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Friday, June 13th - Disease Models

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Lai, Y., Reina-Gonzalez, P., Maor, G., Miller, G. W., Sarkar, S. (2023). Biotin rescues manganese-induced Parkinson's disease phenotypes and neurotoxicity. bioRxiv, PubMed ID: 38045419
Summary:
Occupational exposure to manganese (Mn) induces manganism and has been widely linked as a contributing environmental factor to Parkinson's disease (PD), featuring dramatic signature overlaps between the two in motor symptoms and clinical hallmarks. However, the molecular mechanism underlying such link remains elusive, and for combating PD, effective mechanism-based therapies are lacking. This study developed an adult Drosophila model of Mn toxicity to recapitulate key parkinsonian features, spanning behavioral deficits, neuronal loss, and dysfunctions in lysosome and mitochondria. Global metabolomics was performed on flies at an early stage of toxicity, and metabolism of the B vitamin, biotin (vitamin B (7)) was identified as a master pathway underpinning Mn toxicity with systemic, body-brain increases in Mn-treated groups compared to the controls. Using Btnd (RNAi) mutant flies, we show that biotin depletion exacerbates Mn-induced neurotoxicity, parkinsonism, and mitochondrial dysfunction; while in Mn-exposed wild-type flies, biotin feeding dramatically ameliorates these pathophenotypes. We further show in human induced stem cells (iPSCs)- differentiated midbrain dopaminergic neurons that the supplemented biotin protects against Mn-induced neuronal loss, cytotoxicity, and mitochondrial dysregulation. Finally, human data profiling biotin-related proteins show for PD cases elevated circulating levels of biotin transporters but not of metabolic enzymes compared to healthy controls, suggesting humoral biotin transport as a key event involved in PD. Taken together, these findings identified compensatory biotin pathway as a convergent, systemic driver of Mn toxicity and parkinsonian pathology, providing new basis for devising effective countermeasures against manganism and PD.
Atilano, M. L., Hull, A., Romila, C. A., Adams, M. L., Wildfire, J., Ureña, E., Dyson, M., Ivan-Castillo-Quan, J., Partridge, L., Kinghorn, K. J. (2023). Autophagic dysfunction and gut microbiota dysbiosis cause chronic immune activation in a Drosophila model of Gaucher disease. PLoS Genet, 19(12):e1011063 PubMed ID: 38127816
Summary:
Mutations in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD) and are the greatest known genetic risk factors for Parkinson's disease (PD). Communication between the gut and brain and immune dysregulation are increasingly being implicated in neurodegenerative disorders such as PD. This study shows that flies lacking the Gba1b gene, the main fly orthologue of GBA1, display widespread NF-kB signalling activation, including gut inflammation, and brain glial activation. Intestinal autophagic defects, gut dysfunction, and microbiome dysbiosis were also demonstrated. Remarkably, modulating the microbiome of Gba1b knockout flies, by raising them under germ-free conditions, partially ameliorates lifespan, locomotor and immune phenotypes. Moreover, modulation of the immune deficiency (IMD) pathway is detrimental to the survival of Gba1 deficient flies. It was also revealed that direct stimulation of autophagy by rapamycin treatment achieves similar benefits to germ-free conditions independent of gut bacterial load. Consistent with this, pharmacologically blocking autophagosomal-lysosomal fusion, mimicking the autophagy defects of Gba1 depleted cells, was shown to be sufficient to stimulate intestinal immune activation. Overall, these data elucidate a mechanism whereby an altered microbiome, coupled with defects in autophagy, drive chronic activation of NF-kB signaling in a Gba1 loss-of-function model. It also highlights that elimination of the microbiota or stimulation of autophagy to remove immune mediators, rather than prolonged immunosuppression, may represent effective therapeutic avenues for GBA1-associated disorders.
Chvilicek, M. M., Seguin, A., Lathen, D. R., Titos, I., Cummins-Beebe, P. N., Pabon, M. A., Miscevic, M., Nickel, E. A., Merrill, C. B., Rodan, A. R., Rothenfluh, A. (2023). Large genetic analysis of alcohol resistance and tolerance reveals an inverse correlation and suggests 'true' tolerance mutants. bioRxiv, PubMed ID: 37873285
Summary:
Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce the same behavioral effects. Tolerance is historically not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and between labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. This study has analyzed a large amount of data to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes. Additionally, it was shown that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on this analysis, a method is providing for using a linear regression equation to assess the residuals of potential tolerance mutants. These residuals provide predictive insight into the likelihood of a mutant being a 'true' tolerance mutant, and a framework is offered for understanding the relationship between initial resistance and tolerance.
Liang, Y., Pan, C., Yin, T., Wang, L., Gao, X., Wang, E., Quang, H., Huang, D., Tan, L., Xiang, K., Wang, Y., Alexander, P. B., Li, Q. J., Yao, T. P., Zhang, Z., Wang, X. F. (2024). Branched-Chain Amino Acid Accumulation Fuels the Senescence-Associated Secretory Phenotype. Adv Sci (Weinh), 11(2):e2303489 PubMed ID: 37964763
Summary:
The essential branched-chain amino acids (BCAAs) leucine, isoleucine, and valine play critical roles in protein synthesis and energy metabolism. Despite their widespread use as nutritional supplements, BCAAs' full effects on mammalian physiology remain uncertain due to the complexities of BCAA metabolic regulation. Here a novel mechanism linking intrinsic alterations in BCAA metabolism is identified to cellular senescence and the senescence-associated secretory phenotype (SASP), both of which contribute to organismal aging and inflammation-related diseases. Altered BCAA metabolism driving the SASP is mediated by robust activation of the BCAA transporters Solute Carrier Family 6 Members 14 and 15 as well as downregulation of the catabolic enzyme BCAA transaminase 1 during onset of cellular senescence, leading to highly elevated intracellular BCAA levels in senescent cells. This, in turn, activates the mammalian target of rapamycin complex 1 (mTORC1) to establish the full SASP program. Transgenic Drosophila models further indicate that orthologous BCAA regulators are involved in the induction of cellular senescence and age-related phenotypes in flies, suggesting evolutionary conservation of this metabolic pathway during aging. Finally, experimentally blocking BCAA accumulation attenuates the inflammatory response in a mouse senescence model, highlighting the therapeutic potential of modulating BCAA metabolism for the treatment of age-related and inflammatory diseases.
Yamaguchi, M., Huynh, M. A., Chiyonobu, T., Yoshida, H. (2023). Knockdown of Chronophage in the nervous system mimics features of neurodevelopmental disorders caused by BCL11A/B variants. Exp Cell Res, 433(2):113827 PubMed ID: 37926342
Summary:
Neurodevelopmental disorders (NDD) are a group of disorders that include intellectual disability. Although several genes have been implicated in NDD, the molecular mechanisms underlying its pathogenesis remain unclear. Therefore, it is important to develop novel models to analyze the functions of NDD-causing genes in vivo. Recently, rare pathogenic variants of the B-cell lymphoma/leukemia11A/B (BCL11A/B) gene have been identified in several patients with NDD. Drosophila carries the Chronophage (Cph) gene, which has been predicted to be a homolog of BCL11A/B based on the conservation of the amino acid sequence. This study investigated whether nervous system-specific knockdown of Cph mimics NDD phenotypes in Drosophila. Nervous system-specific knockdown of Cph induced learning and locomotor defects in larvae and epilepsy-like behaviors in adults. The number of synaptic branches was also elevated in the larval neuromuscular junction without a corresponding increase in the number of boutons. Furthermore, the expression levels of putative target genes that are Drosophila homologs of the mammalian BCL11 target genes were decreased in Cph knockdown flies. These results suggest that Cph knockdown flies are a promising model for investigating the pathology of NDD-induced BCL11A/B dysfunction.
Sun, Y., Dai, H., Dai, X., Yin, J., Cui, Y., Liu, X., Gonzalez, G., Yuan, J., Tang, F., Wang, N., Perlegos, A. E., Bonini, N. M., Yang, X. W., Gu, W., Wang, Y. (2023). m(1)A in CAG repeat RNA binds to TDP-43 and induces neurodegeneration. Nature, 623(7987):580-587 PubMed ID: 37938769
Summary:
Microsatellite repeat expansions within genes contribute to a number of neurological diseases such as ALS. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology. This study revealed that the adenosine in CAG repeat RNA can be methylated to N(1)-methyladenosine (m(1)A) by TRMT61A, and that m(1)A can be demethylated by ALKBH3. It was also observed that the m(1)A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m(1)A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m(1)A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, this study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m(1)A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion.

Thursday, June 13th - RNAs and Transposons

Morillo, L., Paternina, T., Alasseur, Q., Genovesio, A., Schwartz, S., Le Hir, H. (2023). Comprhensive mapping of exon junction complex binding sites reveals universal EJC deposition in Drosophila. BMC Biol, 21(1):246 PubMed ID: 37936138
Summary:
The exon junction complex (EJC) is involved in most steps of the mRNA life cycle, ranging from splicing to nonsense-mediated mRNA decay (NMD). It is assembled by the splicing machinery onto mRNA in a sequence-independent manner. A fundamental open question is whether the EJC is deposited onto all exon‒exon junctions or only on a subset of them. Several previous studies have made observations supportive of the latter, yet these have been limited by methodological constraints. This study sought to overcome these limitations via the integration of two different approaches for transcriptome-wide mapping of EJCs. The results revealed that nearly all, if not all, internal exons consistently harbor an EJC in Drosophila, demonstrating that EJC presence is an inherent consequence of the splicing reaction. Furthermore,this study underscores the limitations of eCLIP methods in fully elucidating the landscape of RBP (RNA-binding protein) binding sites. These findings highlight how highly specific (low false positive) methodologies can lead to erroneous interpretations due to partial sensitivity (high false negatives). This study contributes to understanding of EJC deposition and its association with pre-mRNA splicing. The universal presence of EJC on internal exons underscores its significance in ensuring proper mRNA processing. Additionally, these observations highlight the need to consider both specificity and sensitivity in RBP mapping methodologies.
Wang, M., Liang, A. M., Zhou, Z. Z., Pang, T. L., Fan, Y. J., Xu, Y. Z. (2023). Deletions of singular U1 snRNA gene significantly interfere with transcription and 3'-end mRNA formation. PLoS Genet, 19(11):e1011021 PubMed ID: 37917726
Summary:
Small nuclear RNAs (snRNAs) are structural and functional cores of the spliceosome. In metazoan genomes, each snRNA has multiple copies/variants, up to hundreds in mammals. However, the expressions and functions of each copy/variant in one organism have not been systematically studied. Focusing on U1 snRNA genes, this study investigated all five copies in Drosophila melanogaster using two series of constructed strains. Analyses of transgenic flies that each have a U1 promoter-driven gfp revealed that U1:21D is the major and ubiquitously expressed copy, and the other four copies have specificities in developmental stages and tissues. Mutant strains that each have a precisely deleted copy of U1-gene exhibited various extents of defects in fly morphology or mobility, especially deletion of U1:82Eb. Interestingly, splicing was changed at limited levels in the deletion strains, while large amounts of differentially-expressed genes and alternative polyadenylation events were identified, showing preferences in the down-regulation of genes with 1-2 introns and selection of proximal sites for 3'-end polyadenylation. In vitro assays suggested that Drosophila U1 variants pulled down fewer SmD2 proteins compared to the canonical U1. This study demonstrates that all five U1-genes in Drosophila have physiological functions in development and play regulatory roles in transcription and 3'-end formation.
Ghosh, S., Chakraborti, S., Devi, D., Sahu, R., Mandal, S., Mandal, L. (2024). A conserved nutrient responsive axis mediates autophagic degradation of miRNA-mRNA hybrids in blood cell progenitors. Nucleic Acids Res, 52(1):385-403 PubMed ID: 37994707
Summary:
In animals, microRNAs are amongst the primary non-coding RNAs involved in regulating the gene expression of a cell. Most mRNAs in a cell are targeted by one or many miRNAs. Although several mechanisms can be attributed to the degradation of miRNA and mRNA within a cell, but the involvement of autophagy in the clearance of miRNA and its target mRNA is not known. This study discovered a leucine-responsive axis in blood cell progenitors that can mediate an autophagy-directed degradation of miRNA-bound mRNA in Drosophila melanogaster and Homo sapiens. This previously unknown miRNA clearance axis is activated upon amino acid deprivation that can traffic miRNA-mRNA-loaded Argonaute for autophagic degradation in a p62-dependent manner. Thus, this research not only reports a novel axis that can address the turnover of a catalytically active miRISC but also elucidates a slicer-independent mechanism through which autophagy can selectively initiate the clearance of target mRNA.
Zhang, Y., Duan, Y. (2023). Genome-Wide Analysis on Driver and Passenger RNA Editing Sites Suggests an Underestimation of Adaptive Signals in Insects. Genes, 14(10) PubMed ID: 37895300
Summary:
Adenosine-to-inosine (A-to-I) RNA editing leads to a similar effect to A-to-G mutations. RNA editing provides a temporo-spatial flexibility for organisms. Nonsynonymous (Nonsyn) RNA editing in insects is over-represented compared with synonymous (Syn) editing, suggesting adaptive signals of positive selection on Nonsyn editing during evolution. We utilized the brain RNA editome of Drosophila melanogaster to systematically study the LD (r(2)) between editing sites and infer its impact on the adaptive signals of RNA editing. Pairs of editing sites (PESs) were identified from the transcriptome. For CDS PESs of two consecutive editing sites, their occurrence was significantly biased to type-3 PES (Syn-Nonsyn). The haplotype frequency of type-3 PES exhibited a significantly higher abundance of AG than GA, indicating that the rear Nonsyn site is the driver that promotes the editing of the front Syn site (passenger). The exclusion of passenger Syn sites dramatically amplifies the adaptive signal of Nonsyn RNA editing. This study for the first time quantitatively demonstrates that the linkage between RNA editing events comes from hitchhiking effects and leads to the underestimation of adaptive signals for Nonsyn editing. This work provides novel insights for studying the evolutionary significance of RNA editing events.
Luo, Y., He, P., Kanrar, N., Fejes Toth, K., Aravin, A. A. (2023). Maternally inherited siRNAs initiate piRNA cluster formation. Mol Cell, 83(21):3835-3851.e3837 PubMed ID: 37875112
Summary:
PIWI-interacting RNAs (piRNAs) guide transposable element repression in animal germ lines. In Drosophila, piRNAs are produced from heterochromatic loci, called piRNA clusters, which act as information repositories about genome invaders. piRNA generation by dual-strand clusters depends on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, which is deposited on clusters guided by piRNAs, forming a positive feedback loop in which piRNAs promote their own biogenesis. However, how piRNA clusters are formed before cognate piRNAs are present remains unknown. This study reports spontaneous de novo piRNA cluster formation from repetitive transgenic sequences. Cluster formation occurs over several generations and requires continuous trans-generational maternal transmission of small RNAs. Maternally supplied small interfering RNAs (siRNAs) were found to trigger de novo cluster activation in progeny. In contrast, siRNAs are dispensable for cluster function after its establishment. These results reveal an unexpected interplay between the siRNA and piRNA pathways and suggest a mechanism for de novo piRNA cluster formation triggered by siRNAs.
Zhang, S., Wang, R., Zhu, X., Zhang, L., Liu, X., Sun, L. (2023). Characteristics and expression of lncRNA and transposable elements in Drosophila aneuploidy. iScience, 26(12):108494 PubMed ID: 38125016
Summary:
Aneuploidy can globally affect the expression of the whole genome, which is detrimental to organisms. Dosage-sensitive regulators usually have multiple intermolecular interactions, and changes in their stoichiometry are responsible for the dysregulation of the regulatory network. Currently, studies on noncoding genes in aneuploidy are relatively rare. The characteristics and expression profiles of long noncoding RNAs (lncRNAs) and transposable elements (TEs) were studied in aneuploid Drosophila. It is found that lncRNAs and TEs are affected by genomic imbalance and appear to be more sensitive to an inverse dosage effect than mRNAs. Several dosage-sensitive lncRNAs and TEs were detected for their expression patterns during embryogenesis, and their biological functions in the ovary and testes were investigated using tissue-specific RNAi. This study advances understanding of the noncoding sequences in imbalanced genomes and provides a novel perspective for the study of aneuploidy-related human diseases such as cancer.

Wednesday, June 12th - Junctions and Vesicles

Shaheen, A., Richter Gorey, C. L., Sghaier, A., Dason, J. S. (2023). Cholesterol is required for activity-dependent synaptic growth. J Cell Sci, 136(22) PubMed ID: 37902091
Summary:
Changes in cholesterol content of neuronal membranes occur during development and brain aging. Little is known about whether synaptic activity regulates cholesterol levels in neuronal membranes and whether these changes affect neuronal development and function. This study generated transgenic flies that express the cholesterol-binding D4H domain of perfringolysin O toxin and found increased levels of cholesterol in presynaptic terminals of Drosophila larval neuromuscular junctions following increased synaptic activity. Reduced cholesterol impaired synaptic growth and largely prevented activity-dependent synaptic growth. Presynaptic knockdown of adenylyl cyclase phenocopied the impaired synaptic growth caused by reducing cholesterol. Furthermore, the effects of knocking down adenylyl cyclase and reducing cholesterol were not additive, suggesting that they function in the same pathway. Increasing cAMP levels using a dunce mutant with reduced phosphodiesterase activity failed to rescue this impaired synaptic growth, suggesting that cholesterol functions downstream of cAMP. A protein kinase A (PKA) sensor was used to show that reducing cholesterol levels reduced presynaptic PKA activity. Collectively, these results demonstrate that enhanced synaptic activity increased cholesterol levels in presynaptic terminals and that these changes likely activate the cAMP-PKA pathway during activity-dependent growth.
Guangming, G., Mei, C., Qinfeng, Y., Xiang, G., Chenchen, Z., Qingyuan, S., Wei, X., Junhua, G. (2023). Neurexin and neuroligins jointly regulate synaptic degeneration at the Drosophila neuromuscular junction based on TEM studies. Frontiers in cellular neuroscience, 17:1257347 PubMed ID: 38026694
Summary:
The Drosophila larval neuromuscular junction (NMJ) is a well-known model system and is often used to study synapse development. This study shows synaptic degeneration at NMJ boutons, primarily based on transmission electron microscopy (TEM) studies. When degeneration starts, the subsynaptic reticulum (SSR) swells, retracts and folds inward, and the residual SSR then degenerates into a disordered, thin or linear membrane. The axon terminal begins to degenerate from the central region, and the T-bar detaches from the presynaptic membrane with clustered synaptic vesicles to accelerate large-scale degeneration. There are two degeneration modes for clear synaptic vesicles. In the first mode, synaptic vesicles without actin filaments degenerate on the membrane with ultrafine spots and collapse and disperse to form an irregular profile with dark ultrafine particles. In the second mode, clear synaptic vesicles with actin filaments degenerate into dense synaptic vesicles, form irregular dark clumps without a membrane, and collapse and disperse to form an irregular profile with dark ultrafine particles. Last, all residual membranes in NMJ boutons degenerate into a linear shape, and all the residual elements in axon terminals degenerate and eventually form a cluster of dark ultrafine particles. Swelling and retraction of the SSR occurs prior to degradation of the axon terminal, which degenerates faster and with more intensity than the SSR. NMJ bouton degeneration occurs under normal physiological conditions but is accelerated in Drosophila neurexin (dnrx) dnrx273, Drosophila neuroligin (dnlg) dnlg1 and dnlg4 mutants and dnrx83;dnlg3 and dnlg2;dnlg3 double mutants, which suggests that both neurexin and neuroligins play a vital role in preventing synaptic degeneration.
Chen, X., Perry, S., Wang, B., Wang, S., Hu, J., Loxterkamp, E., Dickman, D., Han, C. (2023). Tissue-specific knockout in Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles. bioRxiv, PubMed ID: 37808853
Summary:
Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions in animal development. However, this approach has been successfully applied in only a small number of Drosophila tissues. The Drosophila motor nervous system is an excellent model system for studying the biology of neuromuscular junction (NMJ). To expand tissue-specific CRISPR to the Drosophila motor system, this study presents a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. The efficacy of this toolkit was validated by knocking out known genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. Using these tools, an essential role for SNARE pathways in NMJ maintenance was demonstrated. Furthermore, it was demonstrated that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating the retrograde Gbb signaling. Lastly, axon termini of motoneurons were found to rely on ESCRT-mediated intra-axonal membrane trafficking to lease extracellular vesicles at the NMJ.
Christophers, B., Leahy, S. N., Soffar, D. B., von Saucken, V. E., Broadie, K., Baylies, M. K. (2023). Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission. bioRxiv, PubMed ID: 38045306
Summary:
Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Previous work has shown Drosophila cofilin (DmCFL) knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. It was hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, an RNA sequencing analysis was conducted which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. DmCFL was found to be enriched in the muscle postsynaptic compartment and that DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, no significant changes were found in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology
Xiao, C., M'Angale, P. G., Wang, S., Lemieux, A., Thomson, T. (2023). Identifying new players in structural synaptic plasticity through dArc1 interrogation. iScience, 26(11):108048 PubMed ID: 37876812
Summary:
The formation, expansion, and pruning of synapses, known as structural synaptic plasticity, is needed for learning and memory, and perturbation of plasticity is associated with many neurological disorders and diseases. Previously, it was observed that the Drosophila homolog of Activity-regulated cytoskeleton-associated protein (dArc1), forms a capsid-like structure, associates with its own mRNA, and is transported across synapses. This transfer is needed for structural synaptic plasticity. To identify mRNAs that are modified by dArc1 in presynaptic neuron and postsynaptic muscle, the expression of dArc1 was disrupted, and genomic analysis was performed with deep sequencing. dArc1 was found to affects the expression of genes involved in metabolism, phagocytosis, and RNA-splicing. Through immunoprecipitation this study also identified potential mRNA cargos of dArc1 capsids. This study suggests that dArc1 acts as a master regulator of plasticity by affecting several distinct and highly conserved cellular processes.
Justs, K. A., Sempertegui, S., Riboul, D. V., Oliva, C. D., Durbin, R. J., Crill, S., Stawarski, M., Su, C., Renden, R. B., Fily, Y., Macleod, G. T. (2023). Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals. The Journal of physiology, 601(24):5705-5732 PubMed ID: 37942946
Summary:
Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. In this study, through computer simulation and drawing on previously published empirical data, it was determined that motor neuron terminals in Drosophila larvae experience highly volatile power demands. It might not be surprising then, that the mitochondria in the motor neuron terminals of both Drosophila and mice were found to be heavily decorated with phosphagen kinases - a key element in an energy storage and buffering system well-characterized in fast-twitch muscle fibres. Knockdown of arginine kinase 1 (ArgK1) in Drosophila larval motor neurons led to several bioenergetic deficits, including mitochondrial matrix acidification and a faster decline in the cytosol ATP to ADP ratio during axon burst firing.

