genomes has rapidly expanded, we show the rate of community contributions to RE databases has not kept pace, preventing efficient annotation and high-resolution study of REs in most groups. This study highlight the tremendous opportunity and need for the biodiversity genomics field to embrace REs and suggest collective steps for making progress toward this goal.
Wang, Z., Pu, J., Richards, C., Giannetti, E., Cong, H., Lin, Z., Chung, H. (2023). Evolution of a fatty acyl-CoA elongase underlies desert adaptation in Drosophila. Sci Adv, 9(35):eadg0328 PubMed ID: 37647401
Summary:
Traits that allow species to survive in extreme environments such as hot-arid deserts have independently evolved in multiple taxa. However, the genetic and evolutionary mechanisms underlying these traits have thus far not been elucidated. This study shows that Drosophila mojavensis, a desert-adapted fruit fly species, has evolved high desiccation resistance by producing long-chain methyl-branched cuticular hydrocarbons (mbCHCs) that contribute to a cuticular lipid layer reducing water loss. The ability to synthesize these longer mbCHCs is due to evolutionary changes in a fatty acyl-CoA elongase (mElo). mElo knockout in D. mojavensis led to loss of longer mbCHCs and reduction of desiccation resistance at high temperatures but did not affect mortality at either high temperatures or desiccating conditions individually. Phylogenetic analysis showed that mElo is a Drosophila-specific gene, suggesting that while the physiological mechanisms underlying desert adaptation may be similar between species, the genes involved in these mechanisms may be species or lineage specific.

Thursday, February 15th - Synapse and Vesicles

Bertin, F., Jara-Wilde, J., Auer, B., Köhler-Solis, A., Gonzalez-Silva, C., Thomas, U., Sierralta, J. (2023). Drosophila Atlastin regulates synaptic vesicle mobilization independent of bone morphogenetic protein signaling. Biological research, 56(1):49 PubMed ID: 37710314
Summary:
The endoplasmic reticulum (ER). contacts endosomes in all parts of a motor neuron, including the axon and presynaptic terminal, to move structural proteins, proteins that send signals, and lipids over long distances. Atlastin (Atl), a large GTPase, is required for membrane fusion and the structural dynamics of the ER tubules. Atl mutations are the second most common cause of Hereditary Spastic Paraplegia (HSP), which causes spasticity in both sexes' lower extremities. Through an unknown mechanism, Atl mutations stimulate the BMP (bone morphogenetic protein) pathway in vertebrates and Drosophila. Synaptic defects are caused by atl mutations, which affect the abundance and distribution of synaptic vesicles (SV) in the bouton. It is hypothesized that BMP signaling, does not cause Atl-dependent SV abnormalities in Drosophila. This study shows that atl knockdown in motor neurons (Atl-KD) increases synaptic and satellite boutons in the same way that constitutively activating the BMP-receptor Tkv (thick veins) (Tkv-CA) increases the bouton number. The SV proteins Cysteine string protein (CSP) and vesicular glutamate transporter are reduced in Atl-KD and Tkv-CA larvae. Reducing the activity of the BMP receptor Wishful thinking (wit) can rescue both phenotypes. Unlike Tkv-CA larvae, Atl-KD larvae display altered activity-dependent distributions of CSP staining. Furthermore, Atl-KD larvae display an increased FM 1-43 unload than Control and Tkv-CA larvae. As decreasing wit function does not reduce the phenotype, the hypothesis that BMP signaling is not involved is supported. It was also found that Rab11/CSP colocalization increased in Atl-KD larvae, which supports the concept that late recycling endosomes regulate SV movements. These findings reveal that Atl modulates neurotransmitter release in motor neurons via SV distribution independently of BMP signaling, which could explain the observed SV accumulation and synaptic dysfunction. The data suggest that Atl is involved in membrane traffic as well as formation and/or recycling of the late endosome.
Yin, P., Cai, Y., Cui, T., Berg, A. J., Wang, T., Morency, D. T., Paganelli, P. M., Lok, C., Xue, Y., Vicini, S., Wang, T. (2023). Glial Sphingosine-Mediated Epigenetic Regulation Stabilizes Synaptic Function in Drosophila Models of Alzheimer's Disease. J Neurosci, 43(42):6954-6971 PubMed ID: 37669862
Summary:
Destabilization of neural activity caused by failures of homeostatic regulation has been hypothesized to drive the progression of Alzheimer's Disease (AD). However, the underpinning mechanisms that connect synaptic homeostasis and the disease etiology are yet to be fully understood. This study demonstrated that neuronal overexpression of amyloid β (Aβ) causes abnormal histone acetylation in peripheral glia and completely blocks presynaptic homeostatic potentiation (PHP) at the neuromuscular junction in Drosophila The synaptic deficits caused by Aβ overexpression in motoneurons are associated with motor function impairment at the adult stage. Moreover, a sphingosine analog drug, Fingolimod, was found to ameliorate synaptic homeostatic plasticity impairment, abnormal glial histone acetylation, and motor behavior defects in the Aβ models. It was further demonstrated that perineurial glial sphingosine kinase 2 (Sk2) is not only required for PHP, but also plays a beneficial role in modulating PHP in the Aβ models. Glial overexpression of Sk2 rescues PHP, glial histone acetylation, and motor function deficits that are associated with Aβ in Drosophila Finally, this study showed that glial overexpression of Sk2 restores PHP and glial histone acetylation in a genetic loss-of-function mutant of the Spt-Ada-Gcn5 Acetyltransferase complex, strongly suggesting that Sk2 modulates PHP through epigenetic regulation. Both male and female animals were used in the experiments and analyses in this study. Collectively, this study has provided genetic evidence demonstrating that abnormal glial epigenetic alterations in Aβ models in Drosophila are associated with the impairment of PHP and that the sphingosine signaling pathway displays protective activities in stabilizing synaptic physiology.
Srivastav, S., van der Graaf, K., Singh, P., Utama, A. B., Meyer, M. D., McNew, J. A., Stern, M. (2024). Atl (atlastin) regulates mTor signaling and autophagy in Drosophila muscle through alteration of the lysosomal network. Autophagy, 20(1):131-150 PubMed ID: 37649246
Summary:
The hereditary spastic paraplegias (HSPs) represent a family of genetic disorders comprising at least 72 different genes with the common pathology of progressive locomotor deficits and spasticity. ATL1/SPG3A (atlastin GTPase 1) encodes an ER fusion protein that controls ER morphology, which implicates ER structure as a causal factor in HSP. This study used Drosophila to study effects of decreased atl (atlastin) on properties of the larval body wall muscle. We found that muscle atl loss causes accumulation of aggregates containing polyubiquitin (polyUB), mostly bound to the autophagy receptor ref(2)P/SQSTM1/p62. Muscle atl loss also decreased volume and complexity of the endolysosomal network and decreased lysosome number. To determine effects of these lysosomal deficits on progression through the basal autophagy pathway, Atg8a tagged with both GFP and mCherry in a wild-type and atl mutant background. Numerous structures containing mCherry were found but not GFP fluorescence in wild type, indicating that Atg8a was found mostly in mature autolysosomes. In contrast, muscles lacking atl exhibited significant amounts of GFP signal, indicating failure of autophagosome maturation with acidic lysosomes. Many of these GFP-positive puncta contained the late-endosome marker Rab7 but not Lamp1, indicating that some autophagy cargo was accumulating within amphisomes. It was also found that this autophagy block was accompanied by an inability to activate the mTor kinase. These results provide mechanistic insights into the role of atl in maintaining proper function of the autophagy pathway and suggests that certain pathologies in patients with mutations in ATL1/SPG3A might result from altered MTOR signaling.
Hogan, C. A., Gratz, S. J., Dumouchel, J. L., Thakur, R. S., Delgado, A., Lentini, J. M., Madhwani, K. R., Fu, D., O'Connor-Giles, K. M. (2023). Expanded tRNA methyltransferase family member TRMT9B regulates synaptic growth and function. EMBO reports, 24(10):e56808 PubMed ID: 37642556
Summary:
Nervous system function rests on the formation of functional synapses between neurons. This study has identified TRMT9B as a new regulator of synapse formation and function in Drosophila. TRMT9B has been studied for its role as a tumor suppressor and is one of two metazoan homologs of yeast tRNA methyltransferase 9 (Trm9), which methylates tRNA wobble uridines. Whereas Trm9 homolog ALKBH8 is ubiquitously expressed, TRMT9B is enriched in the nervous system. However, in the absence of animal models, TRMT9B's role in the nervous system has remained unstudied. This study generate null alleles of TRMT9B and found it acts postsynaptically to regulate synaptogenesis and promote neurotransmission. Through liquid chromatography-mass spectrometry, it was found that ALKBH8 catalyzes canonical tRNA wobble uridine methylation, raising the question of whether TRMT9B is a methyltransferase. Structural modeling studies suggest TRMT9B retains methyltransferase function and, in vivo, disruption of key methyltransferase residues blocks TRMT9B's ability to rescue synaptic overgrowth, but not neurotransmitter release. These findings reveal distinct roles for TRMT9B in the nervous system and highlight the significance of tRNA methyltransferase family diversification in metazoans.
Meissner, J. M., Akhmetova, K., Szul, T., Viktorova, E. G., Sha, B., Bhatt, J. M., Lee, E. J., Kahn, R. A., Belov, G. A., Chesnokov, I., Sztul, E. (2023). The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi. Frontiers in cell and developmental biology, 11:1233272 PubMed ID: 37745300
Summary:
Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. This study identified the HDS3 domain as essential for GBF1 membrane association in mammalian cells and documents the critical role of HDS3 during the development of Drosophila melanogaster. Upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. GBF1 interdomain arrangements were illuminated by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on tese data, models of GBF1 engagement on the membrane are proposed to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function.
Boutet, A., Zeledon, C., Emery, G. (2023). ArfGAP1 regulates the endosomal sorting of guidance receptors to promote directed collective cell migration in vivo. iScience, 26(8):107467 PubMed ID: 37599820
Summary:
Chemotaxis drives diverse migrations important for development and involved in diseases, including cancer progression. Using border cells in the Drosophila egg chamber as a model for collective cell migration, this study characterized the role of ArfGAP1 in regulating chemotaxis during this process. ArfGAP1 is required for the maintenance of receptor tyrosine kinases, the guidance receptors, at the plasma membrane. In the absence of ArfGAP1, the level of active receptors is reduced at the plasma membrane and increased in late endosomes. Consequently, clusters with impaired ArfGAP1 activity lose directionality. Furthermore, we found that the number and size of late endosomes and lysosomes are increased in the absence of ArfGAP1. Finally, genetic interactions suggest that ArfGAP1 acts on the kinase and GTPase Lrrk to regulate receptor sorting. Overall, the data indicate that ArfGAP1 is required to maintain guidance receptors at the plasma membrane and promote chemotaxis.

Wednesday, February 14th - Adult Neural Development, Structure and Function

Yoo, J., Dombrovski, M., Mirshahidi, P., Nern, A., LoCascio, S. A., Zipursky, S. L., Kurmangaliyev, Y. Z. (2023). Brain wiring determinants uncovered by integrating connectomes and transcriptomes. Curr Biol, 33(18):3998-4005. PubMed ID: 37647901
Summary:
Advances in brain connectomics have demonstrated the extraordinary complexity of neural circuits. Developing neurons encounter the axons and dendrites of many different neuron types and form synapses with only a subset of them. During circuit assembly, neurons express cell-type-specific repertoires comprising many cell adhesion molecules (CAMs) that can mediate interactions between developing neurites. Many CAM families have been shown to contribute to brain wiring in different ways. It has been challenging, however, to identify receptor-ligand pairs directly matching neurons with their synaptic targets. This study integrated the synapse-level connectome of the neural circuit with the developmental expression patterns and binding specificities of CAMs on pre- and postsynaptic neurons in the Drosophila visual system. To overcome the complexity of neural circuits, focus was placed on pairs of genetically related neurons that make differential wiring choices. In the motion detection circuit, closely related subtypes of T4/T5 neurons choose between alternative synaptic targets in adjacent layers of neuropil. This choice correlates with the matching expression in synaptic partners of different receptor-ligand pairs of the Beat and Side families of CAMs. Genetic analysis demonstrated that presynaptic Side-II and postsynaptic Beat-VI restrict synaptic partners to the same layer. Removal of this receptor-ligand pair disrupts layers and leads to inappropriate targeting of presynaptic sites and postsynaptic dendrites. It is proposde that different Side/Beat receptor-ligand pairs collaborate with other recognition molecules to determine wiring specificities in the fly brain. Combining transcriptomes, connectomes, and protein interactome maps allow unbiased identification of determinants of brain wiring.
Armour, E. M., Thomas, C. M., Greco, G., Bhatnagar, A., Elefant, F. (2023). Experience-dependent Tip60 nucleocytoplasmic transport is regulated by its NLS/NES sequences for neuroplasticity gene control. Molecular and cellular neurosciences, 127:103888 PubMed ID: 37598897
Summary:
Nucleocytoplasmic transport (NCT) in neurons is critical for enabling proteins to enter the nucleus and regulate plasticity genes in response to environmental cues. Such experience-dependent (ED) neural plasticity is central for establishing memory formation and cognitive function and can influence the severity of neurodegenerative disorders like Alzheimer's disease (AD). How histone acetyltransferases (HATs) respond to extracellular cues in the in vivo brain to drive HA-mediated activity-dependent gene control remains unclear. Previous work has demonstrated that extracellular stimulation of rat hippocampal neurons in vitro triggers Tip60 HAT nuclear import with concomitant synaptic gene induction. This study focussed on investigating Tip60 HAT subcellular localization and NCT specifically in neuronal activity-dependent gene control by using the learning and memory mushroom body (MB) region of the Drosophila brain as a powerful in vivo cognitive model system. Immunohistochemistry (IHC) was used to compare the subcellular localization of Tip60 HAT in the Drosophila brain under normal conditions and in response to stimulation of fly brain neurons in vivo either by genetically inducing potassium channels activation or by exposure to natural positive ED conditions. Furthermore, it was found that both inducible and ED condition-mediated neural induction triggered Tip60 nuclear import with concomitant induction of previously identified Tip60 target genes and that Tip60 levels in both the nucleus and cytoplasm were significantly decreased in the well-characterized Drosophila AD model. Mutagenesis of a putative nuclear localization signal (NLS) sequence and nuclear export signal (NES) sequence that were identified in the Drosophila Tip60 protein revealed that both are functionally required for appropriate Tip60 subcellular localization. These results support a model by which neuronal stimulation triggers Tip60 NCT via its NLS and NES sequences to promote induction of activity-dependent neuroplasticity gene transcription and that this process may be disrupted in AD.
Rui, M., Kong, W., Wang, W., Zheng, T., Wang, S., Xie, W. (2023). Droj2 Facilitates Somatosensory Neurite Sculpting via GTP-Binding Protein Arf102F in Drosophila. Int J Mol Sci, 24(17) PubMed ID: 37686022
Summary:
Developmental remodeling of neurite is crucial for the accurate wiring of neural circuits in the developing nervous system in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neuropsychiatric disorders, for instance, autism, Alzheimer's disease (AD), and schizophrenia. However, the molecular underpinnings underlying developmental remodeling are still not fully understood. This study has identified DnaJ-like-2 (Droj2), orthologous to human DNAJA1 and DNAJA4 that is predicted to be involved in protein refolding, as a developmental signal promoting dendrite sculpting of the class IV dendritic arborization (C4da) sensory neuron in Drosophila. It was further shown that Arf102F, a GTP-binding protein previously implicated in protein trafficking, serves downstream of Droj2 to govern neurite pruning of C4da sensory neurons. Intriguingly, these data consistently demonstrate that both Droj2 and Arf102F promote the downregulation of the conserved L1-type cell-adhesion molecule Neuroglian anterior to dendrite pruning. Mechanistically, Droj2 genetically interacts with Arf102F and promotes Neuroglian downregulation to initiate dendrite severing. Taken together, this systematic study sheds light on an unprecedented function of Droj2 and Arf102F in neuronal development.
Takakura, M., Lam, Y. H., Nakagawa, R., Ng, M. Y., Hu, X., Bhargava, P., Alia, A. G., Gu, Y., Wang, Z., Ota, T., Kimura, Y., Morimoto, N., Osakada, F., Lee, A. Y., Leung, D., Miyashita, T., Du, J., Okuno, H., Hirano, Y. (2023). Differential second messenger signaling via dopamine neurons bidirectionally regulates memory retention. PubMed ID: 37639608
Summary:
Memory formation and forgetting unnecessary memory must be balanced for adaptive animal behavior. While cyclic AMP (cAMP) signaling via dopamine neurons induces memory formation, this study reports that cyclic guanine monophosphate (cGMP) signaling via dopamine neurons launches forgetting of unconsolidated memory in Drosophila. Genetic screening and proteomic analyses showed that neural activation induces the complex formation of a histone H3K9 demethylase, Kdm4B, and a GMP synthetase, Bur, which is necessary and sufficient for forgetting unconsolidated memory. Kdm4B/Bur is activated by phosphorylation through NO-dependent cGMP signaling via dopamine neurons, inducing gene expression, including kek2 encoding a presynaptic protein. Accordingly, Kdm4B/Bur activation induced presynaptic changes. These data demonstrate a link between cGMP signaling and synapses via gene expression in forgetting, suggesting that the opposing functions of memory are orchestrated by distinct signaling via dopamine neurons, which affects synaptic integrity and thus balances animal behavior.
Ferreira, A. A. G., Desplan, C. (2023). An Atlas of the Developing Drosophila Visual System Glia and Subcellular mRNA Localization of Transcripts in Single Cells. bioRxiv, PubMed ID: 37609218
Summary:
Glial cells are essential for proper nervous system development and function. To understand glial development and function, glial cells were comprehensively annotated in a single-cell mRNA-sequencing (scRNAseq) atlas of the developing Drosophila visual system.. This allowed study of their developmental trajectories, from larval to adult stages, and to understand how specific types of glia diversify during development. For example, neuropil glia that are initially transcriptionally similar in larvae, split into ensheathing and astrocyte-like glia during pupal stages. Other glial types, such as chiasm glia change gradually during development without splitting into two cell types. The analysis of scRNA-seq led to the discovery that the transcriptome of glial cell bodies can be distinguished from that of their broken processes. The processes contain distinct enriched mRNAs that were validated in vivo. Therefore, this study have identified most glial types in the developing optic lobe and devised a computational approach to identify mRNA species that are localized to cell bodies or cellular processes.
Poe, A. R., Zhu, L., Szuperak, M., McClanahan, P. D., Anafi, R. C., Scholl, B., Thum, A. S., Cavanaugh, D. J., Kayser, M. S. (2023). Developmental emergence of sleep rhythms enables long-term memory in Drosophila. Sci Adv, 9(36):eadh2301 PubMed ID: 37683005
Summary:
In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day. The mechanism through which sleep rhythms emerge, and consequent advantage conferred to a juvenile animal, is unknown. In the second-instar Drosophila larvae (L2), like in human infants, sleep is not under circadian control. This study identified the precise developmental time point when the clock begins to regulate sleep in Drosophila, leading to emergence of sleep rhythms in early third-instars (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousal-promoting Dh44 neurons, bringing arousal under clock control to drive emergence of circadian sleep. Last, this study demonstrated that L3 but not L2 larvae exhibit long-term memory (LTM) of aversive cues and that this LTM depends upon deep sleep generated once sleep rhythms begin. It is proposed that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.

Tuesday, February 13th - Chromatin and DNA

Yuan, Y., Chen, Q., Brovkina, M., Clowney, E. J., Yadlapalli, S. (2023). Clock-dependent chromatin accessibility rhythms regulate circadian transcription. bioRxiv, PubMed ID: 37645872
Summary:
Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) was employed on Drosophila clock neurons to assess genome-wide chromatin accessibility over the circadian cycle. Significant circadian oscillations were observed in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, a complete loss was observed of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible throughout the circadian cycle, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.
Hodkinson, L. J., Smith, C., Comstra, H. S., ...., Zeng, H., Schmidt, C. A., Rieder, L. E. (2023). A bioinformatics screen reveals hox and chromatin remodeling factors at the Drosophila histone locus. BMC genomic data, 24(1):54 PubMed ID: 37735352
Summary:
Cells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation. RESULTS: To expand the list of known HLB factors, we performed a candidate-based screen by mapping 30 publicly available ChIP datasets of 27 unique factors to the Drosophila histone gene array. We identified novel transcription factor candidates, including the Drosophila Hox proteins Ultrabithorax (Ubx), Abdominal-A (Abd-A), and Abdominal-B (Abd-B), suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, six other factors were identified that target the histone gene array: JIL-1, hormone-like receptor 78 (Hr78), the long isoform of female sterile homeotic (1) (fs(1)h) as well as the general transcription factors TBP associated factor 1 (TAF-1), Transcription Factor IIB (TFIIB), and Transcription Factor IIF (TFIIF). This foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, this study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.
Zhu, J. Y., Liu, C., Huang, X., van de Leemput, J., Lee, H. and Han, Z. (2023). H3K36 Di-Methylation Marks, Mediated by Ash1 in Complex with Caf1-55 and MRG15, Are Required during Drosophila Heart Development. J Cardiovasc Dev Dis 10(7). PubMed ID: 37504562
Summary:
Methyltransferases regulate transcriptome dynamics during development and aging, as well as in disease. Various methyltransferases have been linked to heart disease, through disrupted expression and activity, and genetic variants associated with congenital heart disease. However, in vivo functional data for many of the methyltransferases in the context of the heart are limited. This study used the Drosophila model system to investigate different histone 3 lysine 36 (H3K36) methyltransferases for their role in heart development. The data show that Drosophila Ash1 is the functional homolog of human ASH1L in the heart. Both Ash1 and Set2 H3K36 methyltransferases are required for heart structure and function during development. Furthermore, Ash1-mediated H3K36 methylation (H3K36me2) is essential for healthy heart function, which depends on both Ash1-complex components, Caf1-55 and MRG15, together. These findings provide in vivo functional data for Ash1 and its complex, and Set2, in the context of H3K36 methylation in the heart, and support a role for their mammalian homologs, ASH1L with RBBP4 and MORF4L1, and SETD2, during heart development and disease.
Miller, J. M., Prange, S., Ji, H., ...., Hanscom, T., McVey, M., Chiolo, I. (2023). Alternative end-joining results in smaller deletions in heterochromatin relative to euchromatin. bioRxiv, PubMed ID: 37645729
Summary:
Homologous recombination (HR) repair is uniquely regulated in the ericentromeric heterochromatin to enable 'safe' repair while preventing aberrant recombination. In Drosophila cells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR repair. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how alternative EJ (alt-EJ) pathways operate in heterochromatin remains largely uncharacterized. This study induced DSBs in single euchromatic and heterochromatic sites using a new system that combines the DR- white reporter and I-SceI expression in spermatogonia of flies. Using this approach, higher frequency of HR repair is detected in heterochromatin, relative to euchromatin. Further, sequencing of mutagenic repair junctions reveals the preferential use of different EJ pathways across distinct euchromatic and heterochromatic sites. Interestingly, synthesis-dependent microhomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin.
Zion, E. H., Ringwalt, D., Rinaldi, K., Kahney, E. W., Li, Y. and Chen, X. (2023). Old and newly synthesized histones are asymmetrically distributed in Drosophila intestinal stem cell divisions. EMBO Rep 24(7): e56404. PubMed ID: 37255015
Summary:
This study reports that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically partitioned during the division of Drosophila intestinal stem cells (ISCs). Furthermore, the inheritance patterns of old and new H3 and H4 in postmitotic cell pairs correlate with distinct expression patterns of Delta, an important cell fate gene. To understand the biological significance of this phenomenon, a mutant H3T3A was expressed to compromise asymmetric histone inheritance. Under this condition, an increase was observed in Delta-symmetric cell pairs and overpopulated ISC-like, Delta-positive cells. Single-cell RNA-seq assays further indicate that H3T3A expression compromises ISC differentiation. Together, these results indicate that asymmetric histone inheritance potentially contributes to establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings in Drosophila male germline stem cells.
Kleene, V., Corvaglia, V., Chacin, E., Forne, I., Konrad, D. B., Khosravani, P., Douat, C., Kurat, C. F., Huc, I., Imhof, A. (2023). DNA mimic foldamers affect chromatin composition and disturb cell cycle progression. Nucleic Acids Res, 51(18):9629-9642 PubMed ID: 37650653
Summary:
The use of synthetic chemicals to selectively interfere with chromatin and the chromatin-bound proteome represents a great opportunity for pharmacological intervention. Recently, synthetic foldamers that mimic the charge surface of double-stranded DNA have been shown to interfere with selected protein-DNA interactions. However, to better understand their pharmacological potential and to improve their specificity and selectivity, the effect of these molecules on complex chromatin needs to be investigated. Therefore this study systematically studied the influence of the DNA mimic foldamers on the chromatin-bound proteome using an in vitro chromatin assembly extract. These studies show that the foldamer efficiently interferes with the chromatin-association of the origin recognition complex in vitro and in vivo, which leads to a disturbance of cell cycle in cells treated with foldamers. This effect is mediated by a strong direct interaction between the foldamers and the origin recognition complex and results in a failure of the complex to organise chromatin around replication origins. Foldamers that mimic double-stranded nucleic acids thus emerge as a powerful tool with designable features to alter chromatin assembly and selectively interfere with biological mechanisms.

