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Friday June 29th, 2018 - Immune Response

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Shlyakhover, E., Shklyar, B., Hakim-Mishnaevski, K., Levy-Adam, F. and Kurant, E. (2018). Drosophila GATA factor serpent establishes phagocytic ability of embryonic macrophages. Front Immunol 9: 266. PubMed ID: 29568295
During Drosophila embryogenesis, a large number of apoptotic cells are efficiently engulfed and degraded by professional phagocytes, macrophages. Phagocytic receptors Six-Microns-Under (SIMU), Draper (Drpr) and Croquemort (Crq) are specifically expressed in embryonic macrophages and required for their phagocytic function. However, how this function is established during development remains unclear. This study demonstrates that the key regulator of Drosophila embryonic hemocyte differentiation, the transcription factor Serpent (Srp), plays a central role in establishing macrophage phagocytic competence. Srp, a homolog of the mammalian GATA factors, is required and sufficient for the specific expression of SIMU, Drpr and Crq receptors in embryonic macrophages. Moreover, each of these receptors can significantly rescue phagocytosis defects of macrophages in srp mutants, including their distribution in the embryo and engulfment of apoptotic cells. This reveals that the proficiency of macrophages to remove apoptotic cells relies on the expression of SIMU, Crq and/or Drpr. However, Glial Cells Missing (GCM) acting downstream of Srp in the differentiation of hemocytes, is dispensable for their phagocytic function during embryogenesis. Taken together, this study discloses the molecular mechanism underlying the development of macrophages as skilled phagocytes of apoptotic cells.
West, C. and Silverman, N. (2018). p38b and JAK-STAT signaling protect against invertebrate iridescent virus 6 infection in Drosophila. PLoS Pathog 14(5): e1007020. Pubmed ID: 29746571
The fruit fly Drosophila melanogaster is a powerful model system for the study of innate immunity in vector insects as well as mammals. For vector insects, it is particularly important to understand all aspects of their antiviral immune defenses, which could eventually be harnessed to control the transmission of human pathogenic viruses. The immune responses controlling RNA viruses in insects have been extensively studied, but the response to DNA virus infections is poorly characterized. This study reports that infection of Drosophila with the DNA virus Invertebrate iridescent Virus 6 (IIV-6) triggers JAK-STAT signaling and the robust expression of the Turandots, a gene family encoding small secreted proteins. To drive JAK-STAT signaling, IIV-6 infection more immediately induced expression of the unpaireds, a family of IL-6-related cytokine genes, via a pathway that required one of the three Drosophila p38 homologs, p38b. In fact, both Stat92E and p38b were required for the survival of IIV-6 infected flies. In addition, in vitro induction of the unpaireds required an NADPH-oxidase, and in vivo studies demonstrated Nox was required for induction of TotA. These results argue that ROS production, triggered by IIV-6 infection, leads to p38b activation and unpaired expression, and subsequent JAK-STAT signaling, which ultimately protects the fly from IIV-6 infection.
Tokusumi, Y., Tokusumi, T. and Schulz, R. A. (2018). Mechanical stress to Drosophila larvae stimulates a cellular immune response through the JAK/STAT signaling pathway. Biochem Biophys Res Commun. Pubmed ID: 29856996
Acute inflammation can cause serious tissue damage and disease in physiologically-challenged organisms. The precise mechanisms leading to these detrimental effects remain to be determined. This study utilized a reproducible means to induce cellular immune activity in Drosophila larvae in response to mechanical stress. That is, forceps squeeze-administered stress induces lamellocytes, a defensive hemocyte type that normally appears in response to wasp infestation of larvae. The posterior signaling center (PSC) is a cellular microenvironment in the larval hematopoietic lymph gland that is vital for lamellocyte induction upon parasitoid attack. However, the PSC was not required for mechanical stress-induced lamellocyte production. In addition, it was observed that mechanical injury caused a systemic expression of Unpaired3. This cytokine is both necessary and sufficient to activate the cellular immune response to the imposed stress. These findings provide new insights into the communication between injured tissues and immune system induction, using stress-challenged Drosophila larvae as a tractable model system.
Yu, S., Zhang, G. and Jin, L. H. (2018). A high-sugar diet affects cellular and humoral immune responses in Drosophila. Exp Cell Res 368(2): 215-224. Pubmed ID: 29727694
A high-sugar diet (HSD) induces Type 2 diabetes (T2D) and obesity, which severely threaten human health. The Drosophila T2D model has been constructed to study the mechanisms of insulin resistance, diet-induced cardiovascular diseases and other conditions. Innate immunity is the first line of defense against invading pathogens and parasites. However, few studies have focused on the relationship between a HSD and the innate immune response in Drosophila. In this study, flies were fed a high-sucrose diet, and defects were observed in the phagocytosis of latex beads and B. bassiana spores. The actin cytoskeleton was also disrupted in hemocytes from HSD-fed larvae. Furthermore, HSD induced the differentiation of lamellocytes in the lymph gland and circulating hemolymph, which rarely occurs in healthy bodies, via JNK signaling. In addition, the Toll and JNK pathways were excessively activated in the fat bodies of HSD-fed larvae, and a large number of dead cells were observed. Finally, HSD induced the aberrant activation of the innate immune system, including inflammation. These results have established a connection between T2D and the innate immune response.
Zhai, Z., Boquete, J. P. and Lemaitre, B. (2018). Cell-specific Imd-NF-kappaB responses enable simultaneous antibacterial immunity and intestinal epithelial cell shedding upon bacterial infection. Immunity 48(5): 897-910. Pubmed ID: 29752064
Intestinal infection triggers potent immune responses to combat pathogens and concomitantly drives epithelial renewal to maintain barrier integrity. Current models propose that epithelial renewal is primarily driven by damage caused by reactive oxygen species (ROS). This study found that in Drosophila, the Imd-NF-kappaB pathway controlled enterocyte (EC) shedding upon infection, via a mechanism independent of ROS-associated apoptosis. Mechanistically, the Imd pathway synergized with JNK signaling to induce epithelial cell shedding specifically in the context of bacterial infection, requiring also the reduced expression of the transcription factor GATAe. Furthermore, cell-specific NF-kappaB responses enabled simultaneous production of antimicrobial peptides (AMPs) and epithelial shedding in different EC populations. Thus, the Imd-NF-kappaB pathway is central to the intestinal antibacterial response by mediating both AMP production and the maintenance of barrier integrity. Considering the similarities between Drosophila Imd signaling and mammalian TNFR pathway, these findings suggest the existence of an evolutionarily conserved genetic program in immunity-induced epithelial shedding.
Zheng, W., Rus, F., Hernandez, A., Kang, P., Goldman, W., Silverman, N. and Tatar, M. (2018). Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells. BMC Biol 16(1): 60. Pubmed ID: 29855367
Drosophila is a powerful model for the study of factors modulating innate immunity. This study examines the effect of water-loss dehydration on innate immune responsiveness in the Drosophila renal system (Malpighian tubules; MTs), and how this leads to elevated host defense and contributes to immunosenescence. A short period of desiccation-elevated peptidoglycan recognition protein-LC (PGRP-LC) expression in MTs, increased antimicrobial peptide (AMP) gene induction, and protected animals from bacterial infection. Desiccation increased ecdysone synthesis in MTs, while inhibition of ecdysone synthesis or ecdysone receptor expression, specifically within MTs, prevented induction of PGRP-LC and reduced protection from bacterial infection. Additionally, aged flies are constitutively water-stressed and have elevated levels of ecdysone and PGRP-LC. Conversely, adults aged at high relative humidity show less water loss and have reduced expression of PGRP-LC and AMPs. It is concluded that desiccation primes immune responsiveness by elevating PGRP-LC expression specifically in MTs. In response to desiccation, ecdysone is produced in MTs and acts in a paracrine fashion to increase PGRP-LC expression, immune responsiveness, and improve host defense. This activity of the renal system may contribute to the immunosenescence observed in Drosophila.

Thursday, June 28th - Disease Models

Wang, T., Cheng, J., Wang, S., Wang, X., Jiang, H., Yang, Y., Wang, Y., Zhang, C., Liang, W. and Feng, H. (2018). alpha-Lipoic acid attenuates oxidative stress and neurotoxicity via the ERK/Akt-dependent pathway in the mutant hSOD1 related Drosophila model and the NSC34 cell line of amyotrophic lateral sclerosis. Brain Res Bull. Pubmed ID: 29842900
Amyotrophic lateral sclerosis (ALS) is a degenerative disease with a progressive loss of motor neurons in the central nervous system (CNS). However, there are unsolved problems with the therapies for this disease. alpha-Lipoic acid (LA) is a natural, universal antioxidant capable of scavenging hydroxyl radicals as well as regenerating a series of antioxidant enzymes that has been widely used in clinical settings. This study aimed to evaluate the antioxidant and neuroprotective effects of LA in ALS cell and Drosophila models with mutant G85R and G93A hSOD1 genes. The biological effects of LA and the protein levels of several antioxidant factors were examined, as were those of phospho-Akt and phospho-ERK. Furthermore, specific inhibitors of the PI3K/Akt and MEK/ERK signaling pathways were used to analyze their effects on LA-induced antioxidant expression in vivo and in vitro. Evidences showed that the mutant hSOD1 resulted in the increased oxidative stress, abnormal antioxidant signaling and pathological behaviors in motor performance and survival compared with non-mutant hSOD1 models, treatment with LA improved motor activity and survival in transgenic flies, prevented NSC34 cells from mutant hSOD1 or H2O2 induced decreased antioxidant enzymes as well as increased ROS levels. In addition, LA regulated the expression levels of antioxidant proteins in a dose- and periodical time-dependent manner, which might be mediated by ERK/Akt pathway activation and independent from the mutant hSOD1 gene. The observations suggest that LA exerts strong and positive antioxidant and neuroprotective effects through the activation of the ERK-Akt pathway in hSOD1 ALS models.
Saridaki, T., Nippold, M., Dinter, E., Roos, A., Diederichs, L., Fensky, L., Schulz, J. B. and Falkenburger, B. H. (2018). FYCO1 mediates clearance of alpha-synuclein aggregates through a Rab7-dependent mechanism. J Neurochem. Pubmed ID: 29747217
Parkinson disease can be caused by mutations in the alpha-synuclein gene and is characterized by aggregates of alpha-synuclein protein. Overexpression of the small GTPase Rab7 (see Drosophila Rab7) can induce clearance of alpha-synuclein aggregates. This study investigated which Rab7 effectors mediate this effect. To model Parkinson disease the pathogenic A53T mutant of alpha-synuclein was expressed in HEK293T cells and Drosophila melanogaster. The Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP) were investigated. FYCO1-EGFP was found to decorate vesicles containing alpha-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain alpha-synuclein. FYCO1 overexpression reduced the number of cells with alpha-synuclein aggregates, defined as visible particles of EGFP-tagged alpha-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of alpha-synuclein protein as assayed by western blot, increased the disappearance of alpha-synuclein aggregates in time-lapse microscopy, and decreased alpha-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7 induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7 induced aggregate clearance. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of alpha-synuclein aggregates. Extracellular alpha-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased alpha-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-alpha-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates degradation of alpha-synuclein and could be beneficial in patients with Parkinson disease.
Nunez, K. M., Azanchi, R. and Kaun, K. R. (2018). Cue-induced ethanol seeking in Drosophila melanogaster is dose-dependent. Front Physiol 9: 438. Pubmed ID: 29740347
Alcohol use disorder generates devastating social, medical and economic burdens, making it a major global health issue. The persistent nature of memories associated with intoxication experiences often induces cravings and triggers relapse in recovering individuals. Despite recent advances, the neural and molecular mechanisms underlying these memories are complex and not well understood. This makes finding effective pharmacological targets challenging. The investigation of persistent alcohol-associated memories in the fruit fly, Drosophila melanogaster, presents a unique opportunity to gain a comprehensive understanding of the memories for ethanol reward at the level of genes, molecules, neurons and circuits. This study characterizes the dose-dependent nature of ethanol on the expression of memory for an intoxication experience. The concentration of ethanol, number of ethanol exposures, length of ethanol exposures, and timing between ethanol exposures are critical in determining whether ethanol is perceived as aversive or appetitive, and in how long the memory for the intoxicating properties of ethanol last. This study highlights that fruit flies display both acute and persistent memories for ethanol-conditioned odor cues, and that a combination of parameters that determine the intoxication state of the fly influence the seemingly complex retention and expression of memories associated with intoxication. This thorough behavioral characterization provides the opportunity to interrogate the biological underpinnings of these observed preference differences in future studies.
Symonenko, A. V., Roshina, N. V., Krementsova, A. V. and Pasyukova, E. G. (2018). Reduced neuronal transcription of Escargot, the Drosophila gene encoding a Snail-type transcription factor, promotes longevity. Front Genet 9: 151. Pubmed ID: 29760717
In recent years, several genes involved in complex neuron specification networks have been shown to control life span. Previously, several genes have been characterized that encode RNA polymerase II transcription factors and that are involved in the development of the nervous system affect life span in Drosophila melanogaster. Among other genes, escargot (esg) was demonstrated to be causally associated with an increase in the life span of male flies. This study presents new data on the role of esg in life span control. esg affects the life spans of both mated and unmated males and females to varying degrees. By analyzing the survival and locomotion of the esg mutants, this study demonstrated that esg is involved in the control of aging. Increased longevity is caused by decreased esg transcription. In particular, esg knockdown was show to be involved in the nervous system increased life span, directly establishing the involvement of the neuronal esg function in life span control. These data invite attention to the mechanisms regulating the esg transcription rate, which is changed by insertions of DNA fragments of different sizes downstream of the structural part of the gene, indicating the direction of further research. The data agree with the previously made suggestion that alterations in gene expression during development might affect adult lifespan, due to epigenetic patterns inherited in cell lineages or predetermined during the development of the structural and functional properties of the nervous system.
Miguel, L., Avequin, T., Pons, M., Frebourg, T., Campion, D. and Lecourtois, M. (2018). FTLD/ALS-linked TDP-43 mutations do not alter TDP-43's ability to self-regulate its expression in Drosophila. Brain Res. PubMed ID: 29778779
TDP-43 is a major disease-causing protein in amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Today, more than 50 missense mutations in the TARDBP/TDP-43 gene have been described in patients with FTLD/ALS. However, the functional consequences of FTLD/ALS-linked TDP-43 mutations are not fully elucidated. In the physiological state, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Maintaining normal TDP-43 protein levels is critical for proper physiological functions of the cells. This study investigated whether the FTLD/ALS-associated mutations could interfere with TDP-43 protein's capacity to modulate its own protein levels using Drosophila as an experimental model. The data show that FTLD/ALS-associated mutant proteins regulate TDP-43 production with the same efficiency as the wild-type form of the protein. Thus, FTLD/ALS-linked TDP-43 mutations do not alter TDP-43's ability to self-regulate its expression and consequently of the homeostasis of TDP-43 protein levels.
Westfall, S., Lomis, N. and Prakash, S. (2018). Longevity extension in Drosophila through gut-brain communication. Sci Rep 8(1): 8362. Pubmed ID: 29849035
Aging and chronic disease development are multifactorial processes involving the cumulative effects of metabolic distress, inflammation, oxidative stress and mitochondrial dynamics. Recently, variations in the gut microbiota have been associated with age-related phenotypes and probiotics have shown promise in managing chronic disease progression. In this study, novel probiotic and synbiotic formulations are shown to combinatorially extend longevity in male Drosophila melanogaster through mechanisms of gut-brain-axis communication with implications in chronic disease management. Both the probiotic and synbiotic formulations rescued markers of metabolic stress by managing insulin resistance and energy regulatory pathways. Both formulations also ameliorated elevations in inflammation, oxidative stress and the loss of mitochondrial complex integrity. In almost all the measured pathways, the synbiotic formulation has a more robust impact than its individual components insinuating its combinatorial effect. The concomitant action of the gut microbiota on each of the key risk factors of aging and makes it a powerful therapeutic tool against neurodegeneration, diabetes, obesity, cardiovascular disease and other age-related chronic diseases.

Wednesday, June 27th - Small RNAs

Qiu, C., Zhang, Y., Fan, Y. J., Pang, T. L., Su, Y., Zhan, S. and Xu, Y. Z. (2018). HITS-CLIP reveals sex-differential RNA binding and alterative splicing regulation of SRm160 in Drosophila. J Mol Cell Biol. Pubmed ID: 29750417
Serine/arginine (SR)-rich proteins are critical for the regulation of alternative splicing (AS), which generates multiple mRNA isoforms from one gene and provides protein diversity for cell differentiation and tissue development. Genetic evidence suggests that Drosophila genital-specific overexpression of SR-related nuclear matrix protein of 160 kDa (SRm160), an SR protein with a PWI RNA-binding motif, causes defective development only in male flies and results in abnormal male genital structures and abnormal testis. However, the molecular characterization of SRm160 is limited. Using the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) method in two sex-specific embryonic cell lines, S2 from the male and Kc from the female, the genome-wide RNA-binding characteristics were identified of SRm160, which preferred binding to the exonic tri-nucleotide repeats GCA and AAC. This binding was validated through both in vitro gel-shift assay and in vivo splicing of mini-genes, SRm160 levels were found to affects AS of many transcripts. Furthermore, 492 differential binding sites (DBS) of SRm160 were identified varying between the two sex-specific cell lines. Among these DBS-containing genes, splicing factors were highly enriched, including transformer, a key regulator in the sex determination cascade. Analyses of fly mutants demonstrated that the SRm160 level affects AS isoforms of transformer. These findings shed crucial light on SRm160's RNA-binding specificity and regulation of AS in Drosophila sex determination and development.
Ma, F., Lin, P., Chen, Q., Lu, X., Zhang, Y. E. and Wu, C. I. (2018). Direct measurement of pervasive weak repression by microRNAs and their role at the network level. BMC Genomics 19(1): 362. PubMed ID: 29764374
A gene regulatory network (GRN) comprises many weak links that are often regulated by microRNAs. Since miRNAs rarely repress their target genes by more than 30%, doubts have been expressed about the biological relevance of such weak effects. These doubts raise the possibility of under-estimation as miRNA repression is usually estimated indirectly from equilibrium expression levels. To measure miRNA repression directly, transcript synthesis was inhibited in Drosophila larvae and time-course data collected on mRNA abundance, the decline of which reflects transcript degradation. The rate of target degradation in the absence of miR310s, a moderately expressed miRNA family, was found to decrease by 5 to 15%. A conventional analysis that does not remove transcript synthesis yields an estimate of 6.5%, within the range of the new estimates. These data permit further examinations of the repression mechanisms by miRNAs including seed matching types, APA (alternative polyadenylation) sites, effects of other highly-expressed miRNAs and the length of 3'UTR. The direct measurements suggest the latter two factors have a measurable effect on decay rate. The direct measurement confirms pervasive weak repression by miRNAs, supporting the conclusions based on indirect assays. The confirmation suggests that this weak repression may indeed be miRNAs' main function. In this context, the recent proposal is discussed that weak repression is "cumulatively powerful" in stabilizing GRNs.
Parikh, R. Y., Lin, H. and Gangaraju, V. K. (2018). A critical role for nucleoporin 358 (Nup358) in transposon silencing and piRNA biogenesis in Drosophila. J Biol Chem. Pubmed ID: 29735528
Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)-dependent ping-pong cycle generates most germline piRNAs. Loading of anti-sense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. This study shows that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. These results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.
Tsuboyama, K., Tadakuma, H. and Tomari, Y. (2018). Conformational activation of Argonaute by distinct yet coordinated actions of the Hsp70 and Hsp90 chaperone systems. Mol Cell 70(4): 722-729.e724. Pubmed ID: 29775584Journal
Loading of small RNAs into argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. This study utilized single-molecule Forster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. Empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. These data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form.
Huang, Y. C., Moreno, H., Row, S., Jia, D. and Deng, W. M. (2018). Germline silencing of UASt depends on the piRNA pathway. J Genet Genomics. PubMed ID: 29789221
One of the most extensively used techniques in Drosophila is the Gal4/UAS binary system, which allows tissue-specific misexpression or knockdown of specific genes of interest. The original UAS vector, UASt, can only be activated for transgene expression in somatic tissues but not in the germline cells. This study found that the piwi-interacting RNA (piRNA) pathway is involved in suppressing UASt expression in ovarian germline cells. Individually knocking down or mutating components of the piRNA biogenesis pathway (e.g., Piwi, AGO3, Aub, Spn-E, Su(var)2-10, Hsp83, and Vasa) resulted in the expression of the UASt-reporter (GFP or RFP) in the germline. An RNA-seq analysis of small RNAs revealed that the hsp70 promoter of UASt is targeted by piRNAs, and in the aub mutant ovary, the amount of piRNAs targeting the hsp70 promoter is reduced by around 40 folds. In contrast, the SV40 3'UTR of the UASt, which happens to be targeted by the nonsense-mediated RNA decay (NMD) pathway, is not responsible for germline UASt suppression, as UASt-reporters with NMD-insensitive 3'UTR fail to show germline expression. Taken together, these studies reveal a crucial role of the piRNA pathway, potentially via the suppression of the hsp70 promoter, in germline UASt silencing in Drosophila ovaries.
Bakthavachalu, B., Huelsmeier, J., Sudhakaran, I. P., Hillebrand, J., Singh, A., Petrauskas, A., Thiagarajan, D., Sankaranarayanan, M., Mizoue, L., Anderson, E. N., Pandey, U. B., Ross, E., VijayRaghavan, K., Parker, R. and Ramaswami, M. (2018). RNP-granule assembly via Ataxin-2 disordered domains is required for long-term memory and neurodegeneration. Neuron 98(4): 754-766.e754. PubMed ID: 29772202
Human Ataxin-2 is implicated in the cause and progression of amyotrophic lateral sclerosis (ALS) and type 2 spinocerebellar ataxia (SCA-2). In Drosophila, a conserved atx2 gene is essential for animal survival as well as for normal RNP-granule assembly, translational control, and long-term habituation. Like its human homolog, Drosophila Ataxin-2 (Atx2) contains polyQ repeats and additional intrinsically disordered regions (IDRs). Atx2 IDRs, which are capable of mediating liquid-liquid phase transitions in vitro, are essential for efficient formation of neuronal mRNP assemblies in vivo. Remarkably, DeltaIDR mutants that lack neuronal RNP granules show normal animal development, survival, and fertility. However, they show defects in long-term memory formation/consolidation as well as in C9ORF72 dipeptide repeat or FUS-induced neurodegeneration. Together, these findings demonstrate (1) that higher-order mRNP assemblies contribute to long-term neuronal plasticity and memory, and (2) that a targeted reduction in RNP-granule formation efficiency can alleviate specific forms of neurodegeneration.

