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Friday, August 30th, 2019 - Adult Nervous System Function

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Li, X., Abou Tayoun, A., Song, Z., Dau, A., Rien, D., Jaciuch, D., Dongre, S., Blanchard, F., Nikolaev, A., Zheng, L., Bollepalli, M. K., Chu, B., Hardie, R. C., Dolph, P. J. and Juusola, M. (2019). Ca(2+)-activated K(+) channels reduce network excitability, improving adaptability and energetics for transmitting and perceiving sensory information. J Neurosci. PubMed ID: 31350259
Summary:
Ca(2+)-activated K(+) channels (BK and SK) are ubiquitous in synaptic circuits, but their role in network adaptation and sensory perception remains largely unknown. Using electrophysiological and behavioral assays and biophysical modelling, this study discover how visual information transfer in mutants lacking the BK channel (dSlo(-)), SK channel (dSK(-)) or both (dSK (-);;dSlo(-)) is shaped in the female fruit fly (Drosophila melanogaster) R1-R6 photoreceptor-LMC circuits (R-LMC-R system) through synaptic feedforward-feedback interactions and reduced R1-R6 Shaker and Shab K(+) conductances. This homeostatic compensation is specific for each mutant, leading to distinctive adaptive dynamics. These dynamics inescapably increase the energy cost of information and promote the mutants' distorted motion perception, determining the true price and limits of chronic homeostatic compensation in an in vivo genetic animal model. These results reveal why Ca(2+)-activated K(+) channels reduce network excitability (energetics), improving neural adaptability for transmitting and perceiving sensory information.
Mohamed, A. A. M., Hansson, B. S. and Sachse, S. (2019). Third-order neurons in the lateral horn enhance bilateral contrast of odor inputs through contralateral inhibition in Drosophila. Front Physiol 10: 851. PubMed ID: 31354516
Summary:
The survival and reproduction of Drosophila melanogaster depends heavily on its ability to determine the location of an odor source and either to move toward or away from it. Despite the very small spatial separation between the two antennae and the redundancy in sensory neuron projection to both sides of the brain, Drosophila can resolve the concentration gradient by comparing the signal strength between the two antennae. When an odor stimulates the antennae asymmetrically, ipsilateral projection neurons from the first olfactory center are more strongly excited compared to the contralateral ones. However, it remains elusive how higher-order neurons process such asymmetric or lateralized odor inputs. This study monitored and analyzed for the first time the activity patterns of a small cluster of third-order neurons (so-called ventrolateral protocerebrum neurons) to asymmetric olfactory stimulation using two-photon calcium imaging. The data demonstrate that lateralized odors evoke distinct activation of these neurons in the left and right brain hemisphere as a result of contralateral inhibition. Moreover, using laser transection experiments, this contralateral inhibition was shown to be mediated by presynaptic neurons most likely located in the lateral horn. Finally, it is proposed that this inhibitory interaction between higher-order neurons facilitates odor lateralization and plays a crucial role in olfactory navigation behavior of Drosophila, a theory that needs to be experimentally addressed in future studies.
Lee, K. M., Mathies, L. D. and Grotewiel, M. (2019). Alcohol sedation in adult Drosophila is regulated by Cysteine proteinase-1 in cortex glia. Commun Biol 2: 252. PubMed ID: 31286069
Summary:
Although numerous studies have demonstrated that neuronal mechanisms regulate alcohol-related behaviors, very few have investigated the direct role of glia in behavioral responses to alcohol. The results described in this study begin to fill this gap in the alcohol behavior and gliobiology fields. Since Drosophila exhibit conserved behavioral responses to alcohol and their CNS glia are similar to mammalian CNS glia, this study used Drosophila to begin exploring the role of glia in alcohol behavior. Knockdown of Cysteine proteinase-1 (Cp1) in glia increased Drosophila alcohol sedation and that this effect was specific to cortex glia and adulthood. These data implicate Cp1 and cortex glia in alcohol-related behaviors. Cortex glia are functionally homologous to mammalian astrocytes and Cp1 is orthologous to mammalian Cathepsin L. These studies raise the possibility that cathepsins may influence behavioral responses to alcohol in mammals via roles in astrocytes.
Ki, Y. and Lim, C. (2019). Sleep-promoting effects of threonine link amino acid metabolism in Drosophila neuron to GABAergic control of sleep drive. Elife 8. PubMed ID: 31313987
Summary:
Emerging evidence indicates the role of amino acid metabolism in sleep regulation. This study demonstrates sleep-promoting effects of dietary threonine (SPET) in Drosophila. Dietary threonine markedly increased daily sleep amount and decreased the latency to sleep onset in a dose-dependent manner. High levels of synaptic GABA or pharmacological activation of metabotropic GABA receptors (GABAB-R) suppressed SPET. By contrast, synaptic blockade of GABAergic neurons or transgenic depletion of GABAB-R in the ellipsoid body R2 neurons enhanced sleep drive non-additively with SPET. Dietary threonine reduced GABA levels, weakened metabotropic GABA responses in R2 neurons, and ameliorated memory deficits in plasticity mutants. Moreover, genetic elevation of neuronal threonine levels was sufficient for facilitating sleep onset. Taken together, these data define threonine as a physiologically relevant, sleep-promoting molecule that may intimately link neuronal metabolism of amino acids to GABAergic control of sleep drive via the neuronal substrate of sleep homeostasis.
Lyutova, R., Selcho, M., Pfeuffer, M., Segebarth, D., Habenstein, J., Rohwedder, A., Frantzmann, F., Wegener, C., Thum, A. S. and Pauls, D. (2019). Reward signaling in a recurrent circuit of dopaminergic neurons and peptidergic Kenyon cells. Nat Commun 10(1): 3097. PubMed ID: 31308381
Summary:
Dopaminergic neurons in the brain of the Drosophila larva play a key role in mediating reward information to the mushroom bodies during appetitive olfactory learning and memory. Using optogenetic activation of Kenyon cells, evidence is provided that recurrent signaling exists between Kenyon cells and dopaminergic neurons of the primary protocerebral anterior (pPAM) cluster. Optogenetic activation of Kenyon cells paired with odor stimulation is sufficient to induce appetitive memory. Simultaneous impairment of the dopaminergic pPAM neurons abolishes appetitive memory expression. Thus, it is argued that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. This study further shows that this feedback signaling is dependent on short neuropeptide F, but not on acetylcholine known to be important for odor-shock memories in adult flies. These data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization.
Peng, J. J., Lin, S. H., Liu, Y. T., Lin, H. C., Li, T. N. and Yao, C. K. (2019). A circuit-dependent ROS feedback loop mediates glutamate excitotoxicity to sculpt the Drosophila motor system. Elife 8. PubMed ID: 31318331
Summary:
Overproduction of reactive oxygen species (ROS) is known to mediate glutamate excitotoxicity in neurological diseases. However, how ROS burdens can influence neural circuit integrity remains unclear. This study investigated the impact of excitotoxicity induced by depletion of Drosophila Eaat1, an astrocytic glutamate transporter, on locomotor central pattern generator (CPG) activity, neuromuscular junction architecture, and motor function. Glutamate excitotoxicity triggers a circuit-dependent ROS feedback loop to sculpt the motor system. Excitotoxicity initially elevates ROS to inactivate cholinergic interneurons, consequently changing CPG output activity to overexcite motor neurons and muscles. Remarkably, tonic motor neuron stimulation boosts muscular ROS, gradually dampening muscle contractility to feedback-enhance ROS accumulation in the CPG circuit and subsequently exacerbate circuit dysfunction. Ultimately, excess premotor excitation of motor neurons promotes ROS-activated stress signaling to alter neuromuscular junction architecture. Collectively, these results reveal that excitotoxicity-induced ROS can perturb motor system integrity by a circuit-dependent mechanism.

Thursday, August 29th - Cytoskeleton and Junctions

O'Connell, M. E., Sridharan, D., Driscoll, T., Krishnamurthy, I., Perry, W. G. and Applewhite, D. A. (2019). The Drosophila protein, Nausicaa, regulates lamellipodial actin dynamics in a Cortactin-dependent manner. Biol Open 8(6). PubMed ID: 31164339
Summary:
Drosophila CG10915 is an uncharacterized protein coding gene with sequence similarity to human Cortactin-binding protein 2 (CTTNBP2) and Cortactin-binding protein 2 N-terminal-like (CTTNBP2NL). This study has named this gene Nausicaa (naus) and characterize it through a combination of quantitative live-cell total internal reflection fluorescence microscopy, electron microscopy, RNAi depletion and genetics. Naus was found to co-localizes with F-actin and Cortactin in the lamellipodia of Drosophila S2R+ and D25c2 cells and this localization is lost following Cortactin or Arp2/3 depletion or by mutations that disrupt a conserved proline patch found in its mammalian homologs. Using permeabilization activated reduction in fluorescence and fluorescence recovery after photobleaching, it was found that depletion of Cortactin alters Naus dynamics leading to a decrease in its half-life. Furthermore, Naus depletion in S2R+ cells led to a decrease in actin retrograde flow and a lamellipodia characterized by long, unbranched filaments. These alterations to the dynamics and underlying actin architecture also affect D25c2 cell migration and decrease arborization in Drosophila neurons. The hypothesis is presented that Naus functions to slow Cortactin's disassociation from Arp2/3 nucleated branch junctions, thereby increasing both branch nucleation and junction stability.
Baskar, R., Bahkrat, A., Otani, T., Wada, H., Davidov, G., Pandey, H., Gheber, L., Zarivach, R., Hayashi, S. and Abdu, U. (2019). The plus-tip tracking and microtubule stabilizing activities of Javelin-like regulate microtubule organization and cell polarity. FEBS J. PubMed ID: 31152621
Summary:
Cell polarity is essential for building cell asymmetry in all eukaryotic cells. Drosophila oocyte and bristle development require the newly characterized Spn-F protein complex, which includes Spn-F, IKKepsilon, and Javelin-like (Jvl), to establish polarity. Jvl is a novel microtubule (MT)-associated protein; however, the mechanism by which it regulates MT organization is still unknown. This study found that overexpression of Jvl stabilizes MTs and that jvl is needed for stable MT arrangement at the bristle tip and organization of the dynamic MT throughout the bristle shaft. At low levels of expression in cultured cells, Jvl behaved as a microtubule plus-end tracking protein. It was demonstrated that Jvl physically interacts with the highly conserved MT end-binding protein 1 (EB1) using yeast two-hybrid and GST pull-down assays. This interaction is, however, dispensable for Jvl function in oocyte and bristle development. In addition, using a MT-binding assay, Jvl-C terminus was seen to directly bind to MTs. It was also revealed that oocyte developmental arrest caused by Jvl overexpression in the germline can be rescued by mutations in its partners, spn-F and ikkepsilon, suggesting that complex formation with Spn-F and IKKepsilon is required for Jvl function in vivo. In summary, these results show that the microtubule plus-end tracking and stabilizing activities of Jvl are central for controlling cell polarity of oocytes and bristles.
Bajur, A. T., Iyer, K. V. and Knust, E. (2019). Cytocortex-dependent dynamics of Drosophila Crumbs controls junctional stability and tension during germ band retraction. J Cell Sci. PubMed ID: 31300472
Summary:
During morphogenesis epithelia undergo dynamic rearrangements, which requires continuous remodelling of junctions and cell shape, but at the same time mechanisms preserving cell polarity and tissue integrity. Apico-basal polarity is key to localise the machinery that enables cell shape changes. The evolutionarily conserved Drosophila Crumbs protein is critical for maintaining apico-basal polarity and epithelial integrity. How Crumbs is maintained in a dynamically developing embryo remains largely unknown. In this study quantitative fluorescence techniques were applied to show that during germ band retraction, Crumbs dynamics correlates with the morphogenetic activity of the epithelium. Genetic and pharmacological perturbations revealed that the mobile pool of Crumbs is fine-tuned by the actomyosin cortex in a stage dependent manner. Stabilisation of Crumbs at the plasma membrane depends on a proper link to the actomyosin cortex via an intact FERM-domain binding site in its intracellular domain, loss of which leads to increased junctional tension and higher DE-cadherin turnover, resulting in impaired junctional rearrangements. These data define Crumbs as a mediator between polarity and junctional regulation to orchestrate epithelial remodelling in response to changes in actomyosin activity.
Krueger, D., Quinkler, T., Mortensen, S. A., Sachse, C. and De Renzis, S. (2019). Cross-linker-mediated regulation of actin network organization controls tissue morphogenesis. J Cell Biol. PubMed ID: 31253650
Summary:
Contraction of cortical actomyosin networks driven by myosin activation controls cell shape changes and tissue morphogenesis during animal development. In vitro studies suggest that contractility also depends on the geometrical organization of actin filaments. This study analyzed the function of actomyosin network topology in vivo using optogenetic stimulation of myosin-II in Drosophila embryos. Early during cellularization, hexagonally arrayed actomyosin fibers are resilient to myosin-II activation. Actomyosin fibers then acquire a ring-like conformation and become contractile and sensitive to myosin-II. This transition is controlled by Bottleneck, a Drosophila unique protein expressed for only a short time during early cellularization, which this study shows to regulate actin bundling. In addition, it requires two opposing actin cross-linkers, Filamin and Fimbrin. Filamin acts synergistically with Bottleneck to facilitate hexagonal patterning, while Fimbrin controls remodeling of the hexagonal network into contractile rings. Thus, actin cross-linking regulates the spatio-temporal organization of actomyosin contraction in vivo, which is critical for tissue morphogenesis.
Chen, D. Y., Crest, J., Streichan, S. J. and Bilder, D. (2019). Extracellular matrix stiffness cues junctional remodeling for 3D tissue elongation. Nat Commun 10(1): 3339. PubMed ID: 31350387
Summary:
Organs are sculpted by extracellular as well as cell-intrinsic forces, but how collective cell dynamics are orchestrated in response to environmental cues is poorly understood. This study applied advanced image analysis to reveal extracellular matrix-responsive cell behaviors that drive elongation of the Drosophila follicle, a model system in which basement membrane stiffness instructs three-dimensional tissue morphogenesis. Through in toto morphometric analyses of wild type and round egg mutants, this study found that neither changes in average cell shape nor oriented cell division are required for appropriate organ shape. Instead, a major element is the reorientation of elongated cells at the follicle anterior. Polarized reorientation is regulated by mechanical cues from the basement membrane, which are transduced by the Src tyrosine kinase to alter junctional E-cadherin trafficking. This mechanosensitive cellular behavior represents a conserved mechanism that can elongate edgeless tubular epithelia in a process distinct from those that elongate bounded, planar epithelia.
Letizia, A., He, D., Astigarraga, S., Colombelli, J., Hatini, V., Llimargas, M. and Treisman, J. E. (2019). Sidekick is a key component of tricellular adherens junctions that acts to resolve cell rearrangements. Dev Cell. PubMed ID: 31353315
Summary:
Tricellular adherens junctions are points of high tension that are central to the rearrangement of epithelial cells. However, the molecular composition of these junctions is unknown, making it difficult to assess their role in morphogenesis. This study shows that Sidekick, an immunoglobulin family cell adhesion protein, is highly enriched at tricellular adherens junctions in Drosophila. This localization is modulated by tension, and Sidekick is itself necessary to maintain normal levels of cell bond tension. Loss of Sidekick causes defects in cell and junctional rearrangements in actively remodeling epithelial tissues like the retina and tracheal system. The adaptor proteins Polychaetoid and Canoe are enriched at tricellular adherens junctions in a Sidekick-dependent manner; Sidekick functionally interacts with both proteins and directly binds to Polychaetoid. It is suggested that Polychaetoid and Canoe link Sidekick to the actin cytoskeleton to enable tricellular adherens junctions to maintain or transmit cell bond tension during epithelial cell rearrangements.

Wednesday, August 28th - RNA and Transposons

Niu, Y., Liu, Z., Nian, X., Xu, X. and Zhang, Y. (2019). miR-210 controls the evening phase of circadian locomotor rhythms through repression of Fasciclin 2. PLoS Genet 15(7): e1007655. PubMed ID: 31356596
Summary:
Circadian clocks control the timing of animal behavioral and physiological rhythms. Fruit flies anticipate daily environmental changes and exhibit two peaks of locomotor activity around dawn and dusk. microRNAs are small non-coding RNAs that play important roles in post-transcriptional regulation. This study has identified Drosophila miR-210 as a critical regulator of circadian rhythms. Under light-dark conditions, flies lacking miR-210 (miR-210KO) exhibit a dramatic 2 hrs phase advance of evening anticipatory behavior. However, circadian rhythms and molecular pacemaker function are intact in miR-210KO flies under constant darkness. Furthermore, miR-210 determines the evening phase of activity through repression of the cell adhesion molecule Fasciclin 2 (Fas2). Ablation of the miR-210 binding site within the 3' UTR of Fas2 (Fas2DeltamiR-210) by CRISPR-Cas9 advances the evening phase as in miR-210KO. Indeed, miR-210 genetically interacts with Fas2. Moreover, Fas2 abundance is significantly increased in the optic lobe of miR-210KO. In addition, overexpression of Fas2 in the miR-210 expressing cells recapitulates the phase advance behavior phenotype of miR-210KO. Together, these results reveal a novel mechanism by which miR-210 regulates circadian locomotor behavior.
Pajak, A., Laine, I., Clemente, P., El-Fissi, N., Schober, F. A., Maffezzini, C., Calvo-Garrido, J., Wibom, R., Filograna, R., Dhir, A., Wedell, A., Freyer, C. and Wredenberg, A. (2019). Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo. PLoS Genet 15(7): e1008240. PubMed ID: 31365523
Summary:
The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, this study manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC were investigated. The results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together these results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.
Linsalata, A. E., He, F., Malik, A. M., Glineburg, M. R., Green, K. M., Natla, S., Flores, B. N., Krans, A., Archbold, H. C., Fedak, S. J., Barmada, S. J. and Todd, P. K. (2019). DDX3X and specific initiation factors modulate FMR1 repeat-associated non-AUG-initiated translation. EMBO Rep: e47498. PubMed ID: 31347257
Summary:
A CGG trinucleotide repeat expansion in the 5' UTR of FMR1 causes the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). This repeat supports a non-canonical mode of protein synthesis known as repeat-associated, non-AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, a candidate-based screen of eukaryotic initiation factors and RNA helicases was performed in cell-based assays and a Drosophila melanogaster model of FXTAS. Multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5'UTR. These include the DEAD-box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat-induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat-associated neurodegeneration.
Murano, K., Iwasaki, Y. W., Ishizu, H., Mashiko, A., Shibuya, A., Kondo, S., Adachi, S., Suzuki, S., Saito, K., Natsume, T., Siomi, M. C. and Siomi, H. (2019). Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing. Embo j: e102870. PubMed ID: 31368590
Summary:
The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. This study identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. These results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing.
Palsule, G., Gopalan, V. and Simcox, A. (2019). Biogenesis of RNase P RNA from an intron requires co-assembly with cognate protein subunits. Nucleic Acids Res. PubMed ID: 31287870
Summary:
RNase P RNA (RPR), the catalytic subunit of the essential RNase P ribonucleoprotein, removes the 5' leader from precursor tRNAs. The ancestral eukaryotic RPR is a Pol III transcript generated with mature termini. In the branch of the arthropod lineage that led to the insects and crustaceans, however, a new allele arose in which RPR is embedded in an intron of a Pol II transcript and requires processing from intron sequences for maturation. This study demonstrates that the Drosophila intronic-RPR precursor is trimmed to the mature form by the ubiquitous nuclease Rat1/Xrn2 (5') and the RNA exosome (3'). Processing is regulated by a subset of RNase P proteins (Rpps) that protects the nascent RPR from degradation, the typical fate of excised introns. These results indicate that the biogenesis of RPR in vivo entails interaction of Rpps with the nascent RNA to form the RNase P holoenzyme and suggests that a new pathway arose in arthropods by coopting ancient mechanisms common to processing of other noncoding RNAs.
Kobayashi, H., Shoji, K., Kiyokawa, K., Negishi, L. and Tomari, Y. (2019). VCP machinery mediates autophagic degradation of empty Argonaute. Cell Rep 28(5): 1144-1153.e1144. PubMed ID: 31365860
Summary:
The Argonaute subfamily of proteins (AGO) loads microRNAs (miRNAs) to form the effector complex that mediates target gene silencing. Empty AGO, but not miRNA-loaded AGO, is selectively degraded across species. It has been reported that the degradation of empty AGO is part of a quality control pathway that eliminates dysfunctional AGO. However, how empty AGO is degraded remains unclear. This study shows that the empty state of Drosophila Ago1 is degraded by autophagy. Comprehensive LC-MS/MS analyses, together with manipulation of the Ago1 ubiquitination level, revealed that VCP, which mediates selective autophagy, recognizes empty Ago1 via the Ufd1-Npl4 heterodimer. Depletion of VCP-Ufd1-Npl4 machinery impairs degradation of empty Ago1 and miRNA-mediated target gene silencing. These findings reveal a direct link between empty AGO degradation and selective autophagy that ensures efficient miRNA function.