Monday, February 12th - Disease Models

Li, Q., Wang, L., Tang, C., Wang, X., Yu, Z., Ping, X., Ding, M., Zheng, L. (2024). Adipose Tissue Exosome circ_sxc Mediates the Modulatory of Adiposomes on Brain Aging by Inhibiting Brain dme-miR-87-3p. Molecular neurobiology, 61(1):224-238 PubMed ID: 37597108
Summary:
Aging of the brain usually leads to the decline of neurological processes and is a major risk factor for various neurodegenerative diseases, including sleep disturbances and cognitive decline. Adipose tissue exosomes, as adipocyte-derived vesicles, may mediate the regulatory processes of adipose tissue on other organs, including the brain; however, the regulatory mechanisms remain unclear. This study analyzed the sleep-wake behavior of young (10 days) and old (40 days) Drosophila and found that older Drosophila showed increased sleep fragmentation, which is similar to mammalian aging characteristics. To investigate the cross-tissue regulatory mechanisms of adiposity on brain aging, 10-day and 40-day Drosophila adipose tissue exosomes were extracted and circRNAs were identied with age-dependent expression differences by RNA-seq and differential analysis. Furthermore, by combining data from 3 datasets of the GEO database (GSE130158, GSE24992, and GSE184559), circ_sxc that was significantly downregulated with age was finally screened out. Moreover, dme-miR-87-3p, a conserved target of circ_sxc, accumulates in the brain with age and exhibits inhibitory effects in predicted binding relationships with neuroreceptor ligand genes. In summary, the current study showed that the Drosophila brain could obtain circ_sxc by uptake of adipose tissue exosomes which crossed the blood-brain barrier. And circ_sxc suppressed brain miR-87-3p expression through sponge adsorption, which in turn regulated the expression of neurological receptor ligand proteins (5-HT1B, GABA-B-R1, Rdl, Rh7, qvr, NaCP60E) and ensured brain neuronal synaptic signaling normal function of synaptic signaling. However, with aging, this regulatory mechanism is dysregulated by the downregulation of the adipose exosome circ_sxc, which contributes to the brain exhibiting sleep disturbances and other "aging" features.
Sung, H., Lloyd, T. E. (2024). Disrupted endoplasmic reticulum-mediated autophagosomal biogenesis in a Drosophila model of C9-ALS-FTD. Autophagy, 20(1):94-113 PubMed ID: 37599467
Summary:
Macroautophagy/autophagy is a major pathway for the clearance of protein aggregates and damaged organelles, and multiple intracellular organelles participate in the process of autophagy, from autophagosome formation to maturation and degradation. Dysregulation of the autophagy pathway has been implicated in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), however the mechanisms underlying autophagy impairment in these diseases are incompletely understood. Since the expansion of GGGGCC (G4C2) repeats in the first intron of the C9orf72 gene is the most common inherited cause of both ALS and FTD (C9-ALS-FTD), this study investigated autophagosome dynamics in Drosophila motor neurons expressing 30 G4C2 repeats (30 R). In vivo imaging demonstrates that expression of expanded G4C2 repeats markedly impairs biogenesis of autophagosomes at synaptic termini, whereas trafficking and maturation of axonal autophagosomes are unaffected. Motor neurons expressing 30 R display marked disruption in endoplasmic reticulum (ER) structure and dynamics in the soma, axons, and synapses. Disruption of ER morphology with mutations in Rtnl1 (Reticulon-like 1) or atl (atlastin) also impairs autophagosome formation in motor neurons, suggesting that ER integrity is critical for autophagosome formation. Furthermore, live imaging demonstrates that autophagosomes are generated from dynamic ER tubules at synaptic boutons, and this process fails to occur in a C9-ALS-FTD model. Together, these findings suggest that dynamic ER tubules are required for formation of autophagosomes at the neuromuscular junction, and that this process is disrupted by expanded G4C2 repeats that cause ALS-FTD.
Soustelle, L., Aimond, F., Lopez-Andres, C., Brugioti, V., Raoul, C., Layalle, S. (2023). ALS-Associated KIF5A Mutation Causes Locomotor Deficits Associated with Cytoplasmic Inclusions, Alterations of Neuromuscular Junctions, and Motor Neuron Loss. J Neurosci, 43(47):8058-8072 PubMed ID: 37748861
Summary:
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Recently, genome-wide association studies identified KIF5A as a new ALS-causing gene. KIF5A encodes a protein of the kinesin-1 family, allowing the anterograde transport of cargos along the microtubule rails in neurons. In ALS patients, mutations in the KIF5A gene induce exon 27 skipping, resulting in a mutated protein with a new C-terminal region (KIF5A Δ27). To understand how KIF5A Δ27 underpins the disease, this study developed an ALS-associated KIF5A Drosophila model. When selectively expressed in motor neurons, KIF5A Δ27 alters larval locomotion as well as morphology and synaptic transmission at neuromuscular junctions in both males and females. The distribution of mitochondria and synaptic vesicles was found to be profoundly disturbed by KIF5A Δ27 expression. That is consistent with the numerous KIF5A Δ27-containing inclusions observed in motor neuron soma and axons. Moreover, KIF5A Δ27 expression leads to motor neuron death and reduces life expectancy. This in vivo model reveals that a toxic gain of function underlies the pathogenicity of ALS-linked KIF5A mutant.
Cai, Y., Cui, T., Yin, P., Paganelli, P., Vicini, S., Wang, T. (2023). Dysregulated glial genes in Alzheimer's disease are essential for homeostatic plasticity: Evidence from integrative epigenetic and single cell analyses. Aging Cell, 22(11):e13989 PubMed ID: 37712202
Summary:
Synaptic homeostatic plasticity is a foundational regulatory mechanism that maintains the stability of synaptic and neural functions within the nervous system. Impairment of homeostatic regulation has been linked to synapse destabilization during the progression of Alzheimer's disease (AD). Various glial cell types play critical roles in modulating synaptic functions both during the aging process and in the context of AD. This study investigated the impact of glial dysregulation of histone acetylation and transcriptome in AD on synaptic homeostatic plasticity, using computational analysis combined with electrophysiological methods in Drosophila. By integrating snRNA-seq and H3K9ac ChIP-seq data from the same AD patient cohort, this study pinpointed cell type-specific signature genes that were transcriptionally altered by histone acetylation. The role of these glial genes in regulating presynaptic homeostatic potentiation was subsequently investigated in Drosophila. Remarkably, nine glial-specific genes, which were identified through computational methods as targets of H3K9ac and transcriptional dysregulation, were found to be crucial for the regulation of synaptic homeostatic plasticity at the NMJ in Drosophila. This genetic evidence connects abnormal glial transcriptomic changes in AD with the impairment of homeostatic plasticity in the nervous system. In summary, these integrative computational and genetic studies highlight specific glial genes as potential key players in the homeostatic imbalance observed in AD.
Koval, L. A., Proshkina, E. N., Zemskaya, N. V., Solovev, I. A., Schegoleva, E. V., Shaposhnikov, M. V., Moskalev, A. A. (2023). [Drosophila melanogaster Lifespan Is Regulated by nejire Gene Expression in Peripheral Tissues and Nervous System]. Molekuliarnaia biologiia, 57(5):833-852 PubMed ID: 37752649
Summary:
Histone acetyltransferases of the CBP/p300 family are involved in transcriptional regulation and many biological processes (cell proliferation and differentiation, development, and regulation of the stress response and metabolism). Overexpression and knockdown of the nejire (nej) gene (codes for an ortholog of human CBP/p300 proteins) in various tissues (the fat body, intestine, and nervous system) and at various stages of the life cycle (throughout all developmental stages or in adulthood only) were tested for effect on lifespan in the fruit fly Drosophila melanogaster. The activation of nej exerted a positive or a negative effect on the lifespan, depending on the induction mode and the sex. A 6-15% greater lifespan was observed in females with conditional overexpression of nej in the intestine and constitutive overexpression of nej in the nervous system. A decrease (to 44%) or lack of significant changes in lifespan was detected in all other cases observed. In addition, stress response genes (Sod1, Gadd45, Hsp27, Hsp68, and Hif1) were regulated by nej activation. nej knockdown caused a pronounced negative effect on the D. melanogaster lifespan in most variants of the experiment.
Dutta, D., Kanca, O., Byeon, S. K., Marcogliese, P. C., Zuo, Z., Shridharan, R. V., Park, J. H., Lin, G., Ge, M., Heimer, G., Kohler, J. N., Wheeler, M. T., Kaipparettu, B. A., Pandey, A., Bellen, H. J. (2023. A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels. Nature metabolism, 5(9):1595-1614 PubMed ID: 37653044
Summary:
In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. This study shows that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. A defect in Fe-S cluster biogenesis and increased iron levels were observed in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and this study shows that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.

Friday, February 9th - Cytoskeleton

Zhang, X. Q., Yang, R., Jin, L. and Li, G. Q. (2023). Requirement of Snakeskin for normal functions of midgut and Malpighian tubules in Henosepilachna vigintioctopunctata. Arch Insect Biochem Physiol 114(1): e22033. PubMed ID: 37401505
Summary:
Septate junctions (SJs) are located between epithelial cells and play crucial roles in epithelial barrier formation and epithelia cell homeostasis. Nevertheless, the molecular constituents, especially those related to smooth SJs (sSJs), have not been well explored in non-Drosophilid insects. A putative integral membrane protein Snakeskin (Ssk) was identified in a Coleoptera foliar pest Henosepilachna vigintioctopunctata. RNA interference-aided knockdown of Hvssk at the third-instar larval stage arrested larval development. Most resultant larvae failed to shed larval exuviae until their death. Silence of Hvssk at the fourth-instar larvae inhibited the growth and reduced foliage consumption. Dissection and microscopic observation revealed that compromised expression of Hvssk caused obvious phenotypic defects in the midgut. A great number of morphologically abnormal columnar epithelial cells accumulated throughout the midgut lumen. Moreover, numerous vesicles were observed in the malformed cells of the Malpighian tubules (Mt). All the Hvssk depleted larvae remained as prepupae; they gradually darkened and eventually died. Furthermore, depletion of Hvssk at the pupal stage suppressed adult feeding and shortened adult lifespan. These findings demonstrated that Ssk plays a vital role in the integrity and function of both midguts and Mt, and established the conservative roles of Ssk in the formation of epithelial barrier and the homeostasis of epithelial cells in H. vigintioctopunctata.
Salvador-Garcia, D., Jin, L., Hensley, A., Golcuk, M., Gallaud, E., Chaaban, S., Port, F., Vagnoni, A., Planelles-Herrero, V. J., McClintock, M. A., Derivery, E., Carter, A. P., Giet, R., Gur, M., Yildiz, A. and Bullock, S. L. (2023). A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression. bioRxiv. PubMed ID: 37577480
Summary:
The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate in vivo studies of its functions significantly. To address this issue, gene editing was used to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). Mutations associated with human neurological disease were shown to cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. A novel mutation in the microtubule-binding domain (MTBD) of Dhc is described that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. Mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, these data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, these work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.
Wippich, F., Vaishali, Hennrich, M. L. and Ephrussi, A. (2023). Nutritional stress-induced regulation of microtubule organization and mRNP transport by HDAC1 controlled α-tubulin acetylation. Commun Biol 6(1): 776. PubMed ID: 37491525
Summary:
In response to nutritional stress, microtubules in cells of the Drosophila female germline are depleted from the cytoplasm and accumulate cortically. This triggers aggregation of mRNPs into large processing bodies (P-bodies) and oogenesis arrest. This study shows that hyperacetylation of α-tubulin at lysine 40 (K40) alters microtubule dynamics and P-body formation. Depletion of histone deacetylase 1 (HDAC1) by RNAi phenocopies the nutritional stress. response, causing α-tubulin hyperacetylation and accumulation of maternally deposited mRNPs in P-bodies. Through in vitro and in vivo studies, this study identified HDAC1 as a direct regulator of α-tubulin K40 acetylation status. In well-fed flies, HDAC1 maintains low levels of α-tubulin acetylation, enabling the microtubule dynamics required for mRNP transport. Using quantitative phosphoproteomics, nutritional stress-induced changes were identified in protein phosphorylation that act upstream of α-tubulin acetylation, including phosphorylation of HDAC1 at S391, which reduces its ability to deacetylate α-tubulin. These results reveal that Drosophila HDAC1 senses and relays the nutritional status, which regulates germline development through modulation of cytoskeleton dynamics.
Zhao, H., Shi, L., Li, Z., Kong, R., Jia, L., Lu, S., Wang, J. H., Dong, M. Q., Guo, X., Li, Z. (2023). Diamond controls epithelial polarity through the dynactin-dynein complex. Traffic (Copenhagen, Denmark), 24(12):552-563 PubMed ID: 37642208
Summary:
Epithelial polarity is critical for proper functions of epithelial tissues, tumorigenesis, and metastasis. The evolutionarily conserved transmembrane protein Crumbs (Crb) is a key regulator of epithelial polarity. Both Crb protein and its transcripts are apically localized in epithelial cells. However, it remains not fully understood how they are targeted to the apical domain. Using Drosophila ovarian follicular epithelia as a model, it was show that epithelial polarity is lost and Crb protein is absent in the apical domain in follicular cells (FCs) in the absence of Diamond (Dind). Interestingly, Dind is found to associate with different components of the dynactin-dynein complex through co-IP-MS analysis. Dind stabilizes dynactin and depletion of dynactin results in almost identical defects as those observed in dind-defective FCs. Finally, both Dind and dynactin are also required for the apical localization of crb transcripts in FCs. Thus these data illustrate that Dind functions through dynactin/dynein-mediated transport of both Crb protein and its transcripts to the apical domain to control epithelial apico-basal (A/B) polarity.
Song, X., Cui, L., Wu, M., Wang, S., Song, Y., Liu, Z., Xue, Z., Chen, W., Zhang, Y., Li, H., Sun, L., Liang, X. (2023). DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles. J Cell Biol, 222(10). PubMed ID: 37651176
Summary:
Mechanoreceptor cells develop specialized mechanosensory organelles (MOs), where force-sensitive channels and supporting structures are organized in an orderly manner to detect forces. It is intriguing how MOs are formed. This issue was addressed by studying the MOs of fly ciliated mechanoreceptors. The main structure of the MOs is shown to be a compound cytoskeleton formed of short microtubules and electron-dense materials (EDMs). In a knock-out mutant of microtubule associated protein DCX-EMAP, this cytoskeleton is nearly absent, suggesting that DCX-EMAP is required for the formation of the MOs and in turn fly mechanotransduction. Further analysis reveals that DCX-EMAP expresses in fly ciliated mechanoreceptors and localizes to the MOs. Moreover, it plays dual roles by promoting the assembly/stabilization of the microtubules and the accumulation of the EDMs in the MOs. Therefore, DCX-EMAP serves as a core ultrastructural organizer of the MOs, and this finding provides novel molecular insights as to how fly MOs are formed.
Ventura Santos, C., Rogers, S. L., Carter, A. P. (2023). CryoET shows cofilactin filaments inside the microtubule lumen. EMBO reports, 24(11):e57264 PubMed ID: 37702953
Summary:
Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. This study used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin dephosphorylation, an activating modification, was observed in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

Thursday, February 8th - Behavior

Yu, J., Dancausse, S., Paz, M., Faderin, T., Gaviria, M., Shomar, J. W., Zucker, D., Venkatachalam, V. and Klein, M. (2023). Continuous, long-term crawling behavior characterized by a robotic transport system. Elife 12. PubMed ID: 37535068
Summary:
Detailed descriptions of behavior provide critical insight into the structure and function of nervous systems. In Drosophila larvae and many other systems, short behavioral experiments have been successful in characterizing rapid responses to a range of stimuli at the population level. However, the lack of long-term continuous observation makes it difficult to dissect comprehensive behavioral dynamics of individual animals and how behavior (and therefore the nervous system) develops over time. To allow for long-term continuous observations in individual fly larvae, a robotic instrument was engineered that automatically tracks and transports larvae throughout an arena. The flexibility and reliability of its design enables controlled stimulus delivery and continuous measurement over developmental time scales, yielding an unprecedented level of detailed locomotion data. This study utilized the new system's capabilities to perform continuous observation of exploratory search behavior over a duration of 6 hr with and without a thermal gradient present, and in a single larva for over 30 hr. Long-term free-roaming behavior and analogous short-term experiments show similar dynamics that take place at the beginning of each experiment. Finally, characterization of larval thermotaxis in individuals reveals a bimodal distribution in navigation efficiency, identifying distinct phenotypes that are obfuscated when only analyzing population averages.
Venkateswaran, V., Alali, I., Unni, A. P., Weissflog, J., Halitschke, R., Hansson, B. S., Knaden, M. (2023). Carbonyl products of ozone oxidation of volatile organic compounds can modulate olfactory choice behavior in insects. Environ Pollut, 337:122542 PubMed ID: 37717892
Summary:
Insects are a diverse group of organisms that provide important ecosystem services like pollination, pest control, and decomposition and rely on olfaction to perform these services. In the Anthropocene, increasing concentrations of oxidant pollutants such as ozone have been shown to corrupt odor-driven behavior in insects by chemically degrading e.g. flower signals or insect pheromones. The degradation, however, does not only result in a loss of signals, but also in a potential enrichment of oxidation products, predominantly small carbonyls. Whether and how these oxidation products affect insect olfactory perception remains unclear. This study examined the effects of ozone-generated small carbonyls on the olfactory behavior of the vinegar fly Drosophila melanogaster. A broad collection of neurophysiologically relevant odorants were compiled for the fly from databases and literature and predicted the formation of the types of stable small carbonyl products resulting from the odorant's oxidation by ozone. Based on these predictions, the olfactory detection and behavioral impact of the ten most frequently predicted carbonyl products were evaluated in the fly using single sensillum recordings (SSRs) and behavioral tests. The results demonstrate that the fly's olfactory system can detect the oxidation products, which then elicit either attractive or neutral behavioral responses, rather than repulsion. However, certain products alter behavioral choices to an attractive odor source of balsamic vinegar. These findings suggest that the enrichment of small carbonyl oxidation products due to increased ozone levels can affect olfactory guided insect behavior. This study underscores the implications for odor-guided foraging in insects and the essential ecosystem services they offer under carbonyl enriched environments.
Babin, A., Gatti, J. L., Poirie, M. (2023). Bacillus thuringiensis bioinsecticide influences Drosophila oviposition decision. R Soc Open Sci, 10(8):230565 PubMed ID: 37650056
Summary:
Behavioural avoidance has obvious benefits for animals facing environmental stressors such as pathogen-contaminated foods. Most current bioinsecticides are based on the environmental and opportunistic bacterium Bacillus thuringiensis (Bt) that kills targeted insect pests upon ingestion. While food and oviposition avoidance of Bt bioinsecticide by targeted insect species was reported, this remained to be addressed in non-target organisms, especially those affected by chronic exposure to Bt bioinsecticide such as Drosophila species. Here, using a two-choice oviposition test, we showed that female flies of three Drosophila species (four strains of D. melanogaster, D. busckii and D. suzukii) avoided laying eggs in the presence of Bt var. kurstaki bioinsecticide, with potential benefits for the offspring and female's fitness. Avoidance occurred rapidly, regardless of the fraction of the bioinsecticide suspension (spores and toxin crystals versus soluble toxins/compounds) and independently of the female motivation for egg laying. These results suggest that, in addition to recent findings of developmental and physiological alterations upon chronic exposure to non-target Drosophila, this bioinsecticide may modify the competitive interactions between Drosophila species in treated areas and the interactions with their associated natural enemies.
Kobayashi, R., Nakane, S., Tomita, J., Funato, H., Yanagisawa, M., Kume, K. (2023). A phosphorylation-deficient mutant of Sik3, a homolog of Sleepy, alters circadian sleep regulation by PDF neurons in Drosophila. Frontiers in neuroscience, 17:1181555 PubMed ID: 37662102
Summary:
Sleep behavior has been observed from non-vertebrates to humans. Sleepy mutation in mice resulted in a notable increase in sleep and was identified as an exon-skipping mutation of the salt-inducible kinase 3 (Sik3) gene (See Drosophila Sik3), conserved among animals. The skipped exon includes a serine residue that is phosphorylated by protein kinase A. Overexpression of a mutant gene with the conversion of this serine into alanine (Sik3-SA) increased sleep in both mice and the fruit fly Drosophila melanogaster. However, the mechanism by which Sik3-SA increases sleep remains unclear. This study found that Sik3-SA overexpression in all neurons increased sleep under both light-dark (LD) conditions and constant dark (DD) conditions in Drosophila. Additionally, overexpression of Sik3-SA only in PDF neurons, which are a cluster of clock neurons regulating the circadian rhythm, increased sleep during subjective daytime while decreasing the amplitude of circadian rhythm. Furthermore, suppressing Sik3-SA overexpression specifically in PDF neurons in flies overexpressing Sik3-SA in all neurons reversed the sleep increase during subjective daytime. These results indicate that Sik3-SA alters the circadian function of PDF neurons and leads to an increase in sleep during subjective daytime under constant dark conditions.
McKenzie-Smith, G. C., Wolf, S. W., Ayroles, J. F., Shaevitz, J. W. (2023). Capturing continuous, long timescale behavioral changes in Drosophila melanogaster postural data. ArXiv, PubMed ID: 37731659
Summary:
Animal behavior spans many timescales, from short, seconds-scale actions to circadian rhythms over many hours to life-long changes during aging. Most quantitative behavior studies have focused on short-timescale behaviors such as locomotion and grooming. Analysis of these data suggests there exists a hierarchy of timescales; however, the limited duration of these experiments prevents the investigation of the full temporal structure. To access longer timescales of behavior, individual Drosophila melanogaster were continuously recorded at 100 frames per second for up to 7 days at a time in featureless arenas on sucrose-agarose media. The deep learning framework SLEAP was used to produce a full-body postural data set for 47 individuals resulting in nearly 2 billion pose instances. Stereotyped behaviors were identified such as grooming, proboscis extension, and locomotion, and the resulting ethograms were used to explore how the flies' behavior varies across time of day and days in the experiment. Distinct circadian patterns were observed all of the stereotyped behavior, and also changes were seen in behavior over the course of the experiment as the flies weaken and die.
Nguyen, M., Roman, G. W., Soibam, B. (2023). Drosophila genotypes can be predicted from their exploration locomotive trajectories using supervised machine learning. Behavioural processes, 212:104944 PubMed ID: 37717930
Summary:
This study employs supervised machine learning algorithms to test whether locomotive features during exploratory activity in open field arenas can serve as predictors for the genotype of fruit flies. Because of the nonlinearity in locomotive trajectories, traditional statistical methods that are used to compare exploratory activity between genotypes of fruit flies may not reveal all insights. 10-minute-long trajectories of four different genotypes of fruit flies in an open-field arena environment were captured. Turn angles and step size features extracted from the trajectories were used for training supervised learning models to predict the genotype of the fruit flies. Using the first five minute locomotive trajectories, an accuracy of 83% was achieved in differentiating wild-type flies from three other mutant genotypes. Using the final 5 min and the entire ten minute duration decreased the performance indicating that the most variations between the genotypes in their exploratory activity are exhibited in the first few minutes. Feature importance analysis revealed that turn angle is a better predictor than step size in predicting fruit fly genotype. Overall, this study demonstrates that features of trajectories can be used to predict the genotype of fruit flies through supervised machine learning methods.