Tuesday June 26th - Signaling

Ogueta, M., Hardie, R. C. and Stanewsky, R. (2018). Non-canonical phototransduction mediates synchronization of the Drosophila melanogaster circadian clock and retinal light responses. Curr Biol. Pubmed ID: 29779871
The daily light-dark cycles represent a key signal for synchronizing circadian clocks. Both insects and mammals possess dedicated "circadian" photoreceptors but also utilize the visual system for clock resetting. In Drosophila, circadian clock resetting is achieved by the blue-light photoreceptor cryptochrome (CRY), which is expressed within subsets of the brain clock neurons. In addition, rhodopsin-expressing photoreceptor cells contribute to light synchronization. Light resets the molecular clock by CRY-dependent degradation of the clock protein Timeless (TIM), although in specific subsets of key circadian pacemaker neurons, including the small ventral lateral neurons (s-LNvs), TIM and Period (PER) oscillations can be synchronized by light independent of CRY and canonical visual Rhodopsin phototransduction. This study shows that at least three of the seven Drosophila rhodopsins can utilize an alternative transduction mechanism involving the same alpha-subunit of the heterotrimeric G protein operating in canonical visual phototransduction (Gq). Surprisingly, in mutants lacking the canonical phospholipase C-beta (PLC-beta) encoded by the no receptor potential A (norpA) gene, a novel transduction pathway using a different PLC-beta encoded by the Plc21C gene. This novel pathway is important for behavioral clock resetting to semi-natural light-dark cycles and mediates light-dependent molecular synchronization within the s-LNv clock neurons. The same pathway appears to be responsible for norpA-independent light responses in the compound eye. Rhodopsin 5 (Rh5) and Rh6, present in the R8 subset of retinal photoreceptor cells, drive both the long-term circadian and rapid light responses in the eye.
Manning, S. A., Dent, L. G., Kondo, S., Zhao, Z. W., Plachta, N. and Harvey, K. F. (2018). Dynamic fluctuations in subcellular localization of the Hippo pathway effector Yorkie in vivo. Curr Biol 28(10): 1651-1660.e1654. PubMed ID: 29754899
The Hippo pathway is an evolutionarily conserved signaling network that integrates diverse cues to control organ size and cell fate. The central downstream pathway protein in Drosophila is the transcriptional co-activator Yorkie (YAP and TAZ in humans), which regulates gene expression with the Scalloped/TEA domain family member (TEAD) transcription factors. A central regulatory step in the Hippo pathway is phosphorylation of Yorkie by the NDR family kinase Warts, which promotes Yorkie cytoplasmic localization by stimulating association with 14-3-3 proteins. Numerous reports have purported a static model of Hippo signaling whereby, upon Hippo activation, Yorkie/YAP/TAZ become cytoplasmic and therefore inactive, and upon Hippo repression, Yorkie/YAP/TAZ transit to the nucleus and are active. However, there is little appreciation for the dynamics of Yorkie/YAP/TAZ subcellular localization because most studies have been performed in fixed cells and tissues. To address this, live multiphoton microscopy imaging was used to investigate the dynamics of an endogenously tagged Yorkie-Venus protein in growing epithelial organs. The majority of Yorkie was found to rapidly traffic between the cytoplasm and nucleus, rather than being statically localized in either compartment. In addition, discrete cell populations within the same organ display different rates of Yorkie nucleo-cytoplasmic shuttling. By assessing Yorkie dynamics in warts mutant tissue, this study found that the Hippo pathway regulates Yorkie subcellular distribution by regulating its rate of nuclear import. Furthermore, Yorkie's localization fluctuates dramatically throughout the cell cycle, being predominantly cytoplasmic during interphase and, unexpectedly, chromatin enriched during mitosis. Yorkie's association with mitotic chromatin is Scalloped dependent, suggesting a potential role in mitotic bookmarking.
Riehle, M., Tsvetkov, D., Gohlke, B. O., Preissner, R., Harteneck, C., Gollasch, M. and Nurnberg, B. (2018). Molecular basis for the sensitivity of TRP channels to polyunsaturated fatty acids. Naunyn Schmiedebergs Arch Pharmacol. Pubmed ID: 29736621
Transient receptor potential (TRP) channels represent a superfamily of unselective cation channels that are subdivided into seven subfamilies based on their sequence homology and differences in gating and functional properties. Little is known about the molecular mechanisms of TRP channel regulation, particularly of the "canonical" TRP (TRPC) subfamily and their activation by polyunsaturated fatty acids (PUFAs). This study analyzed the structure-function relationship of Drosophila fruit fly TRPC channels. The primary aim was to uncover the molecular basis of PUFA sensitivity of Drosophila TRP-like (TRPL) and TRPgamma channels. Amino acid (aa) sequence alignment of the three Drosophila TRPC channels revealed 50 aa residues highly conserved in PUFA-sensitive TRPL and TRPgamma channels but not in the PUFA-insensitive TRP channel. Substitution of respective aa in TRPL by corresponding aa of TRP identified 18 residues that are necessary for PUFA-mediated activation of TRPL. Most aa positions are located within a stretch comprising transmembrane domains S2-S4, whereas six aa positions have been assigned to the proximal cytosolic C-terminus. Interestingly, residues I465 and S471 are required for activation by 5,8,11,14-eicosatetraynoic acid (ETYA) but not 5,8,11-eicosatriynoic acid (ETI). As proof of concept, a PUFA-sensitive TRP channel was generated by exchanging the corresponding aa from TRPL to TRP. This study demonstrates a specific aa pattern in the transmembrane domains S2-S4 and the proximal C-terminus essential for TRP channel activation by PUFAs.
Sun, Z., Zheng, Y. and Liu, W. (2018). Identification and characterization of a novel calmodulin binding site in Drosophila TRP C-terminus. Biochem Biophys Res Commun 501(2): 434-439. Pubmed ID: 29730291
Transient receptor potential (TRP) channels are a group of essential cation channels involved in many important sensory signal transduction processes, such as light, temperature, tastes and pressure sensing. Drosophila TRP channel is the first discovered family member and plays important roles in photo-transduction in Drosophila. Calmodulin (CaM), an important downstream effector of Ca(2+) signal, was considered as a vital regulator of TRP activities. This study discovered a novel Ca(2+) dependent CaM binding site (TRP 783-862) in between the previously reported two calmodulin binding sites (CBSs). The isothermal titration calorimetry (ITC) and the size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) results showed that the dissociation constant (Kd) between TRP 783-862 and Ca(2+)-CaM is 0.10+/-0.04mμM and their binding stoichiometry is 1:1. In addition, the shortest Ca(2+)-CaM interaction region and core CaM binding sequences in TRP 783-862 were dissected by the boundary mapping and mutagenesis experiments. More interestingly, by comparing the circular dichroism (CD) spectra before and after Ca(2+)-CaM binding, the TRP 783-862 fragment showed Ca(2+)-CaM binding dependent secondary structure changes, indicating that the interaction between CaM and Drosophila TRP channel may have a conformational impact on TRP structure. In summary, by identifying and characterizing a novel CaM binding site in TRP C-terminus, these findings provided a biochemical and structural basis for further in vivo functional studies of Ca(2+)-mediated TRP channel regulation through CaM/TRP interaction.
Ratheesh, A., Biebl, J., Vesela, J., Smutny, M., Papusheva, E., Krens, S. F. G., Kaufmann, W., Gyoergy, A., Casano, A. M. and Siekhaus, D. E. (2018). Drosophila TNF modulates tissue tension in the embryo to facilitate macrophage invasive migration. Dev Cell 45(3): 331-346.e337. Pubmed ID: 29738712
Migrating cells penetrate tissue barriers during development, inflammatory responses, and tumor metastasis. This study examined whether migration in vivo in such three-dimensionally confined environments requires changes in the mechanical properties of the surrounding cells using embryonic Drosophila melanogaster hemocytes, also called macrophages, as a model. Macrophage invasion into the germband through transient separation of the apposing ectoderm and mesoderm requires cell deformations and reductions in apical tension in the ectoderm. Interestingly, the genetic pathway governing these mechanical shifts acts downstream of the only known tumor necrosis factor superfamily member in Drosophila, Eiger, and its receptor, Grindelwald. Eiger-Grindelwald signaling reduces levels of active Myosin in the germband ectodermal cortex through the localization of a Crumbs complex component, Patj (Pals-1-associated tight junction protein). This study therefore elucidates a distinct molecular pathway that controls tissue tension and demonstrate the importance of such regulation for invasive migration in vivo.
Szabo, A., Papin, C., Cornu, D., Chelot, E., Lipinszki, Z., Udvardy, A., Redeker, V., Mayor, U. and Rouyer, F. (2018). Ubiquitylation dynamics of the clock cell proteome and TIMELESS during a circadian cycle. Cell Rep 23(8): 2273-2282. Pubmed ID: 29791839
Circadian clocks have evolved as time-measuring molecular devices to help organisms adapt their physiology to daily changes in light and temperature. Transcriptional oscillations account for a large fraction of rhythmic protein abundance. However, cycling of various posttranslational modifications, such as ubiquitylation, also contributes to shape the rhythmic protein landscape. An in vivo ubiquitin labeling assay was used to investigate the circadian ubiquitylated proteome of Drosophila melanogaster.Cyclic ubiquitylation affects Megator (Mtor), a chromatin-associated nucleoporin that, in turn, feeds back to regulate the core molecular oscillator. Furthermore, the ubiquitin ligase subunits Cullin-3 (Cul-3) and Supernumerary limbs Slbm) cooperate for ubiquitylating the Timeless protein. These findings stress the importance of ubiquitylation pathways in the Drosophila circadian clock and reveal a key component of this system.

Monday, June 25th - Signaling

Luong, D., Perez, L. and Jemc, J. C. (2018). Identification of raw as a regulator of glial development. PLoS One 13(5): e0198161. PubMed ID: 29813126
Glial cells perform numerous functions to support neuron development and function, including axon wrapping, formation of the blood brain barrier, and enhancement of synaptic transmission. A novel gene, raw, has been identified that functions in glia of the central and peripheral nervous systems in Drosophila. Reducing Raw levels in glia results in morphological defects in the brain and ventral nerve cord, as well as defects in neuron function, as revealed by decreased locomotion in crawling assays. Examination of the number of glia along peripheral nerves reveals a reduction in glial number upon raw knockdown. The reduced number of glia along peripheral nerves occurs as a result of decreased glial proliferation. As Raw has been shown to negatively regulate Jun N-terminal kinase (JNK) signaling in other developmental contexts, the expression of a JNK reporter and the downstream JNK target, matrix metalloproteinase 1 (mmp1) was examined, and raw knockdown was found to result in increased reporter activity and Mmp1 levels. These results are consistent with previous studies showing increased Mmp levels lead to nerve cord defects similar to those observed upon raw knockdown. In addition, knockdown of puckered, a negative feedback regulator of JNK signaling, also causes a decrease in glial number. Thus, these studies have resulted in the identification of a new regulator of gliogenesis, and demonstrate that increased JNK signaling negatively impacts glial development.
Pavlowsky, A., Schor, J., Placais, P. Y. and Preat, T. (2018). A GABAergic feedback shapes dopaminergic input on the Drosophila mushroom body to promote appetitive long-term memory. Curr Biol. Pubmed ID: 29779874
Memory consolidation is a crucial step for long-term memory (LTM) storage. However, a clear picture of how memory consolidation is regulated at the neuronal circuit level is still lacking. This study took advantage of the Drosophila olfactory memory center, the mushroom body (MB), to address this question in the context of appetitive LTM. The MB lobes, which are made by the fascicled axons of the MB intrinsic neurons, are organized into discrete anatomical modules, each covered by the terminals of a defined type of dopaminergic neuron (DAN) and the dendrites of a corresponding type of MB output neuron (MBON). An essential role has been revealed of one DAN, the MP1 neuron, in the formation of appetitive LTM. The MP1 neuron is anatomically matched to the GABAergic MBON MVP2, which has been attributed feedforward inhibitory functions recently. This study used behavior experiments and in vivo imaging to challenge the existence of MP1-MVP2 synapses and investigate their role in appetitive LTM consolidation. MP1 and MVP2 neurons form an anatomically and functionally recurrent circuit, which features a feedback inhibition that regulates consolidation of appetitive memory. This circuit involves two opposite type 1 and type 2 dopamine receptors in MVP2 neurons and the metabotropic GABAB-R1 receptor in MP1 neurons. It is proposed that this dual-receptor feedback supports a bidirectional self-regulation of MP1 input to the MB. This mechanism displays striking similarities with the mammalian reward system, in which modulation of the dopaminergic signal is primarily assigned to inhibitory neurons.
Yamada, D., Ishimoto, H., Li, X., Kohashi, T., Ishikawa, Y. and Kamikouchi, A. (2018). GABAergic local interneurons shape female fruit fly response to mating songs. J Neurosci 38(18): 4329-4347. PubMed ID: 29691331
Many animals use acoustic signals to attract a potential mating partner. In fruit flies (Drosophila melanogaster), the courtship pulse song has a species-specific interpulse interval (IPI) that activates mating. Although a series of auditory neurons in the fly brain exhibit different tuning patterns to IPIs, it is unclear how the response of each neuron is tuned. This study examined the neural circuitry regulating the activity of antennal mechanosensory and motor center (AMMC)-B1 neurons, key secondary auditory neurons in the excitatory neural pathway that relay song information. By performing Ca(2+) imaging in female flies, it as found that the IPI selectivity observed in AMMC-B1 neurons differs from that of upstream auditory sensory neurons [Johnston's organ (JO)-B]. Selective knock-down of a GABAA receptor subunit in AMMC-B1 neurons increased their response to short IPIs, suggesting that GABA suppresses AMMC-B1 activity at these IPIs. Connection mapping identified two GABAergic local interneurons that synapse with AMMC-B1 and JO-B. Ca(2+) imaging combined with neuronal silencing revealed that these local interneurons, AMMC-LN and AMMC-B2, shape the response pattern of AMMC-B1 neurons at a 15 ms IPI. Neuronal silencing studies further suggested that both GABAergic local interneurons suppress the behavioral response to artificial pulse songs in flies, particularly those with a 15 ms IPI. Altogether, this study identified a circuit containing two GABAergic local interneurons that affects the temporal tuning of AMMC-B1 neurons in the song relay pathway and the behavioral response to the courtship song. These findings suggest that feedforward inhibitory pathways adjust the behavioral response to courtship pulse songs in female flies.
Im, S. H., Patel, A. A., Cox, D. N. and Galko, M. J. (2018).. Drosophila Insulin receptor regulates the persistence of injury-induced nociceptive sensitization. Dis Model Mech 11(5). PubMed ID: 29752280
Diabetes-associated nociceptive hypersensitivity affects diabetic patients with hard-to-treat chronic pain. Because multiple tissues are affected by systemic alterations in insulin signaling, the functional locus of insulin signaling in diabetes-associated hypersensitivity remains obscure. This study used Drosophila nociception/nociceptive sensitization assays to investigate the role of Insulin receptor (Insulin-like receptor, InR) in nociceptive hypersensitivity. InR mutant larvae exhibited mostly normal baseline thermal nociception (absence of injury) and normal acute thermal hypersensitivity following UV-induced injury. However, their acute thermal hypersensitivity persists and fails to return to baseline, unlike in controls. Remarkably, injury-induced persistent hypersensitivity is also observed in larvae that exhibit either type 1 or type 2 diabetes. Cell type-specific genetic analysis indicates that InR function is required in multidendritic sensory neurons including nociceptive class IV neurons. In these same nociceptive sensory neurons, only modest changes in dendritic morphology were observed in the InR(RNAi) -expressing and diabetic larvae. At the cellular level, InR-deficient nociceptive sensory neurons show elevated calcium responses after injury. Sensory neuron-specific expression of InR rescues the persistent thermal hypersensitivity of InR mutants and constitutive activation of InR in sensory neurons ameliorates the hypersensitivity observed with a type 2-like diabetic state. These results suggest that a sensory neuron-specific function of InR regulates the persistence of injury-associated hypersensitivity. It is likely that this new system will be an informative genetically tractable model of diabetes-associated hypersensitivity.
Banerjee, A. and Roy, J. K. (2018). Bantam regulates the axonal geometry of Drosophila larval brain by modulating actin regulator enabled. Invert Neurosci 18(2): 7. PubMed ID: 29777401
During development, axonogenesis, an integral part of neurogenesis, is based on well-concerted events comprising generation, rearrangement, migration, elongation, and adhesion of neurons. Actin, specifically the crosstalk between the guardians of actin polymerization, like enabled, chickadee, capping protein plays an essential role in crafting several events of axonogenesis. Recent evidences reflect multifaceted role of microRNA during axonogenesis. This study investigated the role of bantam miRNA, a well-established miRNA in Drosophila, in regulating the actin organization during brain development. Immunofluorescence studies showed altered arrangement of neurons and actin filaments whereas both qPCR and western blot revealed elevated expression of enabled, one of the actin modulators in bantam mutant background. Collectively, these results clearly demonstrate that bantam plays an instrumental role in shaping the axon architecture regulating the actin geometry through its modulator enabled.
Dey, S. and Ray, K. (2018). Cholinergic activity is essential for maintaining the anterograde transport of Choline Acetyltransferase in Drosophila. Sci Rep 8(1): 8028. PubMed ID: 29795337
Cholinergic activity is essential for cognitive functions and neuronal homeostasis. Choline Acetyltransferase (ChAT), a soluble protein that synthesizes acetylcholine at the presynaptic compartment, is transported in bulk in the axons by the heterotrimeric Kinesin-2 motor. Axonal transport of soluble proteins is described as a constitutive process assisted by occasional, non-specific interactions with moving vesicles and motor proteins. This study reports that an increase in the influx of Kinesin-2 motor and association between ChAT and the motor during a specific developmental period enhances the axonal entry, as well as the anterograde flow of the protein, in the sensory neurons of intact Drosophila nervous system. Loss of cholinergic activity due to Hemicholinium and Bungarotoxin treatments, respectively, disrupts the interaction between ChAT and Kinesin-2 in the axon, and the episodic enhancement of axonal influx of the protein. Altogether, these observations highlight a phenomenon of synaptic activity-dependent, feedback regulation of a soluble protein transport in vivo, which could potentially define the quantum of its pre-synaptic influx.