Tuesday, August 27th - Synapse and Vesicles

Kikuma, K., Li, X., Perry, S., Li, Q., Goel, P., Chen, C., Kim, D., Stavropoulos, N. and Dickman, D. (2019). Cul3 and insomniac are required for rapid ubiquitination of postsynaptic targets and retrograde homeostatic signaling. Nat Commun 10(1): 2998. PubMed ID: 31278365
Summary:
At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. In this study a forward genetic screen is described to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, it was found that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, it was shown that Peflin, a Ca(2+)-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca(2+) signaling and control of Cul3/Inc activity in the postsynaptic compartment. This study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses.
Lakatos, Z., Lorincz, P., Szabo, Z., Benko, P., Kenez, L. A., Csizmadia, T. and Juhasz, G. (2019). Sec20 is required for autophagic and endocytic degradation independent of golgi-ER retrograde transport. Cells 8(8). PubMed ID: 31344970
Summary:
Endocytosis and autophagy are evolutionarily conserved degradative processes in all eukaryotes. Both pathways converge to the lysosome where cargo is degraded. Improper lysosomal degradation is observed in many human pathologies, so its regulatory mechanisms are important to understand. Sec20/BNIP1 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 1) is a BH3 (Bcl-2 homology 3) domain-containing SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) protein that has been suggested to promote Golgi-ER retrograde transport, mitochondrial fission, apoptosis and mitophagy in yeast and vertebrates. This study shows that loss of Sec20 in Drosophila fat cells causes the accumulation of autophagic vesicles and prevents proper lysosomal acidification and degradation during bulk, starvation-induced autophagy. Furthermore, Sec20 knockdown leads to the enlargement of late endosomes and accumulation of defective endolysosomes in larval Drosophila nephrocytes. Importantly, the loss of Syx18 (Syntaxin 18), one of the known partners of Sec20, led to similar changes in nephrocytes and fat cells. Interestingly. Sec20 appears to function independent of its role in Golgi-ER retrograde transport in regulating lysosomal degradation, as the loss of its other partner SNAREs Use1 (Unconventional SNARE In The ER 1) and Sec22 or tethering factor Zw10 (Zeste white 10), which function together in the Golgi-ER pathway, does not cause defects in autophagy or endocytosis. Thus, these data identify a potential new transport route specific to lysosome biogenesis and function.
Betancourt-Solis, M. A. and McNew, J. A. (2019). Detergent-assisted reconstitution of recombinant Drosophila Atlastin into liposomes for lipid-mixing assays. J Vis Exp(149). PubMed ID: 31329173
Summary:
Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. A method is detailed for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes is described. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion is described.
Liu, K., Jones, S., Minis, A., Rodriguez, J., Molina, H. and Steller, H. (2019). PI31 is an adaptor protein for proteasome transport in axons and required for synaptic development. Dev Cell. PubMed ID: 31327739
Summary:
Protein degradation by the ubiquitin-proteasome system is critical for neuronal function. Neurons utilize microtubule-dependent molecular motors to allocate proteasomes to synapses, but how proteasomes are coupled to motors and how this is regulated to meet changing demand for protein breakdown remain largely unknown. This study shows that the conserved proteasome-binding protein PI31 serves as an adaptor to couple proteasomes with dynein light chain proteins (DYNLL1/2). The inactivation of PI31 inhibited proteasome motility in axons and disrupted synaptic proteostasis, structure, and function. Moreover, phosphorylation of PI31 by p38 MAPK enhanced binding to DYNLL1/2 and promoted the directional movement of proteasomes in axons, suggesting a mechanism to regulate loading of proteasomes onto motors. Inactivation of PI31 in mouse neurons attenuated proteasome movement in axons, indicating this process is conserved. Because mutations affecting PI31 activity are associated with human neurodegenerative diseases, impairment of PI31-mediated axonal transport of proteasomes may contribute to these disorders.
Matusek, T., Therond, P. and Furthauer, M. (2019). Functional analysis of ESCRT-positive extracellular vesicles in the Drosophila wing imaginal disc. Methods Mol Biol 1998: 31-47. PubMed ID: 31250292
Summary:
A large number of studies have shown that proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) can trigger the biogenesis of different types of Extracellular Vesicles (EV). The functions that these vesicular carriers exert in vivo remain, however, poorly understood. This paper describes a series of experimental approaches that were established in the Drosophila wing imaginal disc to study the importance of ESCRT-positive EVs for the extracellular transport of signaling molecules, as exemplified by a functional analysis of the mechanism of secretion and propagation of the major developmental morphogen Hedgehog (Hh). Through the combined use of genetic, cell biological, and imaging approaches, four important aspects of exovesicle biology were investigated: (1) The genetic identification of ESCRT proteins that are specifically required for Hh secretion. (2) The imaging of ESCRT and Hh-positive EVs in the lumenal space of both living and fixed wing imaginal discs. (3) The receptor-mediated capture of Hh-containing EVs on the surface of Hh-receiving cells. (4) The effect of manipulations of ESCRT function on the extracellular pool of Hh ligands.
Ozel, M. N., Kulkarni, A., Hasan, A., Brummer, J., Moldenhauer, M., Daumann, I. M., Wolfenberg, H., Dercksen, V. J., Kiral, F. R., Weiser, M., Prohaska, S., von Kleist, M. and Hiesinger, P. R. (2019). Serial synapse formation through filopodial competition for synaptic seeding factors. Dev Cell. PubMed ID: 31353313
Summary:
Following axon pathfinding, growth cones transition from stochastic filopodial exploration to the formation of a limited number of synapses. How the interplay of filopodia and synapse assembly ensures robust connectivity in the brain has remained a challenging problem. This study developed a new 4D analysis method for filopodial dynamics and a data-driven computational model of synapse formation for R7 photoreceptor axons in developing Drosophila brains. Live data support a "serial synapse formation" model, where at any time point only 1-2 "synaptogenic" filopodia suppress the synaptic competence of other filopodia through competition for synaptic seeding factors. Loss of the synaptic seeding factors Syd-1 and Liprin-alpha leads to a loss of this suppression, filopodial destabilization, and reduced synapse formation. The failure to form synapses can cause the destabilization and secondary retraction of axon terminals. This model provides a filopodial "winner-takes-all" mechanism that ensures the formation of an appropriate number of synapses.

Monday, August 26th - Adult Development

Ogi, S., Matsuda, A., Otsuka, Y., Liu, Z., Satoh, T. and Satoh, A. K. (2019). Syndapin constricts microvillar necks to form a united rhabdomere in Drosophila photoreceptors. Development. PubMed ID: 31371377
Summary:
Drosophila photoreceptors develop from polarized epithelial cells that have apical and basolateral membranes. During morphogenesis, the apical membranes subdivide into a united bundle of photosensory microvilli (rhabdomeres) and a surrounding supporting membrane (stalk). By EMS-induced mutagenesis screening, this study found that the F-Bin/Amphiphysin/Rvs (F-BAR) protein Syndapin is essential for apical membrane segregation. The analysis of the super-resolution microscopy, STORM and the electron microscopy suggest that Syndapin localizes to the neck of the microvilli at the base of the rhabdomere. Syndapin and Moesin are required to constrict the neck of the microvilli to organize the membrane architecture at the base of the rhabdomere, to exclude the stalk membrane. Simultaneous loss of syndapin along with the microvilli adhesion molecule chaoptin significantly enhanced the disruption of stalk-rhabdomere segregation. However, loss of the factors involving endocytosis do not interfere. These results indicated Syndapin is most likely functioning through its membrane curvature properties, and not through endocytic processes for stalk-rhabdomere segregation. Elucidation of the mechanism of this unconventional domain formation will provide novel insights into the field of cell biology.
Cevik, D., Acker, M., Michalski, C. and Jacobs, J. R. (2019). Pericardin, a Drosophila collagen, facilitates accumulation of hemocytes at the heart. Dev Biol. PubMed ID: 31228417
Summary:
Hematopoietic cell lineages support organismal needs by responding to positional and systemic signals that balance proliferative and differentiation events. Drosophila provides an excellent genetic model to dissect these signals, where the activity of cues in the hemolymph or substrate can be traced to determination and differentiation events of well characterized hemocyte types. Plasmatocytes in third instar larvae increase in number in response to infection and in anticipation of metamorphosis. This study characterized hemocyte clustering, proliferation and transdifferentiation on the heart or dorsal vessel. Hemocytes accumulate on the inner foldings of the heart basement membrane, where they move with heart contraction, and are in proximity to the heart ostia and pericardial nephrocytes. The numbers of hemocytes vary, but increase transiently before pupariation, and decrease by 4h before pupa formation. During their accumulation at the heart, plasmatocytes can proliferate and can transdifferentiate into crystal cells. Serrate expressing cells as well as lamellocyte-like, Atilla expressing ensheathing cells are associated with some, but not all hemocyte clusters. Hemocyte aggregation is enhanced by the presence of a heart specific Collagen, Pericardin, but not the associated pericardial cells. The varied and transient number of hemocytes in the pericardial compartment suggests that this is not a hematopoietic hub, but a niche supporting differentiation and rapid dispersal in response to systemic signals.
Flint Brodsly, N., Bitman-Lotan, E., Boico, O., Shafat, A., Monastirioti, M., Gessler, M., Delidakis, C., Rincon-Arano, H. and Orian, A. (2019). The transcription factor Hey and nuclear lamins specify and maintain cell identity. Elife 8. PubMed ID: 31310235
Summary:
The inability of differentiated cells to maintain their identity is a hallmark of age-related diseases. The transcription factor Hey was found to supervise the identity of differentiated enterocytes (ECs) in the adult Drosophila midgut. Lineage tracing established that Hey-deficient ECs are unable to maintain their unique nuclear organization and identity. To supervise cell identity, Hey determines the expression of nuclear lamins, switching from a stem-cell lamin configuration to a differentiated lamin configuration. Moreover, continued Hey expression is required to conserve large-scale nuclear organization. During aging, Hey levels decline, and EC identity and gut homeostasis are impaired, including pathological reprograming and compromised gut integrity. These phenotypes are highly similar to those observed upon acute targeting of Hey or perturbation of lamin expression in ECs in young adults. Indeed, aging phenotypes were suppressed by continued expression of Hey in ECs, suggesting that a Hey-lamin network safeguards nuclear organization and differentiated cell identity.
Lawrence, P. A., Casal, J., Celis, J. F. and Morata, G. (2019). A refutation to 'A new A-P compartment boundary and organizer in holometabolous insect wings'. Sci Rep 9(1): 7049. PubMed ID: 31065001
Summary:
This is a response to a recent report by Abbasi and Marcus who present two main findings: first that study argued that there is an organiser and a compartment boundary within the posterior compartment of the butterfly wing. Second, that study presented evidence for a previously undiscovered lineage boundary near wing vein 5 in Drosophila, a boundary that delineates a "far posterior" compartment. Clones of cells were marked with the yellow mutation and that study reported that these clones always fail to cross a line close to vein 5 on the Drosophila wing. In the current study yellow proved an unusable marker for clones in the wing blade and therefore the matter was reexamined. Clones of cells were marked with multiple wing hairs or forked, and a substantial proportion of these clones were found to cross the proposed lineage boundary near vein 5. As internal controls, these same clones were shown to respect the other two well established compartment boundaries: the anteroposterior compartment boundary is always respected. The dorsoventral boundary is mostly respected, and is crossed only by clones that are induced early in development, consistent with many reports.
Proag, A., Monier, B. and Suzanne, M. (2019). Physical and functional cell-matrix uncoupling in a developing tissue under tension. Development. PubMed ID: 31064785
Summary:
Tissue mechanics play a crucial role in organ development. They rely on the properties of cells and the extracellular matrix (ECM), but the relative physical contribution of cells and ECM to morphogenesis is poorly understood. This study analyzed the behavior of the peripodial epithelium (PE) of the Drosophila leg disc in the light of the dynamics of its cellular and ECM components. The PE undergoes successive changes during leg development, including elongation, opening and removal to free the leg. During elongation, it was found that the ECM and cell layer are progressively uncoupled. Concomitantly, the tension, mainly borne by the ECM at first, builds up in the cell monolayer. Then, each layer of the peripodial epithelium is removed by an independent mechanism: while the ECM layer withdraws following local proteolysis, cellular monolayer withdrawal is independent of ECM degradation and driven by myosin-II-dependent contraction. These results reveal a surprising physical and functional cell-matrix uncoupling in a monolayer epithelium under tension during development.
Martinez-Corrales, G., Cabrero, P., Dow, J. A. T., Terhzaz, S. and Davies, S. A. (2019). Novel roles for GATAe in growth, maintenance and proliferation of cell populations in the Drosophila renal tubule. Development 146(9). PubMed ID: 31036543
Summary:
The GATA family of transcription factors is implicated in numerous developmental and physiological processes in metazoans. In Drosophila melanogaster, five different GATA factor genes (pannier, serpent, grain, GATAd and GATAe) have been reported as essential in the development and identity of multiple tissues, including the midgut, heart and brain. This study presents a novel role for GATAe in the function and homeostasis of the Drosophila renal (Malpighian) tubule. Reduced levels of GATAe gene expression in tubule principal cells induce uncontrolled cell proliferation, resulting in tumorous growth with associated altered expression of apoptotic and carcinogenic key genes. Furthermore, the involvement of GATAe in the maintenance of stellate cells and migration of renal and nephritic stem cells into the tubule was uncovered. These findings of GATAe as a potential master regulator in the events of growth control and cell survival required for the maintenance of the Drosophila renal tubule could provide new insights into the molecular pathways involved in the formation and maintenance of a functional tissue and kidney disease.

Friday, August 23rd - Disease Models

Luthy, K., et al. (2019). TBC1D24-TLDc-related epilepsy exercise-induced dystonia: rescue by antioxidants in a disease model. Brain 142(8): 2319-2335. PubMed ID: 31257402
Summary:
Genetic mutations in TBC1D24 have been associated with multiple phenotypes, with epilepsy being the main clinical manifestation. The TBC1D24 protein consists of the unique association of a Tre2/Bub2/Cdc16 (TBC) domain and a TBC/lysin motif domain/catalytic (TLDc) domain. Through whole genome/exome sequencing compound heterozygous mutations, R360H and G501R, within the TLDc domain, were identified in an index family with a Rolandic epilepsy exercise-induced dystonia phenotype (OMIM). Six patients were discovered to harbour a missense mutation in the subdomain of TLDc between residues 500 and 511. The crystal structure of the conserved Drosophila TLDc domain was solved in this study. The functional consequences were characterized of a strong and a weak TLDc mutation using Drosophila, where TBC1D24/Skywalker regulates synaptic vesicle trafficking. In a Drosophila model, neuronally expressing human TBC1D24, it was demonstrated that the TBC1D24G501R TLDc mutation causes activity-induced locomotion and synaptic vesicle trafficking defects. The neuronal phenotypes of the mutation are consistent with exacerbated oxidative stress sensitivity, which is rescued by treating mutant animals with antioxidants N-acetylcysteine amide or alpha-tocopherol as indicated by restored synaptic vesicle trafficking levels and sustained behavioural activity. These data thus show that mutations in the TLDc domain of TBC1D24 cause Rolandic-type focal motor epilepsy and exercise-induced dystonia. The humanized TBC1D24G501R fly model exhibits sustained activity and vesicle transport defects. It is proposed that the TBC1D24/Sky TLDc domain is a reactive oxygen species sensor mediating synaptic vesicle trafficking rates that, when dysfunctional, causes a movement disorder in patients and flies. The TLDc and TBC domain mutations' response to antioxidant treatment suggests a potential for combining antioxidant-based therapeutic approaches to TBC1D24-associated disorders with previously described lipid-altering strategies for TBC domain mutations.
Li, J. Q., Duan, D. D., Zhang, J. Q., Zhou, Y. Z., Qin, X. M., Du, G. H. and Gao, L. (2019). Bioinformatic prediction of critical genes and pathways involved in longevity in Drosophila melanogaster. Mol Genet Genomics. PubMed ID: 31327054
Summary:
The pursuit of longevity has been the goal of humanity since ancient times. Genetic alterations have been demonstrated to affect lifespan. As increasing numbers of pro-longevity genes and anti-longevity genes have been discovered in Drosophila, screening for functionally important genes among the large number of genes has become difficult. The aim of this study was to explore critical genes and pathways affecting longevity in Drosophila melanogaster. In this study, 168 genes associated with longevity in D. melanogaster were collected from the Human Ageing Genomic Resources (HAGR) database. Network clustering analysis, network topological analysis, and pathway analysis were integrated to identify key genes and pathways. Quantitative real-time PCR (qRT-PCR) was applied to verify the expression of genes in representative pathways and of predicted genes derived from the gene-gene sub-network. The results revealed that six key pathways might be associated with longevity, including the longevity-regulating pathway, the peroxisome pathway, the mTOR-signalling pathway, the FOXO-signalling pathway, the AGE-RAGE-signalling pathway in diabetic complications, and the TGF-beta-signalling pathway. Moreover, the results revealed that six key genes in representative pathways, including Cat, Ry, S6k, Sod, Tor, and Tsc1, and the predicted genes Jra, Kay, and Rheb exhibited significant expression changes in ageing D. melanogaster strain w(1118) compared to young ones. Overall, these results revealed that six pathways and six key genes might play pivotal roles in regulating longevity, and three interacting genes might be implicated in longevity. The results will not only provide new insight into the mechanisms of longevity, but also provide novel ideas for network-based approaches for longevity-related research.
Higham, J. P., Malik, B. R., Buhl, E., Dawson, J. M., Ogier, A. S., Lunnon, K. and Hodge, J. J. L. (2019). Alzheimer's disease associated genes Ankyrin and Tau cause shortened lifespan and memory loss in Drosophila. Front Cell Neurosci 13: 260. PubMed ID: 31244615
Summary:
Alzheimer's disease (AD) is the most common form of dementia and is characterized by intracellular neurofibrillary tangles of hyperphosphorylated Tau, including the 0N4R isoform and accumulation of extracellular amyloid beta (Abeta) plaques. However, less than 5% of AD cases are familial, with many additional risk factors contributing to AD including aging, lifestyle, the environment and epigenetics. Recent epigenome-wide association studies (EWAS) of AD have identified a number of loci that are differentially methylated in the AD cortex. Indeed, hypermethylation and reduced expression of the Ankyrin 1 (ANK1) gene in AD has been reported in the cortex in numerous different post-mortem brain cohorts. Little is known about the normal function of ANK1 in the healthy brain, nor the role it may play in AD. This study has generated Drosophila models to allow functional characterization of Drosophila Ank2, the ortholog of human ANK1 and to determine its interaction with human Tau and Abeta. Expression of human Tau 0N4R or the oligomerizing Abeta 42 amino acid peptide caused shortened lifespan, degeneration, disrupted movement, memory loss, and decreased excitability of memory neurons with co-expression tending to make the pathology worse. Drosophila with reduced neuronal Ank2 expression have shortened lifespan, reduced locomotion, reduced memory and reduced neuronal excitability similar to flies overexpressing either human Tau 0N4R or Abeta42. Therefore, this study shows that the mis-expression of Ank2 can drive disease relevant processes and phenocopy some features of AD. Therefore, it is proposed targeting human ANK1 may have therapeutic potential. This represents the first study to characterize an AD-relevant gene nominated from EWAS.
Hope, K. A., Flatten, D., Cavitch, P., May, B., Sutcliffe, J. S., O'Donnell, J. and Reiter, L. T. (2019). The Drosophila gene Sulfateless modulates autism-like behaviors. Front Genet 10: 574. PubMed ID: 31316544
Summary:
Major challenges to identifying genes that contribute to autism spectrum disorder (ASD) risk include the availability of large ASD cohorts, the contribution of many genes overall, and small effect sizes attributable to common gene variants. An alternative approach is to use a model organism to detect alleles that impact ASD-relevant behaviors and ask whether homologous human genes infer ASD risk. This study used the Drosophila genetic reference panel (DGRP) as a tool to probe for perturbation in naturally occurring behaviors in Drosophila melanogaster that are analogous to three behavior domains: impaired social communication, social reciprocity and repetitive behaviors or restricted interests. Using 40 of the available DGRP lines, single nucleotide polymorphisms (SNPs) were identified in or near genes controlling these behavior domains, including ASD gene orthologs (neurexin 4 and neuroligin 2), an intellectual disability (ID) gene homolog (kirre), and a gene encoding a heparan sulfate (HS) modifying enzyme called sulfateless (sfl). SNPs in sfl were associated with all three ASD-like behaviors. Using RNAi knock-down of neuronal sfl expression, significant changes were observed in expressive and receptive communication during mating, decreased grooming behavior, and increased social spacing. These results suggest a role for HS proteoglycan synthesis and/or modification in normal social communication, repetitive behavior, and social interaction in flies. Finally, using the DGRP to directly identify genetic effects relevant to a neuropsychiatric disorder further demonstrates the utility of the Drosophila system in the discovery of genes relevant to human disease.
Hunt, L. C., Stover, J., Haugen, B., Shaw, T. I., Li, Y., Pagala, V. R., Finkelstein, D., Barton, E. R., Fan, Y., Labelle, M., Peng, J. and Demontis, F. (2019). A key role for the ubiquitin ligase UBR4 in myofiber hypertrophy in Drosophila and mice. Cell Rep 28(5): 1268-1281. PubMed ID: 31365869
Summary:
Skeletal muscle cell (myofiber) atrophy is a detrimental component of aging and cancer that primarily results from muscle protein degradation via the proteasome and ubiquitin ligases. Transcriptional upregulation of some ubiquitin ligases contributes to myofiber atrophy, but little is known about the role that most other ubiquitin ligases play in this process. To address this question, RNAi screening in Drosophila was used to identify the function of > 320 evolutionarily conserved ubiquitin ligases in myofiber size regulation in vivo. Whereas RNAi for some ubiquitin ligases induces myofiber atrophy, loss of others (including the N-end rule ubiquitin ligase UBR4) promotes hypertrophy. In Drosophila and mouse myofibers, loss of UBR4 induces hypertrophy via decreased ubiquitination and degradation of a core set of target proteins, including the HAT1/RBBP4/RBBP7 histone-binding complex. Together, this study defines the repertoire of ubiquitin ligases that regulate myofiber size and the role of UBR4 in myofiber hypertrophy.
Johnson, S. L., Blount, J. R., Libohova, K., Ranxhi, B., Paulson, H. L., Tsou, W. L. and Todi, S. V. (2019). Differential toxicity of ataxin-3 isoforms in Drosophila models of Spinocerebellar Ataxia Type 3. Neurobiol Dis: 104535. PubMed ID: 31310802
Summary:
The most commonly inherited dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3), is caused by a CAG repeat expansion that encodes an abnormally long polyglutamine (polyQ) repeat in the disease protein ataxin-3, a deubiquitinase. Two major full-length isoforms of ataxin-3 exist, both of which contain the same N-terminal portion and polyQ repeat, but differ in their C-termini; one (denoted here as isoform 1) contains a motif that binds ataxin-3's substrate, ubiquitin, whereas the other (denoted here as isoform 2) has a hydrophobic tail. This study took advantage of the fruit fly Drosophila melanogaster to model SCA3 and to examine the toxicity of each ataxin-3 isoform. The assays reveal isoform 1 to be markedly more toxic than isoform 2 in all fly tissues. Reduced toxicity from isoform 2 is due to much lower protein levels as a result of its expedited degradation. Additional studies indicate that isoform 1 is more aggregation-prone than isoform 2 and that the C-terminus of isoform 2 is critical for its enhanced proteasomal degradation. According to these results, although both full-length ataxin-3 isoforms are toxic, isoform 1 is likely the primary contributor to SCA3 due to its presence at higher levels. Isoform 2, as a result of rapid degradation that is dictated by its tail, is unlikely to be a key player in this disease. These findings provide new insight into the biology of this ataxia and the cellular processing of the underlying disease protein.