Monday, June 10th - Gonads

Takashima, Y. A., Majane, A. C., Begun, D. J. (2023). Evolution of secondary cell number and position in the Drosophila accessory gland. PLoS One, 18(10):e0278811 PubMed ID: 37878630
Summary:
In animals with internal fertilization, males transfer gametes and seminal fluid during copulation, both of which are required for successful reproduction. In Drosophila and other insects, seminal fluid is produced in the paired accessory gland (AG), the ejaculatory duct, and the ejaculatory bulb. The D. melanogaster AG has emerged as an important model system for this component of male reproductive biology. Seminal fluid proteins produced in the Drosophila AG are required for proper storage and use of sperm by the females, and are also critical for establishing and maintaining a suite of short- and long-term postcopulatory female physiological responses that promote reproductive success. The Drosophila AG is composed of two main cell types. The majority of AG cells, which are referred to as main cells, are responsible for production of many seminal fluid proteins. A minority of cells, about 4%, are referred to as secondary cells. These cells, which are restricted to the distal tip of the D. melanogaster AG, may play an especially important role in the maintenance of the long-term female post-mating response. Many studies of Drosophila AG evolution have suggested that the proteins produced in the gland evolve quickly, as does the transcriptome. This study investigated the evolution of secondary cell number and position in the AG in a collection of eight species spanning the entire history of the Drosophila genus. A heretofore underappreciated rapid evolutionary rate was documented for both number and position of these specialized AG cells, raising several questions about the developmental, functional, and evolutionary significance of this variation.
Anderson, J., Henikoff, S., Ahmad, K. (2023). Chromosome-specific maturation of the epigenome in the Drosophila male germline. bioRxiv, PubMed ID: 37873332
Summary:
Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, active chromatin features were profiled in the testes from wildtype and meiotic arrest mutants, and this was integrated with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, widespread patterns associated with regulation of the sex chromosomes were detailed. The results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome.
Wenzel, M., Aquadro, C. F. (2023). Wolbachia infection at least partially rescues the fertility and ovary defects of several new Drosophila melanogaster bag of marbles protein-coding mutants. bioRxiv, PubMed ID: 37645949
Summary:
The D. melanogaster protein coding gene bag of marbles (bam) plays a key role in early male and female reproduction by forming complexes with partner proteins to promote differentiation in gametogenesis. Like another germline gene, Sex lethal, bam genetically interacts with the endosymbiont Wolbachia, as Wolbachia rescues the reduced fertility of a bam hypomorphic mutant. This study explored the specificity of the bam-Wolbachia interaction by generating 22 new bam mutants, with ten mutants displaying fertility defects. Nine of these mutants trend towards rescue by the w Mel Wolbachia variant, with eight statistically significant at the fertility and/or cytological level. In some cases, fertility was increased a striking 20-fold. There is no specificity between the rescue and the known binding regions of bam, suggesting w Mel does not interact with one singular bam partner to rescue the reproductive phenotype. Whether Mel interacts with bam in a non-specific way, by increasing bam transcript levels or acting upstream in germline stem cells. A fertility assessment of a bam RNAi knockdown mutant reveals that w Mel rescue is specific to functionally mutant bam alleles and no obvious evidence was found of w Mel interaction with germline stem cells in bam mutants.
Francois, C. M., Pihl, T., Dunoyer de Segonzac, M., Herault, C., Hudry, B. (2023). Metabolic regulation of proteome stability via N-terminal acetylation controls male germline stem cell differentiation and reproduction. Nat Commun, 14(1):6737 PubMed ID: 37872135
Summary:
The molecular mechanisms connecting cellular metabolism with differentiation remain poorly understood. This study found that metabolic signals contribute to stem cell differentiation and germline homeostasis during Drosophila melanogaster spermatogenesis. External citrate, originating outside the gonad, was found to fuel the production of Acetyl-coenzyme A by germline ATP-citrate lyase (dACLY). This pathway is essential during the final spermatogenic stages, where a high Acetyl-coenzyme A level promotes N-terminal acetyltransferase B (NatB)-dependent N-terminal protein acetylation. Using genetic and biochemical experiments, this study established that N-terminal acetylation shields key target proteins, essential for spermatid differentiation, from proteasomal degradation by the ubiquitin ligase dUBR1. This work uncovers crosstalk between metabolism and proteome stability that is mediated via protein post-translational modification. It is proposed that this system coordinates the metabolic state of the organism with gamete production. More broadly, modulation of proteome turnover by circulating metabolites may be a conserved regulatory mechanism to control cell functions.
Guntur, A. R., Smith, J. E., Brahmandam, A., DeBauche, P., Cronmiller, C., Lundell, M. J. (2023). ZFH-2 is required for Drosophila ovarian follicle development and is expressed at the band/interband boundaries of polytene chromosomes. Dev Biol, 504:1-11 PubMed ID: 37666353
Summary:
The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. This study provides the first evidence that ZFH-2 has a role in oogenesis. ZFH-2 is expressed in the wild-type ovary, and a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. It was also shown that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. ZFH-2 was shown to be located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190.
Roach, T. V., Lenhart, K. F. (2023). Mating-induced ecdysone in the testis disrupts soma-germline contacts and stem cell cytokinesis. bioRxiv, PubMed ID: 37905121
Summary:
Germline maintenance relies on adult stem cells to continually replenish lost gametes over a lifetime and respond to external cues altering the demands on the tissue. Mating worsens germline homeostasis over time, yet a negative impact on stem cell behavior has not been explored. Using extended live imaging of the Drosophila testis stem cell niche, this study found that short periods of mating in young males disrupts cytokinesis in germline stem cells (GSCs). This defect leads to failure of abscission, preventing release of differentiating cells from the niche. GSC abscission failure was found to be caused by increased ecdysone hormone signaling induced upon mating, which leads to disrupted somatic encystment of the germline. Abscission failure is rescued by isolating males from females but recurs with resumption of mating. Importantly, reiterative mating also leads to increased GSC loss, requiring increased restoration of stem cells via symmetric renewal and de-differentiation. Together, these results suggest a model whereby acute mating results in hormonal changes that negatively impact GSC cytokinesis but preserves the stem cell population.

Friday, June 7th - Cytoskeleton and Junctions

Madan, V., Albacete-Albacete, L., Jin, L., Scaturro, P., Watson, J. L., Muschalik, N., Begum, F., Boulanger, J., Bauer, K., Kiebler, M. A., Derivery, E., Bullock, S. L. (2023). HEATR5B associates with dynein-dynactin and promotes motility of AP1-bound endosomal membranes. The EMBO journal, 42(23):e114473 PubMed ID: 37872872
Summary:
The microtubule motor dynein mediates polarised trafficking of a wide variety of organelles, vesicles and macromolecules. These functions are dependent on the dynactin complex, which helps recruit cargoes to dynein's tail and activates motor movement. How the dynein-dynactin complex orchestrates trafficking of diverse cargoes is unclear. This study identified HEATR5B, an interactor of the adaptor protein-1 (AP1) clathrin adaptor complex, as a novel player in dynein-dynactin function. HEATR5B was recovered in a biochemical screen for proteins whose association with the dynein tail is augmented by dynactin. HEATR5B binds directly to the dynein tail and dynactin and stimulates motility of AP1-associated endosomal membranes in human cells. It was also demonstrated that the Drosophila HEATR5B homologue is an essential gene that selectively promotes dynein-based transport of AP1-bound membranes to the Golgi apparatus. As HEATR5B lacks the coiled-coil architecture typical of dynein adaptors, these data point to a non-canonical process orchestrating motor function on a specific cargo. It was additionally shown that HEATR5B promotes association of AP1 with endosomal membranes independently of dynein. Thus, HEATR5B co-ordinates multiple events in AP1-based trafficking.
McParland, E. D., Amber Butcher, T., Gurley, N. J., Johnson, R. I., Slep, K. C., Peifer, M. (2023). The Dilute domain of Canoe is not essential for Canoe's role in linking adherens junctions to the cytoskeleton but contributes to robustness of morphogenesis. bioRxiv, PubMed ID: 37905001
Summary:
Robust linkage between cell-cell adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The multidomain protein Drosophila Canoe and its mammalian homolog Afadin are critical for this linkage, and in their absence many events of morphogenesis fail. To define underlying mechanisms, this study took Canoe apart, using Drosophila as a model. Canoe and Afadin share five folded protein domains, followed by a large intrinsically disordered region. The largest of these folded domains is the Dilute domain, which is found in Canoe/Afadin, their paralogs, and members of the MyosinV family. To define the roles of Canoe's Dilute domain this study combined biochemical, genetic and cell biological assays. Use of the AlphaFold tools revealed the predicted structure of the Canoe/Afadin Dilute domain, providing similarities and contrasts with that of MyosinV. The biochemical data suggest one potential shared function: the ability to dimerize. We next generated Drosophila mutants with the Dilute domain cleanly deleted. Surprisingly, these mutants are viable and fertile, and CanoeΔDIL protein localizes to adherens junctions and is enriched at junctions under tension. However, when we reduce the dose of Canoe&DeltaDIL protein in a sensitized assay, it becomes clear it does not provide full wildtype function. Further, canoe&DeltalDIL mutants have defects in pupal eye development, another process that requires orchestrated cell rearrangements. Together, these data reveal the robustness in AJ-cytoskeletal connections during multiple embryonic and postembryonic events, and the power of natural selection to maintain protein structure even in robust systems.
Zhu, H., O'Shaughnessy, B. (2023). Actomyosin pulsing rescues embryonic tissue folding from disruption by myosin fluctuations. Research square, PubMed ID: 37886516
Summary:
During early development, myosin II mechanically reshapes and folds embryo tissue. A much studied example is ventral furrow formation in Drosophila, marking the onset of gastrulation. Furrowing is driven by contraction of actomyosin networks on apical cell surfaces, but how the myosin patterning encodes tissue shape is unclear, and elastic models failed to reproduce essential features of experimental cell contraction profiles. The myosin patterning exhibits substantial cell-to-cell fluctuations with pulsatile time-dependence, a striking but unexplained feature of morphogenesis in many organisms. Using biophysical modeling this study found that viscous forces offer the principal resistance to actomyosin-driven apical constriction. In consequence, tissue shape is encoded in the direction-dependent curvature of the myosin patterning which orients an anterior-posterior furrow. Tissue contraction is highly sensitive to cell-to-cell myosin fluctuations, explaining furrowing failure in genetically perturbed embryos whose fluctuations are temporally persistent. In wild-type embryos this disastrous outcome is averted by pulsatile myosin time-dependence, which rescues furrowing by eliminating high frequencies in the fluctuation power spectrum. This low pass filter mechanism may underlie the usage of actomyosin pulsing in diverse morphogenetic processes across many organisms.

LaFountain, J. R., Jr., Seaman, C. E., Cohan, C. S., Oldenbourg, R. (2023). Sliding of antiparallel microtubules drives bipolarization of monoastral spindles. PubMed ID: 37812128
Summary:
Time-lapse imaging with liquid crystal polarized light (LC-PolScope) and fluorescent speckle microscopy (FSM) enabled this study of spindle microtubules in monoastral spindles that were produced in crane-fly spermatocytes through flattening-induced centrosome displacement. Monoastral spindles are found in several other contexts: after laser ablation of one of a cell's two centrosomes, in Drosophila "urchin" mutants, in Sciara males, and in RNAi variants of Drosophila S2 cells. In all cases, just one pole has a centrosome (the astral pole); the other lacks a centrosome (the anastral pole). Thus, the question: How is the anastral half-spindle, lacking a centrosome, constructed? It was learned that monoastral spindles are assembled in two phases: Phase I assembles the astral half-spindle composed of centrosomal microtubules, and Phase II assembles microtubules of the anastral half through extension of new microtubule polymerization outward from the spindle's equatorial mid-zone. That process uses plus ends of existing centrosomal microtubules as guiding templates to assemble anastral microtubules of opposite polarity. Anastral microtubules slide outward with their minus ends leading, thereby establishing proper bipolarity just like in normal biastral spindles that have two centrosomes.
Zhu, Z., Becam, I., Tovey, C. A., Elfarkouchi, A., Yen, E. C., Bernard, F., Guichet, A., Conduit, P. T. (2023). Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes. J Cell Biol, 222(10) PubMed ID: 37698931
Summary:
Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. This study shows that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. These data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated.
Li, L., Zhang, N., Beati, S. A. H., De Las Heras Chanes, J., di Pietro, F., Bellaiche, Y., Muller, H. J., Grosshans, J. (2024). Kinesin-1 patterns Par-1 and Rho signaling at the cortex of syncytial embryos of Drosophila. J Cell Biol, 223(1) PubMed ID: 37955925
Summary:
The cell cortex of syncytial Drosophila embryos is patterned into cap and intercap regions by centrosomes, specific sets of proteins that are restricted to their respective regions by unknown mechanisms. This study found that Kinesin-1 is required for the restriction of plus- and minus-ends of centrosomal and non-centrosomal microtubules to the cap region, marked by EB1 and Patronin/Shot, respectively. Kinesin-1 also directly or indirectly restricts proteins and Rho signaling to the intercap, including the RhoGEF Pebble, Dia, Myosin II, Capping protein-α, and the polarity protein Par-1. Furthermore, we found that Par-1 is required for cap restriction of Patronin/Shot, and vice versa Patronin, for Par-1 enrichment at the intercap. In summary, these data support a model that Kinesin-1 would mediate the restriction of centrosomal and non-centrosomal microtubules to a region close to the centrosomes and exclude Rho signaling and Par-1. In addition, mutual antagonistic interactions would refine and maintain the boundary between cap and intercap and thus generate a distinct cortical pattern.

Thursday, June 6th - Drosophila as a Model of Human Diseases

Filosevic Vujnovic, A., Saftic Martinovic, L., Medija, M., Andretic Waldowski, R. (2023). Distinct and Dynamic Changes in the Temporal Profiles of Neurotransmitters in Drosophila melanogaster Brain following Volatilized Cocaine or Methamphetamine Administrations. Pharmaceuticals (Basel, Switzerland), 16(10) PubMed ID: 37895961
Summary:
Due to similarities in genetics, cellular response, and behavior, Drosophila is used as a model organism in addiction research. A well-described behavioral response examined in flies is the induced increase in locomotor activity after a single dose of volatilized cocaine (vCOC) and volatilized methamphetamine (vMETH), the sensitivity, and the escalation of the locomotor response after the repeated dose, the locomotor sensitization. However, knowledge about how vCOC and vMETH affect different neurotransmitter systems over time is scarce. This study used LC-MS/MS to systematically examine changes in the concentration of neurotransmitters, metabolites and non-metabolized COC and METH in the whole head homogenates of male flies one to seven hours after single and double vCOC or vMETH administrations. vMETH leads to complex changes in the levels of examined substances over time, while vCOC strongly and briefly increases concentrations of dopamine, tyramine and octopamine followed by a delayed degradation into N-acetyl dopamine and N-acetyl tyramine. The first exposure to psychostimulants leads to significant and dynamic changes in the concentrations relative to the second administration when they are more stable over several hours. Further investigations are needed to understand neurochemical and molecular changes post-psychostimulant administration.
Min, Y., Wang, X., Is, O., Patel, T. A., Gao, J., Reddy, J. S., Quicksall, Z. S., Nguyen, T., Lin, S., Tutor-New, F. Q., Chalk, J. L., Mitchell, A. O., Crook, J. E., Nelson, P. T., Van Eldik, L. J., Golde, T. E., Carrasquillo, M. M., Dickson, D. W., Zhang, K., Allen, M., Ertekin-Taner, N. (2023). Cross species systems biology discovers glial DDR2, STOM, and KANK2 as therapeutic targets in progressive supranuclear palsy. Nat Commun, 14(1):6801 PubMed ID: 37919278
Summary:
Progressive supranuclear palsy (PSP) is a neurodegenerative parkinsonian disorder characterized by cell-type-specific tau lesions in neurons and glia. Prior work uncovered transcriptome changes in human PSP brains, although their cell-specificity is unknown. Further, systematic data integration and experimental validation platforms to prioritize brain transcriptional perturbations as therapeutic targets in PSP are currently lacking. This study, combined bulk tissue (n = 408) and single nucleus RNAseq (n = 34) data from PSP and control brains with transcriptome data from a mouse tauopathy and experimental validations in Drosophila tau models for systematic discovery of high-confidence expression changes in PSP with therapeutic potential. This study discovered, replicated, and annotated thousands of differentially expressed genes in PSP, many of which reside in glia-enriched co-expression modules and cells. DDR2, STOM, and KANK2 were prioritized as promising therapeutic targets in PSP with striking cross-species validations. These findings and data are shared via interactive application tool PSP RNAseq Atlas . These findings reveal robust glial transcriptome changes in PSP, provide a cross-species systems biology approach, and a tool for therapeutic target discoveries in PSP with potential application in other neurodegenerative diseases.
He, M. F., Liu, C. Q., Zhang, X. X., Lin, Y. M., Mao, Y. L., Qiao, J. D. (2023). Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy. J Vis Exp, (200) PubMed ID: 37902337
Summary:
Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.
Al-Ayari, E. A., Shehata, M. G., El-Hadidi, M., Shaalan, M. G. (2023). In silico SNP prediction of selected protein orthologues in insect models for Alzheimer's, Parkinson's, and Huntington's diseases. Sci Rep, 13(1):18986 PubMed ID: 37923901
Summary:
Alzheimer's, Parkinson's, and Huntington's are the most common neurodegenerative diseases that are incurable and affect the elderly population. Discovery of effective treatments for these diseases is often difficult, expensive, and serendipitous. Previous comparative studies on different model organisms have revealed that most animals share similar cellular and molecular characteristics. The meta-SNP tool includes four different integrated tools (SIFT, PANTHER, SNAP, and PhD-SNP) was used to identify non synonymous single nucleotide polymorphism (nsSNPs). Prediction of nsSNPs was conducted on three representative proteins for Alzheimer's, Parkinson's, and Huntington's diseases; APPl in Drosophila melanogaster, LRRK1 in Aedes aegypti, and VCPl in Tribolium castaneum. With the possibility of using insect models to investigate neurodegenerative diseases. It is concluded from the protein comparative analysis between different insect models and nsSNP analyses that D. melanogaster is the best model for Alzheimer's representing five nsSNPs of the 21 suggested mutations in the APPl protein. Aedes aegypti is the best model for Parkinson's representing three nsSNPs in the LRRK1 protein. Tribolium castaneum is the best model for Huntington's disease representing 13 SNPs of 37 suggested mutations in the VCPl protein. This study aimed to improve human neural health by identifying the best insect to model Alzheimer's, Parkinson's, and Huntington's.
Nithianandam, V., Bukhari, H., Leventhal, M. J., Battaglia, R. A., Dong, X., Fraenkel, E., Feany, M. B. (2023). Integrative analysis reveals a conserved role for the amyloid precursor protein in proteostasis during aging. Nat Commun, 14(1):7034 PubMed ID: 37923712
Summary:
Aβ peptides derived from the amyloid precursor protein (APP) have been strongly implicated in the pathogenesis of Alzheimer's disease. However, the normal function of APP and the importance of that role in neurodegenerative disease is less clear. The Drosophila ortholog of APP, Appl, was uncovered in an unbiased forward genetic screen for neurodegeneration mutants. Comprehensive single cell transcriptional and proteomic studies of Appl mutant flies were performed to investigate Appl function in the aging brain. An unexpected role was found for Appl in control of multiple cellular pathways, including translation, mitochondrial function, nucleic acid and lipid metabolism, cellular signaling and proteostasis. A role for Appl in regulating autophagy through TGFβ signaling was mechanistically defined, and the broader relevance of these findings was documented using mouse genetic, human iPSC and in vivo tauopathy models. The results demonstrate a conserved role for APP in controlling age-dependent proteostasis with plausible relevance to Alzheimer's disease.
Xu, J., Deng, Z., Shang, S., Wang, C., Han, H. (2023). FUNDC1 collaborates with PINK1 in regulating mitochondrial Fission and compensating for PINK1 deficiency. Biochem Biophys Res Commun, 687:149210 PubMed ID: 37931419
Summary:
Parkinson's disease is presently thought to have its molecular roots in the alteration of PINK1-mediated mitophagy and mitochondrial dynamics. Finding new suppressors of the pathway is essential for developing cutting-edge treatment approaches. This study shows that FUNDC1 suppressed PINK1 mutant phenotypes in Drosophila. The restoration of PINK1-deficient phenotypes through FUNDC1 is not reliant on its LC3-binding motif Y (18)L (21) or autophagy-related pathway. Moreover, the absence of Drp1 affects the phenotypic restoration of PINK1 mediated by FUNDC1 in flies. In summary, these findings have unveiled a fresh mechanism through which FUNDC1 compensates for the loss of PINK1, operating independently of autophagy but exerting its influence via interaction with Drp1.

Wednesday, June 5th - Homologs of Drosophila Proteins

Cowell, L. M., King, M., West, H., Broadsmith, M., Genever, P., Pownall, M. E., Isaacs, H. V. (2023). Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development. PLoS One, 18(10):e0286040 PubMed ID: 37856433
Summary:
Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. It is proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor.This study investigated the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. Xenopus Cic was characterized and was shown to be widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with our hypothesis, we show that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. Two of these genes were investigated as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b, which encode a leucine zipper transcription factor and a ras family GTPase, respectively. Cic consensus binding sites were identified in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. We show that expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, it was shown that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. The data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding.
Torres, H. M., Hinojosa, L., VanCleave, A. M., Rodezno, T., Westendorf, J. J., Tao, J. (2023). Hdac1 and Hdac2 positively regulate Notch1 gain-of-function pathogenic signaling in committed osteoblasts of male mice. Birth defects research, PubMed ID: 37921375
Summary:
Skeletal development requires precise extrinsic and intrinsic signals to regulate processes that form and maintain bone and cartilage. Notch1 is a highly conserved signaling receptor that regulates cell fate decisions by controlling the duration of transcriptional bursts. Epigenetic molecular events reversibly modify DNA and histone tails by influencing the spatial organization of chromatin and can fine-tune the outcome of a Notch1 transcriptional response. Histone deacetylase 1 and 2 (HDAC1 and HDAC2) are chromatin modifying enzymes that mediate osteoblast differentiation. While an HDAC1-Notch interaction has been studied in vitro and in Drosophila, its role in mammalian skeletal development and disorders is unclear. Osteosclerosis is a bone disorder with an abnormal increase in the number of osteoblasts and excessive bone formation. This study, tested whether Hdac1/2 contribute to the pathogenesis of osteosclerosis in a murine model of the disease owing to conditionally cre-activated expression of the Notch1 intracellular domain in immature osteoblasts. RESULTS: Importantly, selective homozygous deletions of Hdac1/2 in osteoblasts partially alleviate osteosclerotic phenotypes (Col2.3kb-Cre; TG(RosaN1ICD/+) ; Hdac1(flox/flox) ; Hdac2(flox/flox) ) with a 40% decrease in bone volume and a 22% decrease in trabecular thickness in 4 weeks old when compared to male mice with heterozygous deletions of Hdac1/2 (Col2.3 kb-Cre; TG(RosaN1ICD/+) ; Hdac1(flox/+) ; Hdac2(flox/+) ). Osteoblast-specific deletion of Hdac1/2 in male and female mice results in no overt bone phenotype in the absence of the Notch1 gain-of-function (GOF) allele. These results provide evidence that Hdac1/2 contribute to Notch1 pathogenic signaling in the mammalian skeleton. This study on epigenetic regulation of Notch1 GOF-induced osteosclerosis may facilitate further mechanistic studies of skeletal birth defects caused by Notch-related GOF mutations in human patients, such as Adams-Oliver disease, congenital heart disease, and lateral meningocele syndrome.
Sauty, S. M., Yankulov, K. (2023). Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-modal active/silent state at the sub-telomeres of S. cerevisiae.Epigenetics & chromatin, 16(1):40 PubMed ID: 37858268
Summary:
Classical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. This study used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the different roles of PCNA and histone chaperones in the stability and the propagation of repressed states at the sub-telomeres of S. cerevisiae. Position dependent transient repression or bi-modal expression of reporter genes is shown at the VIIL sub-telomere. Mutations in the replicative clamp POL30 (PCNA) or the deletion of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the deletion of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. The physical interaction of CAF1 and RRM3 with PCNA was analyzed, and the implications of these findings for understanding of the stability and transmission of the epigenetic state of the genes id discussed. There are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. Yhe roles of CAF1, RRM3 and ASF1 in these modes of gene repression were analyzed. It is suggestd that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing.
Duan, D., Lyu, W., Chai, P., Ma, S., Wu, K., Wu, C., Xiong, Y., Sestan, N., Zhang, K., Koleske, A. J. (2023). Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation. Curr Biol, 33(21):4582-4598.e4510 PubMed ID: 37858340
Summary:
Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both in vitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. This study reports that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. Abl2 was found to promote MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ688-790. It is proposed that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue.
Kushwaha, A., Thakur, M. K. (2024). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Molecular neurobiology. 61(1):487-497 PubMed ID: 37626270
Summary:
Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ Suv39h1 (a mammalian ortholog of the Drosophila melanogaster SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It has been reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. In the current study, an attempt was made to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. It was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia.
Liu, Q., Bell, B. J., Kim, D. W., Lee, S. S., Keles, M. F., Liu, Q., Blum, I. D., Wang, A. A., Blank, E. J., Xiong, J., Bedont, J. L., Chang, A. J., Issa, H., Cohen, J. Y., Blackshaw, S., Wu, M. N. (2023). A clock-dependent brake for rhythmic arousal in the dorsomedial hypothalamus. Nat Commun, 14(1):6381 PubMed ID: 37821426
Summary:
Circadian clocks generate rhythms of arousal, but the underlying molecular and cellular mechanisms remain unclear. In Drosophila, the clock output molecule WIDE AWAKE (WAKE) labels rhythmic neural networks and cyclically regulates sleep and arousal. This study shows, in a male mouse model, that mWAKE/ANKFN1 labels a subpopulation of dorsomedial hypothalamus (DMH) neurons involved in rhythmic arousal and acts in the DMH to reduce arousal at night. In vivo Ca(2+) imaging reveals elevated DMH(mWAKE) activity during wakefulness and rapid eye movement (REM) sleep, while patch-clamp recordings show that DMH(mWAKE) neurons fire more frequently at night. Chemogenetic manipulations demonstrate that DMH(mWAKE) neurons are necessary and sufficient for arousal. Single-cell profiling coupled with optogenetic activation experiments suggest that GABAergic DMH(mWAKE) neurons promote arousal. Surprisingly, the data suggest that mWAKE acts as a clock-dependent brake on arousal during the night, when mice are normally active. mWAKE levels peak at night under clock control, and loss of mWAKE leads to hyperarousal and greater DMH(mWAKE) neuronal excitability specifically at night. These results suggest that the clock does not solely promote arousal during an animal's active period, but instead uses opposing processes to produce appropriate levels of arousal in a time-dependent manner.