Friday, June 22nd - Transcriptional Regulation

Datta, R. R., Ling, J., Kurland, J., Ren, X., Xu, Z., Yucel, G., Moore, J., Shokri, L., Baker, I., Bishop, T., Struffi, P., Levina, R., Bulyk, M. L., Johnston, R. J., Jr. and Small, S. (2018). A feed-forward relay integrates the regulatory activities of Bicoid and Orthodenticle via sequential binding to suboptimal sites. Genes Dev 32(9-10): 723-736. PubMed ID: 29764918
The K50 (lysine at amino acid position 50) homeodomain (HD) protein Orthodenticle (Otd) is critical for anterior patterning and brain and eye development in most metazoans. In Drosophila melanogaster, another K50HD protein, Bicoid (Bcd), has evolved to replace Otd's ancestral function in embryo patterning. Bcd is distributed as a long-range maternal gradient and activates transcription of a large number of target genes, including otd. Otd and Bcd bind similar DNA sequences in vitro, but how their transcriptional activities are integrated to pattern anterior regions of the embryo is unknown. This study defines three major classes of enhancers that are differentially sensitive to binding and transcriptional activation by Bcd and Otd. Class 1 enhancers are initially activated by Bcd, and activation is transferred to Otd via a feed-forward relay (FFR) that involves sequential binding of the two proteins to the same DNA motif. Class 2 enhancers are activated by Bcd and maintained by an Otd-independent mechanism. Class 3 enhancers are never bound by Bcd, but Otd binds and activates them in a second wave of zygotic transcription. The specific activities of enhancers in each class are mediated by DNA motif variants preferentially bound by Bcd or Otd and the presence or absence of sites for cofactors that interact with these proteins. These results define specific patterning roles for Bcd and Otd and provide mechanisms for coordinating the precise timing of gene expression patterns during embryonic development.
Manee, M. M., Jackson, J. and Bergman, C. M. (2018). Conserved noncoding elements influence the transposable element landscape in Drosophila. Genome Biol Evol. PubMed ID: 29850787
Highly conserved noncoding elements (CNEs) constitute a significant proportion of the genomes of multicellular eukaryotes. The function of most CNEs remains elusive, but growing evidence indicates they are under some form of purifying selection. Noncoding regions in many species also harbor large numbers of transposable element (TE) insertions, which are typically lineage specific and depleted in exons because of their deleterious effects on gene function or expression. However, it is currently unknown whether the landscape of TE insertions in noncoding regions is random or influenced by purifying selection on CNEs. This study combine comparative and population genomic data in Drosophila melanogaster to show that abundance of TE insertions in intronic and intergenic CNEs is reduced relative to random expectation, supporting the idea that selective constraints on CNEs eliminate a proportion of TE insertions in noncoding regions. However, no evidence was found for differences in the allele frequency spectra for polymorphic TE insertions in CNEs versus those in unconstrained spacer regions, suggesting that the distribution of fitness effects acting on observable TE insertions is similar across different functional compartments in noncoding DNA. The results provide evidence that selective constraints on CNEs contribute to shaping the landscape of TE insertion in eukaryotic genomes, and provide further evidence that CNEs are indeed functionally constrained and not simply mutational cold spots.
Zandvakili, A., Campbell, I., Gutzwiller, L. M., Weirauch, M. T. and Gebelein, B. (2018). Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity. PLoS Genet 14(4): e1007289. PubMed ID: 29617378
Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. This study shows that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, a method was developed that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, it was found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs.
Xu, K., Liu, X., Wang, Y., Wong, C. and Song, Y. (2018). Temporospatial induction of homeodomain gene cut dictates natural lineage reprogramming. Elife 7. PubMed ID: 29714689
Understanding how cellular identity naturally interconverts with high efficiency and temporospatial precision is crucial for regenerative medicine. This study revealed a natural midgut-to-renal lineage conversion event during Drosophila metamorphosis and identified the evolutionarily-conserved homeodomain protein Cut as a master switch in this process. A steep Wnt/Wingless morphogen gradient intersects with a pulse of steroid hormone ecdysone to induce cut expression in a subset of midgut progenitors and reprogram them into renal progenitors. Molecularly, ecdysone-induced temporal factor Broad physically interacts with cut enhancer-bound Wnt pathway effector TCF/beta-catenin and likely bridges the distant enhancer and promoter region of cut through its self-association. Such long-range enhancer-promoter looping could subsequently trigger timely cut transcription. These results therefore led the autnors to a propose an unexpected poising-and-bridging mechanism whereby spatial and temporal cues intersect, likely via chromatin looping, to turn on a master transcription factor and dictate efficient and precise lineage reprogramming.
Webber, J. L., Zhang, J., Massey, A., Sanchez-Luege, N. and Rebay, I. (2018). Collaborative repressive action of the antagonistic ETS transcription factors Pointed and Yan fine-tunes gene expression to confer robustness in Drosophila. Development. PubMed ID: 29848501
The acquisition of cellular identity during development depends on precise spatiotemporal regulation of gene expression, with combinatorial interactions between transcription factors, accessory proteins and the basal transcription machinery together translating complex signaling inputs into appropriate gene expression outputs. The Drosophila ETS family transcription factors Yan and Pointed, whose opposing repressive and activating inputs orchestrate numerous cell fate transitions downstream of receptor tyrosine kinase signaling, provide one of the premier systems for studying this process. Current models describe the differentiative transition as a switch from Yan-mediated repression to Pointed-mediated activation of common target genes. This paper describes a new layer of regulation whereby Yan and Pointed co-occupy regulatory elements to coordinately repress gene expression, with Pointed unexpectedly required for the genome-wide occupancy of both Yan and the corepressor Groucho. Using even-skipped as a test-case, synergistic genetic interactions between Pointed, Groucho, Yan and components of the RNA polymerase II pausing machinery suggest Pointed integrates multiple scales of repressive regulation to confer robustness. It is speculated that this mechanism may be used broadly to fine-tune the expression of many developmentally critical genes.
Jacobs, J., Atkins, M., Davie, K., Imrichova, H., Romanelli, L., Christiaens, V., Hulselmans, G., Potier, D., Wouters, J., Taskiran, II, Paciello, G., Gonzalez-Blas, C. B., Koldere, D., Aibar, S., Halder, G. and Aerts, S. (2018). The transcription factor Grainy head primes epithelial enhancers for spatiotemporal activation by displacing nucleosomes. Nat Genet. PubMed ID: 29867222
Transcriptional enhancers function as docking platforms for combinations of transcription factors (TFs) to control gene expression. How enhancer sequences determine nucleosome occupancy, TF recruitment and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains, this study found that SNPs affecting binding sites of the TF Grainy head (Grh) causally determine the accessibility of epithelial enhancers. Deletion and ectopic expression of Grh cause loss and gain of DNA accessibility, respectively. However, although Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, it was shown that human Grh homologs-GRHL1, GRHL2 and GRHL3-function similarly. It is concluded that Grh binding is necessary and sufficient for the opening of epithelial enhancers but not for their activation. This data support a model positing that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.

Thursday, June 21st - Disease Models

Lam, A., Karekar, P., Shah, K., Hariharan, G., Fleyshman, M., Kaur, H., Singh, H. and Gururaja Rao, S. (2018). Drosophila voltage-gated calcium channel alpha1-subunits regulate cardiac function in the aging heart. Sci Rep 8(1): 6910. PubMed ID: 29720608
Ion channels maintain numerous physiological functions and regulate signaling pathways. They are the key targets for cellular reactive oxygen species (ROS), acting as signaling switches between ROS and ionic homeostasis. A paraquat (PQ) screen was carried out in Drosophila to identify ion channels regulating the ROS handling and survival in Drosophila melanogaster. The screen has revealed that α1-subunits (D-type, T-type, and cacophony) of voltage-gated calcium channels (VGCCs) handle PQ-mediated ROS stress differentially in a gender-based manner. Since ROS are also involved in determining the lifespan, it was discovered that the absence of T-type and cacophony decreased the lifespan while the absence of D-type maintained a similar lifespan to that of the wild-type strain. VGCCs are also responsible for electrical signaling in cardiac cells. The cardiac function of each mutant was evaluated through optical coherence tomography (OCT), which revealed that alpha1-subunits of VGCCs are essential in maintaining cardiac rhythmicity and cardiac function in an age-dependent manner. The results establish specific roles of alpha1-subunits of VGCCs in the functioning of the aging heart.
Kushimura, Y., Azuma, Y., Mizuta, I., Muraoka, Y., Kyotani, A., Yoshida, H., Tokuda, T., Mizuno, T. and Yamaguchi, M. (2018). Loss-of-function mutation in Hippo suppressed enlargement of lysosomes and neurodegeneration caused by dFIG4 knockdown. Neuroreport 29(10): 856-862. PubMed ID: 29742619
Charcot-Marie-Tooth disease (CMT) is the most common hereditary neuropathy, and more than 80 CMT-causing genes have been identified to date. CMT4J is caused by a loss-of-function mutation in the Factor-Induced-Gene 4 (FIG4) gene, the product of which plays important roles in endosome-lysosome homeostasis. It was hypothesized that Mammalian sterile 20-like kinase (MST) 1 and 2, tumor-suppressor genes, are candidate modifiers of CMT4J. The interactions were examined between dFIG4 and Hippo (hpo), Drosophila counterparts of FIG4 and MSTs, respectively, using the Drosophila CMT4J model with the knockdown of dFIG4. The loss-of-function allele of hpo improved the rough eye morphology, locomotive dysfunction accompanied by structural defects in the presynaptic terminals of motoneurons, and the enlargement of lysosomes caused by the knockdown of dFIG4. Therefore, this study identified hpo as a modifier of phenotypes induced by the knockdown of dFIG4. These results in Drosophila may provide an insight into the pathogenesis of CMT4J and contribute toward the development of disease-modifying therapy for CMT. The regulation of endosome-lysosome homeostasis was also identified as a novel probable function of Hippo/MST.
Das, T. K., Esernio, J. and Cagan, R. (2018). Restraining network response to targeted cancer therapies improves efficacy and reduces cellular resistance. Cancer Res. PubMed ID: 29844121
A key tool of cancer therapy has been targeted inhibition of oncogene addicted pathways. However, efficacy has been limited by progressive emergence of resistance as transformed cells adapt. This study used Drosophila to dissect response to targeted therapies. Treatment with a range of kinase inhibitors led to hyperactivation of overall cellular networks, resulting in emergent resistance and expression of stem cell markers including Sox2. Genetic and drug screens revealed that inhibitors of histone deacetylases, proteasome, and Hsp90 family of proteins restrained this network hyperactivation. These 'network brake' cocktails, used as adjuncts, prevented emergent resistance and promoted cell death at subtherapeutic doses. These results highlight a general response of cells-transformed and normal-to targeted therapies that leads to resistance and toxicity. Pairing targeted therapeutics with subtherapeutic doses of broad acting 'network brake' drugs may provide a means of extending therapeutic utility while reducing whole body toxicity.
Akiyama, T., User, S. D. and Gibson, M. C. (2018). Somatic clones heterozygous for recessive disease alleles of BMPR1A exhibit unexpected phenotypes in Drosophila. Elife 7. PubMed ID: 29745898
The majority of mutations studied in animal models are designated as recessive based on the absence of visible phenotypes in germline heterozygotes. Accordingly, genetic studies primarily rely on homozygous loss-of-function to determine gene requirements, and a conceptually-related 'two-hit model' remains the central paradigm in cancer genetics. This study investigated pathogenesis due to somatic mutation in epithelial tissues, a process that predominantly generates heterozygous cell clones. To study somatic mutation in Drosophila, inducible alleles were generated that mimic human Juvenile polyposis-associated BMPR1A mutations. Unexpectedly, four of these mutations had no phenotype in heterozygous carriers but exhibited clear tissue-level effects when present in somatic clones of heterozygous cells. It is concluded that these alleles are indeed recessive when present in the germline, but nevertheless deleterious when present in heterozygous clones. This unforeseen effect, deleterious heteromosaicism, suggests a 'one-hit' mechanism for disease initiation that may explain some instances of pathogenesis associated with spontaneous mutation.

Wednesday, June 20th - Oogenesis

Duan, T. and Geyer, P. K. (2018). Spermiogenesis and male fertility require the function of Suppressor of Hairy-Wing in somatic cyst cells of Drosophila. Genetics. PubMed ID: 29739818
Drosophila Suppressor of Hairy-wing [Su(Hw)] protein is an example of a multivalent transcription factor. Although best known for its role in establishing the chromatin insulator of the gypsy retrotransposon, Su(Hw) functions as an activator and repressor at non-gypsy genomic sites. It remains unclear how the different regulatory activities of Su(Hw) are utilized during development. Motivated from observations of spatially restricted expression of Su(Hw) in the testis, this study investigated the role of Su(Hw) in spermatogenesis to advance an understanding of its developmental contributions as an insulator, repressor and activator protein. It was discovered that Su(Hw) is required for sustained male fertility. Although dynamics of Su(Hw) expression coincide with changes in nuclear architecture and activation of co-regulated testis-specific gene clusters, loss of Su(Hw) does not disrupt meiotic chromosome pairing or transcription of testis-specific genes, suggesting that Su(Hw) has minor architectural or insulator functions in the testis. Instead, Su(Hw) has a prominent role as a repressor of neuronal genes, consistent with suggestions that Su(Hw) is a functional homologue of mammalian REST, a repressor of neuronal genes in non-neuronal tissues. Su(Hw) regulates transcription in both germline and somatic cells. Surprisingly, the essential spermatogenesis function of Su(Hw) resides in somatic cyst cells, implying context-specific consequences due to loss of this transcription factor. Together, these studies highlight that Su(Hw) has a major developmental function as a transcriptional repressor, with the impact of its loss dependent upon the cell-specific factors.
Kline, A., Curry, T. and Lewellyn, L. (2018). The Misshapen kinase regulates the size and stability of the germline ring canals in the Drosophila egg chamber. Dev Biol. PubMed ID: 29753016
Intercellular bridges are conserved structures that allow neighboring cells to exchange cytoplasmic material; defects in intercellular bridges can lead to infertility in many organisms. This study used the Drosophila egg chamber to study the mechanisms that regulate intercellular bridges. Within the developing egg chamber, the germ cells (15 nurse cells and 1 oocyte) are connected to each other through intercellular bridges called ring canals, which expand over the course of oogenesis to support the transfer of materials from the nurse cells to the oocyte. The ring canals are enriched in actin and actin binding proteins, and many proteins have been identified that localize to the germline ring canals and control their expansion and stability. This study demonstrates a novel role for the Ste20 family kinase, Misshapen (Msn), in regulation of the size of the germline ring canals. Msn localizes to ring canals throughout most of oogenesis, and depletion of Msn led to the formation of larger ring canals. Over-expression of Msn decreased ring canal diameter, and expression of a membrane tethered form of Msn caused ring canal detachment and nurse cell fusion. Altering the levels or localization of Msn also led to changes in the actin cytoskeleton and altered the localization of E-cadherin, which suggests that Msn could be indirectly limiting ring canal size by altering the structure or dynamics of the actin cytoskeleton and/or adherens junctions.
Cho, Y., Lai, C. M., Lin, K. Y. and Hsu, H. J. (2018). A targeted RNAi screen reveals Drosophila female-sterile genes That control the size of germline stem cell niche during development. G3 (Bethesda). PubMed ID: 29764959
Adult stem cells maintain tissue homeostasis. This unique capability largely depends on the stem cell niche, a specialized microenvironment, which preserves stem cell identity through physical contacts and secreted factors. In many cancers, latent tumor cell niches are thought to house stem cells and aid tumor initiation. However, in developing tissue and cancer it is unclear how the niche is established. The well-characterized germline stem cells (GSCs) and niches in the Drosophila melanogaster ovary provide an excellent model to address this fundamental issue. As such, this study conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. Feminization factors (Transformer, Sex lethal, and Virilizer), a histone methyltransferase (Enhancer of Zeste), a transcriptional machinery component (Enhancer of yellow 1), a chromatin remodeling complex member (Enhancer of yellow 3) and a chromosome passenger complex constituent (Incenp) were identified as potentially functioning in the control of niche size. The identification of these molecules highlights specific molecular events that are critical for niche formation and will provide a basis for future studies to fully understand the mechanisms of GSC recruitment and maintenance.
Shukla, V., Dhiman, N., Nayak, P., Dahanukar, N., Deshpande, G. and Ratnaparkhi, G. (2018). Stonewall and Brickwall: Two partially redundant determinants required for the maintenance of female germline in Drosophila. G3 (Bethesda). PubMed ID: 29669801
Proper specification of germline stem cells (GSCs) in Drosophila ovaries depends on niche derived non-autonomous signaling and cell autonomous components of transcriptional machinery. Stonewall (Stwl), a MADF-BESS family protein, is one of the cell intrinsic transcriptional regulators involved in the establishment and/or maintenance of GSC fate in Drosophila ovaries. This stufy reports identification and functional characterization of another member of the same protein family, CG3838/Brickwall (Brwl) with analogous functions. Loss of function alleles of brwl exhibit age dependent progressive degeneration of the developing ovarioles and loss of GSCs. Supporting the conclusion that the structural deterioration of mutant egg chambers is a result of apoptotic cell death, activated caspase levels are considerably elevated in brwl(-) ovaries. Moreover, as in the case of stwl mutants, on several instances, loss of brwl activity results in fusion of egg chambers and misspecification of the oocyte. Importantly, brwl phenotypes can be partially rescued by germline specific over-expression of stwl arguing for overlapping yet distinct functional capabilities of the two proteins. Taken together with the phylogenetic analysis, these data suggest that brwl and stwl likely share a common MADF-BESS ancestor and they are expressed in overlapping spatiotemporal domains to ensure robust development of the female germline.
Su, T. Y., Nakato, E., Choi, P. Y. and Nakato, H. (2018). Drosophila glypicans regulate follicle stem cell maintenance and niche competition. Genetics. PubMed ID: 29632032
Adult stem cells reside in specialized microenvironments, called niches, which provide signals for stem cells to maintain their undifferentiated and self-renewing state. To maintain stem cell quality, several types of stem cells are known to be regularly replaced by progenitor cells through niche competition. However, the cellular and molecular bases for stem cell competition for niche occupancy are largely unknown. This study shows that two Drosophila members of the glypican family of heparan sulfate proteoglycans (HSPGs), Dally and Dally-like (Dlp), differentially regulate follicle stem cell (FSC) maintenance and FSC competitiveness for niche occupancy. Lineage analyses of glypican mutant FSC clones showed that dally is essential for normal FSC maintenance. In contrast, dlp is a hyper-competitive mutation: dlp mutant FSC progenitors often eventually occupy the entire epithelial sheet. RNAi knockdown experiments showed that Dally and Dlp play both partially redundant and distinct roles in regulating Jak/Stat, Wg and Hh signaling in FSCs. The Drosophila FSC system offers a powerful genetic model to study the mechanisms by which HSPGs exert specific functions in stem cell replacement and competition.
Xu, R., Li, J., Zhao, H., Kong, R., Wei, M., Shi, L., Bai, G. and Li, Z. (2018). Self-restrained regulation of stem cell niche activity by niche components in the Drosophila testis. Dev Biol. PubMed ID: 29679558
Most, if not all, stem cells reside in a defined microenvironment, called the niche. Short-ranged niche signal must be tightly controlled to be active only inside the niche to maintain the proper balance of stem cell self-renewal verse differentiation. However, how niche components restrict localized niche signal activation remains largely unknown. This study finds that Thickveins (Tkv, a type I receptor of the Dpp signaling pathway) in cyst stem cells (CySCs) of the testis niche prevents Dpp signaling activation outside of the niche. Tkv functions as Dpp trap/sink to spatially restrain Dpp signaling inside the niche. This self-restrained regulation of niche activity by Tkv in CySCs is independent of the canonical Dpp signaling pathway. These data demonstrate the critical roles of niche components (CySCs) in the self-restrained regulation of niche activity, which could be shed light on niche activity regulation in general.