Thursday, August 22nd - Signaling

Chai, F., Xu, W., Musoke, T., Tarabelsi, G., Assaad, S., Freedman, J., Peterson, R., Piotrowska, K., Byrnes, J., Rogers, S. and Veraksa, A. (2019). Structure-function analysis of beta-arrestin Kurtz reveals a critical role of receptor interactions in downregulation of GPCR signaling in vivo. Dev Biol. PubMed ID: 31325455
Summary:
Arrestins control signaling via the G protein coupled receptors (GPCRs), serving as both signal terminators and transducers. Previous studies identified several structural elements in arrestins that contribute to their functions as GPCR regulators. However, the importance of these elements in vivo is unclear, and the developmental roles of arrestins are not well understood. An in vivo structure-function analysis of Kurtz (Krz), the single ortholog of mammalian beta-arrestins in the Drosophila genome, was carried out. A combination of Krz mutations affecting the GPCR-phosphosensing and receptor core-binding ("finger loop") functions (Krz-KKVL/A) resulted in a complete loss of Krz activity during development. Endosome recruitment and bioluminescence resonance energy transfer (BRET) assays revealed that the KKVL/A mutations abolished the GPCR-binding ability of Krz. The isolated "finger loop" mutation (Krz-VL/A), while having a negligible effect on GPCR internalization, severely affected Krz function, suggesting that tight receptor interactions are necessary for proper termination of signaling in vivo. Genetic analysis as well as live imaging demonstrated that mutations in Krz led to hyperactivity of the GPCR Mist (also known as Mthl1), which is activated by its ligand Folded gastrulation (Fog) and is responsible for cellular contractility and epithelial morphogenesis. Krz mutations affected two developmental events that are under the control of Fog-Mist signaling: gastrulation and morphogenesis of the wing. Overall, these data reveal the functional importance in vivo of direct beta-arrestin/GPCR binding, which is mediated by the recognition of the phosphorylated receptor tail and receptor core interaction. These Krz-GPCR interactions are critical for setting the correct level of Fog-Mist signaling during epithelial morphogenesis.
Ji, T., Zhang, L., Deng, M., Huang, S., Wang, Y., Pham, T. T., Smith, A. A., Sridhar, V., Cabernard, C., Wang, J. and Yan, Y. (2019). Dynamic MAPK signaling activity underlies a transition from growth arrest to proliferation in Drosophila scribble mutant tumors. Dis Model Mech. PubMed ID: 31371383
Summary:
Human tumors exhibit plasticity and evolving capacity over time. It is difficult to study the mechanisms of how tumors change over time in human patients, in particular during the early stages when a few oncogenic cells are barely detectable. This study used a Drosophila tumor model caused by loss of Scribble (Scrib), a highly conserved apicobasal cell polarity gene, to investigate the spatial-temporal dynamics of early tumorigenesis events. The fly scrib mutant tumors have been successfully used to model many aspects of tumorigenesis processes. However, it is still unknown whether the fly scrib mutant tumors exhibit plasticity and evolvability along the temporal axis. This study found that the scrib mutant tumors display different growth rates and cell cycle profiles over time, indicative of a growth arrest-to-proliferation transition as the scrib mutant tumors progress. Longitudinal bulk and single-cell transcriptomic analysis of the scrib mutant tumors revealed that the MAPK pathway, including the JNK and ERK signaling activities, shows quantitative changes over time. High JNK signaling activity causes G2/M cell cycle arrest in the early scrib mutant tumors. In addition, JNK signaling activity displays a radial polarity with the JNK(high) cells located at the periphery of the scrib mutant tumors, providing an inherent mechanism that leads to an overall JNK signaling activity decrease over time. The ERK signaling activity, in contrast to JNK activity, increases over time and promotes growth in the late-stage scrib mutant tumors. Finally, high JNK signaling activity represses ERK signaling activity in the early scrib mutant tumors. Together, these data demonstrated that dynamic MAPK signaling activity, fueled by intratumor heterogeneity derived from tissue topological differences, drives a growth arrest-to-proliferation transition in the scrib mutant tumors.
Li, P., Ma, Z., Yu, Y., Hu, X., Zhou, Y. and Song, H. (2019). FER promotes cell migration via regulating JNK activity. Cell Prolif: e12656. PubMed ID: 31264309
Summary:
Cell migration has a key role in cancer metastasis, which contributes to drug resistance and tumour recurrence. Better understanding of the mechanisms involved in this process will potentially reveal new drug targets for cancer therapy. Fer is a non-receptor protein tyrosine kinase aberrantly expressed in various human cancers, whereas its role in tumour progression remains elusive. Transgenic flies and epigenetic analysis were employed to investigate the role of Drosophila Fer (FER) in cell migration and underlying mechanisms. Co-immunoprecipitation assay was used to monitor the interaction between FER and Drosophila JNK (Bsk). The conservation of Fer in regulating JNK signalling was explored in mammalian cancer and non-cancer cells. Overexpression of FER triggered cell migration and activated JNK signalling in the Drosophila wing disc. Upregulation and downregulation in the basal activity of Bsk exacerbated and eliminated FER-mediated migration, respectively. In addition, loss of FER blocked signal transduction of the JNK pathway. Specifically, FER interacted with and promoted the activity of Bsk, which required both the kinase domain and the C-terminal of Bsk. Lastly, Fer regulated JNK activities in mammalian cells. This study reveals FER as a positive regulator of JNK-mediated cell migration and suggests its potential role as a therapeutic target for cancer metastasis.
Mu, Y., Tian, Y., Zhang, Z. C. and Han, J. (2019). Metallophosphoesterase regulates light-induced rhodopsin endocytosis by promoting an association between arrestin and the adaptor protein AP2. J Biol Chem. PubMed ID: 31324721
Summary:
Light-induced endocytosis of rhodopsin in the retina is critical for preventing photoreceptor hyperactivity and for the survival of photoreceptor cells. In Drosophila, this process is mediated by arrestin1 (Arr1). Because Arr1 lacks a clathrin-binding domain required for receptor internalization and the C-terminal sequence that interacts with the beta-subunit of the clathrin adaptor protein AP2, but the mechanism of how Arr1 mediates endocytosis of the major rhodopsin Rh1 is unclear. Using several approaches, including Arr-binding and pull-down assays, immunofluorescence techniques, and EM imaging, this study found that Drosophila metallophosphoesterase (dMPPE) is involved in light-induced rhodopsin endocytosis. It was observed that the photoreceptor cells of a dmppe mutant exhibit impaired light-induced rhodopsin endocytosis and that this impairment is independent of dMPPE phosphoesterase activity. Furthermore, dMPPE directly interacted with Arr1 and promoted the association of Arr1 with AP2. Of note, genetic dmppe deletion largely prevented retinal degeneration in norpA (encoding phospholipase C) mutants, which were previously reported to contribute to retinal degeneration, by suppressing Rh1 endocytosis. These findings demonstrate that Arr1 interacts with AP2 and that dMPPE functions as a critical regulator in Rh1 endocytosis and retinal degeneration.
Munkacsy, E., Chocron, E. S., Quintanilla, L., Gendron, C. M., Pletcher, S. D. and Pickering, A. M. (2019). Neuronal-specific proteasome augmentation via Prosbeta5 overexpression extends lifespan and reduces age-related cognitive decline. Aging Cell: e13005. PubMed ID: 31334599
Summary:
Cognitive function declines with age throughout the animal kingdom, and increasing evidence shows that disruption of the proteasome system contributes to this deterioration. The proteasome has important roles in multiple aspects of the nervous system, including synapse function and plasticity, as well as preventing cell death and senescence. Previous studies have shown neuronal proteasome depletion and inhibition can result in neurodegeneration and cognitive deficits, but it is unclear if this pathway is a driver of neurodegeneration and cognitive decline in aging. This study reports that overexpression of the proteasome beta5 subunit enhances proteasome assembly and function. Significantly, it was shown that neuronal-specific proteasome augmentation slows age-related declines in measures of learning, memory, and circadian rhythmicity. Surprisingly, neuronal-specific augmentation of proteasome function also produces a robust increase of lifespan in Drosophila melanogaster. These findings appear specific to the nervous system; ubiquitous proteasome overexpression increases oxidative stress resistance but does not impact lifespan and is detrimental to some healthspan measures. These findings demonstrate a key role of the proteasome system in brain aging.
Bageritz, J., Willnow, P., Valentini, E., Leible, S., Boutros, M. and Teleman, A. A. (2019). Gene expression atlas of a developing tissue by single cell expression correlation analysis. Nat Methods 16(8): 750-756. PubMed ID: 31363221
Summary:
The Drosophila wing disc has been a fundamental model system for the discovery of key signaling pathways and for understanding of developmental processes. However, a complete map of gene expression in this tissue is lacking. To obtain a gene expression atlas in the wing disc, single cell RNA sequencing (scRNA-seq) was used, and a method was developed for analyzing scRNA-seq data based on gene expression correlations rather than cell mapping. This enables computation of expression maps for all detected genes in the wing disc and to discover 824 genes with spatially restricted expression patterns. This approach identifies clusters of genes with similar expression patterns and functional relevance. As proof of concept, the previously unstudied gene CG5151 was characterized and was show to regulate Wnt signaling. This method will enable the leveraging of scRNA-seq data for generating expression atlases of undifferentiated tissues during development.

Wednesday, August 21st - Behavior

Agrawal, S. and Dickinson, M. H. (2019). The effects of target contrast on Drosophila courtship. J Exp Biol. PubMed ID: 31315932
Summary:
Many animals use visual cues like object shape, color, and motion to detect and pursue conspecific mates. Contrast is another possibly informative visual cue, but has not been studied in great detail. This study presents male Drosophila melanogaster with small, fly-sized, moving objects painted either black, white, or grey to test if they use contrast cues to identify mates. Males were found to frequently chase grey objects and rarely chased white or black objects. Although males started chasing black objects as often as grey objects, the resulting chases were much shorter. To test whether the attraction to grey objects was mediated via contrast, black and grey behavioral chambers were fabricated. However, wildtype males almost never chased any objects in these darkly colored chambers. To circumvent this limitation, baseline levels of chasing were increased by thermogenetically activating P1 neurons to promote courtship. Males with thermogenetically activated P1 neurons maintained a similar preference for grey objects despite elevated levels of courtship behavior. When placed in a black chamber, males with activated P1 neurons switched their preference and chased black objects more than grey objects. Whether males use contrast cues to orient to particular parts of the female's body during courtship was also tested. When presented with moving objects painted two colors, males positioned themselves next to the grey half regardless of whether the other half was painted black or white. These results suggest that males can use contrast to recognize potential mates and to position themselves during courtship.
De Araujo, L. I., Karsten, M. and Terblanche, J. S. (2019). Exploring thermal flight responses as predictors of flight ability and geographic range size in Drosophila. Comp Biochem Physiol A Mol Integr Physiol 236: 110532. PubMed ID: 31351148
Summary:
Thermal flight performance curves (TFPCs) may be a useful proxy for determining dispersal on daily timescales in winged insect species. To better understand how flight performance may be correlated with geographic range extent and potential latitudinal climate variability, the thermal performance curves of flight ability was estimated in 11 Drosophilidae species (in 4 degrees C increments across 16-28 degrees C) after standard laboratory rearing for two generations. Whether key morphological, evolutionary or ecological factors (e.g. species identity, sex, body mass, wing loading, geographic range size) predicted traits of TFPCs (including optimum temperature, maximum performance, thermal breadth of performance) or flight ability (success/failure to fly) was tested. Although several parameters of TFPCs varied among species, these were typically not statistically significant probably owing to the relatively small pool of species assessed and the limited trait variation detected. The best explanatory model of these flight responses across species included significant positive effects of test temperature and wing area. However, the rank of geographic distribution breadth and phylogeny failed to explain significant variation in most of the traits, except for thermal performance breadth, of thermal flight performance curves among these 11 species. Future studies that employ a wider range of Drosophilidae species, especially if coupled with fine-scale estimates of species' environmental niches, would be useful.
DeAngelis, B. D., Zavatone-Veth, J. A. and Clark, D. A. (2019). The manifold structure of limb coordination in walking Drosophila. Elife 8. PubMed ID: 31250807
Summary:
Terrestrial locomotion requires animals to coordinate their limb movements to efficiently traverse their environment. While previous studies in hexapods have reported that limb coordination patterns can vary substantially, the structure of this variability is not yet well understood. This study characterized the symmetric and asymmetric components of variation in walking kinematics in the genetic model organism Drosophila. Drosophila were found to use a single continuum of coordination patterns without evidence for preferred configurations. Spontaneous symmetric variability was associated with modulation of a single control parameter-stance duration-while asymmetric variability consisted of small, limb-specific modulations along multiple dimensions of the underlying symmetric pattern. Commands that modulated walking speed, originating from artificial neural activation or from the visual system, evoked modulations consistent with spontaneous behavior. These findings suggest that Drosophila employ a low-dimensional control architecture, which provides a framework for understanding the neural circuits that regulate hexapod legged locomotion.
Gupta, T., Howe, S. E., Zorman, M. L. and Lockwood, B. L. (2019). Aggression and discrimination among closely versus distantly related species of Drosophila. R Soc Open Sci 6(6): 190069. PubMed ID: 31312482
Summary:
Fighting between different species is widespread in the animal kingdom, yet this phenomenon has been relatively understudied in the field of aggression research. Particularly lacking are studies that test the effect of genetic distance, or relatedness, on aggressive behaviour between species. This study characterized male-male aggression within and between species of fruit flies across the Drosophila phylogeny. Male Drosophila are shown to discriminate between conspecifics and heterospecifics and show a bias for the target of aggression that depends on the genetic relatedness of opponent males. Specifically, males of closely related species treated conspecifics and heterospecifics equally, whereas males of distantly related species were overwhelmingly aggressive towards conspecifics. This is the first study to quantify aggression between Drosophila species and to establish a behavioural bias for aggression against conspecifics versus heterospecifics. The results suggest that future study of heterospecific aggression behaviour in Drosophila is warranted to investigate the degree to which these trends in aggression among species extend to broader behavioural, ecological and evolutionary contexts.
Zhou, Y., Cao, L. H., Sui, X. W., Guo, X. Q. and Luo, D. G. (2019). Mechanosensory circuits coordinate two opposing motor actions in Drosophila feeding. Sci Adv 5(5): eaaw5141. PubMed ID: 31131327
Summary:
Mechanoreception detects physical forces in the senses of hearing, touch, and proprioception. This study shows that labellar mechanoreception wires two motor circuits to facilitate and terminate Drosophila feeding. Using patch-clamp recordings, Mechanosensory neurons (MSNs) in taste pegs of the inner labella and taste bristles of the outer labella were identified, both of which rely on the same mechanoreceptor, NOMPC (no mechanoreceptor potential C), to transduce mechanical deflection. Connecting with distinct brain motor circuits, bristle MSNs drive labellar spread to facilitate feeding and peg MSNs elicit proboscis retraction to terminate feeding. Bitter sense modulates these two mechanosensory circuits in opposing manners, preventing labellar spread by bristle MSNs and promoting proboscis retraction by peg MSNs. Together, these labeled-line circuits enable labellar peg and bristle MSNs to use the same mechanoreceptors to direct opposing feeding actions and differentially integrate gustatory information in shaping feeding decisions.
Konig, C., Khalili, A., Niewalda, T., Gao, S. and Gerber, B. (2019). An optogenetic analogue of second-order reinforcement in Drosophila. Biol Lett 15(7): 20190084. PubMed ID: 31266421
Summary:
In insects, odours are coded by the combinatorial activation of ascending pathways, including their third-order representation in mushroom body Kenyon cells. Kenyon cells also receive intersecting input from ascending and mostly dopaminergic reinforcement pathways. Indeed, in Drosophila, presenting an odour together with activation of the dopaminergic mushroom body input neuron PPL1-01 leads to a weakening of the synapse between Kenyon cells and the approach-promoting mushroom body output neuron MBON-11. As a result of such weakened approach tendencies, flies avoid the shock-predicting odour in a subsequent choice test. Thus, increased activity in PPL1-01 stands for punishment, whereas reduced activity in MBON-11 stands for predicted punishment. Given that punishment-predictors can themselves serve as punishments of second order, whether presenting an odour together with the optogenetic silencing of MBON-11 would lead to learned odour avoidance was tested, and this was found to be the case. In turn, the optogenetic activation of MBON-11 together with odour presentation led to learned odour approach. Thus, manipulating activity in MBON-11 can be an analogue of predicted, second-order reinforcement.