Tuesday, June 4th - Enhancers and Transcriptional Regulation

Chaubal, A., Waldern, J. M., Taylor, C., Laederach, A., Marzluff, W. F., Duronio, R. J. (2023). Coordinated expression of replication-dependent histone genes from multiple loci promotes histone homeostasis in Drosophila. Mol Biol Cell, 34(12):ar118 PubMed ID: 37647143
Summary:
Production of large amounts of histone proteins during S phase is critical for proper chromatin formation and genome integrity. This process is achieved in part by the presence of multiple copies of replication dependent (RD) histone genes that occur in one or more clusters in metazoan genomes. In addition, RD histone gene clusters are associated with a specialized nuclear body, the histone locus body (HLB), which facilitates efficient transcription and 3' end-processing of RD histone mRNA. How all five RD histone genes within these clusters are coordinately regulated such that neither too few nor too many histones are produced, a process referred to as histone homeostasis, is not fully understood. This study explored the mechanisms of coordinate regulation between multiple RD histone loci in Drosophila melanogaster and Drosophila virilis. Evidence is provided for functional competition between endogenous and ectopic transgenic histone arrays located at different chromosomal locations in D. melanogaster that helps maintain proper histone mRNA levels. Consistent with this model, in both species it was found that individual histone gene arrays can independently assemble an HLB that results in active histone transcription. These findings suggest a role for HLB assembly in coordinating RD histone gene expression to maintain histone homeostasis.
Masuda, L. H. P., Sabino, A. U., Reinitz, J., Ramos, A. F., Machado-Lima, A., Andrioli, L. P. (2024). Global repression by tailless during segmentation. Dev Biol, 505:11-23 PubMed ID: 37879494
Summary:
The early expression of tll in two gap domains in the segmentation cascade of Drosophila is unusual even for most other insects. This study investigated tll regulation on pair-rule stripes. With ectopic misexpression of tll unexpected repression was detected of almost all pair-rule stripes of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz). Examining Tll embryonic ChIP-chip data with regions mapped as Cis-Regulatory Modules (CRMs) of pair-rule stripes this study verified Tll interactions to these regions. With the ChIP-chip data Tll interactions to the CRMs of gap domains and in the misexpression assay, Tll-mediated repression on Kruppel (Kr), kni (kni) and giant (gt) according to their differential sensitivity to Tll. These results with gap genes confirmed previous data from the literature and argue against indirect repression roles of Tll in the striped pattern. Moreover, the prediction of Tll binding sites in the CRMs of eve stripes and the mathematical modeling of their removal using an experimentally validated theoretical framework shows effects on eve stripes compatible with the absence of a repressor binding to the CRMs. In addition, modeling increased tll levels in the embryo results in the differential repression of eve stripes, agreeing well with the results of the misexpression assay. In genetic assays we investigated eve 5, that is strongly repressed by the ectopic domain and representative of more central stripes not previously implied to be under direct regulation of tll. While this stripe is little affected in tll-, its posterior border is expanded in gt- but detected with even greater expansion in gt-;tll-. The paper ends with a discussion of tll with key roles in combinatorial repression mechanisms to contain the expression of medial patterns of the segmentation cascade in the extremities of the embryo.
Ramalingam, V., Yu, X., Slaughter, B. D., Unruh, J. R., Brennan, K. J., Onyshchenko, A., Lange, J. J., Natarajan, M., Buck, M., Zeitlinger, J. (2023). Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development. Nat Commun, 14(1):5862 PubMed ID: 37735176
Summary:
While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor that is one of the more than 25 different splice isoforms from the lola locus, this study showed that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.
Lovero, D., Porcelli, D., Giordano, L., Lo Giudice, C., Picardi, E., Pesole, G., Pignataro, E., Palazzo, A., Marsano, R. M. (2023). Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors. Biology, 12(8) PubMed ID: 37627011
Summary:
Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. This work have analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. The conservation of a short sequence is highlighted in the genes encoding subunits of the V-ATPase complex, and their assembly factors were analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding knowledge on the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization.
Eggers, N., Gkountromichos, F., Krause, S., Campos-Sparr, A., Becker, P. B. (2023). Physical interaction between MSL2 and CLAMP assures direct cooperativity and prevents competition at composite binding sites. Nucleic Acids Res, 51(17):9039-9054 PubMed ID: 37602401
Summary:
MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. This study explored the nature of the cooperative binding to these GA-rich, composite sequence elements in reconstituted naive embryonic chromatin. The cooperativity was found to requires physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. The cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in the reconstitution, such as roX2 lncRNA.
Haroush, N., Levo, M., Wieschaus, E. F., Gregor, T. (2023). Functional analysis of the Drosophila eve locus in response to non-canonical combinations of gap gene expression levels. Dev Cell, 58(23):2789-2801.e2785 PubMed ID: 37890488
Summary:
Transcription factor combinations play a key role in shaping cellular identity. However, the precise relationship between specific combinations and downstream effects remains elusive. This relationship was examined within the context of the Drosophila eve locus, which is controlled by gap genes. Spatiotemporal levels of four gap genes was measured in heterozygous and homozygous gap mutant embryos, and they were correlated with the striped eve activity pattern. Although changes in gap gene expression extend beyond the manipulated gene, the spatial patterns of Eve expression closely mirror canonical activation levels in wild type. Interestingly, some combinations deviate from the wild-type repertoire but still drive eve activation. Although in homozygous mutants some Eve stripes exhibit partial penetrance, stripes consistently emerge at reproducible positions, even with varying gap gene levels. The findings suggest a robust molecular canalization of cell fates in gap mutants and provide insights into the regulatory constraints governing multi-enhancer gene loci.

Monday, June 3rd - Larval and Adult Neural Structurel, Development and Function

Mitchell, J. W., Midillioglu, I., Schauer, E., Wang, B., Han, C., Wildonger, J. (2023). Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons. PLoS Genet, 19(11):e1011025 PubMed ID: 37943859
Summary:
Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. This study investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. CRISPR-Cas9 genome engineering approaches were used to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, an over-active channel mutant was found to severely reduce dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. The data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on these data, it is proposed that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites.
Zhao, A., Nern, A., Koskela, S., Dreher, M., Erginkaya, M., Laughland, C. W., Ludwigh, H., Thomson, A., Hoeller, J., Parekh, R., Romani, S., Bock, D. D., Chiappe, E., Reiser, M. B. (2023). A comprehensive neuroanatomical survey of the Drosophila Lobula Plate Tangential Neurons with predictions for their optic flow sensitivity. bioRxiv, PubMed ID: 37904921
Summary:
Flying insects exhibit remarkable navigational abilities controlled by their compact nervous systems. Optic flow, the pattern of changes in the visual scene induced by locomotion, is a crucial sensory cue for robust self-motion estimation, especially during rapid flight. The best-known optic-flow sensitive neurons are the large tangential cells of the dipteran lobula plate. Most of these studies have focused on the large, Horizontal and Vertical System neurons, yet the lobula plate houses a much larger set of 'optic-flow' sensitive neurons, many of which have been challenging to unambiguously identify or to reliably target for functional studies. This study reports the comprehensive reconstruction and identification of the Lobula Plate Tangential Neurons in an Electron Microscopy (EM) volume of a whole Drosophila brain. This catalog of 58 LPT neurons (per brain hemisphere) contains many neurons that are described here for the first time and provides a basis for systematic investigation of the circuitry linking self-motion to locomotion control. Leveraging computational anatomy methods, It was estimated the visual motion receptive fields of these neurons, and their tuning was compared to the visual consequence of body rotations and translational movements. These neurons were matched, in most cases on a one-for-one basis, to stochastically labeled cells in genetic driver lines, to the mirror-symmetric neurons in the same EM brain volume, and to neurons in an additional EM data set. Using cell matches across data sets, the integration of optic flow patterns by neurons downstream of the LPTs were analyzed, Most central brain neurons establish sharper selectivity for global optic flow patterns than their input neurons. Furthermore, self-motion information extracted from optic flow was found to be processed in distinct regions of the central brain, pointing to diverse foci for the generation of visual behaviors
Jovanoski, K. D., Duquenoy, L., Mitchell, J., Kapoor, I., Treiber, C. D., Croset, V., Dempsey, G., Parepalli, S., Cognigni, P., Otto, N., Felsenberg, J., Waddell, S. (2023). Dopaminergic systems create reward seeking despite adverse consequences. Nature, 623(7986):356-365 PubMed ID: 37880370
Summary:
Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking. This study describes dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. It is proposed that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals.
Ganguly, I., Heckman, E. L., Litwin-Kumar, A., Clowney, E. J., Behnia, R. (2023). Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body. bioRxiv, PubMed ID: 37873086
Summary:
The arthropod mushroom body is well-studied as an expansion layer that represents olfactory stimuli and links them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their tuning and function are poorly understood. This study used the FlyWire adult whole-brain connectome to identify inputs to visual Kenyon cells. The types of visual neurons identified are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual projection neurons presynaptic to Kenyon cells receive input from large swathes of visual space, while local visual interneurons, providing smaller fractions of input, receive more spatially restricted signals that may be tuned to specific features of the visual scene. Like olfactory Kenyon cells, visual Kenyon cells receive sparse inputs from different combinations of visual channels, including inputs from multiple optic lobe neuropils. The sets of inputs to individual visual Kenyon cells are consistent with random sampling of available inputs. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the expansion coding properties appear different, with a specific repertoire of visual inputs projecting onto a relatively small number of visual Kenyon cells.
Anthoney, N., Tainton-Heap, L., Luong, H., Notaras, E., Kewin, A. B., Zhao, Q., Perry, T., Batterham, P., Shaw, P. J., van Swinderen, B. (2023). Experimentally induced active and quiet sleep engage non-overlapping transcriptional programs in Drosophila. Elife, 12 PubMed ID: 37910019
Summary:
Sleep in mammals can be broadly classified into two different physiological categories: rapid eye movement (REM) sleep and slow-wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. This study compared two commonly used approaches for studying sleep experimentally in Drosophila: optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, gaboxadol. These different sleep-induction methods have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions.
Yang, H. H., Brezovec, L. E., Capdevila, L. S., Vanderbeck, Q. X., Adachi, A., Mann, R. S., Wilson, R. I. (2023). Fine-grained descending control of steering in walking Drosophila. bioRxiv, PubMed ID: 37904997
Summary:

. . Locomotion involves rhythmic limb movement patterns that originate in circuits outside the brain. Purposeful locomotion requires descending commands from the brain, but how these commands are structured is not understood. This study investigated this issue, focusing on the control of steering in walking Drosophila. First, different limb "gestures" are described associated with different steering maneuvers. Next, a set of descending neurons was identified whose activity predicts steering. Focusing on two descending cell types downstream from distinct brain networks, this study showed that they evoke specific limb gestures: one lengthens strides on the outside of a turn, while the other attenuates strides on the inside of a turn. Notably, a single descending neuron can have opposite effects during different locomotor rhythm phases, and networks were identified positioned to implement this phase-specific gating. Together, these results show how purposeful locomotion emerges from brain cells that drive specific, coordinated modulations of low-level patterns.

Friday, May 31st - Physiology and Metabolism

Alassaf, M., Rajan, A. (2023). Diet-induced glial insulin resistance impairs the clearance of neuronal debris in Drosophila brain. PLoS Biol, 21(11):e3002359 PubMed ID: 37934726
Summary:
Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Using a Drosophila model, this study shows that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, this study showed that genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, this study has established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris.
Brener, A., Lorber, D., Reuveny, A., Toledano, H., Porat-Kuperstein, L., Lebenthal, Y., Weizman, E., Olender, T., Volk, T. (2023). Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae. Cells, 12(19) PubMed ID: 37830547
Summary:
The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. THE findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating these findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases.
Li, J., Dang, P., Li, Z., Zhao, T., Cheng, D., Pan, D., Yuan, Y., Song, W. (2023). Peroxisomal ERK mediates Akh/glucagon action and glycemic control. Cell Rep, 42(10):113200 PubMed ID: 37796662
Summary:
The enhanced response of glucagon and its Drosophila homolog, adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. This study found that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. Mammalian glucagon-induced hepatic ERK peroxisomal translocation was observed in diabetic subjects. Therefore, it is concluded that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control.
Zhao, Y., Johansson, E., Duan, J., Han, Z., Alenius, M. (2023). Fat- and sugar-induced signals regulate sweet and fat taste perception in Drosophila. Cell Rep, 42(11):113387 PubMed ID: 37934669
Summary:
This study investigated the interplay between taste perception and macronutrients. While sugar's and protein's self-regulation of taste perception is known, the role of fat remains unclear.In Drosophila, fat overconsumption reduces fatty acid taste in favor of sweet perception. Conversely, sugar intake increases fatty acid perception and suppresses sweet taste. Genetic investigations show that the sugar signal, gut-secreted Hedgehog, suppresses sugar taste and enhances fatty acid perception. Fat overconsumption induces Unpaired 2 (Upd2) secretion from adipose tissue to the hemolymph. This study revealed taste neurons take up Upd2, which triggers Domeless suppression of fatty acid perception. It was further shown that the downstream JAK/STAT signaling enhances sweet perception and, via Socs36E, fine-tunes Domeless activity and the fatty acid taste perception. Together, these results show that sugar regulates Hedgehog signaling and fat induces Upd2 signaling to balance nutrient intake and to regulate sweet and fat taste perception.
Brischigliaro, M., Cabrera-Orefice, A., Arnold, S., Viscomi, C., Zeviani, M., Fernández-Vizarra, E. (2023). Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes. Elife, 12 PubMed ID: 37823874
Summary:
Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. This study shows that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, it is concluded that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis.
Bobrovskikh, M. A., Gruntenko, N. E. (2023). The Role of 20-Hydroxyecdysone in the Control of Carbohydrate Levels in Drosophila melanogaster under Short-Term Heat Stress. Doklady Biochemistry and biophysics, 511(1):162-165 PubMed ID: 37833600
Summary:
It is known that 20-hydroxyecdysone is one of the most important hormonal regulators of development, reproduction and adaptation to unfavorable conditions in insects. This study shows for the first time that exogenous 20-hydroxyecdysone increases the content of two main insect carbohydrates, trehalose and glucose, in Drosophila melanogaster females both in normal conditions and under short-term heat stress. It was found that the levels of both trehalose and glucose increase after 39 min of heat exposure and return to their original levels after 1.5 h. A scheme of hormonal regulation of carbohydrate content under heat stress, involving 20-hydroxyecdysone, juvenile hormone, and dopamine, is suggested.

Thursday, May 30th - Signaling

Alassaf, M., Rajan, A. (2023). Diet-induced glial insulin resistance impairs the clearance of neuronal debris in Drosophila brain. PLoS Biol, 21(11):e3002359 PubMed ID: 37934726
Summary:
Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Using a Drosophila model, this study shows that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, this study showed that genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, this study has established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris.
Brener, A., Lorber, D., Reuveny, A., Toledano, H., Porat-Kuperstein, L., Lebenthal, Y., Weizman, E., Olender, T., Volk, T. (2023). Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae. Cells, 12(19) PubMed ID: 37830547
Summary:
The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. THE findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating these findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases.
Li, J., Dang, P., Li, Z., Zhao, T., Cheng, D., Pan, D., Yuan, Y., Song, W. (2023). Peroxisomal ERK mediates Akh/glucagon action and glycemic control. Cell Rep, 42(10):113200 PubMed ID: 37796662
Summary:
The enhanced response of glucagon and its Drosophila homolog, adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. This study found that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. Mammalian glucagon-induced hepatic ERK peroxisomal translocation was observed in diabetic subjects. Therefore, it is concluded that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control.
Zhao, Y., Johansson, E., Duan, J., Han, Z., Alenius, M. (2023). Fat- and sugar-induced signals regulate sweet and fat taste perception in Drosophila. Cell Rep, 42(11):113387 PubMed ID: 37934669
Summary:
This study investigated the interplay between taste perception and macronutrients. While sugar's and protein's self-regulation of taste perception is known, the role of fat remains unclear.In Drosophila, fat overconsumption reduces fatty acid taste in favor of sweet perception. Conversely, sugar intake increases fatty acid perception and suppresses sweet taste. Genetic investigations show that the sugar signal, gut-secreted Hedgehog, suppresses sugar taste and enhances fatty acid perception. Fat overconsumption induces Unpaired 2 (Upd2) secretion from adipose tissue to the hemolymph. This study revealed taste neurons take up Upd2, which triggers Domeless suppression of fatty acid perception. It was further shown that the downstream JAK/STAT signaling enhances sweet perception and, via Socs36E, fine-tunes Domeless activity and the fatty acid taste perception. Together, these results show that sugar regulates Hedgehog signaling and fat induces Upd2 signaling to balance nutrient intake and to regulate sweet and fat taste perception.
Brischigliaro, M., Cabrera-Orefice, A., Arnold, S., Viscomi, C., Zeviani, M., Fernández-Vizarra, E. (2023). Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes. Elife, 12 PubMed ID: 37823874
Summary:
Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. This study shows that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, it is concluded that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis.
Bobrovskikh, M. A., Gruntenko, N. E. (2023). The Role of 20-Hydroxyecdysone in the Control of Carbohydrate Levels in Drosophila melanogaster under Short-Term Heat Stress. Doklady Biochemistry and biophysics, 511(1):162-165 PubMed ID: 37833600
Summary:
It is known that 20-hydroxyecdysone is one of the most important hormonal regulators of development, reproduction and adaptation to unfavorable conditions in insects. This study shows for the first time that exogenous 20-hydroxyecdysone increases the content of two main insect carbohydrates, trehalose and glucose, in Drosophila melanogaster females both in normal conditions and under short-term heat stress. It was found that the levels of both trehalose and glucose increase after 39 min of heat exposure and return to their original levels after 1.5 h. A scheme of hormonal regulation of carbohydrate content under heat stress, involving 20-hydroxyecdysone, juvenile hormone, and dopamine, is suggested.

Thursday, May 30th - Signaling

Yarikipati, P., Jonusaite, S., Pleinis, J. M., Dominicci Cotto, C., Sanchez-Hernandez, D., Morrison, D. E., Goyal, S., Schellinger, J., Penalva, C., Curtiss, J., Rodan, A. R., Jenny, A. (2023). Unanticipated domain requirements for Drosophila Wnk kinase in vivo. PLoS Genet, 19(10):e1010975 PubMed ID: 37819975
Summary:
WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate anonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. This study shows that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. The only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. These data thus provide novel insights into unexpected in vivo roles of specific WNK domains.
Brown, J., Su, T. T. (2024). E2F1, DIAP1, and the presence of a homologous chromosome promote while JNK inhibits radiation-induced loss of heterozygosity in Drosophila melanogaster. Genetics, 226(1) PubMed ID: 37874851
Summary:
Loss of heterozygosity (LOH) can occur when a heterozygous mutant cell loses the remaining wild-type allele to become a homozygous mutant. LOH can have physiological consequences if, for example, the affected gene encodes a tumor suppressor. Fluorescent reporters were used to study the mechanisms of LOH induction by X-rays, a type of ionizing radiation (IR), in Drosophila melanogaster larval wing discs. IR is used to treat more than half of patients with cancer, so understanding its effects is of biomedical relevance. Quantitative analysis of IR-induced LOH at different positions between the telomere and the centromere on the X chromosome showed a strong sex dependence and the need for a recombination-proficient homologous chromosome, whereas, paradoxically, position along the chromosome made little difference in LOH incidence. It is proposed that published data documenting high recombination frequency within centromeric heterochromatin on the X chromosome can explain these data. Using a focused screen, E2F1 was identified as a key promotor of LOH and further testing suggests a mechanism involving its role in cell-cycle regulation. The loss of a transcriptional repressor was leveraged through LOH to express transgenes specifically in cells that have already acquired LOH. This approach identified JNK signaling and apoptosis as key determinants of LOH maintenance. These studies reveal previously unknown mechanisms for the generation and elimination of cells with chromosome aberrations after exposure to IR.
Zhang, J., Liu, Y., Wang, C., Vander Kooi, C. W., Jia, J. (2023). Phosphatidic acid binding to Patched contributes to the inhibition of Smoothened and Hedgehog signaling in Drosophila wing development. Science signaling, 16(807):eadd6834. PubMed ID: 37847757
Summary:
Hedgehog (Hh) signaling controls growth and patterning during embryonic development and homeostasis in adult tissues. Hh binding to the receptor Patched (Ptc) elicits intracellular signaling by relieving Ptc-mediated inhibition of the transmembrane protein Smoothened (Smo). This study uncovered a role for the lipid phosphatidic acid (PA) in the regulation of the Hh pathway in Drosophila melanogaster. Deleting the Ptc C-terminal tail or mutating the predicted PA-binding sites within it prevented Ptc from inhibiting Smo in wing discs and in cultured cells. The C-terminal tail of Ptc directly interacted with PA in vitro, an association that was reduced by Hh, and increased the amount of PA at the plasma membrane in cultured cells. Smo also interacted with PA in vitro through a binding pocket located in the transmembrane region, and mutating residues in this pocket reduced Smo activity in vivo and in cells. By genetically manipulating PA amounts in vivo or treating cultured cells with PA, this study demonstrated that PA promoted Smo activation.These findings suggest that Ptc may sequester PA in the absence of Hh and release it in the presence of Hh, thereby increasing the amount of PA that is locally available to promote Smo activation.
Spencer, Z. T., Ng, V. H., Benchabane, H., Siddiqui, G. S., Duwadi, D., Maines, B., Bryant, J. M., Schwarzkopf, A., Yuan, K., Kassel, S. N., Mishra, A., Pimentel, A., Lebensohn, A. M., Rohatgi, R., Gerber, S. A., Robbins, D. J., Lee, E., Ahmed, Y. (2023). The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6. Nat Commun, 14(1):6174 PubMed ID: 37798281
Summary:
The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. This study demonstrates that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/&alpha-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient.
Li, C., Zhu, X., Sun, X., Guo, X., Li, W., Chen, P., Shidlovskii, Y. V., Zhou, Q., Xue, L. (2023). Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis. Cell division, 18(1):16 PubMed ID: 37794497
Summary:
The c-Jun N-terminal kinase (JNK) pathway is an evolutionarily conserved regulator of cell death, which is essential for coordinating tissue homeostasis. This study characterized the Drosophila Ste20-like kinase Slik as a novel modulator of JNK pathway-mediated apoptotic cell death. First, ectopic JNK signaling-triggered cell death is enhanced by slik depletion whereas suppressed by Slik overexpression. Second, loss of slik activates JNK signaling, which results in enhanced apoptosis and impaired tissue homeostasis. In addition, genetic epistasis analysis suggests that Slik acts upstream of or in parallel to Hep to regulate JNK-mediated apoptotic cell death. Moreover, Slik is necessary and sufficient for preventing physiologic JNK signaling-mediated cell death in development. Furthermore, introduction of STK10, the human ortholog of Slik, into Drosophila restores slik depletion-induced cell death and compromised tissue homeostasis. Lastly, knockdown of STK10 in human cancer cells also leads to JNK activation, which is cancelled by expression of Slik. This study has uncovered an evolutionarily conserved role of Slik/STK10 in blocking JNK signaling, which is required for cell death inhibition and tissue homeostasis maintenance in development.
Tokamov, S. A., Buiter, S., Ullyot, A., Scepanovic, G., Williams, A. M., Fernandez-Gonzalez, R., Horne-Badovinac, S., Fehon, R. G. (2024). Cortical tension promotes Kibra degradation via Par-1. Mol Biol Cell, 35(1):ar2 PubMed ID: 37903240
Summary:
The Hippo pathway is an evolutionarily conserved regulator of tissue growth. Multiple Hippo signaling components are regulated via proteolytic degradation. However, how these degradation mechanisms are themselves modulated remains unexplored. Kibra is a key upstream pathway activator that promotes its own ubiquitin-mediated degradation upon assembling a Hippo signaling complex. This study demonstrates that Hippo complex-dependent Kibra degradation is modulated by cortical tension. Using classical genetic, osmotic, and pharmacological manipulations of myosin activity and cortical tension, it was shown that increasing cortical tension leads to Kibra degradation, whereas decreasing cortical tension increases Kibra abundance. This study also implicates Par-1 in regulating Kib abundance downstream of cortical tension. Par-1 promotes ubiquitin-mediated Kib degradation in a Hippo complex-dependent manner and is required for tension-induced Kib degradation. Collectively, these results reveal a previously unknown molecular mechanism by which cortical tension affects Hippo signaling and provide novel insights into the role of mechanical forces in growth control.