Tuesday June 19th

Vagnoni, A. and Bullock, S. L. (2018). A cAMP/PKA/Kinesin-1 axis promotes the axonal transport of mitochondria in aging Drosophila neurons. Curr Biol 28(8): 1265-1272.e1264. PubMed ID: 29606421
Mitochondria play fundamental roles within cells, including energy provision, calcium homeostasis, and the regulation of apoptosis. The transport of mitochondria by microtubule-based motors is critical for neuronal structure and function. This process allows local requirements for mitochondrial functions to be met and also facilitates recycling of these organelles. An age-related reduction in mitochondrial transport has been observed in neurons of mammalian and non-mammalian organisms, and has been proposed to contribute to the broader decline in neuronal function that occurs during aging. However, the factors that influence mitochondrial transport in aging neurons are poorly understood. This study provides evidence using the tractable Drosophila wing nerve system that the cyclic AMP/protein kinase A (cAMP/PKA) pathway promotes the axonal transport of mitochondria in adult neurons. The level of the catalytic subunit of PKA decreases during aging, and acute activation of the cAMP/PKA pathway in aged flies strongly stimulates mitochondrial motility. Thus, the age-related impairment of transport is reversible. The expression of many genes is increased by PKA activation in aged flies. However, the results indicate that elevated mitochondrial transport is due in part to upregulation of the heavy chain of the kinesin-1 motor, the level of which declines during aging. This study identifies evolutionarily conserved factors that can strongly influence mitochondrial motility in aging neurons.
Kweon, S. H., Lee, J., Lim, C. and Choe, J. (2018). High-amplitude circadian rhythms in Drosophila driven by Calcineurin-mediated post-translational control of sarah. Genetics. PubMed ID: 29724861
Post-translational control is a crucial mechanism for circadian timekeeping. Evolutionarily conserved kinases and phosphatases have been implicated in circadian phosphorylation and degradation of clock-relevant proteins, which sustain high-amplitude rhythms with 24 hr periodicity in animal behaviors and physiology. This study reports a novel clock function of the heterodimeric Ca(2+)/calmodulin-dependent phosphatase calcineurin and its regulator sarah (sra) in Drosophila. Genomic deletion of the sra locus dampened circadian locomotor activity rhythms in free-running constant dark after entrainment in light-dark cycles. Poor rhythms in sra mutant behaviors were accompanied by lower expression of two oscillating clock proteins, PERIOD (PER) and TIMELESS (TIM), at the post-transcriptional level. RNA interference-mediated sra depletion in circadian pacemaker neurons was sufficient to phenocopy loss-of-function mutation in sra. On the other hand, a constitutively active form of the catalytic calcineurin subunit, Pp2B-14D(ACT), shortened circadian periodicity in locomotor behaviors and phase-advanced PER and TIM rhythms when overexpressed in clock neurons. Heterozygous sra deletion induced behavioral arrhythmicity in Pp2B-14D(ACT) flies, whereas sra overexpression rescued short periods in these animals. Finally, pharmacological inhibition of calcineurin in either wild-type flies or clock-less S2 cells decreased the levels of PER and TIM, likely by facilitating their proteasomal degradation. Taken together, these data suggest that sra negatively regulates calcineurin by cell-autonomously titrating calcineurin-dependent stabilization of PER and TIM proteins, thereby sustaining high-amplitude behavioral rhythms in Drosophila.
Top, D., O'Neil, J. L., Merz, G. E., Dusad, K., Crane, B. R. and Young, M. W. (2018). CK1/Doubletime activity delays transcription activation in the circadian clock. Elife 7. PubMed ID: 29611807
In the Drosophila circadian clock, Period (PER) and Timeless (TIM) proteins inhibit Clock-mediated transcription of per and tim genes until PER is degraded by Doubletime/CK1 (DBT)-mediated phosphorylation, establishing a negative feedback loop. Multiple regulatory delays within this feedback loop ensure ~24 hr periodicity. Of these delays, the mechanisms that regulate delayed PER degradation (and Clock reactivation) remain unclear. This study shows that phosphorylation of certain DBT target sites within a central region of PER affect PER inhibition of Clock and the stability of the PER/TIM complex. The results indicate that phosphorylation of PER residue S589 stabilizes and activates PER inhibitory function in the presence of TIM, but promotes PER degradation in its absence. The role of DBT in regulating PER activity, stabilization and degradation ensures that these events are chronologically and biochemically linked, and contributes to the timing of an essential delay that influences the period of the circadian clock.
Katsukawa, M., Ohsawa, S., Zhang, L., Yan, Y. and Igaki, T. (2018). Serpin facilitates tumor-suppressive cell competition by blocking Toll-mediated Yki activation in Drosophila. Curr Biol. PubMed ID: 29804808
Normal epithelial tissue exerts an intrinsic tumor-suppressive effect against oncogenically transformed cells. In Drosophila imaginal epithelium, clones of oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg) are eliminated by cell competition when surrounded by wild-type cells. In this study, a genetic screen in Drosophila identified Serpin5 (Spn5), a secreted negative regulator of Toll signaling, as a crucial factor for epithelial cells to eliminate scrib mutant clones from epithelium. Downregulation of Spn5 in wild-type cells leads to elevation of Toll signaling in neighboring scrib cells. Strikingly, forced activation of Toll signaling or Toll-related receptor (TRR) signaling in scrib clones transforms scrib cells from losers to supercompetitors, resulting in tumorous overgrowth of mutant clones. Mechanistically, Toll activation in scrib clones leads to c-Jun N-terminal kinase (JNK) activation and F-actin accumulation, which cause strong activation of the Hippo pathway effector Yorkie that blocks cell death and promotes cell proliferation. These data suggest that Spn5 secreted from normal epithelial cells acts as a component of the extracellular surveillance system that facilitates elimination of pre-malignant cells from epithelium.
Blount, J. R., Libohova, K., Marsh, G. B., Sutton, J. R. and Todi, S. V. (2018). Expression and regulation of deubiquitinase-resistant, unanchored ubiquitin chains in Drosophila. Sci Rep 8(1): 8513. PubMed ID: 29855490
The modifier protein, ubiquitin (Ub) regulates various cellular pathways by controlling the fate of substrates to which it is conjugated. Ub moieties are also conjugated to each other, forming chains of various topologies. In cells, poly-Ub is attached to proteins and also exists in unanchored form. Accumulation of unanchored poly-Ub is thought to be harmful and quickly dispersed through dismantling by deubiquitinases (DUBs). This study asked whether disassembly by DUBs is necessary to control unanchored Ub chains in vivo. Drosophila melanogaster lines were generated that express Ub chains non-cleavable into mono-Ub by DUBs. These chains are rapidly modified with different linkages and represent various types of unanchored species. Unanchored poly-Ub is not devastating in Drosophila, under normal conditions or during stress. The DUB-resistant, free Ub chains are degraded by the proteasome, at least in part through the assistance of VCP and its cofactor, p47. Also, unanchored poly-Ub that cannot be cleaved by DUBs can be conjugated en bloc, in vivo. These results indicate that unanchored poly-Ub species need not be intrinsically toxic; they can be controlled independently of DUB-based disassembly by being degraded, or through conjugation onto other proteins.
Fisher, K. H., Fragiadaki, M., Pugazhendhi, D., Bausek, N., Arredondo, M. A., Thomas, S. J., Brown, S. and Zeidler, M. P. (2018). A genome-wide RNAi screen identifies MASK as a positive regulator of cytokine receptor stability. J Cell Sci. PubMed ID: 29848658
Cytokine receptors often act via the Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT) pathway to form a signalling cascade essential for processes such as haematopoiesis, immune responses and tissue homeostasis. In order to transduce ligand activation, cytokine receptors must dimerise. However, mechanisms regulating their dimerisation are poorly understood. In order to better understand the processes regulating cytokine receptor levels, activity and dimerisation, the highly conserved JAK/STAT pathway in Drosophila was used that acts via a single receptor, known as Domeless. A genome-wide RNAi screen was performed in Drosophila cells, identifying MASK as a positive regulator of Domeless dimerisation and protein levels. MASK is able to regulate receptor levels and JAK/STAT signalling both in vitro and in vivo. The human homologue, ANKHD1, is also able to regulate JAK/STAT signalling and the levels of a subset of pathway receptors in human cells. Taken together, these results identify MASK as a novel regulator of cytokine receptor levels, and suggest functional conservation, which may have implications for human health.

Monday, June 18 - Chromatin

Horard, B., Sapey-Triomphe, L., Bonnefoy, E. and Loppin, B. (2018). ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization. Epigenetics Chromatin 11(1): 19. PubMed ID: 29751847
Anti-Silencing Factor 1 (ASF1) is a conserved H3-H4 histone chaperone involved in both Replication-Coupled and Replication-Independent (RI) nucleosome assembly pathways. At DNA replication forks, ASF1 plays an important role in regulating the supply of H3.1/2 and H4 to the CAF-1 chromatin assembly complex. ASF1 also provides H3.3-H4 dimers to HIRA and DAXX chaperones for RI nucleosome assembly. The early Drosophila embryo is an attractive system to study chromatin assembly in a developmental context. The formation of a diploid zygote begins with the unique, genome-wide RI assembly of paternal chromatin following sperm protamine eviction. Then, within the same cytoplasm, syncytial embryonic nuclei undergo a series of rapid, synchronous S and M phases to form the blastoderm embryo. This study has investigated the implication of ASF1 in these two distinct assembly processes. Depletion of the maternal pool of ASF1 with a specific shRNA induces a fully penetrant, maternal effect embryo lethal phenotype. Unexpectedly, despite the depletion of ASF1 protein to undetectable levels, asf1 knocked-down (KD) embryos can develop to various stages, thus demonstrating that ASF1 is not absolutely required for the amplification of cleavage nuclei. Remarkably, it was found that ASF1 is required for the formation of the male pronucleus, although ASF1 protein does not reside in the decondensing sperm nucleus. In asf1 KD embryos, HIRA localizes to the male nucleus but is only capable of limited and insufficient chromatin assembly. Finally, it was shown that the conserved HIRA B domain, which is involved in ASF1-HIRA interaction, is dispensable for female fertility. It is concluded that ASF1 is critically required to load H3.3-H4 dimers on the HIRA complex prior to histone deposition on paternal DNA.
Ellis, K., Wardwell-Ozgo, J., Moberg, K. H. and Yedvobnick, B. (2018). The domino SWI2/SNF2 gene product represses cell death in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 29752350
The Drosophila domino locus encodes DNA-dependent ATPases of the SWI2/SNF2 class. This class of chromatin remodeler is associated with an array of cellular activities encompassing transcription, replication, repair and recombination. Moreover, domino was observed initially to maintain a repressive chromatin state via genetic interaction studies with homeotic genes. Although domino mutations were also characterized with a cell death phenotype, its association with a death pathway has not been investigated. This study has used targeted RNA interference to depress domino function in the wing. Resultant wing damage phenotypes were found to be enhanced through overexpression of pro-apoptotic loci, and suppressed through loss of function of these loci. Loss of wing margin and blade tissue was correlated with activation of the effector Caspase Dcp-1, a marker for apoptosis. The affected wing regions also exhibited lower levels of the DIAP1 protein, an inhibitor of apoptosis. The lower level of DIAP1 protein was not correlated with an effect on the activity of a DIAP1 gene transgenic reporter (thread-LacZ), suggesting that loss of DIAP1 occurred post transcriptionally. In some cases excessive cell proliferation within the targeted tissue, measured through BrdU incorporation, was also observed. Finally, a transgenic reporter construct was used to monitor the chromatin state upstream of the proapoptotic reaper locus. In genotypes exhibiting targeted domino loss and wing phenotypes, increased reporter activity was observed only in the affected areas. These data support the conclusion that domino normally functions to maintain pro-apoptotic genes in a repressed state.
Funikov, S. Y., Kulikova, D. A., Krasnov, G. S., Rezvykh, A. P., Chuvakova, L. N., Shostak, N. G., Zelentsova, E. S., Blumenstiel, J. P. and Evgen'ev, M. B. (2018). Spontaneous gain of susceptibility suggests a novel mechanism of resistance to hybrid dysgenesis in Drosophila virilis. PLoS Genet 14(5): e1007400. PubMed ID: 29813067
Syndromes of hybrid dysgenesis (HD) have been critical for understanding of the transgenerational maintenance of genome stability by piRNA. HD in D. virilis represents a special case of HD since it includes simultaneous mobilization of a set of TEs that belong to different classes. The standard explanation for HD is that eggs of the responder strains lack an abundant pool of piRNAs corresponding to the asymmetric TE families transmitted solely by sperm. However, there are several strains of D. virilis that lack asymmetric TEs, but exhibit a "neutral" cytotype that confers resistance to HD. To characterize the mechanism of resistance to HD, a comparative analysis of the landscape of ovarian small RNAs was performed in strains that vary in their resistance to HD mediated sterility. It was demonstrated that resistance to HD cannot be solely explained by a maternal piRNA pool that matches the assemblage of TEs that likely cause HD. In support of this, a cytotype shift from neutral (N) to susceptible (M) was observed in a strain devoid of all major TEs implicated in HD. This shift occurred in the absence of significant change in TE copy number and expression of piRNAs homologous to asymmetric TEs. Instead, this shift is associated with a change in the chromatin profile of repeat sequences unlikely to be causative of paternal induction. Overall, these data suggest that resistance to TE-mediated sterility during HD may be achieved by mechanisms that are distinct from the canonical syndromes of HD.
Hirai, K., Wang, Z., Miura, K., Hayashi, T., Awasaki, T., Wada, M., Keira, Y., Ishikawa, H. O. and Sawamura, K. (2018). Genetic analyses of Elys mutations in Drosophila show maternal-effect lethality and interactions with nucleoporin genes. G3 (Bethesda). PubMed ID: 29773558
ELYS determines the subcellular localizations of Nucleoporins (Nups) during interphase and mitosis. This study made loss-of-function mutations of Elys in Drosophila melanogaster and found that ELYS is dispensable for zygotic viability and male fertility but the maternal supply is necessary for embryonic development. Subsequent to fertilization, mitotic progression of the embryos produced by the mutant females is severely disrupted at the first cleavage division, accompanied by irregular behavior of mitotic centrosomes. The Nup160 introgression from D. simulans shows close resemblance to that of the Elys mutations, suggesting a common role for those proteins in the first cleavage division. Genetic experiments indicated critical interactions between ELYS and three Nup107-160 subcomplex components; hemizygotes of either Nup37, Nup96 or Nup160 were lethal in the genetic background of the Elys mutation. Not only Nup96 and Nup160 but also Nup37 of D. simulans behave as recessive hybrid incompatibility genes with D. melanogaster. An evolutionary analysis indicated positive natural selection in the ELYS-like domain of ELYS. This study proposes that genetic incompatibility between Elys and Nups may lead to reproductive isolation between D. melanogaster and D. simulans, although direct evidence is necessary.
Haines, J. E. and Eisen, M. B. (2018). Patterns of chromatin accessibility along the anterior-posterior axis in the early Drosophila embryo. PLoS Genet 14(5): e1007367. PubMed ID: 29727464
As the Drosophila embryo transitions from the use of maternal RNAs to zygotic transcription, domains of open chromatin, with relatively low nucleosome density and specific histone marks, are established at promoters and enhancers involved in patterned embryonic transcription. However it remains unclear how regions of activity are established during early embryogenesis, and if they are the product of spatially restricted or ubiquitous processes. To shed light on this question, chromatin accessibility across the anterior-posterior axis (A-P) of early Drosophila melanogaster embryos was probed by applying a transposon based assay for chromatin accessibility (ATAC-seq) to anterior and posterior halves of hand-dissected, cellular blastoderm embryos. Genome-wide chromatin accessibility is highly similar between the two halves, with regions that manifest significant accessibility in one half of the embryo almost always accessible in the other half, even for promoters that are active in exclusively one half of the embryo. These data support previous studies that show that chromatin accessibility is not a direct result of activity, and point to a role for ubiquitous factors or processes in establishing chromatin accessibility at promoters in the early embryo. However, in concordance with similar works, this study found that at enhancers active exclusively in one half of the embryo, a significant skew was found towards greater accessibility in the region of their activity, highlighting the role of patterning factors such as Bicoid in this process.
Kovac, K., Sauer, A., Macinkovic, I., Awe, S., Finkernagel, F., Hoffmeister, H., Fuchs, A., Muller, R., Rathke, C., Langst, G. and Brehm, A. (2018). Tumour-associated missense mutations in the dMi-2 ATPase alters nucleosome remodelling properties in a mutation-specific manner. Nat Commun 9(1): 2112. PubMed ID: 29844320
ATP-dependent chromatin remodellers are mutated in more than 20% of human cancers. The consequences of these mutations on enzyme function are poorly understood. This study characterised the effects of CHD4 mutations identified in endometrial carcinoma on the remodelling properties of dMi-2, the highly conserved Drosophila homologue of CHD4. Mutations from different patients have surprisingly diverse defects on nucleosome binding, ATPase activity and nucleosome remodelling. Unexpectedly, both mutations that decrease and increase the enzyme activity were identified. The results define the chromodomains and a novel regulatory region as essential for nucleosome remodelling. Genetic experiments in Drosophila demonstrate that expression of cancer-derived dMi-2 mutants misregulates differentiation of epithelial wing structures and produces phenotypes that correlate with their nucleosome remodelling properties. These results help to define the defects of CHD4 in cancer at the mechanistic level and provide the basis for the development of molecular approaches aimed at restoring their activity.