Tuesday, August 20th - Adult neural development and function

Bridi, J. C., Ludlow, Z. N. and Hirth, F. (2019). Lineage-specific determination of ring neuron circuitry in the central complex of Drosophila. Biol Open 8(7). PubMed ID: 31285267
Summary:
The ellipsoid body (EB) of the Drosophila central complex mediates sensorimotor integration and action selection for adaptive behaviours. Insights into its physiological function are steadily accumulating, however the developmental origin and genetic specification have remained largely elusive. This study identified two stem cells in the embryonic neuroectoderm as precursor cells of neuronal progeny that establish EB circuits in the adult brain. Genetic tracing of embryonic neuroblasts ppd5 and mosaic analysis with a repressible cell marker identified lineage-related progeny as Pox neuro (Poxn)-expressing EB ring neurons, R1-R4. During embryonic brain development, engrailed function is required for the initial formation of Poxn-expressing ppd5-derived progeny. Postembryonic determination of R1-R4 identity depends on lineage-specific Poxn function that separates neuronal subtypes of ppd5-derived progeny into hemi-lineages with projections either terminating in the EB ring neuropil or the superior protocerebrum (SP). Poxn knockdown in ppd5-derived progeny results in identity transformation of engrailed-expressing hemi-lineages from SP to EB-specific circuits. In contrast, lineage-specific knockdown of engrailed leads to reduced numbers of Poxn-expressing ring neurons. These findings establish neuroblasts ppd5-derived ring neurons as lineage-related sister cells that require engrailed and Poxn function for the proper formation of EB circuitry in the adult central complex of Drosophila.
Kadas, D., Duch, C. and Consoulas, C. (2019). Postnatal increases in axonal conduction velocity of an identified Drosophila interneuron require fast sodium, L-type calcium and Shaker potassium channels. eNeuro. PubMed ID: 31253715
Summary:
During early postnatal life, speed up of signal propagation through many central and peripheral neurons has been associated with an increase in axon diameter or/and myelination. This study shows that axonal action potential conduction velocity in the Drosophila giant fiber interneuron (GF), that is required for fast long distance signal conduction through the escape circuit, is increased by 80% during the first day of adult life. Genetic manipulations indicate that this postnatal increase in action potential conduction velocity in the unmyelinated GF axon is likely owed to adjustments of ion channel expression or properties rather than axon diameter increases. Specifically, targeted RNAi knockdown of either Para fast voltage-gated sodium, Shaker potassium (Kv1 homologue), or surprisingly, L-type like calcium channels counteracts postnatal increases in GF axonal conduction velocity. By contrast, the calcium-dependent potassium channel Slowpoke (BK) is not essential for postnatal speeding, though it also significantly increases conduction velocity. Therefore, this study has identified multiple ion channels that function to support fast axonal action potential conduction velocity, but only a subset of these are regulated during early postnatal life to maximize conduction velocity. Despite its large diameter ( approximately 7microm) and postnatal regulation of multiple ionic conductances, mature GF axonal conduction velocity is still 20-60 times slower than that of vertebrate Abeta sensory axons and alpha motoneurons, thus unraveling the limits of long range information transfer speed through invertebrate circuits.
Couturier, L., Mazouni, K., Corson, F. and Schweisguth, F. (2019). Regulation of Notch output dynamics via specific E(spl)-HLH factors during bristle patterning in Drosophila. Nat Commun 10(1): 3486. PubMed ID: 31375669
Summary:
The stereotyped arrangement of sensory bristles on the adult fly thorax arises from a self-organized process, in which inhibitory Notch signaling both delimits proneural stripes and singles out sensory organ precursor cells (SOPs). A dynamic balance between proneural factors and Enhancer of split-HLH (E(spl)-HLH) Notch targets underlies patterning, but how this is regulated is unclear. This study identified two classes of E(spl)-HLH factors, whose expression both precedes and delimits proneural activity, and is dependent on proneural activity and required for proper SOP spacing within the stripes, respectively. These two classes are partially redundant, since a member of the second class, that is normally cross-repressed by members of the first class, can functionally compensate for their absence. The regulation of specific E(spl)-HLH genes by proneural factors amplifies the response to Notch as SOPs are being selected, contributing to patterning dynamics in the notum, and likely operates in other developmental contexts.
Curt, J. R., Yaghmaeian Salmani, B. and Thor, S. (2019). Anterior CNS expansion driven by brain transcription factors. Elife 8. PubMed ID: 31271353
Summary:
During CNS development, there is prominent expansion of the anterior region, the brain. In Drosophila, anterior CNS expansion emerges from three rostral features: (1) increased progenitor cell generation, (2) extended progenitor cell proliferation, (3) more proliferative daughters. This study finds that tailless (mouse Nr2E1/Tlx), otp/Rx/hbn (Otp/Arx/Rax) and Doc1/2/3 (Tbx2/3/6) are important for brain progenitor generation. These genes, and earmuff (FezF1/2), are also important for subsequent progenitor and/or daughter cell proliferation in the brain. Brain TF co-misexpression can drive brain-profile proliferation in the nerve cord, and can reprogram developing wing discs into brain neural progenitors. Brain TF expression is promoted by the PRC2 complex, acting to keep the brain free of anti-proliferative and repressive action of Hox homeotic genes. Hence, anterior expansion of the Drosophila CNS is mediated by brain TF driven 'super-generation' of progenitors, as well as 'hyper-proliferation' of progenitor and daughter cells, promoted by PRC2-mediated repression of Hox activity.
Green, J., Vijayan, V., Mussells Pires, P., Adachi, A. and Maimon, G. (2019). A neural heading estimate is compared with an internal goal to guide oriented navigation. Nat Neurosci. PubMed ID: 31332373
Summary:
Goal-directed navigation is thought to rely on the activity of head-direction cells, but how this activity guides moment-to-moment actions remains poorly understood. This study characterize how heading neurons in the Drosophila central complex guide moment-to-moment navigational behavior. An innate, heading-neuron-dependent, tethered navigational behavior was established where walking flies maintain a straight trajectory along a specific angular bearing for hundreds of body lengths. While flies perform this task, chemogenetics was used to transiently rotate their neural heading estimate and observe that the flies slow down and turn in a direction that aims to return the heading estimate to the angle it occupied before stimulation. These results support a working model in which the fly brain quantitatively compares an internal estimate of current heading with an internal goal heading and uses the sign and magnitude of the difference to determine which way to turn, how hard to turn and how fast to walk forward.
Khuong, T. M., Wang, Q. P., Manion, J., Oyston, L. J., Lau, M. T., Towler, H., Lin, Y. Q. and Neely, G. G. (2019). Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila. Sci Adv 5(7): eaaw4099. PubMed ID: 31309148
Summary:
Injury can lead to devastating and often untreatable chronic pain. While acute pain perception (nociception) evolved more than 500 million years ago, virtually nothing is known about the molecular origin of chronic pain. This study provides the first evidence that nerve injury leads to chronic neuropathic sensitization in insects. Mechanistically, peripheral nerve injury triggers a loss of central inhibition that drives escape circuit plasticity and neuropathic allodynia. At the molecular level, excitotoxic signaling within GABAergic (gamma-aminobutyric acid) neurons required the acetylcholine receptor nAChRalpha1 and led to caspase-dependent death of GABAergic neurons. Conversely, disruption of GABA signaling was sufficient to trigger allodynia without injury. Last, the conserved transcription factor Twist was identified as a critical downstream regulator driving GABAergic cell death and neuropathic allodynia. Together, this study has defined how injury leads to allodynia in insects, and describe a primordial precursor to neuropathic pain may have been advantageous, protecting animals after serious injury.

Monday, August 19th - RNA and Transposons

Issa, A. R., Picao-Osorio, J., Rito, N., Chiappe, M. E. and Alonso, C. R. (2019). A single microRNA-Hox gene module controls equivalent movements in biomechanically distinct forms of Drosophila. Curr Biol. PubMed ID: 31327720
Summary:
Movement is the main output of the nervous system. It emerges during development to become a highly coordinated physiological process essential to survival and adaptation of the organism to the environment. Similar movements can be observed in morphologically distinct developmental stages of an organism, but it is currently unclear whether or not these movements have a common molecular cellular basis. This study explores this problem in Drosophila, focusing on the roles played by the microRNA (miRNA) locus miR-iab4/8, which has been previously shown to be essential for the normal corrective response displayed by the fruit fly larva when turned upside down (self-righting). This study shows that miR-iab4 is required for normal self-righting across all three Drosophila larval stages. Unexpectedly, it was also discovered that this miRNA is essential for normal self-righting behavior in the adult fly, an organism with different morphology, neural constitution, and biomechanics. Through the combination of gene expression, optical imaging, and quantitative behavioral approaches, evidence is provided that miR-iab4 exerts its effects on adult self-righting behavior in part through repression of the Hox gene Ultrabithorax (Ubx) in a specific set of adult motor neurons, the NB2-3/lin15 neurons. The results show that miRNA controls the function, rather than the morphology, of these neurons and demonstrate that post-developmental changes in Hox gene expression can modulate behavior in the adult. This work reveals that a common miRNA-Hox genetic module can be re-deployed in different neurons to control functionally equivalent movements in biomechanically distinct organisms and describes a novel post-developmental role of the Hox genes in adult neural function.
Guo, Y. E., et al. (2019). Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature. PubMed ID: 31391587
Summary:
The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. This study investigated whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. The hypophosphorylated C-terminal domain of Pol II was found to be incorporated into mediator condensates; phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. It was also found that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference.
Hirakata, S., Ishizu, H., Fujita, A., Tomoe, Y. and Siomi, M. C. (2019). Requirements for multivalent Yb body assembly in transposon silencing in Drosophila. EMBO Rep 20(7): e47708. PubMed ID: 31267711
Summary:
Female sterile (1) Yb (Yb) is a primary component of Yb bodies, perinuclear foci considered to be the site of PIWI-interacting RNA (piRNA) biogenesis in Drosophila ovarian somatic cells (OSCs). Yb consists of three domains: Helicase C-terminal (Hel-C), RNA helicase, and extended Tudor (eTud) domains. Previous work has shown that the RNA helicase domain is necessary for Yb-RNA interaction, Yb body formation, and piRNA biogenesis. This study investigated the functions of Hel-C and eTud and reveald that Hel-C is dedicated to Yb-Yb homotypic interaction, while eTud is necessary for Yb-RNA association, as is the RNA helicase domain. All of these domains are indispensable for Yb body formation and transposon-repressing piRNA production. Strikingly, however, genic piRNAs unrelated to transposon silencing are produced in OSCs where Yb bodies are disassembled. It was also revealed that Yb bodies are liquid-like multivalent condensates whose assembly depends on Yb-Yb homotypic interaction and Yb binding particularly with flamenco RNA transcripts, the source of transposon-repressing piRNAs. New insights into Yb body assembly and biological relevance of Yb bodies in transposon silencing have emerged.
Dennis, C., Brasset, E. and Vaury, C. (2019). flam piRNA precursors channel from the nucleus to the cytoplasm in a temporally regulated manner along Drosophila oogenesis. Mob DNA 10: 28. PubMed ID: 31312260
Summary:
PIWI-interacting RNAs (piRNAs) are the effectors of transposable element silencing in the reproductive apparatus. In Drosophila ovarian somatic cells, piRNAs arise from long RNA precursors presumably processed within cytoplasmic Yb-bodies. This study shows that the nucleo-cytoplasmic traffic of piRNA precursors encoded by the flamenco locus is subjected to a spatio-temporal regulation. Precursor RNAs first gather in a single nuclear focus, Dot COM, close to the nuclear periphery, and transit through the membrane before being delivered to the cytoplasmic Yb-bodies. Early in oogenesis, flamenco transcripts are rapidly transferred to the cytoplasm making their initial nuclear gathering in Dot COM too transient to be visualized. As oogenesis proceeds, the cytoplasmic delivery steadily decreases concomitantly with the decrease in the protein levels of Armi and Yb, two components of the Yb-bodies. Both events lead to a reduction of Yb-body assembly in late stages of oogenesis, which likely results in a drop in piRNA production. These findings show a spatio-temporal regulation of the piRNA biogenesis in the follicle cells of Drosophila ovaries, that involves coordinated control of both piRNA precursors and components of the piRNA processing machinery. This newly unveiled regulation establishes another level of complexity in the production of piRNAs and suggests a stage-dependent involvement of the piRNA biogenesis in the mechanism of transposable elements silencing along oogenesis.
Gerlach, S. U., Sander, M., Song, S. and Herranz, H. (2019). The miRNA bantam regulates growth and tumorigenesis by repressing the cell cycle regulator tribbles. Life Sci Alliance 2(4). PubMed ID: 31331981
Summary:
One of the fundamental issues in biology is understanding how organ size is controlled. Tissue growth has to be carefully regulated to generate well-functioning organs, and defects in growth control can result in tumor formation. The Hippo signaling pathway is a universal growth regulator and has been implicated in cancer. In Drosophila, the Hippo pathway acts through the miRNA bantam to regulate cell proliferation and apoptosis. Even though the bantam targets regulating apoptosis have been determined, the target genes controlling proliferation have not been identified thus far. This study identifies the gene tribbles as a direct bantam target gene. Tribbles limits cell proliferation by suppressing G2/M transition. This study shows that tribbles regulation by bantam is central in controlling tissue growth and tumorigenesis. This study was expanded to other cell cycle regulators and shows that deregulated G2/M transition can collaborate with oncogene activation driving tumor formation.
Cassidy, J. J., Bernasek, S. M., Bakker, R., Giri, R., Pelaez, N., Eder, B., Bobrowska, A., Bagheri, N., Nunes Amaral, L. A. and Carthew, R. W. (2019). Repressive gene regulation synchronizes development with cellular metabolism. Cell. PubMed ID: 31353220
Summary:
Metabolic conditions affect the developmental tempo of animals. Developmental gene regulatory networks (GRNs) must therefore synchronize their dynamics with a variable timescale. This study found that layered repression of genes couples GRN output with variable metabolism. When repressors of transcription or mRNA and protein stability are lost, fewer errors in Drosophila development occur when metabolism is lowered. This study demonstrates the universality of this phenomenon by eliminating the entire microRNA family of repressors. Development to maturity can be largely rescued when metabolism is reduced. Using a mathematical model that replicates GRN dynamics, lowering metabolism was found to suppress the emergence of developmental errors by curtailing the influence of auxiliary repressors on GRN output. This study experimentally shows that gene expression dynamics are less affected by loss of repressors when metabolism is reduced. Thus, layered repression provides robustness through error suppression and may provide an evolutionary route to a shorter reproductive cycle.

Friday, August 15th - Evolution

Loehlin, D. W., Ames, J. R., Vaccaro, K. and Carroll, S. B. (2019). A major role for noncoding regulatory mutations in the evolution of enzyme activity. Proc Natl Acad Sci U S A 116(25): 12383-12389. PubMed ID: 31152141
Summary:
The quantitative evolution of protein activity is a common phenomenon, yet little is known about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. To determine the types of genetic changes underlying the quantitative evolution of protein activity, this study dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, this study found that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5' and 3' untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, both regulatory and coding substitutions were found to contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.
Sarikaya, D. P., Church, S. H., Lagomarsino, L. P., Magnacca, K. N., Montgomery, S. L., Price, D. K., Kaneshiro, K. Y. and Extavour, C. G. (2019). Reproductive capacity evolves in response to ecology through common changes in cell number in Hawaiian Drosophila. Curr Biol 29(11): 1877-1884. PubMed ID: 31130459
Summary:
Lifetime reproductive capacity is a critical fitness component. In insects, female reproductive capacity is largely determined by the number of ovarioles, the egg-producing subunits of the ovary. Recent work has provided insights into ovariole number regulation in Drosophila melanogaster. However, whether mechanisms discovered under laboratory conditions explain evolutionary variation in natural populations is an outstanding question. This study investigated potential effects of ecology on the developmental processes underlying ovariole number evolution among Hawaiian Drosophila, a large adaptive radiation wherein the highest and lowest ovariole numbers of the family have evolved within 25 million years. Previous studies proposed that ovariole number correlated with oviposition substrate but sampled largely one clade of these flies and were limited by a provisional phylogeny and the available comparative methods. This hypothesis was tested by applying phylogenetic modeling to an expanded sampling of ovariole numbers and substrate types and shows support for these predictions across all major groups of Hawaiian Drosophila, wherein ovariole number variation is best explained by adaptation to specific substrates. Furthermore, oviposition substrate evolution is linked to changes in the allometric relationship between body size and ovariole number. Finally, evidence is provided that the major changes in ovarian cell number that regulate D. melanogaster ovariole number also regulate ovariole number in Hawaiian drosophilids. Thus, evidence is provided that this remarkable adaptive radiation is linked to evolutionary changes in a key reproductive trait regulated at least partly by variation in the same developmental parameters that operate in the model species D. melanogaster.
Brand, C. L., Wright, L. and Presgraves, D. C. (2019). Positive selection and functional divergence at meiosis genes that mediate crossing over across the Drosophila phylogeny. G3 (Bethesda). PubMed ID: 31362974
Summary:
Meiotic crossing over ensures proper segregation of homologous chromosomes and generates genotypic diversity. Despite these functions, little is known about the genetic factors and population genetic forces involved in the evolution of recombination rate differences among species. The dicistronic meiosis gene, mei-217/mei-218, mediates most of the species differences in crossover rate and patterning during female meiosis between the closely related fruitfly species, Drosophila melanogaster and D. mauritiana The MEI-218 protein is one of several meiosis-specific mini-chromosome maintenance (mei-MCM) proteins that form a multi-protein complex essential to crossover formation, whereas the BLM helicase acts as an anti-crossover protein. This study examined the molecular evolution of five genes - mei218, the other three known members of the mei-MCM complex, and Blm-over the phylogenies of three Drosophila species groups-melanogaster, obscura, and virilis. Transgenic assays in D. melanogaster were used to test if molecular evolution at mei218 has functional consequences for crossing over using alleles from the distantly related species D. pseudoobscura and D. virilis. This molecular evolutionary analyses reveal recurrent positive selection at two mei-MCM genes. Transgenic assays show that sequence divergence among mei-218 alleles from D. melanogaster, D. pseudoobscura, and D. virilis has functional consequences for crossing over. In a D. melanogaster genetic background, the D. pseudoobscura mei218 allele nearly rescues wildtype crossover rates but alters crossover patterning, whereas the D. virilis mei218 allele conversely rescues wildtype crossover patterning but not crossover rates. These experiments demonstrate functional divergence at mei218 and suggest that crossover rate and patterning are separable functions.
Ellison, C. and Bachtrog, D. (2019). Recurrent gene co-amplification on Drosophila X and Y chromosomes. PLoS Genet 15(7): e1008251. PubMed ID: 31329593
Summary:
Y chromosomes often contain amplified genes which can increase dosage of male fertility genes and counteract degeneration via gene conversion. This study identified genes with increased copy number on both X and Y chromosomes in various species of Drosophila, a pattern that has previously been associated with sex chromosome drive involving the Slx and Sly gene families in mice. Recurrent X/Y co-amplification appears to be an important evolutionary force that has shaped gene content evolution of sex chromosomes in Drosophila. This study also demonstrates that convergent acquisition and amplification of testis expressed gene families are common on Drosophila sex chromosomes, and especially on recently formed ones, and one putative novel X/Y co-amplification system was carefully characterized. Co-amplification of the S-Lap1/GAPsec gene pair on both the X and the Y chromosome occurred independently several times in members of the D. obscura group, where this normally autosomal gene pair is sex-linked due to a sex chromosome-autosome fusion. Several evolutionary scenarios were explored that would explain this pattern of co-amplification. Investigation of gene expression and short RNA profiles at the S-Lap1/GAPsec system suggest that, like Slx/Sly in mice, these genes may be remnants of a cryptic sex chromosome drive system, however additional transgenic experiments will be necessary to validate this model. Regardless of whether sex chromosome drive is responsible for this co-amplification, the findings suggest that recurrent gene duplications between X and Y sex chromosomes could have a widespread effect on genomic and evolutionary patterns, including the epigenetic regulation of sex chromosomes, the distribution of sex-biased genes, and the evolution of hybrid sterility.
Helleu, Q., Courret, C., Ogereau, D., Burnham, K. L., Chaminade, N., Chakir, M., Aulard, S. and Montchamp-Moreau, C. (2019). Sex-Ratio meiotic drive shapes the evolution of the Y chromosome in Drosophila simulans. Mol Biol Evol. PubMed ID: 31290972
Summary:
The recent emergence and spread of X-linked segregation distorters - called "Paris" system - in the worldwide species Drosophila simulans has elicited the selection of drive-resistant Y chromosomes. This study investigated the evolutionary history of 386 Y chromosomes originating from 29 population samples collected over a period of twenty years, showing a wide continuum of phenotypes when tested against the Paris distorters, from high sensitivity to complete resistance (males sire approximately 95% to approximately 40% female progeny). Analyzing around 13 kb of Y-linked gene sequences in a representative subset of nine Y chromosomes, only three polymorphic sites resulting in three haplotypes were found. Remarkably, one of the haplotypes is associated with resistance. This haplotype is fixed in all samples from Sub-Saharan Africa, the region of origin of the drivers. Exceptionally, with the spread of the drivers in Egypt and Morocco, it was possible to record the replacement of the sensitive lineage by the resistant haplotype in real time, within only a few years. In addition, in situ hybridization, using satellite DNA probes, was performed on a subset of 21 Y chromosomes from six locations. In contrast to the low molecular polymorphism, this revealed extensive structural variation suggestive of rapid evolution, either neutral or adaptive. Moreover, the results show that intragenomic conflicts can drive astonishingly rapid replacement of Y chromosomes and suggest that the emergence of Paris segregation distorters in East Africa occurred less than half a century ago.
Hunt, L. C., Jiao, J., Wang, Y. D., Finkelstein, D., Rao, D., Curley, M., Robles-Murguia, M., Shirinifard, A., Pagala, V. R., Peng, J., Fan, Y. and Demontis, F. (2019). Circadian gene variants and the skeletal muscle circadian clock contribute to the evolutionary divergence in longevity across Drosophila populations. Genome Res 29(8): 1262-1276. PubMed ID: 31249065
Summary:
Organisms use endogenous clocks to adapt to the rhythmicity of the environment and to synchronize social activities. Although the circadian cycle is implicated in aging, it is unknown whether natural variation in its function contributes to differences in lifespan between populations and whether the circadian clock of specific tissues is key for longevity. This study sequenced the genomes of Drosophila melanogaster strains with exceptional longevity that were obtained via multiple rounds of selection from a parental strain. Comparison of genomic, transcriptomic, and proteomic data revealed that changes in gene expression due to intergenic polymorphisms are associated with longevity and preservation of skeletal muscle function with aging in these strains. Analysis of transcription factors differentially modulated in long-lived versus parental strains indicates a possible role of circadian clock core components. Specifically, there is higher period and timeless and lower cycle expression in the muscle of strains with delayed aging compared to the parental strain. These changes in the levels of circadian clock transcription factors lead to changes in the muscle circadian transcriptome, which includes genes involved in metabolism, proteolysis, and xenobiotic detoxification. Moreover, a skeletal muscle-specific increase in timeless expression extends lifespan and recapitulates some of the transcriptional and circadian changes that differentiate the long-lived from the parental strains. Altogether, these findings indicate that the muscle circadian clock is important for longevity and that circadian gene variants contribute to the evolutionary divergence in longevity across populations.