Wednesday, May 29th - Apoptosis and Autophagy

Leung, H. H., Mansour, C., Rousseau, M., Nakhla, A., Kiselyov, K., Venkatachalam, K., Wong, C. O. (2024). Drosophila tweety facilitates autophagy to regulate mitochondrial homeostasis and bioenergetics in Glia. Glia, 72(2):433-451 PubMed ID: 37870193
Summary:
Mitochondria support the energetic demands of the cells. Autophagic turnover of mitochondria serves as a critical pathway for mitochondrial homeostasis. It is unclear how bioenergetics and autophagy are functionally connected. This study identified an endolysosomal membrane protein that facilitates autophagy to regulate ATP production in glia. Drosophila tweety (tty) was determined to be highly expressed in glia and localized to endolysosomes. Diminished fusion between autophagosomes and endolysosomes in tty-deficient glia was rescued by expressing the human Tweety Homolog 1 (TTYH1). Loss of tty in glia attenuated mitochondrial turnover, elevated mitochondrial oxidative stress, and impaired locomotor functions. The cellular and organismal defects were partially reversed by antioxidant treatment. Live-cell imaging of genetically encoded metabolite sensors was performed to determine the impact of tty and autophagy deficiencies on glial bioenergetics. tty-deficient glia exhibited reduced mitochondrial pyruvate consumption accompanied by a shift toward glycolysis for ATP production. Likewise, genetic inhibition of autophagy in glia resulted in a similar glycolytic shift in bioenergetics. Furthermore, the survival of mutant flies became more sensitive to starvation, underlining the significance of tty in the crosstalk between autophagy and bioenergetics. Together, these findings uncover the role for tty in mitochondrial homeostasis via facilitating autophagy, which determines bioenergetic balance in glia.
Yoo, J., Dombrovski, M., Mirshahidi, P., Nern, A., LoCascio, S. A., Zipursky, S. L., Kurmangaliyev, Y. Z. (2023). Brain wiring determinants uncovered by integrating connectomes and transcriptomes. Curr Biol, 33(18):3998-4005. PubMed ID: 37647901
Summary:
Advances in brain connectomics have demonstrated the extraordinary complexity of neural circuits. Developing neurons encounter the axons and dendrites of many different neuron types and form synapses with only a subset of them. During circuit assembly, neurons express cell-type-specific repertoires comprising many cell adhesion molecules (CAMs) that can mediate interactions between developing neurites. Many CAM families have been shown to contribute to brain wiring in different ways. It has been challenging, however, to identify receptor-ligand pairs directly matching neurons with their synaptic targets. This study integrated the synapse-level connectome of the neural circuit with the developmental expression patterns and binding specificities of CAMs on pre- and postsynaptic neurons in the Drosophila visual system. To overcome the complexity of neural circuits, focus was placed on pairs of genetically related neurons that make differential wiring choices. In the motion detection circuit, closely related subtypes of T4/T5 neurons choose between alternative synaptic targets in adjacent layers of neuropil. This choice correlates with the matching expression in synaptic partners of different receptor-ligand pairs of the Beat and Side families of CAMs. Genetic analysis demonstrated that presynaptic Side-II and postsynaptic Beat-VI restrict synaptic partners to the same layer. Removal of this receptor-ligand pair disrupts layers and leads to inappropriate targeting of presynaptic sites and postsynaptic dendrites. It is proposed that different Side/Beat receptor-ligand pairs collaborate with other recognition molecules to determine wiring specificities in the fly brain. Combining transcriptomes, connectomes, and protein interactome maps allow unbiased identification of determinants of brain wiring.
Willot, Q., du Toit, A., de Wet, S., Huisamen, E. J., Loos, B., Terblanche, J. S. (2023). Exploring the connection between autophagy and heat-stress tolerance in Drosophila melanogaster. Proceedings Biological sciences, 290(2006):20231305 PubMed ID: 37700658
Summary:

Mechanisms aimed at recovering from heat-induced damages are closely associated with the ability of ectotherms to survive exposure to stressful temperatures. Autophagy, a ubiquitous stress-responsive catabolic process, has recently gained renewed attention as one of these mechanisms. By increasing the turnover of cellular structures as well as the clearance of long-lived protein and protein aggregates, the induction of autophagy has been linked to increased tolerance to a range of abiotic stressors in diverse ectothermic organisms. However, whether a link between autophagy and heat-tolerance exists in insect models remains unclear despite broad ecophysiological implications thereof. This study explored the putative association between autophagy and heat-tolerance using Drosophila melanogaster as a model. It was hypothesized that (1) heat-stress would cause an increase of autophagy in flies' tissues, and (2) rapamycin exposure would trigger a detectable autophagic response in adults and increase their heat-tolerance. In line with this hypothesis, it is reported that flies exposed to heat-stress present signs of protein aggregation and appear to trigger an autophagy-related homoeostatic response as a result. It was further shown that rapamycin feeding causes the systemic effect associated with target of rapamycin (TOR) inhibition, induces autophagy locally in the fly gut, and increases the heat-stress tolerance of individuals. These results argue in favour of a substantial contribution of autophagy to the heat-stress tolerance mechanisms of insects.

Cachoux, V. M. L., Balakireva, M., Gracia, M., Bosveld, F., Lopez-Gay, J. M., Maugarny, A., Gaugue, I., di Pietro, F., Rigaud, S. U., Noiret, L., Guirao, B., Bellaiche, Y. (2023). Epithelial apoptotic pattern emerges from global and local regulation by cell apical area. Curr Biol, 33(22):4807-4826.e4806 PubMed ID: 37827152
Summary:
Geometry is a fundamental attribute of biological systems, and it underlies cell and tissue dynamics. Cell geometry controls cell-cycle progression and mitosis and thus modulates tissue development and homeostasis. In sharp contrast and despite the extensive characterization of the genetic mechanisms of caspase activation, little is known about whether and how cell geometry controls apoptosis commitment in developing tissues. This study combined multiscale time-lapse microscopy of developing Drosophila epithelium, quantitative characterization of cell behaviors, and genetic and mechanical perturbations to determine how apoptosis is controlled during epithelial tissue development. Early in cell lives and well before extrusion, apoptosis commitment is linked to two distinct geometric features: a small apical area compared with other cells within the tissue and a small relative apical area with respect to the immediate neighboring cells. These global and local geometric characteristics are shown to be sufficient to recapitulate the tissue-scale apoptotic pattern. Furthermore, the coupling between these two geometric features and apoptotic cells is shown to be dependent on the Hippo/YAP and Notch pathways. Overall, by exploring the links between cell geometry and apoptosis commitment, this work provides important insights into the spatial regulation of cell death in tissues and improves understanding of the mechanisms that control cell number and tissue size.
Yamada, T., Yoshinari, Y., Tobo, M., Habara, O., Nishimura, T. (2023). Nacα protects the larval fat body from cell death by maintaining cellular proteostasis in Drosophila. Nat Commun, 14(1):5328 PubMed ID: 37658058
Summary:
Protein homeostasis (proteostasis) is crucial for the maintenance of cellular homeostasis. Impairment of proteostasis activates proteotoxic and unfolded protein response pathways to resolve cellular stress or induce apoptosis in damaged cells. However, the responses of individual tissues to proteotoxic stress and evoking cell death program have not been extensively explored in vivo. This study shows that a reduction in Nascent polypeptide-associated complex protein alpha subunit (Nacα) specifically and progressively induces cell death in Drosophila fat body cells. Nacα mutants disrupt both ER integrity and the proteasomal degradation system, resulting in caspase activation through JNK and p53. Although forced activation of the JNK and p53 pathways was insufficient to induce cell death in the fat body, the reduction of Nacα sensitized fat body cells to intrinsic and environmental stresses. Reducing overall protein synthesis by mTor inhibition or Minute mutants alleviated the cell death phenotype in Nacα mutant fat body cells. This work revealed that Nacα is crucial for protecting the fat body from cell death by maintaining cellular proteostasis, thus demonstrating the coexistence of a unique vulnerability and cell death resistance in the fat body.
Pai, Y. L., Lin, Y. J., Peng, W. H., Huang, L. T., Chou, H. Y., Wang, C. H., Chien, C. T., Chen, G. C. (2023). The deubiquitinase Leon/USP5 interacts with Atg1/ULK1 and antagonizes autophagy. Cell Death Dis, 14(8):540 PubMed ID: 37607937
Summary:
Accumulating evidence has shown that the quality of proteins must be tightly monitored and controlled to maintain cellular proteostasis. Misfolded proteins and protein aggregates are targeted for degradation through the ubiquitin proteasome (UPS) and autophagy-lysosome systems. The ubiquitination and deubiquitinating enzymes (DUBs) have been reported to play pivotal roles in the regulation of the UPS system. However, the function of DUBs in the regulation of autophagy remain to be elucidated. This study found that knockdown of Leon/USP5 caused a marked increase in the formation of autophagosomes and autophagic flux under well-fed conditions. Genetic analysis revealed that overexpression of Leon suppressed Atg1-induced cell death in Drosophila. Immunoblotting assays further showed a strong interaction between Leon/USP5 and the autophagy initiating kinase Atg1/ULK1. Depletion of Leon/USP5 led to increased levels of Atg1/ULK1. These findings indicate that Leon/USP5 is an autophagic DUB that interacts with Atg1/ULK1, negatively regulating the autophagic process.

Tuesday, May 28th - Cancer, Tumors and Growth/h3>

Khalili, D., Mohammed, M., Kunc, M., Sindlerova, M., Ankarklev, J., Theopold, U. (2023). Single-cell sequencing of tumor-associated macrophages in a Drosophila model. Frontiers in immunology, 14:1243797 PubMed ID: 37795097
Summary:
Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized. This study used a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (Ras(V12)) to study dysplastic growth during early tumor progression. Single-cell RNA-sequencing was performed of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells. Five distinct hemocyte clusters were identified. In addition to Ras(V12) larvae, a tumor model was included where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells-they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors-antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, evidence is provided for transcript transfer between hemocytes and possibly other tissues. Taken together, these data support the usefulness of Drosophila to study the response against tumors at the organismic level.
Yu, K., Ramkumar, N., Wong, K. K. L., Tettweiler, G., Verheyen, E. M. (2023). The AMPK-like protein kinases Sik2 and Sik3 interact with Hipk and induce synergistic tumorigenesis in a Drosophila cancer model. Frontiers in cell and developmental biology, 11:1214539 PubMed ID: 37854071
Summary:
Homeodomain-interacting protein kinases (Hipks) regulate cell proliferation, apoptosis, and tissue development. Overexpression of Hipk in Drosophila causes tumorigenic phenotypes in larval imaginal discs. This study found that depletion of Salt-inducible kinases Sik2 or Sik3 can suppress Hipk-induced overgrowth. Furthermore, co-expression of constitutively active forms of Sik2 or Sik3 with Hipk caused significant tissue hyperplasia and tissue distortion, indicating that both Sik2 and Sik3 can synergize with Hipk to promote tumorous phenotypes, accompanied by elevated dMyc, Armadillo/β-catenin, and the Yorkie target gene expanded. Larvae expressing these hyperplastic growths also display an extended larval phase, characteristic of other Drosophila tumour models. Examination of total protein levels from fly tissues showed that Hipk proteins were reduced when Siks were depleted through RNAi, suggesting that Siks may regulate Hipk protein stability and/or activity. Conversely, expression of constitutively active Siks with Hipk leads to increased Hipk protein levels. Furthermore, Hipk can interact with Sik2 and Sik3 by co-immunoprecipitation. Co-expression of both proteins leads to a mobility shift of Hipk protein, suggesting it is post-translationally modified. In summary, this research demonstrates a novel function of Siks in synergizing with Hipk to promote tumour growth.
Bosch, P. S., Cho, B., Axelrod, J. D. (2023). . Flamingo participates in multiple models of cell competition. bioRxiv, PubMed ID: 37790459
Summary:
The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. This study shows that in Drosophila, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in pre-malignant cells as they grow among wildtype cells, in healthy cells to eliminate pre-malignant cells, and by supercompetitors to occupy excessive territory within wildtype tissues. "Would-be" winners that lack Fmi are unable to over-proliferate, and instead become losers. This study demonstrate that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well defined functions of Fmi.
Pranoto, I. K. A., Lee, J., Kwon, Y. V. (2023). The roles of the native cell differentiation program aberrantly recapitulated in Drosophila intestinal tumors. Cell Rep, 42(10):113245 PubMed ID: 37837622
Summary:
Many tumors recapitulate the developmental and differentiation program of their tissue of origin, a basis for tumor cell heterogeneity. Although stem-cell-like tumor cells are well studied, the roles of tumor cells undergoing differentiation remain to be elucidated. This study employed Drosophila genetics to demonstrate that the differentiation program of intestinal stem cells is crucial for enabling intestinal tumors to invade and induce non-tumor-autonomous phenotypes. The differentiation program that generates absorptive cells is aberrantly recapitulated in the intestinal tumors generated by activation of the Yap1 ortholog Yorkie. Inhibiting it allows stem-cell-like tumor cells to grow but suppresses invasiveness and reshapes various phenotypes associated with cachexia-like wasting by altering the expression of tumor-derived factors. This study provides insight into how a native differentiation program determines a tumor's capacity to induce advanced cancer phenotypes and suggests that manipulating the differentiation programs co-opted in tumors might alleviate complications of cancer, including cachexia.
Quintero, M., Bangi, E. (2023). Disruptions in cell fate decisions and transformed enteroendocrine cells drive intestinal tumorigenesis in Drosophila. Cell Rep, 42(11):113370 PubMed ID: 37924517
Summary:
Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between different cell types ensures precise regulation of stem cell behavior and cell fate decisions. These cell-cell interactions are often disrupted during tumorigenesis, but mechanisms by which they are co-opted to support tumor growth in different genetic contexts are poorly understood. This study introduces PromoterSwitch, a genetic platform established to generate large, transformed clones derived from individual adult Drosophila intestinal stem/progenitor cells. This study showed that cancer-driving genetic alterations representing common colon tumor genome landscapes disrupt cell fate decisions within transformed tissue and result in the emergence of abnormal cell fates. It was also shown that transformed enteroendocrine cells, a differentiated, hormone-secreting cell lineage, support tumor growth by regulating intestinal stem cell proliferation through multiple genotype-dependent mechanisms, which represent potential vulnerabilities that could be exploited for therapy.
Zheng, J., Guo, Y., Shi, C., Yang, S., Xu, W., Ma, X. (2023). Differential Ire1 determines loser cell fate in tumor-suppressive cell competition. Cell Rep, 42(11):113303 PubMed ID: 37924514
Summary:
Tumor-suppressive cell competition (TSCC) is a conserved surveillance mechanism in which neighboring cells actively eliminate oncogenic cells. Despite overwhelming studies showing that the unfolded protein response (UPR) is dysregulated in various tumors, it remains debatable whether the UPR restrains or promotes tumorigenesis. Using Drosophila eye epithelium as a model, this study uncovered a surprising decisive role of the Ire1 branch of the UPR in regulating cell polarity gene scribble (scrib) loss-induced TSCC. Both mutation and hyperactivation of Ire1 accelerate elimination of scrib clones via inducing apoptosis and autophagy, respectively. Unexpectedly, relative Ire1 activity is also crucial for determining loser cell fate, as dysregulating Ire1 signaling in the surrounding healthy cells reversed the "loser" status of scrib clones by decreasing their apoptosis. Furthermore, it was shown that Ire1 is required for cell competition in mammalian cells. Together, these findings provide molecular insights into scrib-mediated TSCC and highlight Ire1 as a key determinant of loser cell fate.

Friday, May 24th - Disease Models

Yan, L., Zhou, J., Yuan, L., Ye, J., Zhao, X., Ren, G., Chen, H. (2023). Silibinin alleviates intestinal inflammation via inhibiting JNK signaling in Drosophila. Frontiers in pharmacology, 14:1246960 PubMed ID: 37781701
Summary:
Inflammatory bowel diseases (IBDs) are characterized by chronic relapsing intestinal inflammation that causes digestive system dysfunction. For years, researchers have been working to find more effective and safer therapeutic strategies to treat these diseases. Silibinin (SIL), a flavonoid compound extracted from the seeds of milk thistle plants, possesses multiple biological activities and is traditionally applied to treat liver diseases. SIL is also widely used in the treatment of a variety of inflammatory diseases attributed to its excellent antioxidant and anti-inflammatory effects. However, the efficacy of SIL against IBDs and its mechanisms remain unclear. This study, using Drosophila melanogaster as a model organism, found that SIL can effectively relieve intestinal inflammation caused by dextran sulfate sodium (DSS). The results suggested that SIL supplementation can inhibit the overproliferation of intestinal stem cells (ISCs) induced by DSS, protect intestinal barrier function, acid-base balance, and intestinal excretion function, reduce intestinal reactive oxygen species (ROS) levels and inflammatory stress, and extend the lifespan of Drosophila. Furthermore, this study demonstrated that SIL ameliorates intestinal inflammation via modulating the c-Jun N-terminal kinase (JNK) signaling pathway in Drosophila. This research aims to provide new insight into the treatment of IBDs.
Aalto, A. L., Saadabadi, A., Lindholm, F., Kietz, C., Himmelroos, E., Marimuthu, P., Salo-Ahen, O. M. H., Eklund, P., Meinander, A. (2023). Stilbenoid compounds inhibit NF-κB-mediated inflammatory responses in the Drosophila intestine. Frontiers in immunology, 14:1253805 PubMed ID: 37809071
Summary:
Stilbenoid compounds have been described to have anti-inflammatory properties in animal models in vivo, and have been shown to inhibit Ca2+-influx through the transient receptor potential ankyrin 1 (TrpA1). To study how stilbenoid compounds affect inflammatory signaling in vivo, this study has utilized the fruit fly, Drosophila melanogaster, as a model system. To induce intestinal inflammation, flies were fed with the intestinal irritant dextran sodium sulphate (DSS). DSS was found to induce severe changes in the bacteriome of the Drosophila intestine, and that this dysbiosis causes activation of the NF-κB transcription factor Relish. Advantage was taken of the DSS-model to study the anti-inflammatory properties of the stilbenoid compounds pinosylvin (PS) and pinosylvin monomethyl ether (PSMME). With the help of in vivo approaches, PS and PSMME were found to be to be transient receptor ankyrin 1 (TrpA1)-dependent antagonists of NF-κB-mediated intestinal immune responses in Drosophila. This study has also computationally predicted the putative antagonist binding sites of these compounds at Drosophila TrpA1. Taken together, this study showed that the stilbenoids PS and PSMME have anti-inflammatory properties in vivo in the intestine and can be used to alleviate chemically induced intestinal inflammation in Drosophila.
Fan, X., Huang, T., Wang, S., Yang, Z., Song, W., Zeng, Y., Tong, Y., Cai, Y., Yang, D., Zeng, B., Zhang, M., Ni, Q., Li, Y., Li, D., Yang, M. (2023). The adaptor protein 14-3-3zeta modulates intestinal immunity and aging in Drosophila. J Biol Chem, 299(12):105414 PubMed ID: 37918806
Summary:
The proteins that coordinate the complex transcriptional networks of aging have not been completely documented. Protein 14-3-3zeta is an adaptor protein that coordinates signaling and transcription factor networks, but its function in aging is not fully understood. This study showed that the protein expression of 14-3-3zeta gradually increased during aging. High levels of 14-3-3zeta led to shortened lifespan and imbalance of intestinal immune homeostasis in Drosophila, but the decrease in 14-3-3zeta protein levels by RNAi was able to significantly promote the longevity and intestinal immune homeostasis of fruit flies. Importantly, it was demonstrated that adult-onset administration of TIC10, a compound that reduces the aging-related AKT and ">extracellular signal-regulated kinase (ERK) signaling pathways, rescues the shortened lifespan of 14-3-3zeta-overexpressing flies. This finding suggests that 14-3-3zeta plays a critical role in regulating the aging process. This study elucidates the role of 14-3-3zeta in natural aging and provides the rationale for subsequent 14-3-3zeta-based antiaging research.
Diaw, S. H., Borsche, M., Streubel-Gallasch, L., Dulovic-Mahlow, M., Hermes, J., Lenz, I., Seibler, P., Klein, C., Bruggemann, N., Vos, M., Lohmann, K. (2023). Characterization of the pathogenic α-Synuclein Variant V15A in Parkinson´s disease. NPJ Parkinson's disease. 9(1):148 PubMed ID: 37903765
Summary:
Despite being a major component of Lewy bodies and Lewy neurites, pathogenic variants in the gene encoding alpha-Synuclein (α-Syn) are rare. To date, only four missense variants in the SNCA gene, encoding α-Syn have unequivocally been shown to be disease-causing. This study describes a Parkinson´s disease patient with early cognitive decline carrying an as yet not fully characterized variant in SNCA. Different cellular models, including stably transfected neuroblastoma (SH-SY5Y) cell cultures, induced pluripotent stem cell (iPSC)-derived neuronal cultures, and generated a Drosophila model to elucidate the impact of the p.V15A variant on α-Syn function and aggregation properties compared to other known pathogenic variants. This study demonstrated that p.V15A increased the aggregation potential of α-Syn and the levels of apoptotic markers, and impaired the mitochondrial network. Moreover, p.V15A affects the flying ability and survival of mutant flies. Thus, this study provides supporting evidence for the pathogenicity of the p.V15A variant, suggesting its inclusion in genetic testing approaches.
Wang, C. W., Clemot, M., Hashimoto, T., Diaz, J. A., Goins, L. M., Goldstein, A. S., Nagaraj, R., Banerjee, U. (2023). A conserved mechanism for JNK-mediated loss of Notch function in advanced prostate cancer. Science signaling, 16(810):eabo5213 PubMed ID: 37934809
Summary:
ysregulated Notch signaling is a common feature of cancer; however, its effects on tumor initiation and progression are highly variable, with Notch having either oncogenic or tumor-suppressive functions in various cancers. To better understand the mechanisms that regulate Notch function in cancer, Notch signaling was studied in a Drosophila tumor model, prostate cancer-derived cell lines, and tissue samples from patients with advanced prostate cancer. Increased activity of the Src-JNK pathway in tumors inactivated Notch signaling because of JNK pathway-mediated inhibition of the expression of the gene encoding the Notch S2 cleavage protease, Kuzbanian, which is critical for Notch activity. Consequently, inactive Notch accumulated in cells, where it was unable to transcribe genes encoding its target proteins, many of which have tumor-suppressive activities. These findings suggest that Src-JNK activity in tumors predicts Notch activity status and that suppressing Src-JNK signaling could restore Notch function in tumors, offering opportunities for diagnosis and targeted therapies for a subset of patients with advanced prostate cancer.
Zane, F., Bouzid, H., Sosa Marmol, S., Brazane, M., Besse, S., Molina, J. L., Cansell, C., Aprahamian, F., Durand, S., Ayache, J., Antoniewski, C., Todd, N., Carre, C., Rera, M. (2023). Smurfness-based two-phase model of ageing helps deconvolve the ageing transcriptional signature. Aging Cell, 22(11):e13946 PubMed ID: 37822253
Summary:
Ageing is characterised at the molecular level by six transcriptional 'hallmarks of ageing', that are commonly described as progressively affected as time passes. By contrast, the 'Smurf' assay, which assesses food intake by the co-ingestion of a blue dye, which detects increased intestinal permeability. Performing whole body total RNA sequencing, it was found that Smurfness distinguishes transcriptional changes associated with chronological age from those associated with biological age. Transcriptional heterogeneity increases with chronological age in non-Smurf individuals preceding the other five hallmarks of ageing that are specifically associated with the Smurf state. Using this approach, this study devised targeted pro-longevity genetic interventions delaying entry in the Smurf state. It is anticipated that increased attention to the evolutionary conserved Smurf phenotype will bring about significant advances in understanding of the mechanisms of ageing.