Wednesday, February 7th - Adult physiology and metabolism

Strunov, A., Schoenherr, C. and Kapun, M. (2023). Wolbachia has subtle effects on thermal preference in highly inbred Drosophila melanogaster which vary with life stage and environmental conditions. Sci Rep 13(1): 13792. PubMed ID: 37612420
Summary:
Temperature fluctuations are challenging for ectotherms which are not able to regulate body temperature by physiological means and thus have to adjust their thermal environment via behavior. However, little is yet known about whether microbial symbionts influence thermal preference (T(p)) in ectotherms by modulating their physiology. Several recent studies have demonstrated substantial effects of Wolbachia infections on host T(p) in different Drosophila species. These data indicate that the direction and strength of thermal preference variation is strongly dependent on host and symbiont genotypes and highly variable among studies. By employing highly controlled experiments, this study investigated the impact of several environmental factors including humidity, food quality, light exposure, and experimental setup that may influence T(p) measurements in adult Drosophila melanogaster flies. Additionally, the effects of Wolbachia infection on T(p) of Drosophila was assessed at different developmental stages, which has not been done before. Only subtle effects were found of Wolbachia on host T(p) which are strongly affected by experimental variation in adult, but not during juvenile life stages. These in-depth analyses show that environmental variation has a substantial influence on T(p) which demonstrates the necessity of careful experimental design and cautious interpretations of T(p) measurements together with a thorough description of the methods and equipment used to conduct behavioral studies.
Ren, M., Xu, Y., Phoon, C. K. L., Erdjument-Bromage, H., Neubert, T. A., Schlame, M. (2023). Cardiolipin prolongs the lifetimes of respiratory proteins in Drosophila flight muscle. J Biol Chem, 299(10):105241 PubMed ID: 37690688
Summary:
Respiratory complexes and cardiolipins have exceptionally long lifetimes. The fact that they co-localize in mitochondrial cristae raises the question of whether their longevities have a common cause and whether the longevity of OXPHOS proteins is dependent on cardiolipin. To address these questions, a method was developed to measure side-by-side the half-lives of proteins and lipids in wild-type Drosophila and cardiolipin-deficient mutants. Adult flies were fed with stable isotope-labeled precursors ((13)C(6)(15)N(2)-lysine or (13)C(6)-glucose), and the relative abundance of heavy isotopomers in protein and lipid species was determined by mass spectrometry. To minimize the confounding effects of tissue regeneration, this analysis was restricted to the thorax, the bulk of which consists of post-mitotic flight muscles. Analysis of 680 protein and 45 lipid species showed that the subunits of respiratory complexes I-V and the carriers for phosphate and ADP/ATP were among the longest-lived proteins (average half-life of 48 ± 16 days) while the molecular species of cardiolipin were the longest-lived lipids (average half-life of 27 ± 6 days). The remarkable longevity of these crista residents was not shared by all mitochondrial proteins, especially not by those residing in the matrix and the inner boundary membrane. Ablation of cardiolipin synthase, which causes replacement of cardiolipin by phosphatidylglycerol, and ablation of tafazzin, which causes partial replacement of cardiolipin by monolyso-cardiolipin, decreased the lifetimes of the respiratory complexes. Ablation of tafazzin also decreased the lifetimes of the remaining cardiolipin species. These data suggest that an important function of cardiolipin in mitochondria is to protect respiratory complexes from degradation.
Bozkurt, B., Terlemez, G., Sezgin, E. (2023). Basidiomycota species in Drosophila gut are associated with host fat metabolism. Sci Rep, 13(1):13807 PubMed ID: 37612350
Summary:
The importance of bacterial microbiota on host metabolism and obesity risk is well documented. However, the role of fungal microbiota on host storage metabolite pools is largely unexplored. This study investigated the role of microbiota on D. melanogaster fat metabolism, and examine interrelatedness between fungal and bacterial microbiota, and major metabolic pools. Fungal and bacterial microbiota profiles, fat, glycogen, and trehalose metabolic pools are measured in a context of genetic variation represented by whole genome sequenced inbred Drosophila Genetic Reference Panel (DGRP) samples. Increasing Basidiomycota, Acetobacter persici, Acetobacter pomorum, and Lactobacillus brevis levels correlated with decreasing triglyceride levels. Host genes and biological pathways, identified via genome-wide scans, associated with Basidiomycota and triglyceride levels were different suggesting the effect of Basidiomycota on fat metabolism is independent of host biological pathways that control fungal microbiota or host fat metabolism. Although triglyceride, glycogen and trehalose levels were highly correlated, microorganisms' effect on triglyceride pool were independent of glycogen and trehalose levels. Multivariate analyses suggested positive interactions between Basidiomycota, A. persici, and L. brevis that collectively correlated negatively with fat and glycogen pools. In conclusion, fungal microbiota can be a major player in host fat metabolism. Interactions between fungal and bacterial microbiota may exert substantial control over host storage metabolite pools and influence obesity risk.
Dornan, A. J., Halberg, K. V., Beuter, L. K., Davies, S. A., Dow, J. A. T. (2023). Compromised junctional integrity phenocopies age-dependent renal dysfunction in Drosophila Snakeskin mutants. J Cell Sci, 136(19) PubMed ID: 37694602
Summary:
Transporting epithelia provide a protective barrier against pathogenic insults while allowing the controlled exchange of ions, solutes and water with the external environment. In invertebrates, these functions depend on formation and maintenance of 'tight' septate junctions (SJs). However, the mechanism by which SJs affect transport competence and tissue homeostasis, and how these are modulated by ageing, remain incompletely understood. This study demonstrated that the Drosophila renal (Malpighian) tubules undergo an age-dependent decline in secretory capacity, which correlates with mislocalisation of SJ proteins and progressive degeneration in cellular morphology and tissue homeostasis. Acute loss of the SJ protein Snakeskin in adult tubules induced progressive changes in cellular and tissue architecture, including altered expression and localisation of junctional proteins with concomitant loss of cell polarity and barrier integrity, demonstrating that compromised junctional integrity is sufficient to replicate these ageing-related phenotypes. Taken together, this work demonstrates a crucial link between epithelial barrier integrity, tubule transport competence, renal homeostasis and organismal viability, as well as providing novel insights into the mechanisms underpinning ageing and renal disease.
Williams-Simon, P. A., Oster, C., Moaton, J. A., Ghidey, R., Ng'oma, E., Middleton, K. M., Zars, T. and King, E. G. (2023). Naturally segregating genetic variants contribute to thermal tolerance in a D. melanogaster model system. bioRxiv. PubMed ID: 37461510
Summary:
Thermal tolerance is a fundamental physiological complex trait for survival in many species. This study took a multipronged approach to dissect the genetic architecture that controls thermal tolerance in natural populations using the Drosophila Synthetic Population Resource (DSPR) as a model system. First, quantitative genetics and Quantitative Trait Loci (QTL) mapping were used to identify major effect regions within the genome that influences thermal tolerance, then integrated RNA-sequencing to identify differences in gene expression, and lastly, the RNAi system was used to 1) alter tissue-specific gene expression and 2) functionally validate the findings. This powerful integration of approaches not only allows for the identification of the genetic basis of thermal tolerance but also the physiology of thermal tolerance in a natural population, which ultimately elucidates thermal tolerance through a fitness-associated lens.
Swope, S. D., Jones, T. W., Mellina, K. N., Nichols, S. J., DiAngelo, J. R. (2023). Arc1 : a regulator of triglyceride homeostasis in male Drosophila. Micro Publication Biology. PubMed ID: 37675078
Summary:
Achieving metabolic homeostasis is necessary for survival, and many genes are required to control organismal metabolism. A genetic screen in Drosophila larvae identified putative fat storage genes including Arc1. Arc1 has been shown to act in neurons to regulate larval lipid storage; however, whether Arc1 functions to regulate adult metabolism is unknown. Arc1esm18 males store more fat than controls while both groups eat similar amounts. Arc1esm18 flies express more brummer lipase and less of the glycolytic enzyme triose phosphate isomerase, which may contribute to excess fat observed in these mutants. These results suggest that Arc1 regulates adult Drosophila lipid homeostasis.

Tuesday, February 6th - Autophagy

Qin, B., Yu, S., Chen, Q., Jin, L. H. (2023). Atg2 Regulates Cellular and Humoral Immunity in Drosophila. Insects, 14(8) PubMed ID: 37623416
Summary:
Autophagy is a process that promotes the lysosomal degradation of cytoplasmic proteins and is highly conserved in eukaryotic organisms. Autophagy maintains homeostasis in organisms and regulates multiple developmental processes, and autophagy disruption is related to human diseases. However, the functional roles of autophagy in mediating innate immune responses are largely unknown. This study sought to understand how Atg2, an autophagy-related gene, functions in the innate immunity of Drosophila melanogaster. The results showed that a large number of melanotic nodules were produced upon inhibition of Atg2. In addition, inhibiting Atg2 suppressed the phagocytosis of latex beads, Staphylococcus aureus and Escherichia coli; the proportion of Nimrod C1 (one of the phagocytosis receptors)-positive hemocytes also decreased. Moreover, inhibiting Atg2 altered actin cytoskeleton patterns, showing longer filopodia but with decreased numbers of filopodia. The expression of AMP-encoding genes was altered by inhibiting Atg2. Drosomycin was upregulated, and the transcript levels of Attacin-A, Diptericin and Metchnikowin were decreased. Finally, the above alterations caused by the inhibition of Atg2 prevented flies from resisting invading pathogens, showing that flies with low expression of Atg2 were highly susceptible to Staphylococcus aureus and Erwinia carotovora carotovora 15 infections. In conclusion, Atg2 regulated both cellular and humoral innate immunity in Drosophila. We have identified Atg2 as a crucial regulator in mediating the homeostasis of immunity, which further established the interactions between autophagy and innate immunity.
Szinyakovics, J., Keresztes, F., Kiss, E. A., Falcsik, G., Vellai, T. and Kovacs, T. (2023). Potent New Targets for Autophagy Enhancement to Delay Neuronal Ageing. Cells 12(13). PubMed ID: 37443788
Summary:
Autophagy is a lysosomal-dependent degradation process of eukaryotic cells responsible for breaking down unnecessary and damaged intracellular components. This study aimed to uncover new regulatory points where autophagy could be specifically activated and tested these potential drug targets in neurodegenerative disease models of Drosophila melanogaster. One possible way to activate autophagy is by enhancing autophagosome-lysosome fusion that creates the autolysosome in which the enzymatic degradation happens. The HOPS (homotypic fusion and protein sorting) and SNARE (Snap receptor) protein complexes regulate the fusion process. The HOPS complex forms a bridge between the lysosome and autophagosome with the assistance of small GTPase proteins. Thus, small GTPases are essential for autolysosome maturation, and among these proteins, Rab2 (Ras-associated binding 2), Rab7, and Arl8 (Arf-like 8) are required to degrade the autophagic cargo. For these experiments, Drosophila melanogaster was used as a model organism. Nerve-specific small GTPases were silenced and overexpressed. The effects were examined of these genetic interventions on lifespan, climbing ability, and autophagy. Finally, the activation of small GTPases was also studied in a Parkinson's disease model. The results revealed that GTP-locked, constitutively active Rab2 (Rab2-CA) and Arl8 (Arl8-CA) expression reduces the levels of the autophagic substrate p62/Ref(2)P in neurons, extends lifespan, and improves the climbing ability of animals during ageing. However, Rab7-CA expression dramatically shortens lifespan and inhibits autophagy. Rab2-CA expression also increases lifespan in a Parkinson's disease model fly strain overexpressing human mutant (A53T) α-synuclein protein. Data provided by this study suggests that Rab2 and Arl8 serve as potential targets for autophagy enhancement in the Drosophila nervous system.
Zhang, S., Yi, S., Wang, L., Li, S., Wang, H., Song, L., Ou, J., Zhang, M., Wang, R., Wang, M., Zheng, Y., Yang, K., Liu, T. and Ho, M. S. (2023). Cyclin-G-associated kinase GAK/dAux regulates autophagy initiation via ULK1/Atg1 in glia. Proc Natl Acad Sci U S A 120(29): e2301002120. PubMed ID: 37428930
Summary:
Autophagy is a major means for the elimination of protein inclusions in neurons in neurodegenerative diseases such as Parkinson's disease (PD). Yet, the mechanism of autophagy in the other brain cell type, glia, is less well characterized and remains largely unknown. This study presents evidence that the PD risk factor, Cyclin-G-associated kinase (GAK)/Drosophila homolog Auxilin (dAux), is a component in glial autophagy. The lack of GAK/dAux increases the autophagosome number and size in adult fly glia and mouse microglia, and generally up-regulates levels of components in the initiation and PI3K class III complexes. GAK/dAux interacts with the master initiation regulator UNC-51-like autophagy activating kinase 1/Atg1 via its uncoating domain and regulates the trafficking of Atg1 and Atg9 to autophagosomes, hence controlling the onset of glial autophagy. On the other hand, lack of GAK/dAux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/dAux might play additional roles. Importantly, dAux contributes to PD-like symptoms including dopaminergic neurodegeneration and locomotor function in flies. These findings identify an autophagy factor in glia; considering the pivotal role of glia under pathological conditions, targeting glial autophagy is potentially a therapeutic strategy for PD.
Bierlein, M., Charles, J., Polisuk-Balfour, T., Bretscher, H., Rice, M., Zvonar, J., Pohl, D., Winslow, L., Wasie, B., Deurloo, S., Van Wert, J., Williams, B., Ankney, G., Harmon, Z., Dann, E., Azuz, A., Guzman-Vargas, A., Kuhns, E., Neufeld, T. P., O'Connor, M. B., Amissah, F., Zhu, C. C. (2023). Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster). Genetics, 225(2) PubMed ID: >37594076
Summary:
Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. This report shows that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies but the lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Overexpression of wild-type Atg1 in adult fly muscle or adipocytes reduces the lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. This research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation.
Pino-Jimenez, B., Giannios, P. and Casanova, J. (2023). Polyploidy-associated autophagy promotes larval tracheal histolysis at Drosophila metamorphosis. Autophagy: 1-10. PubMed ID: 37424089
Summary:
Polyploidy is an extended phenomenon in biology. However, its physiological significance and whether it defines specific cell behaviors is not well understood. Polyploidy connection to macroautophagy/autophagy was examined, using the larval respiratory system of Drosophila as a model. This system comprises cells with the same function yet with notably different ploidy status, namely diploid progenitors and their polyploid larval counterparts, the latter destined to die during metamorphosis. An association was identified between polyploidy and autophagy and higher endoreplication status was found to correlate with elevated autophagy. Finally, it is reported that tissue histolysis in the trachea during Drosophila metamorphosis is mediated by autophagy, which triggers the apoptosis of polyploid cells.
Barrio, L., Gaspar, A. E., Muzzopappa, M., Ghosh, K., Romao, D., Clemente-Ruiz, M., Milan, M. (2023). Chromosomal instability-induced cell invasion through caspase-driven DNA damage. Curr Biol, 33(20):4446-4457.e4445 PubMed ID: 37751744
Summary:
Chromosomal instability (CIN), an increased rate of changes in chromosome structure and number, is observed in most sporadic human carcinomas with high metastatic activity. This study used a Drosophila epithelial model to show that DNA damage, as a result of the production of lagging chromosomes during mitosis and aneuploidy-induced replicative stress, contributes to CIN-induced invasiveness. A sub-lethal role of effector caspases in invasiveness was unraveled by enhancing CIN-induced DNA damage and identify the JAK/STAT signaling pathway as an activator of apoptotic caspases through transcriptional induction of pro-apoptotic genes. Evidence is provided that an autocrine feedforward amplification loop mediated by Upd3-a cytokine with homology to interleukin-6 and a ligand of the JAK/STAT signaling pathway-contributes to amplifying the activation levels of the apoptotic pathway in migrating cells, thus promoting CIN-induced invasiveness. This work sheds new light on the chromosome-signature-independent effects of CIN in metastasis.

Monday, February 5th - Splicing, RNAs, and RNAi

Wang, Y., Li, H., Liu, X., Gao, L., Fan, Y., Zhu, K. Y. and Zhang, J. (2023). Three alternative splicing variants of Loquacious play different roles in miRNA- and siRNA-mediated RNAi pathways in Locusta migratoria. RNA Biol 20(1): 323-333. PubMed ID: 37310197
Summary:
RNA interference (RNAi) is a specific post-transcriptional gene-silencing phenomenon, which plays an important role in the regulation of gene expression and the protection from transposable elements in eukaryotic organisms. In Drosophila melanogaster, RNAi can be induced by microRNA (miRNA), endogenous small interfering RNA (siRNA), or exogenous siRNA. However, the biogenesis of miRNA and siRNA in these RNAi pathways is aided by the double-stranded RNA binding proteins (dsRBPs) Loquacious (Loqs)-PB, Loqs-PD or R2D2. This studyidentified three alternative splicing variants of Loqs, namely Loqs-PA, -PB, and -PC in the orthopteran Locusta migratoria. in vitro and in vivo experiments were performed to study the roles of the three Loqs variants in the miRNA- and siRNA-mediated RNAi pathways. The results show that Loqs-PB assists the binding of pre-miRNA to Dicer-1 to lead to the cleavage of pre-miRNA to yield matured miRNA in the miRNA-mediated RNAi pathway. In contrast, different Loqs proteins participate in different siRNA-mediated RNAi pathways. In exogenous siRNA-mediated RNAi pathway, binding of Loqs-PA or LmLoqs-PB to exogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2, whereas in endogenous siRNA-mediated RNAi pathway, binding of Loqs-PB or Loqs-PC to endogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2. These findings provide new insights into the functional importance of different Loqs proteins derived from alternative splicing variants of Loqs in achieving high RNAi efficiency in different RNAi pathways in insects.
Yadav, A. K., Butler, C., Yamamoto, A., Patil, A. A., Lloyd, A. L. and Scott, M. J. (2023). CRISPR/Cas9-based split homing gene drive targeting doublesex for population suppression of the global fruit pest Drosophila suzukii. Proc Natl Acad Sci U S A 120(25): e2301525120. PubMed ID: 37307469
Summary:
Genetic-based methods offer environmentally friendly species-specific approaches for control of insect pests. One method, CRISPR homing gene drive that target genes essential for development, could provide very efficient and cost-effective control. While significant progress has been made in developing homing gene drives for mosquito disease vectors, little progress has been made with agricultural insect pests. This study reports the development and evaluation of split homing drives that target the doublesex (dsx) gene in Drosophila suzukii, an invasive pest of soft-skinned fruits. The drive component, consisting of dsx single guide RNA and DsRed genes, was introduced into the female-specific exon of dsx, which is essential for function in females but not males. However, in most strains, hemizygous females were sterile and produced the male dsx transcript. With a modified homing drive that included an optimal splice acceptor site, hemizygous females from each of the four independent lines were fertile. High transmission rates of the DsRed gene (94 to 99%) were observed with a line that expressed Cas9 with two nuclear localization sequences from the D. suzukii nanos promoter. Mutant alleles of dsx with small in-frame deletions near the Cas9 cut site were not functional and thus would not provide resistance to drive. Finally, mathematical modeling showed that the strains could be used for suppression of lab cage populations of D. suzukii with repeated releases at relatively low release ratios (1:4). These results indicate that the split CRISPR homing gene drive strains could potentially provide an effective means for control of D. suzukii populations.
Warden, M. S., DeRose, E. F., Tamayo, J. V., Mueller, G. A., Gavis, E. R. and Hall, T. M. T. (2023). The translational repressor Glorund uses interchangeable RNA recognition domains to recognize Drosophila nanos. Nucleic Acids Res. PubMed ID: 37427795
Summary:
The Drosophila melanogaster protein Glorund (Glo) represses nanos (nos) translation and uses its quasi-RNA recognition motifs (qRRMs) to recognize both G-tract and structured UA-rich motifs within the nos translational control element (TCE). It has been shown previously that each of the three qRRMs is multifunctional, capable of binding to G-tract and UA-rich motifs, yet if and how the qRRMs combine to recognize the nos TCE remained unclear. This study determined solution structures of a nos TCEI_III RNA containing the G-tract and UA-rich motifs. The RNA structure demonstrated that a single qRRM is physically incapable of recognizing both RNA elements simultaneously. In vivo experiments further indicated that any two qRRMs are sufficient to repress nos translation. interactions of Glo qRRMs were probed with TCEI_III RNA using NMR paramagnetic relaxation experiments. The in vitro and in vivo data support a model whereby tandem Glo qRRMs are indeed multifunctional and interchangeable for recognition of TCE G-tract or UA-rich motifs. This study illustrates how multiple RNA recognition modules within an RNA-binding protein may combine to diversify the RNAs that are recognized and regulated.
Zhai, R., Ruan, K., Perez, G. F., Kubat, M., Liu, J., Hofacker, I. and Wuchty, S. (2023). MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS): a global mechanism for the regulation of alternative splicing. Res Sq. PubMed ID: 37546804
Summary:
While RNA secondary structures are critical to regulate alternative splicing of long-range pre-mRNA, the factors that modulate RNA structure and interfere with the recognition of the splice sites are largely unknown. Previously, a small, non-coding microRNA was identified that sufficiently affects stable stem structure formation of Nmnat pre-mRNA to regulate the outcomes of alternative splicing. However, the fundamental question remains whether such microRNA-mediated interference with RNA secondary structures is a global molecular mechanism for regulating mRNA splicing. A bioinformatic pipeline was designed and refined to predict candidate microRNAs that potentially interfere with pre-mRNA stem-loop structures, and splicing predictions were experimentally verified of three different long-range pre-mRNAs in the Drosophila model system. Specifically, it was observed that microRNAs can either disrupt or stabilize stem-loop structures to influence splicing outcomes. Fhiz study suggests that MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing, increases the repertoire of microRNA function and further indicates cellular complexity of post-transcriptional regulation.
Lee, S., Kim, N., Jang, D., Kim, H. K., Kim, J., Jeon, J. W., Lim, D. H. (2023). Ecdysone-induced microRNA miR-276a-3p controls developmental growth by targeting the insulin-like receptor in Drosophila. Insect Mol Biol, 32(6):703-715 PubMed ID: 37702106
Summary:
Animal growth is controlled by a variety of external and internal factors during development. The steroid hormone ecdysone plays a critical role in insect development by regulating the expression of various genes. In this study, it was found that fat body-specific expression of miR-276a, an ecdysone-responsive microRNA (miRNA), led to a decrease in the total mass of the larval fat body, resulting in significant growth reduction in Drosophila. Changes in miR-276a expression also affected the proliferation of Drosophila S2 cells. Furthermore, it was found that the insulin-like receptor (InR) is a biologically relevant target gene regulated by miR-276a-3p. In addition, its was found that miR-276a-3p is upregulated by the canonical ecdysone signalling pathway involving the ecdysone receptor and broad complex. A reduction in cell proliferation caused by ecdysone was compromised by blocking miR-276a-3p activity. Thus, these results suggest that miR-276a-3p is involved in ecdysone-mediated growth reduction by controlling InR expression in the insulin signalling pathway.
Wilinski, D. and Dus, M. (2023). N(6)-adenosine methylation controls the translation of insulin mRNA. Nat Struct Mol Biol. PubMed ID: 37488356
Summary:
Control of insulin mRNA translation is crucial for energy homeostasis, but the mechanisms remain largely unknown. This study discovered that insulin mRNAs across invertebrates, vertebrates and mammals feature the modified base N(6)-methyladenosine (m(6)A). In flies, this RNA modification enhances insulin mRNA translation by promoting the association of the transcript with polysomes. Depleting m(6)A in Drosophila melanogaster insulin 2 mRNA (dilp2) directly through specific 3' untranslated region (UTR) mutations, or indirectly by mutating the m(6)A writer Mettl3, decreases dilp2 protein production, leading to aberrant energy homeostasis and diabetic-like phenotypes. Together, these findings reveal adenosine mRNA methylation as a key regulator of insulin protein synthesis with notable implications for energy balance and metabolic disease.

Friday, February 2nd - Adult Neural Development and Function

Garcia-Vaquero, M. L., Heim, M., Flix, B., Pereira, M., Palin, L., Marques, T. M., Pinto, F. R., de Las Rivas, J., Voigt, A., Besse, F., Gama-Carvalho, M. (2023). Analysis of asymptomatic Drosophila models for ALS and SMA reveals convergent impact on functional protein complexes linked to neuro-muscular degeneration. BMC Genomics, 24(1):576 PubMed ID: 37759179
Summary:
Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 (TBPH) and FUS. There is currently no model to explain the resulting motor neuron dysfunction. This study generated parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, a computational approach was devised based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown. Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through the integrative approach it was possible to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes. These results show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings.
Upadhyay, A., Chhangani, D., Rao, N. R., Kofler, J., Vassar, R., Rincon-Limas, D. E., Savas, J. N. (2023). Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity. Molecular neurodegeneration, 18(1):61 PubMed ID: 37710351
Summary:
The accumulation of amyloid beta (β) peptides in fibrils is prerequisite for Alzheimer's disease (AD). understanding of the proteins that promote Aβ (see Drosophila jmuller/appl1.htm) fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. To investigate how amyloid fibrils form and cause neurotoxicity in AD brain, this study developed a robust biochemical strategy. The success of these purifications was benchmarked using electron microscopy, amyloid dyes, and a large panel of Aβ immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed comparison oc amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic Aβ42 flies, and Aβ42 seeded cultured neurons. Amyloid fibrils were found to be primarily composed by Aβ42 and unexpectedly harbor Aβ38 but generally lacked Aβ40 peptides. Multidimensional quantitative proteomics allowed redefinition of the fibril proteome by identifying 20 new amyloid-associated proteins. Notably, 57 previously reported plaque-associated proteins were identified. A panel of these proteins was validated as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 was found to be tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, a transgenic Aβ42 fly model was used to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Here, it was possible to functionally validate 20 genes as modifiers of Aβ42 toxicity in vivo. These discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.
Stewart, T. M., Foley, J. R., Holbert, C. E., Khomutov, M., Rastkari, N., Tao, X., Khomutov, A. R., Zhai, R. G., Casero, R. A. (2023). Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome. EMBO molecular medicine, 15(11):e17833 PubMed ID: 37702369
Summary:
Snyder-Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. The current studies aimed to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. The repurposing of 2-difluoromethylornithine (DFMO), an FDA-approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells is reported. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.
Luedke, K. P., Yoshino, J., Yin, C., Jiang, N., Huang, J. M., Huynh, K., Parrish, J. Z. (2023). Dendrite intercalation between epidermal cells tunes nociceptor sensitivity to mechanical stimuli in Drosophila larvae. bioRxiv PubMed ID: 37745567
Summary:
An animal's skin provides a first point of contact with the sensory environment, including noxious cues that elicit protective behavioral responses. Nociceptive somatosensory neurons densely innervate and intimately interact with epidermal cells to receive these cues. This study identified a role for dendrite intercalation between epidermal cells in tuning sensitivity of Drosophila larvae to noxious mechanical stimuli. In wild-type larvae, dendrites of nociceptive class IV da neurons intercalate between epidermal cells at apodemes, which function as body wall muscle attachment sites. From a genetic screen miR-14 was identified as a regulator of dendrite positioning in the epidermis: miR-14 is expressed broadly in the epidermis but not in apodemes, and miR-14 inactivation leads to excessive apical dendrite intercalation between epidermal cells. miR-14 regulates expression and distribution of the epidermal Innexins ogre and Inx2 and that these epidermal gap junction proteins restrict epidermal dendrite intercalation. Finally, it was found that altering the extent of epidermal dendrite intercalation had corresponding effects on nociception: increasing epidermal intercalation sensitized larvae to noxious mechanical inputs and increased mechanically evoked calcium responses in nociceptive neurons, whereas reducing epidermal dendrite intercalation had the opposite effects. Altogether, these studies identify epidermal dendrite intercalation as a mechanism for mechanical coupling of nociceptive neurons to the epidermis, with nociceptive sensitivity tuned by the extent of intercalation.
Zhu, B. and Liu, S. (2023). Preservation of ∼12-hour ultradian rhythms of gene expression of mRNA and protein metabolism in the absence of canonical circadian clock. bioRxiv PubMed ID: 37205336
Summary:
Besides the ∼24-hour circadian rhythms, ∼12-hour ultradian rhythms of gene expression, metabolism and behaviors exist in animals ranging from crustaceans to mammals. Three major hypotheses were proposed on the origin and mechanisms of regulation of ∼12-hour rhythms, namely that they are not cell-autonomous and controlled by a combination of the circadian clock and environmental cues, that they are regulated by two anti-phase circadian transcriptional factors in a cell-autonomous manner, or that they are established by a cell-autonomous ∼12-hour oscillator. To distinguish among these possibilities, a post-hoc analysis was performed of two high temporal resolution transcriptome dataset in animals and cells lacking the canonical circadian clock. In both the liver of BMAL1 knockout mice and Drosophila S2 cells, robust and prevalent ∼12-hour rhythms of gene expression were observed, enriched in fundamental processes of mRNA and protein metabolism that show large convergence with those identified in wild-type mice liver. Bioinformatics analysis further predicted ELF1 (Drosophila Ecdysone-induced protein 74EF) and ATF6B (Drosophila Atf6) as putative transcription factors regulating the ∼12-hour rhythms of gene expression independently of the circadian clock in both fly and mice. These findings provide additional evidence to support the existence of an evolutionarily conserved 12-hour oscillator that controls ∼12-hour rhythms of gene expression of protein and mRNA metabolism in multiple species.
Zsindely, N., Nagy, G., Siagi, F., Farkas, A. and Bodai, L. (2023). Dysregulated miRNA and mRNA Expression Affect Overlapping Pathways in a Huntington's Disease Model. Int J Mol Sci 24(15). PubMed ID: 37569316
Summary:
Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the Huntingtin gene. Transcriptional dysregulation is one of the main cellular processes affected by mutant Huntingtin (mHtt). This study investigated the alterations in miRNA and mRNA expression levels in a Drosophila model of HD by RNA sequencing and assessed the functional effects of misregulated miRNAs in vivo. in head samples of HD flies, the level of 32 miRNAs were found to change significantly; half of these were upregulated, while the other half were downregulated. After comparing miRNA and mRNA expression data, similarities were discovered in the impacted molecular pathways. Additionally, it was observed that the putative targets of almost all dysregulated miRNAs were overrepresented were tested among the upregulated mRNAs. The effects were tested of overexpression of five misregulated miRNAs in the HD model, and it was found that while mir-10 and mir-219 enhanced, mir-137, mir-305, and mir-1010 ameliorated mHtt-induced phenotypes. Based on these results, it is proposed that while altered expression of mir-10, mir-137, and mir-1010 might be part of HD pathology, the upregulation of mir-305 might serve as a compensatory mechanism as a response to mHtt-induced transcriptional dysregulation.