Thursday, August 15th - Chromatin

Hartmann, M., Umbanhowar, J. and Sekelsky, J. (2019). Centromere-proximal meiotic crossovers in Drosophila melanogaster are suppressed by both highly-repetitive heterochromatin and proximity to the centromere. Genetics. PubMed ID: 31345993
Summary:
Crossovers are essential in meiosis of most organisms to ensure the proper segregation of chromosomes, but improper placement of crossovers can result in nondisjunction and aneuploidy in progeny. In particular, crossovers near the centromere can cause nondisjunction. Centromere-proximal crossovers are suppressed by what is termed the centromere effect, but the mechanism is unknown. This study investigated contributions to centromere-proximal crossover suppression in Drosophila melanogaster. A large number of centromere-proximal crossovers were mapped; crossovers were found to be essentially absent from the highly repetitive (HR)heterochromatin surrounding the centromere but occur at a low frequency within the less-repetitive (LR)heterochromatic region and adjacent euchromatin. Previous research suggested that flies that lack the Bloom syndrome helicase (Blm) lose meiotic crossover patterning, including the centromere effect. Mapping of centromere-proximal crossovers in Blm mutants reveals that the suppression within the HRheterochromatin is intact, but the distance-dependent centromere effect is lost. It is concluded that centromere-proximal crossovers are suppressed by two separable mechanisms: an HR-heterochromatin effect that completely suppresses crossovers in the HRheterochromatin, and the centromere effect, which suppresses crossovers with a dissipating effect with distance from the centromere.
Porcelli, D., Fischer, B., Russell, S. and White, R. (2019). Chromatin accessibility plays a key role in selective targeting of Hox proteins. Genome Biol 20(1): 115. PubMed ID: 31159833
Summary:
Hox transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. While the different Hox family members show clear functional specificity in vivo, they all show similar binding specificity in vitro and a satisfactory understanding of in vivo Hox target selectivity is still lacking. Using transient transfection in Kc167 cells, this study systematically analyze the binding of all eight Drosophila Hox proteins. Hox proteins were found to show considerable binding selectivity in vivo even in the absence of canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility, being highest in less accessible chromatin. Individual Hox proteins exhibit different propensities to bind less accessible chromatin, and high binding selectivity is associated with high-affinity binding regions, leading to a model where Hox proteins derive binding selectivity through affinity-based competition with nucleosomes. Extradenticle/Homothorax cofactors generally facilitate Hox binding, promoting binding to regions in less accessible chromatin but with little effect on the overall selectivity of Hox targeting. These cofactors collaborate with Hox proteins in opening chromatin, in contrast to the pioneer factor, Glial cells missing, which facilitates Hox binding by independently generating accessible chromatin regions. These studies indicate that chromatin accessibility plays a key role in Hox selectivity. It is proposed that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of in vivo genomic targets for different Hox proteins.
Singh, A., Paul, M. S., Dutta, D., Mutsuddi, M. and Mukherjee, A. (2019). Regulation of Notch signaling by a chromatin modeling protein Hat-trick. Development. PubMed ID: 31142544
Summary:
Notch signaling plays pleiotropic role in astounding variety of cellular processes including cell fate determination, differentiation, proliferation and apoptosis. The increasingly complex regulatory mechanisms of Notch signaling account for the multitude of functions exhibited by Notch during development. This study identified Hat-trick (Htk), a DNA binding protein, as an interacting partner of Notch-ICD in a yeast two-hybrid screen and their physical interaction was further validated by co-immunoprecipitation experiments. htk genetically interacts with Notch pathway components in trans-heterozygous combinations. Loss of htk function in htk mutant somatic clones showed down-regulation of Notch targets, whereas over-expression of htk caused ectopic expression of Notch target, without affecting the level of Notch protein. Immunocytochemical analysis has demonstrated that Htk co-localizes with over-expressed Notch-ICD in the same nuclear compartment. This study has shown that Htk cooperates with Notch-ICD and Suppressor of Hairless to form activation complex and binds to the regulatory sequences of Notch downstream targets, Enhancer of Split complex genes to direct their expression. Taken together, these results suggest a novel mode of regulation of Notch signaling by a chromatin modeling protein Htk.
Ghavi-Helm, Y., Jankowski, A., Meiers, S., Viales, R. R., Korbel, J. O. and Furlong, E. E. M. (2019). Highly rearranged chromosomes reveal uncoupling between genome topology and gene expression. Nat Genet 51(8): 1272-1282. PubMed ID: 31308546
Summary:
Chromatin topology is intricately linked to gene expression, yet its functional requirement remains unclear. This study comprehensively assessed the interplay between genome topology and gene expression using highly rearranged chromosomes (balancers) spanning ~75% of the Drosophila genome. Using transheterozyte (balancer/wild-type) embryos, allele-specific changes were measured in topology and gene expression in cis, while minimizing trans effects. Through genome sequencing, eight large nested inversions, smaller inversions, duplications and thousands of deletions were identified. These extensive rearrangements caused many changes to chromatin topology, disrupting long-range loops, topologically associating domains (TADs) and promoter interactions, yet these are not predictive of changes in expression. Gene expression is generally not altered around inversion breakpoints, indicating that mis-appropriate enhancer-promoter activation is a rare event. Similarly, shuffling or fusing TADs, changing intra-TAD connections and disrupting long-range inter-TAD loops does not alter expression for the majority of genes. These results suggest that properties other than chromatin topology ensure productive enhancer-promoter interactions.
Bozler, J., Kacsoh, B. Z. and Bosco, G. (2019). Transgeneratonal inheritance of ethanol preference is caused by maternal NPF repression. Elife 8. PubMed ID: 31287057
Summary:
Rapid or even anticipatory adaptation to environmental conditions can provide a decisive fitness advantage to an organism. The memory of recurring conditions could also benefit future generations; however, neuronally-encoded behavior isn't thought to be inherited across generations. This study tested the possibility that environmentally triggered modifications could allow 'memory' of parental experiences to be inherited. In Drosophila melanogaster, exposure to predatory wasps leads to inheritance of a predisposition for ethanol-rich food for five generations. Inhibition of Neuropeptide-F (NPF) activates germline caspases required for transgenerational ethanol preference. Further, inheritance of low NPF expression in specific regions of F1 brains is required for the transmission of this food preference: a maternally derived NPF locus is necessary for this phenomenon, implicating a maternal epigenetic mechanism of NPF-repression. Given the conserved signaling functions of NPF and its mammalian NPY homolog in drug and alcohol disorders, these observations raise the intriguing possibility of NPY-related transgenerational effects in humans.
Kuhn, T. M., Pascual-Garcia, P., Gozalo, A., Little, S. C. and Capelson, M. (2019). Chromatin targeting of nuclear pore proteins induces chromatin decondensation. J Cell Biol. PubMed ID: 31366666
Summary:
Nuclear pore complexes have emerged in recent years as chromatin-binding nuclear scaffolds, able to influence target gene expression. However, how nucleoporins (Nups) exert this control remains poorly understood. This study shows that ectopically tethering Drosophila Nups, especially Sec13, to chromatin is sufficient to induce chromatin decondensation. This decondensation is mediated through chromatin-remodeling complex PBAP, as PBAP is both robustly recruited by Sec13 and required for Sec13-induced decondensation. This phenomenon is not correlated with localization of the target locus to the nuclear periphery, but is correlated with robust recruitment of Nup Elys. Furthermore, this study identified a biochemical interaction between endogenous Sec13 and Elys with PBAP, and a role for endogenous Elys in global as well as gene-specific chromatin decompaction. Together, these findings reveal a functional role and mechanism for specific nuclear pore components in promoting an open chromatin state.

Wednesday, August 14th

Duxbury, E. M. L. and Chapman, T. (2019). Sex-specific responses of lifespan and fitness to variation in developmental versus adult diets in D. melanogaster. J Gerontol A Biol Sci Med Sci. PubMed ID: 31362304
Summary:
Nutritional variation across the lifetime can have significant and sex-specific impacts upon fitness. Using Drosophila melanogaster, these impacts were measured by testing the effects on lifespan and reproductive success of high or low yeast content in developmental versus adult diets, separately for each sex. Two hypotheses were tested: that dietary mismatches between development and adulthood are costly and that any such costs are sex-specific. Overall, the results revealed the rich and complex responses of each sex to dietary variation across the lifetime. Contrary to the first hypothesis, dietary mismatches between developmental and adult life stages were not universally costly. Where costs of nutritional variation across the life course did occur, they were sex-, context- and trait-specific, consistent with hypothesis 2. Effects of mismatches between developmental and adult diets on reproductive success were found in females but not males. Adult diet was the main determinant of survival, and lifespan was significantly longer on high yeast adult food, in comparison to low, in both sexes. Developing on a high yeast diet also benefited adult female lifespan and reproductive success, regardless of adult diet. In contrast, a high yeast developmental diet was only beneficial for male lifespan when it was followed by low yeast adult food. Adult diet affected mating frequency in opposing directions, with males having higher mating frequency on high and females on low, with no interaction with developmental diet for either sex. The results emphasize the importance of sex differences and of the directionality of dietary mismatches in the responses to nutritional variation.
Fiocca, K., Barrett, M., Waddell, E. A., Viveiros, J., McNair, C., O'Donnell, S. and Marenda, D. R. (2019). Mannitol ingestion causes concentration-dependent, sex-biased mortality in adults of the fruit fly (Drosophila melanogaster). PLoS One 14(5): e0213760. PubMed ID: 31150400
Summary:
Mannitol, a sugar alcohol used in commercial food products, has been previously shown to induce sex-biased mortality in female Drosophila melanogaster when ingested at a single concentration (1 M). It is hypothesized that sex differences in energy needs, related to reproductive costs, contributed to the increased mortality observed in females compared to males. To test this, the longevity was compared of actively mating and non-mating flies fed increasing concentrations of mannitol. It was also asked whether mannitol-induced mortality was concentration-dependent for both males and females, and if mannitol's sex-biased effects were consistent across concentrations. Females and males both showed concentration-dependent increases in mortality, but female mortality was consistently higher at concentrations of 0.75 M and above. Additionally, fly longevity decreased further for both sexes when housed in mixed sex vials as compared to single sex vials. This suggests that the increased energetic demands of mating and reproduction for both sexes increased the ingestion of mannitol. Finally, larvae raised on mannitol produced expected adult sex ratios, suggesting that sex-biased mortality due to the ingestion of mannitol occurs only in adults. It is concluded that sex and reproductive status differences in mannitol ingestion drive sex-biased differences in adult fly mortality.
Gerofotis, C. D., Kouloussis, N. A., Koukougiannidou, C., Papadopoulos, N. T., Damos, P., Koveos, D. S. and Carey, J. R. (2019). Age, sex, adult and larval diet shape starvation resistance in the Mediterranean fruit fly: an ecological and gerontological perspective. Sci Rep 9(1): 10704. PubMed ID: 31341198
Summary:
The ability of an animal to withstand periods of food deprivation is a key driver of invasion success (biodiversity), adaptation to new conditions, and a crucial determinant of senescence in populations. Starvation resistance (SR) is a highly plastic trait and varies in relation to environmental and genetic variables. However, beyond Drosophila, SR has been studied poorly. Exploiting an interesting model species in invasion and ageing studies-the Mediterranean fruit fly (Ceratitis capitata)- this study investigated how age, food and gender, shape SR in this species. SR was measured in adults feeding in rich and poor dietary conditions that had been reared either on natural hosts or artificial larval diet, for every single day across their lifespan. This study defined which factor is the most significant determinant of SR, and potential links between SR and ageing were explored. SR was found to declines with age, and age-specific patterns are shaped in relation to adult and larval diet. Females exhibited higher SR than males. Age and adult diet were the most significant determinants of SR, followed by gender and the larval diet. Starvation resistance proved to be a weak predictor of functional ageing. Possible underlying mechanisms, ecological and gerontological significance and potential applied benefits are discussed.
Chandel, I., Baker, R., Nakamura, N. and Panin, V. (2019). Live imaging and analysis of muscle contractions in Drosophila embryo. J Vis Exp(149). PubMed ID: 31355800
Summary:
Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. Previously work has shown that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. This study presents a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching; this method was used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. Body posture was also examined and advantage was taken of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.
Estrella, M. A., Du, J., Chen, L., Rath, S., Prangley, E., Chitrakar, A., Aoki, T., Schedl, P., Rabinowitz, J. and Korennykh, A. (2019). The metabolites NADP(+) and NADPH are the targets of the circadian protein Nocturnin (Curled). Nat Commun 10(1): 2367. PubMed ID: 31147539
Summary:
Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, a mass spectrometry screen was conducted, and NOCT was found to specifically and directly convert the dinucleotide NADP(+) into NAD(+) and NADPH into NADH. Further, it was demonstrated that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. The 2.7 A crystal structure of the human NOCT*NADPH complex, revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2'-terminal phosphate productively for removal. Evidence is provided for NOCT targeting to mitochondria and it is proposed that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism.
Duy Binh, T., T, L. A. P., Nishihara, T., Thanh Men, T. and Kamei, K. (2019). The function of Lipin in the wing development of Drosophila melanogaster. Int J Mol Sci 20(13). PubMed ID: 31277421
Summary:
Lipin is evolutionarily conserved from yeast to mammals. Although its roles in lipid metabolism in adipocyte tissue, skeletal muscle, and the liver, and as a transcriptional co-activator are known, its functions during development are still under investigation. This study analyzed the role of Drosophila lipin (dLipin) in development. Specifically, the tissue-selective knockdown of dLipin in the wing pouch led to an atrophied wing. Elevated DNA damage was observed in the wing imaginal disc of dLipin-knockdown flies. dLipin dysfunction induced accumulation of cells in S phase and significantly reduced the number of mitotic cells, indicating DNA damage-induced activation of the G2/M checkpoint. Reduced expression of cyclin B, which is critical for the G2 to M transition, was observed in the margin of the wing imaginal disc of dLipin-knockdown flies. The knockdown of dLipin led to increased apoptotic cell death in the wing imaginal disc. Thus, these results suggest that dLipin is involved in DNA replication during normal cell cycle progression in wing development of Drosophila melanogaster.

Tuesday, August 13th - Disease Models

Dobson, A. J., Boulton-McDonald, R., Houchou, L., Svermova, T., Ren, Z., Subrini, J., Vazquez-Prada, M., Hoti, M., Rodriguez-Lopez, M., Ibrahim, R., Gregoriou, A., Gkantiragas, A., Bahler, J., Ezcurra, M. and Alic, N. (2019). Longevity is determined by ETS transcription factors in multiple tissues and diverse species. PLoS Genet 15(7): e1008212. PubMed ID: 31356597
Summary:
Ageing populations pose one of the main public health crises of our time. Reprogramming gene expression by altering the activities of sequence-specific transcription factors (TFs) can ameliorate deleterious effects of age. This explore how a circuit of TFs coordinates pro-longevity transcriptional outcomes, which reveals a multi-tissue and multi-species role for an entire protein family: the E-twenty-six (ETS) TFs. In Drosophila, reduced insulin/IGF signalling (IIS) extends lifespan by coordinating activation of Aop, an ETS transcriptional repressor, and Foxo, a Forkhead transcriptional activator. Aop and Foxo bind the same genomic loci, and this study shows that, individually, they effect similar transcriptional programmes in vivo. In combination, Aop can both moderate or synergise with Foxo, dependent on promoter context. Moreover, Foxo and Aop oppose the gene-regulatory activity of Pnt, an ETS transcriptional activator. Directly knocking down Pnt recapitulates aspects of the Aop/Foxo transcriptional programme and is sufficient to extend lifespan. The lifespan-limiting role of Pnt appears to be balanced by a requirement for metabolic regulation in young flies, in which the Aop-Pnt-Foxo circuit determines expression of metabolic genes, and Pnt regulates lipolysis and responses to nutrient stress. Molecular functions are often conserved amongst ETS TFs, prompting examination of whether other Drosophila ETS-coding genes may also affect ageing. This study shows that five out of eight Drosophila ETS TFs play a role in fly ageing, acting from a range of organs and cells including the intestine, adipose and neurons. This study expands the repertoire of lifespan-limiting ETS TFs in C. elegans, confirming their conserved function in ageing and revealing that the roles of ETS TFs in physiology and lifespan are conserved throughout the family, both within and between species.
Fenckova, M., Blok, L. E. R., Asztalos, L., Goodman, D. P., Cizek, P., Singgih, E. L., Glennon, J. C., IntHout, J., Zweier, C., Eichler, E. E., von Reyn, C. R., Bernier, R. A., Asztalos, Z. and Schenck, A. (2019). Habituation learning is a widely affected mechanism in Drosophila models of intellectual disability and autism spectrum disorders. Biol Psychiatry 86(4): 294-305. PubMed ID: 31272685
Summary:
Although habituation is one of the most ancient and fundamental forms of learning, its regulators and its relevance for human disease are poorly understood. This study manipulated the orthologs of 286 genes implicated in intellectual disability (ID) with or without comorbid autism spectrum disorder (ASD) specifically in Drosophila neurons, and these models were tested in light-off jump habituation. Neuronal substrates underlying the identified habituation deficits were dissected and genotype-phenotype annotations, gene ontologies, and interaction networks were integrated to determine the clinical features and molecular processes that are associated with habituation deficits. >100 genes required for habituation learning were identified. For 93 of these genes, a role in habituation learning was previously unknown. These genes characterize ID disorders with macrocephaly and/or overgrowth and comorbid ASD. Moreover, individuals with ASD from the Simons Simplex Collection carrying damaging de novo mutations in these genes exhibit increased aberrant behaviors associated with inappropriate, stereotypic speech. At the molecular level, ID genes required for normal habituation are enriched in synaptic function and converge on Ras/mitogen-activated protein kinase (Ras/MAPK) signaling. Both increased Ras/MAPK signaling in gamma-aminobutyric acidergic (GABAergic) neurons and decreased Ras/MAPK signaling in cholinergic neurons specifically inhibit the adaptive habituation response. This work supports the relevance of habituation learning to ASD, identifies an unprecedented number of novel habituation players, supports an emerging role for inhibitory neurons in habituation, and reveals an opposing, circuit-level-based mechanism for Ras/MAPK signaling. These findings establish habituation as a possible, widely applicable functional readout and target for pharmacologic intervention in ID/ASD.
Donde, A., Sun, M., Ling, J. P., Braunstein, K. E., Pang, B., Wen, X., Cheng, X., Chen, L. and Wong, P. C. (2019). Splicing repression is a major function of TDP-43 in motor neurons. Acta Neuropathol. PubMed ID: 31332509
Summary:
Nuclear depletion of TDP-43, an essential RNA binding protein, may underlie neurodegeneration in amyotrophic lateral sclerosis (ALS). As several functions have been ascribed to this protein, the critical role(s) of TDP-43 in motor neurons that may be compromised in ALS remains unknown. This study shows that TDP-43 mediated splicing repression, which serves to protect the transcriptome by preventing aberrant splicing, is central to the physiology of motor neurons. Expression in Drosophila TDP-43 knockout models of a chimeric repressor, comprised of the RNA recognition domain of TDP-43 fused to an unrelated splicing repressor, RAVER1, attenuated motor deficits and extended lifespan. Likewise, AAV9-mediated delivery of this chimeric rescue repressor to mice lacking TDP-43 in motor neurons delayed the onset, slowed the progression of motor symptoms, and markedly extended their lifespan. In treated mice lacking TDP-43 in motor neurons, aberrant splicing was significantly decreased and accompanied by amelioration of axon degeneration and motor neuron loss. This AAV9 strategy allowed long-term expression of the chimeric repressor without any adverse effects. These findings establish that splicing repression is a major function of TDP-43 in motor neurons and strongly support the idea that loss of TDP-43-mediated splicing fidelity represents a key pathogenic mechanism underlying motor neuron loss in ALS.
Ghimirie, S., Terhzaz, S., Cabrero, P., Romero, M. F., Davies, S. and Dow, J. A. T. (2019). Targeted renal knockdown of Na(+)/H(+) exchanger regulatory factor Sip1 produces uric acid nephrolithiasis in Drosophila. Am J Physiol Renal Physiol. PubMed ID: 31364377
Summary:
Nephrolithiasis is one of the most common kidney diseases with poorly understood pathophysiology, but experimental study has been hindered by lack of experimentally tractable models. Drosophila melanogaster is a useful model organism for renal diseases because of genetic and functional similarities of Malpighian (renal) tubules with the human kidney. This study demonstrates the function of Sip1 (SRY-interacting protein 1) gene, an orthologue of human NHERF1 in Drosophila MTs, and its impact on nephrolithiasis. Abundant birefringent calculi were observed in Sip1 mutant flies, and the phenotype was also observed in renal stellate cell-specific RNAi Sip1 knockdowns in otherwise normal flies, confirming a renal aetiology. This phenotype was abolished in rosy flies (which model human xanthinuria) and by the xanthine oxidase inhibitor allopurinol, suggesting that the calculi were of uric acid. This was confirmed by direct assay for urate. Stones rapidly dissolved when the tubule was bathed in alkaline media, suggesting that Sip1 knockdown was acidifying the tubule. SIP1 was shown to co-locate with Na(+)/H(+) exchanger NHE2, and with moesin, in stellate cells; and so a model was developed in which Sip1 normally regulates NHE2 activity and thus luminal pH. Drosophila renal tubule thus offers a useful model for urate nephrolithiasis.
Cha, S. J., Choi, H. J., Kim, H. J., Choi, E. J., Song, K. H., Im, D. S. and Kim, K. (2019). Parkin expression reverses mitochondrial dysfunction in fused in sarcoma-induced amyotrophic lateral sclerosis. Insect Mol Biol. PubMed ID: 31290213
Summary:
Fused in sarcoma (FUS) is a DNA/RNA-binding protein associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. The exact molecular mechanisms by which FUS results in neurotoxicity have not yet been fully elucidated. This study found that parkin is a genetic suppressor of defective phenotypes induced by exogenous human wild type FUS in Drosophila. Although parkin overexpression did not modulate the FUS protein expression level, the locomotive defects in FUS-expressing larvae and adult flies were rescued by parkin expression. FUS expression in muscle tissues resulted in a reduction of the levels and assembly of mitochondrial complex I and III subunits, as well as decreased ATP. Remarkably, expression of parkin suppressed these mitochondrial dysfunctions. These results indicate parkin as a neuroprotective regulator of FUS-induced proteinopathy by recovering the protein levels of mitochondrial complexes I and III. These findings on Parkin-mediated neuroprotection may expand understanding of FUS-induced ALS pathogenesis.
George, J. and Jacobs, H. T. (2019). Minimal effects of spargel (PGC-1) overexpression in a Drosophila mitochondrial disease model. Biol Open 8(7). PubMed ID: 31292108
Summary:
PGC-1alpha and its homologues have been proposed to act as master regulators of mitochondrial biogenesis in animals. Most relevant studies have been conducted in mammals, where interpretation is complicated by the fact that there are three partially redundant members of the gene family. In Drosophila, only a single PGC-1 homologue, spargel (srl), is present in the genome. This study analyzed the effects of srl overexpression on phenotype and on gene expression in tko25t, a recessive bang-sensitive mutant with a global defect in oxidative phosphorylation, resulting from a deficiency of mitochondrial protein synthesis. In contrast to previous reports, this study found that substantial overexpression of srl throughout development had only minimal effects on the tko25t mutant phenotype. Copy number of mtDNA was unaltered and srl overexpression produced no systematic effects on a representative set of transcripts related to mitochondrial OXPHOS and other metabolic enzymes, although these were influenced by sex and genetic background. This study provides no support to the concept of Spargel as a global regulator of mitochondrial biogenesis, at least in the context of the tko25t model.