Thursday, May 23nd - Cell Cycle

Koury, S. A. (2023). Female meiotic drive shapes the distribution of rare inversion polymorphisms in Drosophila melanogaster. Genetics. PubMed ID: 37616566
Summary:
In all species, new chromosomal inversions are constantly being formed by spontaneous rearrangement and then stochastically eliminated from natural populations. In Drosophila, when new chromosomal inversions overlap with a pre-existing inversion in the population, their rate of elimination becomes a function of the relative size, position, and linkage phase of the gene rearrangements. These altered dynamics result from complex meiotic behavior wherein overlapping inversions generate asymmetric dyads that cause both meiotic drive/drag and segmental aneuploidy. In this context, patterns in rare inversion polymorphisms of a natural population can be modeled from the fundamental genetic processes of forming asymmetric dyads via crossing-over in meiosis I and preferential segregation from asymmetric dyads in meiosis II. In this study a mathematical model of crossover-dependent female meiotic drive is developed and parameterized with published experimental data from Drosophila melanogaster laboratory constructs. This mechanism is demonstrated to favor smaller, distal inversions and accelerate the elimination of larger, proximal inversions. Simulated sampling experiments indicate that the paracentric inversions directly observed in natural population surveys of Drosophila melanogaster are a biased subset that both maximizes meiotic drive and minimizes the frequency of lethal zygotes caused by this cytogenetic mechanism. Incorporating this form of selection into a population genetic model accurately predicts the shift in relative size, position, and linkage phase for rare inversions found in this species. The model and analysis presented in this study suggest that this weak form of female meiotic drive is an important process influencing the genomic distribution of rare inversion polymorphisms.
Haseeb, M. A., Weng, K. A., Bickel, S. E. (2023). Chromatin-associated cohesin turns over extensively and forms new cohesive linkages during meiotic prophase. bioRxiv, PubMed ID: 37645916
Summary:
In dividing cells, accurate chromosome segregation depends on sister chromatid cohesion, protein linkages that are established during DNA replication. Faithful chromosome segregation in oocytes requires that cohesion, first established in S phase, remain intact for days to decades, depending on the organism. Premature loss of meiotic cohesion in oocytes leads to the production of aneuploid gametes and contributes to the increased incidence of meiotic segregation errors as women age (maternal age effect). The prevailing model is that cohesive linkages do not turn over in mammalian oocytes. However, it has been previously reported that cohesion-related defects arise in Drosophila oocytes when individual cohesin subunits (see SMC1) cohesin regulators are knocked down after meiotic S phase. This study use two strategies to express a tagged cohesin subunit exclusively during mid-prophase in Drosophila oocytes and demonstrate that newly expressed cohesin is used to form de novo linkages after meiotic S phase. Moreover, nearly complete turnover of chromosome-associated cohesin occurs during meiotic prophase, with faster replacement on the arms than at the centromeres. Unlike S-phase cohesion establishment, the formation of new cohesive linkages during meiotic prophase does not require acetylation of conserved lysines within the Smc3 head. These findings indicate that maintenance of cohesion between S phase and chromosome segregation in Drosophila oocytes requires an active cohesion rejuvenation program that generates new cohesive linkages during meiotic prophase.
Warecki, B. and Tao, L. (2023). Centralspindlin-mediated transport of RhoGEF positions the cleavage plane for cytokinesis.. Sci Signal 16(792): eadh0601. PubMed ID: 37402224
Summary:
During cytokinesis, the cell membrane furrows inward along a cleavage plane. The positioning of the cleavage plane is critical to faithful cell division and is determined by the Rho guanine nucleotide exchange factor (RhoGEF)-mediated activation of the small guanosine triphosphatase RhoA and the conserved motor protein complex centralspindlin. This study explored whether and how centralspindlin mediates the positioning of RhoGEF. In dividing neuroblasts from Drosophila melanogaster, it was observed that immediately before cleavage, first centralspindlin and then RhoGEF localized to the sites where cleavage subsequently initiated. Using in vitro assays with purified Drosophila proteins and stabilized microtubules, it was found that centralspindlin directly transported RhoGEF as cargo along single microtubules and sequestered it at microtubule plus-ends for prolonged periods of time. In addition, the binding of RhoGEF to centralspindlin appeared to stimulate centralspindlin motor activity. Thus, the motor activity and microtubule association of centralspindlin can translocate RhoGEF to areas where microtubule plus-ends are abundant, such as at overlapping astral microtubules, to locally activate RhoA and accurately position the cleavage plane during cell division.
Sperling, A. L., Fabian, D. K., Garrison, E. and Glover, D. M. (2023). A genetic basis for facultative parthenogenesis in Drosophila. Curr Biol. PubMed ID: 37516115
Summary:
Facultative parthenogenesis enables sexually reproducing organisms to switch between sexual and asexual parthenogenetic reproduction. To gain insights into this phenomenon, the genomes of sexually reproducing and parthenogenetic strains of Drosophila mercatorum were sequenced, and differences were identified in the gene expression in their eggs. Then whether manipulating the expression of candidate gene homologs identified in Drosophila mercatorum could lead to facultative parthenogenesis in the non-parthenogenetic species Drosophila melanogaster was tested. This identified a polygenic system whereby increased expression of the mitotic protein kinase polo and decreased expression of a desaturase, Desat2, caused facultative parthenogenesis in the non-parthenogenetic species that was enhanced by increased expression of Myc. The genetically induced parthenogenetic Drosophila melanogaster eggs exhibit de novo centrosome formation, fusion of the meiotic products, and the onset of development to generate predominantly triploid offspring. Thus, this study demonstrated a genetic basis for sporadic facultative parthenogenesis in an animal.
Bakshi, A., Iturra, F. E., Alamban, A., Rosas-Salvans, M., Dumont, S., Aydogan, M. G. (2023). Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes. Cell, 186(21):4694-4709. PubMed ID: 37832525
Summary:
Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. This study demonstrates in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, evidence is presented that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. It is postulated that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.
Baker, C. C., Gallicchio, L., Matias, N. R., Porter, D. F., Parsanian, L., Taing, E., Tam, C., Fuller, M. T. (2023). Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase. Development, 150(22) PubMed ID: 37882771
Summary:
During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. This study showa that the spermatocyte-specific protein Lutin (Lut) is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.

Wednesday, May 22nd - Immune Response

Jin, Q., Wang, Y., Yin, H., Jiang, H. (2023). Two clip-domain serine protease homologs, cSPH35 and cSPH242, act as a cofactor for prophenoloxidase-1 activation in Drosophila melanogaster. Frontiers in immunology, 14:1244792 PubMed ID: 37781370
Summary:
Insect phenoloxidases (POs) catalyze phenol oxygenation and o-diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high M(r) cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila, PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. This study produced the recombinant cSPH35 and cSPH242 precursors, activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila PPO1 activation by MP2 (i.e., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta. In the presence of high M(r) complexes of the cSPHs, PO at a high specific activity of 260 U/μg was generated in vitro. To complement the in vitro analysis, hemolymph PO activity levels were measured in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/μl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults.
Tafesh-Edwards, G., Eleftherianos, I. (2023). The Drosophila melanogaster prophenoloxidase system participates in immunity against Zika virus infection.. Eur J Immunol, 53(12):e2350632 PubMed ID: 37793051
Summary:
Drosophila melanogaster relies on an evolutionarily conserved innate immune system to protect itself from a wide range of pathogens, making it a convenient genetic model to study various human pathogenic viruses and host antiviral immunity. This study explored for the first time the contribution of the Drosophila phenoloxidase (PO) system to host survival and defenses against Zika virus (ZIKV) infection by analyzing the role of mutations in the three prophenoloxidase (PPO) genes in female and male flies. Only PPO1 and PPO2 genes contribute to host survival and appear to be upregulated following ZIKV infection in Drosophila. Also, data suggesting that a complex regulatory system exists between Drosophila PPOs, potentially allowing for a sex-dependent compensation of PPOs by one another or other immune responses such as the Toll, Imd, and JAK/STAT pathways. Furthermore, this study showed that PPO1 and PPO2 are essential for melanization in the hemolymph and the wound site in flies upon ZIKV infection. These results reveal an important role played by the melanization pathway in response to ZIKV infection, hence highlighting the importance of this pathway in insect host defense against viral pathogens and potential vector control strategies to alleviate ZIKV outbreaks.
Elguero, J. E., Liu, G., Tiemeyer, K., Bandyadka, S., Gandevia, H., Duro, L., Yan, Z., McCall, K. (2023). Defective phagocytosis leads to neurodegeneration through systemic increased innate immune signaling. iScience, 26(10):108052 PubMed ID: 37854687
Summary:
In nervous system development, disease, and injury, neurons undergo programmed cell death, leaving behind cell corpses that are removed by phagocytic glia. Altered glial phagocytosis has been implicated in several neurological diseases including Alzheimer's disease. To untangle the links between glial phagocytosis and neurodegeneration, Drosophila mutants lacking the phagocytic receptor Draper were investigated. Loss of Draper leads to persistent neuronal cell corpses and age-dependent neurodegeneration. Whether the phagocytic defects observed in draper mutants lead to chronic increased immune activation that promotes neurodegeneration was investigate. It was found that the antimicrobial peptide Attacin-A is highly upregulated in the fat body of aged draper mutants and that the inhibition of the Immune deficiency (Imd) pathway in the glia and fat body of draper mutants led to reduced neurodegeneration. Taken together, these findings indicate that phagocytic defects lead to neurodegeneration via increased immune signaling, both systemically and locally in the brain.
Pan, W., Yao, X., Lin, L., Liu, X., Jin, P., Ma, F. (2023). The Relish/miR-275/Dredd mediated negative feedback loop is crucial to restoring immune homeostasis of Drosophila Imd pathway. Insect biochemistry and molecular biology, 162:104013 PubMed ID: 37804878
Summary:
The NF-κB/Relish, as a core transcription factor of Drosophila immune deficiency (Imd) pathway, activates the transcriptions of antimicrobial peptides (AMPs) to combat gram-negative bacterial infections, but its role in regulating miRNA expression during immune response has less been reported. This study describes a negative feedback loop of Imd signaling mediated by Relish/miR-275/Dredd that controls Drosophila immune homeostasis after Escherichia coli (E. coli) infection. The results demonstrate that Relish may directly activate the transcription of miR-275 via binding to its promoter in vitro and vivo, particularly miR-275 further inhibits the expression of Dredd through binding to its 3'UTR to negatively control Drosophila Imd immune response. Remarkably, the ectopic expression of miR-275 significantly reduces Drosophila lifespan. More importantly, this work uncovers a new mechanism by which Relish can flexibly switch its role to maintain Drosophila immune response and homeostasis during infection. Collectively, this study not only reveals the functional duality of Relish in regulating immune response of Drosophila Imd pathway, but also provides a new insight into the maintenance of animal innate immune homeostasis.
Arias-Rojas, A., Frahm, D., Hurwitz, R., Brinkmann, V., Iatsenko, I. (2023). Resistance to host antimicrobial peptides mediates resilience of gut commensals during infection and aging in Drosophila. Proc Natl Acad Sci U S A, 120(36):e2305649120 PubMed ID: 37639605
Summary:
Resilience to short-term perturbations, like inflammation, is a fundamental feature of microbiota, yet the underlying mechanisms of microbiota resilience are incompletely understood. This study shows that Lactiplantibacillus plantarum, a major Drosophila commensal, stably colonizes the fruit fly gut during infection and is resistant to Drosophila antimicrobial peptides (AMPs). By transposon screening, L. plantarum mutants sensitive to AMPs were identified. These mutants were impaired in peptidoglycan O-acetylation or teichoic acid D-alanylation, resulting in increased negative cell surface charge and higher affinity to cationic AMPs. AMP-sensitive mutants were cleared from the gut after infection and aging-induced gut inflammation in wild-type, but not in AMP-deficient flies, suggesting that resistance to host AMPs is essential for commensal resilience in an inflamed gut environment. Thus, this work reveals that in addition to the host immune tolerance to the microbiota, commensal-encoded resilience mechanisms are necessary to maintain the stable association between host and microbiota during inflammation.
Cai, H., Li, L., Slavik, K. M., Huang, J., Yin, T., Ai, X., Hedelin, L., Haas, G., Xiang, Z., Yang, Y., Li, X., Chen, Y., Wei, Z., Deng, H., Chen, D., Jiao, R., Martins, N., Meignin, C., Kranzusch, P. J., Imler, J. L. (2023). The virus-induced cyclic dinucleotide 2'3'-c-di-GMP mediates STING-dependent antiviral immunity in Drosophila. Immunity, 56(9):1991-2005 PubMed ID: 37659413
Summary:
In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP, which triggers STING-dependent immunity. In Drosophila melanogaster, two cGAS-like receptors (cGLRs) produce 3'2'-cGAMP and 2'3'-cGAMP to activate STING. CDN-mediated immunity was explored in 14 Drosophila species covering 50 million years of evolution; 2'3'-cGAMP and 3'2'-cGAMP failed to control infection by Drosophila C virus in D. serrata and two other species. Diverse CDNs produced in a cGLR-dependent manner were descovered in response to viral infection in D. melanogaster, including 2'3'-c-di-GMP. This CDN was a more potent STING agonist than cGAMP in D. melanogaster and it also activated a strong antiviral transcriptional response in D. serrata. These results shed light on the evolution of cGLRs in flies and provide a basis for understanding the function and regulation of this emerging family of pattern recognition receptors in animal innate immunity.

Tuesday, May 21st - Enzymes and protein expression, evolution, structure, and function

Yushkova, E. (2024). . Interaction effect of mutations in the genes (piwi and aub) of the Argonaute family and hobo transposons on the integral survival parameters of Drosophila melanogaster. Biogerontology, 25(1):131-146 PubMed ID: 37864608
Summary:
The Argonaute family genes (piwi and aub) involved in the production of small RNAs are responsible for the regulation of many cellular processes, including the suppression of genome instability, modulation of gene activity, and transposable elements. Dysfunction of these genes and the associated activation of transposable elements adversely affect reproductive development and quality of life. The role of transposons in contrast to retrotransposons and their interaction with genes of the Argonaute family in aging processes have not been studied. This study considers a scenario in which the piwi and aub genes in the presence of functional hobo transposons can modify the effects from the level of DNA damage to lifespan. The simultaneous presence of mutation (piwi or aub) and hobo (regardless of size) in the genome has practically no effect or (less often) leads to a decrease in the level of DNA damage in ovarian cells. A high level of sterility and low ovarian reserve were noted mainly with a combination of mutations and full-sized hobo elements. The combination of these two genetic factors negatively affects the fertility of young females and embryonic survival. Isolated cases of restoration of reproductive functions with age were noted but only in females that had low fertility in the early period of life. The presence of hobo transposons contributed to an increase in the lifespan of both mutant and non-mutant females. Dysfunction of the piwi and aub genes (without hobo) can reduce the lifespan of both sexes. Together, each mutation and hobo transposons act antagonistically/additively (in females) and synergistically/antagonistically (in males) to change the lifespan. In parameters of locus-specific instability, hobo activation was more pronounced in piwi gene dysfunction. The results obtained complement data on the study of new functions of Argonaute family genes and their interactions with transposable elements in the aging process.
Krzywinska, E., Ribeca, P., Ferretti, L., Hammond, A., Krzywinski, J. (2023). A novel factor modulating X chromosome dosage compensation in Anopheles. Curr Biol, 33(21):4697-4703. PubMed ID: 37774706
Summary:
Dosage compensation (DC), a process countering chromosomal imbalance in individuals with heteromorphic sex chromosomes, has been molecularly characterized only in mammals, Caenorhabditis elegans, and fruit flies. In Drosophila melanogaster males, it is achieved by an approximately 2-fold hypertranscription of the monosomic X chromosome mediated by the MSL complex. The complex is not assembled on female X chromosomes because production of its key protein MSL-2 is prevented due to intron retention and inhibition of translation by Sex-lethal, a female-specific protein operating at the top of the sex determination pathway.) It remains unclear how DC is mechanistically regulated in other insects. In the malaria mosquito Anopheles gambiae, an approximately 2-fold hypertranscription of the male X also occurs by a yet-unknown molecular mechanism distinct from that in D. melanogaster. This study shows that a male-specifically spliced gene call 007, which arose by a tandem duplication in the Anopheles ancestral lineage, is involved in the control of DC in males. Homozygous 007 knockouts lead to a global downregulation of the male X, phenotypically manifested by a slower development compared to wild-type mosquitoes or mutant females-however, without loss of viability or fertility. In females, a 007 intron retention promoted by the sex determination protein Femaleless, known to prevent hypertranscription from both X chromosomes introduces a premature termination codon apparently rendering the female transcripts non-productive. In addition to providing a unique perspective on DC evolution, the 007, with its conserved properties, may represent an important addition to a genetic toolbox for malaria vector control.
Liu, M., Xie, X. J., Li, X., Ren, X., Sun, J., Lin, Z., Hemba-Waduge, R. U., Ji, J. Y. (2023). Transcriptional coupling of telomeric retrotransposons with the cell cycle. bioRxiv, PubMed ID: 37808851
Summary:
Instead of employing telomerases to safeguard chromosome ends, dipteran species maintain their telomeres by transposition of telomeric-specific retrotransposons (TRs): in Drosophila , these are HeT-A, TART, and TAHRE. Previous studies have shown how these TRs create tandem repeats at chromosome ends, but the exact mechanism controlling TR transcription has remained unclear. This study reports the identification of multiple subunits of the transcription cofactor Mediator complex and transcriptional factors Scalloped (Sd, the TEAD homolog in flies) and E2F1-Dp as novel regulators of TR transcription and telomere length in Drosophila . Depletion of multiple Mediator subunits, Dp, or Sd increased TR expression and telomere length, while over-expressing E2F1-Dp or knocking down the E2F1 regulator Rbf1 (Retinoblastoma-family protein 1) stimulated TR transcription, with Mediator and Sd affecting TR expression through E2F1-Dp. The CUT&RUN analysis revealed direct binding of CDK8, Dp, and Sd to telomeric repeats. These findings highlight the essential role of the Mediator complex in maintaining telomere homeostasis by regulating TR transcription through E2F1-Dp and Sd, revealing the intricate coupling of TR transcription with the host cell-cycle machinery, thereby ensuring chromosome end protection and genomic stability during cell division.
Kour, S., Fortuna, T., Anderson, E. N., Mawrie, D., Bilstein, J., Sivasubramanian, R., Ward, C., Roy, R., Rajasundaram, D., Sterneckert, J., Pandey, U. B. (2023). Drosha-dependent microRNAs modulate FUS-mediated neurodegeneration in vivo. Nucleic Acids Res, 51(20):11258-11276 PubMed ID: 37791873
Summary:
Mutations in the Fused in Sarcoma (FUS) gene (see Drosophila Cabeza) cause the familial and progressive form of amyotrophic lateral sclerosis (ALS). FUS is a nuclear RNA-binding protein involved in RNA processing and the biogenesis of a specific set of microRNAs. This study reports that Drosha and two previously uncharacterized Drosha-dependent miRNAs are strong modulators of FUS expression and prevent the cytoplasmic segregation of insoluble mutant FUS in vivo. Depletion of Drosha mitigates FUS-mediated degeneration, survival and motor defects in Drosophila. Mutant FUS strongly interacts with Drosha and causes its cytoplasmic mis-localization into the insoluble FUS inclusions. Reduction in Drosha levels increases the solubility of mutant FUS. Interestingly, it was found that two Drosha dependent microRNAs, miR-378i and miR-6832-5p, which differentially regulate the expression, solubility and cytoplasmic aggregation of mutant FUS in iPSC neurons and mammalian cells. More importantly, this study reports different modes of action of these miRNAs against mutant FUS. Whereas miR-378i may regulate mutant FUS inclusions by preventing G3BP-mediated stress granule formation, miR-6832-5p may affect FUS expression via other proteins or pathways. Overall, this research reveals a possible association between ALS-linked FUS mutations and the Drosha-dependent miRNA regulatory circuit, as well as a useful perspective on potential ALS treatment via microRNAs.
Matzkin, L. M., Bono, J. M., Pigage, H. K., Allan, C. W., Diaz, F., McCoy, J. R., Green, C. C., Callan, J. B., Delahunt, S. P. (2023). Females translate male mRNA transferred during mating. bioRxiv, PubMed ID: 37790342
Summary:
Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. This study develop an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. Strong evidence was found for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction.
Coronado-Zamora, M., Gonzalez, J. (2023). Transposons contribute to the functional diversification of the head, gut, and ovary transcriptomes across Drosophila natural strains. Genome research, 33(9):1541-1553 PubMed ID: 37793782
Summary:
Transcriptomes are dynamic, with cells, tissues, and body parts expressing particular sets of transcripts. Transposable elements (TEs) are a known source of transcriptome diversity; however, studies often focus on a particular type of chimeric transcript, analyze single body parts or cell types, or are based on incomplete TE annotations from a single reference genome. This work has implemented a method based on de novo transcriptome assembly that minimizes the potential sources of errors while identifying a comprehensive set of gene-TE chimeras. This method was applied to the head, gut, and ovary dissected from five Drosophila melanogaster natural strains, with individual reference genomes available. ~19% of body part-specific transcripts are gene-TE chimeras. Overall, chimeric transcripts contribute a mean of 43% to the total gene expression, and they provide protein domains for DNA binding, catalytic activity, and DNA polymerase activity. This comprehensive data set is a rich resource for follow-up analysis. Moreover, because TEs are present in virtually all species sequenced to date, their role in spatially restricted transcript expression is likely not exclusive to the species analyzed in this work.

Monday, May 20th - Enzymes and protein expression, evolution, structure, and function

Sayeesh, P. M., Iguchi, M., Suemoto, Y., Inoue, J., Inomata, K., Ikeya, T., Ito, Y. (2023). Interactions of the N- and C-Terminal SH3 Domains of Drosophila Drk with the Proline-Rich Peptides from Sos and Dos. Int J Mol Sci, 24(18) PubMed ID: 37762438
Summary:
Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. A previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, NMR titration experiments were conducted using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. The results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. Docking simulations were performed based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, the various modes in the interactions of Drk with Sos/Dos are discussed.
Li, J., Zheng, H., Hou, J., Chen, J., Zhang, F., Yang, X., Jin, F., Xi, Y. (2023). X-linked RBBP7 mutation causes maturation arrest and testicular tumors. J Clin Invest, 133(20) PubMed ID: 37843278
Summary:
Maturation arrest (MA) is a subtype of non-obstructive azoospermia, and male infertility is a known risk factor for testicular tumors. However, the genetic basis for many affected individuals remains unknown. This study identified a deleterious hemizygous variant of X-linked retinoblastoma-binding protein 7 (RBBP7) as a potential key cause of MA, which was also found to be associated with the development of Leydig cell tumors. This mutation resulted in premature protein translation termination, affecting the sixth WD40 domain of the RBBP7 and the interaction of the mutated RBBP7 with histone H4. Decreased BRCA1 and increased γH2AX were observed in the proband. In mouse spermatogonial and pachytene spermatocyte-derived cells, deprivation of rbbp7 led to cell cycle arrest and apoptosis. In Drosophila, knockdown of RBBP7/Caf1-55 in germ cells resulted in complete absence of germ cells and reduced testis size, whereas knockdown of RBBP7/Caf1-55 in cyst cells resulted in hyperproliferative testicular cells. Interestingly, male infertility caused by Caf1-55 deficiency was rescued by ectopic expression of wild-type human RBBP7 but not mutant variants, suggesting the importance of RBBP7 in spermatogenesis. This study provides insights into the mechanisms underlying the co-occurrence of MA and testicular tumors and may pave the way for innovative genetic diagnostics of these 2 diseases.
Yoon, H. J., Price, B. E., Parks, R. K., Ahn, S. J., Choi, M. Y. (2023). Diuretic hormone 31 activates two G protein-coupled receptors with differential second messengers for diuresis in Drosophila suzukii. Insect biochemistry and molecular biology, 162:104025 PubMed ID: 37813200
Summary:
Diuretic hormones (DHs) bind to G protein-coupled receptors (GPCRs), regulating water and ion balance to maintain homeostasis in animals. Two distinct DHs are known in insects: calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. This study identified and characterized DH31 and two DH31 GPCR variants, DH31-Ra and DH31-Rb, from spotted-wing drosophila, Drosophila suzukii, a globally prevalent vinegar fly causing severe damage to small fruits. Both GPCRs are active, but DH31-Ra is the dominant receptor based on gene expression analyses and DH31 peptide binding affinities. A notable difference between the two variants lies in 1) the GPCR structures of their C-termini and 2) the utilization of second messengers, and the amino acid sequences of the two variants are identical. DH31-Ra contains 12 additional amino acids, providing different intracellular C-terminal configurations. DH31-Ra utilizes both cAMP and Ca(2+) as second messengers, whereas DH31-Rb utilizes only cAMP; this is the first time reported for an insect CT-like DH31 peptide. DH31 stimulated fluid secretion in D. suzukii adults, and secretion increased in a dose-dependent manner. However, when the fly was injected with a mixture of DH31 and CAPA, an anti-diuretic hormone, fluid secretion was suppressed. The structures are discussed of the DH31 receptors and the differential signaling pathways, including second messengers, involved in fly diuresis. These findings provide fundamental insights into the characterization of D. suzukii DH31 and DH31-Rs, and facilitate the identification of potential biological targets for D. suzukii management.
Gupta, K., Chakrabarti, S., Janardan, V., Gogia, N., Banerjee, S., Srinivas, S., Mahishi, D., Visweswariah, S. S. (2023). Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response. PLoS Genet, 19(9):e1010962 PubMed ID: 37733787
Summary:
Evolutionarily conserved genes often play critical roles in organismal physiology. This study describes multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped (Metallophosphoesterase domain containing), the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. It was observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.
Abbasi Yeganeh, F., Rastegarpouyani, H., Li, J., Taylor, K. A. (2023). Structure of the Drosophila melanogaster Flight Muscle Myosin Filament at 4.7 A Resolution Reveals New Details of Non-Myosin Proteins. Int J Mol Sci, 24(19) PubMed ID: 37834384
Summary:
Striated muscle thick filaments are composed of myosin II and several non-myosin proteins which define the filament length and modify its function. Myosin II has a globular N-terminal motor domain comprising its catalytic and actin-binding activities and a long α-helical, coiled tail that forms the dense filament backbone. Myosin alone polymerizes into filaments of irregular length, but striated muscle thick filaments have defined lengths that, with thin filaments, define the sarcomere structure. The motor domain structure and function are well understood, but the myosin filament backbone is not. This study reports on the structure of the flight muscle thick filaments from Drosophila melanogaster at 4.7 Å resolution, which eliminates previous ambiguities in non-myosin densities. The full proximal S2 region is resolved, as are the connecting densities between the Ig domains of stretchin-klp. The proteins, flightin, and myofilin are resolved in sufficient detail to build an atomic model based on an AlphaFold prediction. The results suggest a method by which flightin and myofilin cooperate to define the structure of the thick filament and explains a key myosin mutation that affects flightin incorporation. Drosophila is a genetic model organism for which These results can define strategies for functional testing.
Keller, S. H., Deng, H., Lim, B. (2023). Regulation of the dynamic RNA Pol II elongation rate in Drosophila embryos. Cell Rep, 42(10):113225 PubMed ID: 37837623
Summary:
An increasing number of studies have shown the key role that RNA polymerase II (RNA Pol II) elongation plays in gene regulation. This study systematically examine how various enhancers, promoters, and gene body composition influence the RNA Pol II elongation rate through a single-cell-resolution live imaging assay. By using reporter constructs containing 5' MS2 and 3' PP7 repeating stem loops, this study quantified the rate of RNA Pol II elongation in live Drosophila embryos. Promoters and exonic gene lengths were found to have no effect on elongation rate, while enhancers and the presence of long introns may significantly change how quickly RNA Pol II moves across a gene. Furthermore, it was observed in multiple constructs that the RNA Pol II elongation rate accelerates after the transcriptional onset of nuclear cycle 14 in Drosophila embryos. This study provides a single-cell view of various mechanisms that affect the dynamic RNA Pol II elongation rate, ultimately affecting the rate of mRNA production.