Wednesday, January 31st - Gonads

Zheng, Y., Mao, B., Wang, Q., Duan, X., Chen, M. Y., Shen, W., Li, C. and Wang, Y. F. (2023). Quantitative proteomics and phosphoproteomics reveal insights into mechanisms of ocnus function in Drosophila testis development. BMC Genomics 24(1): 283. PubMed ID: 37237333
Summary:
Testis has the largest number of proteins and tissue-specific proteins in animals. Knockdown of ocnus (ocn), a testis-specific gene, has been shown to result in much smaller testis with no germ cells in Drosophila. Through iTRAQ quantitative proteomics sequencing, 606 proteins were identified as having a significant and at least a 1.5-fold change in expression after ocn knockdown in fly testes; 85 were up-regulated and 521 were down-regulated. Among the differential expressed proteins (DEPs) were proteins that affected generation of precursor metabolites and energy, metabolic process, and mitochondrial transport. Protein-protein interaction (PPI) analyses of DEPs showed that several kinases and/or phosphatases interacted with Ocn. qRT-PCR confirmed 12 genes appeared in both DEGs and DEPs were significantly down-regulated after ocn knockdown in testes. Furthermore, 153 differentially expressed phosphoproteins (DEPPs), including 72 up-regulated and 94 down-regulated ones were also identified. In addition to those DEPPs associated with spermatogenesis, the other DEPPs were enriched in actin filament-based process, protein folding, and mesoderm development. Some DEPs and DEPPs were involved in Notch, JAK/STAT, and cell death pathways. The differences in protein abundance in the ocn knockdown flies might not necessarily be the direct result of differential gene regulation due to the inactivation of ocn. Nevertheless, these results suggest that the expression of ocn is essential for Drosophila testis development and that its down-regulation disturbs key signaling pathways related to cell survival and differentiation.
Tu, R., Tang, X. A., Xu, R., Ping, Z., Yu, Z. and Xie, T. (2023). Gap junction-transported cAMP from the niche controls stem cell progeny differentiation. Proc Natl Acad Sci U S A 120(35): e2304168120. PubMed ID: 37603749
Summary:
The niche has been shown to control stem cell self-renewal in different tissue types and organisms. Recently, a separate niche has been proposed to control stem cell progeny differentiation, called the differentiation niche. However, it remains poorly understood whether and how the differentiation niche directly signals to stem cell progeny to control their differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells contribute to two separate niche compartments for controlling both germline stem cell (GSC) self-renewal and progeny differentiation. This study shows that IGS cells express Inx2 protein, which forms gap junctions (GJs) with germline-specific Zpg protein to control stepwise GSC lineage development, including GSC self-renewal, germline cyst formation, meiotic double-strand DNA break formation, and oocyte specification. Germline-specific Zpg and IGS-specific Inx2 knockdowns cause similar defects in stepwise GSC development. Additionally, secondary messenger cAMP is transported from IGS cells to GSCs and their progeny via GJs to activate PKA signaling for controlling stepwise GSC development. Therefore, this study demonstrates that the niche directly controls GSC progeny differentiation via the GJ-cAMP-PKA signaling axis, which provides important insights into niche control of stem cell differentiation and highlights the importance of GJ-transported cAMP in tissue regeneration. This may represent a general strategy for the niche to control adult stem cell development in various tissue types and organisms since GJs and cAMP are widely distributed.
Santos, I. B., Wainman, A., Garrido-Maraver, J., Pires, V., Riparbelli, M. G., Kovacs, L., Callaini, G., Glover, D. M. and Tavares A, A. (2023). Mob4 is essential for spermatogenesis in Drosophila melanogaster. Genetics 224(4). PubMed ID: 37259670
Summary:
Gamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature sperm. In Drosophila melanogaster, individualization of interconnected spermatids requires the formation of individualization complexes that synchronously move along the sperm bundles. This study showed that Mob4, a member of the Mps-one binder family, is essential for male fertility but has no detectable role in female fertility. Mob4 was shown to be required for proper axonemal structure and its loss leads to male sterility associated with defective spermatid individualization and absence of mature sperm in the seminal vesicles. Transmission electron micrographs of developing spermatids following mob4RNAi revealed expansion of the outer axonemal microtubules such that the 9 doublets no longer remained linked to each other and defective mitochondrial organization. Mob4 is a STRIPAK component, and male fertility is similarly impaired upon depletion of the STRIPAK components, Strip and Cka. Expression of the human Mob4 gene rescues all phenotypes of Drosophila mob4 downregulation, indicating that the gene is evolutionarily and functionally conserved. Together, this suggests that Mob4 contributes to the regulation of the microtubule- and actin-cytoskeleton during spermatogenesis through the conserved STRIPAK complex. This study advances the understanding of male infertility by uncovering the requirement for Mob4 in sperm individualization.
Ryniawec, J. M., Hannaford, M. R., Zibrat, M. E., Fagerstrom, C. J., Galletta, B. J., Aguirre, S. E., Guice, B. A., Dean, S. M., Rusan, N. M., Rogers, G. C. (2023). Cep104 is a component of the centriole distal tip complex that regulates centriole growth and contributes to Drosophila spermiogenesis. Curr Biol, 33(19):4202-4216 PubMed ID: 37729913
Summary:
Proper centrosome number and function relies on the accurate assembly of centrioles, barrel-shaped structures that form the core duplicating elements of the organelle. The growth of centrioles is regulated in a cell cycle-dependent manner; while new daughter centrioles elongate during the S/G2/M phase, mature mother centrioles maintain their length throughout the cell cycle. Centriole length is controlled by the synchronized growth of the microtubules that ensheathe the centriole barrel. Although proteins exist that target the growing distal tips of centrioles, such as Cep97, these proteins are generally thought to suppress centriolar microtubule growth, suggesting that distal tips may also contain unidentified counteracting factors that facilitate microtubule polymerization. Currently, a mechanistic understanding of how distal tip proteins balance microtubule growth and shrinkage to either promote daughter centriole elongation or maintain centriole length is lacking. Using a proximity-labeling screen in Drosophila cells, this study identified Cep104 as a novel component of a group of evolutionarily conserved proteins that were collectively refered to as the distal tip complex (DTC). Cep104 was shown to regulats centriole growth and promote centriole elongation through its microtubule-binding TOG domain. Furthermore, analysis of Cep104 null flies revealed that Cep104 and Cep97 cooperate during spermiogenesis to align spermatids and coordinate individualization. Lastly, the complete DTC interactome was mapped, and Cep97 is the central scaffolding unit required to recruit DTC components to the distal tip of centrioles.
Yildirim, K., van Nierop, Y. S. P. and Lohmann, I. (2023). Analysis of Bub3 and Nup75 in the Drosophila male germline lineage. Cells Dev 175: 203863. PubMed ID: 37286104
Summary:
Extensive communication at the stem cell-niche interface and asymmetric stem cell division is key for the homeostasis of the Drosophila male germline stem cell system. To improve understanding of these processes, the function of the mitotic checkpoint complex (MCC) component Bub3 and the nucleoporin Nup75, a component of the nuclear pore complex realizing the transport of signalling effector molecules to the nucleus, were analyzed in the Drosophila testis. By lineage-specific interference, it was found that the two genes control germline development and maintenance. Bub3 is continuously required in the germline, as its loss results in the beginning in an over-proliferation of early germ cells and later on in loss of the germline. The absence of the germline lineage in such testes has dramatic cell non-autonomous consequences, as cells co-expressing markers of hub and somatic cyst cell fates accumulate and populate in extreme cases the whole testis. This analysis of Nups showed that some of them are critical for lineage maintenance, as their depletion results in the loss of the affected lineage. In contrast, Nup75 plays a role in controlling proliferation of early germ cells but not differentiating spermatogonia and seems to be involved in keeping hub cells quiescent. In sum, this analysis shows that Bub3 and Nup75 are required for male germline development and maintenance.
Knudsen, C., Woo Seuk, K., Izumikawa, T., Nakato, E., Akiyama, T., Kinoshita-Toyoda, A., Haugstad, G., Yu, G., Toyoda, H., Nakato, H. (2023). Chondroitin sulfate is required for follicle epithelial integrity and organ shape maintenance in Drosophila. Development, 150(17) PubMed ID: 37694610
Summary:
Heparan sulfate (HS) and chondroitin sulfate (CS) are evolutionarily conserved glycosaminoglycans that are found in most animal species, including the genetically tractable model organism Drosophila. In contrast to extensive in vivo studies elucidating co-receptor functions of Drosophila HS proteoglycans (PGs), only a limited number of studies have been conducted for those of CSPGs. To investigate the global function of CS in development, mutants were generated for Chondroitin sulfate synthase (Chsy), which encodes the Drosophila homolog of mammalian chondroitin synthase 1, a crucial CS biosynthetic enzyme. Characterizations of the Chsy mutants indicated that a fraction survive to adult stage, which allowed analysis of the morphology of the adult organs. In the ovary, Chsy mutants exhibited altered stiffness of the basement membrane and muscle dysfunction, leading to a gradual degradation of the gross organ structure as mutant animals aged. These observations show that normal CS function is required for the maintenance of the structural integrity of the ECM and gross organ architecture.

Tuesday, January 30th - Adult Neural Structure and Function

Boyan, G., Williams, L., Ehrhardt, E. (2023). Central projections from Johnston's organ in the locust: Axogenesis and brain neuroarchitecture. Dev Genes Evol, 233(2):147-159 PubMed ID: 37695323
Summary:
Johnston's organ (Jo) acts as an antennal wind-sensitive and/or auditory organ across a spectrum of insect species and its axons universally project to the brain. In the locust, this pathway is already present at mid-embryogenesis but the process of fasciculation involved in its construction has not been investigated. Terminal projections into the fine neuropilar organization of the brain also remain unresolved, information essential not only for understanding the neural circuitry mediating Jo-mediated behavior but also for providing comparative data offering insights into its evolution. In this study, neuron-specific, axon-specific, and epithelial domain labels were employed to show that the pathway to the brain of the locust is built in a stepwise manner during early embryogenesis as processes from Jo cell clusters in the pedicel fasciculate first with one another, and then with the two tracts constituting the pioneer axon scaffold of the antenna. A comparison of fasciculation patterns confirms that projections from cell clusters of Jo stereotypically associate with only one axon tract according to their location in the pedicellar epithelium, consistent with a topographic plan. At the molecular level, all neuronal elements of the Jo pathway to the brain express the lipocalin Lazarillo, a cell surface epitope that regulates axogenesis in the primary axon scaffold itself, and putatively during fasciculation of the Jo projections to the brain. Central projections from Jo first contact the primary axon scaffold of the deutocerebral brain at mid-embryogenesis, and in the adult traverse mechanosensory/motor neuropils similar to those in Drosophila. These axons then terminate among protocerebral commissures containing premotor interneurons known to regulate flight behavior.
Zhuravlev, A. V., Zalomaeva, E. S., Egozova, E. S., Sokurova, V. V., Nikitina, E. A. and Savvateeva-Popova, E. V. (2023). LIM-kinase 1 effects on memory abilities and male courtship song in Drosophila depend on the neuronal type.. Vavilovskii Zhurnal Genet Selektsii 27(3): 250-263. PubMed ID: 37293442
Summary:
The signal pathway of actin remodeling, including LIM-kinase 1 (LIMK1) and its substrate cofilin, regulates multiple processes in neurons of vertebrates and invertebrates. Drosophila melanogaster is widely used as a model object for studying mechanisms of memory formation, storage, retrieval and forgetting. Previously, active forgetting in Drosophila was investigated in the standard Pavlovian olfactory conditioning paradigm. The role of specific dopaminergic neurons (DAN) and components of the actin remodeling pathway in different forms of forgetting was shown. This research investigated the role of LIMK1 in Drosophila memory and forgetting in the conditioned courtship suppression paradigm (CCSP). In the Drosophila brain, LIMK1 and p-cofilin levels appeared to be low in specific neuropil structures, including the mushroom body (MB) lobes and the central complex. At the same time, LIMK1 was observed in cell bodies, such as DAN clusters regulating memory formation in CCSP. GAL4 x UAS binary system was applied to induce limk1 RNA interference in different types of neurons. The hybrid strain with limk1 interference in MB lobes and glia showed an increase in 3-h short-term memory (STM), without significant effects on long-term memory. limk1 interference in cholinergic neurons (CHN) impaired STM, while its interference in DAN and serotoninergic neurons (SRN) also dramatically impaired the flies' learning ability. By contrast, limk1 interference in fruitless neurons (FRN) resulted in increased 15-60 min STM, indicating a possible LIMK1 role in active forgetting. Males with limk1 interference in CHN and FRN also showed the opposite trends of courtship song parameters changes. Thus, LIMK1 effects on the Drosophila male memory and courtship song appeared to depend on the neuronal type or brain structure.
Zhu, J., Boivin, J. C., Pang, S., Xu, C. S., Lu, Z., Saalfeld, S., Hess, H. F. and Ohyama, T. (2023). Comparative connectomics and escape behavior in larvae of closely related Drosophila species. Curr Biol 33(12): 2491-2503. PubMed ID: 37285846
Summary:
Evolution has generated an enormous variety of morphological, physiological, and behavioral traits in animals. How do behaviors evolve in different directions in species equipped with similar neurons and molecular components? This study adopted a comparative approach to investigate the similarities and differences of escape behaviors in response to noxious stimuli and their underlying neural circuits between closely related drosophilid species. Drosophilids show a wide range of escape behaviors in response to noxious cues, including escape crawling, stopping, head casting, and rolling. This study found that D. santomea, compared with its close relative D. melanogaster, shows a higher probability of rolling in response to noxious stimulation. To assess whether this behavioral difference could be attributed to differences in neural circuitry, focused ion beam-scanning electron microscope volumes of the ventral nerve cord of D. santomea were generated to reconstruct the downstream partners of mdIV, a nociceptive sensory neuron in D. melanogaster. Along with partner interneurons of mdVI (including Basin-2, a multisensory integration neuron necessary for rolling) previously identified in D. melanogaster, two additional partners of mdVI were identified in D. santomea. Finally, this study showed that joint activation of one of the partners (Basin-1) and a common partner (Basin-2) in D. melanogaster increased rolling probability, suggesting that the high rolling probability in D. santomea is mediated by the additional activation of Basin-1 by mdIV. These results provide a plausible mechanistic explanation for how closely related species exhibit quantitative differences in the likelihood of expressing the same behavior.
Zhang, Y., Zhang, Y., Shen, C., Hao, S., Duan, W., Liu, L. and Wei, H. (2023). . Ionizing radiation alters functional neurotransmission in Drosophila larvae. Front Cell Neurosci 17: 1151489. PubMed ID: 37484822
Summary:
Patients undergoing cranial ionizing radiation therapy for brain malignancies are at increased risk of long-term neurocognitive decline, which is poorly understood and currently untreatable. Although the molecular pathogenesis has been intensively researched in many organisms, whether and how ionizing radiation alters functional neurotransmission remains unknown. This is the first study addressing physiological changes in neurotransmission after ionizing radiation exposure. To elucidate the cellular mechanisms of radiation damage, using calcium imaging, the effects were analyzed of ionizing radiation on the neurotransmitter-evoked responses of prothoracicotropic hormone (PTTH)-releasing neurons in Drosophila larvae, which play essential roles in normal larval development. The neurotransmitters dopamine and tyramine decreased intracellular calcium levels of PTTH neurons in a dose-dependent manner. In gamma irradiated third-instar larvae, a dose of 25 Gy increased the sensitivity of PTTH neurons to dopamine and tyramine, and delayed development, possibly in response to abnormal functional neurotransmission. This irradiation level did not affect the viability and arborization of PTTH neurons and successful survival to adulthood. Exposure to a 40-Gy dose of gamma irradiation decreased the neurotransmitter sensitivity, physiological viability and axo-dendritic length of PTTH neurons. These serious damages led to substantial developmental delays and a precipitous reduction in the percentage of larvae that survived to adulthood. These results demonstrate that gamma irradiation alters neurotransmitter-evoked responses, indicating synapses are vulnerable targets of ionizing radiation. The current study provides new insights into ionizing radiation-induced disruption of physiological neurotransmitter signaling, which should be considered in preventive therapeutic interventions to reduce risks of neurological deficits after photon therapy.
Petsakou, A., Liu, Y., Liu, Y., Comjean, A., Hu, Y., Perrimon, N. (2023). Cholinergic neurons trigger epithelial Ca(2+) currents to heal the gut. Nature, 623(7985):122-131. PubMed ID: 37722602 ID:
Summary:
A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the aetiology of chronic disorders such as inflammatory bowel diseases and cancer. This study used the Drosophila midgut to investigate this and discovered that during regeneration a subpopulation of cholinergic neurons triggers Ca(2+) currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. It was found that downregulation of the conserved cholinergic enzyme Acetylcholine esterase in the gut epithelium enables acetylcholine from specific Eiger (TNF in mammals)-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca(2+), which spreads in the epithelium through Innexin2-Innexin7 gap junctions, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki (YAP in humans) activation, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel disease. Altogether, the conserved cholinergic pathway facilitates epithelial Ca(2+) currents that heal the intestinal epithelium. These findings demonstrate nerve- and bioelectric-dependent intestinal regeneration and advance current understanding of how a tissue returns to homeostasis after injury.
Long, D. R., Kinser, A., Olalde-Welling, A., Brewer, L., Lim, J., Matheny, D., Long, B., Roossien, D. H. (2023). 5-HT1A regulates axon outgrowth in a subpopulation of Drosophila serotonergic neurons. Developmental neurobiology. 83(7-8):268-281 PubMed ID: 37714743
Summary:
Serotonergic neurons produce extensively branched axons that fill most of the central nervous system, where they modulate a wide variety of behaviors. Many behavioral disorders have been correlated with defective serotonergic axon morphologies. Proper behavioral output therefore depends on the precise outgrowth and targeting of serotonergic axons during development. To direct outgrowth, serotonergic neurons utilize serotonin as a signaling molecule prior to it assuming its neurotransmitter role. This process, termed serotonin autoregulation, regulates axon outgrowth, branching, and varicosity development of serotonergic neurons. However, the receptor that mediates serotonin autoregulation is unknown. This study asked if serotonin receptor 5-HT1A plays a role in serotonergic axon outgrowth and branching. Using cultured Drosophila serotonergic neurons, this study found that exogenous serotonin reduced axon length and branching only in those expressing 5-HT1A. Pharmacological activation of 5-HT1A led to reduced axon length and branching, whereas the disruption of 5-HT1A rescued outgrowth in the presence of exogenous serotonin. Altogether this suggests that 5-HT1A is a serotonin autoreceptor in a subpopulation of serotonergic neurons and initiates signaling pathways that regulate axon outgrowth and branching during Drosophila development.

Friday, January 26th - Signaling

Yu, D., Dai, Q., Wang, Z., Hou, S. X. and Sun, L. V. (2023). ARF1 maintains intestinal homeostasis by modulating gut microbiota and stem cell function. Life Sci 328: 121902. PubMed ID:
Summary:
The small GTPase protein
ARF1 has been shown to be involved in the lipolysis pathway and to selectively kill stem cells in Drosophila melanogaster. However, the role of ARF1 in mammalian intestinal homeostasis remains elusive. This study aimed to explore the role of ARF1 in intestinal epithelial cells (IECs) and reveal the possible mechanism. IEC-specific ARF1 deletion mouse model was used to evaluate the role of ARF1 in intestine. Immunohistochemistry and immunofluorescence analyses were performed to detect specific cell type markers, and intestinal organoids were cultured to assess intestinal stem cell (ISC) proliferation and differentiation. Fluorescence in situ hybridization, 16S rRNA-seq analysis, and antibiotic treatments were conducted to elucidate the role of gut microbes in ARF1-mediated intestinal function and the underlying mechanism. Colitis was induced in control and ARF1-deficient mice by dextran sulfate sodium (DSS). RNA-seq was performed to elucidate the transcriptomic changes after ARF1 deletion. ARF1 was shown to be essential for ISC proliferation and differentiation. Loss of ARF1 increased susceptibility to DSS-induced colitis and gut microbial dysbiosis. Gut microbiota depletion by antibiotics could rescue the intestinal abnormalities to a certain extent. Furthermore, RNA-seq analysis revealed alterations in multiple metabolic pathways. This work is the first to elucidate the essential role of ARF1 in regulating gut homeostasis, and provides novel insights into the pathogenesis of intestinal diseases and potential therapeutic targets.
Zhang, J., She, M., Dai, Y., Nie, X., Tang, M. and Zeng, Q. (2023). Lmpt regulates the function of Drosophila muscle by acting as a repressor of Wnt signaling. Gene 876: 147514. PubMed ID: 37245676
Summary:
LIM domain is considered to be important in mediating protein-protein interactions, and members of the LIM protein family can co-regulate tissue-specific gene expression by interacting with different transcription factors. However, its exact function in vivo remains unclear. This study demonstrates that the LIM protein family member Lmpt may act as a cofactor that interacts with other transcription factors to regulate cellular functions. This study generated Lmpt knockdown Drosophila (Lmpt-KD) using the UAS-Gal4 system. The lifespan and motility of Lmpt-KD Drosophila was assessed, and the expression of muscle-related and metabolism-related genes was analyzed using qRT-PCR. Additionally, Western blot and Top-Flash luciferase reporter assay were used to evaluate the level of the Wnt signaling pathway. This study revealed that knockdown of the Lmpt gene in Drosophila resulted in a shortened lifespan and reduced motility. A significant increase was observed in oxidative free radicals in the fly gut. Furthermore, qRT-PCR analysis indicated that knockdown of Lmpt led to decreased expression of muscle-related and metabolism-related genes in Drosophila, suggesting that Lmpt plays a crucial role in maintaining muscle and metabolic functions. Finally, it was found that reduction of Lmpt significantly upregulated the expression of Wnt signaling pathway proteins. These results demonstrate that Lmpt is essential for motility and survival in Drosophila and acts as a repressor in Wnt signaling.
Zhu, R., Santat, L. A., Markson, J. S., Nandagopal, N., Gregrowicz, J. and Elowitz, M. B. (2023). Reconstitution of morphogen shuttling circuits. Sci Adv 9(28): eadf9336. PubMed ID: 37436981
Summary:
Developing tissues form spatial patterns by establishing concentration gradients of diffusible signaling proteins called morphogens. The bone morphogenetic protein (BMP) morphogen pathway uses a family of extracellular modulators to reshape signaling gradients by actively 'shuttling' ligands to different locations. It has remained unclear what circuits are sufficient to enable shuttling, what other patterns they can generate, and whether shuttling is evolutionarily conserved. Using a synthetic, bottom-up approach, this study compared the spatiotemporal dynamics of different extracellular circuits. Three proteins-Chordin (Drosophila Sog), Twsg (Drosophila Tsg), and the BMP-1 protease (Drosophila Tolloid)-successfully displaced gradients by shuttling ligands away from the site of production. A mathematical model explained the different spatial dynamics of this and other circuits. Last, combining mammalian and Drosophila components in the same system suggests that shuttling is a conserved capability. Together, these results reveal principles through which extracellular circuits control the spatiotemporal dynamics of morphogen signaling.
Torres, A. Y., Nano, M., Campanale, J. P., Deak, S., Montell, D. J. (2023). Activated Src kinase promotes cell cannibalism in Drosophila. J Cell Biol, 222(11) PubMed ID: >37747450
Summary:
Src family kinases (SFKs) are evolutionarily conserved proteins acting downstream of receptors and regulating cellular processes including proliferation, adhesion, and migration. Elevated SFK expression and activity correlate with progression of a variety of cancers. Using the Drosophila melanogaster border cells as a model, this study reports that localized activation of a Src kinase promotes an unusual behavior: engulfment of one cell by another. By modulating Src expression and activity in the border cell cluster, it was found that increased Src kinase activity, either by mutation or loss of a negative regulator, is sufficient to drive one cell to engulf another living cell. A molecular mechanism was elucidated that requires integrins, the kinases SHARK and FAK, and Rho family GTPases, but not the engulfment receptor Draper. It is proposed that cell cannibalism is a result of aberrant phagocytosis, where cells with dysregulated Src activity fail to differentiate between living and dead or self versus non-self, thus driving this malignant behavior.
Tokamov, S. A., Nouri, N., Rich, A., Buiter, S., Glotzer, M., Fehon, R. G. (2023). Apical polarity and actomyosin dynamics control Kibra subcellular localization and function in Drosophila Hippo signaling. Dev Cell, 58(19): 1864-1879 PubMed ID: 37729921>
Summary:
The Hippo pathway is an evolutionarily conserved regulator of tissue growth that integrates inputs from both polarity and actomyosin networks. An upstream activator of the Hippo pathway, Kibra, localizes at the junctional and medial regions of the apical cortex in epithelial cells, and medial accumulation promotes Kibra activity. This study demonstrates that cortical Kibra distribution is controlled by a tug-of-war between apical polarity and actomyosin dynamics. This study show sthat while the apical polarity network, in part via atypical protein kinase C (aPKC), tethers Kibra at the junctional cortex to silence its activity, medial actomyosin flows promote Kibra-mediated Hippo complex formation at the medial cortex, thereby activating the Hippo pathway. This study provides a mechanistic understanding of the relationship between the Hippo pathway, polarity, and actomyosin cytoskeleton, and it offers novel insights into how fundamental features of epithelial tissue architecture can serve as inputs into signaling cascades that control tissue growth, patterning, and morphogenesis.
Zhou, Y., Guan, J., Meng, G., Fan, W., Ge, C., Niu, C., Cheng, Y., Fu, Y., Lu, Y. and Wei, Y. (2023). The RagA GTPase protects young egg chambers in Drosophila. Cell Rep 42(6): 112631. PubMed ID: 37302067
Summary:
The preservation of female fertility under unfavorable conditions is essential for animal reproduction. Inhibition of the target of rapamycin complex 1 (TORC1) is indispensable for Drosophila young egg chamber maintenance under nutrient starvation. This study shows that knockdown of RagA results in young egg chamber death independent of TORC1 hyperactivity. RagA RNAi ovaries have autolysosomal acidification and degradation defects, which make the young egg chambers sensitive to autophagosome augmentation. Meanwhile, RagA RNAi ovaries have nuclear-localized Mitf, which promotes autophagic degradation and protects young egg chambers under stress. Interestingly, GDP-bound RagA rescues autolysosome defects, while GTP-bound RagA rescues Mitf nuclear localization in RagA RNAi young egg chambers. Moreover, Rag GTPase activity, rather than TORC1 activity, controls Mitf cellular localization in the Drosophila germ line. This work suggests that RagA separately controls autolysosomal acidification and Mitf activity in the Drosophila young egg chambers.