Monday, August 12th - Cell Cycle

Wang, L. I., Das, A. and McKim, K. S. (2019). Sister centromere fusion during meiosis I depends on maintaining cohesins and destabilizing microtubule attachments. PLoS Genet 15(5): e1008072. PubMed ID: 31150390
Summary:
Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. This study found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. This study has demonstrated that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, this study proposes that two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments.
Sun, M. S., Weber, J., Blattner, A. C., Chaurasia, S. and Lehner, C. F. (2019). MNM and SNM maintain but do not establish achiasmate homolog conjunction during Drosophila male meiosis. PLoS Genet 15(5): e1008162. PubMed ID: 31136586
Summary:
The first meiotic division reduces genome ploidy. This requires pairing of homologous chromosomes into bivalents that can be bi-oriented within the spindle during prometaphase I. Thereafter, pairing is abolished during late metaphase I, and univalents are segregated apart onto opposite spindle poles during anaphase I. In contrast to canonical meiosis, homologous chromosome pairing does not include the formation of a synaptonemal complex and of cross-overs in spermatocytes of Drosophila melanogaster. The alternative pairing mode in these cells depends on mnm and snm. These genes are required exclusively in spermatocytes specifically for successful conjunction of chromosomes into bivalents. Available evidence suggests that MNM and SNM might be part of a physical linkage that directly conjoins chromosomes. This study analyzed this notion was analyzed further. Temporal variation in delivery of mnm and snm function was realized by combining various transgenes with null mutant backgrounds. The observed phenotypic consequences provide strong evidence that MNM and SNM contribute directly to chromosome linkage. Premature elimination of these proteins results in precocious bivalent splitting. Delayed provision results in partial conjunction defects that are more pronounced in autosomal bivalents compared to the sex chromosome bivalent. Overall, these findings suggest that MNM and SNM cannot re-establish pairing of chromosomes into bivalents if provided after a chromosome-specific time point of no return. When delivered before this time point, they fortify preformed linkages in order to preclude premature bivalent splitting by the disruptive forces that drive chromosome territory formation during spermatocyte maturation and chromosome condensation during entry into meiosis I.
Chik, J. K., Moiseeva, V., Goel, P. K., Meinen, B. A., Koldewey, P., An, S., Mellone, B. G., Subramanian, L. and Cho, U. S. (2019). Structures of CENP-C cupin domains at regional centromeres reveal unique patterns of dimerization and recruitment functions for the inner pocket. J Biol Chem. PubMed ID: 31366733
Summary:
The successful assembly and regulation of the kinetochore are critical for the equal and accurate segregation of genetic material during the cell cycle. Centromere protein C (CENP-C), a conserved inner kinetochore component, has been broadly characterized as a scaffolding protein and is required for the recruitment of multiple kinetochore proteins to the centromere. At its C-terminus, CENP-C harbors a conserved cupin domain that has an established role in protein dimerization. Although the crystal structure of the Saccharomyces cerevisiae Mif2CENP-C cupin domain has been determined, centromeric organization and kinetochore composition vary greatly between S. cerevisiae (point centromere) and other eukaryotes (regional centromere). Therefore, whether the structural and functional role of the cupin domain is conserved throughout evolution, requires investigation. This study reports the crystal structures of the Schizosaccharomyces pombe and Drosophila melanogaster CENP-C cupin domains at 2.52 Å and 1.81 Å resolutions, respectively. While the central jelly roll architecture is conserved among the three determined CENP-C cupin domain structures, the cupin domains from organisms with regional centromeres contain additional structural features that aid in dimerization. Moreover, this study found that the S. pombe Cnp3CENP-C jelly roll fold harbors an inner binding pocket that is used to recruit the meiosis-specific protein Moa1. In summary, these results unveil the evolutionarily conserved and unique features of the CENP-C cupin domain and uncover the mechanism by which it functions as a recruitment factor.
Bivik Stadler, C., Arefin, B., Ekman, H. and Thor, S. (2019). PIP degron-stabilized Dacapo/p21(Cip1) and mutations in ago act in an anti- versus pro-proliferative manner, yet both trigger an increase in Cyclin E levels. Development 146(13). PubMed ID: 31289041
Summary:
During cell cycle progression, the activity of the CycE-Cdk2 complex gates S-phase entry. CycE-Cdk2 is inhibited by CDK inhibitors (CKIs) of the Cip/Kip family, which include the human p21(Cip1) and Drosophila Dacapo (Dap) proteins. Both the CycE and Cip/Kip family proteins are under elaborate control via protein degradation, mediated by the Cullin-RING ligase (CRL) family of ubiquitin ligase complexes. The CRL complex SCF(Fbxw7/Ago) targets phosphorylated CycE, whereas p21(Cip1) and Dap are targeted by the CRL4(Cdt2) complex, binding to the PIP degron. The role of CRL-mediated degradation of CycE and Cip/Kip proteins during CNS development is not well understood. This study analyses the role of ago (Fbxw7)-mediated CycE degradation, and of Dap and p21(Cip1) degradation during Drosophila CNS development. ago mutants display over-proliferation, accompanied by elevated CycE expression levels. By contrast, expression of PIP degron mutant Dap and p21(Cip1) transgenes inhibit proliferation. However, surprisingly, this is also accompanied by elevated CycE levels. Hence, ago mutation and PIP degron Cip/Kip transgenic expression trigger opposite effects on proliferation, but similar effects on CycE levels.
Campbell, K., Rossi, F., Adams, J., Pitsidianaki, I., Barriga, F. M., Garcia-Gerique, L., Batlle, E., Casanova, J. and Casali, A. (2019). Collective cell migration and metastases induced by an epithelial-to-mesenchymal transition in Drosophila intestinal tumors. Nat Commun 10(1): 2311. PubMed ID: 31127094
Summary:
Metastasis underlies the majority of cancer-related deaths yet remains poorly understood due, in part, to the lack of models in vivo. This study shows that expression of the EMT master inducer Snail in primary adult Drosophila intestinal tumors leads to the dissemination of tumor cells and formation of macrometastases. Snail drives an EMT in tumor cells, which, although retaining some epithelial markers, subsequently break through the basal lamina of the midgut, undergo a collective migration and seed polyclonal metastases. While metastases re-epithelialize over time, this study found that early metastases are remarkably mesenchymal, discarding the requirement for a mesenchymal-to-epithelial transition for early stages of metastatic growth. These results demonstrate the formation of metastases in adult flies, and identify a key role for partial-EMTs in driving it. This model opens the door to investigate the basic mechanisms underlying metastasis, in a powerful in vivo system suited for rapid genetic and drug screens.

Friday, August 9th - Transcriptional Regulation

Chen, X., Ye, Y., Gu, L., Sun, J., Du, Y., Liu, W. J., Li, W., Zhang, X. and Jiang, C. (2019). H3K27me3 signal in the cis regulatory elements reveals the differentiation potential of progenitors during Drosophila neuroglial development. Genomics Proteomics Bioinformatics. PubMed ID: 31195140
Summary:
Drosophila neural development undergoes extensive chromatin remodeling and precise epigenetic regulation. However, the roles of chromatin remodeling in establishment and maintenance of cell identity during cell fate transition remain enigmatic. This study compared the changes in gene expression, as well as the dynamics of nucleosome positioning and key histone modifications between the four major neural cell types during Drosophila neural development. The neural progenitors could be separated from the terminally differentiated cells based on their gene expression profiles, whereas nucleosome distribution in the flanking regions of transcription start sites fails to identify the relationships between the progenitors and the differentiated cells. H3K27me3 signal in promoters and enhancers can not only distinguish the progenitors from the differentiated cells but also identify the differentiation path of the neural stem cells (NSCs) to the intermediate progenitor cells to the glial cells. In contrast, H3K9ac signal fails to identify the differentiation path, although it activates distinct sets of genes with neuron-specific and glia-related functions during the differentiation of the NSCs into neurons and glia, respectively. Together, this study provides novel insights into the crucial roles of chromatin remodeling in determining cell type during Drosophila neural development.
Zubair, A., Rosen, I. G., Nuzhdin, S. V. and Marjoram, P. (2019). Bayesian model selection for the Drosophila gap gene network. BMC Bioinformatics 20(1): 327. PubMed ID: 31195954
Summary:
The gap gene system controls the early cascade of the segmentation pathway in Drosophila melanogaster as well as other insects. Owing to its tractability and key role in embryo patterning, this system has been the focus for both computational modelers and experimentalists. The gap gene expression dynamics can be considered strictly as a one-dimensional process and modeled as a system of reaction-diffusion equations. The Bayesian framework offers a means of doing formal model evaluation. This study demonstrates how this framework can be used to compare different models of gene expression. Focus was placed on the Papatsenko-Levine formalism, which exploits a fractional occupancy based approach to incorporate activation of the gap genes by the maternal genes and cross-regulation by the gap genes themselves. The Bayesian approach provides insight about relationship between system parameters. In the regulatory pathway of segmentation, the parameters for number of binding sites and binding affinity have a negative correlation. The model selection analysis supports a stronger binding affinity for Bicoid compared to other regulatory edges, as shown by a larger posterior mean. The procedure doesn't show support for activation of Kruppel by Bicoid. This study provides an efficient solver for the general representation of the Papatsenko-Levine model and demonstrates the utility of Bayes factor for evaluating candidate models for spatial pattering models. In addition, by using the parallel tempering sampler, the convergence of Markov chains can be remarkably improved and robust estimates of Bayes factors obtained.
Lo, C. A. and Chen, B. E. (2019). Parental allele-specific protein expression in single cells in vivo. Dev Biol. PubMed ID: 31194972
Summary:
Allelic expression from each parent-of-origin is important as a backup and to ensure that enough protein products of a gene are produced. Thus far, it is not known how each cell throughout a tissue differs in parental allele expression at the level of protein synthesis. This study measured the expression of the Ribosomal protein L13a from both parental alleles simultaneously in single cells in the living animal. Genome-edited Drosophila were used that have a quantitative reporter of protein synthesis inserted into the endogenous Rpl13a locus. Individual cells can have large (>10-fold) differences in protein expression between the two parental alleles. Cells can produce protein from only one allele oftentimes, and time-lapse imaging of protein production from each parental allele in each cell showed that the imbalance in expression from one parental allele over the other can invert over time. This study also identified the histone methyltransferase EHMT as involved in the protein synthesis dynamics within cells.
Lambert, S. A., Yang, A. W. H., Sasse, A., Cowley, G., Albu, M., Caddick, M. X., Morris, Q. D., Weirauch, M. T. and Hughes, T. R. (2019). Similarity regression predicts evolution of transcription factor sequence specificity. Nat Genet 51(6): 981-989. PubMed ID: 31133749
Summary:
Transcription factor (TF) binding specificities (motifs) are essential for the analysis of gene regulation. Accurate prediction of TF motifs is critical, because it is infeasible to assay all TFs in all sequenced eukaryotic genomes. There is ongoing controversy regarding the degree of motif diversification among related species that is, in part, because of uncertainty in motif prediction methods. This study describes similarity regression, a significantly improved method for predicting motifs, which was used to update and expand the Cis-BP database. Similarity regression inherently quantifies TF motif evolution, and shows that previous claims of near-complete conservation of motifs between human and Drosophila are inflated, with nearly half of the motifs in each species absent from the other, largely due to extensive divergence in C2H2 zinc finger proteins. It is concluded that diversification in DNA-binding motifs is pervasive, and a new tool and updated resource are presented to study TF diversity and gene regulation across eukaryotes.
Lovato, C. V., Lovato, T. L. and Cripps, R. M. (2019). Crossveinless is a direct transcriptional target of Trachealess and Tango in Drosophila tracheal precursor cells. PLoS One 14(6): e0217906. PubMed ID: 31158257
Summary:
Understanding the transcriptional pathways controlling tissue-specific gene expression is critical to unraveling the complex regulatory networks that underlie developmental mechanisms. This study assessed how the Drosophila crossveinless (cv) gene, that encodes a BMP-binding factor, is transcriptionally regulated in the developing embryonic tracheal system. An upstream regulatory region of cv was identified that promotes reporter gene expression in the tracheal precursors. It was further demonstrated that this promoter region is directly responsive to the basic, helix-loop-helix-PAS domain factors Trachealess (Trh) and Tango (Tgo), that function to specify tracheal fate. Moreover, cv expression in embryos is lost in trh mutants, and the integrity of the Trh/Tgo binding sites are required for promoter-lacZ expression. These findings for the first time elucidate the transcriptional regulation of one member of a family of BMP binding proteins, that have diverse functions in animal development.
Bhardwaj, V., Semplicio, G., Erdogdu, N. U., Manke, T. and Akhtar, A. (2019). MAPCap allows high-resolution detection and differential expression analysis of transcription start sites. Nat Commun 10(1): 3219. PubMed ID: 31363093
Summary:
The position, shape and number of transcription start sites (TSS) are critical determinants of gene regulation. Most methods developed to detect TSSs and study promoter usage are, however, of limited use in studies that demand quantification of expression changes between two or more groups. This study combined high-resolution detection of transcription start sites and differential expression analysis using a simplified TSS quantification protocol, MAPCap (Multiplexed Affinity Purification of Capped RNA) along with the software icetea. Applying MAPCap on developing Drosophila melanogaster embryos and larvae, stage and sex-specific promoter and enhancer activity was detected, and the effect of mutants of maleless (MLE) helicase at X-chromosomal promoters was quantified. It was observed that MLE mutation leads to a median 1.9 fold drop in expression of X-chromosome promoters and affects the expression of several TSSs with a sexually dimorphic expression on autosomes. These results provide quantitative insights into promoter activity during dosage compensation.