Thursday, May 16th - Drosophila as a model for human diseases

Bennett, C. L., Dastidar, S., Arnold, F. J., McKinstry, S. U., Stockford, C., Freibaum, B. D., Sopher, B. L., Wu, M., Seidner, G., Joiner, W., Taylor, J. P., West, R. J. H., La Spada, A. R. (2023). Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity. Acta neuropathologica communications, 11(1):164 PubMed ID: 37845749
Summary:
Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. This study considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, SETX fly lines were generated, and the effect of SETX in flies expressing either (G4C2)(58) repeats or glycine-arginine-50 [GR(50)] DPRs was evaluated. Dramatic suppression of disease phenotypes was observed in (G4C2)(58) and GR(50) Drosophila models, and a striking relocalization of GR(50) out of the nucleolus was detected in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. It is noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, the nucleolus in cells expressing GR-DPRs was directly assessed, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS.
Swinter, K., Salah, D., Rathnayake, R., Gunawardena, S. (2023). PolyQ-Expansion Causes Mitochondria Fragmentation Independent of Huntingtin and Is Distinct from Traumatic Brain Injury (TBI)/Mechanical Stress-Mediated Fragmentation Which Results from Cell Death. Cells, 12(19) PubMed ID: 37830620
Summary:
Mitochondrial dysfunction has been reported in many Huntington's disease (HD) models; however, it is unclear how these defects occur. This study tested the hypothesis that excess pathogenic huntingtin (HTT) impairs mitochondrial homeostasis, using Drosophila genetics and pharmacological inhibitors in HD and polyQ-expansion disease models and in a mechanical stress-induced traumatic brain injury (TBI) model. Expression of pathogenic HTT caused fragmented mitochondria compared to normal HTT, but HTT did not co-localize with mitochondria under normal or pathogenic conditions. Expression of pathogenic polyQ (127Q) alone or in the context of Machado Joseph Disease (MJD) caused fragmented mitochondria. While mitochondrial fragmentation was not dependent on the cellular location of polyQ accumulations, the expression of a chaperone protein, excess of mitofusin (MFN), or depletion of dynamin-related protein 1 (DRP1) rescued fragmentation. Intriguingly, a higher concentration of nitric oxide (NO) was observed in polyQ-expressing larval brains and inhibiting NO production rescued polyQ-mediated fragmented mitochondria, postulating that DRP1 nitrosylation could contribute to excess fission. Furthermore, while excess PI3K, which suppresses polyQ-induced cell death, did not rescue polyQ-mediated fragmentation, it did rescue fragmentation caused by mechanical stress/TBI. Together, these observations suggest that pathogenic polyQ alone is sufficient to cause DRP1-dependent mitochondrial fragmentation upstream of cell death, uncovering distinct physiological mechanisms for mitochondrial dysfunction in polyQ disease and mechanical stress.
Roth, J. R., de Moraes, R. C. M., Xu, B. P., Crawley, S. R., Khan, M. A., Melkani, G. C. (2023). Rapamycin reduces neuronal mutant huntingtin aggregation and ameliorates locomotor performance in Drosophila. Frontiers in aging neuroscience, 15:1223911 PubMed ID: 37823007
Summary:
Huntington's disease (HD) is a neurodegenerative disease characterized by movement and cognitive dysfunction. HD is caused by a CAG expansion in exon 1 of the HTT gene that leads to a polyglutamine (PQ) repeat in the huntingtin protein, which aggregates in the brain and periphery. Previously, Drosophila models were used to determine that Htt-PQ aggregation in the heart causes shortened lifespan and cardiac dysfunction that is ameliorated by promoting chaperonin function or reducing oxidative stress. The role of neuronal mutant huntingtin and how it affects peripheral function was further studied in this project. Normal (Htt-PQ25) or expanded mutant (Htt-PQ72) exon 1 of huntingtin was overexpressed in Drosophila neurons, and mutant huntingtin was found to cause age-dependent Htt-PQ aggregation in the brain and could cause a loss of synapsin. To determine if this neuronal dysfunction led to peripheral dysfunction,a negative geotaxis assay was performed to measure locomotor performance, and neuronal mutant huntingtin was found to cause an age-dependent decrease in locomotor performance. Next, it was found that rapamycin reduced Htt-PQ aggregation in the brain. These results demonstrate the role of neuronal Htt-PQ in dysfunction in models of HD, suggest that brain-periphery crosstalk could be important to the pathogenesis of HD, and show that rapamycin reduces mutant huntingtin aggregation in the brain.
Catterson, J. H., Minkley, L., Aspe, S., Judd-Mole, S., Moura, S., Dyson, M. C., Rajasingam, A., Woodling, N. S., Atilano, M. L., Ahmad, M., Durrant, C. S., Spires-Jones, T. L., Partridge, L. (2023). Protein retention in the endoplasmic reticulum rescues A β toxicity in Drosophila. Neurobiology of aging, 132:154-174 PubMed ID: 37837732
Summary:
Amyloid β (A β; see Drosophila Appl) accumulation is a hallmark of Alzheimer's disease. In adult Drosophila brains, human A β overexpression harms climbing and lifespan. It's uncertain whether A β is intrinsically toxic or activates downstream neurodegeneration pathways. This study uncovers a novel protective role against A β toxicity: intra-endoplasmic reticulum (ER) protein accumulation with a focus on laminin and collagen subunits. Despite high A β, laminin B1 (LanB1) overexpression robustly counters toxicity, suggesting a potential A β resistance mechanism. Other laminin subunits and collagen IV also alleviate A β toxicity; combining them with LanB1 augments the effect. Imaging reveals ER retention of LanB1 without altering A β secretion. LanB1's rescue function operates independently of the IRE1α/XBP1 ER stress response. ER-targeted GFP overexpression also mitigates A β toxicity, highlighting broader ER protein retention advantages. Proof-of-principle tests in murine hippocampal slices using mouse Lamb1 demonstrate ER retention in transduced cells, indicating a conserved mechanism. Though ER protein retention generally harms, it could paradoxically counter neuronal A β toxicity, offering a new therapeutic avenue for Alzheimer's disease.
Han, J. E., Kang, K. H., Kim, H., Hong, Y. B., Choi, B. O., Koh, H. (2023). PINK1 and Parkin rescue motor defects and mitochondria dysfunction induced by a patient-derived HSPB3 mutant in Drosophila models. Biochem Biophys Res Commun, 682:71-76 PubMed ID: 37804589
Summary:
Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with the α-crystalline domain that is critical to their chaperone activity. Within the sHSP family, three (HSPB1, HSPB3, and HSPB8) proteins are linked with inherited peripheral neuropathies, including distal hereditary motor neuropathy (dHMN) and Charco-Marie-Tooth disease (CMT). This study introduced the HSPB3 Y118H (HSPB3(Y118H)) mutant gene identified from the CMT2 family in Drosophila. With a missense mutation on its α-crystalline domain, this human HSPB3 mutant gene induced a loss of motor activity accompanied by reduced mitochondrial membrane potential in fly neuronal tissues. Moreover, mitophagy, a critical mechanism of mitochondrial quality control, is downregulated in fly motor neurons expressing HSPB3(Y118H). Surprisingly, ">PINK1 and Parkin, the core regulators of mitophagy, successfully rescued these motor and mitochondrial abnormalities in HSPB3 mutant flies. Results from the first animal model of HSPB3 mutations suggest that mitochondrial dysfunction plays a critical role in HSPB3-associated human pathology.
Nil, Z., Deshwar, A. R., Huang, Y., Barish, S., ..., Yamamoto, S., Costain, G., Bellen, H. J. (2023). Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies. American journal of human genetics, 110(11):1919-1937 PubMed ID: 37827158
Summary:
Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L (homolog of Drosophila Grappa) is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. This study has identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. Thesee results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease.

Wednesday, May 15th - Homologs of Drosophila Genes

Duan, D., Lyu, W., Chai, P., Ma, S., Wu, K., Wu, C., Xiong, Y., Sestan, N., Zhang, K., Koleske, A. J. (2023). Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation. Curr Biol, 33(21):4582-4598.e4510 PubMed ID: 37858340
Summary:
Abl family kinases (see Drosophila Abl) are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both in vitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. This study reports that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ-790. It is proposed that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue.
Cowell, L. M., King, M., West, H., Broadsmith, M., Genever, P., Pownall, M. E., Isaacs, H. V. (2023). Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development. PLoS One, 18(10):e0286040 PubMed ID: 37856433
Summary:
Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf; see Drosophila Breathless) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. It has been proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor. This study investigated the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. Xenopus Cic was characterized and it was shown to be widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with the hypothesis, it was shown that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. Two of these genes were investigated as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b (see Drosophila Ras oncogene at 85D), which encode a leucine zipper transcription factor and a ras family GTPase, respectively. Cic consensus binding sites were identified in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. Expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, it was shown that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. These data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding.
Sauty, S. M., Yankulov, K. (2023). Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-modal active/silent state at the sub-telomeres of S. cerevisiae. Epigenetics & chromatin, 16(1):40 PubMed ID: 37858268
Summary:
Classical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. This study used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the different roles of PCNA and histone chaperones in the stability and the propagation of repressed states are shown to at the sub-telomeres of S. cerevisiae. Position dependent transient repression or bi-modal expression of reporter genes were documented at the VIIL sub-telomere. This study also showed that mutations in the replicative clamp POL30 (PCNA) or the deletion of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the deletion of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. The physical interaction of CAF1 and RRM3 with PCNA was analyzed and the implications of these findings for understanding of the stability and transmission of the epigenetic state of the genes are discussed. There are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. This study characterised the roles of CAF1, RRM3 and ASF1 in these modes of gene repression. It is suggested that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing.
Liu, Q., Bell, B. J., Kim, D. W., Lee, S. S., Keles, M. F., Liu, Q., Blum, I. D., Wang, A. A., Blank, E. J., Xiong, J., Bedont, J. L., Chang, A. J., Issa, H., Cohen, J. Y., Blackshaw, S., Wu, M. N. (2023). A clock-dependent brake for rhythmic arousal in the dorsomedial hypothalamus. Nat Commun, 14(1):6381 PubMed ID: 37821426
Summary:
Circadian clocks generate rhythms of arousal, but the underlying molecular and cellular mechanisms remain unclear. In Drosophila, the clock output molecule WIDE AWAKE (WAKE) labels rhythmic neural networks and cyclically regulates sleep and arousal. This study shows, in a male mouse model, that mWAKE/ANKFN1 labels a subpopulation of dorsomedial hypothalamus (DMH) neurons involved in rhythmic arousal and acts in the DMH to reduce arousal at night. In vivo Ca(2+) imaging reveals elevated DMH(mWAKE) activity during wakefulness and rapid eye movement (REM) sleep, while patch-clamp recordings show that DMH(mWAKE) neurons fire more frequently at night. Chemogenetic manipulations demonstrate that DMH(mWAKE) neurons are necessary and sufficient for arousal. Single-cell profiling coupled with optogenetic activation experiments suggest that GABAergic DMH(mWAKE) neurons promote arousal. Surprisingly, the data suggest that mWAKE acts as a clock-dependent brake on arousal during the night, when mice are normally active. mWAKE levels peak at night under clock control, and loss of mWAKE leads to hyperarousal and greater DMH(mWAKE) neuronal excitability specifically at night. These results suggest that the clock does not solely promote arousal during an animal's active period, but instead uses opposing processes to produce appropriate levels of arousal in a time-dependent manner.
Adiji, O. A., McConnell, B. S., Parker, M. W. (2024). The origin recognition complex requires chromatin tethering by a hypervariable intrinsically disordered region that is functionally conserved from sponge to man. Nucleic Acids Res, 52(8):4344-4360 PubMed ID: 38381902
Summary:
The first step toward eukaryotic genome duplication is loading of the replicative helicase onto chromatin. This 'licensing' step initiates with the recruitment of the origin recognition complex (ORC) to chromatin, which is thought to occur via ORC's ATP-dependent DNA binding and encirclement activity. However, previous work has shown that ATP binding is dispensable for the chromatin recruitment of fly ORC, raising the question of how metazoan ORC binds chromosomes. This study shows that the intrinsically disordered region (IDR) of fly Orc1 is both necessary and sufficient for recruitment of ORC to chromosomes in vivo and demonstrate that this is regulated by IDR phosphorylation. Consistently, it was found that the IDR confers the ORC holocomplex with ATP-independent DNA binding activity in vitro. Using phylogenetic analysis, the surprising observation was made that metazoan Orc1 IDRs have diverged so markedly that they are unrecognizable as orthologs and yet these compositionally homologous sequences are functionally conserved. Altogether, these data suggest that chromatin is recalcitrant to ORC's ATP-dependent DNA binding activity, necessitating IDR-dependent chromatin tethering, which is proposed to poise ORC to opportunistically encircle nucleosome-free regions as they become available.
Kushwaha, A., Thakur, M. K. (2024). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Molecular neurobiology 61(1):487-497 PubMed ID: 37626270
Summary:
Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ Suv39h1 (a mammalian ortholog of the Drosophila melanogaster SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It has been previously reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. In the current study an attempt was made to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. This attempt was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia.

Tuesday, May 14th - Adult neural structure, development and function

Banach-Latapy, A., Rincheval, V., Briand, D., Guenal, I., Speder, P. (2023). Differential adhesion during development establishes individual neural stem cell niches and shapes adult behaviour in Drosophila. PLoS Biol, 21(11):e3002352 PubMed ID: 37943883
Summary:
Neural stem cells (NSCs) reside in a defined cellular microenvironment, the niche, which supports the generation and integration of newborn neurons. The mechanisms building a sophisticated niche structure around NSCs and their functional relevance for neurogenesis are yet to be understood. In the Drosophila larval brain, the cortex glia (CG) encase individual NSC lineages in membranous chambers, organising the stem cell population and newborn neurons into a stereotypic structure. CG were found to wrap around lineage-related cells regardless of their identity, showing that lineage information builds CG architecture. It was then discovered that a mechanism of temporally controlled differential adhesion using conserved complexes supports the individual encasing of NSC lineages. An intralineage adhesion through homophilic Neuroglian interactions provides strong binding between cells of a same lineage, while a weaker interaction through Neurexin-IV and Wrapper exists between NSC lineages and CG. Loss of Neuroglian results in NSC lineages clumped together and in an altered CG network, while loss of Neurexin-IV/Wrapper generates larger yet defined CG chamber grouping several lineages together. Axonal projections of newborn neurons are also altered in these conditions. Further, the loss of these 2 adhesion complexes specifically during development is linked to locomotor hyperactivity in the resulting adults. Altogether, these findings identify a belt of adhesions building a neurogenic niche at the scale of individual stem cell and provide the proof of concept that niche properties during development shape adult behaviour.
Zhao, A., Nern, A., Koskela, S., Dreher, M., Erginkaya, M., Laughland, C. W., Ludwigh, H., Thomson, A., Hoeller, J., Parekh, R., Romani, S., Bock, D. D., Chiappe, E., Reiser, M. B. (2023). A comprehensive neuroanatomical survey of the Drosophila Lobula Plate Tangential Neurons with predictions for their optic flow sensitivity. bioRxiv, PubMed ID: 37904921
Summary:
Flying insects exhibit remarkable navigational abilities controlled by their compact nervous systems. Optic flow, the pattern of changes in the visual scene induced by locomotion, is a crucial sensory cue for robust self-motion estimation, especially during rapid flight. The best-known optic-flow sensitive neurons are the large tangential cells of the dipteran lobula plate. This study reports the comprehensive reconstruction and identification of the Lobula Plate Tangential Neurons in an Electron Microscopy (EM) volume of a whole Drosophila brain. This catalog of 58 LPT neurons (per brain hemisphere) contains many neurons that are described here for the first time and provides a basis for systematic investigation of the circuitry linking self-motion to locomotion control. Leveraging computational anatomy methods, it is estimated the visual motion receptive fields of these neurons and compared their tuning to the visual consequence of body rotations and translational movements. These neurons were matched, in most cases on a one-for-one basis, to stochastically labeled cells in genetic driver lines, to the mirror-symmetric neurons in the same EM brain volume, and to neurons in an additional EM data set. Using cell matches across data sets, the integration of optic flow patterns by neurons downstream of the LPTs was analyzed and most central brain neurons were found to establish sharper selectivity for global optic flow patterns than their input neurons. Furthermore, it was found that self-motion information extracted from optic flow is processed in distinct regions of the central brain, pointing to diverse foci for the generation of visual behaviors.
Jovanoski, K. D., Duquenoy, L., Mitchell, J., Kapoor, I., Treiber, C. D., Croset, V., Dempsey, G., Parepalli, S., Cognigni, P., Otto, N., Felsenberg, J., Waddell, S. (2023). Dopaminergic systems create reward seeking despite adverse consequences. Nature, 623(7986):356-365 PubMed ID: 37880370
Summary:
Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking. Although dysfunction of the dopaminergic valuation system of the brain is known to contribute towards unconstrained reward seeking, the underlying reasons for this behaviour are unclear. This study describes dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. It is proposed that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals.
Ganguly, I., Heckman, E. L., Litwin-Kumar, A., Clowney, E. J., Behnia, R. (2023). Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body. bioRxiv, PubMed ID: 37873086
Summary:
The arthropod mushroom body is well-studied as an expansion layer that represents olfactory stimuli and links them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their tuning and function are poorly understood. This study used the FlyWire adult whole-brain connectome to identify inputs to visual Kenyon cells. The types of visual neurons identified are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual projection neurons presynaptic to Kenyon cells receive input from large swathes of visual space, while local visual interneurons, providing smaller fractions of input, receive more spatially restricted signals that may be tuned to specific features of the visual scene. Like olfactory Kenyon cells, visual Kenyon cells receive sparse inputs from different combinations of visual channels, including inputs from multiple optic lobe neuropils. The sets of inputs to individual visual Kenyon cells are consistent with random sampling of available inputs. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the expansion coding properties appear different, with a specific repertoire of visual inputs projecting onto a relatively small number of visual Kenyon cells.
Wang, C. M., Wu, C. Y., Lin, C. E., Hsu, M. C., Lin, J. C., Huang, C. C., Lien, T. Y., Lin, H. K., Chang, T. W., Chiang, H. C. (2023). Forgotten memory storage and retrieval in Drosophila. Nat Commun, 14(1):7153 PubMed ID: 37935667
Summary:
Inaccessibility of stored memory in ensemble cells through the forgetting process causes animals to be unable to respond to natural recalling cues. While accumulating evidence has demonstrated that reactivating memory-stored cells can switch cells from an inaccessible state to an accessible form and lead to recall of previously learned information, the underlying cellular and molecular mechanisms remain elusive. The current study used Drosophila as a model to demonstrate that the memory of one-trial aversive olfactory conditioning, although inaccessible within a few hours after learning, is stored in KCαβ and retrievable after mild retraining. One-trial aversive conditioning triggers protein synthesis to form a long-lasting cellular memory trace, approximately 20 days, via creb in KCαβ, and a transient cellular memory trace, approximately one day, via orb in MBON-α3. PPL1-α3 negatively regulates forgotten one-trial conditioning memory retrieval. The current study demonstrated that KCαβ, PPL1-α3, and MBON-α3 collaboratively regulate the formation of forgotten one-cycle aversive conditioning memory formation and retrieval.
Yang, H. H., Brezovec, L. E., Capdevila, L. S., Vanderbeck, Q. X., Adachi, A., Mann, R. S., Wilson, R. I. (2023). Fine-grained descending control of steering in walking Drosophila. bioRxiv, PubMed ID: 37904997
Summary:
Locomotion involves rhythmic limb movement patterns that originate in circuits outside the brain. Purposeful locomotion requires descending commands from the brain, but it is not understood how these commands are structured. Focusing on the control of steering in walking Drosophila. First, different limb "gestures" associated with different steering maneuvers are described. Next, this study identified a set of descending neurons whose activity predicts steering. Focusing on two descending cell types downstream from distinct brain networks, this study shows that they evoke specific limb gestures: one lengthens strides on the outside of a turn, while the other attenuates strides on the inside of a turn. Notably, a single descending neuron can have opposite effects during different locomotor rhythm phases, and networks positioned to implement this phase-specific gating were identified. Together, these results show how purposeful locomotion emerges from brain cells that drive specific, coordinated modulations of low-level patterns.