Friday, June 15th - Junctions and cytoskeleton

Weiner, A. T., Seebold, D. Y., Michael, N. L., Guignet, M., Feng, C., Follick, B., Yusko, B. A., Wasilko, N. P., Torres-Gutierrez, P. and Rolls, M. M. (2018). Identification of proteins required for precise positioning of Apc2 in dendrites. G3 (Bethesda). PubMed ID: 29602811
In Drosophila neurons, uniform minus-end-out polarity in dendrites is maintained in part by kinesin-2-mediated steering of growing microtubules at branch points. Apc links the kinesin motor to growing microtubule plus ends and Apc2 recruits Apc to branch points where it functions. Because Apc2 acts to concentrate other steering proteins to branch points, it is of interest to understand how Apc2 is targeted. From an initial broad candidate RNAi screen, Miro (a mitochondrial transport protein), Ank2, Axin, spastin and Rac1 were required to position Apc2-GFP at dendrite branch points. YFP-Ank2-L8, Axin-GFP and mitochondria also localized to branch points suggesting the screen identified relevant proteins. By performing secondary screens, it was found that energy production by mitochondria was key for Apc2-GFP positioning and spastin acted upstream of mitochondria. Ank2 seems to act independently from other players, except its membrane partner, Neuroglian (Nrg). Rac1 likely acts through Arp2/3 to generate branched actin to help recruit Apc2-GFP. Axin can function in a variety of wnt signaling pathways, one of which includes heterotrimeric G proteins and Frizzleds. Knockdown of Gas, Gao, Fz and Fz2, reduced targeting of Apc2 and Axin to branch points. Overall these data suggest that mitochondrial energy production, Nrg/Ank2, branched actin generated by Arp2/3 and Fz/G proteins/Axin function as four modules that control localization of the microtubule regulator Apc2 to its site of action in dendrite branch points.
Daniel, E., Daude, M., Kolotuev, I., Charish, K., Auld, V. and Le Borgne, R. (2018). Coordination of septate junctions assembly and completion of cytokinesis in proliferative epithelial tissues. Curr Biol [Epub ahead of print]. PubMed ID: 29706514
How permeability barrier function is maintained when epithelial cells divide is largely unknown. This study has investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. Following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. It is proposed that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia.
Herzmann, S., Gotzelmann, I., Reekers, L. F. and Rumpf, S. (2018). Spatial regulation of microtubule disruption during dendrite pruning in Drosophila. Development [Epub ahead of print]. PubMed ID: 29712642
Large scale neurite pruning is an important specificity mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their larval dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions, but how this spatial information is encoded is not clear. Dendrite severing is preceded by local breakdown of dendritic microtubules through PAR-1-mediated inhibition of Tau. This study investigated spatial aspects of microtubule breakdown during dendrite pruning. Live imaging of fluorescently tagged tubulin shows that microtubule breakdown first occurs at proximal dendritic branchpoints, followed by breakdown at more distal branchpoints, suggesting that the process is triggered by a signal emanating from the soma. In fly dendrites, microtubules are arranged in uniformly oriented arrays where all plus ends face towards the soma. Mutants in kinesin-1 and -2, which are required for uniform microtubule orientation, cause defects in microtubule breakdown and dendrite pruning. These data suggest that the local microtubule organization at branch points determines where microtubule breakdown occurs. Local microtubule organization may therefore contribute spatial information for severing sites during dendrite pruning.
Shukla, V. K., Maheshwari, D., Jain, A., Tripathi, S., Kumar, D. and Arora, A. (2018). Structure, dynamics, and biochemical characterization of ADF/cofilin Twinstar from Drosophila melanogastor. Biochim Biophys Acta. PubMed ID: 29709602
Twinstar is an ADF/Cofilin family protein, which is expressed by the tsr gene in Drosophila melanogaster. Twinstar is one of the main regulators of actin cytoskeleton remodelling and is essential for vital cellular processes like cytokinesis and endocytosis. This study has characterized the structure and dynamics of Twinstar by solution NMR spectroscopy. The solution structure of Twinstar shows characteristic ADF-H fold with well-formed G/F-site and F-site for interaction with actin. The structure possesses an extended F-loop, which is rigid at the base, but flexible towards its apical region. Twinstar shares similar dynamics for the G/F-site with C. elegans homologs, UNC-60A and UNC-60B. However, the dynamics of its F-loop are different from its C. elegans homologs. Twinstar shows strong affinity for ADP-G-Actin and ATP-G-Actin with Kds of ~7.6nM and ~0.4mμM, respectively. It shows mild F-actin depolymerizing activity and stable interaction with F-actin with a Kd of ~5.0mμM. It inhibits the rate of the nucleotide exchange in a dose dependent manner. On the basis of structure, dynamics, and biochemical activity, Twinstar can be taken to execute its biochemical role by facilitating directional growth and maintenance of length of actin filaments. This study characterizes the structure, backbone dynamics, and biochemical activities of Twinstar of Drosophila, which provides an insight into the regulation of actin dynamics in the member of phylum insecta.
Kosakamoto, H., Fujisawa, Y., Obata, F. and Miura, M. (2018). High expression of A-type lamin in the leading front is required for Drosophila thorax closure. Biochem Biophys Res Commun 499(2): 209-214. PubMed ID: 29559239
Tissue closure involves the coordinated unidirectional movement of a group of cells without loss of cell-cell contact. However, the molecular mechanisms controlling the tissue closure are not fully understood. This study demonstrates that Lamin C, the sole A-type lamin in Drosophila, contributes to the process of thorax closure in pupa. High expression of Lamin C was observed at the leading front of the migrating wing imaginal discs. Live imaging analysis revealed that knockdown of Lamin C in the thorax region affected the coordinated movement of the leading front, resulting in incomplete tissue fusion required for formation of the adult thorax. The closure defect due to knockdown of Lamin C correlated with insufficient accumulation of F-actin at the front. This study indicates a link between A-type lamin and the cell migration behavior during tissue closure.
Qin, X., Hannezo, E., Mangeat, T., Liu, C., Majumder, P., Liu, J., Choesmel-Cadamuro, V., McDonald, J. A., Liu, Y., Yi, B. and Wang, X. (2018). A biochemical network controlling basal myosin oscillation. Nat Commun 9(1): 1210. PubMed ID: 29572440
The actomyosin cytoskeleton, a key stress-producing unit in epithelial cells, oscillates spontaneously in a wide variety of systems. Although much of the signal cascade regulating myosin activity has been characterized, the origin of such oscillatory behavior is still unclear. This study shows that basal myosin II oscillation in Drosophila ovarian epithelium is not controlled by actomyosin cortical tension, but instead relies on a biochemical oscillator involving ROCK and myosin phosphatase. Key to this oscillation is a diffusive ROCK flow, linking junctional Rho1 to medial actomyosin cortex, and dynamically maintained by a self-activation loop reliant on ROCK kinase activity. In response to the resulting myosin II recruitment, myosin phosphatase is locally enriched and shuts off ROCK and myosin II signals. Coupling Drosophila genetics, live imaging, modeling, and optogenetics, this study uncovered an intrinsic biochemical oscillator at the core of myosin II regulatory network, shedding light on the spatio-temporal dynamics of force generation.

Thursday, June 14th - Apoptosis

Hilu-Dadia, R., Hakim-Mishnaevski, K., Levy-Adam, F. and Kurant, E. (2018). Draper-mediated JNK signaling is required for glial phagocytosis of apoptotic neurons during Drosophila metamorphosis. Glia [Epub ahead of print]. PubMed ID: 29520845
Development of the central nervous system involves elimination of superfluous neurons through apoptosis and subsequent phagocytosis. In Drosophila, this occurs mainly during three developmental stages: embryogenesis, metamorphosis and emerging adult. Two transmembrane glial phagocytic receptors, SIMU (homolog of the mammalian Stabilin-2) and Draper (homolog of the mammalian MEGF10 and Jedi), mediate glial phagocytosis of apoptotic neurons during embryogenesis. However, less is known about the removal of apoptotic neurons during later stages of development. This study shows that during metamorphosis, Draper plays a critical role in apoptotic cell clearance by glia, whereas SIMU, which is mostly expressed in pupal macrophages outside the brain, is not involved in glial phagocytosis. Draper was found to activate Drosophila c-Jun N-terminal kinase (dJNK) signaling predominantly in the ensheathing glia and astrocytes, where it is required for efficient removal of apoptotic neurons. These data suggest that besides the dJNK pathway, Draper also triggers an additional signaling pathway capable of removing apoptotic neurons in the pupal brain. This study thus reveals that SIMU unexpectedly is not involved in glial phagocytosis of apoptotic neurons during metamorphosis and highlights the novel role of dJNK signaling in developmental apoptotic cell clearance downstream of Draper.
Kang, I., Choi, Y., Jung, S., Lim, J. Y., Lee, D., Gupta, S., Moon, W. and Shin, C. (2018). Identification of target genes regulated by the Drosophila histone methyltransferase Eggless reveals a role of Decapentaplegic in apoptotic signaling. Sci Rep 8(1): 7123. PubMed ID: 29740006
Epigenetic gene regulation is essential for developmental processes. Eggless (Egg), the Drosophila orthologue of the mammalian histone methyltransferase, SETDB1, is known to be involved in the survival and differentiation of germline stem cells and piRNA cluster transcription during Drosophila oogenesis; however the detailed mechanisms remain to be determined. Using high-throughput RNA sequencing this study investigated target genes regulated by Egg in an unbiased manner. Egg was shown to play diverse roles in particular piRNA pathway gene expression, some long non-coding RNA expression, apoptosis-related gene regulation, and Decapentaplegic (Dpp) signaling during Drosophila oogenesis. Furthermore, using genetic and cell biological approaches, this study demonstrate that ectopic upregulation of dpp caused by loss of Egg in the germarium can trigger apoptotic cell death through activation of two pro-apoptotic genes, reaper and head involution defective. A model is proposed in which Egg regulates germ cell differentiation and apoptosis through canonical and noncanonical Dpp pathways in Drosophila oogenesis.
Amcheslavsky, A., Wang, S., Fogarty, C. E., Lindblad, J. L., Fan, Y. and Bergmann, A. (2018). Plasma membrane localization of apoptotic caspases for non-apoptotic functions. Dev Cell 45(4): 450-464.e453. PubMed ID: 29787709
Caspases are best characterized for their function in apoptosis. However, they also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), where caspases release mitogens for compensatory proliferation independently of their apoptotic role. This study reports that the unconventional myosin, Myo1D, which is known for its involvement in left/right development, is an important mediator of AiP in Drosophila. Mechanistically, Myo1D translocates the initiator caspase Dronc to the basal side of the plasma membrane of epithelial cells where Dronc promotes the activation of the NADPH-oxidase Duox for reactive oxygen species generation and AiP in a non-apoptotic manner. It is proposed that the basal side of the plasma membrane constitutes a non-apoptotic compartment for caspases. Finally, Myo1D promotes tumor growth and invasiveness of the neoplastic scrib Ras(V12) model. Together, these studies have identified a new function of Myo1D for AiP and tumorigenesis and reveal a mechanism by which cells sequester apoptotic caspases in a non-apoptotic compartment at the plasma membrane.
Dutta, D., Singh, A., Paul, M. S., Sharma, V., Mutsuddi, M. and Mukherjee, A. (2018). Deltex interacts with Eiger and consequently influences the cell death in Drosophila melanogaster. Cell Signal 49: 17-29. PubMed ID: 29775737
TNF-JNK signaling is one of the highly conserved signaling pathways that regulate a broad spectrum of cellular processes including proliferation and apoptosis. Eiger, the sole homologue of TNF in Drosophila, initiates the TNF-JNK pathway to induce cell death. Previously, Deltex (Dx) has been identified as a Notch signaling component that regulates vesicular trafficking of Notch. This study investigated the interaction between these two proteins in order to identify how Dx influences the activity of Eiger. Dx is found to act as a novel modulator of JNK-mediated cell death inducing activity of Eiger. Additionally, it was observed that dx genetically interacts with eiger during wing development, and these two proteins, Dx and Eiger, colocalize in the cytoplasm. This analysis reveals that Dx is involved in the cytoplasmic relocalization of Eiger from the cell membrane, thereby influencing Eiger-mediated JNK-activation process. Moreover, this study demonstrated that Dx potentiates the activity of Eiger to downregulate Notch signaling pathway by retaining the Notch protein in the cytoplasm. Together, these findings reveal a novel role of Dx to modulate the signaling activity of Eiger and subsequent JNK-mediated cell death.
Cornelissen, T., Vilain, S., Vints, K., Gounko, N., Verstreken, P. and Vandenberghe, W. (2018). Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila. Elife 7. PubMed ID: 29809156
Mutations in the genes for PINK1 and parkin cause Parkinson's disease. PINK1 and parkin cooperate in the selective autophagic degradation of damaged mitochondria (mitophagy) in cultured cells. However, evidence for their role in mitophagy in vivo is still scarce. This study generated a Drosophila model expressing the mitophagy probe mt-Keima. Using live mt-Keima imaging and correlative light and electron microscopy (CLEM) it was shown that mitophagy occurs in muscle cells and dopaminergic neurons in vivo, even in the absence of exogenous mitochondrial toxins. Mitophagy increases with aging, and this age-dependent rise is abrogated by PINK1 or parkin deficiency. Knockdown of the Drosophila homologues of the deubiquitinases USP15 and, to a lesser extent, USP30, rescues mitophagy in the parkin-deficient flies. These data demonstrate a crucial role for parkin and PINK1 in age-dependent mitophagy in Drosophila in vivo.
Sinha, N. K., Iwasa, J., Shen, P. S. and Bass, B. L. (2018). Dicer uses distinct modules for recognizing dsRNA termini. Science 359(6373): 329-334. PubMed ID: 29269422
Invertebrates rely on Dicer to cleave viral double-stranded RNA (dsRNA), and Drosophila Dicer-2 distinguishes dsRNA substrates by their termini. Blunt termini promote processive cleavage, while 3' overhanging termini are cleaved distributively. To understand this discrimination, this study used cryo-electron microscopy to solve structures of Drosophila Dicer-2 alone and in complex with blunt dsRNA. Whereas the Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini, unexpectedly, this study found that the helicase domain is required for binding blunt, but not 3' overhanging, termini. The study further showed that blunt dsRNA is locally unwound and threaded through the helicase domain in an adenosine triphosphate-dependent manner. These studies reveal a previously unrecognized mechanism for optimizing antiviral defense and set the stage for the discovery of helicase-dependent functions in other Dicers.

Wednesday, June 13th - Synapse

Robinson, S. W., Bourgognon, J. M., Spiers, J. G., Breda, C., Campesan, S., Butcher, A., Mallucci, G. R., Dinsdale, D., Morone, N., Mistry, R., Smith, T. M., Guerra-Martin, M., Challiss, R. A. J., Giorgini, F. and Steinert, J. R. (2018). Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability. PLoS Biol 16(4): e2003611. PubMed ID: 29630591
Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. This study interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. NO was found to compromise synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, these data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release.
Kopke, D. L. and Broadie, K. (2018). FM dye cycling at the synapse: Comparing high potassium depolarization, electrical and channelrhodopsin stimulation. J Vis Exp(135). PubMed ID: 29889207
FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a >40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. This study compared and contrastd the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K(+)] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. These advantages and disadvantages are discussed to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. Comparisons are provided of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.
Sato, S., Ueno, K., Saitoe, M. and Sakai, T. (2018). Synaptic depression induced by postsynaptic cAMP production in the Drosophila mushroom body calyx. J Physiol. PubMed ID: 29659025
Synaptic plasticity has been studied to reveal the molecular and cellular mechanisms of associative and nonassociative learning. The fruit fly Drosophila melanogaster can be used to identify the molecular mechanisms of synaptic plasticity because vast genetic information or tools are available. By ex vivo Ca(2+) imaging of an isolated cultured Drosophila brain, this study examined the novel activity-dependent synaptic depression between the projection neurons of the antennal lobe (AL) and mushroom body (MB). Ex vivo Ca(2+) imaging analysis revealed that electrical stimulation of AL elicits Ca(2+) responses in the dendritic (calyx) and axonal (alpha lobe) regions of MB neurons, and the responses are reduced after repetitive AL stimulation. Since the cAMP signalling pathway plays an important role in synaptic plasticity in invertebrates and vertebrates, this study examined whether the reduction of Ca(2+) responses is also regulated by the cAMP signalling pathway. The expression of rutabaga (rut), which encodes Ca(2+) /calmodulin-dependent adenylyl cyclase, was essential for the reduction of Ca(2+) responses in the calyx and alpha lobe. Furthermore, imaging analysis using a fluorescence resonance energy transfer-based cAMP indicator revealed that the cAMP level increased in the wild-type calyx during repetitive AL stimulation, whereas it decreased in rut1 mutant flies with a loss-of-function mutation of rut. Thus, this study suggests that an increase in postsynaptic cAMP level during repetitive AL stimulation contributes to the attenuation of inputs at the AL-MB synapses.
Stocker, B., et al. (2018). Structural and molecular properties of insect type II motor axon terminals. Front Syst Neurosci 12: 5. PubMed ID: 29615874
A comparison between the axon terminals of octopaminergic efferent dorsal or ventral unpaired median neurons in either desert locusts (Schistocerca) or fruit flies (Drosophila across skeletal muscles reveals many similarities. In both species the octopaminergic axon forms beaded fibers where the boutons or varicosities form type II terminals in contrast to the neuromuscular junction (NMJ) or type I terminals. These type II terminals are immunopositive for both tyramine and octopamine and, in contrast to the type I terminals, which possess clear synaptic vesicles, only contain dense core vesicles. These dense core vesicles contain octopamine as shown by immunogold methods. With respect to the cytomatrix and active zone peptides the type II terminals exhibit active zone-like accumulations of the scaffold protein Bruchpilot (BRP) only sparsely in contrast to the many accumulations of BRP identifying active zones of NMJ type I terminals. In the fruit fly larva marked dynamic changes of octopaminergic fibers have been reported after short starvation which not only affects the formation of new branches ("synaptopods") but also affects the type I terminals or NMJs via octopamine-signaling. Starvation experiments of Drosophila-larvae revealed a time-dependency of the formation of additional branches. Whereas after 2 h of starvation a decrease was found in "synaptopods", the increase is significant after 6 h of starvation. In addition, evidence is provided that the release of octopamine from dendritic and/or axonal type II terminals uses a similar synaptic machinery to glutamate release from type I terminals of excitatory motor neurons. Indeed, blocking this canonical synaptic release machinery via RNAi induced downregulation of BRP in neurons with type II terminals leads to flight performance deficits similar to those observed for octopamine mutants or flies lacking this class of neurons.
Goel, P. and Dickman, D. (2018). Distinct homeostatic modulations stabilize reduced postsynaptic receptivity in response to presynaptic DLK signaling. Nat Commun 9(1): 1856. PubMed ID: 29748610
Synapses are constructed with the stability to last a lifetime, yet sufficiently flexible to adapt during injury. Although fundamental pathways that mediate intrinsic responses to neuronal injury have been defined, less is known about how synaptic partners adapt. This study investigated responses in the postsynaptic cell to presynaptic activation of the injury-related Dual Leucine Zipper Kinase pathway at the Drosophila neuromuscular junction. The postsynaptic compartment reduces neurotransmitter receptor levels, thus depressing synaptic strength. Interestingly, this diminished state is stabilized through distinct modulations to two postsynaptic homeostatic signaling systems. First, a retrograde response normally triggered by reduced receptor levels is silenced, preventing a compensatory enhancement in presynaptic neurotransmitter release. However, when global presynaptic release is attenuated, a postsynaptic receptor scaling mechanism persists to adaptively stabilize this diminished neurotransmission state. Thus, the homeostatic set point of synaptic strength is recalibrated to a reduced state as synapses acclimate to injury.
West, R. J. H., Briggs, L., Perona Fjeldstad, M., Ribchester, R. R. and Sweeney, S. T. (2018). Sphingolipids regulate neuromuscular synapse structure and function in Drosophila. J Comp Neurol. PubMed ID: 29761896
Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function and degeneration. Serine Palmitoyl-transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to approximately 50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig-domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. This data points to a role for sphingolipids in the regulation and fine-tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg.