Thursday August 8th - Adult neural development and function

Awata, H., Takakura, M., Kimura, Y., Iwata, I., Masuda, T. and Hirano, Y. (2019). The neural circuit linking mushroom body parallel circuits induces memory consolidation in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 31337675
Summary:
Memory consolidation is augmented by repeated learning following rest intervals, which is known as the spacing effect. Although the spacing effect has been associated with cumulative cellular responses in the neurons engaged in memory, this study reports the neural circuit-based mechanism for generating the spacing effect in the memory-related mushroom body (MB) parallel circuits in Drosophila. To investigate the neurons activated during the training, expression was monitored of phosphorylation of mitogen-activated protein kinase (MAPK), ERK [phosphorylation of extracellular signal-related kinase (pERK)]. In an olfactory spaced training paradigm, pERK expression in one of the parallel circuits, consisting of gammam neurons, was progressively inhibited via dopamine. This inhibition resulted in reduced pERK expression in a postsynaptic GABAergic neuron that, in turn, led to an increase in pERK expression in a dopaminergic neuron specifically in the later session during spaced training, suggesting that disinhibition of the dopaminergic neuron occurs during spaced training. The dopaminergic neuron was significant for gene expression in the different MB parallel circuits consisting of alpha/betas neurons for memory consolidation. These results suggest that the spacing effect-generating neurons and the neurons engaged in memory reside in the distinct MB parallel circuits and that the spacing effect can be a consequence of evolved neural circuit architecture.
Lopez-Bellido, R., Puig, S., Huang, P. J., Tsai, C. R., Turner, H. N., Galko, M. J. and Gutstein, H. B. (2019). Growth factor signaling regulates mechanical nociception in flies and vertebrates. J Neurosci. PubMed ID: 31138657
Summary:
Mechanical sensitization is one of the most difficult clinical pain problems to treat. However, the molecular and genetic bases of mechanical nociception are unclear. This study developed a Drosophila model of mechanical nociception to investigate the ion channels and signaling pathways that regulate mechanical nociception. Von Frey filaments were fabricated that span the sub-threshold to high noxious range for Drosophila larvae. Utilizing these, pressure (force/area) rather than force per se was found to be the main determinant of aversive rolling responses to noxious mechanical stimuli. The RTK PDGF/VEGF receptor (Pvr) and its ligands (Pvfs 2 and 3) are required for mechanical nociception and normal dendritic branching. Pvr is expressed and functions in class IV sensory neurons, while Pvf2 and Pvf3 are produced by multiple tissues. Constitutive overexpression of Pvr and its ligands or inducible overexpression of Pvr led to mechanical hypersensitivity that could be partially separated from morphological effects. Genetic analyses revealed that the Piezo and Pain ion channels are required for mechanical hypersensitivity observed upon ectopic activation of Pvr signaling. Platelet-derived growth factor (PDGF), but not vascular endothelial growth factor (VEGF) peptides caused mechanical hypersensitivity in rats. Pharmacological inhibition of vascular endothelial growth factor receptor type 2 (VEGFR-2) signaling attenuated mechanical nociception in rats, suggesting a conserved role for PDGF and VEGFR-2 signaling in regulating mechanical nociception. VEGFR2 inhibition also attenuated morphine analgesic tolerance in rats. The results reveal that a conserved RTK signaling pathway regulates baseline mechanical nociception in flies and rats.
Abdusselamoglu, M. D., Eroglu, E., Burkard, T. R. and Knoblich, J. A. (2019). The transcription factor odd-paired regulates temporal identity in transit-amplifying neural progenitors via an incoherent feed-forward loop. Elife 8. PubMed ID: 31329099
Summary:
Neural progenitors undergo temporal patterning to generate diverse neurons in a chronological order. This process is well-studied in the developing Drosophila brain and conserved in mammals. During larval stages, intermediate neural progenitors (INPs) serially express Dichaete (D), grainyhead (Grh) and eyeless (Ey/Pax6), but how the transitions are regulated is not precisely understood. In this study a method was developed to isolate transcriptomes of INPs in their distinct temporal states to identify a complete set of temporal patterning factors. This analysis identifies odd-paired (opa), as a key regulator of temporal patterning. Temporal patterning is initiated when the SWI/SNF complex component Osa induces D and its repressor Opa at the same time but with distinct kinetics. Then, high Opa levels repress D to allow Grh transcription and progress to the next temporal state. It is proposed that Osa and its target genes opa and D form an incoherent feedforward loop (FFL) and a new mechanism allowing the successive expression of temporal identities.
Badwan, B. A., Creamer, M. S., Zavatone-Veth, J. A. and Clark, D. A. (2019). Dynamic nonlinearities enable direction opponency in Drosophila elementary motion detectors. Nat Neurosci 22(8): 1318-1326. PubMed ID: 31346296
Summary:
Direction-selective neurons respond to visual motion in a preferred direction. They are direction-opponent if they are also inhibited by motion in the opposite direction. In flies and vertebrates, direction opponency has been observed in second-order direction-selective neurons, which achieve this opponency by subtracting signals from first-order direction-selective cells with opposite directional tunings. This study reports direction opponency in Drosophila that emerges in first-order direction-selective neurons, the elementary motion detectors T4 and T5. This opponency persists when synaptic output from these cells is blocked, suggesting that it arises from feedforward, not feedback, computations. These observations exclude a broad class of linear-nonlinear models that have been proposed to describe direction-selective computations. However, they are consistent with models that include dynamic nonlinearities. Simulations of opponent models suggest that direction opponency in first-order motion detectors improves motion discriminability by suppressing noise generated by the local structure of natural scenes.
Berntsson, O., Rodriguez, R., Henry, L., Panman, M. R., Hughes, A. J., Einholz, C., Weber, S., Ihalainen, J. A., Henning, R., Kosheleva, I., Schleicher, E. and Westenhoff, S. (2019). Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions. Sci Adv 5(7): eaaw1531. PubMed ID: 31328161
Summary:
Cryptochromes are blue-light photoreceptor proteins, which provide input to circadian clocks. The cryptochrome from Drosophila melanogaster (DmCry) modulates the degradation of Timeless and itself. It is unclear how light absorption by the chromophore and the subsequent redox reactions trigger these events. This study nano- to millisecond time-resolved x-ray solution scattering to reveal the light-activated conformational changes in DmCry and the related (6-4) photolyase. DmCry undergoes a series of structural changes, culminating in the release of the carboxyl-terminal tail (CTT). The photolyase has a simpler structural response. The CTT release in DmCry depends on pH. Mutation of a conserved histidine, important for the biochemical activity of DmCry, does not affect transduction of the structural signal to the CTT. Instead, molecular dynamics simulations suggest that it stabilizes the CTT in the resting-state conformation. This structural photocycle unravels the first molecular events of signal transduction in an animal cryptochrome.
Latcheva, N. K., Viveiros, J. M. and Marenda, D. R. (2019). The Drosophila chromodomain protein Kismet activates steroid hormone receptor transcription to govern axon pruning and memory in vivo. iScience 16: 79-93. PubMed ID: 31153043
Summary:
Axon pruning is critical for sculpting precise neural circuits. Although axon pruning has been described in the literature for decades, relatively little is known about the molecular and cellular mechanisms that govern axon pruning in vivo. This study shows that the epigenetic reader Kismet (Kis) is required for developmental axon pruning in Drosophila mushroom bodies. Kis binds to cis-regulatory elements of the steroid hormone receptor Ecdysone receptor (Ecr) gene and is necessary for activating expression of EcR-B1. Kis promotes the active H3K36 di- and tri-methylation and H4K16 acetylation histone marks at the Ecr locus. Transgenic EcR-B1 can rescue axon pruning and memory defects associated with loss of Kis, and the histone deacetylase inhibitor SAHA also rescues these phenotypes. EcR protein abundance is the cell-autonomous, rate-limiting step required to initiate axon pruning in Drosophila, and the data suggest this step is under the epigenetic control of Kis.

Wednesday, August 7th - RNA and Transposons

Bennick, R. A., Nagengast, A. A. and DiAngelo, J. R. (2019). The SR proteins SF2 and RBP1 regulate triglyceride storage in the fat body of Drosophila. Biochem Biophys Res Commun 516(3): 928-933. PubMed ID: 31277943
Summary:
In Western societies where food is abundant, these excess nutrients are stored as fats mainly in adipose tissue. Fats are stored in structures known as lipid droplets, and a genome-wide screen performed in Drosophila cells has identified several genes that are important for the formation of these droplets. One group of genes found during this screen included those that regulate mRNA splicing. Previous work has identified some splicing factors that play a role in regulating fat storage; however, the full complement of splicing proteins that regulate lipid metabolism is still unknown. In this study, the levels of a number of serine-arginine (SR) domain containing splicing factors (RSF1, RBP1, RBP1-like, SF2 and Srp-54) were decreased using RNAi in the adult fat body to assess their role in the control of Drosophila metabolism. Decreasing SF2 and RBP1 showed increased triglycerides, while inducing RNAi towards RSF1, RBP1-Like and Srp-54 had no effect on triglycerides. Interestingly, the increased triglyceride phenotype in the SF2-RNAi flies was due to an increase in the amount of fat stored per cell while the RBP1-RNAi flies have more fat cells. In addition, the splicing of the beta-oxidation enzyme, CPT1, was altered in the SF2-RNAi flies potentially promoting the increased triglycerides in these animals. Together, this study identifies novel splicing factors responsible for the regulation of lipid storage in the Drosophila fat body and contributes to understanding of the mechanisms, which influence the regulation of fat storage in adipose-like cells.
Saint-Leandre, B., Nguyen, S. C. and Levine, M. T. (2019). Diversification and collapse of a telomere elongation mechanism. Genome Res 29(6): 920-931. PubMed ID: 31138619
Summary:
In most eukaryotes, telomerase counteracts chromosome erosion by adding repetitive sequence to terminal ends. Drosophila melanogaster instead relies on specialized retrotransposons that insert exclusively at telomeres. This exchange of goods between host and mobile element-wherein the mobile element provides an essential genome service and the host provides a hospitable niche for mobile element propagation-has been called a "genomic symbiosis." However, these telomere-specialized, jockey family retrotransposons may actually evolve to "selfishly" overreplicate in the genomes that they ostensibly serve. Under this model, rapid diversification is expected of telomere-specialized retrotransposon lineages and, possibly, the breakdown of this ostensibly symbiotic relationship. This study reports data consistent with both predictions. Searching the raw reads of the 15-Myr-old melanogaster species group, de novo jockey retrotransposon consensus sequences were generated, and phylogenetic tree-building was used to delineate four distinct telomere-associated lineages. Recurrent gains, losses, and replacements account for this retrotransposon lineage diversity. In Drosophila biarmipes, telomere-specialized elements have disappeared completely. De novo assembly of long reads and cytogenetics confirmed this species-specific collapse of retrotransposon-dependent telomere elongation. Instead, telomere-restricted satellite DNA and DNA transposon fragments occupy its terminal ends. It is infered that D. biarmipes relies instead on a recombination-based mechanism conserved from yeast to flies to humans. Telomeric retrotransposon diversification and disappearance suggest that persistently "selfish" machinery shapes telomere elongation across Drosophila rather than completely domesticated, symbiotic mobile elements.
Zhu, L., Liao, S. E. and Fukunaga, R. (2019). Drosophila Regnase-1 RNase is required for mRNA and miRNA profile remodelling during larva-to-adult metamorphosis. RNA Biol: 1-15. PubMed ID: 31195914
Summary:
Metamorphosis is an intricate developmental process in which large-scale remodelling of mRNA and microRNA (miRNA) profiles leads to orchestrated tissue remodelling and organogenesis. Whether, which, and how, ribonucleases (RNases) are involved in the RNA profile remodelling during metamorphosis remain unknown. Human Regnase-1 (also known as MCPIP1 and Zc3h12a) RNase remodels RNA profile by cleaving specific RNAs and is a crucial modulator of immune-inflammatory and cellular defence. This study characterized Drosophila CG10889, which was named Drosophila Regnase-1, an ortholog of human Regnase-1. The larva-to-adult metamorphosis in Drosophila includes two major transitions, larva-to-pupa and pupa-to-adult. regnase-1 knockout flies developed until the pupa stage but could not complete pupa-to-adult transition, dying in puparium case. Regnase-1 RNase activity is required for completion of pupa-to-adult transition as transgenic expression of wild-type Drosophila Regnase-1, but not the RNase catalytic-dead mutants, rescued the pupa-to-adult transition in regnase-1 knockout. High-throughput RNA sequencing revealed that regnase-1 knockout flies fail to remodel mRNA and miRNA profiles during the larva-to-pupa transition. Thus, this study has uncovered the roles of Drosophila Regnase-1 in the larva-to-adult metamorphosis and large-scale remodelling of mRNA and miRNA profiles during this metamorphosis process.
Munafo, M., Manelli, V., Falconio, F. A., Sawle, A., Kneuss, E., Eastwood, E. L., Seah, J. W. E., Czech, B. and Hannon, G. J. (2019). Daedalus and Gasz recruit Armitage to mitochondria, bringing piRNA precursors to the biogenesis machinery. Genes Dev. PubMed ID: 31123065
Summary:
The Piwi-interacting RNA (piRNA) pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialized machinery that converts long single-stranded precursors into small RNAs of approximately 25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. This study investigated a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and the correct localization of the indispensable piRNA biogenesis factor Armitage (Armi). Gasz and Daed interact with each other and likely provide a mitochondrial "anchoring platform" to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. The data suggest that Armi initially identifies piRNA precursors in nuage/Yb bodies in a manner that depends on Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing; namely, how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
Zhang, Z., So, K., Peterson, R., Bauer, M., Ng, H., Zhang, Y., Kim, J. H., Kidd, T. and Miura, P. (2019). Elav-mediated exon skipping and alternative polyadenylation of the Dscam1 gene are required for axon outgrowth. Cell Rep 27(13): 3808-3817. PubMed ID: 31242415
Summary:
Many metazoan genes express alternative long 3' UTR isoforms in the nervous system, but their functions remain largely unclear. In Drosophila melanogaster, the Dscam1 gene generates short and long (Dscam1-L) 3' UTR isoforms because of alternative polyadenylation (APA). This study found that the RNA-binding protein Embryonic Lethal Abnormal Visual System (Elav) impacts Dscam1 biogenesis at two levels, including regulation of long 3' UTR biogenesis and skipping of an upstream exon (exon 19). MinION long-read sequencing confirmed the connectivity of this alternative splicing event to the long 3' UTR. Knockdown or CRISPR deletion of Dscam1-L impaired axon outgrowth in Drosophila. The Dscam1 long 3' UTR was found to be required for correct Elav-mediated skipping of exon 19. Elav thus co-regulates APA and alternative splicing to generate specific Dscam1 transcripts that are essential for neural development. This coupling of APA to alternative splicing might represent a new class of regulated RNA processing.
Zhu, L., Liao, S. E., Ai, Y. and Fukunaga, R. (2019). RNA methyltransferase BCDIN3D is crucial for female fertility and miRNA and mRNA profiles in Drosophila ovaries. PLoS One 14(5): e0217603. PubMed ID: 31145769
Summary:
RNA methyltransferases post-transcriptionally add methyl groups to RNAs, which can regulate their fates and functions. Human BCDIN3D (Bicoid interacting 3 domain containing RNA methyltransferase) has been reported to specifically methylate the 5'-monophosphates of pre-miR-145 and cytoplasmic tRNAHis. Methylation of the 5'-monophosphate of pre-miR-145 blocks its cleavage by the miRNA generating enzyme Dicer, preventing generation of miR-145. Elevated expression of BCDIN3D has been associated with poor prognosis in breast cancer. However, the biological functions of BCDIN3D and its orthologs remain unknown. This study examined the biological and molecular functions of CG1239, a Drosophila ortholog of BCDIN3D. Ovary-specific knockdown of Drosophila BCDIN3D causes female sterility. High-throughput sequencing revealed that miRNA and mRNA profiles are dysregulated in BCDIN3D knockdown ovaries. Pathway analysis showed that many of the dysregulated genes are involved in metabolic processes, ribonucleoprotein complex regulation, and translational control. These results reveal BCDIN3D's biological role in female fertility and its molecular role in defining miRNA and mRNA profiles in ovaries.

Tuesday, August 6th - Oogenesis and Spermatogenesis

Viktorinova, I., Haase, R., Pietzsch, T., Henry, I. and Tomancak, P. (2019). Analysis of actomyosin dynamics at local cellular and tissue scales using time-lapse movies of cultured Drosophila egg chambers. J Vis Exp(148). PubMed ID: 31205315
Summary:
Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development. Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis. Thus, to overcome this limitation, this study cultured Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, a manual is provided detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, users are provided with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers' circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (>50 cells). Finally, the limitations of these approaches are pinpointed at both the local cellular and tissue scales and its potential future development and possible applications are discussed.
Weiss, I. and Bohrmann, J. (2019). Electrochemical patterns during Drosophila oogenesis: ion-transport mechanisms generate stage-specific gradients of pH and membrane potential in the follicle-cell epithelium. BMC Dev Biol 19(1): 12. PubMed ID: 31226923
Summary:
Alterations of bioelectrical properties of cells and tissues are known to function as wide-ranging signals during development, regeneration and wound-healing in several species. The Drosophila follicle-cell epithelium provides an appropriate model system for studying the potential role of electrochemical signals, like intracellular pH (pHi) and membrane potential (Vmem), during development. This study analysed stage-specific gradients of pHi and Vmem. Distinct alterations were found of pHi- and Vmem-patterns during stages 8 to 12 of oogenesis. To determine the roles of relevant ion-transport mechanisms in regulating pHi and Vmem and in establishing stage-specific antero-posterior and dorso-ventral gradients, inhibitors were used of Na+/H+-exchangers and Na+-channels, V-ATPases, ATP-sensitive K+-channels, voltage-dependent L-type Ca2+-channels, Cl(-)-channels and Na+/K+/2Cl(-)-cotransporters. Either pHi or Vmem or both parameters were affected by each tested inhibitor. While the inhibition of Na+/H+-exchangers and amiloride-sensitive Na+-channels or of V-ATPases resulted in relative acidification, inhibiting the other ion-transport mechanisms led to relative alkalisation. The most prominent effects on pHi were obtained by inhibiting Na+/K+/2Cl(-)-cotransporters or ATP-sensitive K+-channels. Vmem was most efficiently hyperpolarised by inhibiting voltage-dependent L-type Ca2+-channels or ATP-sensitive K+-channels, whereas the impact of the other ion-transport mechanisms was smaller. In case of very prominent effects of inhibitors on pHi and/or Vmem, strong influences were found on the A-P and D-V pHi- and/or Vmem-gradients. These data show that in the Drosophila follicle-cell epithelium stage-specific pHi- and Vmem-gradients develop which result from the activity of several ion-transport mechanisms. These gradients are supposed to represent important bioelectrical cues during oogenesis, e.g., by serving as electrochemical prepatterns in modifying cell polarity and cytoskeletal organisation.
Senos Demarco, R., Uyemura, B. S., D'Alterio, C. and Jones, D. L. (2019). Mitochondrial fusion regulates lipid homeostasis and stem cell maintenance in the Drosophila testis. Nat Cell Biol 21(6): 710-720. PubMed ID: 31160709
Summary:
The capacity of stem cells to self-renew or differentiate has been attributed to distinct metabolic states. A genetic screen targeting regulators of mitochondrial dynamics revealed that mitochondrial fusion is required for the maintenance of male germline stem cells (GSCs) in Drosophila melanogaster. Depletion of Mitofusin (dMfn) or Opa1 led to dysfunctional mitochondria, activation of Target of rapamycin (TOR) and a marked accumulation of lipid droplets. Enhancement of lipid utilization by the mitochondria attenuated TOR activation and rescued the loss of GSCs that was caused by inhibition of mitochondrial fusion. Moreover, constitutive activation of the TOR-pathway target and lipogenesis factor Sterol regulatory element binding protein (SREBP) also resulted in GSC loss, whereas inhibition of SREBP rescued GSC loss triggered by depletion of dMfn. These findings highlight a critical role for mitochondrial fusion and lipid homeostasis in GSC maintenance, providing insight into the potential impact of mitochondrial and metabolic diseases on the function of stem and/or germ cells.
Shropshire, J. D. and Bordenstein, S. R. (2019). Two-By-One model of cytoplasmic incompatibility: Synthetic recapitulation by transgenic expression of cifA and cifB in Drosophila. PLoS Genet 15(6): e1008221. PubMed ID: 31242186
Summary:
Wolbachia are maternally inherited bacteria that infect arthropod species worldwide and are deployed in vector control to curb arboviral spread using cytoplasmic incompatibility (CI). CI kills embryos when an infected male mates with an uninfected female, but the lethality is rescued if the female and her embryos are likewise infected. Two phage WO genes, cifAwMel and cifBwMel from the wMel Wolbachia deployed in vector control, transgenically recapitulate variably penetrant CI, and one of the same genes, cifAwMel, rescues wild type CI. The proposed Two-by-One genetic model predicts that CI and rescue can be recapitulated by transgenic expression alone and that dual cifAwMel and cifBwMel expression can recapitulate strong CI. This study used hatch rate and gene expression analyses in transgenic Drosophila melanogaster to demonstrate that CI and rescue can be synthetically recapitulated in full, and strong, transgenic CI comparable to wild type CI is achievable. These data explicitly validate the Two-by-One model in wMel-infected D. melanogaster, establish a robust system for transgenic studies of CI in a model system, and represent the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products.
Wilcockson, S. G. and Ashe, H. L. (2019). Drosophila ovarian germline stem cell cytocensor projections dynamically receive and attenuate BMP signaling. Dev Cell. PubMed ID: 31178401
Summary:
In the Drosophila ovarian germline, Bone Morphogenetic Protein (BMP) signals released by niche cells promote germline stem cell (GSC) maintenance. Although BMP signaling is known to repress expression of a key differentiation factor, it remains unclear whether BMP-responsive transcription also contributes positively to GSC identity. This study has identified the GSC transcriptome using RNA sequencing (RNA-seq), including the BMP-induced transcriptional network. Based on these data, evidence is provided that GSCs form two types of cellular projections. Genetic manipulation and live ex vivo imaging reveal that both classes of projection allow GSCs to access a reservoir of Dpp held away from the GSC-niche interface. Moreover, microtubule-rich projections, termed "cytocensors", form downstream of BMP and have additional functionality, which is to attenuate BMP signaling. In this way, cytocensors allow dynamic modulation of signal transduction to facilitate differentiation following GSC division. This ability of cytocensors to attenuate the signaling response expands the repertoire of functions associated with signaling projections.
Basar, M. A., Williamson, K., Roy, S. D., Finger, D. S., Ables, E. T. and Duttaroy, A. (2019). Spargel/dPGC-1 is essential for oogenesis and nutrient-mediated ovarian growth in Drosophila. Dev Biol. PubMed ID: 31251895
Summary:
Dietary proteins are crucial for oogenesis. Target of Rapamycin (TOR) is a major nutrient sensor controlling organismal growth and fertility, but the downstream effectors of TOR signaling remain largely uncharacterized. Drosophila Spargel/dPGC-1 has been identified as a terminal effector of the TOR-TSC pathway, and this study now reports that Spargel connects nutrition to oogenesis. Spargel is expressed predominantly in the ovaries of adult flies, and germline spargel knockdown inhibits cyst growth, ultimately leading to egg chamber degeneration and female sterility. In situ staining demonstrated nuclear localization of Spargel in the nurse cells and follicle cells of the ovariole. Furthermore, Spargel/dPGC-1 expression is influenced by dietary yeast concentration and TOR signaling, suggesting Spargel/dPGC-1 might transmit nutrient-mediated signals into ovarian growth. It is proposed that potentiating Spargel/dPGC-1 expression in the ovary is instrumental in nutrient-mediated regulation of oogenesis.