Thursday, January 25th - Evolution

Zivanovic, G., Arenas, C. and Mestres, F. (2023). The Adaptive Value of Chromosomal Inversions and Climatic Change-Studies on the Natural Populations of Drosophila subobscura from the Balkans. Insects 14(7). PubMed ID: 37504602
Summary:
The adaptive value of the Drosophila subobscura chromosomal inversion polymorphism with regard to environmental effects is well-known. However, the specific details of the inversion adaptations to the global warming scenario deserve to be analyzed. Toward this aim, polymorphism and karyotypes were studied in 574 individuals from Petnica (Serbia) in annual samples taken in June for the period 2019-2022. Comparing the results of Petnica (Cfa: humid subtropical climate) with those from Avala (Serbia: Cfb, temperate oceanic climate) and Font Groga (Barcelona, Spain; Csa: hot-summer Mediterranean climate), significant differences were observed for their chromosomal polymorphism. In Petnica, inversions from U and E chromosomes mainly reacted significantly with regard to temperature, humidity, and rainfall. Moreover, the inversion polymorphism from Petnica (2019-2022) was compared with that from 1995. In this period, a significant increase in mean and maximum temperature was observed. However, to properly explain the observed variations of inversions over time, it was necessary to carefully analyze annual seasonal changes and particular heat wave episodes. Interestingly, yearly fluctuations of U chromosome 'warm'-adapted inversions corresponded with opposite changes in 'non-thermal' inversions. Perhaps these types of inversions were not correctly defined with regard to thermal adaptation, or these fluctuations were also due to adaptations to other physical and/or biological variables. Finally, a joint study of chromosomal inversion polymorphism from many Balkan populations of D. subobscura indicated that different climatic regions presented distinct composition, including thermal-adapted inversions.
Vedanayagam, J., Herbette, M., Mudgett, H., Lin, C. J., Lai, C. M., McDonough-Goldstein, C., Dorus, S., Loppin, B., Meiklejohn, C., Dubruille, R. and Lai, E. C. (2023). Essential and recurrent roles for hairpin RNAs in silencing de novo sex chromosome conflict in Drosophila simulans. PLoS Biol 21(6): e3002136. PubMed ID: 37289846
Summary:
Meiotic drive loci distort the normally equal segregation of alleles, which benefits their own transmission even in the face of severe fitness costs to their host organism. However, relatively little is known about the molecular identity of meiotic drivers, their strategies of action, and mechanisms that can suppress their activity. This study presents data from the fruitfly Drosophila simulans that address these questions. A family of de novo, protamine-derived X-linked selfish genes (the Dox gene family: Distorter on the X-which is found on the X chromosome and kills Y chromosome-bearing sperm) was demonstrated to be silenced by a pair of newly emerged hairpin RNA (hpRNA) small interfering RNA (siRNA)-class loci, Nmy and Tmy. In the w[XD1] genetic background, knockout of nmy derepresses Dox and MDox in testes and depletes male progeny, whereas knockout of tmy causes misexpression of PDox genes and renders males sterile. Importantly, genetic interactions between nmy and tmy mutant alleles reveal that Tmy also specifically maintains male progeny for normal sex ratio. The Dox loci are functionally polymorphic within D. simulans, such that both nmy-associated sex ratio bias and tmy-associated sterility can be rescued by wild-type X chromosomes bearing natural deletions in different Dox family genes. Finally, using tagged transgenes of Dox and PDox2, the first experimental evidence is provided that Dox family genes encode proteins that are strongly derepressed in cognate hpRNA mutants. Altogether, these studies support a model in which protamine-derived drivers and hpRNA suppressors drive repeated cycles of sex chromosome conflict and resolution that shape genome evolution and the genetic control of male gametogenesis.
Kannan, A., Dugand, R. J., Appleton, N. C., Chenoweth, S. F., Sgro, C. M., McGuigan, K. (2023). Environmental dependence of mutational (co)variances of adaptive traits. Evolution, 77(11):2341-2351. PubMed ID: 37668059
Summary:
Standing genetic variation, and capacity to adapt to environment change, will ultimately depend on the fitness effects of mutations across the range of environments experienced by contemporary, panmictic, populations. This study investigated how mild perturbations in diet and temperature affect mutational (co)variances of traits that evolve under climatic adaptation, and contribute to individual fitness in Drosophila serrata. Egg-to-adult viability, development time and wing size were assessed of 64 lines that had diverged from one another via spontaneous mutation over 30 generations of brother-sister mating. The results suggested most mutations have directionally concordant (i.e., synergistic) effects in all environments and both sexes. However, elevated mutational variance under reduced macronutrient conditions suggested environment-dependent variation in mutational effect sizes for development time. Evidence was also observed for antagonistic effects under standard versus reduced macronutrient conditions, where these effects were further contingent on temperature (for development time) or sex (for size). Diet also influenced the magnitude and sign of mutational correlations between traits, although this result was largely due to a single genotype (line), which may reflect a rare, large effect mutation. Overall, these results suggest environmental heterogeneity and environment-dependency of mutational effects could contribute to the maintenance of genetic variance.
Vedanayagam, J., Lin, C. J., Papareddy, R., Nodine, M., Flynt, A. S., Wen, J. and Lai, E. C. (2023). Regulatory logic of endogenous RNAi in silencing de novo genomic conflicts. PLoS Genet 19(6): e1010787. PubMed ID: 37343034
Summary:
Although the biological utilities of endogenous RNAi (endo-RNAi) have been largely elusive, recent studies reveal its critical role in the non-model fruitfly Drosophila simulans to suppress selfish genes, whose unchecked activities can severely impair spermatogenesis. In particular, hairpin RNA (hpRNA) loci generate endo-siRNAs that suppress evolutionary novel, X-linked, meiotic drive loci. The consequences of deleting even a single hpRNA (Nmy) in males are profound, as such individuals are nearly incapable of siring male progeny. In this study, comparative genomic analyses of D. simulans and D. melanogaster mutants of the core RNAi factor dcr-2 reveal a substantially expanded network of recently-emerged hpRNA-target interactions in the former species. The de novo hpRNA regulatory network in D. simulans provides insight into molecular strategies that underlie hpRNA emergence and their potential roles in sex chromosome conflict. In particular, these data support the existence of ongoing rapid evolution of Nmy/Dox-related networks, and recurrent targeting of testis HMG-box loci by hpRNAs. Importantly, the impact of the endo-RNAi network on gene expression flips the convention for regulatory networks, since strong derepression of targets of the youngest hpRNAs was observed, but only mild effects on the targets of the oldest hpRNAs. These data suggest that endo-RNAi are especially critical during incipient stages of intrinsic sex chromosome conflicts, and that continual cycles of distortion and resolution may contribute to speciation.
Yıldırım, B. and Vogl, C. (2023). Purifying selection against spurious splicing signals contributes to the base composition evolution of the polypyrimidine tract. J Evol Biol. PubMed ID: 37564008
Summary:
Among eukaryotes, the major spliceosomal pathway is highly conserved. While long introns may contain additional regulatory sequences, the ones in short introns seem to be nearly exclusively related to splicing. Although these regulatory sequences involved in splicing are well-characterized, little is known about their evolution. At the 3' end of introns, the splice signal nearly universally contains the dimer AG, which consists of purines, and the polypyrimidine tract upstream of this 3' splice signal is characterized by over-representation of pyrimidines. If the over-representation of pyrimidines in the polypyrimidine tract is also due to avoidance of a premature splicing signal, it is hypothesize that AG should be the most under-represented dimer. Through the use of DNA-strand asymmetry patterns, this prediction was confirmed in fruit flies of the genus Drosophila and by comparing the asymmetry patterns to a presumably neutrally evolving region, the selection strength acting on each motif was quantified. Moreover, the inference and simulation method of this study revealed that the best explanation for the base composition evolution of the polypyrimidine tract is the joint action of purifying selection against a spurious 3' splice signal and the selection for pyrimidines. Patterns of asymmetry in other eukaryotes indicate that avoidance of premature splicing similarly affects the nucleotide composition in their polypyrimidine tracts.
Chen, J., Gish, C. M., Fransen, J. W., Salazar-Gatzimas, E., Clark, D. A., Borghuis, B. G. (2023). Direct comparison reveals algorithmic similarities in fly and mouse visual motion detection. iScience, 26(10):107928 PubMed ID: 37810236
Summary:
Evolution has equipped vertebrates and invertebrates with neural circuits that selectively encode visual motion. While similarities in the computations performed by these circuits in mouse and fruit fly have been noted, direct experimental comparisons have been lacking. Because molecular mechanisms and neuronal morphology in the two species are distinct, this study directly compared motion encoding in these two species at the algorithmic level, using matched stimuli and focusing on a pair of analogous neurons, the mouse ON starburst amacrine cell (ON SAC) and Drosophila T4 neurons. It was found that the cells share similar spatiotemporal receptive field structures, sensitivity to spatiotemporal correlations, and tuning to sinusoidal drifting gratings, but differ in their responses to apparent motion stimuli. Both neuron types showed a response to summed sinusoids that deviates from models for motion processing in these cells, underscoring the similarities in their processing and identifying response features that remain to be explained.

Monday, January 22nd - Enhancers and Transcriptional Regulation

Thompson, K. D., Suber, W., Nicholas, R., Arnosti, D. N. (2023). Long-range repression by ecdysone receptor on complex enhancers of the insulin receptor gene. Fly (Austin), 17(1):2242238. PubMed ID: 37621079
Summary:
The insulin signalling pathway is evolutionarily conserved throughout metazoans, playing key roles in development, growth, and metabolism. Misregulation of this pathway is associated with a multitude of disease states including diabetes, cancer, and neurodegeneration. The human insulin receptor gene (INSR) is widely expressed throughout development and was previously described as a 'housekeeping' gene. Yet, there is abundant evidence that this gene is expressed in a cell-type specific manner, with dynamic regulation in response to environmental signals. The Drosophila insulin-like receptor gene (InR) is homologous to the human INSR gene and was previously shown to be regulated by multiple transcriptional elements located primarily within the introns of the gene. These elements were roughly defined in ~1.5 kbp segments, but an understanding of the potential detailed mechanisms of their regulation is lacking. The substructure of these cis-regulatory elements was characterized in Drosophila S2 cells, focusing on regulation through the ecdysone receptor (EcR) and the dFOXO transcription factor. By identifying specific locations of activators and repressors within 300 bp subelements, this study showed that some previously identified enhancers consist of relatively compact clusters of activators, while others have a distributed architecture not amenable to further reduction. In addition, these assays uncovered a long-range repressive action of unliganded EcR. The complex transcriptional circuitry likely endows InR with a highly flexible and tissue-specific response to tune insulin signalling. Further studies will provide insights to demonstrate the impact of natural variation in this gene's regulation, applicable to human genetic studies.
Nowling, R. J., Njoya, K., Peters, J. G., Riehle, M. M. (2023). Prediction accuracy of regulatory elements from sequence varies by functional sequencing technique. Frontiers in cellular and infection microbiology, 13:1182567 PubMed ID: >37600946
Summary:
Various sequencing based approaches are used to identify and characterize the activities of cis-regulatory elements in a genome-wide fashion. The activities of cis-regulatory elements such as enhancers, promoters, and repressors are determined by their sequence and secondary processes such as chromatin accessibility, DNA methylation, and bound histone markers. This study used machine learning models to evaluate the accuracy with which cis-regulatory elements identified by various commonly used sequencing techniques can be predicted by their underlying sequence alone to distinguish between cis-regulatory activity that is reflective of sequence content versus secondary processes. Models trained and evaluated on D. melanogaster sequences identified through DNase-seq and STARR-seq are significantly more accurate than models trained on sequences identified by H3K4me1, H3K4me3, and H3K27ac ChIP-seq, FAIRE-seq, and ATAC-seq. These results suggest that the activity detected by DNase-seq and STARR-seq can be largely explained by underlying DNA sequence, independent of secondary processes. Experimentally, a subset of DNase-seq and H3K4me1 ChIP-seq sequences were tested for enhancer activity using luciferase assays and compared with previous tests performed on STARR-seq sequences. The experimental data indicated that STARR-seq sequences are substantially enriched for enhancer-specific activity, while the DNase-seq and H3K4me1 ChIP-seq sequences are not. Taken together, these results indicate that the DNase-seq approach identifies a broad class of regulatory elements of which enhancers are a subset and the associated data are appropriate for training models for detecting regulatory activity from sequence alone, STARR-seq data are best for training enhancer-specific sequence models, and H3K4me1 ChIP-seq data are not well suited for training and evaluating sequence-based models for cis-regulatory element prediction.
Sloutskin, A., Itzhak, D., Vogler, G., Ideses, D., Alter, H., Shachar, H., Doniger, T., Frasch, M., Bodmer, R., Duttke, S. H. and Juven-Gershon, T. (2023). A single DPE core promoter motif contributes to in vivo transcriptional regulation and affects cardiac function. bioRxiv. PubMed ID: 37398300
Summary:
Transcription is initiated at the core promoter, which confers specific functions depending on the unique combination of core promoter elements. The downstream core promoter element (DPE) is found in many genes related to heart and mesodermal development. However, the function of these core promoter elements has thus far been studied primarily in isolated, in vitro or reporter gene settings. tinman (tin) encodes a key transcription factor that regulates the formation of the dorsal musculature and heart. Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, this study showed that a substitution mutation of the functional tin DPE motif within the natural context of the core promoter results in a massive perturbation of Tinman's regulatory network orchestrating dorsal musculature and heart formation. Mutation of endogenous tin DPE reduced the expression of tin and distinct target genes, resulting in significantly reduced viability and an overall decrease in adult heart function. This study has demonstrated the feasibility and importance of characterizing DNA sequence elements in vivo in their natural context, and accentuate the critical impact a single DPE motif has during Drosophila embryogenesis and functional heart formation.
van den Berg, L., Kokki, K., Wowro, S. J., Petricek, K. M., Deniz, O., Stegmann, C. A., Robciuc, M., Teesalu, M., Melvin, R. G., Nieminen, A. I., Schupp, M. and Hietakangas, V. (2023). Sugar-responsive inhibition of Myc-dependent ribosome biogenesis by Clockwork orange. Cell Rep 42(7): 112739. PubMed ID: 37405919
Summary:
The ability to feed on a sugar-containing diet depends on a gene regulatory network controlled by the intracellular sugar sensor Mondo/ChREBP-Mlx, which remains insufficiently characterized. This study presents a genome-wide temporal clustering of sugar-responsive gene expression in Drosophila larvae. Gene expression programs responding to sugar feeding were identified, including downregulation of ribosome biogenesis genes, known targets of Myc. Clockwork orange (CWO), a component of the circadian clock, is found to be a mediator of this repressive response and to be necessary for survival on a high-sugar diet. CWO expression is directly activated by Mondo-Mlx, and it counteracts Myc through repression of its gene expression and through binding to overlapping genomic regions. CWO mouse ortholog BHLHE41 has a conserved role in repressing ribosome biogenesis genes in primary hepatocytes. Collectively, these data uncover a cross-talk between conserved gene regulatory circuits balancing the activities of anabolic pathways to maintain homeostasis during sugar feeding.
Zhao, J., Perkins, M. L., Norstad, M. and Garcia, H. G. (2023). A bistable autoregulatory module in the developing embryo commits cells to binary expression fates. Curr Biol 33(14): 2851-2864 PubMed ID: 37453424
Summary:
Bistable autoactivation has been proposed as a mechanism for cells to adopt binary fates during embryonic development. However, it is unclear whether the autoactivating modules found within developmental gene regulatory networks are bistable, unless their parameters are quantitatively determined. This study combined in vivo live imaging with mathematical modeling to dissect the binary cell fate dynamics of the fruit fly pair-rule gene fushi tarazu (ftz), which is regulated by two known enhancers: the early (non-autoregulating) element and the autoregulatory element. Live imaging of transcription and protein concentration in the blastoderm revealed that binary Ftz fates are achieved as Ftz expression rapidly transitions from being dictated by the early element to the autoregulatory element. Moreover, this study discovered that Ftz concentration alone is insufficient to activate the autoregulatory element, and that this element only becomes responsive to Ftz at a prescribed developmental time. Based on these observations,a dynamical systems model was developed, and its kinetic parameters were quantitated directly from experimental measurements. This model demonstrated that the ftz autoregulatory module is indeed bistable and that the early element transiently establishes the content of the binary cell fate decision to which the autoregulatory module then commits. Further in silico analysis revealed that the autoregulatory element locks the Ftz fate quickly, within 35 min of exposure to the transient signal of the early element. Overall, this work confirms the widely held hypothesis that autoregulation can establish developmental fates through bistability and, most importantly, provides a framework for the quantitative dissection of cellular decision-making.
Moudgil, A., Sobti, R. C., Kaur, T. (2023). In-silico identification and comparison of transcription factor binding sites cluster in anterior-posterior patterning genes in Drosophila melanogaster and Tribolium castaneum. PLoS One, 18(8):e0290035 PubMed ID: 37590227
Summary:
The cis-regulatory data that help in transcriptional regulation is arranged into modular pieces of a few hundred base pairs called CRMs (cis-regulatory modules) and numerous binding sites for multiple transcription factors are prominent characteristics of these cis-regulatory modules. The present study was designed to localize transcription factor binding site (TFBS) clusters on twelve Anterior-posterior (A-P) genes in Tribolium castaneum and compare them to their orthologous gene enhancers in Drosophila melanogaster. Out of the twelve A-P patterning genes, six were gap genes (Kruppel, Knirps, Tailless, Hunchback, Giant, and Caudal) and six were pair rule genes (Hairy, Runt, Even-skipped, Fushi-tarazu, Paired, and Odd-skipped). The genes along with 20 kb upstream and downstream regions were scanned for TFBS clusters using the Motif Cluster Alignment Search Tool (MCAST), a bioinformatics tool that looks for set of nucleotide sequences for statistically significant clusters of non-overlapping occurrence of a given set of motifs. The motifs used in the current study were Hunchback, Caudal, Giant, Kruppel, Knirps, and Even-skipped. The results of the MCAST analysis revealed the maximum number of TFBS for Hunchback, Knirps, Caudal, and Kruppel in both D. melanogaster and T. castaneum, while Bicoid TFBS clusters were found only in D. melanogaster. The size of all the predicted TFBS clusters was less than 1kb in both insect species. These sequences revealed more transversional sites (Tv) than transitional sites (Ti) and the average Ti/Tv ratio was 0.75.

Friday, January 19th - Disease Models

Yue, W., Deng, X., Wang, Z., Jiang, M., Hu, R., Duan, Y., Wang, Q., Cui, J. and Fang, Y. (2023). Inhibition of the MEK/ERK pathway suppresses immune overactivation and mitigates TDP-43 toxicity in a Drosophila model of ALS. Immun Ageing 20(1): 27. PubMed ID: 37340309
Summary:
TDP-43 is an important DNA/RNA-binding protein that is associated with age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, its pathomechanism is not fully understood. In a transgenic RNAi screen using Drosophila as a model, this study uncovered that knockdown (KD) of Dsor1 (the Drosophila MAPK kinase dMEK) suppressed TDP-43 toxicity without altering TDP-43 phosphorylation or protein levels. Further investigation revealed that the Dsor1 downstream gene rl (dERK) was abnormally upregulated in TDP-43 flies, and neuronal overexpression of dERK induced profound upregulation of antimicrobial peptides (AMPs). A robust immune overactivation was pbserved in TDP-43 flies, which could be suppressed by downregulation of the MEK/ERK pathway in TDP-43 fly neurons. Furthermore, neuronal KD of abnormally increased AMPs improved the motor function of TDP-43 flies. On the other hand, neuronal KD of Dnr1, a negative regulator of the Drosophila immune deficiency (IMD) pathway, activated the innate immunity and boosted AMP expression independent of the regulation by the MEK/ERK pathway, which diminished the mitigating effect of RNAi-dMEK on TDP-43 toxicity. Finally, this study showed that an FDA-approved MEK inhibitor trametinib markedly suppressed immune overactivation, alleviated motor deficits and prolonged the lifespan of TDP-43 flies, but did not exhibit a lifespan-extending effect in Alzheimer disease (AD) or spinocerebellar ataxia type 3 (SCA3) fly models. Together, these findings suggest an important role of abnormal elevation of the MEK/ERK signaling and innate immunity in TDP-43 pathogenesis and propose trametinib as a potential therapeutic agent for ALS and other TDP-43-related diseases.
Yamada, M., Nitta, Y., Uehara, T., Suzuki, H., Miya, F., Takenouchi, T., Tamura, M., Ayabe, S., Yoshiki, A., Maeno, A., Saga, Y., Furuse, T., Yamada, I., Okamoto, N., Kosaki, K. and Sugie, A. (2023). Heterozygous loss-of-function DHX9 variants are associated with neurodevelopmental disorders: Human genetic and experimental evidences. Eur J Med Genet 66(8): 104804. PubMed ID: 37369308
Summary:
DExH-box helicases are involved in unwinding of RNA and DNA. Among the 16 DExH-box genes, monoallelic variants of DHX16, DHX30, DHX34, and DHX37 are known to be associated with neurodevelopmental disorders. In particular, DHX30 is well established as a causative gene for neurodevelopmental disorders. Germline variants of DHX9, the closest homolog of DHX30, have not been reported until now as being associated with congenital disorders in humans, except that one de novo heterozygous variant, p.(Arg1052Gln) of the gene was identified during comprehensive screening in a patient with autism; unfortunately, the phenotypic details of this individual are unknown. This study reports a patient with a heterozygous de novo missense variant, p.(Gly414Arg) of DHX9 who presented with a short stature, intellectual disability, and ventricular non-compaction cardiomyopathy. The variant was located in the glycine codon of the ATP-binding site, G-C-G-K-T. To assess the pathogenicity of these variants, transgenic Drosophila lines were generated expressing human wild-type and mutant DHX9 proteins (Drosophila homolog: maleless): 1) the mutant proteins showed aberrant localization both in the nucleus and the cytoplasm; 2) ectopic expression of wild-type protein in the visual system led to the rough eye phenotype, whereas expression of the mutant proteins had minimal effect; 3) overexpression of the wild-type protein in the retina led to a reduction in axonal numbers, whereas expression of the mutant proteins had a less pronounced effect. Furthermore, in a gene-editing experiment of Dhx9 G416 to R416, corresponding to p.(Gly414Arg) in humans, heterozygous mice showed a reduced body size, reduced emotionality, and cardiac conduction abnormality. In conclusion, this study has established that heterozygosity for a loss-of-function variant of DHX9 can lead to a new neurodevelopmental disorder.
Yu, M., Ye, H., De-Paula, R. B., Mangleburg, C. G., Wu, T., Lee, T. V., Li, Y., Duong, D., Phillips, B., Cruchaga, C., Allen, G. I., Seyfried, N. T., Al-Ramahi, I., Botas, J. and Shulman, J. M. (2023). Functional screening of lysosomal storage disorder genes identifies modifiers of alpha-synuclein neurotoxicity. PLoS Genet 19(5): e1010760. PubMed ID: 37200393
Summary:
Heterozygous variants in the glucocerebrosidase (GBA; see Drosophila Gba1a) gene are common and potent risk factors for Parkinson's disease (PD). GBA also causes the autosomal recessive lysosomal storage disorder (LSD), Gaucher disease, and emerging evidence from human genetics implicates many other LSD genes in PD susceptibility. This study has systemically tested 86 conserved fly homologs of 37 human LSD genes for requirements in the aging adult Drosophila brain and for potential genetic interactions with neurodegeneration caused by α-synuclein (αSyn), which forms Lewy body pathology in PD. The screen identifies 15 genetic enhancers of αSyn-induced progressive locomotor dysfunction, including knockdown of fly homologs of GBA and other LSD genes with independent support as PD susceptibility factors from human genetics (SCARB2, SMPD1, CTSD, GNPTAB, SLC17A5). For several genes, results from multiple alleles suggest dose-sensitivity and context-dependent pleiotropy in the presence or absence of αSyn. Homologs of two genes causing cholesterol storage disorders, Npc1a / NPC1 and Lip4 / LIPA, were independently confirmed as loss-of-function enhancers of αSyn-induced retinal degeneration. The enzymes encoded by several modifier genes are upregulated in αSyn transgenic flies, based on unbiased proteomics, revealing a possible, albeit ineffective, compensatory response. Overall, these results reinforce the important role of lysosomal genes in brain health and PD pathogenesis, and implicate several metabolic pathways, including cholesterol homeostasis, in αSyn-mediated neurotoxicity.
Zhou, H., Wang, J. and Wen, T. (2023). The molecular neural mechanism underlying the acceleration of brain aging due to Dcf1 deficiency. Mol Cell Neurosci 126: 103884. PubMed ID: 37506857
Summary:
Owing to the continuous increase in human life expectancy, the management of aging-related diseases has become an urgent issue. The brain dominates the central nervous system; therefore, brain aging is a key area of aging-related research. Previous work uncovered that dendritic cell factor 1 (Dcf1) maintains the stemness of neural stem cells and its expression in Drosophila can prolong lifespan, suggesting an association between Dcf1 and aging; however, the specific underlying neural mechanism remains unclear. The present study showed for the first time that hippocampal neurogenesis is decreased in aged Dcf1(-/-) mice, which leads to a decrease in the number of brain neurons and an increased number of senescent cells. Moreover, astrocytes proliferate abnormally and express elevated mRNA levels of aging-related factors, in addition to displaying increased activation of Akt and Foxo3a. Finally, behavioral tests confirm that aged Dcf1(-/-) mice exhibit a significant decline in cognitive abilities related to learning and memory. In conclusion, this study revealed a novel mechanism underlying brain aging triggered by Dcf1 deficiency at the molecular, cellular, tissue, and behavioral levels, providing a new perspective for the exploration of brain aging.
Xu, W., Rustenhoven, J., Nelson, C. A., Dykstra, T., Ferreiro, A., Papadopoulos, Z., Burnham, C. D., Dantas, G., Fremont, D. H. and Kipnis, J. (2023). A novel immune modulator IM33 mediates a glia-gut-neuronal axis that controls lifespan. Neuron. PubMed ID: 37582366
Summary:
Aging is a complex process involving various systems and behavioral changes. Altered immune regulation, dysbiosis, oxidative stress, and sleep decline are common features of aging, but their interconnection is poorly understood. Using Drosophila, this study discovered that IM33, a novel immune modulator, and its mammalian homolog, secretory leukocyte protease inhibitor (SLPI), are upregulated in old flies and old mice, respectively. Knockdown of IM33 in glia elevates the gut reactive oxygen species (ROS) level and alters gut microbiota composition, including increased Lactiplantibacillus plantarum abundance, leading to a shortened lifespan. Additionally, dysbiosis induces sleep fragmentation through the activation of insulin-producing cells in the brain, which is mediated by the binding of Lactiplantibacillus plantarum-produced DAP-type peptidoglycan to the peptidoglycan recognition protein LE (PGRP-LE) receptor. Therefore, IM33 plays a role in the glia-microbiota-neuronal axis, connecting neuroinflammation, dysbiosis, and sleep decline during aging. Identifying molecular mediators of these processes could lead to the development of innovative strategies for extending lifespan.
Zhang, F., Wang, L., Jin, J., Pang, Y., Shi, H., Fang, Z., Wang, H., Du, Y., Hu, Y., Zhang, Y., Ding, X. and Zhu, Z. (2023). Insights into the genetic influences of the microbiota on the life span of a host. Front Microbiol 14: 1138979. PubMed ID: 37601381
Summary:
Escherichia coli (E. coli) mutant strains have been reported to extend the life span of Caenorhabditis elegans (C. elegans). However, the specific mechanisms through which the genes and pathways affect aging are not yet clear. In this study, Drosophila melanogaster (fruit fly) was fed various E. coli single-gene knockout strains to screen mutant strains with an extended lifespan. The results showed that D. melanogaster fed with E. coli purE had the longest mean lifespan, which was verified by C. elegans. RNA-sequencing and analysis was conducted of C. elegans fed with E. coli purE (a single-gene knockout mutant) to further explore the underlying molecular mechanism. Differential gene expression (DGE) analysis, enrichment analysis, and gene set enrichment analysis (GSEA) to screen vital genes and modules with significant changes in overall expression. These results suggest that E. coli mutant strains may affect the host lifespan by regulating the protein synthesis rate (cfz-2) and ATP level (catp-4). To conclude, this study could provide new insights into the genetic influences of the microbiota on the life span of a host and a basis for developing anti-aging probiotics and drugs.