Monday, May 13th - Transcriptional Regulation

Chaubal, A., Waldern, J. M., Taylor, C., Laederach, A., Marzluff, W. F., Duronio, R. J. (2023). Coordinated expression of replication-dependent histone genes from multiple loci promotes histone homeostasis in Drosophila. Mol Biol Cell, 34(12):ar118 PubMed ID: 37647143
Summary:
Production of large amounts of histone proteins during S phase is critical for proper chromatin formation and genome integrity. This process is achieved in part by the presence of multiple copies of replication dependent (RD) histone genes that occur in one or more clusters in metazoan genomes. In addition, RD histone gene clusters are associated with a specialized nuclear body, the histone locus body (HLB), which facilitates efficient transcription and 3' end-processing of RD histone mRNA. How all five RD histone genes within these clusters are coordinately regulated such that neither too few nor too many histones are produced, a process referred to as histone homeostasis, is not fully understood. This study explored the mechanisms of coordinate regulation between multiple RD histone loci in Drosophila melanogaster and Drosophila virilis. Evidence ia provided for functional competition between endogenous and ectopic transgenic histone arrays located at different chromosomal locations in D. melanogaster that helps maintain proper histone mRNA levels. Consistent with this model, in both species it was found that individual histone gene arrays can independently assemble an HLB that results in active histone transcription. These findings suggest a role for HLB assembly in coordinating RD histone gene expression to maintain histone homeostasis.
Khanbabei, A., Segura, L., Petrossian, C., Lemus, A., Cano, I., Frazier, C., Halajyan, A., Ca, D., Loza-Coll, M. (2024). Experimental validation and characterization of putative targets of Escargot and STAT, two master regulators of the intestinal stem cells in Drosophila melanogaster. Dev Biol, 505:148-163 PubMed ID: 37952851
Summary:
Many organs contain adult stem cells (ASCs) to replace cells due to damage, disease, or normal tissue turnover. ASCs can divide asymmetrically, giving rise to a new copy of themselves (self-renewal) and a sister that commits to a specific cell type (differentiation). Decades of research have led to the identification of pleiotropic genes whose loss or gain of function affect diverse aspects of normal ASC biology. Genome-wide screens of these so-called genetic "master regulator" (MR) genes, have pointed to hundreds of putative targets that could serve as their downstream effectors. This study experimentally validated and characterized the regulation of several putative targets of Escargot (Esg) and the Signal Transducer and Activator of Transcription (Stat92E, a.k.a. STAT), two known MRs in Drosophila intestinal stem cells (ISCs). The results indicate that regardless of bioinformatic predictions, most experimentally validated targets show a profile of gene expression that is consistent with co-regulation by both Esg and STAT, fitting a rather limited set of co-regulatory modalities. A bioinformatic analysis of proximal regulatory sequences in specific subsets of co-regulated targets identified additional transcription factors that might cooperate with Esg and STAT in modulating their transcription. Lastly, in vivo manipulations of validated targets rarely phenocopied the effects of manipulating Esg and STAT, suggesting the existence of complex genetic interactions among downstream targets of these two MR genes during ISC homeostasis.
Eggers, N., Gkountromichos, F., Krause, S., Campos-Sparr, A., Becker, P. B. (2023). Physical interaction between MSL2 and CLAMP assures direct cooperativity and prevents competition at composite binding sites. Nucleic Acids Res, 51(17):9039-9054 PubMed ID: 37602401
Summary:
MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. This study explored the nature of the cooperative binding to these GA-rich, composite sequence elements in reconstituted naive embryonic chromatin. The cooperativity was found to require physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. The cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in the reconstitution, such as roX2 lncRNA.
Aguilera, J., Duan, J., Lee, S. M., Ray, M., Larschan, E. (2023). The CLAMP GA-binding transcription factor regulates heat stress-induced transcriptional repression by associating with 3D loop anchors. bioRxiv, PubMed ID: 37873306
Summary:
In order to survive when exposed to heat stress (HS), organisms activate stress response genes and repress constitutive gene expression to prevent the accumulation of potentially toxic RNA and protein products. Although many studies have elucidated the mechanisms that drive HS-induced activation of stress response genes across species, little is known about repression mechanisms or how genes are targeted for activation versus repression context-specifically. The mechanisms of heat stress-regulated activation have been well-studied in Drosophila, in which the GA-binding transcription factor GAF is important for activating genes upon heat stress. This study shows that a functionally distinct GA-binding transcription factor (TF) protein, CLAMP (Chromatin-linked adaptor for MSL complex proteins), is essential for repressing constitutive genes upon heat stress but not activation of the canonical heat stress pathway. HS induces loss of CLAMP-associated 3D chromatin loop anchors associated with different combinations of GA-binding TFs prior to HS if a gene becomes repressed versus activated. Overall, this study demonstrated that CLAMP promotes repression of constitutive genes upon HS, and repression and activation are associated with the loss of CLAMP-associated 3D chromatin loops bound by different combinations of GA-binding TFs.
Ramalingam, V., Yu, X., Slaughter, B. D., Unruh, J. R., Brennan, K. J., Onyshchenko, A., Lange, J. J., Natarajan, M., Buck, M., Zeitlinger, J. (2023). Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development. Nat Commun, 14(1):5862 PubMed ID: 37735176
Summary:
While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-, a Drosophila zinc finger transcription factor that has one of the more than 25 different splice isoforms from the lolaI locus, this study shows that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.
Lovero, D., Porcelli, D., Giordano, L., Lo Giudice, C., Picardi, E., Pesole, G., Pignataro, E., Palazzo, A., Marsano, R. M. (2023). Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors. Biology, 12(8) PubMed ID: 37627011
Summary:
Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. This work analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. The conservation is highlighted of a short sequence in the genes encoding subunits of the V-ATPase complex and their assembly factors analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding knowledge of the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization.

Friday, May 10th - Adult physiology and metabolism

Vujnovic, A. F., Martinovic, L., S., Medija, M., Waldowski, R. A. (2023). Distinct and Dynamic Changes in the Temporal Profiles of Neurotransmitters in Drosophila melanogaster Brain following Volatilized Cocaine or Methamphetamine Administrations. Pharmaceuticals (Basel, Switzerland), 16(10) PubMed ID: 37895961
Summary:
Due to similarities in genetics, cellular response, and behavior, Drosophila is used as a model organism in addiction research. A well-described behavioral response examined in flies is the induced increase in locomotor activity after a single dose of volatilized cocaine (vCOC) and volatilized methamphetamine (vMETH), the sensitivity, and the escalation of the locomotor response after the repeated dose, the locomotor sensitization. However, knowledge about how vCOC and vMETH affect different neurotransmitter systems over time is scarce. This study used LC-MS/MS to systematically examine changes in the concentration of neurotransmitters, metabolites and non-metabolized COC and METH in the whole head homogenates of male flies one to seven hours after single and double vCOC or vMETH administrations. vMETH leads to complex changes in the levels of examined substances over time, while vCOC strongly and briefly increases concentrations of dopamine, tyramine and octopamine followed by a delayed degradation into N-acetyl dopamine and N-acetyl tyramine. The first exposure to psychostimulants leads to significant and dynamic changes in the concentrations relative to the second administration when they are more stable over several hours. Further investigations are needed to understand neurochemical and molecular changes post-psychostimulant administration.
Alassaf, M., Rajan, A. (2023). Diet-induced glial insulin resistance impairs the clearance of neuronal debris in Drosophila brain. PLoS Biol, 21(11):e3002359 PubMed ID: 37934726
Summary:
Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Using a Drosophila model this study shows that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, this study has established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris.
Brener, A., Lorber, D., Reuveny, A., Toledano, H., Porat-Kuperstein, L., Lebenthal, Y., Weizman, E., Olender, T., Volk, T. (2023). Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae. Cells, 12(19) PubMed ID: 37830547
Summary:
The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi(1)) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. The findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating these findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases.
Torre, M., Bukhari, H., Nithianandam, V., Zanella, C. A., Mata, D. A., Feany, M. B. (2023). A Drosophila model relevant to chemotherapy-related cognitive impairment. Sci Rep, 13(1):19290 PubMed ID: 37935827
Summary:
Chemotherapy-related cognitive impairment (CRCI) is a common adverse effect of treatment and is characterized by deficits involving multiple cognitive domains including memory. Despite the significant morbidity of CRCI and the expected increase in cancer survivors over the coming decades, the pathophysiology of CRCI remains incompletely understood, highlighting the need for new model systems to study CRCI. Given the powerful array of genetic approaches and facile high throughput screening ability in Drosophila, the goal of this study was to validate a Drosophila model relevant to CRCI. The chemotherapeutic agents cisplatin, cyclophosphamide, and doxorubicin were administered to adult Drosophila. Neurologic deficits were observed with all tested chemotherapies, with doxorubicin and in particular cisplatin also resulting in memory deficits. Histologic and immunohistochemical analysis was performed of cisplatin-treated Drosophila tissue, demonstrating neuropathologic evidence of increased neurodegeneration, DNA damage, and oxidative stress. Thus, the Drosophila model relevant to CRCI recapitulates clinical, radiologic, and histologic alterations reported in chemotherapy patients. This new Drosophila model can be used for mechanistic dissection of pathways contributing to CRCI (and chemotherapy-induced neurotoxicity more generally) and pharmacologic screens to identify disease-modifying therapies.
Brischigliaro, M., Cabrera-Orefice, A., Arnold, S., Viscomi, C., Zeviani, M., Fernandez-Vizarra, E. (2023). Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes. Elife, 12 PubMed ID: 37823874
Summary:
Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. This study show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, it is concluded that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis.
Li, J., Dang, P., Li, Z., Zhao, T., Cheng, D., Pan, D., Yuan, Y., Song, W. (2023). Peroxisomal ERK mediates Akh/glucagon action and glycemic control. Cell Rep, 42(10):113200 PubMed ID: 37796662
Summary:
he enhanced response of glucagon and its Drosophila homolog, Adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. This study found that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. Mammalian glucagon-induced hepatic ERK peroxisomal translocation was also observed in diabetic subjects. Therefore, these results conclude that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control.

Thursday, May 9th - Tumors, Cancer and Growth

Khalili, D., Mohammed, M., Kunc, M., Sindlerova, M., Ankarklev, J., Theopold, U. (2023). Single-cell sequencing of tumor-associated macrophages in a Drosophila model. Frontiers in immunology, 14:1243797 PubMed ID: 37795097
Summary:
Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized. This study used a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (Ras(V12)) to study dysplastic growth during early tumor progression. Single-cell RNA-sequencing was performed of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells. Five distinct hemocyte clusters were identified. In addition to Ras(V12) larvae, a tumor model was included where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells-they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors-antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, evidence is provided for transcript transfer between hemocytes and possibly other tissues. Taken together, these data support the usefulness of Drosophila to study the response against tumors at the organismic level.
Yu, K., Ramkumar, N., Wong, K. K. L., Tettweiler, G., Verheyen, E. M. (2023). The AMPK-like protein kinases Sik2 and Sik3 interact with Hipk and induce synergistic tumorigenesis in a Drosophila cancer model. Frontiers in cell and developmental biology, 11:1214539 PubMed ID: 37854071.
Summary:
Homeodomain-interacting protein kinases (Hipks) regulate cell proliferation, apoptosis, and tissue development. Overexpression of Hipk in Drosophila causes tumorigenic phenotypes in larval imaginal discs. This study found that depletion of Salt-inducible kinases Sik2 or Sik3 can suppress Hipk-induced overgrowth. Furthermore, co-expression of constitutively active forms of Sik2 or Sik3 with Hipk caused significant tissue hyperplasia and tissue distortion, indicating that both Sik2 and Sik3 can synergize with Hipk to promote tumorous phenotypes, accompanied by elevated dMyc, Armadillo/β-catenin, and the Yorkie target gene expanded. Larvae expressing these hyperplastic growths also display an extended larval phase, characteristic of other Drosophila tumour models. Examination of total protein levels from fly tissues showed that Hipk proteins were reduced when Siks were depleted through RNAi, suggesting that Siks may regulate Hipk protein stability and/or activity. Conversely, expression of constitutively active Siks with Hipk leads to increased Hipk protein levels. Furthermore, Hipk can interact with Sik2 and Sik3 by co-immunoprecipitation. Co-expression of both proteins leads to a mobility shift of Hipk protein, suggesting it is post-translationally modified. In summary, this research demonstrates a novel function of Siks in synergizing with Hipk to promote tumour growth.
Bosch, P. S., Cho, B., Axelrod, J. D. (2023). Flamingo participates in multiple models of cell competition. bioRxiv, PubMed ID: 37790459
Summary:
The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. This study shows that in Drosophila, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in pre-malignant cells as they grow among wildtype cells, in healthy cells to eliminate pre-malignant cells, and by supercompetitors to occupy excessive territory within wildtype tissues. "Would-be" winners that lack Fmi are unable to over-proliferate, and instead become losers. This study demonstrates that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well defined functions of Fmi.
Pranoto, I. K. A., Lee, J., Kwon, Y. V. (2023). The roles of the native cell differentiation program aberrantly recapitulated in Drosophila intestinal tumors. Cell Rep, 42(10):113245 PubMed ID: 37837622ID:
Summary:
Many tumors recapitulate the developmental and differentiation program of their tissue of origin, a basis for tumor cell heterogeneity. Although stem-cell-like tumor cells are well studied, the roles of tumor cells undergoing differentiation remain to be elucidated. This study employ Drosophila genetics to demonstrate that the differentiation program of intestinal stem cells is crucial for enabling intestinal tumors to invade and induce non-tumor-autonomous phenotypes. The differentiation program that generates absorptive cells is aberrantly recapitulated in the intestinal tumors generated by activation of the Yap1 ortholog Yorkie. Inhibiting it allows stem-cell-like tumor cells to grow but suppresses invasiveness and reshapes various phenotypes associated with cachexia-like wasting by altering the expression of tumor-derived factors. This study provides insight into how a native differentiation program determines a tumor's capacity to induce advanced cancer phenotypes and suggests that manipulating the differentiation programs co-opted in tumors might alleviate complications of cancer, including cachexia.
Zheng, J., Guo, Y., Shi, C., Yang, S., Xu, W., Ma, X. (2023). Differential Ire1 determines loser cell fate in tumor-suppressive cell competition. Cell Rep, 42(11):113303 PubMed ID: 37924514
Summary:
Tumor-suppressive cell competition (TSCC) is a conserved surveillance mechanism in which neighboring cells actively eliminate oncogenic cells. Despite overwhelming studies showing that the unfolded protein response (UPR) is dysregulated in various tumors, it remains debatable whether the UPR restrains or promotes tumorigenesis. Using Drosophila eye epithelium as a model, this study uncovered a surprising decisive role of the Ire1 branch of the UPR in regulating cell polarity gene scribble (scrib) loss-induced TSCC. Both mutation and hyperactivation of Ire1 accelerate elimination of scrib clones via inducing apoptosis and autophagy, respectively. Unexpectedly, relative Ire1 activity is also crucial for determining loser cell fate, as dysregulating Ire1 signaling in the surrounding healthy cells reversed the "loser" status of scrib clones by decreasing their apoptosis. Furthermore, it was shown that Ire1 is required for cell competition in mammalian cells. Together, these findings provide molecular insights into scrib-mediated TSCC and highlight Ire1 as a key determinant of loser cell fate.
Quintero, M., Bangi, E. (2023). Disruptions in cell fate decisions and transformed enteroendocrine cells drive intestinal tumorigenesis in Drosophila. Cell Rep, 42(11):113370 PubMed ID: 37924517
Summary:
Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between different cell types ensures precise regulation of stem cell behavior and cell fate decisions. These cell-cell interactions are often disrupted during tumorigenesis, but mechanisms by which they are co-opted to support tumor growth in different genetic contexts are poorly understood. This study introduced PromoterSwitch, a genetic platform established to generate large, transformed clones derived from individual adult Drosophila intestinal stem/progenitor cells. Cancer-driving genetic alterations representing common colon tumor genome landscapes were shown to disrupt cell fate decisions within transformed tissue and result in the emergence of abnormal cell fates. It was also shown that transformed enteroendocrine cells, a differentiated, hormone-secreting cell lineage, support tumor growth by regulating intestinal stem cell proliferation through multiple genotype-dependent mechanisms, which represent potential vulnerabilities that could be exploited for therapy.

Wednesday, May 8th - Drosophila as a model for human diseases

Min, Y., Wang, X., Is, O., Patel, T. A., Gao, J., Reddy, J. S., Quicksall, Z. S., Nguyen, T., Lin, S., Tutor-New, F. Q., Chalk, J. L., Mitchell, A. O., Crook, J. E., Nelson, P. T., Van Eldik, L. J., Golde, T. E., Carrasquillo, M. M., Dickson, D. W., Zhang, K., Allen, M., Ertekin-Taner, N. (2023). Cross species systems biology discovers glial DDR2, STOM, and KANK2 as therapeutic targets in progressive supranuclear palsy. Nat Commun, 14(1):6801 PubMed ID: 37919278
Summary:
Progressive supranuclear palsy (PSP) is a neurodegenerative parkinsonian disorder characterized by cell-type-specific tau lesions in neurons and glia. Prior work uncovered transcriptome changes in human PSP brains, although their cell-specificity is unknown. Further, systematic data integration and experimental validation platforms to prioritize brain transcriptional perturbations as therapeutic targets in PSP are currently lacking. This study combined bulk tissue (n = 408) and single nucleus RNAseq (n = 34) data from PSP and control brains with transcriptome data from a mouse tauopathy and experimental validations in Drosophila tau models for systematic discovery of high-confidence expression changes in PSP with therapeutic potential. Thousands of differentially expressed genes were discovered, replicated, and annotated in PSP, many of which reside in glia-enriched co-expression modules and cells. DDR2, STOM, and KANK2 were were prioritized as promising therapeutic targets in PSP with striking cross-species validations. These findings and data are shared via an interactive application tool PSP RNAseq Atlas. The findings reveal robust glial transcriptome changes in PSP, provide a cross-species systems biology approach, and a tool for therapeutic target discoveries in PSP with potential application in other neurodegenerative diseases.
Yan, L., Zhou, J., Yuan, L., Ye, J., Zhao, X., Ren, G., Chen, H. (2023). Silibinin alleviates intestinal inflammation via inhibiting JNK signaling in Drosophila. Frontiers in pharmacology, 14:1246960 PubMed ID: 37781701
Summary:
Inflammatory bowel diseases (IBDs) are characterized by chronic relapsing intestinal inflammation that causes digestive system dysfunction. For years, researchers have been working to find more effective and safer therapeutic strategies to treat these diseases. Silibinin (SIL), a flavonoid compound extracted from the seeds of milk thistle plants, possesses multiple biological activities and is traditionally applied to treat liver diseases. SIL is also widely used in the treatment of a variety of inflammatory diseases attributed to its excellent antioxidant and anti-inflammatory effects. However, the efficacy of SIL against IBDs and its mechanisms remain unclear. This study, using Drosophila melanogaster as a model organism, found that SIL can effectively relieve intestinal inflammation caused by dextran sulfate sodium (DSS). The results suggested that SIL supplementation can inhibit the overproliferation of intestinal stem cells (ISCs) induced by DSS, protect intestinal barrier function, acid-base balance, and intestinal excretion function, reduce intestinal reactive oxygen species (ROS) levels and inflammatory stress, and extend the lifespan of Drosophila. Furthermore, this study demonstrated that SIL ameliorates intestinal inflammation via modulating the c-Jun N-terminal kinase (JNK) signaling pathway in Drosophila. This research aims to provide new insight into the treatment of IBDs.
Aalto, A. L., Saadabadi, A., Lindholm, F., Kietz, C., Himmelroos, E., Marimuthu, P., Salo-Ahen, O. M. H., Eklund, P., Meinander, A. (2023). Stilbenoid compounds inhibit NF-κB-mediated inflammatory responses in the Drosophila intestine. Frontiers in immunology, 14:1253805 PubMed ID: 37809071
Summary:
Stilbenoid compounds have been described to have anti-inflammatory properties in animal models in vivo, and have been shown to inhibit Ca2+-influx through the transient receptor potential ankyrin 1 (TrpA1). To study how stilbenoid compounds affect inflammatory signaling in vivo, the fruit fly, Drosophila melanogaster, was used as a model system. To induce intestinal inflammation in the fly, flies were fed with the intestinal irritant dextran sodium sulphate (DSS). DSS was found to induce severe changes in the bacteriome of the Drosophila intestine, and that this dysbiosis causes activation of the NF-κB transcription factor Relish. The DSS-model was used to study the anti-inflammatory properties of the stilbenoid compounds pinosylvin (PS) and pinosylvin monomethyl ether (PSMME). With the help of in vivo approaches, PS and PSMME were identified as transient receptor ankyrin 1 (TrpA1)-dependent antagonists of NF-κB-mediated intestinal immune responses in Drosophila were computationally predicted. The putative antagonist binding sites of these compounds at Drosophila TrpA1. Taken together, this study showed that the stilbenoids PS and PSMME have anti-inflammatory properties in vivo in the intestine and can be used to alleviate chemically induced intestinal inflammation in Drosophila.
Wang, C. W., Clemot, M., Hashimoto, T., Diaz, J. A., Goins, L. M., Goldstein, A. S., Nagaraj, R., Banerjee, U. (2023). A conserved mechanism for JNK-mediated loss of Notch function in advanced prostate cancer. Science signaling, 16(810):eabo5213 PubMed ID: 37934809
Summary:
Dysregulated Notch signaling is a common feature of cancer; however, its effects on tumor initiation and progression are highly variable, with Notch having either oncogenic or tumor-suppressive functions in various cancers. To better understand the mechanisms that regulate Notch function in cancer, Notch signaling was studied in a Drosophila tumor model, prostate cancer-derived cell lines, and tissue samples from patients with advanced prostate cancer. It was demonstrated that increased activity of the Src-JNK pathway in tumors inactivated Notch signaling because of JNK pathway-mediated inhibition of the expression of the gene encoding the Notch S2 cleavage protease, Kuzbanian, which is critical for Notch activity. Consequently, inactive Notch accumulated in cells, where it was unable to transcribe genes encoding its target proteins, many of which have tumor-suppressive activities. These findings suggest that Src-JNK activity in tumors predicts Notch activity status and that suppressing Src-JNK signaling could restore Notch function in tumors, offering opportunities for diagnosis and targeted therapies for a subset of patients with advanced prostate cancer.
Diaw, S. H., Borsche, M., Streubel-Gallasch, L., Dulovic-Mahlow, M., Hermes, J., Lenz, I., Seibler, P., Klein, C., Bruggemann, N., Vos, M., Lohmann, K. (2023). Characterization of the pathogenic alpha-Synuclein Variant V15A in Parkinson´s disease. NPJ Parkinson's disease, 9(1):148 PubMed ID: 37903765
Summary:
Despite being a major component of Lewy bodies and Lewy neurites, pathogenic variants in the gene encoding alpha-Synuclein (α-Syn) are rare. To date, only four missense variants in the SNCA gene, encoding α-Syn have unequivocally been shown to be disease-causing. This study describes a Parkinson´s disease patient with early cognitive decline carrying an as yet not fully characterized variant in SNCA (NM_001146055: c.44T > C, p.V15A). Different cellular models, including stably transfected neuroblastoma (SH-SY5Y) cell cultures, induced pluripotent stem cell (iPSC)-derived neuronal cultures, and a Drosophila model was generated to elucidate the impact of the p.V15A variant on α-Syn function and aggregation properties compared to other known pathogenic variants. This study demonstrated that p.V15A increased the aggregation potential of α-Syn and the levels of apoptotic markers, and impaired the mitochondrial network. Moreover, p.V15A affects the flying ability and survival of mutant flies. Thus, this study provides supporting evidence for the pathogenicity of the p.V15A variant, suggesting its inclusion in genetic testing approaches.
Zane, F., Bouzid, H., Sosa Marmol, S., Brazane, M., Besse, S., Molina, J. L., Cansell, C., Aprahamian, F., Durand, S., Ayache, J., Antoniewski, C., Todd, N., Carre, C., Rera, M. (2023). Smurfness-based two-phase model of ageing helps deconvolve the ageing transcriptional signature. Aging Cell, 22(11):e13946 PubMed ID: 37822253
Summary:
Ageing is characterised at the molecular level by six transcriptional 'hallmarks of ageing', that are commonly described as progressively affected as time passes. By contrast, the 'Smurf' assay, which assesses food intake by the co-ingestion of a blue dye, which is not absorbed by the digestive tract, separates high-and-constant-mortality risk individuals from healthy, zero-mortality risk individuals, based on increased intestinal permeability. Performing whole body total RNA sequencing, it was found that Smurfness distinguishes transcriptional changes associated with chronological age from those associated with biological age. Transcriptional heterogeneity is shown to increase with chronological age in non-Smurf individuals preceding the other five hallmarks of ageing that are specifically associated with the Smurf state. Using this approach, targeted pro-longevity genetic interventions delaying entry in the Smurf state were devised. It is anticipated that increased attention to the evolutionary conserved Smurf phenotype will bring about significant advances in understanding of the mechanisms of ageing.