Tuesday, June 12th - Adult Development

Zhang, T., Song, W., Li, Z., Qian, W., Wei, L., Yang, Y., Wang, W., Zhou, X., Meng, M., Peng, J., Xia, Q., Perrimon, N. and Cheng, D. (2018). Kruppel homolog 1 represses insect ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes. Proc Natl Acad Sci U S A 115(15): 3960-3965. PubMed ID: 29567866
In insects, juvenile hormone (JH) and the steroid hormone ecdysone have opposing effects on regulation of the larval-pupal transition. Although increasing evidence suggests that JH represses ecdysone biosynthesis during larval development, the mechanism underlying this repression is not well understood. This study demonstrates that the expression of the Kruppel homolog 1 (Kr-h1), a gene encoding a transcription factor that mediates JH signaling, in ecdysone-producing organ prothoracic gland (PG) represses ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes in both Drosophila and Bombyx. Application of a JH mimic on ex vivo cultured PGs from Drosophila and Bombyx larvae induces Kr-h1 expression and inhibits the transcription of steroidogenic enzymes. In addition, PG-specific knockdown of Drosophila Kr-h1 promotes-while overexpression hampers-ecdysone production and pupariation. It was further found that Kr-h1 inhibits the transcription of steroidogenic enzymes by directly binding to their promoters to induce promoter DNA methylation. Finally, it was shown that Kr-h1 does not affect DNA replication in Drosophila PG cells and that the reduction of PG size mediated by Kr-h1 overexpression can be rescued by feeding ecdysone. Taken together, these data indicate direct and conserved Kr-h1 repression of insect ecdysone biosynthesis in response to JH stimulation, providing insights into mechanisms underlying the antagonistic roles of JH and ecdysone.
Rotstein, B., Post, Y., Reinhardt, M., Lammers, K., Buhr, A., Heinisch, J. J., Meyer, H. and Paululat, A. (2018). Distinct domains in the matricellular protein Lonely heart are crucial for cardiac extracellular matrix formation and heart function in Drosophila. J Biol Chem [Epub ahead of print]. PubMed ID: 29599288
The correct balance between stiffness and elasticity is essential to the function of numerous tissues, and depends on ECM constituents (the matrisome). However, despite its physiological relevance, the matrisome composition and organization remain poorly understood. Previously, it was reported that the ADAMTS-like protein Lonely heart (Loh) is critical for recruiting the type IV collagen-like protein Pericardin to the cardiac ECM. Thus study utilized Drosophila as a simple and genetically amenable invertebrate model for studying Loh-mediated recruitment of tissue-specific ECM components such as Pericardin to the ECM. Focus was placed on the functional relevance of distinct Loh domains to protein localization and Pericardin recruitment. Analysis of Loh deletion constructs revealed that one thrombospondin type 1 repeat (TSR1-1), which has an embedded WXXW motif, is critical for anchoring Loh to the ECM. Two other thrombospondin repeats, TSR1-2 and TSR1-4, the latter containing a CXXTCXXG motif, appeared to be dispensable for tethering Loh to the ECM, but were crucial for proper interaction with and recruitment of Pericardin. Moreover, the results also suggested that Pericardin in the cardiac ECM primarily ensures the structural integrity of the heart, rather than increasing tissue flexibility. In conclusion, this work provides new insight into the roles of thrombospondin type 1 repeats and advances understanding of cardiac ECM assembly and function.
Hinaux, H., Bachem, K., Battistara, M., Rossi, M., Xin, Y., Jaenichen, R., Le Poul, Y., Arnoult, L., Kobler, J. M., Grunwald Kadow, I. C., Rodermund, L., Prud'homme, B. and Gompel, N. (2018). Revisiting the developmental and cellular role of the pigmentation gene yellow in Drosophila using a tagged allele. Dev Biol [Epub ahead of print]. PubMed ID: 29634916
This study of pigment temporality focusing on yellow, a gene involved in the formation of black melanin. A protein-tagged yellow allele was developed in the fruit fly Drosophila melanogaster, which allowed precise description of Yellow expression pattern at the tissue and cellular levels throughout development. Yellow expressed in the pupal epidermis in patterns prefiguring black pigmentation. It was also found that Yellow expressed in a few central neurons from the second larval instar to adult stages, including a subset of neurons adjacent to the clock neurons marked by the gene Pdf. The dynamics of Yellow expression domain and subcellular localization were specifically examined in relationship to pigment formation. In particular, it was shown how a late step of re-internalization is regulated by the large low-density lipoprotein receptor-related protein Megalin. Finally a new function for Yellow is presented in the establishment of sharp pigmentation pattern boundaries, whereby this protein may assume a structural role, anchoring pigment deposits or pigmentation enzymes in the cuticle.
Riddiford, L. M., Truman, J. W. and Nern, A. (2018). Juvenile hormone reveals mosaic developmental programs in the metamorphosing optic lobe of Drosophila melanogaster. Biol Open 7(4). PubMed ID: 29618455
The development of the adult optic lobe (OL) of Drosophila melanogaster is directed by a wave of ingrowth of the photoreceptors over a 2-day period at the outset of metamorphosis, which is accompanied by the appearance of the pupal-specific transcription factor Broad-Z3 (Br-Z3) and expression of early drivers in OL neurons. During this time, there are pulses of ecdysteroids that time the metamorphic events. At the outset, the transient appearance of juvenile hormone (JH) prevents precocious development of the OL caused by the ecdysteroid peak that initiates pupariation, but the artificial maintenance of JH after this time misdirects subsequent development. Axon ingrowth, Br-Z3 appearance and the expression of early drivers were unaffected, but aspects of later development such as the dendritic expansion of the lamina monopolar neurons and the expression of late drivers were suppressed. This effect of the exogenous JH mimic (JHM) pyriproxifen is lost by 24 h after pupariation. Part of this effect of JHM is due to its suppression of the appearance of ecdysone receptor EcR-B1 that occurs after pupation and during early adult development.
Del Bel, L. M., Griffiths, N., Wilk, R., Wei, H. C., Blagoveshchenskaya, A., Burgess, J., Polevoy, G., Price, J. V., Mayinger, P. and Brill, J. A. (2018). The phosphoinositide phosphatase Sac1 regulates cell shape and microtubule stability in the developing Drosophila eye. Development 145(11). PubMed ID: 29752385
Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). This study using a novel temperature-sensitive (ts) allele, showed that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo Together, these data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner.
ADe Las Heras, J. M., Garcia-Cortes, C., Foronda, D., Pastor-Pareja, J. C., Shashidhara, L. S. and Sanchez-Herrero, E. (2018)c. The Drosophila Hox gene Ultrabithorax controls appendage shape by regulating extracellular matrix dynamics. Development. PubMed ID: 29853618
Although the specific form of an organ is frequently important for its function, the mechanisms underlying organ shape are largely unknown. In Drosophila, the wings and halteres, homologous appendages of the second and third thoracic segments, respectively, bear different forms: wings are flat whereas halteres are globular and yet both characteristic shapes are essential for a normal flight. The Hox gene Ultrabithorax governs the difference between wing and haltere development, but how Ultrabithorax function in the appendages prevents or allows flat or globular shapes is unknown. This study shows that Ultrabithorax down-regulates Matrix metalloproteinase1 expression in the haltere pouch at early pupal stage, which in turn prevents the rapid clearance of Collagen IV compared to the wing disc. This difference is instrumental in determining cell shape changes, expansion of the disc and apposition of dorsal and ventral layers, all of these phenotypic traits being characteristic of wing pouch development. These results suggest that Ultrabithorax regulates organ shape by controlling Matrix metalloproteinase1 expression and the extent and timing of extracellular matrix degradation.

Monday, July 11th - Embryonic Development

Hemmi, N., Akiyama-Oda, Y., Fujimoto, K. and Oda, H. (2018). A quantitative study of the diversity of stripe-forming processes in an arthropod cell-based field undergoing axis formation and growth. Dev Biol [Epub ahead of print]. PubMed ID: 29551694
Evolutionary Homolog Study
The spider Parasteatoda tepidariorum embryo, which has a hedgehog (hh)-dependent mechanism of axis formation, offers a cell-based field where the stripes of Pt-hh (a hh homolog) expression dynamically develop in accordance with axis formation and growth, with the patterning processes varying among the regions of the field. In this study, using cell labeling, the future body subdivisions were masked to the germ disc in the spider embryo and provided substantial evidence for the occurrence of kinetic waves of Pt-hh expression in the presumptive head and opisthosomal (or abdominal) regions of the embryonic field. Notably, combined with cell tracking, it was shown that surface cells at and near the center of the germ disc persist in the posterior portion of the field from where Pt-hh stripes sequentially arise, suggesting the operation of ordered oscillations of Pt-hh expression. A quantitative analysis of forming/formed Pt-hh stripes was conducted using serially timed fixation of sibling embryos. By utilizing length measurements that reflect the axis growth of the embryonic field, the pattern dynamics were reconstructed, that captured repeated splitting of Pt-hh stripes and oscillations of Pt-hh expression in the presumptive head and opisthosomal regions, respectively. In the intermediate thoracic region, three stripes of Pt-hh expression showed a late appearance, with the segmental units specified much earlier by another mechanism. Analyses provided quantitative estimates related to axis growth and stripe-splitting and oscillation events, including the periods of the patterning cycles. This work characterizes the diversity of stripe-forming processes in a cell-based field in a common spatiotemporal framework and highlights the contrasting dynamics of splitting versus oscillation. The cellular and quantitative data presented in this study provide the foundation for experimental, theoretical and evolutionary studies of cell-based pattern formation, especially body axis segmentation in arthropods.
Hernandez de Madrid, B. and Casanova, J. (2018). GATA factor genes in the Drosophila midgut embryo. PLoS One 13(3): e0193612. PubMed ID: 29518114
The Drosophila GATA factor gene serpent (srp) is required for the early differentiation of the anterior and posterior midgut primordia. In particular, srp is sufficient and necessary for the primordial gut cells to undertake an epithelial-to-mesenchimal transition (EMT). Two other GATA factor genes, dGATAe and grain (grn), are also specifically expressed in the midgut. On the one hand, dGATAe expression is activated by srp. Embryos homozygous for a deficiency uncovering dGATAe were shown to lack the expression of some differentiated midgut genes. Moreover, ectopic expression of dGATAe was sufficient to drive the expression of some of these differentiation marker genes, thus establishing the role of dGATAe in the regulation of their expression. However, due to the gross abnormalities associated with this deficiency, it was not possible to assess whether, similarly to srp, dGATAe might play a role in setting the midgut morphology. To further investigate this role, a dGATAe mutant was generated. On the other hand, grn is expressed in the midgut primordia around stage 11 and remains expressed until the end of embryogenesis. Yet, no midgut function has been described for grn. As for dGATAe, midgut grn expression is dependent on srp; conversely, dGATAe and grn expression are independent of each other. These results also indicate that, unlike srp, dGATAe and grn,/i> are not responsible for setting the general embryonic midgut morphology. Analysed midgut genes whose expression is lacking in embryos homozygous for a deficiency uncovering dGATAe are indeed dGATAe-dependent genes. Conversely, no midgut gene was found to be grn-dependent, with the exception of midgut repression of the proventriculus iroquois (iro) gene. In conclusion, these results clarify the expression patterns and function of the GATA factor genes expressed in the embryonic midgut.
Verd, B., Clark, E., Wotton, K. R., Janssens, H., Jimenez-Guri, E., Crombach, A. and Jaeger, J. (2018). A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila. PLoS Biol 16(2): e2003174. PubMed ID: 29451884
Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). This study used data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, the current simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. These results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects.
Wilmes, A. C., Klinke, N., Rotstein, B., Meyer, H. and Paululat, A. (2018). Biosynthesis and assembly of the Collagen IV-like protein Pericardin in Drosophila melanogaster. Biol Open 7(4). PubMed ID: 29685999
In Drosophila, formation of the cardiac extracellular matrix (ECM) starts during embryogenesis. Assembly and incorporation of structural proteins such as Collagen IV, Pericardin, and Laminin A, B1, and B2 into the cardiac ECM is critical to the maintenance of heart integrity and functionality. The cardiac ECM connects the heart tube with the alary muscles; thus, the ECM contributes to a flexible positioning of the heart within the animal's body. Moreover, the cardiac ECM holds the larval pericardial nephrocytes in close proximity to the heart tube and the inflow tract, which is assumed to be critical to efficient haemolymph clearance. Mutations in either structural ECM constituents or ECM receptors cause breakdown of the ECM network upon ageing, with disconnection of the heart tube from alary muscles becoming apparent at larval stages. Finally, the heart becomes non-functional. This study characterised existing and new pericardin mutants and investigated biosynthesis, secretion, and assembly of Pericardin in matrices. Two new pericardin alleles, which turned out to be a null (pericardin3-548) and a hypomorphic allele (pericardin3-21), were identifed. Both mutants could be rescued with a genomic duplication of a fosmid coding for the pericardin locus. Biochemical analysis revealed that Pericardin is highly glycosylated and forms redox-dependent multimers. Multimer formation is remarkably reduced in animals deficient for the prolyl-4 hydroxylase cluster at 75D3-4.
Fraire-Zamora, J. J., Jaeger, J. and Solon, J. (2018). Two consecutive microtubule-based epithelial seaming events mediate dorsal closure in the scuttle fly Megaselia abdita. Elife 7. PubMed ID: 29537962
Evolution of morphogenesis is generally associated with changes in genetic regulation. This study reports evidence indicating that dorsal closure, a conserved morphogenetic process in dipterans, evolved as the consequence of rearrangements in epithelial organization rather than signaling regulation. In Drosophila melanogaster, dorsal closure consists of a two-tissue system where the contraction of extraembryonic amnioserosa and a JNK/Dpp-dependent epidermal actomyosin cable result in microtubule-dependent seaming of the epidermis. Dorsal closure in Megaselia abdita, a three-tissue system comprising serosa, amnion and epidermis, differs in morphogenetic rearrangements despite conservation of JNK/Dpp signaling. In addition to an actomyosin cable, M. abdita dorsal closure is driven by the rupture and contraction of the serosa and the consecutive microtubule-dependent seaming of amnion and epidermis. This study indicates that the evolutionary transition to a reduced system of dorsal closure involves simplification of the seaming process without changing the signaling pathways of closure progression.
Clark, E. and Peel, A. D. (2018). Evidence for the temporal regulation of insect segmentation by a conserved sequence of transcription factors. Development. PubMed ID: 29724758
Long-germ insects, such as the fruit fly Drosophila melanogaster, pattern their segments simultaneously, whereas short-germ insects, such as the beetle Tribolium castaneum, pattern their segments sequentially, from anterior to posterior. While the two modes of segmentation at first appear quite distinct, much of this difference might simply reflect developmental heterochrony. This study now shows that, in both Drosophila and Tribolium, segment patterning occurs within a common framework of sequential Caudal, Dichaete, and Odd-paired expression. In Drosophila these transcription factors are expressed like simple timers within the blastoderm, while in Tribolium they form wavefronts that sweep from anterior to posterior across the germband. In Drosophila, all three are known to regulate pair-rule gene expression and influence the temporal progression of segmentation. It is proposed that these regulatory roles are conserved in short-germ embryos, and that therefore the changing expression profiles of these genes across insects provide a mechanistic explanation for observed differences in the timing of segmentation. In support of this hypothesis it was demonstrated that Odd-paired is essential for segmentation in Tribolium, contrary to previous reports.

Friday, June 8th - Behavior

Soto-Padilla, A., Ruijsink, R., Sibon, O. C. M., van Rijn, H. and Billeter, J. C. (2018). Thermosensory perception regulates speed of movement in response to temperature changes in Drosophila melanogaster. J Exp Biol. PubMed ID: 29650755
Temperature influences physiology and behavior of all organisms. For ectotherms, which lack central temperature regulation, temperature adaptation requires sheltering from or moving to a heat source. As temperature constrains the rate of metabolic reactions, it can directly affect ectotherm physiology and thus behavioral performance. This direct effect is particularly relevant for insects whose small body readily equilibrates with ambient temperature. In fact, models of enzyme kinetics applied to insect behavior predict performance at different temperatures, suggesting that thermal physiology governs behavior. However, insects also possess thermosensory neurons critical for locating preferred temperatures, showing cognitive control. This suggests that temperature-related behavior can emerge directly from a physiological effect, indirectly as consequence of thermosensory processing, or through both. To separate the roles of thermal physiology and cognitive control, this study developed an arena that allows fast temperature changes in time and space, and in which animals' movements are automatically quantified. Wild-type and thermosensory receptor mutants Drosophila melanogaster were exposed to a dynamic temperature environment, and their movements were tracked. The locomotor speed of wild-type flies closely matched models of enzyme kinetics, but the behavior of thermosensory mutants did not. Mutations in thermosensory receptor dTrpA1 (Transient receptor potential) expressed in the brain resulted in a complete lack of response to temperature changes, while mutation in peripheral thermosensory receptor Gr28b(D) resulted in diminished response. It is concluded that flies react to temperature through cognitive control, informed by interactions between various thermosensory neurons, whose behavioral output resembles that of enzyme kinetics.
Chen, C., Xu, M., Anantaprakorn, Y., Rosing, M. and Stanewsky, R. (2018). nocte is required for integrating light and temperature inputs in circadian clock neurons of Drosophila. Curr Biol 28(10): 1595-1605.e1593. PubMed ID: 29754901
Circadian clocks organize biological processes to occur at optimized times of day and thereby contribute to overall fitness. While the regular daily changes of environmental light and temperature synchronize circadian clocks, extreme external conditions can bypass the temporal constraints dictated by the clock. Despite advanced knowledge about how the daily light-dark changes synchronize the clock, relatively little is known with regard to how the daily temperature changes influence daily timing and how temperature and light signals are integrated. In Drosophila, a network of approximately 150 brain clock neurons exhibit 24-hr oscillations of clock gene expression to regulate daily activity and sleep. This study shows that a temperature input pathway from peripheral sensory organs, which depends on the gene nochte, targets specific subsets of these clock neurons to synchronize molecular and behavioral rhythms to temperature cycles. Strikingly, while nocte1 mutant flies synchronize normally to light-dark cycles at constant temperatures, the combined presence of light-dark and temperature cycles inhibits synchronization. nocte1 flies exhibit altered siesta sleep, suggesting that the sleep-regulating clock neurons are an important target for nocte-dependent temperature input, which dominates a parallel light input into these cells. In conclusion, this study reveals a nocte-dependent temperature input pathway to central clock neurons and shows that this pathway and its target neurons are important for the integration of sensory light and temperature information in order to temporally regulate activity and sleep during daily light and temperature cycles.
Zhang, M., Li, X., Zheng, H., Wen, X., Chen, S., Ye, J., Tang, S., Yao, F., Li, Y. and Yan, Z. (2018). Brv1 is required for Drosophila larvae to sense gentle touch. Cell Rep 23(1): 23-31. PubMed ID: 29617663
How touch is sensed is fundamental for many physiological processes. However, the underlying mechanism and molecular identity for touch sensation are largely unknown. This study reports on defective gentle-touch behavioral responses in brv1 loss-of-function Drosophila larvae. RNAi and Ca(2+) imaging confirmed the involvement of Brv1 in sensing touch and demonstrated that Brv1 mediates the mechanotransduction of class III dendritic arborization neurons. Electrophysiological recordings further revealed that the expression of Brv1 protein in HEK293T cells gives rise to stretch-activated cation channels. Purified Brv1 protein reconstituted into liposomes were found to sense stretch stimuli. In addition, co-expression studies suggested that Brv1 amplifies the response of mechanosensitive ion channel NOMPC (no mechanoreceptor potential C) to touch stimuli. Altogether, these findings demonstrate a molecular entity that mediates the gentle-touch response in Drosophila larvae, providing insights into the molecular mechanisms of touch sensation.
Snellings, Y., Herrera, B., Wildemann, B., Beelen, M., Zwarts, L., Wenseleers, T. and Callaerts, P. (2018). The role of cuticular hydrocarbons in mate recognition in Drosophila suzukii. Sci Rep 8(1): 4996. PubMed ID: 29567945
Cuticular hydrocarbons (CHCs) play a central role in the chemical communication of many insects. In Drosophila suzukii, an economically important pest insect, very little is known about chemical communication and the possible role of CHCs. This study identified 60 CHCs of Drosophila suzukii and studied their changes in function of age (maturation), sex and interactions with the opposite sex. Age (maturation) was identified is the key factor driving changes in the CHC profiles. The effect on courtship behaviour and mating of six CHCs, five of which were positively associated with maturation and one negatively. The results of these experiments demonstrate that four of the major CHC peaks with a chain length of 23 carbons, namely 9-tricosene (9-C23:1), 7-tricosene (7-C23:1), 5-tricosene (5-C23:1) and tricosane (n-C23), negatively regulated courtship and mating, even though all these compounds were characteristic for sexually mature flies. The study then goes on to show that this effect on courtship and mating is likely due to the disruption of the natural ratios in which these hydrocarbons occur in Drosophila suzukii. Overall, these results provide key insights into the cuticular hydrocarbon signals that play a role in D. suzukii mate recognition.
Wu, B., Ma, L., Zhang, E., Du, J., Liu, S., Price, J., Li, S. and Zhao, Z. (2018). Sexual dimorphism of sleep regulated by juvenile hormone signaling in Drosophila. PLoS Genet 14(4): e1007318. PubMed ID: 29617359
Sexually dimorphic phenotypes are a universal phenomenon in animals. In the model animal fruit fly Drosophila, males and females exhibit long- and short-sleep phenotypes, respectively. However, the mechanism is still a mystery. This study showed that juvenile hormone (JH) is involved in regulation of sexually dimorphic sleep in Drosophila, in which gain of JH function enlarges differences of the dimorphic sleep phenotype with higher sleep in males and lower sleep in females, while loss of JH function blurs these differences and results in feminization of male sleep and masculinization of female sleep. Further studies indicate that germ cell-expressed (GCE), one of the JH receptors, mediates the response in the JH pathway because the sexually dimorphic sleep phenotypes cannot be rescued by JH hormone in a gce deletion mutant. The JH-GCE regulated sleep dimorphism is generated through the sex differentiation-related genes -fruitless (fru) and doublesex (dsx) in males and sex-lethal (sxl), transformer (tra) and doublesex (dsx) in females. These are the "switch" genes that separately control the sleep pattern in males and females. Moreover, analysis of sleep deprivation and circadian behaviors showed that the sexually dimorphic sleep induced by JH signals is a change of sleep drive and independent of the circadian clock. Furthermore, this study found that JH seems to also play an unanticipated role in antagonism of an aging-induced sleep decrease in male flies. Taken together, these results indicate that the JH signal pathway is critical for maintenance of sexually dimorphic sleep by regulating sex-relevant genes.
Zer-Krispil, S., Zak, H., Shao, L., Ben-Shaanan, S., Tordjman, L., Bentzur, A., Shmueli, A. and Shohat-Ophir, G. (2018). Ejaculation induced by the activation of Crz neurons is rewarding to Drosophila males. Curr Biol. PubMed ID: 29681474
The reward system is a collection of circuits that reinforce behaviors necessary for survival. Given the importance of reproduction for survival, actions that promote successful mating induce pleasurable feeling and are positively reinforced. This principle is conserved in Drosophila, where successful copulation is naturally rewarding to male flies, induces long-term appetitive memories, increases brain levels of neuropeptide F (NPF, the fly homolog of neuropeptide Y), and prevents ethanol, known otherwise as rewarding to flies, from being rewarding. It is not clear which of the multiple sensory and motor responses performed during mating induces perception of reward. Sexual interactions with female flies that do not reach copulation are not sufficient to reduce ethanol consumption, suggesting that only successful mating encounters are rewarding. This study uncoupled the initial steps of mating from its final steps and tested the ability of ejaculation to mimic the rewarding value of full copulation. Ejaculation was produced by activating neurons that express the neuropeptide corazonin (CRZ) and subsequently measured different aspects of reward. Activating Crz-expressing neurons is rewarding to male flies, as they choose to reside in a zone that triggers optogenetic stimulation of Crz neurons and display conditioned preference for an odor paired with the activation. Reminiscent of successful mating, repeated activation of Crz neurons increases npf levels and reduces ethanol consumption. These results demonstrate that ejaculation stimulated by Crz/Crz-receptor signaling serves as an essential part of the mating reward mechanism in Drosophila.