Wednesday, January 17th - Enzymes and Protein Expression, Evolution, Structure and Function

Weingartner, K. A., Tran, T., Tripp, K. W. and Kavran, J. M. (2023). Dimerization and autophosphorylation of the MST family of kinases are controlled by the same set of residues. Biochem J 480(15): 1165-1182. PubMed ID: 37459121
Summary:
The Hippo pathway controls tissue growth and regulates stem cell fate through the activities of core kinase cassette that begins with the Sterile 20-like kinase MST1/2. Activation of MST1/2 relies on trans-autophosphorylation but the details of the mechanisms regulating that reaction are not fully elucidated. Proposals include dimerization as a first step and include multiple models for potential kinase-domain dimers. Efforts to verify and link these dimers to trans-autophosphorylation were unsuccessful. The link between dimerization and trans-autophosphorylation for MST2 and the entire family of MST kinases was explored. Crystal lattice contacts of structures of MST kinases was examined, and an ensemble of kinase-domain dimers compatible with trans-autophosphorylation was identified. These dimers share a common dimerization interface comprised of the activation loop and αG-helix while the arrangements of the kinase-domains within the dimer varied depending on their activation state. The dimerization interface was identfied, and its function was determined using MST2. Variants bearing alanine substitutions of the αG-helix prevented dimerization of the MST2 kinase domain both in solution and in cells. These substitutions also blocked autophosphorylation of full-length MST2 and its Drosophila homolog Hippo in cells. These variants retain the same secondary structure as wild-type and capacity to phosphorylate a protein substrate, indicating the loss of MST2 activation can be directly attributed to a loss of dimerization rather than loss of either fold or catalytic function. Together this data functionally links dimerization and autophosphorylation for MST2 and suggests this activation mechanism is conserved across both species and the entire MST family.
Zhang, Y., Huang, Q., Sheng, C., Liu, G., Zhang, K., Jia, Z., Tang, T., Mao, X., Jones, A. K., Han, Z. and Zhao, C. (2023). G3'MTMD3 in the insect GABA receptor subunit, RDL, confers resistance to broflanilide and fluralaner. PLoS Genet 19(6): e1010814. PubMed ID: 37384781
Summary:
Meta-diamides (e.g. broflanilide) and isoxazolines (e.g. fluralaner) are novel insecticides that target the resistant to dieldrin (RDL) subunit of insect γ-aminobutyric acid receptors (GABARs). This study used in silico analysis to identify residues that are critical for the interaction between RDL and these insecticides. Substitution of glycine at the third position (G3') in the third transmembrane domain (TMD3) with methionine (G3'M TMD3), which is present in vertebrate GABARs, had the strongest effect on fluralaner binding. This was confirmed by expression of RDL from the rice stem borer, Chilo suppressalis (CsRDL) in oocytes of the African clawed frog, Xenopus laevis, where the G3'MTMD3 mutation almost abolished the antagonistic action of fluralaner. Subsequently, G3'MTMD3 was introduced into the Rdl gene of the fruit fly, Drosophila melanogaster, using the CRISPR/Cas9 system. Larvae of heterozygous lines bearing G3'MTMD3 did not show significant resistance to avermectin, fipronil, broflanilide, and fluralaner. However, larvae homozygous for G3'MTMD3 were highly resistant to broflanilide and fluralaner whilst still being sensitive to fipronil and avermectin. Also, homozygous lines showed severely impaired locomotivity and did not survive to the pupal stage, indicating a significant fitness cost associated with G3'MTMD3. Moreover, the M3'GTMD3 mutation in the mouse Mus musculus α1β2 GABAR increased sensitivity to fluralaner. Taken together, these results provide convincing in vitro and in vivo evidence for both broflanilide and fluralaner acting on the same amino acid site, as well as insights into potential mechanisms leading to target-site resistance to these insecticides. In addition, these findings could guide further modification of isoxazolines to achieve higher selectivity for the control of insect pests with minimal effects on mammals.
Tomihara, K. and Kiuchi, T. (2023). Disruption of a BTB-ZF transcription factor causes female sterility and melanization in the larval body of the silkworm, Bombyx mori. Insect Biochem Mol Biol 159: 103982. PubMed ID: 37356736
Summary:
The dilute black (bd) of the silkworm Bombyx mori is a recessive mutant that produces a grayish-black color in the larval integument, instead of the characteristic white color found in wild-type larvae. In addition, eggs produced by bd females are sterile due to a deficiency in the micropylar apparatus. This study identified candidate genes responsible for the bd phenotype using publicly available RNA-seq data. One of these candidate genes was homologous to the maternal gene required for meiosis (mamo) of Drosophila melanogaster, which encodes a broad-complex, tramtrack, and bric-à-brac-zinc finger (BTB-ZF) transcription factor essential for female fertility. In three independent bd strains, the expression of the B. mori mamo (Bmmamo) was downregulated in the larval integument. Using a CRISPR/Cas9-mediated knockout strategy, it was found that Bmmamo knockout mutants exhibit a grayish-black color in the larval integument and female infertility. Moreover, larvae obtained from the complementation cross between bd/+ mutants and heterozygous knockouts for the Bmmamo also exhibited a grayish-black color, indicating that Bmmamo is responsible for the bd phenotype. Gene expression analysis using Bmmamo knockout mutants suggested that the BmMamo protein suppresses the expression of melanin synthesis genes. Previous comparative genome analysis revealed that the Bmmamo was selected during silkworm domestication, and this study found that Bmmamo expression in the larval integument is higher in B. mori than in the wild silkworm B. mandarina, suggesting that the Bmmamo is involved in domestication-associated pigmentation changes of the silkworm.
Zhao, J., Lin, L., Hadiatullah, H., Chen, W., Huang, J., Wu, S., Murayama, T. and Yuchi, Z. (2023). Characterization of Six Diamide Insecticides on Ryanodine Receptor: Resistance and Species Selectivity. J Agric Food Chem 71(29): 11001-11007. PubMed ID: 37462137
Summary:
Ryanodine receptor (RyR) has been used as an insecticide target to control many destructive agricultural pests. The effectiveness of these insecticides has been limited by the spread of resistance mutations identified in pest RyRs, but the detailed molecular impacts of the individual mutations on the activity of different diamide compounds have not been fully explored. This study created five HEK293 cell lines stably expressing wild type rabbit RyR1, wild type Spodoptera frugiperda RyR (Sf RyR), or Sf RyR carrying different resistance mutations, including G4891E, G4891E/I4734M, and Y4867F, respectively. R-CEPIA1er, a genetically encoded fluorescent protein, was also introduced in these cell lines to report the Ca(2+) concentration in the endoplasmic reticulum. The activities of six commercial diamide insecticides were systematically characterized against different RyRs using the time-lapse fluorescence assay. Among them, cyantraniliprole (CYAN) displayed the highest activity against all three resistant Sf RyRs. The good performance of CYAN was confirmed by the toxicity assay using gene-edited Drosophila expressing the mutant RyRs, in which CYAN showed the lowest LD(50) value for the double resistant mutant. In addition, their acitivty were compared between mammalian and insect RyRs and found that flubendiamide has the best insect-selectivity. The mechanism of the anti-resistance property and selectivity of the compounds was proposed based on the structural models generated by homology modeling and molecular docking. These findings provide insights into the mechanism of insect resistance and guidance for developing effective RyR agonists that can selectively target resistant pests.
Scott, H., Dong, L., Stevenson, A., MacDonald, A. I., Srinivasan, S., Massimi, P., Banks, L., Martin, P. E., Johnstone, S. R. and Graham, S. V. (2023). The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes. J Cell Sci 136(11). PubMed ID: 37288673
Summary:
Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1 for which there is no ortholog) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, this study identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. This study shows that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. These data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.
Zarubin, M., Azorskaya, T., Kuldoshina, O., Alekseev, S., Mitrofanov, S. and Kravchenko, E. (2023). The tardigrade Dsup protein enhances radioresistance in Drosophila melanogaster and acts as an unspecific repressor of transcription. iScience 26(7): 106998. PubMed ID: 37534176
Summary:
Tardigrades are free-living tiny invertebrates belonging to the phylum Tardigrada. They are considered to be close relatives of arthropods and worms. The tardigrade-unique damage suppressor protein (Dsup) can protect DNA from ionizing radiation and reactive oxygen species (ROS). This study generated Dsup-expressing lines of Drosophila melanogaster and demonstrated that Dsup increased the survival rate after γ-ray irradiation and hydrogen peroxide treatment in flies too, but reduced the level of their locomotor activity. The transcriptome analyses of Dsup-expressing lines revealed a significant number of differentially expressed genes, >99% of which were down-regulated. Moreover, Dsup could bind RNA. These findings suggest that Dsup can act not only as a DNA protector but also as a non-specific transcriptional repressor and RNA-binding protein, that may lead to disturbance of a number of biological processes in D. melanogaster. The obtained data demonstrate features of the Dsup protein action in non-tardigrade organisms and can be used to understand the impact of other unspecific DNA/RNA-binding proteins on ROS and radiation resistance, gene expression, and epigenetic processes.

Tuesday, January 16th, Adult Development

Vuong, L. T. and Mlodzik, M. (2023). Wg/Wnt-signaling induced nuclear translocation of β-catenin is attenuated by a β-catenin peptide through its interaction with IFT-A in development and cancer cells. bioRxiv. PubMed ID: 37398005
Summary:
Wnt/Wingless (Wg) signaling is critical for many developmental patterning processes and linked to diseases, including cancer. Canonical Wnt-signaling is mediated by β-catenin, Armadillo/Arm in Drosophila transducing signal activation to a nuclear response. The IFT-A/Kinesin-2 complex is required to promote the nuclear translocation of β-catenin/Arm. This study defined a small conserved N-terminal Arm/β-catenin (Arm (34-87)) peptide, which binds IFT140, as a dominant interference tool to attenuate Wg/Wnt-signaling in vivo. Expression of Arm (34-87) is sufficient to antagonize endogenous Wnt/Wg-signaling activation resulting in marked reduction of Wg-signaling target gene expression. This effect is modulated by endogenous levels of Arm and IFT140, with the Arm (34-87) effect being enhanced or suppressed, respectively. Arm (34-87) thus inhibits Wg/Wnt-signaling by interfering with the nuclear translocation of endogenous Arm/β-catenin. Importantly, this mechanism is conserved in mammals with the equivalent β-catenin (34-87) peptide blocking nuclear translocation and pathway activation, including in cancer cells. This work indicates that Wnt-signaling can be regulated by a defined N-terminal peptide of Arm/β-catenin, and thus this might serve as an entry point for potential therapeutic applications to attenuate Wnt/β-catenin signaling.
Wen, D., Chen, Z., Wen, J. and Jia, Q. (2023). Sterol Regulation of Development and 20-Hydroxyecdysone Biosynthetic and Signaling Genes in Drosophila melanogaster. Cells 12(13). PubMed ID: 37443773
Summary:
Ecdysteroids are crucial in regulating the growth and development of insects. In the fruit fly Drosophila melanogaster, both C(27) and C(28) ecdysteroids have been identified. While the biosynthetic pathway of the C(27) ecdysteroid 20-hydroxyecdysone (20E) from cholesterol is relatively well understood, the biosynthetic pathway of C(28) ecdysteroids from C(28) or C(29) dietary sterols remains unknown. This study found that different dietary sterols (including the C(27) sterols cholesterol and 7-dehydrocholesterol, the C(28) sterols brassicasterol, campesterol, and ergosterol, and the C(29) sterols β-sitosterol, α-spinasterol, and stigmasterol) differentially affected the expression of 20E biosynthetic genes to varying degrees, but similarly activated 20E primary response gene expression in D. melanogaster Kc cells. It was also found that a single dietary sterol was sufficient to support D. melanogaster growth and development. Furthermore, the expression levels of some 20E biosynthetic genes were significantly altered, whereas the expression of 20E signaling primary response genes remained unaffected when flies were reared on lipid-depleted diets supplemented with single sterol types. Overall, this study provided preliminary clues to suggest that the same enzymatic system responsible for the classical C(27) ecdysteroid 20E biosynthetic pathway also participated in the conversion of C(28) and C(29) dietary sterols into C(28) ecdysteroids.
Williams, A. M. and Horne-Badovinac, S. (2023). Fat2 polarizes Lar and Sema5c to coordinate the motility of collectively migrating epithelial cells. J Cell Sci. PubMed ID: 37593878
Summary:
Migrating epithelial cells globally align their migration machinery to achieve tissue-level movement. Biochemical signaling across leading-trailing cell-cell interfaces can promote this alignment by partitioning migratory behaviors like protrusion and retraction to opposite sides of the interface. However, how signaling proteins become organized at interfaces to accomplish this is poorly understood. The follicular epithelial cells of Drosophila melanogaster have two signaling modules at their leading-trailing interfaces-one composed of the atypical cadherin Fat2 and the receptor tyrosine phosphatase Lar, and one composed of Semaphorin5c and its receptorPlexin A. This study shows that these modules form one interface signaling system with Fat2 at its core. Trailing edge-enriched Fat2 concentrates both Lar and Semaphorin5c at cells' leading edges, but Lar and Semaphorin5c play little role in Fat2's localization. Fat2 is also more stable at interfaces than Lar and Semaphorin5c. Once localized, Lar and Semaphorin5c act in parallel to promote collective migration. It is proposed that Fat2 serves as the organizer this interface signaling system by coupling and polarizing the distributions of multiple effectors that work together to align the migration machinery of neighboring cells.
Xue, M., Cong, F., Zheng, W., Xu, R., Liu, X., Bao, H., Sung, Y. Y., Xi, Y., He, F., Ma, J., Yang, X. and Ge, W. (2023). Loss of Paip1 causes translation reduction and induces apoptotic cell death through ISR activation and Xrp1. Cell Death Discov 9(1): 288. PubMed ID: 37543696
Summary:
Regulation of protein translation initiation is tightly associated with cell growth and survival. This study identified Paip1, the Drosophila homolog of the translation initiation factor PAIP1 and analyzed its role during development. Through genetic analysis, this study found that loss of Paip1 causes reduced protein translation and pupal lethality. Furthermore, tissue specific knockdown of Paip1 results in apoptotic cell death in the wing imaginal disc. Paip1 depletion leads to increased proteotoxic stress and activation of the integrated stress response (ISR) pathway. Mechanistically, it was shown that loss of Paip1 promotes phosphorylation of eIF2α via the kinase PERK, leading to apoptotic cell death. Moreover, Paip1 depletion upregulates the transcription factor gene Xrp1, which contributes to apoptotic cell death and eIF2α phosphorylation. It was further shown that loss of Paip1 leads to an increase in Xrp1 translation mediated by its 5'UTR. These findings uncover a novel mechanism that links translation impairment to tissue homeostasis and establish a role of ISR activation and Xrp1 in promoting cell death.
Troost, T., Binshtok, U., Sprinzak, D. and Klein, T. (2023). Cis-inhibition suppresses basal Notch signaling during sensory organ precursor selection. Proc Natl Acad Sci U S A 120(23): e2214535120. PubMed ID: 37252950
Summary:
The emergence of the sensory organ precursor (SOP) from an equivalence group in Drosophila is a paradigm for studying single-cell fate specification through Notch-mediated lateral inhibition. Yet, it remains unclear how only a single SOP is selected from a relatively large group of cells. A critical aspect of SOP selection is controlled by cis-inhibition (CI), whereby the Notch ligands,Delta (Dl), cis-inhibit Notch receptors in the same cell. Based on the observation that the mammalian ligand Dl-like 1 cannot cis-inhibit Notch in Drosophila, this study probed the role of CI in vivo. A mathematical model was developed for SOP selection where Dl activity is independently regulated by the ubiquitin ligases Neuralized and Mindbomb1. This study showed theoretically and experimentally that Mindbomb1 induces basal Notch activity, which is suppressed by CI. These results highlight the trade-off between basal Notch activity and CI as a mechanism for singling out a SOP from a large equivalence group.
Yost, P. P., Al-Nouman, A. and Curtiss, J. (2023). The Rap1 small GTPase affects cell fate or survival and morphogenetic patterning during Drosophila melanogaster eye development. Differentiation 133: 12-24. PubMed ID: 37437447
Summary:
The Drosophila melanogaster eye has been instrumental for determining both how cells communicate with one another to determine cell fate, as well as cell morphogenesis and patterning. This study describes the effects of the small GTPase Rap1 on the development of multiple cell types in the D. melanogaster eye. Although Rap1 has previously been linked to RTK-Ras-MAPK signaling in eye development, this study demonstrate that manipulation of Rap1 activity is modified by increase or decrease of Delta/Notch signaling during several events of cell fate specification in eye development. In addition, it was demonstrated that manipulating Rap1 function either in primary pigment cells or in interommatidial cells affects cone cell contact switching, primary pigment cell enwrapment of the ommatidial cluster, and sorting of secondary and tertiary pigment cells. These data suggest that Rap1 has roles in both ommatidial cell recruitment/survival and in ommatidial morphogenesis in the pupal stage. They lay groundwork for future experiments on the role of Rap1 in these events.

Tuesday, January 16th - Signaling

Vuong, L. T. and Mlodzik, M. (2023). Wg/Wnt-signaling induced nuclear translocation of β-catenin is attenuated by a β-catenin peptide through its interaction with IFT-A in development and cancer cells. bioRxiv. PubMed ID: 37398005
Summary:
Wnt/Wingless (Wg) signaling is critical for many developmental patterning processes and linked to diseases, including cancer. Canonical Wnt-signaling is mediated by β-catenin, Armadillo/Arm in Drosophila transducing signal activation to a nuclear response. The IFT-A/Kinesin-2 complex is required to promote the nuclear translocation of β-catenin/Arm. This study defined a small conserved N-terminal Arm/β-catenin (Arm (34-87)) peptide, which binds IFT140, as a dominant interference tool to attenuate Wg/Wnt-signaling in vivo. Expression of Arm (34-87) is sufficient to antagonize endogenous Wnt/Wg-signaling activation resulting in marked reduction of Wg-signaling target gene expression. This effect is modulated by endogenous levels of Arm and IFT140, with the Arm (34-87) effect being enhanced or suppressed, respectively. Arm (34-87) thus inhibits Wg/Wnt-signaling by interfering with the nuclear translocation of endogenous Arm/β-catenin. Importantly, this mechanism is conserved in mammals with the equivalent β-catenin (34-87) peptide blocking nuclear translocation and pathway activation, including in cancer cells. This work indicates that Wnt-signaling can be regulated by a defined N-terminal peptide of Arm/β-catenin, and thus this might serve as an entry point for potential therapeutic applications to attenuate Wnt/β-catenin signaling.
Wen, D., Chen, Z., Wen, J. and Jia, Q. (2023). Sterol Regulation of Development and 20-Hydroxyecdysone Biosynthetic and Signaling Genes in Drosophila melanogaster. Cells 12(13). PubMed ID: 37443773
Summary:
Ecdysteroids are crucial in regulating the growth and development of insects. In the fruit fly Drosophila melanogaster, both C(27) and C(28) ecdysteroids have been identified. While the biosynthetic pathway of the C(27) ecdysteroid 20-hydroxyecdysone (20E) from cholesterol is relatively well understood, the biosynthetic pathway of C(28) ecdysteroids from C(28) or C(29) dietary sterols remains unknown. This study found that different dietary sterols (including the C(27) sterols cholesterol and 7-dehydrocholesterol, the C(28) sterols brassicasterol, campesterol, and ergosterol, and the C(29) sterols β-sitosterol, α-spinasterol, and stigmasterol) differentially affected the expression of 20E biosynthetic genes to varying degrees, but similarly activated 20E primary response gene expression in D. melanogaster Kc cells. It was also found that a single dietary sterol was sufficient to support D. melanogaster growth and development. Furthermore, the expression levels of some 20E biosynthetic genes were significantly altered, whereas the expression of 20E signaling primary response genes remained unaffected when flies were reared on lipid-depleted diets supplemented with single sterol types. Overall, this study provided preliminary clues to suggest that the same enzymatic system responsible for the classical C(27) ecdysteroid 20E biosynthetic pathway also participated in the conversion of C(28) and C(29) dietary sterols into C(28) ecdysteroids.
Williams, A. M. and Horne-Badovinac, S. (2023). Fat2 polarizes Lar and Sema5c to coordinate the motility of collectively migrating epithelial cells. J Cell Sci. PubMed ID: 37593878
Summary:
Migrating epithelial cells globally align their migration machinery to achieve tissue-level movement. Biochemical signaling across leading-trailing cell-cell interfaces can promote this alignment by partitioning migratory behaviors like protrusion and retraction to opposite sides of the interface. However, how signaling proteins become organized at interfaces to accomplish this is poorly understood. The follicular epithelial cells of Drosophila melanogaster have two signaling modules at their leading-trailing interfaces-one composed of the atypical cadherin Fat2 and the receptor tyrosine phosphatase Lar, and one composed of Semaphorin5c and its receptorPlexin A. This study shows that these modules form one interface signaling system with Fat2 at its core. Trailing edge-enriched Fat2 concentrates both Lar and Semaphorin5c at cells' leading edges, but Lar and Semaphorin5c play little role in Fat2's localization. Fat2 is also more stable at interfaces than Lar and Semaphorin5c. Once localized, Lar and Semaphorin5c act in parallel to promote collective migration. It is proposed that Fat2 serves as the organizer this interface signaling system by coupling and polarizing the distributions of multiple effectors that work together to align the migration machinery of neighboring cells.
Xue, M., Cong, F., Zheng, W., Xu, R., Liu, X., Bao, H., Sung, Y. Y., Xi, Y., He, F., Ma, J., Yang, X. and Ge, W. (2023). Loss of Paip1 causes translation reduction and induces apoptotic cell death through ISR activation and Xrp1. Cell Death Discov 9(1): 288. PubMed ID: 37543696
Summary:
Regulation of protein translation initiation is tightly associated with cell growth and survival. This study identified Paip1, the Drosophila homolog of the translation initiation factor PAIP1 and analyzed its role during development. Through genetic analysis, this study found that loss of Paip1 causes reduced protein translation and pupal lethality. Furthermore, tissue specific knockdown of Paip1 results in apoptotic cell death in the wing imaginal disc. Paip1 depletion leads to increased proteotoxic stress and activation of the integrated stress response (ISR) pathway. Mechanistically, it was shown that loss of Paip1 promotes phosphorylation of eIF2α via the kinase PERK, leading to apoptotic cell death. Moreover, Paip1 depletion upregulates the transcription factor gene Xrp1, which contributes to apoptotic cell death and eIF2α phosphorylation. It was further shown that loss of Paip1 leads to an increase in Xrp1 translation mediated by its 5'UTR. These findings uncover a novel mechanism that links translation impairment to tissue homeostasis and establish a role of ISR activation and Xrp1 in promoting cell death.
Troost, T., Binshtok, U., Sprinzak, D. and Klein, T. (2023). Cis-inhibition suppresses basal Notch signaling during sensory organ precursor selection. Proc Natl Acad Sci U S A 120(23): e2214535120. PubMed ID: 37252950
Summary:
The emergence of the sensory organ precursor (SOP) from an equivalence group in Drosophila is a paradigm for studying single-cell fate specification through Notch-mediated lateral inhibition. Yet, it remains unclear how only a single SOP is selected from a relatively large group of cells. A critical aspect of SOP selection is controlled by cis-inhibition (CI), whereby the Notch ligands, elta (Dl), cis-inhibit Notch receptors in the same cell. Based on the observation that the mammalian ligand Dl-like 1 cannot cis-inhibit Notch in Drosophila, this study probed the role of CI in vivo. A mathematical model was developed for SOP selection where Dl activity is independently regulated by the ubiquitin ligases Neuralized and Mindbomb1. This study showed theoretically and experimentally that Mindbomb1 induces basal Notch activity, which is suppressed by CI. These results highlight the trade-off between basal Notch activity and CI as a mechanism for singling out a SOP from a large equivalence group.
Yost, P. P., Al-Nouman, A. and Curtiss, J. (2023). The Rap1 small GTPase affects cell fate or survival and morphogenetic patterning during Drosophila melanogaster eye development. Differentiation 133: 12-24. PubMed ID: 37437447
Summary:
The Drosophila melanogaster eye has been instrumental for determining both how cells communicate with one another to determine cell fate, as well as cell morphogenesis and patterning. This study describes the effects of the small GTPase Rap1 on the development of multiple cell types in the D. melanogaster eye. Although Rap1 has previously been linked to RTK-Ras-MAPK signaling in eye development, this study demonstrate that manipulation of Rap1 activity is modified by increase or decrease of Delta/Notch signaling during several events of cell fate specification in eye development. In addition, it was demonstrated that manipulating Rap1 function either in primary pigment cells or in interommatidial cells affects cone cell contact switching, primary pigment cell enwrapment of the ommatidial cluster, and sorting of secondary and tertiary pigment cells. These data suggest that Rap1 has roles in both ommatidial cell recruitment/survival and in ommatidial morphogenesis in the pupal stage. They lay groundwork for future experiments on the role of Rap1 in these events.