Tuesday, May 7th - Embryonic Neural Development

Mitchell, J. W., Midillioglu, I., Schauer, E., Wang, B., Han, C., Wildonger, J. (2023). Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons. PLoS Genet, 19(11):e1011025 PubMed ID: 37943859
Summary:
Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. This study investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. CRISPR-Cas9 genome engineering approaches were used to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, it was found that an over-active channel mutant severely reduces dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. These data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on these data, it is proposed that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites.
Hsiao, Y. L., Chen, H. W., Chen, K. H., Tan, B. C., Chen, C. H. and Pi, H. (2023). Actin-related protein 6 facilitates proneural protein-induced gene activation for rapid neural differentiation. Development 150(5). PubMed ID: 36897355
Summary:
Neurogenesis is initiated by basic helix-loop-helix proneural proteins. This study showed that Actin-related protein 6 (Arp6), a core component of the H2A.Z exchange complex SWR1, interacts with proneural proteins and is crucial for efficient onset of proneural protein target gene expression. Arp6 mutants exhibit reduced transcription in sensory organ precursors (SOPs) downstream of the proneural protein patterning event. This leads to retarded differentiation and division of SOPs and smaller sensory organs. These phenotypes are also observed in proneural gene hypomorphic mutants. Hypomorphic ac sc mutant recapitulates Arp6 mutant phenotypes. Arp6 also interacted with Sc and Atonal (Ato) but failed to interact with the proneural protein heterodimeric partner Daughterless. Proneural protein expression is not reduced in Arp6 mutants. Enhanced proneural gene expression fails to rescue retarded differentiation in Arp6 mutants, suggesting that Arp6 acts downstream of or in parallel with proneural proteins. H2A.Z mutants display Arp6-like retardation in SOPs. Transcriptomic analyses demonstrate that loss of Arp6 and H2A.Z preferentially decreases expression of proneural protein-activated genes. H2A.Z enrichment in nucleosomes around the transcription start site before neurogenesis correlates highly with greater activation of proneural protein target genes by H2A.Z. It is proposed that upon proneural protein binding to E-box sites, H2A.Z incorporation around the transcription start site allows rapid and efficient activation of target genes, promoting rapid neural differentiation.
Singh, B. N., Tran, H., Kramer, J., Kirishenko, E., Changela, N., Wang, F., Feng, Y., Kumar, D., Tu, M., Lan, J., Bizet, M., Fuks, F. and Steward, R. (2023). Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates the axon guidance genes robo2 and slit in Drosophila. Res Sq. PubMed ID: 36824980
Summary:
Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases enzymes catalyzing the transition of 5mC to 5hmC in DNA and have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila because Tet knock-out animals do not reach adulthood. The identification is described of Tet-target genes in the embryo and larval brain by determining Tet DNA-binding sites throughout the genome and by mapping the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC-modified sites can be found along the entire transcript and are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are frequently involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and are sensitized to reduced levels of slit. Both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. These results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs, primarily in developing nerve cells.
Sullivan, K. G., Bashaw, G. J. (2023). Commissureless acts as a substrate adapter in a conserved Nedd4 E3 ubiquitin ligase pathway to promote axon growth across the midline. bioRxiv. PubMed ID: 37905056
Summary:
In both vertebrates and invertebrates, commissural neurons prevent premature responsiveness to the midline repellant Slit by downregulating surface levels of its receptor Roundabout1 (Robo1). In Drosophila, Commissureless (Comm) plays a critical role in this process; however, there is conflicting data on the underlying molecular mechanism. This study demonstrated that the conserved PY motifs in the cytoplasmic domain of Comm are required allow the ubiquitination and lysosomal degradation of Robo1. Disruption of these motifs prevents Comm from localizing to Lamp1 positive late endosomes and to promote axon growth across the midline in vivo. In addition, a role for Nedd4 in midline crossing was identified. Genetic analysis shows that nedd4 mutations result in midline crossing defects in the Drosophila embryonic nerve cord, which can be rescued by introduction of exogenous Nedd4. Biochemical evidence shows that Nedd4 incorporates into a three-member complex with Comm and Robo in a PY motif-dependent manner. Finally, genetic evidence is presented that Nedd4 acts with Comm in the embryonic nerve cord to downregulate Robo1 levels. Taken together, these findings demonstrate that Comm promotes midline crossing in the nerve cord by facilitating Robo ubiquitination by Nedd4, ultimately leading to its degradation.
Carranza, A., Howard, L. J., Brown, H. E., Ametepe, A. S. and Evans, T. A. (2023). Slit-independent guidance of longitudinal axons by Drosophila Robo3. bioRxiv. PubMed ID: 37214810
Summary:
Drosophila Robo3 is a member of the evolutionarily conserved Roundabout (Robo) receptor family and one of three Drosophila Robo paralogs. During embryonic ventral nerve cord development, Robo3 does not participate in canonical Slit-dependent midline repulsion, but instead regulates the formation of longitudinal axon pathways at specific positions along the medial-lateral axis. Longitudinal axon guidance by Robo3 is hypothesized to be Slit dependent, but this has not been directly tested. In this study a series of Robo3 variants was created in which the N-terminal Ig1 domain is deleted or modified, in order to characterize the functional importance of Ig1 and Slit binding for Robo3's axon guidance activity. Robo3 is shown to require its Ig1 domain for interaction with Slit and for proper axonal localization in embryonic neurons, but deleting Ig1 from Robo3 only partially disrupts longitudinal pathway formation. Robo3 variants with modified Ig1 domains that cannot bind Slit retain proper localization and fully rescue longitudinal axon guidance. These results indicate that Robo3 guides longitudinal axons independently of Slit, and that sequences both within and outside of Ig1 contribute to this Slit-independent activity.
Karkali, K., Saunders, T. E., Panayotou, G. and Martín-Blanco, E. (2023). JNK signaling in pioneer neurons organizes ventral nerve cord architecture in Drosophila embryos. Nat Commun 14(1): 675. PubMed ID: 36750572
Summary:
Morphogenesis of the Central Nervous System (CNS) is a complex process that obeys precise architectural rules. Yet, the mechanisms dictating these rules remain unknown. Analyzing morphogenesis of the Drosophila embryo Ventral Nerve Cord (VNC), this study observe that a tight control of JNK signaling is essential for attaining the final VNC architecture. JNK signaling in a specific subset of pioneer neurons autonomously regulates the expression of Fasciclin 2 (Fas 2) and Neurexin IV (Nrx IV) adhesion molecules, probably via the transcription factor zfh1. Interfering at any step in this cascade affects fasciculation along pioneer axons, leading to secondary cumulative scaffolding defects during the structural organization of the axonal network. The global disorder of architectural landmarks ultimately influences nervous system condensation. In summary, these data point to JNK signaling in a subset of pioneer neurons as a key element underpinning VNC architecture, revealing critical milestones on the mechanism of control of its structural organization.

Monday, May 6th - Cell Cycle

Haseeb, M. A., Weng, K. A., Bickel, S. E. (2023). Chromatin-associated cohesin turns over extensively and forms new cohesive linkages during meiotic prophase. bioRxiv, PubMed ID: 37645916
Summary:
In dividing cells, accurate chromosome segregation depends on sister chromatid cohesion, protein linkages that are established during DNA replication. Faithful chromosome segregation in oocytes requires that cohesion, first established in S phase, remain intact for days to decades, depending on the organism. Premature loss of meiotic cohesion in oocytes leads to the production of aneuploid gametes and contributes to the increased incidence of meiotic segregation errors as women age (maternal age effect). The prevailing model is that cohesive linkages do not turn over in mammalian oocytes. However, it has been reported that cohesion-related defects arise in Drosophila oocytes when individual cohesin subunits (see Verthandi) or cohesin regulators are knocked down after meiotic S phase. This study used two strategies to express a tagged cohesin subunit exclusively during mid-prophase in Drosophila oocytes and demonstrate that newly expressed cohesin is used to form de novo linkages after meiotic S phase. Moreover, nearly complete turnover of chromosome-associated cohesin occurs during meiotic prophase, with faster replacement on the arms than at the centromeres. Unlike S-phase cohesion establishment, the formation of new cohesive linkages during meiotic prophase does not require acetylation of conserved lysines within the Smc3 head. These findings indicate that maintenance of cohesion between S phase and chromosome segregation in Drosophila oocytes requires an active cohesion rejuvenation program that generates new cohesive linkages during meiotic prophase.
Koury, S. A. (2023). Female meiotic drive shapes the distribution of rare inversion polymorphisms in Drosophila melanogaster. Genetics. PubMed ID: 37616566
Summary:
In all species, new chromosomal inversions are constantly being formed by spontaneous rearrangement and then stochastically eliminated from natural populations. In Drosophila, when new chromosomal inversions overlap with a pre-existing inversion in the population, their rate of elimination becomes a function of the relative size, position, and linkage phase of the gene rearrangements. These altered dynamics result from complex meiotic behavior wherein overlapping inversions generate asymmetric dyads that cause both meiotic drive/drag and segmental aneuploidy. In this context, patterns in rare inversion polymorphisms of a natural population can be modeled from the fundamental genetic processes of forming asymmetric dyads via crossing-over in meiosis I and preferential segregation from asymmetric dyads in meiosis II. A mathematical model was developed of crossover-dependent female meiotic drive and was parameterized with published experimental data from Drosophila melanogaster laboratory constructs. This mechanism is demonstrated to favor smaller, distal inversions and accelerate the elimination of larger, proximal inversions. Simulated sampling experiments indicate that the paracentric inversions directly observed in natural population surveys of Drosophila melanogaster are a biased subset that both maximizes meiotic drive and minimizes the frequency of lethal zygotes caused by this cytogenetic mechanism. Incorporating this form of selection into a population genetic model accurately predicts the shift in relative size, position, and linkage phase for rare inversions found in this species. The model and analysis presented in this study suggest that this weak form of female meiotic drive is an important process influencing the genomic distribution of rare inversion polymorphisms.
Warecki, B. and Tao, L. (2023). Centralspindlin-mediated transport of RhoGEF positions the cleavage plane for cytokinesis. Sci Signal 16(792): eadh0601. PubMed ID: 37402224
Summary:
During cytokinesis, the cell membrane furrows inward along a cleavage plane. The positioning of the cleavage plane is critical to faithful cell division and is determined by the Rho guanine nucleotide exchange factor (RhoGEF)-mediated activation of the small guanosine triphosphatase RhoA and the conserved motor protein complex centralspindlin. This study explored whether and how centralspindlin mediates the positioning of RhoGEF. In dividing neuroblasts from Drosophila melanogaster, immediately before cleavage, first centralspindlin and then RhoGEF localized to the sites where cleavage subsequently initiated. Using in vitro assays with purified Drosophila proteins and stabilized microtubules, it was found that centralspindlin directly transported RhoGEF as cargo along single microtubules and sequestered it at microtubule plus-ends for prolonged periods of time. In addition, the binding of RhoGEF to centralspindlin appeared to stimulate centralspindlin motor activity. Thus, the motor activity and microtubule association of centralspindlin can translocate RhoGEF to areas where microtubule plus-ends are abundant, such as at overlapping astral microtubules, to locally activate RhoA and accurately position the cleavage plane during cell division.
Baker, C. C., Gallicchio, L., Matias, N. R., Porter, D. F., Parsanian, L., Taing, E., Tam, C., Fuller, M. T. (2023). Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase. Development, 150(22) PubMed ID: 37882771
Summary:
During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. This study shows that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.
Sperling, A. L., Fabian, D. K., Garrison, E. and Glover, D. M. (2023). A genetic basis for facultative parthenogenesis in Drosophila. Curr Biol. PubMed ID: 37516115
Summary:
Facultative parthenogenesis enables sexually reproducing organisms to switch between sexual and asexual parthenogenetic reproduction. To gain insights into this phenomenon, the genomes of sexually reproducing and parthenogenetic strains of Drosophila mercatorum were sequenced, and differences were identified in the gene expression in their eggs. Then whether manipulating the expression of candidate gene homologs identified in Drosophila mercatorum could lead to facultative parthenogenesis in the non-parthenogenetic species Drosophila melanogaster was tested. This identified a polygenic system whereby increased expression of the mitotic protein kinase polo and decreased expression of a desaturase, Desat2, caused facultative parthenogenesis in the non-parthenogenetic species that was enhanced by increased expression of Myc. The genetically induced parthenogenetic Drosophila melanogaster eggs exhibit de novo centrosome formation, fusion of the meiotic products, and the onset of development to generate predominantly triploid offspring. Thus, this study demonstrated a genetic basis for sporadic facultative parthenogenesis in an animal.
Bakshi, A., Iturra, F. E., Alamban, A., Rosas-Salvans, M., Dumont, S., Aydogan, M. G. (2023). Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes. Cell, 186(21):4694-4709. PubMed ID: 37832525
Summary:
Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. This study demonstrates in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, evidence is presented that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. It is postulated that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

Friday, May 3rd - RNAs and Transposons

Krzywinska, E., Ribeca, P., Ferretti, L., Hammond, A., Krzywinski, J. (2023). A novel factor modulating X chromosome dosage compensation in Anopheles. Curr Biol, 33(21):4697-4703. PubMed ID: 37774706
Summary:
Dosage compensation (DC), a process countering chromosomal imbalance in individuals with heteromorphic sex chromosomes, has been molecularly characterized only in mammals, Caenorhabditis elegans, and fruit flies. In Drosophila melanogaster males, it is achieved by an approximately 2-fold hypertranscription of the monosomic X chromosome mediated by the MSL complex. The complex is not assembled on female X chromosomes because production of its key protein MSL-2 is prevented due to intron retention and inhibition of translation by Sex-lethal, a female-specific protein operating at the top of the sex determination pathway. It remains unclear how DC is mechanistically regulated in other insects. In the malaria mosquito Anopheles gambiae, an approximately 2-fold hypertranscription of the male X also occurs by a yet-unknown molecular mechanism distinct from that in D. melanogaster. This study shows that a male-specifically spliced gene called 007, which arose by a tandem duplication in the Anopheles ancestral lineage, is involved in the control of DC in males. Homozygous 007 knockouts lead to a global downregulation of the male X, phenotypically manifested by a slower development compared to wild-type mosquitoes or mutant females-however, without loss of viability or fertility. In females, a 007 intron retention promoted by the sex determination protein Femaleless, known to prevent hypertranscription from both X chromosomes, introduces a premature termination codon apparently rendering the female transcripts non-productive. In addition to providing a unique perspective on DC evolution, the 007, with its conserved properties, may represent an important addition to a genetic toolbox for malaria vector control.
Yushkova, E. (2024). Interaction effect of mutations in the genes (piwi and aub) of the Argonaute family and hobo transposons on the integral survival parameters of Drosophila melanogaster. Biogerontology, 25(1):131-146 PubMed ID: 37864608
Summary:
The Argonaute family genes (piwi and aub) involved in the production of small RNAs are responsible for the regulation of many cellular processes, including the suppression of genome instability, modulation of gene activity, and transposable elements. Dysfunction of these genes and the associated activation of transposable elements adversely affect reproductive development and quality of life. The role of transposons in contrast to retrotransposons and their interaction with genes of the Argonaute family in aging processes have not been studied. This study considers a scenario in which the piwi and aub genes in the presence of functional hobo transposons can modify the effects from the level of DNA damage to lifespan. The simultaneous presence of mutation (piwi or aub) and hobo (regardless of size) in the genome has practically no effect or (less often) leads to a decrease in the level of DNA damage in ovarian cells. A high level of sterility and low ovarian reserve were noted mainly with a combination of mutations and full-sized hobo elements. The combination of these two genetic factors negatively affects the fertility of young females and embryonic survival. Isolated cases of restoration of reproductive functions with age were noted but only in females that had low fertility in the early period of life. The presence of hobo transposons contributed to an increase in the lifespan of both mutant and non-mutant females. Dysfunction of the piwi and aub genes (without hobo) can reduce the lifespan of both sexes. Together, each mutation and hobo transposons act antagonistically/additively (in females) and synergistically/antagonistically (in males) to change the lifespan. In parameters of locus-specific instability, hobo activation was more pronounced in piwi gene dysfunction. The results obtained complement data on the study of new functions of Argonaute family genes and their interactions with transposable elements in the aging process.
Kour, S., Fortuna, T., Anderson, E. N., Mawrie, D., Bilstein, J., Sivasubramanian, R., Ward, C., Roy, R., Rajasundaram, D., Sterneckert, J., Pandey, U. B. (2023). Drosha-dependent microRNAs modulate FUS-mediated neurodegeneration in vivo. Nucleic Acids Res, 51(20):11258-11276 PubMed ID: 37791873
Summary:
Mutations in the Fused in Sarcoma (FUS) gene cause the familial and progressive form of amyotrophic lateral sclerosis (ALS). FUS is a nuclear RNA-binding protein involved in RNA processing and the biogenesis of a specific set of microRNAs. This study reports that Drosha and two previously uncharacterized Drosha-dependent miRNAs are strong modulators of FUS expression and prevent the cytoplasmic segregation of insoluble mutant FUS in vivo. Depletion of Drosha mitigates FUS-mediated degeneration, survival and motor defects in Drosophila. Mutant FUS strongly interacts with Drosha and causes its cytoplasmic mis-localization into the insoluble FUS inclusions. Reduction in Drosha levels increases the solubility of mutant FUS. Interestingly, two Drosha dependent microRNAs, miR-378i and miR-6832-5p, which differentially regulate the expression, solubility and cytoplasmic aggregation of mutant FUS in iPSC neurons and mammalian cells. More importantly, different modes of action are reported of these miRNAs against mutant FUS. Whereas miR-378i may regulate mutant FUS inclusions by preventing G3BP-mediated stress granule formation, miR-6832-5p may affect FUS expression via other proteins or pathways. Overall, this research reveals a possible association between ALS-linked FUS mutations and the Drosha-dependent miRNA regulatory circuit, as well as a useful perspective on potential ALS treatment via microRNAs.
Liu, M., Xie, X. J., Li, X., Ren, X., Sun, J., Lin, Z., Hemba-Waduge, R. U., Ji, J. Y. (2023). Transcriptional coupling of telomeric retrotransposons with the cell cycle. bioRxiv, PubMed ID: 37808851
Summary:
Instead of employing telomerases to safeguard chromosome ends, dipteran species maintain their telomeres by transposition of telomeric-specific retrotransposons (TRs): in Drosophila , these are HeT-A, TART, and TAHRE. Previous studies have shown how these TRs create tandem repeats at chromosome ends, but the exact mechanism controlling TR transcription has remained unclear. This study reports the identification of multiple subunits of the transcription cofactor Mediator complex and transcriptional factors Scalloped (Sd, the TEAD homolog in flies) and E2F1-Dp as novel regulators of TR transcription and telomere length in Drosophila. Depletion of multiple Mediator subunits, Dp, or Sd increased TR expression and telomere length, while over-expressing E2F1-Dp or knocking down the E2F1 regulator Rbf1 (Retinoblastoma-family protein 1) stimulated TR transcription, with Mediator and Sd affecting TR expression through E2F1-Dp. The CUT&RUN analysis revealed direct binding of CDK8, Dp, and Sd to telomeric repeats. These findings highlight the essential role of the Mediator complex in maintaining telomere homeostasis by regulating TR transcription through E2F1-Dp and Sd, revealing the intricate coupling of TR transcription with the host cell-cycle machinery, thereby ensuring chromosome end protection and genomic stability during cell division.
Matzkin, L. M., Bono, J. M., Pigage, H. K., Allan, C. W., Diaz, F., McCoy, J. R., Green, C. C., Callan, J. B., Delahunt, S. P. (2023). Females translate male mRNA transferred during mating. bioRxiv, PubMed ID: 37790342
Summary:
Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. This study developed an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. Strong evidence is found for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction.
Coronado-Zamora, M., Gonzalez, J. (2023). Transposons contribute to the functional diversification of the head, gut, and ovary transcriptomes across Drosophila natural strains. Genome research, 33(9):1541-1553 PubMed ID: 37793782
Summary:
Transcriptomes are dynamic, with cells, tissues, and body parts expressing particular sets of transcripts. Transposable elements (TEs) are a known source of transcriptome diversity; however, studies often focus on a particular type of chimeric transcript, analyze single body parts or cell types, or are based on incomplete TE annotations from a single reference genome. This work has implemented a method based on de novo transcriptome assembly that minimizes the potential sources of errors while identifying a comprehensive set of gene-TE chimeras. This method was applied to the head, gut, and ovary dissected from five Drosophila melanogaster natural strains, with individual reference genomes available. ~19% of body part-specific transcripts were found to be gene-TE chimeras. Overall, chimeric transcripts contribute a mean of 43% to the total gene expression, and they provide protein domains for DNA binding, catalytic activity, and DNA polymerase activity. This comprehensive data set is a rich resource for follow-up analysis. Moreover, because TEs are present in virtually all species sequenced to date, their role in spatially restricted transcript expression is likely not exclusive to the species analyzed in this work.

Thursday, May 2nd - Enzyme and protein expression, evolution, structure, and function

Yoon, H. J., Price, B. E., Parks, R. K., Ahn, S. J., Choi, M. Y. (2023). Diuretic hormone 31 activates two G protein-coupled receptors with differential second messengers for diuresis in Drosophila suzukii. Insect biochemistry and molecular biology, 162:104025 PubMed ID: 37813200
Summary:
Diuretic hormones (DHs) bind to G protein-coupled receptors (GPCRs), regulating water and ion balance to maintain homeostasis in animals. Two distinct DHs are known in insects: calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. This study identified and characterized DH31 and two DH31 GPCR variants, DH31-Ra and DH31-Rb, from spotted-wing drosophila, Drosophila suzukii, a globally prevalent vinegar fly causing severe damage to small fruits. Both GPCRs are active, but DH31-Ra is the dominant receptor based on gene expression analyses and DH31 peptide binding affinities. A notable difference between the two variants lies in 1) the GPCR structures of their C-termini and 2) the utilization of second messengers, and the amino acid sequences of the two variants are identical. DH31-Ra contains 12 additional amino acids, providing different intracellular C-terminal configurations. DH31-Ra utilizes both cAMP and Ca(2+) as second messengers, whereas DH31-Rb utilizes only cAMP; this is the first time reported for an insect CT-like DH31 peptide. DH31 stimulated fluid secretion in D. suzukii adults, and secretion increased in a dose-dependent manner. However, when the fly was injected with a mixture of DH31 and CAPA, an anti-diuretic hormone, fluid secretion was suppressed. The structures of the DH31 receptors and the differential signaling pathways, including second messengers, involved in fly diuresis, are discussed. These findings provide fundamental insights into the characterization of D. suzukii DH31 and DH31-Rs, and facilitate the identification of potential biological targets for D. suzukii management.
Yarikipati, P., Jonusaite, S., Pleinis, J. M., Dominicci Cotto, C., Sanchez-Hernandez, D., Morrison, D. E., Goyal, S., Schellinger, J., Penalva, C., Curtiss, J., Rodan, A. R., Jenny, A. (2023). Unanticipated domain requirements for Drosophila Wnk kinase in vivo. PLoS Genet, 19(10):e1010975 PubMed ID: 37819975
Summary:
WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate canonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. This study shows that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. The only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. These data thus provide novel insights into unexpected in vivo roles of specific WNK domains.
Sayeesh, P. M., Iguchi, M., Suemoto, Y., Inoue, J., Inomata, K., Ikeya, T., Ito, Y. (2023). Interactions of the N- and C-Terminal SH3 Domains of Drosophila Drk with the Proline-Rich Peptides from Sos and Dos. Int J Mol Sci, 24(18) PubMed ID: 37762438
Summary:
Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. A previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, NMR titration experiments were conducted using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. The results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. Docking simulations were performed based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, the various modes in the interactions of Drk with Sos/Dos are discussed.
Gupta, K., Chakrabarti, S., Janardan, V., Gogia, N., Banerjee, S., Srinivas, S., Mahishi, D., Visweswariah, S. S. (2023). Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response. PLoS Genet, 19(9):e1010962 PubMed ID: 37733787
Summary:
Evolutionarily conserved genes often play critical roles in organismal physiology. This study describes multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. It was observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.
Keller, S. H., Deng, H., Lim, B. (2023). Regulation of the dynamic RNA Pol II elongation rate in Drosophila embryos. Cell Rep, 42(10):113225 PubMed ID: 37837623
Summary:
An increasing number of studies have shown the key role that RNA polymerase II (RNA Pol II) elongation plays in gene regulation. This study systematically examine how various enhancers, promoters, and gene body composition influence the RNA Pol II elongation rate through a single-cell-resolution live imaging assay. By using reporter constructs containing 5' MS2 and 3' PP7 repeating stem loops, the rate of RNA Pol II elongation in live Drosophila embryos was quantified. Promoters and exonic gene lengths have no effect on elongation rate, while enhancers and the presence of long introns may significantly change how quickly RNA Pol II moves across a gene. Furthermore, it was observed in multiple constructs that the RNA Pol II elongation rate accelerates after the transcriptional onset of nuclear cycle 14 in Drosophila embryos. This study provides a single-cell view of various mechanisms that affect the dynamic RNA Pol II elongation rate, ultimately affecting the rate of mRNA production.
Viola, C. M., Frittmann, O., Jenkins, H. T., Shafi, T., De Meyts, P., Brzozowski, A. M. (2023). Structural conservation of insulin/IGF signalling axis at the insulin receptors level in Drosophila and humans. Nat Commun, 14(1):6271 PubMed ID: 37805602
Summary:
The insulin-related hormones regulate key life processes in Metazoa, from metabolism to growth, lifespan and aging, through an evolutionarily conserved insulin signalling axis (IIS). In humans the IIS axis is controlled by insulin, two insulin-like growth factors, two isoforms of the insulin receptor (hIR-A and -B), and its homologous IGF-1R. In Drosophila, this signalling engages seven insulin-like hormones (DILP1-7) and a single receptor (dmIR). This report describes the cryoEM structure of the dmIR ectodomain:DILP5 complex, revealing high structural homology between dmIR and hIR. The excess of DILP5 yields dmIR complex in an asymmetric 'T' conformation, similar to that observed in some complexes of human IRs. However, dmIR binds three DILP5 molecules in a distinct arrangement, showing also dmIR-specific features. This work adds structural support to evolutionary conservation of the IIS axis at the IR level, and also underpins a better understanding of an important model organism.
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