Thursday, June 7th - Cell cycle

Vergassola, M., Deneke, V. E. and Di Talia, S. (2018). Mitotic waves in the early embryogenesis of Drosophila: Bistability traded for speed. Proc Natl Acad Sci U S A 115(10): E2165-e2174. PubMed ID: 29449348
Early embryogenesis of most metazoans is characterized by rapid and synchronous cleavage divisions. Chemical waves of Cdk1 activity were previously shown to spread across Drosophila embryos, and the underlying molecular processes were dissected. This paper presents the theory of the physical mechanisms that control Cdk1 waves in Drosophila. The in vivo dynamics of Cdk1 are captured by a transiently bistable reaction-diffusion model, where time-dependent reaction terms account for the growing level of cyclins and Cdk1 activation across the cell cycle. Two distinct regimes were identified. The first one is observed in mutants of the mitotic switch. There, waves are triggered by the classical mechanism of a stable state invading a metastable one. Conversely, waves in wild type reflect a transient phase that preserves the Cdk1 spatial gradients while the overall level of Cdk1 activity is swept upward by the time-dependent reaction terms. This unique mechanism generates a wave-like spreading that differs from bistable waves for its dependence on dynamic parameters and its faster speed. Namely, the speed of "sweep" waves strikingly decreases as the strength of the reaction terms increases and scales as the powers 3/4, -1/2, and 7/12 of Cdk1 molecular diffusivity, noise amplitude, and rate of increase of Cdk1 activity in the cell-cycle S phase, respectively. Theoretical predictions are supported by numerical simulations and experiments that couple quantitative measurements of Cdk1 activity and genetic perturbations of the accumulation rate of cyclins. Finally, this analysis bears upon the inhibition required to suppress Cdk1 waves at the cell-cycle pause for the maternal-to-zygotic transition.
Chaurasia, S. and Lehner, C. F. (2018). Dynamics and control of sister kinetochore behavior during the meiotic divisions in Drosophila spermatocytes. PLoS Genet 14(5): e1007372. PubMed ID: 29734336
Sister kinetochores are connected to the same spindle pole during meiosis I and to opposite poles during meiosis II. The molecular mechanisms controlling the distinct behavior of sister kinetochores during the two meiotic divisions are poorly understood. To study kinetochore behavior during meiosis, time lapse imaging was optimized with Drosophila spermatocytes, enabling kinetochore tracking with high temporal and spatial resolution through both meiotic divisions. The correct bipolar orientation of chromosomes within the spindle proceeds rapidly during both divisions. Stable bi-orientation of the last chromosome is achieved within ten minutes after the onset of kinetochore-microtubule interactions. Analyses of mnm and tef mutants, where univalents instead of bivalents are present during meiosis I, indicate that the high efficiency of normal bi-orientation depends on pronounced stabilization of kinetochore attachments to spindle microtubules by the mechanical tension generated by spindle forces upon bi-orientation. Except for occasional brief separation episodes, sister kinetochores are so closely associated that they cannot be resolved individually by light microscopy during meiosis I, interkinesis and at the start of meiosis II. Permanent evident separation of sister kinetochores during M II depends on spindle forces resulting from bi-orientation. In mnm and tef mutants, sister kinetochore separation can be observed already during meiosis I in bi-oriented univalents. Interestingly, however, this sister kinetochore separation is delayed until the metaphase to anaphase transition and depends on the Fzy/Cdc20 activator of the anaphase-promoting complex/cyclosome. It is proposed that univalent bi-orientation in mnm and tef mutants exposes a release of sister kinetochore conjunction that occurs also during normal meiosis I in preparation for bi-orientation of dyads during meiosis II.
Clemente, G. D., Hannaford, M. R., Beati, H., Kapp, K., Januschke, J., Griffis, E. R. and Muller, H. J. (2018). Requirement of the Dynein-adaptor Spindly for mitotic and post-mitotic functions in Drosophila. J Dev Biol 6(2). PubMed ID: 29615558
Spindly was originally identified as a specific regulator of Dynein activity at the kinetochore. In early prometaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. This study presents loss- and gain-of-function studies of Spindly using transgenic RNAi in Drosophila. Knock-down of Spindly in the female germ line results in mitotic arrest during embryonic cleavage divisions. The requirements were investagated of Spindly protein domains for its localisation and function; the carboxy-terminal region was shown to controls Spindly localisation in a cell-type specific manner. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells, Spindly protein levels were altered in migrating cells, and it was found that ovarian border cell migration is sensitive to the levels of Spindly protein. This study uncovers novel functions of Spindly and a differential, functional requirement for its carboxy-terminal region in Drosophila.
Kim, M., Tang, J. P. and Moon, N. S. (2018). An alternatively spliced form affecting the Marked Box domain of Drosophila E2F1 is required for proper cell cycle regulation. PLoS Genet 14(2): e1007204. PubMed ID: 29420631
Across metazoans, cell cycle progression is regulated by E2F family transcription factors that can function as either transcriptional activators or repressors. For decades, the Drosophila E2F family has been viewed as a streamlined RB/E2F network, consisting of one activator (dE2F1) and one repressor (dE2F2). This study reports that an uncharacterized isoform of dE2F1, hereon called dE2F1b, plays an important function during development and is functionally distinct from the widely-studied dE2F1 isoform, dE2F1a. dE2F1b contains an additional exon that inserts 16 amino acids to the evolutionarily conserved Marked Box domain. Analysis of de2f1b-specific mutants generated via CRISPR/Cas9 indicates that dE2F1b is a critical regulator of the cell cycle during development. This is particularly evident in endocycling salivary glands in which a tight control of dE2F1 activity is required. Interestingly, close examination of mitotic tissues such as eye and wing imaginal discs suggests that dE2F1b plays a repressive function as cells exit from the cell cycle. Evidence is also provided demonstrating that dE2F1b differentially interacts with RBF1 and alters the recruitment of RBF1 and dE2F1 to promoters. Collectively, these data suggest that dE2F1b is a novel member of the E2F family, revealing a previously unappreciated complexity in the Drosophila RB/E2F network.
Lim, D. H., Lee, S., Han, J. Y., Choi, M. S., Hong, J. S., Seong, Y., Kwon, Y. S. and Lee, Y. S. (2018). Ecdysone-responsive microRNA-252-5p controls the cell cycle by targeting Abi in Drosophila. Faseb J: fj201701185RR. PubMed ID: 29543534
The steroid hormone ecdysone has a central role in the developmental transitions of insects through its control of responsive protein-coding and microRNA (miRNA) gene expression. However, the complete regulatory network controlling the expression of these genes remains to be elucidated. In this study, cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 (Ago1) protein, the core effector of the miRNA pathway, was performed in Drosophila S2 cells. Regulatory interactions between miRNAs and their cognate targets were substantially altered by Ago1 in response to ecdysone signaling. Additionally, during the larva-to-adult metamorphosis, miR-252-5p was up-regulated via the canonical ecdysone-signaling pathway. Evidence is provided that miR-252-5p targets Abelson interacting protein (Abi) to decrease the protein levels of cyclins A and B, controlling the cell cycle. Overall, these data suggest a potential role for the ecdysone/miR-252-5p/Abi regulatory axis partly in cell-cycle control during metamorphosis in Drosophila.
Kizhedathu, A., Bagul, A. V. and Guha, A. (2018). Negative regulation of G2-M by ATR (mei-41)/Chk1(Grapes) facilitates tracheoblast growth and tracheal hypertrophy in Drosophila. Elife 7. PubMed ID: 29658881
Imaginal progenitors in Drosophila are known to arrest in G2 during larval stages and proliferate thereafter. This study investigated the mechanism and implications of G2 arrest in progenitors of the adult thoracic tracheal epithelium (tracheoblasts). Tracheoblasts were shown to pause in G2 for ~48-56 h and grow in size over this period. Surprisingly, tracheoblasts arrested in G2 express drivers of G2-M like Cdc25/String (Stg). Mechanisms that prevent G2-M are also in place in this interval. Tracheoblasts activate Checkpoint Kinase 1/Grapes (Chk1/Grp) in an ATR/mei-41-dependent manner. Loss of ATR/Chk1 led to precocious mitotic entry ~24-32 h earlier. These divisions were apparently normal as there was no evidence of increased DNA damage or cell death. However, induction of precocious mitoses impaired growth of tracheoblasts and the tracheae they comprise. It is proposed that ATR/Chk1 negatively regulate G2-M in developing tracheoblasts and that G2 arrest facilitates cellular and hypertrophic organ growth.

Wednesday, June 6th - Chromatin

Adhikari, P., Orozco, D., Randhawa, H. and Wolf, F. W. (2018). Mef2 induction of the immediate early gene Hr38/Nr4a is terminated by Sirt1 to promote ethanol tolerance. Genes Brain Behav: e12486. PubMed ID: 29726098
Drug naive animals given a single dose of ethanol show changed responses to subsequent doses, including the development of ethanol tolerance and ethanol preference. These simple forms of behavioral plasticity are due in part to changes in gene expression and neuronal properties. Surprisingly little is known about how ethanol initiates changes in gene expression or what the changes do. This study demonstrate a role in ethanol plasticity for Hr38, the sole Drosophila homolog of the mammalian Nr4a1/2/3 class of immediate early response transcription factors. Acute ethanol exposure induces transient expression of Hr38 and other immediate early neuronal activity genes. Ethanol activates the Mef2 transcriptional activator to induce Hr38, and the Sirt1 histone/protein deacetylase is required to terminate Hr38 induction. Loss of Hr38 decreases ethanol tolerance and causes precocious but short-lasting ethanol preference. Similarly, reduced Mef2 activity in all neurons or specifically in the mushroom body alpha/beta neurons decreases ethanol tolerance; Sirt1 promotes ethanol tolerance in these same neurons. Genetically decreasing Hr38 expression levels in Sirt1 null mutants restores ethanol tolerance, demonstrating that both induction and termination of Hr38 expression are important for behavioral plasticity to proceed. These data demonstrate that Hr38 functions as an immediate early transcription factor that promotes ethanol behavioral plasticity.
McGurk, M. P. and Barbash, D. A. (2018). Double insertion of transposable elements provides a substrate for the evolution of satellite DNA. Genome Res 28(5): 714-725. PubMed ID: 29588362
Eukaryotic genomes are replete with repeated sequences in the form of transposable elements (TEs) dispersed across the genome or as satellite arrays, large stretches of tandemly repeated sequences. Many satellites clearly originated as TEs, but it is unclear how mobile genetic parasites can transform into megabase-sized tandem arrays. Comprehensive population genomic sampling is needed to determine the frequency and generative mechanisms of tandem TEs, at all stages from their initial formation to their subsequent expansion and maintenance as satellites. The best available population resources, short-read DNA sequences, are often considered to be of limited utility for analyzing repetitive DNA due to the challenge of mapping individual repeats to unique genomic locations. A new pipeline called ConTExt has been developed that demonstrates that paired-end Illumina data can be successfully leveraged to identify a wide range of structural variation within repetitive sequence, including tandem elements. By analyzing 85 genomes from five populations of Drosophila melanogaster, it was discovered that TEs commonly form tandem dimers. The results further suggest that insertion site preference is the major mechanism by which dimers arise and that, consequently, dimers form rapidly during periods of active transposition. This abundance of TE dimers has the potential to provide source material for future expansion into satellite arrays, and one such copy number expansion of the DNA transposon hobo was found to approximately 16 tandem copies in a single line. The very process that defines TEs-transposition-thus regularly generates sequences from which new satellites can arise.
Pokholkova, G. V., Demakov, S. A., Andreenkov, O. V., Andreenkova, N. G., Volkova, E. I., Belyaeva, E. S. and Zhimulev, I. F. (2018). Tethering of CHROMATOR and dCTCF proteins results in decompaction of condensed bands in the Drosophila melanogaster polytene chromosomes but does not affect their transcription and replication timing. PLoS One 13(4): e0192634. PubMed ID: 29608600
Insulator proteins are central to domain organization and gene regulation in the genome. This study used ectopic tethering of CHROMATOR (CHRIZ/CHRO) and dCTCF to pre-defined regions of the genome to dissect the influence of these proteins on local chromatin organization, to analyze their interaction with other key chromatin proteins and to evaluate the effects on transcription and replication. Specifically, using UAS-GAL4DBD system, CHRO and dCTCF were artificially recruited into highly compacted polytene chromosome bands that share the features of silent chromatin type known as intercalary heterochromatin (IH). This led to local chromatin decondensation, formation of novel DHSes and recruitment of several "open chromatin" proteins. CHRO tethering resulted in the recruitment of CP190 and Z4 (Putzig), whereas dCTCF tethering attracted CHRO, CP190, and Z4. Importantly, formation of a local stretch of open chromatin did not result in the reactivation of silent marker genes yellow and mini-white immediately adjacent to the targeting region (UAS), nor did RNA polII become recruited into this chromatin. The decompacted region retained late replicated, similarly to the wild-type untargeted region.
Sadasivam, D. A. and Huang, D. H. (2018). Feedback regulation by antagonistic epigenetic factors potentially maintains homeostasis in Drosophila. J Cell Sci [Epub ahead of print]. PubMed ID: 29661849
Polycomb group (PcG) repressors confer epigenetically heritable silencing on key regulatory genes through histone H3 trimethylation on lysine 27 (H3K27me3). How the silencing state withstands antagonistic activities from co-expressed trithorax group (trxG) activators is unclear. Using overexpression of Trx H3K4 methylase to perturb the silenced state, this study found a dynamic process triggered in a stepwise fashion to neutralize the inductive impacts from excess Trx. Shortly after Trx overexpression, there are global increases in H3K4 trimethylation and RNA polymerase II phosphorylation, marking active transcription. Subsequently, these patterns diminish when dSet1, an abundant H3K4 methylase involved in productive transcription, is reduced. Concomitantly, the global H3K27me3 level is markedly reduced, corresponding to an increase of dUtx demethylase. Finally, excess Pc repressive complex 1 (PRC1) is induced and located to numerous ectopic chromosomal sites independently of H3K27me3 and several key recruitment factors. The observation that PRC1 becomes almost completely co-localized with Trx provides new aspects of recruitment and antagonistic interaction. It is proposed that these events represent a feedback circuitry ensuring the stability of the silenced state.
Bobkov, G. O. M., Gilbert, N. and Heun, P. (2018). Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation. J Cell Biol [Epub ahead of print]. PubMed ID: 29626011
Centromeres are essential for chromosome segregation and are specified epigenetically by the presence of the histone H3 variant CENP-A. In flies and humans, replenishment of the centromeric mark is uncoupled from DNA replication and requires the removal of H3 "placeholder" nucleosomes. Although transcription at centromeres has been previously linked to the loading of new CENP-A, the underlying molecular mechanism remains poorly understood. This study used Drosophila melanogaster tissue culture cells to show that centromeric presence of actively transcribing RNA polymerase II temporally coincides with de novo deposition of dCENP-A. Using a newly developed dCENP-A loading system that is independent of acute transcription, it was found that short inhibition of transcription impaired dCENP-A incorporation into chromatin. Interestingly, initial targeting of dCENP-A to centromeres was unaffected, revealing two stability states of newly loaded dCENP-A: a salt-sensitive association with the centromere and a salt-resistant chromatin-incorporated form. This suggests that transcription-mediated chromatin remodeling is required for the transition of dCENP-A to fully incorporated nucleosomes at the centromere.
Wang, S., Stoops, E., Cp, U., Markus, B., Reuveny, A., Ordan, E. and Volk, T. (2018). Mechanotransduction via the LINC complex regulates DNA replication in myonuclei. J Cell Biol. PubMed ID: 29650775
Nuclear mechanotransduction has been implicated in the control of chromatin organization; however, its impact on functional contractile myofibers is unclear. This study found that deleting components of the linker of nucleoskeleton and cytoskeleton (LINC) complex in Drosophila melanogaster larval muscles abolishes the controlled and synchronized DNA endoreplication, typical of nuclei across myofibers, resulting in increased and variable DNA content in myonuclei of individual myofibers. Moreover, perturbation of LINC-independent mechanical input after knockdown of beta-Integrin in larval muscles similarly led to increased DNA content in myonuclei. Genome-wide RNA-polymerase II occupancy analysis in myofibers of the LINC mutant klar indicated an altered binding profile, including a significant decrease in the chromatin regulator barrier-to-autointegration factor (BAF) and the contractile regulator Troponin C. Importantly, muscle-specific knockdown of BAF led to increased DNA content in myonuclei, phenocopying the LINC mutant phenotype. It is propose that mechanical stimuli transmitted via the LINC complex act via BAF to regulate synchronized cell-cycle progression of myonuclei across single myofibers.