Friday, January 11th - Behaviors

Xie, S. M., Lai, J. X., Liu, C. Q., Zhang, X. X., Lin, Y. M., Lan, Q. W., Hong, D. Y., Chen, X. C., Qiao, J. D. and Mao, Y. L. (2023). UBR4 deficiency causes male sterility and testis abnormal in Drosophila. Front Endocrinol (Lausanne) 14: 1165825. PubMed ID: 37529615
Summary:
It has been established that UBR4 encodes E3 ubiquitin ligase, which determines the specificity of substrate binding during protein ubiquitination and has been associated with various functions of the nervous system but not the reproductive system. This study explored the role of UBR4 on fertility with a Drosophila model. Different Ubr4 knockdown flies were established using the UAS/GAL4 activating sequence system. Fertility, hatchability, and testis morphology were studied, and bioinformatics analyses were conducted. The results indicated that UBR4 deficiency could induce male sterility and influent egg hatchability in Drosophila. Ubr4 deficiency affected the testis during morphological analysis. Proteomics analysis indicated 188 upregulated proteins and 175 downregulated proteins in the testis of Ubr4 knockdown flies. Gene Ontology analysis revealed significant upregulation of CG11598 and Sfp65A, and downregulation of Pelota in Ubr4 knockdown flies. These proteins were involved in the biometabolic or reproductive process in Drosophila. These regulated proteins are important in testis generation and sperm storage promotion. Bioinformatics analysis verified that expression of UBR4 was low in cryptorchidism patients, which further supported the important role of UBR4 in male fertility. Overall, these findings suggest that UBR4 deficiency could promote male infertility and may be involved in the protein modification of UBR4 by upregulating Sfp65A and CG11598, whereas downregulating Pelota protein expression.
Salim, S., Hussain, S., Banu, A., Gowda, S. B. M., Ahammad, F., Alwa, A., Pasha, M. and Mohammad, F. (2023). The ortholog of human ssDNA-binding protein SSBP3 influences neurodevelopment and autism-like behaviors in Drosophila melanogaster. PLoS Biol 21(7): e3002210. PubMed ID: 37486945
Summary:
The 1p32.3 microdeletion is implicated in many neurodevelopmental disorders. One deleted gene is the single-stranded DNA-binding protein 3 (SSBP3); its Drosophila ortholog is Ssdp). This study investigated consequences of Ssdp manipulations. SSBP3 and Ssdp are expressed in excitatory neurons in the brain. Ssdp overexpression caused morphological alterations in Drosophila wing, mechanosensory bristles, and head. Ssdp manipulations also affected the neuropil brain volume and glial cell number in larvae and adult flies. Moreover, Ssdp overexpression led to differential changes in synaptic density in specific brain regions. Decreased levels of armadillo were observed in the heads of Ssdp overexpressing flies, as well as a decrease in armadillo and wingless expression in the larval wing discs, implicating the involvement of the canonical Wnt signaling pathway in Ssdp function. RNA sequencing revealed perturbation of oxidative stress-related pathways in heads of Ssdp overexpressing flies. Furthermore, Ssdp overexpressing brains showed enhanced reactive oxygen species (ROS) and altered neuronal mitochondrial morphology. Flies with elevated levels of Ssdp exhibited behavior defects, which were phenocopied in pan-neuronal Ssdp knockdown flies, suggesting that Ssdp is dosage sensitive. Notably, Ssdp knockdown exclusively in adult flies did not produce behavioral and functional defects. Finally, optogenetic manipulation of Ssdp-expressing neurons altered autism-associated behaviors. This study proposes SSBP3 as a critical gene in the 1p32.3 microdeletion/duplication genomic region and sheds light on the functional role of Ssdp in neurodevelopmental processes in Drosophila.
Sun, Z., Nystul, T. G. and Zhong, G. (2023). Single-cell RNA sequencing identifies eggplant as a regulator of germ cell development in Drosophila. EMBO Rep: e56475. PubMed ID: 37603128

Drosophila ovarian germline stem cells (GSCs) are a powerful model for stem cell research. In this study, single-cell RNA sequencing (scRNA-seq), an RNAi screen and bioinformatic analysis, was used to identify genes involved in germ cell differentiation, including 34 genes with upregulated expression during early germ cell development and 19 genes that may regulate germ cell differentiation. Among these, a gene named eggplant (eggpl - CG32814) is highly expressed in GSCs and downregulated in early daughter cells. RNAi knockdown of eggpl causes germ cell proliferation and differentiation defects. In flies fed a rich yeast diet, the expression of eggpl is significantly lower and knockdown or knockout of eggpl phenocopies a rich diet. In addition, eggpl knockdown suppresses the reduction in germ cell proliferation caused by inhibition of the insulin effector PI3K. These findings suggest that downregulation of eggpl links nutritional status to germ cell proliferation and differentiation. Collectively, this study provides new insights into the signaling networks that regulate early germ cell development and identifies eggpl as a key player in this process.

Roy, S. D., Nagarajan, S., Jalal, M. S., Basar, M. A. and Duttaroy, A. (2023). New mutant alleles for Spargel/dPGC-1 highlights the function of Spargel RRM domain in oogenesis and expands the role of Spargel in embryogenesis and intracellular transport. G3 (Bethesda). PubMed ID: 37369430
Summary:
Energy metabolism in vertebrates is controlled by three members of the PGC-1 (PPAR γ- coactivator 1) family, transcriptional coactivators that shape responses to physiological stimuli by interacting with the nuclear receptors and other transcription factors. Multiple evidence now supports that Spargel protein found in insects and ascidians is the ancestral form of vertebrate PGC-1's. This study undertook functional analysis of srl gene in Drosophila, asking about the requirement of Spargel per se during embryogenesis and its RNA binding domains. CRISPR- engineered srl gene deletion turned out to be an amorphic allele that is late embryonic/early larval lethal and Spargel protein missing its RNA binding domain (SrlΔRRM) negatively affects female fertility. Overexpression of wild-type Spargel in transgenic flies expedited the growth of egg chambers. On the other hand, oogenesis is blocked in a dominant-negative fashion in the presence of excess Spargel lacking its RRM domains. Finally, aggregation of Notch proteins was observed in egg chambers of srl mutant flies, suggesting that Spargel is involved in intracellular transport of Notch proteins. Taken together, this study claims that these new mutant alleles of spargel are emerging powerful tools for revealing new biological functions for Spargel, an essential transcription coactivator in both Drosophila and mammals.
Wooten, M., Takushi, B., Ahmad, K. and Henikoff, S. (2023).. Aclarubicin stimulates RNA polymerase II elongation at closely spaced divergent promoters. Sci Adv 9(24): eadg3257. PubMed ID: 37315134

Anthracyclines are a class of widely prescribed anticancer drugs that disrupt chromatin by intercalating into DNA and enhancing nucleosome turnover. To understand the molecular consequences of anthracycline-mediated chromatin disruption, Cleavage Under Targets and Tagmentation (CUT&Tag) was used to profile RNA polymerase II during anthracycline treatment in Drosophila cells. Treatment with the anthracycline aclarubicin leads to elevated levels of RNA polymerase II and changes in chromatin accessibility. It was found that promoter proximity and orientation affect chromatin changes during aclarubicin treatment, as closely spaced divergent promoter pairs show greater chromatin changes when compared to codirectionally oriented tandem promoters. It was also found that aclarubicin treatment changes the distribution of noncanonical DNA G-quadruplex structures both at promoters and at G-rich pericentromeric repeats. This work suggests that the cancer-killing activity of aclarubicin is driven by the disruption of nucleosomes and RNA polymerase II.

Vdovina, Y. A., Kurshakova, M. M., Georgieva, S. G. and Kopytova, D. V. (2023). PCID2 Subunit of the Drosophila TREX-2 Complex Has Two RNA-Binding Regions. Curr Issues Mol Biol 45(7): 5662-5676. PubMed ID: 37504273
Summary:
Drosophila PCID2 is a subunit of the TREX-2 mRNA nuclear export complex. Although the complex has long been studied in eukaryotes, it is still unclear how TREX-2 interacts with mRNA in multicellular organisms. Here, the interaction between Drosophila PCID2 and the ras2 RNA was studied by EMSA. The C-terminal region of the WH domain of PCID2 specifically binds the 3'-noncoding region of the ras2 RNA. While the same region of PCID2 interacts with the Xmas-2 subunit of the TREX-2 complex, PCID2 interacts with RNA independently of Xmas-2. An additional RNA-binding region (M region) was identified in the N-terminal part of the PCI domain and found to bind RNA nonspecifically. Point mutations of evolutionarily conserved amino acid residues in this region completely abolish the PCID2-RNA interaction, while a deletion of the C-terminal domain only partly decreases it. Thus, the specific interaction of PCID2 with RNA requires nonspecific PCID2-RNA binding.

Wednesday, January 10th - Chromatin

Tolokh, I. S., Kinney, N. A., Sharakhov, I. V. and Onufriev, A. V. (2023). Strong interactions between highly dynamic lamina-associated domains and the nuclear envelope stabilize the 3D architecture of Drosophila interphase chromatin. Epigenetics Chromatin 16(1): 21. PubMed ID: 37254161
Summary:
Interactions among topologically associating domains (TADs), and between the nuclear envelope (NE) and lamina-associated domains (LADs) are expected to shape various aspects of three-dimensional (3D) chromatin structure and dynamics. This study has developed a dynamical model of D. melanogaster nuclei at TAD resolution that explicitly accounts for four distinct epigenetic classes of TADs and LAD-NE interactions. The model is parameterized to reproduce the experimental Hi-C map of the wild type (WT) nuclei; it describes time evolution of the chromatin over the G1 phase of the interphase. Predicted positioning of all LADs at the NE is highly dynamic-the same LAD can attach, detach and move far away from the NE multiple times during interphase. The probabilities of LADs to be in contact with the NE vary by an order of magnitude, despite all having the same affinity to the NE in the model. These probabilities are mostly determined by a highly variable local linear density of LADs along the genome, which also has the same strong effect on the predicted positioning of individual TADs -- higher probability of a TAD to be near NE is largely determined by a higher linear density of LADs surrounding this TAD. The distribution of LADs along the chromosome chains plays a notable role in maintaining a non-random average global structure of chromatin. Relatively high affinity of LADs to the NE in the WT nuclei substantially reduces sensitivity of the global radial chromatin distribution to variations in the strength of TAD-TAD interactions compared to the lamin depleted nuclei, where a small (0.5 kT) increase of cross-type TAD-TAD interactions doubles the chromatin density in the central nucleus region. A dynamical model of the entire fruit fly genome makes multiple genome-wide predictions of biological interest. The distribution of LADs along the chromatin chains affects their probabilities to be in contact with the NE and radial positioning of highly mobile TADs, playing a notable role in creating a non-random average global structure of the chromatin. It is conjectured that an important role of attractive LAD-NE interactions is to stabilize global chromatin structure against inevitable cell-to-cell variations in TAD-TAD interactions.
Tortora, M. M. C., Brennan, L. D., Karpen, G. and Jost, D. (2023). HP1-driven phase separation recapitulates the thermodynamics and kinetics of heterochromatin condensate formation. Proc Natl Acad Sci U S A 120(33): e2211855120. PubMed ID: 37549295
Summary:
The spatial segregation of pericentromeric heterochromatin (PCH) into distinct, membrane-less nuclear compartments involves the binding of Heterochromatin Protein 1 (HP1) to H3K9me2/3-rich genomic regions. While HP1 exhibits liquid-liquid phase separation properties in vitro, its mechanistic impact on the structure and dynamics of PCH condensate formation in vivo remains largely unresolved. Using a minimal theoretical framework, this study systematically investigate the mutual coupling between self-interacting HP1-like molecules and the chromatin polymer. The specific affinity of HP1 for H3K9me2/3 loci was shown to facilitate coacervation in nucleo and promotes the formation of stable PCH condensates at HP1 levels far below the concentration required to observe phase separation in purified protein assays in vitro. These heterotypic HP1-chromatin interactions give rise to a strong dependence of the nucleoplasmic HP1 density on HP1-H3K9me2/3 stoichiometry, consistent with the thermodynamics of multicomponent phase separation. The dynamical cross talk between HP1 and the viscoelastic chromatin scaffold also leads to anomalously slow equilibration kinetics, which strongly depend on the genomic distribution of H3K9me2/3 domains and result in the coexistence of multiple long-lived, microphase-separated PCH compartments. The morphology of these complex coacervates is further found to be governed by the dynamic establishment of the underlying H3K9me2/3 landscape, which may drive their increasingly abnormal, aspherical shapes during cell development. These findings compare favorably to 4D microscopy measurements of HP1 condensate formation in live Drosophila embryos and suggest a general quantitative model of PCH formation based on the interplay between HP1-based phase separation and chromatin polymer mechanics.
Prozzillo, Y., Fattorini, G., Ferreri, D., Leo, M., Dimitri, P. and Messina, G. (2023). Knockdown of DOM/Tip60 Complex Subunits Impairs Male Meiosis of Drosophila melanogaster. Cells 12(10). PubMed ID: 37408183
Summary:
ATP-dependent chromatin remodeling complexes are involved in nucleosome sliding and eviction and/or the incorporation of histone variants into chromatin to facilitate several cellular and biological processes, including DNA transcription, replication and repair. The DOM/TIP60 chromatin remodeling complex of Drosophila melanogaster contains 18 subunits, including DOMINO (DOM), an ATPase that catalyzes the exchange of the canonical H2A with its variant (H2A.V), and TIP60, a lysine-acetyltransferase that acetylates H4, H2A and H2A.V histones. In recent decades, experimental evidence has shown that ATP-dependent chromatin remodeling factors, in addition to their role in chromatin organization, have a functional relevance in cell division. In particular, emerging studies suggested the direct roles of ATP-dependent chromatin remodeling complex subunits in controlling mitosis and cytokinesis in both humans and D. melanogaster. However, little is known about their possible involvement during meiosis. The results of this work show that the knockdown of 12 of DOM/TIP60 complex subunits generates cell division defects that, in turn, cause total/partial sterility in Drosophila males, providing new insights into the functions of chromatin remodelers in cell division control during gametogenesis.
Rajan, A., Anhezini, L., Rives-Quinto, N., Chhabra, J. Y., Neville, M. C., Larson, E. D., Goodwin, S. F., Harrison, M. M. and Lee, C. Y. (2023). Low-level repressive histone marks fine-tune gene transcription in neural stem cells. PubMed ID: 37314324
Summary:
Coordinated regulation of gene activity by transcriptional and translational mechanisms poise stem cells for a timely cell-state transition during differentiation. Although important for all stemness-to-differentiation transitions, mechanistic understanding of the fine-tuning of gene transcription is lacking due to the compensatory effect of translational control. This study used intermediate neural progenitor (INP) identity commitment to define the mechanisms that fine-tune stemness gene transcription in fly neural stem cells (neuroblasts). This study demonstrated that the transcription factor Fruitless(C) (Fru(C)) binds cis-regulatory elements of most genes uniquely transcribed in neuroblasts. Loss of fru(C) function alone has no effect on INP commitment but drives INP dedifferentiation when translational control is reduced. Fru(C) negatively regulates gene expression by promoting low-level enrichment of the repressive histone mark H3K27me3 in gene cis-regulatory regions. Identical to fru(C) loss-of-function, reducing Polycomb Repressive Complex 2 activity increases stemness gene activity. It is proposed that low-level H3K27me3 enrichment fine-tunes gene transcription in stem cells, a mechanism likely conserved from flies to humans.
Tang, M., Regadas, I., Belikov, S., Shilkova, O., Xu, L., Wernersson, E., Liu, X., Wu, H., Bienko, M. and Mannervik, M. (2023). Separation of transcriptional repressor and activator functions in Drosophila HDAC3. Development 150(15). PubMed ID: 37455638
Summary:
The histone deacetylase HDAC3 is associated with the NCoR/SMRT co-repressor complex, and its canonical function is in transcriptional repression, but it can also activate transcription. This study shows that the repressor and activator functions of HDAC3 can be genetically separated in Drosophila. A lysine substitution in the N terminus (K26A) disrupts its catalytic activity and activator function, whereas a combination of substitutions (HEBI) abrogating the interaction with SMRTER enhances repressor activity beyond wild type in the early embryo. It is concluded that the crucial functions of HDAC3 in embryo development involve catalytic-dependent gene activation and non-enzymatic repression by several mechanisms, including tethering of loci to the nuclear periphery.
Williamson, I., Boyle, S., Grimes, G. R., Friman, E. T. and Bickmore, W. A. (2023). Dispersal of PRC1 condensates disrupts polycomb chromatin domains and loops. Life Sci Alliance 6(10). PubMed ID: 37487640
Summary:
Polycomb repressive complex 1 (PRC1) strongly influences 3D genome organization, mediating local chromatin compaction and clustering of target loci. Several PRC1 subunits have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. This study used 1,6-hexanediol, which can disrupt liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation, this study showed that PRC1-mediated chromatin compaction and clustering of targeted genomic loci-at different length scales-can be reversibly disrupted by the addition and subsequent removal of 1,6-hexanediol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to reduced PRC1 occupancy detected by chromatin immunoprecipitation following 1,6-hexanediol treatment as the addition of 2,5-hexanediol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules may have a role in polycomb-mediated genome organization.

Monday, January 8th - Stress

Wen, D., Xie, J., Yuan, Y., Shen, L., Yang, Y. and Chen, W. (2023). The endogenous antioxidant ability of royal jelly in Drosophila is independent of Keap1/Nrf2 by activating oxidoreductase activity. Insect Sci. PubMed ID: 37632209
Summary:
Royal jelly (RJ) is a biologically active substance secreted by the hypopharyngeal and mandibular glands of worker honeybees. It is widely claimed that RJ reduces oxidative stress. However, the antioxidant activity of RJ has mostly been determined by in vitro chemical detection methods or by external administration drugs that cause oxidative stress. Whether RJ can clear the endogenous production of reactive oxygen species (ROS) in cells remains largely unknown. This study systematically investigated the antioxidant properties of RJ using several endogenous oxidative stress models of Drosophila. RJ was found to enhance sleep quality of aging Drosophila, which is decreased due to an increase of oxidative damage with age. RJ supplementation improved survival and suppressed ROS levels in gut cells of flies upon exposure to hydrogen peroxide or to the neurotoxic agent paraquat. Moreover, RJ supplementation moderated levels of ROS in endogenous gut cells and extended lifespan after exposure of flies to heat stress. Sleep deprivation leads to accumulation of ROS in the gut cells, and RJ attenuated the consequences of oxidative stress caused by sleep loss and prolonged lifespan. Mechanistically, RJ prevented cell oxidative damage caused by heat stress or sleep deprivation, with the antioxidant activity in vivo independent of Keap1/Nrf2 signaling. RJ supplementation activated oxidoreductase activity in the guts of flies, suggesting its ability to inhibit endogenous oxidative stress and maintain health, possibly in humans.
Kiparaki, M. and Baker, N. E. (2023). Ribosomal protein mutations and cell competition: autonomous and nonautonomous effects on a stress response. Genetics 224(3). PubMed ID: 37267156
Summary:
Ribosomal proteins (Rps) are essential for viability. Genetic mutations affecting Rp genes were first discovered in Drosophila, where they represent a major class of haploinsufficient mutations. One mutant copy gives rise to the dominant "Minute" phenotype, characterized by slow growth and small, thin bristles. Wild-type (WT) and Minute cells compete in mosaics, that is, Rp+/- are preferentially lost when their neighbors are of the wild-type genotype. Many features of Rp gene haploinsufficiency (i.e. Rp+/- phenotypes) are mediated by a transcriptional program. In Drosophila, reduced translation and slow growth are under the control of Xrp1, a bZip-domain transcription factor induced in Rp mutant cells that leads ultimately to the phosphorylation of eIF2α; and consequently inhibition of most translation. Rp mutant phenotypes are also mediated transcriptionally in yeast and in mammals. In mammals, the Impaired Ribosome Biogenesis Checkpoint activates p53. Recent findings link Rp mutant phenotypes to other cellular stresses, including the DNA damage response and endoplasmic reticulum stress. It is suggested that cell competition results from nonautonomous inputs to stress responses, bringing decisions between adaptive and apoptotic outcomes under the influence of nearby cells. In Drosophila, cell competition eliminates aneuploid cells in which loss of chromosome leads to Rp gene haploinsufficiency. The effects of Rp gene mutations on the whole organism, in Minute flies or in humans with Diamond-Blackfan Anemia, may be inevitable consequences of pathways that are useful in eliminating individual cells from mosaics. Alternatively, apparently deleterious whole organism phenotypes might be adaptive, preventing even more detrimental outcomes. In mammals, for example, p53 activation appears to supress oncogenic effects of Rp gene haploinsufficiency.
Krama, T., Bahhir, D., Ots, L., Popovs, S., Bartkevics, V., Pugajeva, I., Krams, R., Merivee, E., Must, A., Rantala, M. J., Krams, I. and Joers, P. (2023). A diabetes-like biochemical and behavioural phenotype of Drosophila induced by predator stress. Proc Biol Sci 290(2002): 20230442. PubMed ID: 37403506
Summary:
Predation can have both lethal and non-lethal effects on prey. The non-lethal effects of predation can instil changes in prey life history, behaviour, morphology and physiology, causing adaptive evolution. The chronic stress caused by sustained predation on prey is comparable to chronic stress conditions in humans. Conditions like anxiety, depression, and post-traumatic stress syndrome have also been implicated in the development of metabolic disorders such as obesity and diabetes. This study found that predator stress induced during larval development in fruit flies Drosophila melanogaster impairs carbohydrate metabolism by systemic inhibition of Akt protein kinase, which is a central regulator of glucose uptake. However, Drosophila grown with predators survived better under direct spider predation in the adult phase. Administration of metformin and 5-hydroxytryptophan (5-HTP), a precursor of the neurotransmitter serotonin, reversed these effects. These results demonstrate a direct link between predator stress and metabolic impairment, suggesting that a diabetes-like biochemical phenotype may be adaptive in terms of survival and reproductive success. This study provides a novel animal model to explore the mechanisms responsible for the onset of these metabolic disorders, which are highly prevalent in human populations.
Lidsky, P. V., Yuan, J., Lashkevich, K. A., Dmitriev, S. E. and Andino, R. (2023). Monitoring integrated stress response in live Drosophila. bioRxiv. PubMed ID: 37502856
Summary:
Cells exhibit stress responses to various environmental changes. Among these responses, the integrated stress response (ISR) plays a pivotal role as a crucial stress signaling pathway. While extensive ISR research has been conducted on cultured cells, understanding of its implications in multicellular organisms remains limited, largely due to the constraints of current techniques that hinder the ability to track and manipulate the ISR in vivo. To overcome these limitations, this study has successfully developed an internal ribosome entry site (IRES)-based fluorescent reporter system. This innovative reporter enables labelling of Drosophila cells, within the context of a living organism, that exhibit eIF2 phosphorylation-dependent translational shutoff - a characteristic feature of the ISR and viral infections. Through this methodology, this study has unveiled tissue- and cell-specific regulation of stress response in Drosophila flies and have even been able to detect stressed tissues in vivo during virus and bacterial infections. To further validate the specificity of the reporter, this study has engineered ISR-null eIF2αS50A mutant flies for stress response analysis. The results shed light on the tremendous potential of this technique for investigating a broad range of developmental, stress, and infection-related experimental conditions. Combining the reporter tool with ISR-null mutants establishes Drosophila as an exceptionally powerful model for studying the ISR in the context of multicellular organisms.
Hu, D., Li, W., Wang, J., Peng, Y., Yun, Y. and Peng, Y. (2023). Interaction of High Temperature stress and Wolbachia Infection on the Biological Characteristic of Drosophila melanogaster. Insects 14(6). PubMed ID: 37367374
Summary:
It was reported that temperature affects the distribution of Wolbachia in the host, but only a few papers reported the effect of the interaction between high temperature and Wolbachia on the biological characteristic of the host. This study found four treatment Drosophila melanogaster groups: Wolbachia-infected flies in 25 °C (W(+)M), Wolbachia-infected flies in 31 °C (W(+)H), Wolbachia-uninfected flies in 25 °C (W(-)M), Wolbachia-uninfected flies in 31 °C (W(-)H), and the interaction effect of temperature and Wolbachia infection on the biological characteristic of D. melanogaster in F(1), F(2) and F(3) generations was detected. Both temperature and Wolbachia infection had significant effects on the development and survival rate of D. melanogaster. High temperature and Wolbachia infection had interaction effect on hatching rate, developmental durations, emergence rate, body weight and body length of F(1), F(2) and F(3) flies, and the interaction effect also existed on oviposition amount of F(3) flies, and on pupation rate of F(2) and F(3) flies. High temperature stress reduced the Wolbachia vertical transmission efficiency between generations. These results indicated that high temperature stress and Wolbachia infection had negative effects on the morphological development of D. melanogaster.
Li, X., Li, M., Xue, X. and Wang, X. (2023). Proteomic analysis reveals oxidative stress-induced activation of Hippo signaling in thiamethoxam-exposed Drosophila. Chemosphere 338: 139448. PubMed ID: 37437626
Summary:
Thiamethoxam (THIA) is a widely used neonicotinoid insecticide. However, the toxicity and defense mechanisms activated in THIA-exposed insects are unclear. This study used isobaric tags for relative and absolute quantitation (iTRAQ) proteomics technology to identify changes in protein expression in THIA-exposed Drosophila. It was found that the antioxidant proteins Cyp6a23 and Dys were upregulated, whereas vir-1 was downregulated, which may have been detoxification in response to THIA exposure. Prx5 downregulation promoted the generation of reactive oxygen species. Furthermore, the accumulation of reactive oxygen species led to the induction of antioxidant defenses in THIA-exposed Drosophila, thereby enhancing the levels of oxidative stress markers (e.g., superoxide dismutase, glutathione S-transferase, and glutathione) and reducing catalase expression. Furthermore, the Hippo signaling transcription coactivator Yki was inactivated by THIA. Thise results suggesting that Hippo signaling may be necessary to promote insect survival in response to neonicotinoid insecticide toxicity.