Tuesday, June 5th - Drosophila Disease Models

Machamer, J. B., Woolums, B. M., Fuller, G. G. and Lloyd, T. E. (2018). FUS causes synaptic hyperexcitability in Drosophila dendritic arborization neurons. Brain Res [Epub ahead of print]. PubMed ID: 29625118
Mutations in the nuclear localization signal of the RNA binding protein FUS cause both Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). These mutations result in a loss of FUS from the nucleus and the formation of FUS-containing cytoplasmic aggregates in patients. To better understand the role of cytoplasmic FUS mislocalization in the pathogenesis of ALS, this study identified a population of cholinergic neurons in Drosophila that recapitulate these pathologic hallmarks. Expression of mutant FUS or the Drosophila homolog, Cabeza (Caz), in class IV dendritic arborization neurons results in cytoplasmic mislocalization and axonal transport to presynaptic terminals. Interestingly, overexpression of FUS or Caz causes the progressive loss of neuronal projections, reduction of synaptic mitochondria, and the appearance of large calcium transients within the synapse. Additionally, overexpression of mutant but not wild type FUS results in a reduction in presynaptic Synaptotagmin, an integral component of the neurotransmitter release machinery, and mutant Caz specifically disrupts axonal transport and induces hyperexcitability. These results suggest that FUS/Caz overexpression disrupts neuronal function through multiple mechanisms, and that ALS-causing mutations impair the transport of synaptic vesicle proteins and induce hyperexcitability.
Papanikolopoulou, K., Grammenoudi, S., Samiotaki, M. and Skoulakis, E. M. C. (2018). Differential effects of 14-3-3 dimers on Tau phosphorylation, stability and toxicity in vivo. Hum Mol Genet. PubMed ID: 29659825
Tauopathies involve aberrant phosphorylation and aggregation of the neuronal protein Tau. The largely neuronal 14-3-3 proteins are also elevated in the Central Nervous System (CNS) and Cerebrospinal Fluid of Tauopathy patients, suggesting functional linkage. This study used the Drosophila system to investigate in vivo whether 14-3-3s are causal or synergistic with Tau accumulation in precipitating pathogenesis. Both Drosophila 14-3-3 proteins interact with human wild type and mutant Tau on multiple sites irrespective of their phosphorylation state. 14-3-3 dimers regulate steady state phosphorylation of both Wild Type and the R406W mutant Tau, but they are not essential for toxicity of either variant. Moreover, 14-3-3 elevation itself is not pathogenic, but recruitment of dimers on accumulating Wild Type Tau increases its steady state levels ostensibly by occluding access to proteases in a phosphorylation-dependent manner. In contrast, the R406W mutant, which lacks a putative 14-3-3 binding site, responds differentially to elevation of each 14-3-3 isoform. Although excess 14-3-3zeta stabilizes the mutant protein, elevated D14-3-3epsilon has a destabilizing effect probably because of altered 14-3-3 dimer composition. These collective data demonstrate the complexity of 14-3-3/Tau interactions in vivo and suggest that 14-3-3 attenuation is not appropriate ameliorative treatment of Tauopathies. Finally, it is suggestd that 'bystander' 14-3-3s are recruited by accumulating Tau with the consequences depending on the composition of available dimers within particular neurons and the Tau variant.
Panikker, P., Xu, S. J., Zhang, H., Sarthi, J., Beaver, M., Sheth, A., Akhter, S. and Elefant, F. (2018). Restoring Tip60 HAT/HDAC2 balance in the neurodegenerative brain relieves epigenetic transcriptional repression and reinstates cognition. J Neurosci. PubMed ID: 29654189
Cognitive decline is a debilitating hallmark during pre-clinical stages of AD yet causes remain unclear. As histone acetylation homeostasis is critical for mediating epigenetic gene control throughout neuronal development, it was postulated that its misregulation contributes to cognitive impairment preceding AD pathology. This study shows that disruption of Tip60 HAT/HDAC2 homeostasis occurs early in the brain of an AD associated amyloid precursor protein (APP) Drosophila model and triggers epigenetic repression of neuroplasticity genes well before Abeta plaques form in male and female larvae. Repressed genes display enhanced HDAC2 binding and reduced Tip60 and histone acetylation enrichment. Increasing Tip60 in the AD associated APP brain restores Tip60 HAT/HDAC2 balance by decreasing HDAC2 levels, reverses neuroepigenetic alterations to activate synaptic plasticity genes, and reinstates brain morphology and cognition. Such Drosophila neuroplasticity gene epigenetic signatures are conserved in male and female mouse hippocampus and their expression and Tip60 function is compromised in hippocampus from AD patients. It is advocated that Tip60 HAT/HDAC2 mediated epigenetic gene disruption is a critical initial step in AD that is reversed by restoring Tip60 in the brain.
Ristic, G., Sutton, J. R., Libohova, K. and Todi, S. V. (2018). Toxicity and aggregation of the polyglutamine disease protein, ataxin-3 is regulated by its binding to VCP/p97 in Drosophila melanogaster. Neurobiol Dis [Epub ahead of print]. PubMed ID: 29704548
Among the nine dominantly inherited, age-dependent neurodegenerative diseases caused by abnormal expansion in the polyglutamine (polyQ) repeat of otherwise unrelated proteins is Spinocerebellar Ataxia Type 3 (SCA3). SCA3 is caused by polyQ expansion in the deubiquitinase (DUB), ataxin-3. Molecular sequelae related to SCA3 remain unclear. This study sought to understand the role of protein context in SCA3 by focusing on the interaction between this DUB and Valosin-Containing Protein (VCP). VCP is bound directly by ataxin-3 through an arginine-rich area preceding the polyQ repeat. The importance of this interaction was examined in ataxin-3-dependent degeneration in Drosophila melanogaster. Assays with new isogenic fly lines expressing pathogenic ataxin-3 with an intact or mutated VCP-binding site show that disrupting the ataxin-3-VCP interaction delays the aggregation of the toxic protein in vivo. Importantly, early on flies that express pathogenic ataxin-3 with a mutated VCP-binding site are indistinguishable from flies that do not express any SCA3 protein. Also, reducing levels of VCP through RNA-interference has a similar, protective effect to mutating the VCP-binding site of pathogenic ataxin-3. Based on in vivo pulse-chases, aggregated species of ataxin-3 are highly stable, in a manner independent of VCP-binding. Collectively, these results highlight an important role for the ataxin-3-VCP interaction in SCA3, based on a model that posits a seeding effect from VCP on pathogenic ataxin-3 aggregation and subsequent toxicity.
Jonson, M., Nystrom, S., Sandberg, A., Carlback, M., Michno, W., Hanrieder, J., Starkenberg, A., Nilsson, K. P. R., Thor, S. and Hammarstrom, P. (2018). Aggregated Abeta1-42 is selectively toxic for neurons, whereas glial cells produce mature fibrils with low toxicity in Drosophila. Cell Chem Biol [Epub ahead of print]. PubMed ID: 29657084
The basis for selective vulnerability of certain cell types for misfolded proteins (MPs) in neurodegenerative diseases is largely unknown. This knowledge is crucial for understanding disease progression in relation to MPs spreading in the CNS. This issue was addressed in Drosophila by cell-specific expression of human Abeta1-42 associated with Alzheimer's disease. Expression of Abeta1-42 in various neurons resulted in concentration-dependent severe neurodegenerative phenotypes, and intraneuronal ring-tangle-like aggregates with immature fibril properties when analyzed by aggregate-specific ligands. Unexpectedly, expression of Abeta1-42 from a pan-glial driver produced a mild phenotype despite massive brain load of Abeta1-42 aggregates, even higher than in the strongest neuronal driver. Glial cells formed more mature fibrous aggregates, morphologically distinct from aggregates found in neurons, and was mainly extracellular. Theses findings implicate that Abeta1-42 cytotoxicity is both cell and aggregate morphotype dependent.
Zhang, S., Feng, R., Li, Y., Gan, L., Zhou, F., Meng, S., Li, Q. and Wen, T. (2018). Degradation of alpha-synuclein by dendritic cell factor 1 delays neurodegeneration and extends lifespan in Drosophila. Neurobiol Aging 67: 67-74. PubMed ID: 29649746
Parkinson's disease (PD) is a common neurodegenerative disease associated with the progressive loss of dopaminergic neurons in the substantia nigra. Proteinaceous depositions of alpha-synuclein (alpha-syn) and its mutations, A30P and A53T, are one important characteristic of PD. However, little is known about their aggregation and degradation mechanisms. Dendritic cell factor 1 (DCF1) is a membrane protein that plays important roles in nerve development in mouse. This study aimed to show that DCF1 overexpression in a PD Drosophila model significantly ameliorates impaired locomotor behavior in third instar larvae and normalizes neuromuscular junction growth. Furthermore, climbing ability also significantly increased in adult PD Drosophila. More importantly, the lifespan dramatically extended by an average of approximately 23%, and surprisingly, DCF1 could prevent alpha-syn-induced dopaminergic neuron loss by aggregating alpha-syn in the dorsomedial region of Drosophila. Mechanistically, it was confirmed that DCF1 could degrade alpha-syn both in vivo and in vitro. These findings revealed an important role of DCF1 in PD process and may provide new potential strategies for developing drugs to treat neurodegenerative diseases.

Monday, June 4th - Adult physiology

Wang, X., Yin, S., Yang, Z. and Zhou, B. (2018). Drosophila multicopper oxidase 3 is a potential ferroxidase involved in iron homeostasis. Biochim Biophys Acta. PubMed ID: 29684424
Multicopper oxidases (MCOs) are a specific group of enzymes that contain multiple copper centers through which different substrates are oxidized. Main members of MCO family include ferroxidases, ascorbate oxidases, and laccases. MCO type of ferroxidases is key to iron transport across the plasma membrane. In Drosophila, there are four potential multiple oxidases, MCO1-4. No convincing evidence has been presented so far to indicate any of these, or even any insect multicopper oxidase, to be a ferroxidase. This study shows Drosophila MCO3 (dMCO3) is highly likely a bona fide ferroxidase. In vitro activity assay with insect-cell-expressed dMCO3 demonstrated it has potent ferroxidase activity. Meanwhile, the ascorbate oxidase and laccase activities of dMCO3 are much less significant. dMCO3 expression in vivo, albeit at low levels, appears mostly extracellular, reminiscent of mammalian ceruloplasmin in the serum. A null dMCO3 mutant, generated by CRISPR/Cas9 technology, showed disrupted iron homeostasis, evidenced by increased iron level and reduced metal importer Mvl expression. Notably, dMCO3-null flies phenotypically are largely normal at normal or iron stressed-conditions. The likely existence of a similar iron efflux apparatus is speculated as the mammalian ferroportin/ferroxidase in Drosophila. However, its importance to fly iron homeostasis is greatly minimized, which is instead dominated by another iron efflux avenue mediated by the ZIP13-ferritin axis along the ER/Golgi secretion pathway.
Gates, H., Urbina, B. and French, R. (2018). Developmental ethanol exposure causes reduced feeding and reveals a critical role for Neuropeptide F in survival. Front Physiol 9: 237. PubMed ID: 29623043
Food intake is necessary for survival, and natural reward circuitry has evolved to help ensure that animals ingest sufficient food to maintain development, growth, and survival. Drugs of abuse, including alcohol, co-opt the natural reward circuitry in the brain, and this is a major factor in the reinforcement of drug behaviors leading to addiction. At the junction of these two aspects of reward are alterations in feeding behavior due to alcohol consumption. In particular, developmental alcohol exposure (DAE) results in a collection of physical and neurobehavioral disorders collectively referred to as Fetal Alcohol Spectrum Disorder (FASD). The deleterious effects of DAE include intellectual disabilities and other neurobehavioral changes, including altered feeding behaviors. This study used Drosophila melanogaster as a genetic model organism to study the effects of DAE on feeding behavior and the expression and function of Neuropeptide F. Addition of a defined concentration of ethanol to food leads to reduced feeding at all stages of development. Further, genetic conditions that reduce or eliminate NPF signaling combine with ethanol exposure to further reduce feeding, and the distribution of NPF is altered in the brains of ethanol-supplemented larvae. Most strikingly, it was found that the vast majority of flies with a null mutation in the NPF receptor die early in larval development when reared in ethanol, and provide evidence that this lethality is due to voluntary starvation. Collectively, this study found a critical role for NPF signaling in protecting against altered feeding behavior induced by developmental ethanol exposure.
Ohashi, H. and Sakai, T. (2018). Leucokinin signaling regulates hunger-driven reduction of behavioral responses to noxious heat in Drosophila. Biochem Biophys Res Commun 499(2): 221-226. PubMed ID: 29559237
In the fruitfly Drosophila melanogaster, hunger has a significant impact on its sensory systems and brain functions, and consequently modifies related behaviors. However, it remains unclarified whether hunger affects nociceptive behavioral responses to heat stimuli. This study shows that food deprivation reduces responses to noxious heat in wild-type flies. The neuropeptide Leucokinin (Lk) and its receptor (Lkr) are essential for the reduction of responses to noxious heat. Temporal silencing of Lk-expressing neurons and a knockout mutation of Lkr generated using the CRISPR/Cas9 system inhibited the reduction of responses to noxious heat. Thus, these results reveal that hunger induces reduction of responses to noxious heat through the Lk/Lkr signaling pathway in Drosophila.
Lam, A., Karekar, P., Shah, K., Hariharan, G., Fleyshman, M., Kaur, H., Singh, H. and Gururaja Rao, S. (2018). Drosophila voltage-gated calcium channel alpha1-subunits regulate cardiac function in the aging heart. Sci Rep 8(1): 6910. PubMed ID: 29720608
This study has carried out a paraquat (PQ) screen in Drosophila to identify ion channels regulating the ROS handling and survival in Drosophila melanogaster. The screen has revealed that alpha1-subunits (D-type, T-type, and cacophony) of voltage-gated calcium channels (VGCCs) handle PQ-mediated ROS stress differentially in a gender-based manner. Since ROS are also involved in determining the lifespan, this study discovered that the absence of T-type and cacophony decreased the lifespan while the absence of D-type maintained a similar lifespan to that of the wild-type strain. VGCCs are also responsible for electrical signaling in cardiac cells. The cardiac function of each mutant was evaluated through optical coherence tomography (OCT), which revealed that alpha1-subunits of VGCCs are essential in maintaining cardiac rhythmicity and cardiac function in an age-dependent manner. These results establish specific roles of alpha1-subunits of VGCCs in the functioning of the aging heart.
Lirakis, M., Dolezal, M. and Schlotterer, C. (2018). Redefining reproductive dormancy in Drosophila as a general stress response to cold temperatures. J Insect Physiol 107: 175-185. PubMed ID: 29649483
Organisms regularly encounter unfavorable conditions and the genetic adaptations facilitating survival have been of long-standing interest to evolutionary biologists. Despite dormancy being a well-studied adaptation to facilitate overwintering, there is still considerable controversy about the distribution of dormancy among natural populations and between species in Drosophila. The current definition of dormancy as developmental arrest of oogenesis at the previtellogenic stage (stage 7) distinguishes dormancy from general stress related block of oogenesis at early vitellogenic stages (stages 8 - 9). In an attempt to resolve this, reproductive dormancy in D. melanogaster and D. simulans was scrutinized. WDormancy shows the same hallmarks of arrest of oogenesis at stage 9, as described for other stressors and propose a new classification for dormancy. Applying this modified classification, this study showed that both species express dormancy in cosmopolitan and African populations, further supporting that dormancy uses an ancestral pathway induced by environmental stress. While significant differences were found between individuals and the two Drosophila species in their sensitivity to cold temperature stress, it is also noted that extreme temperature stress (8 degrees C) resulted in very strong dormancy incidence, which strongly reduced the differences seen at less extreme temperatures. It is concluded that dormancy in Drosophila should not be considered a special trait, but is better understood as a generic stress response occurring at low temperatures.
Liang, Y., Liu, C., Lu, M., Dong, Q., Wang, Z., Wang, Z., Xiong, W., Zhang, N., Zhou, J., Liu, Q., Wang, X. and Wang, Z. (2018). Calorie restriction is the most reasonable anti-ageing intervention: a meta-analysis of survival curves. Sci Rep 8(1): 5779. PubMed ID: 29636552
Despite technological advances, the survival records from longevity experiments remain the most indispensable tool in ageing-related research. A variety of interventions, including medications, genetic manipulations and calorie restriction (CR), have been demonstrated to extend the lifespan of several species. Surprisingly, few systematic studies have investigated the differences among these anti-ageing strategies using survival data. This study conductd a comprehensive and comparative meta-analysis of numerous published studies on Caenorhabditis elegans and Drosophila. CR and genetic manipulations were found to be generally more effective than medications at extending the total lifespan in both models, and CR can improve the ageing pattern of C. elegans. The survival variation for different anti-ageing medications was examined and determined that hypoglycaemic agents and antioxidants are advantageous despite only moderately increasing the overall lifespan; therefore, these two types of medications are promising CR mimetics. Analysis of genetic manipulations also indicated that the genes or pathways that extend lifespan in a healthier pattern are associated with CR. These results suggest that CR or CR mimetics may be the most reasonable and potentially beneficial anti-ageing strategy.

Friday June 1st - Cell Signaling

Schaefer, K. N., Bonello, T. T., Zhang, S., Williams, C. E., Roberts, D. M., McKay, D. J. and Peifer, M. (2018). Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo. PLoS Genet 14(4): e1007339. PubMed ID: 29641560
Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis. The tumor suppressors Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. This study manipulated Axin and APC2 levels. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. In the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. These data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.
Sriskanthadevan-Pirahas, S., Lee, J. and Grewal, S. S. (2018). The EGF/Ras pathway controls growth in Drosophila via ribosomal RNA synthesis. Dev Biol 439(1):19-29. PubMed ID: 29660312
The Ras small G-protein is a conserved regulator of cell and tissue growth during animal development. Studies in Drosophila have shown how Ras can stimulate a RAF-MEK-ERK signalling pathway to control cell growth and proliferation in response to Epidermal Growth Factor (EGF) stimulation. This work has also defined several transcription factors that can function as downstream growth effectors of the EGF/Ras/ERK pathway by stimulating mRNA transcription. This study reports on stimulation of RNA polymerase I (Pol I)-mediated ribosomal RNA (rRNA) synthesis as a growth effector of Ras/ERK signalling in Drosophila. Ras/ERK signalling promotes an increase in nucleolar size in larval wing discs, which is indicative of increased ribosome synthesis. Activation of Ras/ERK signalling promotes rRNA synthesis both in vivo and in cultured Drosophila S2 cells. Ras signalling can regulate the expression of the Pol I transcription factor TIF-IA, and that this regulation requires dMyc. Finally, TIF-IA-mediated rRNA synthesis was found to be required for Ras/ERK signalling to drive proliferation in both larval and adult Drosophila tissues. These findings indicate that Ras signalling can promote ribosome synthesis in Drosophila, and that this is one mechanism that contributes to the growth effects of the Ras signalling pathway.
Tan, K. L., Haelterman, N. A., Kwartler, C. S., Regalado, E. S., Lee, P. T., Nagarkar-Jaiswal, S., Guo, D. C., Duraine, L., Wangler, M. F., Bamshad, M. J., Nickerson, D. A., Lin, G., Milewicz, D. M. and Bellen, H. J. (2018). Ari-1 regulates myonuclear organization together with Parkin and is associated with aortic aneurysms. Dev Cell 45(2): 226-244.e228. PubMed ID: 29689197
Nuclei are actively positioned and anchored to the cytoskeleton via the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. This study identified mutations in the Parkin-like E3 ubiquitin ligase Ariadne-1 (Ari-1) that affect the localization and distribution of LINC complex members in Drosophila. ari-1 mutants exhibit nuclear clustering and morphology defects in larval muscles. Ari-1 mono-ubiquitinates the core LINC complex member Koi. Surprisingly, functional redundancy was found between Parkin and Ari-1: increasing Parkin expression rescues ari-1 mutant phenotypes and vice versa. It was further shown that rare variants in the human homolog of ari-1 (ARIH1) are associated with thoracic aortic aneurysms and dissections, conditions resulting from smooth muscle cell (SMC) dysfunction. Human ARIH1 rescues fly ari-1 mutant phenotypes, whereas human variants found in patients fail to do so. In addition, SMCs obtained from patients display aberrant nuclear morphology. Hence, ARIH1 is critical in anchoring myonuclei to the cytoskeleton.
Tang, H. W., Hu, Y., Chen, C. L., Xia, B., Zirin, J., Yuan, M., Asara, J. M., Rabinow, L. and Perrimon, N. (2018). The TORC1-regulated CPA complex rewires an RNA processing network to drive autophagy and metabolic reprogramming. Cell Metab 27(5): 1040-1054.e1048. PubMed ID: 29606597
Nutrient deprivation induces autophagy through inhibiting TORC1 activity. This study describes a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein levels and activities via the cleavage and polyadenylation (CPA) complex. TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, it was found that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism.
Urbano, J. M., Naylor, H. W., Scarpa, E., Muresan, L. and Sanson, B. (2018). Suppression of epithelial folding at actomyosin-enriched compartment boundaries downstream of Wingless signalling in Drosophila. Development 145(8). PubMed ID: 29691225
Epithelial folding shapes embryos and tissues during development. This study investigated the coupling between epithelial folding and actomyosin-enriched compartmental boundaries. The mechanistic relationship between the two is unclear, because actomyosin-enriched boundaries are not necessarily associated with folds. Also, some cases of epithelial folding occur independently of actomyosin contractility. Shallow folds called parasegment grooves that form at boundaries between anterior and posterior compartments in the early Drosophila embryo were investigated. Formation of these folds requires the presence of an actomyosin enrichment along the boundary cell-cell contacts. These enrichments, which require Wingless signalling, increase interfacial tension not only at the level of the adherens junctions but also along the lateral surfaces. Epithelial folding is normally under inhibitory control because different genetic manipulations, including depletion of the Myosin II phosphatase Flapwing, increase the depth of folds at boundaries. Fold depth correlates with the levels of Bazooka (Baz), the Par-3 homologue, along the boundary cell-cell contacts. Moreover, Wingless and Hedgehog signalling have opposite effects on fold depth at the boundary that correlate with changes in Baz planar polarity.
Tokusumi, T., Tokusumi, Y. and Schulz, R. A. (2018). The mir-7 and bag of marble genes regulate Hedgehog pathway signaling in blood cell progenitors in Drosophila larval lymph glands. Genesis e23210. PubMed ID: 29663653
Hedgehog (Hh) pathway signaling is crucial for the maintenance of blood cell progenitors in the lymph gland hematopoietic organ present in Drosophila third instar larvae. Previous studies have likewise shown the importance of the mir-7 and bag of marbles (bam) genes in maintaining the progenitor state. Thus attempts were made to investigate a possible interaction between the Hh pathway and mir-7/bam in the prohemocyte population within this hematopoietic tissue. Gain of function mir-7 was able to rescue a blood cell progenitor depletion phenotype caused by Patched (Ptc) inhibition of Hh pathway signaling in these cells. Similarly, expression of a dominant/negative version of Ptc was able to rescue the severe reduction of prohemocytes due to bam loss of function. Furthermore, it was demonstrated that Suppressor of fused [Su(fu)], another known inhibitor of Hh signaling, likely serves as a translational repression target of the mir-7 miRNA. These results suggest the mir-7/bam combination regulates the Hh signaling network through repression of Su(fu) to maintain hemocyte progenitors in the larval lymph gland.
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