Thursday, January 4th - Synapse, ER, and Vesicles

Sung, H. and Lloyd, T. E. (2023). Disrupted endoplasmic reticulum-mediated autophagosomal biogenesis in a Drosophila model of C9-ALS-FTD. Autophagy. PubMed ID: 37599467
Summary:
Macroautophagy/autophagy is a major pathway for the clearance of protein aggregates and damaged organelles, and multiple intracellular organelles participate in the process of autophagy, from autophagosome formation to maturation and degradation. Dysregulation of the autophagy pathway has been implicated in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), however the mechanisms underlying autophagy impairment in these diseases are incompletely understood. Since the expansion of GGGGCC (G(4)C(2)) repeats in the first intron of the C9orf72 gene is the most common inherited cause of both ALS and FTD (C9-ALS-FTD), this study investigated autophagosome dynamics in Drosophila motor neurons expressing 30 G(4)C(2) repeats (30 R). In vivo imaging demonstrates that expression of expanded G(4)C(2) repeats markedly impairs biogenesis of autophagosomes at synaptic termini, whereas trafficking and maturation of axonal autophagosomes are unaffected. Motor neurons expressing 30 R display marked disruption in endoplasmic reticulum (ER) structure and dynamics in the soma, axons, and synapses. Disruption of ER morphology with mutations in Rtnl1 (Reticulon-like 1) or atl (atlastin) also impairs autophagosome formation in motor neurons, suggesting that ER integrity is critical for autophagosome formation. Furthermore, live imaging demonstrates that autophagosomes are generated from dynamic ER tubules at synaptic boutons, and this process fails to occur in a C9-ALS-FTD model. Together, these findings suggest that dynamic ER tubules are required for formation of autophagosomes at the neuromuscular junction, and that this process is disrupted by expanded G(4)C(2) repeats that cause ALS-FTD.
Yang, M., Zinkgraf, M., Fitzgerald-Cook, C., Harrison, B. R., Putzier, A., Promislow, D. E. L. and Wang, A. M. (2023). Using Drosophila to identify naturally occurring genetic modifiers of Aβ42- and tau-induced toxicity. G3 (Bethesda). PubMed ID: 37311212
Summary:
Alzheimer's disease (AD) is characterized by two pathological proteins, amyloid beta 42 (Aβ42) and tau. The majority of AD cases in the population are sporadic and late-onset AD (LOAD), which exhibits high levels of heritability. While several genetic risk factors for LOAD have been identified and replicated in independent studies, including the ApoE ε4 allele, the great majority of the heritability of LOAD remains unexplained, likely due to the aggregate effects of a very large number of genes with small effect size, as well as to biases in sample collection and statistical approaches. This study presents an unbiased forward genetic screen in Drosophila looking for naturally occurring modifiers of Aβ42- and tau-induced ommatidial degeneration. The results identify 14 significant SNPs, which map to 12 potential genes in 8 unique genomic regions. These hits that are significant after genome-wide correction identify genes involved in neuronal development, signal transduction and organismal development. Looking more broadly at suggestive hits (P < 10-5), significant enrichment was seen in genes associated with neurogenesis, development and growth as well as significant enrichment in genes whose orthologs have been identified as significantly or suggestively associated with AD in human GWAS studies. These latter genes include ones whose orthologs are in close proximity to regions in the human genome that are associated with AD, but where a causal gene has not been identified. Together, these results illustrate the potential for complementary and convergent evidence provided through multi-trait GWAS in Drosophila to supplement and inform human studies, helping to identify the remaining heritability and novel modifiers of complex diseases.
Sharma, A., Narasimha, K., Manjithaya, R. and Sheeba, V. (2023). Restoration of Sleep and Circadian Behavior by Autophagy Modulation in Huntington's Disease. J Neurosci 43(26): 4907-4925. PubMed ID: 37268416
Summary:
Circadian and sleep defects are well documented in Huntington's disease (HD). Modulation of the autophagy pathway has been shown to mitigate toxic effects of mutant Huntingtin (HTT) protein. However, it is not clear whether autophagy induction can also rescue circadian and sleep defects. Using a genetic approach, this study expressed human mutant HTT protein in a subset of Drosophila circadian neurons and sleep center neurons. In this context, the contribution was examined of autophagy in mitigating toxicity caused by mutant HTT protein. Targeted overexpression of an autophagy gene, Atg8a in male flies, induces autophagy pathway and partially rescues several HTT-induced behavioral defects, including sleep fragmentation, a key hallmark of many neurodegenerative disorders. Using cellular markers and genetic approaches, it was demonstrated that indeed the autophagy pathway is involved in behavioral rescue. Surprisingly, despite behavioral rescue and evidence for the involvement of the autophagy pathway, the large visible aggregates of mutant HTT protein were not eliminated. The rescue in behavior is associated with increased mutant protein aggregation and possibly enhanced output from the targeted neurons, resulting in the strengthening of downstream circuits. Overall, this study suggests that, in the presence of mutant HTT protein, Atg8a induces autophagy and improves the functioning of circadian and sleep circuits.
Tan, F. H. P., Ting, A. C. J., Najimudin, N., Watanabe, N., Shamsuddin, S., Zainuddin, A., Osada, H. and Azzam, G. (2023). 3-[[(3S)-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl]amino]propanoic acid (THICAPA) is protective against Aβ42-induced toxicity in vitro and in an Alzheimer's Disease Drosophila. J Gerontol A Biol Sci Med Sci. PubMed ID: 37453137
Summary:
Alzheimer's disease (AD) is the most prevalent type of dementia globally. The accumulation of amyloid-beta (Aβ) extracellular senile plaques in the brain is one of the hallmark mechanisms found in AD. Aβ42 is the most damaging and aggressively aggregating Aβ isomer produced in the brain. Although Aβ42 has been extensively researched as a crucial peptide connected to the development of the characteristic amyloid fibrils in AD, the specifics of its pathophysiology are still unknown. Therefore, the main objective was to identify novel compounds that could potentially mitigate the negative effects of Aβ42. 3-[[(3S)-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl]amino]propanoic acid (THICAPA) was identified as a ligand for Aβ42 and for reducing fibrillary Aβ42 aggregation. THICAPA also improved cell viability when administered to PC12 neuronal cells that were exposed to Aβ42. Additionally, this compound diminished Aβ42 toxicity in the current AD Drosophila model by rescuing the rough eye phenotype, prolonging the lifespan and enhancing motor functions. Through Next-generation RNA-sequencing, immune response pathways were downregulated in response to THICAPA treatment. Thus, this study suggests THICAPA as a possible disease-modifying treatment for AD.
Wu, T., Deger, J. M., Ye, H., Guo, C., Dhindsa, J., Pekarek, B. T., Al-Ouran, R., Liu, Z., Al-Ramahi, I., Botas, J. and Shulman, J. M. (2023). Tau polarizes an aging transcriptional signature to excitatory neurons and glia. Elife 12. PubMed ID: 37219079
Summary:
Aging is a major risk factor for Alzheimer's disease (AD), and cell-type vulnerability underlies its characteristic clinical manifestations. Longitudinal, single-cell RNA-sequencing was performed in Drosophila with pan-neuronal expression of human tau, which forms AD neurofibrillary tangle pathology. Whereas tau- and aging-induced gene expression strongly overlap (93%), they differ in the affected cell types. In contrast to the broad impact of aging, tau-triggered changes are strongly polarized to excitatory neurons and glia. Further, tau can either activate or suppress innate immune gene expression signatures in a cell-type-specific manner. Integration of cellular abundance and gene expression pinpoints nuclear factor kappa B signaling in neurons as a marker for cellular vulnerability. This study also highlights the conservation of cell-type-specific transcriptional patterns between Drosophila and human postmortem brain tissue. Overall, these results create a resource for dissection of dynamic, age-dependent gene expression changes at cellular resolution in a genetically tractable model of tauopathy.
Suzuki, M., Kuromi, H., Shindo, M., Sakata, N., Niimi, N., Fukui, K., Saitoe, M. and Sango, K. (2023). A Drosophila model of diabetic neuropathy reveals a role of proteasome activity in the glia. iScience 26(6): 106997. PubMed ID: 37378316
Summary:
Diabetic peripheral neuropathy (DPN) is the most common chronic, progressive complication of diabetes mellitus. The main symptom is sensory loss; the molecular mechanisms are not fully understood. This study found that Drosophila fed a high-sugar diet, which induces diabetes-like phenotypes, exhibit impairment of noxious heat avoidance. The impairment of heat avoidance was associated with shrinkage of the leg neurons expressing the Drosophila transient receptor potential channel Painless. Using a candidate genetic screening approach proteasome modulator 9b (CG9588)/ was identified as one of the modulators of impairment of heat avoidance. It waS further showN that proteasome inhibition in the glia reversed the impairment of noxious heat avoidance, and heat-shock proteins and endolysosomal trafficking in the glia mediated the effect of proteasome inhibition. These results establish Drosophila as a useful system for exploring molecular mechanisms of diet-induced peripheral neuropathy and propose that the glial proteasome is one of the candidate therapeutic targets for DPN.

Wednesday, January 3rd- Synapse, ER, and Vesicles

Miao, H., Millage, M., Rollins, K. R. and Blankenship, J. T. (2023). A Rab39-Klp98A-Rab35 endocytic recycling pathway is essential for rapid Golgi-dependent furrow ingression. Development 150(16). PubMed ID: 37590130
Summary:
Ingression of the plasma membrane is an essential part of the cell topology-distorting repertoire and a key element in animal cell cytokinesis. Many embryos have rapid cleavage stages in which they are furrowing powerhouses, quickly forming and disassembling cleavage furrows on timescales of just minutes. Previous work has shown that cytoskeletal proteins and membrane trafficking coordinate to drive furrow Ingression, but where these membrane stores are derived from and how they are directed to furrowing processes has been less clear. This study identified an extensive Rab35/Rab4>Rab39/Klp98A>gt;trans-Golgi network (TGN) endocytic recycling pathway necessary for fast furrow ingression in the Drosophila embryo. Rab39 is present in vesiculotubular compartments at the TGN where it receives endocytically derived cargo through a Rab35/Rab4-dependent pathway. A Kinesin-3 family member, Klp98A, drives the movements and tubulation activities of Rab39, and disruption of this Rab39-Klp98A-Rab35 pathway causes deep furrow ingression defects and genomic instability. These data suggest that an endocytic recycling pathway rapidly remobilizes membrane cargo from the cell surface and directs it to the trans-Golgi network to permit the initiation of new cycles of cleavage furrow formation.
Maruzs, T., Feil-Borcsok, D., Lakatos, E., Juhasz, G., Blastyak, A., Hargitai, D., Jean, S., Lorincz, P. and Juhasz, G. (2023). Interaction of the sorting nexin 25 homologue Snazarus with Rab11 balances endocytic and secretory transport and maintains the ultrafiltration diaphragm in nephrocytes. Mol Biol Cell 34(9): ar87. PubMed ID: 37314856
Summary:
Proper balance of exocytosis and endocytosis is important for the maintenance of plasma membrane lipid and protein homeostasis. This is especially critical in human podocytes and the podocyte-like Drosophila nephrocytes that both use a delicate diaphragm system with evolutionarily conserved components for ultrafiltration. This study shows that the sorting nexin 25 homologue Snazarus (Snz) binds to Rab11 and localizes to Rab11-positive recycling endosomes in Drosophila nephrocytes, unlike in fat cells where it is present in plasma membrane/lipid droplet/endoplasmic reticulum contact sites. loss of Snz leads to redistribution of Rab11 vesicles from the cell periphery and increases endocytic activity in nephrocytes. These changes are accompanied by defects in diaphragm protein distribution that resemble those seen in Rab11 gain-of-function cells. Of note, co-overexpression of Snz rescues diaphragm defects in Rab11 overexpressing cells, whereas snz knockdown in Rab11 overexpressing nephrocytes or simultaneous knockdown of snz and tbc1d8b encoding a Rab11 GTPase-activating protein (GAP) leads to massive expansion of the lacunar system that contains mislocalized diaphragm components: Sns and Pyd/ZO-1. Loss of Snz enhances while its overexpression impairs secretion, which, together with genetic epistasis analyses, suggest that Snz counteracts Rab11 to maintain the diaphragm via setting the proper balance of exocytosis and endocytosis.
Maddison, D. C., Malik, B., Amadio, L., Bis-Brewer, D. M., Zuchner, S., Peters, O. M. and Smith, G. A. (2023). COPI-regulated mitochondria-ER contact site formation maintains axonal integrity. Cell Rep 42(8): 112883. PubMed ID: 37498742
Summary:
Coat protein complex I (COPI) is best known for its role in Golgi-endoplasmic reticulum (ER) trafficking, responsible for the retrograde transport of ER-resident proteins. The ER is crucial to neuronal function, regulating Ca(2+) homeostasis and the distribution and function of other organelles such as endosomes, peroxisomes, and mitochondria via functional contact sites. This study demonstrates that disruption of COPI results in mitochondrial dysfunction in Drosophila axons and human cells. The ER network is also disrupted, and the neurons undergo rapid degeneration. Mitochondria-ER contact sites (MERCS) are decreased in COPI-deficient axons, leading to Ca(2+) dysregulation, heightened mitophagy, and a decrease in respiratory capacity. Reintroducing MERCS is sufficient to rescue not only mitochondrial distribution and Ca(2+) uptake but also ER morphology, dramatically delaying neurodegeneration. This work demonstrates an important role for COPI-mediated trafficking in MERC formation, which is an essential process for maintaining axonal integrity.
Mohylyak, I., Bengochea, M., Pascual-Caro, C., Asfogo, N., Fonseca-Topp, S., Danda, N., Atak, Z. K., De Waegeneer, M., Placais, P. Y., Preat, T., Aerts, S., Corti, O., de Juan-Sanz, J. and Hassan, B. A. (2023). Developmental transcriptional control of mitochondrial homeostasis is required for activity-dependent synaptic connectivity. bioRxiv. PubMed ID: 37333418
Summary:
During neuronal circuit formation, local control of axonal organelles ensures proper synaptic connectivity. Whether this process is genetically encoded is unclear and if so, its developmental regulatory mechanisms remain to be identified. It was hypothesized that developmental transcription factors regulate critical parameters of organelle homeostasis that contribute to circuit wiring. This study combined cell type-specific transcriptomics with a genetic screen to discover such factors. Telomeric Zinc finger-Associated Protein (TZAP; CG7101) as a temporal developmental regulator of neuronal mitochondrial homeostasis genes, including Pink1. In Drosophila, loss of dTzap function during visual circuit development leads to loss of activity-dependent synaptic connectivity, that can be rescued by Pink1 expression. At the cellular level, loss of dTzap/TZAP leads to defects in mitochondrial morphology, attenuated calcium uptake and reduced synaptic vesicle release in fly and mammalian neurons. These findings highlight developmental transcriptional regulation of mitochondrial homeostasis as a key factor in activity-dependent synaptic connectivity.
Rozenfeld, E., Ehmann, N., Manoim, J. E., Kittel, R. J. and Parnas, M. (2023). Homeostatic synaptic plasticity rescues neural coding reliability. Nat Commun 14(1): 2993. PubMed ID: 37225688
Summary:
To survive, animals must recognize reoccurring stimuli. This necessitates a reliable stimulus representation by the neural code. While synaptic transmission underlies the propagation of neural codes, it is unclear how synaptic plasticity can maintain coding reliability. By studying the olfactory system of Drosophila melanogaster, this study aimed to obtain a deeper mechanistic understanding of how synaptic function shapes neural coding in the live, behaving animal. It was shown that the properties of the active zone (AZ), the presynaptic site of neurotransmitter release, are critical for generating a reliable neural code. Reducing neurotransmitter release probability of olfactory sensory neurons disrupts both neural coding and behavioral reliability. Strikingly, a target-specific homeostatic increase of AZ numbers rescues these defects within a day. These findings demonstrate an important role for synaptic plasticity in maintaining neural coding reliability and are of pathophysiological interest by uncovering an elegant mechanism through which the neural circuitry can counterbalance perturbations. .
Wang, R., Fortier, T. M., Chai, F., Miao, G., Shen, J. L., Restrepo, L. J., DiGiacomo, J. J., Velentzas, P. D. and Baehrecke, E. H. (2023). PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development. Cell. PubMed ID: 37633267
Summary:
Selective clearance of organelles, including endoplasmic reticulum (ER) and mitochondria, by autophagy plays an important role in cell health. This study describes a developmentally programmed selective ER clearance by autophagy. This study shows that Parkinson's disease-associated PINK1, as well as Atl, Rtnl1, and Trp1 receptors, regulate ER clearance by autophagy. The E3 ubiquitin ligase Parkin functions downstream of PINK1 and is required for mitochondrial clearance while having the opposite function in ER clearance. By contrast, Keap1 and the E3 ubiquitin ligase Cullin3 function downstream of PINK1 to regulate ER clearance by influencing Rtnl1 and Atl. PINK1 regulates a change in Keap1 localization and Keap1-dependent ubiquitylation of the ER-phagy receptor Rtnl1 to facilitate ER clearance. Thus, PINK1 regulates the selective clearance of ER and mitochondria by influencing the balance of Keap1- and Parkin-dependent ubiquitylation of substrates that determine which organelle is removed by autophagy

Tuesday, January 2nd - Signal Transduction

Sokolowski, D. J., Vasquez, O. E., Wilson, M. D., Sokolowski, M. B. and Anreiter, I. (2023). Transcriptomic effects of the foraging gene shed light on pathways of pleiotropy and plasticity. Ann N Y Acad Sci 1526(1): 99-113. PubMed ID: 37350250
Summary:
>Genes are often pleiotropic and plastic in their expression, features which increase and diversify the functionality of the genome. The foraging (for) gene in Drosophila melanogaster is highly pleiotropic and a long-standing model for studying individual differences in behavior and plasticity from ethological, evolutionary, and genetic perspectives. Its pleiotropy is known to be linked to its complex molecular structure; however, the downstream pathways and interactors remain mostly elusive. To uncover these pathways and interactors and gain a better understanding of how pleiotropy and plasticity are achieved at the molecular level, this study explored the effects of different for alleles on gene expression at baseline and in response to 4 h of food deprivation, using RNA sequencing analysis in different Drosophila larval tissues. The results show tissue-specific transcriptomic dynamics influenced by for allelic variation and food deprivation, as well as genotype by treatment interactions. Differentially expressed genes yielded pathways linked to previously described for phenotypes and several potentially novel phenotypes. Together, these findings provide putative genes and pathways through which for might regulate its varied phenotypes in a pleiotropic, plastic, and gene-structure-dependent manner.
Shieh, B. H., Sun, W. and Ferng, D. (2023). A conventional PKC critical for both the light-dependent and the light-independent regulation of the actin cytoskeleton in Drosophila photoreceptors. J Biol Chem 299(6): 104822. PubMed ID: 37201584
Summary:
Pkc53E is the second conventional protein kinase C (PKC) gene expressed in Drosophila photoreceptors; it encodes at least six transcripts generating four distinct protein isoforms including Pkc53E-B whose mRNA is preferentially expressed in photoreceptors. By characterizing transgenic lines expressing Pkc53E-B-GFP, this study showed Pkc53E-B is localized in the cytosol and rhabdomeres of photoreceptors, and the rhabdomeric localization appears dependent on the diurnal rhythm. A loss of function of pkc53E-B leads to light-dependent retinal degeneration. Interestingly, the knockdown of pkc53E also impacted the actin cytoskeleton of rhabdomeres in a light-independent manner. Here the Actin-GFP reporter is mislocalized and accumulated at the base of the rhabdomere, suggesting that Pkc53E regulates depolymerization of the actin microfilament. The light-dependent regulation of Pkc53E was demonstrated and it was demonstrated that activation of Pkc53 E can be independent of the phospholipase C PLCβ4/NorpA as degeneration of norpA(P24) photoreceptors was enhanced by a reduced Pkc53E activity. It was further shown that the activation of Pkc53E may involve the activation of Plc21C by Gqα. Taken together, Pkc53E-B appears to exert both constitutive and light-regulated activity to promote the maintenance of photoreceptors possibly by regulating the actin cytoskeleton.
Roberto, G. M., Boutet, A., Keil, S. and Emery, G. (2023). Dual regulation of Misshapen by Tao and Rap2l promotes collective cell migration. bioRxiv. PubMed ID: 37503122
Summary:
Collective cell migration occurs in various biological processes such as development, wound healing and metastasis. During Drosophila oogenesis, border cells (BC) form a cluster that migrates collectively inside the egg chamber. The Ste20-like kinase Misshapen (Msn) is a key regulator of BC migration coordinating the restriction of protrusion formation and contractile forces within the cluster. This study demonstrates that the kinase Tao acts as an upstream activator of Msn in BCs. Depletion of Tao significantly impedes BC migration and produces a phenotype similar to Msn loss-of-function. Furthermore, it was shown that the localization of Msn relies on its CNH domain, which interacts with the small GTPase Rap2l. These findings indicate that Rap2l promotes the trafficking of Msn to the endolysosomal pathway. When Rap2l is depleted, the levels of Msn increase in the cytoplasm and at cell-cell junctions between BCs. Overall, these data suggest that Rap2l ensures that the levels of Msn are higher at the periphery of the cluster through the targeting of Msn to the degradative pathway. Together, this study identified two distinct regulatory mechanisms that ensure the appropriate distribution and activation of Msn in BCs.
Townson, J. M., Gomez-Lamarca, M. J., Santa Cruz Mateos, C. and Bray, S. J. (2023). OptIC-Notch reveals mechanism that regulates receptor interactions with CSL. Development 150(11). PubMed ID: 37294169
Summary:
Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes, forming a complex with the DNA-binding transcription factor CSL [CBF1/Su(H)/LAG-1] and co-activator Mastermind. However, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe the mechanisms involved, an optogenetic approach was designed to control NICD release (OptIC-Notch) and monitored the subsequent complex formation and target gene activation. Strikingly, it was observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane φWφP motif is key to sequestration, this motif was masked with a second light-sensitive domain (OptIC-Notch{ω}), which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light-controlled activation. These results demonstrate that exposure of the φWφP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry.
Prakash, A., Monteith, K. M., Bonnet, M. and Vale, P. F. (2023). Duox and Jak/Stat signalling influence disease tolerance in Drosophila during Pseudomonas entomophila infection. Dev Comp Immunol 147: 104756. PubMed ID: 37302730
Summary:
Disease tolerance describes an infected host's ability to maintain health independently of the ability to clear microbe loads. The Jak/Stat pathway plays a pivotal role in humoral innate immunity by detecting tissue damage and triggering cellular renewal, making it a candidate tolerance mechanism. This study found that in Drosophila melanogaster infected with Pseudomonas entomophila disrupting ROS-producing dual oxidase (duox) or the negative regulator of Jak/Stat Socs36E, render male flies less tolerant. Another negative regulator of Jak/Stat, G9a - which has previously been associated with variable tolerance of viral infections - did not affect the rate of mortality with increasing microbe loads compared to flies with functional G9a, suggesting it does not affect tolerance of bacterial infection as in viral infection. These findings highlight that ROS production and Jak/Stat signalling influence the ability of flies to tolerate bacterial infection sex-specifically and may therefore contribute to sexually dimorphic infection outcomes in Drosophila.
Pierini, G. and Dahmann, C. (2023). Hedgehog morphogen gradient is robust towards variations in tissue morphology in Drosophila. Sci Rep 13(1): 8454. PubMed ID: 37231029
Summary:
During tissue development, gradients of secreted signaling molecules known as morphogens provide cells with positional information. The mechanisms underlying morphogen spreading have been widely studied, however, it remains largely unexplored whether the shape of morphogen gradients is influenced by tissue morphology. This study developed an analysis pipeline to quantify the distribution of proteins within a curved tissue. This analyses was applied to the Hedgehog morphogen gradient in the Drosophila wing and eye-antennal imaginal discs, which are flat and curved tissues, respectively. Despite a different expression profile, the slope of the Hedgehog gradient was comparable between the two tissues. Moreover, inducing ectopic folds in wing imaginal discs did not affect the slope of the Hedgehog gradient. Suppressing curvature in the eye-antennal imaginal disc also did not alter the Hedgehog gradient slope but led to ectopic Hedgehog expression. In conclusion, through the development of an analysis pipeline that allows quantifying protein distribution in curved tissues, this study showed that the Hedgehog gradient is robust towards variations in tissue morphology.
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