Monday, August 5th - Disease Models

Wu, S., Tan, K. J., Govindarajan, L. N., Stewart, J. C., Gu, L., Ho, J. W. H., Katarya, M., Wong, B. H., Tan, E. K., Li, D., Claridge-Chang, A., Libedinsky, C., Cheng, L. and Aw, S. S. (2019). Fully automated leg tracking of Drosophila neurodegeneration models reveals distinct conserved movement signatures. PLoS Biol 17(6): e3000346. PubMed ID: 31246996
Summary:
Some neurodegenerative diseases, like Parkinsons Disease (PD) and Spinocerebellar ataxia 3 (SCA3), are associated with distinct, altered gait and tremor movements that are reflective of the underlying disease etiology. Drosophila melanogaster models of neurodegeneration have led to understanding of the molecular mechanisms of disease. However, it is unknown whether specific gait and tremor dysfunctions also occur in fly disease mutants. To answer this question, a machine-learning image-analysis program, Feature Learning-based LImb segmentation and Tracking (FLLIT), was developed that automatically tracks leg claw positions of freely moving flies recorded on high-speed video, producing a series of gait measurements. Notably, unlike other machine-learning methods, FLLIT generates its own training sets and does not require user-annotated images for learning. Using FLLIT, high-throughput and high-resolution analysis of gait and tremor features were carried out in Drosophila neurodegeneration mutants for the first time. Fly models of PD and SCA3 exhibited markedly different walking gait and tremor signatures, which recapitulated characteristics of the respective human diseases. Selective expression of mutant SCA3 in dopaminergic neurons led to a gait signature that more closely resembled those of PD flies. This suggests that the behavioral phenotype depends on the neurons affected rather than the specific nature of the mutation. Different mutations produced tremors in distinct leg pairs, indicating that different motor circuits were affected. Using this approach, fly models can be used to dissect the neurogenetic mechanisms that underlie movement disorders.
Rybina, O. Y., Schelkunov, M. I., Veselkina, E. R., Sarantseva, S. V., Krementsova, A. V., Vysokikh, M. Y., Melentev, P. A., Volodina, M. A. and Pasyukova, E. G. (2019). Knockdown of the neuronal gene Lim3 at the early stages of development affects mitochondrial function and lifespan in Drosophila. Mech Ageing Dev 181: 29-41. PubMed ID: 31158363
Summary:
Understanding the molecular mechanisms underlying variation in lifespan is central to ensure long life. Lim3 encoding a homolog of the vertebrate Lhx3/4 transcription factors plays a key role in Drosophila neuron development. This study demonstrated that Lim3 knockdown early in life decreased survival of adult flies. To study the mechanisms underlying this effect, embryonic Lim3 targets were identified using combined RNA-seq and RT-qPCR analyses complemented by in silico analysis of Lim3 binding sites. Though genes with neuronal functions were revealed as Lim3 targets, the characteristics of neurons were not affected by Lim3 depletion. Many of the direct and indirect Lim3 target genes were associated with mitochondrial function, ATP-related activity, redox processes and antioxidant defense. Consistent with the observed changes in the embryonic transcription of these genes, ROS levels were increased in embryos, which could cause changes in the transcription of indirect Lim3 targets known to affect lifespan.It is hypothesized that altered mitochondrial activity is crucial for the decrease of adult lifespan caused by Lim3 knockdown early in life. In adults that encountered Lim3 depletion early in life, the transcription of several genes remained altered, and mitochondrial membrane potential, ATP level and locomotion were increased, confirming the existence of carry-over effects.
Villanueva, J. E., Livelo, C., Trujillo, A. S., Chandran, S., Woodworth, B., Andrade, L., Le, H. D., Manor, U., Panda, S. and Melkani, G. C. (2019). Time-restricted feeding restores muscle function in Drosophila models of obesity and circadian-rhythm disruption. Nat Commun 10(1): 2700. PubMed ID: 31221967
Summary:
Pathological obesity can result from genetic predisposition, obesogenic diet, and circadian rhythm disruption. Obesity compromises function of muscle, which accounts for a majority of body mass. Behavioral intervention that can counteract obesity arising from genetic, diet or circadian disruption and can improve muscle function holds untapped potential to combat the obesity epidemic. This study shows that Drosophila melanogaster subject to obesogenic challenges exhibits metabolic disease phenotypes in skeletal muscle; sarcomere disorganization, mitochondrial deformation, upregulation of Phospho-AKT level, aberrant intramuscular lipid infiltration, and insulin resistance. Imposing time-restricted feeding (TRF) paradigm in which flies were fed for 12 h during the day counteracts obesity-induced dysmetabolism and improves muscle performance by suppressing intramuscular fat deposits, Phospho-AKT level, mitochondrial aberrations, and markers of insulin resistance. Importantly, TRF was effective even in an irregular lighting schedule mimicking shiftwork. Hence, TRF is an effective dietary intervention for combating metabolic dysfunction arising from multiple causes.
Doktor, B., Damulewicz, M. and Pyza, E. (2019). Effects of MUL1 and PARKIN on the circadian clock, brain and behaviour in Drosophila Parkinson's disease models. BMC Neurosci 20(1): 24. PubMed ID: 31138137
Summary:
Mutants which carry mutations in genes encoding mitochondrial ligases MUL1 and PARKIN are convenient Drosophila models of Parkinson's disease (PD). In several studies it has been shown that in Parkinson's disease sleep disturbance occurs, which may be the result of a disturbed circadian clock. This study found that the ROS level was higher, while the anti-oxidant enzyme SOD1 level was lower in mul1(A6) and park(1) mutants than in the white mutant used as a control. Moreover, mutations of both ligases affected circadian rhythms and the clock. The expression of clock genes per, tim and clock and the level of PER protein were changed in the mutants. Moreover, expression of ATG5, an autophagy protein also involved in circadian rhythm regulation, was decreased in the brain and in PDF-immunoreactive large ventral lateral clock neurons. The observed changes in the molecular clock resulted in a longer period of locomotor activity rhythm, increased total activity and shorter sleep at night. Finally, the lack of both ligases led to decreased longevity and climbing ability of the flies. It is concluded that all of the changes observed in the brains of these Drosophila models of PD, in which mitochondrial ligases MUL1 and PARKIN do not function, may explain the mechanisms of some neurological and behavioural symptoms of PD.
Schinaman, J. M., Rana, A., Ja, W. W., Clark, R. I. and Walker, D. W. (2019). Rapamycin modulates tissue aging and lifespan independently of the gut microbiota in Drosophila. Sci Rep 9(1): 7824. PubMed ID: 31127145
Summary:
The FDA approved drug rapamycin can prolong lifespan in diverse species and delay the onset of age-related disease in mammals. However, a number of fundamental questions remain unanswered regarding the mechanisms by which rapamycin modulates age-related pathophysiology and lifespan. Alterations in the gut microbiota can impact host physiology, metabolism and lifespan. While recent studies have shown that rapamycin treatment alters the gut microbiota in aged animals, the causal relationships between rapamycin treatment, microbiota dynamics and aging are not known. Using Drosophila as a model organism, this study shows that rapamycin-mediated alterations in microbiota dynamics in aged flies are associated with improved markers of intestinal and muscle aging. Critically, however, this study shows that the beneficial effects of rapamycin treatment on tissue aging and lifespan are not dependent upon the microbiota. Indeed, germ-free flies show delayed onset of intestinal barrier dysfunction, improved proteostasis in aged muscles and a significant lifespan extension upon rapamycin treatment. In contrast, genetic inhibition of autophagy impairs the ability of rapamycin to mediate improved gut health and proteostasis during aging. These results indicate that rapamycin-mediated modulation of the microbiota in aged animals is not causally required to slow tissue and organismal aging.
Andreazza, S., Samstag, C. L., Sanchez-Martinez, A., Fernandez-Vizarra, E., Gomez-Duran, A., Lee, J. J., Tufi, R., Hipp, M. J., Schmidt, E. K., Nicholls, T. J., Gammage, P. A., Chinnery, P. F., Minczuk, M., Pallanck, L. J., Kennedy, S. R. and Whitworth, A. J. (2019). Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila. Nat Commun 10(1): 3280. PubMed ID: 31337756
Summary:
Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. This study describes a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research.

Friday, April 2nd - Signaling

Popow, O., Paulo, J. A., Tatham, M. H., Volk, M. S., Rojas-Fernandez, A., Loyer, N., Newton, I. P., Januschke, J., Haigis, K. M. and Nathke, I. (2019). Identification of endogenous Adenomatous polyposis coli interaction partners and beta-catenin-independent targets by proteomics. Mol Cancer Res. PubMed ID: 31160382
Summary:
Adenomatous Polyposis Coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the Wnt signaling pathway by promoting the degradation of beta-catenin, but the extent to which APC exerts Wnt/beta-catenin-independent tumor suppressive activity is unclear. To identify interaction partners and beta-catenin-independent targets of endogenous, full-length APC, label-free and multiplexed tandem mass tag-based mass spectrometry was applied. Affinity enrichment-mass spectrometry identified more than 150 previously unidentified APC interaction partners. Moreover, a global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of beta-catenin. Combining these two analyses, Misshapen-like kinase 1 (MINK1) was identifed as a putative substrate of an APC-containing destruction complex. The interaction was validated between endogenous MINK1 and APC and further confirmed the negative - and beta-catenin-independent -regulation of MINK1 by APC. Increased Mink1/Msn levels were also observed in mouse intestinal tissue and Drosophila follicular cells expressing mutant Apc/APC when compared to wild-type tissue/cells. Collectively, the results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease. The tumor suppressive function of APC - the most frequently mutated gene in colorectal cancer - is mainly attributed to its role in beta-catenin/Wnt signaling. This study substantially expands the list of APC interaction partners and reveals that approximately half of the changes in the cellular proteome induced by loss of APC function are mediated by beta-catenin-independent mechanisms.
Yoon, W., Hwang, S. H., Lee, S. H. and Chung, J. (2019). Drosophila ADCK1 is critical for maintaining mitochondrial structures and functions in the muscle. PLoS Genet 15(5): e1008184. PubMed ID: 31125351
Summary:
The function of AarF domain-containing kinase 1 (ADCK1) has not been thoroughly revealed. This study identified that ADCK1 utilizes YME1-like 1 ATPase (YME1L1) to control optic atrophy 1 (OPA1) and inner membrane mitochondrial protein (IMMT) in regulating mitochondrial dynamics and cristae structure. It was firstly observed that a serious developmental impairment occurred in Drosophila ADCK1 (dADCK1) deletion mutant, resulting in premature death before adulthood. By using temperature sensitive ubiquitously expression driver tub-Gal80ts/tub-Gal4 or muscle-specific expression driver mhc-Gal4, severely defective locomotive activities and structural abnormality in the muscle were observed along with increased mitochondrial fusion in the dADCK1 knockdown flies. Moreover, decreased mitochondrial membrane potential, ATP production and survival rate along with increased ROS and apoptosis in the flies further demonstrated that the structural abnormalities of mitochondria induced by dADCK1 knockdown led to their functional abnormalities. Consistent with the ADCK1 loss-of-function data in Drosophila, ADCK1 over-expression induced mitochondrial fission and clustering in addition to destruction of the cristae structure in Drosophila and mammalian cells. Interestingly, knockdown of YME1L1 rescued the phenotypes of ADCK1 over-expression. Furthermore, genetic epistasis from fly genetics and mammalian cell biology experiments led to a discovery the interactions among IMMT, OPA1 and ADCK1. Collectively, these results established a mitochondrial signaling pathway composed of ADCK1, YME1L1, OPA1 and IMMT, which has essential roles in maintaining mitochondrial morphologies and functions in the muscle.
Perochon, J., Grandon, B., Roche, D., Wintz, C., Demay, Y., Mignotte, B., Szuplewski, S. and Gaumer, S. (2019). The endoplasmic reticulum unfolded protein response varies depending on the affected region of the tissue but independently from the source of stress. Cell Stress Chaperones. PubMed ID: 31144193
Summary:
Accumulation of unfolded proteins and calcium dyshomeostasis induces endoplasmic reticulum (ER) stress, which can be resolved by the unfolded protein response (UPR). Previous work has shown that activation of the PERK/ATF4 branch of the UPR, by overexpressing Presenilin in part of the vestigial domain of Drosophila wing imaginal discs, induces both a caspase-dependent apoptosis and a Slpr/JNK/Dilp8-dependent developmental delay that allows compensation of cell death in the tissue. Recently, dDad1 depletion in Drosophila in engrailed-expressing cells of wing imaginal discs was also reported to activate the PERK/ATF4 branch but induced Mekk1/JNK-dependent apoptosis. This study assessed whether the stressed cell location in the wing imaginal disc could explain these differences in response to chronic ER stress or whether the stress source could be responsible for the signaling discrepancy. To address this question, this study overexpressed a Rhodopsin-1 mutant prone to aggregate either in vestigial- or engrailed-expressing cells. Similar responses were obtained to the Presenilin overexpression in the vestigial domain and to the dDad1 depletion in the engrailed domain. Therefore, the consequences of a PERK/ATF4 branch activation depend on the position of the cell in the Drosophila wing imaginal disc, suggesting interactions of PERK signaling with developmental pathways involved in the determination or maintenance of wing domains.
Sharma, S. K., Ghosh, S., Geetha, A. R., Mandal, S. and Mandal, L. (2019).. Cell adhesion-mediated actomyosin assembly regulates the activity of Cubitus interruptus for hematopoietic progenitor maintenance in Drosophila. Genetics. PubMed ID: 31138608
Summary:
The actomyosin network is involved in crucial cellular processes including morphogenesis, cell adhesion, apoptosis, proliferation, differentiation and collective cell migration in Drosophila, C. elegans and mammals. This study demonstrates that Drosophila larval blood stem-like progenitors require actomyosin activity for their maintenance. Genetic loss of actomyosin network from the progenitors caused a decline in their number. Likewise, the progenitor population increased upon sustained actomyosin activation via phosphorylation by Rho-associated kinase. This study shows that actomyosin positively regulates larval blood progenitors by controlling the maintenance factor Cubitus interruptus (Ci). Overexpression of the maintenance signal via a constitutively activated construct (ci.HA) failed to sustain Ci-155 in the absence of actomyosin components like Zipper (zip) and Squash (sqh), thus favoring protein kinase A (PKA)-independent regulation of Ci activity. Furthermore, this study demonstrates that a change in cortical actomyosin assembly mediated by DE-cadherin modulates Ci activity, thereby determining progenitor status. Thus, loss of cell adhesion and downstream actomyosin activity results in desensitization of the progenitors to Hh signaling, leading to their differentiation. These data reveal how cell adhesion and actomyosin network cooperate to influence patterning, morphogenesis, and maintenance of the hematopoietic stem-like progenitor pool in the developing Drosophila hematopoietic organ.
Xie, Q., Wu, B., Li, J., Xu, C., Li, H., Luginbuhl, D. J., Wang, X., Ward, A. and Luo, L. (2019). Transsynaptic Fish-lips signaling prevents misconnections between nonsynaptic partner olfactory neurons. Proc Natl Acad Sci U S A. PubMed ID: 31341080
Summary:
Understanding of the mechanisms of neural circuit assembly is far from complete. Identification of wiring molecules with novel mechanisms of action will provide insights into how complex and heterogeneous neural circuits assemble during development. In the Drosophila olfactory system, 50 classes of olfactory receptor neurons (ORNs) make precise synaptic connections with 50 classes of partner projection neurons (PNs). In this study, an RNA interference screen for cell surface molecules was performed and the leucine-rich repeat-containing transmembrane protein known as Fish-lips (Fili) was identified as a novel wiring molecule in the assembly of the Drosophila olfactory circuit. Fili contributes to the precise axon and dendrite targeting of a small subset of ORN and PN classes, respectively. Cell-type-specific expression and genetic analyses suggest that Fili sends a transsynaptic repulsive signal to neurites of nonpartner classes that prevents their targeting to inappropriate glomeruli in the antennal lobe.
Niraula, P. and Kim, M. S. (2019). N-Acetylcysteine extends lifespan of Drosophila via modulating ROS scavenger gene expression. Biogerontology 20(4): 533-543. PubMed ID: 31115735
Summary:
N-Acetylcysteine (NAc) has been shown to play a diversity of favorable health-related roles (e.g., antioxidant, paracetamol antidote, mucolytics, neuroprotective agent). This study evaluated the health-promoting properties of NAc, particularly its ability to modulate organismal longevity. It is noted that 1 mg/ml NAc prolonged the lifespan of Drosophila. Furthermore, it was observed that NAc increased the capability of these flies to resist environmental stresses measured by starvation and paraquat stress assays. In an effort to reveal cellular mechanisms behind this interesting phenomenon, qPCR was performed, uncovering that transcript levels of catalase and phospholipid hydroperoxide glutathione peroxidase-key enzymes to fend off reactive oxygen species (ROS) assaults, were up-regulated. Correspondingly, enzyme activities of catalase and glutathione peroxidase were increased as well. Combined, it is hoped that this research helps broaden the spectrum of clinical application for NAc so that one may eventually determine if NAc is a potentially useful anti-aging agent by encouraging others to scrutinize the hidden health benefits of NAc.

Thursday, August 1st - Synapse and Vesicles

Shi, Q., Lin, Y. Q., Saliba, A., Xie, J., Neely, G. G. and Banerjee, S. (2019). Tubulin polymerization promoting protein, Ringmaker, and MAP1B Homolog Futsch coordinate microtubule organization and synaptic growth. Front Cell Neurosci 13: 192. PubMed ID: 31156389
Summary:
Drosophila Ringmaker (Ringer) is homologous to the human Tubulin Polymerization Promoting Proteins (TPPPs) that are implicated in the stabilization and bundling of microtubules (MTs) that are particularly important for neurons and are also implicated in synaptic organization and plasticity. No in vivo functional data exist that have addressed the role of TPPP in synapse organization in any system. This study presents the phenotypic and functional characterization of ringer mutants during Drosophila larval neuromuscular junction (NMJ) synaptic development. ringer mutants show reduced synaptic growth and transmission and display phenotypic similarities and genetic interactions with the Drosophila homolog of vertebrate Microtubule Associated Protein (MAP)1B, futsch. Immunohistochemical and biochemical analyses show that individual and combined loss of Ringer and Futsch cause a significant reduction in MT loops at the NMJs and reduced acetylated-tubulin levels. Presynaptic over-expression of Ringer and Futsch causes elevated levels of acetylated-tubulin and significant increase in NMJ MT loops. These results indicate that Ringer and Futsch regulate synaptic MT organization in addition to synaptic growth. Together these findings may inform studies on the close mammalian homolog, TPPP, and provide insights into the role of MTs and associated proteins in synapse growth and organization.
Sunderhaus, E. R., Law, A. D. and Kretzschmar, D. (2019). ER responses play a key role in Swiss-Cheese/Neuropathy Target Esterase-associated neurodegeneration. Neurobiol Dis: 104520. PubMed ID: 31233884
Summary:
Swiss Cheese (SWS) is the Drosophila orthologue of Neuropathy Target Esterase (NTE), a phospholipase that when mutated has been shown to cause a spectrum of disorders in humans. Loss of SWS in Drosophila also causes locomotion deficits, age-dependent neurodegeneration, and an increase in lysophosphatidylcholine (LPC) and phosphatidylcholine (PC). SWS is localized to the Endoplasmic Reticulum (ER), and recently, it has been shown that perturbing the membrane lipid composition of the ER can lead to the activation of ER stress response through the inhibition of the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA). sws flies showed an activated ER stress response as determined by elevated levels of the chaperone GRP78 and by increased splicing of XBP, an ER transcription factor that activates transcriptional ER stress responses. To address whether ER stress plays a role in the degenerative and behavioral phenotypes detected in sws1, this study overexpressed XBP1 or treated the flies with tauroursodeoxycholic acid (TUDCA), a chemical known to attenuate ER stress-mediated cell death. Both manipulations suppressed the locomotor deficits and neurodegeneration of sws1. In addition, sws1 flies showed reduced SERCA levels and expressing additional SERCA also suppressed the sws1-related phenotypes. This suggests that the disruption in lipid compositions and its effect on SERCA are inducing ER stress, aimed to ameliorate the deleterious effects of sws1. This includes the effects on lipid composition because XBP1 and SERCA expression also reduced the LPC levels in sws1. Promoting cytoprotective ER stress pathways may therefore provide a therapeutic approach to alleviate the neurodegeneration and motor symptoms seen in NTE-associated disorders.
Mao, Y., Tu, R., Huang, Y., Mao, D., Yang, Z., Lau, P. K., Wang, J., Ni, J., Guo, Y. and Xie, T. (2019). The exocyst functions in niche cells to promote germline stem cell differentiation by directly controlling EGFR membrane trafficking. Development. PubMed ID: 31142545
Summary:
The niche controls stem cell self-renewal and differentiation in animal tissues. Although the exocyst is known to be important for protein membrane trafficking and secretion, its role in stem cells and niches has never been reported. This study shows that the exocyst functions in the niche to promote germline stem cell (GSC) progeny differentiation in the Drosophila ovary by directly regulating EGFR membrane trafficking and signaling. Inactivating exocyst components in inner germarial sheath cells, which form the differentiation niche, causes a severe GSC differentiation defect. The exocyst is required for maintaining niche cells and preventing BMP signaling in GSC progeny by promoting EGFR membrane targeting and signaling through direct association with EGFR. Finally, it is also required for EGFR membrane targeting, recycling and signaling in human cells. Therefore, this study has revealed a novel function of the exocyst in niche cells to promote stem cell progeny differentiation by directly controlling EGFR membrane trafficking and signaling in vivo and has also provided important insight into how the niche controls stem cell progeny differentiation at the molecular level.
Shaw, H. S., Cameron, S. A., Chang, W. T. and Rao, Y. (2019). The conserved IgSF9 protein Borderless regulates axonal transport of presynaptic components and color vision in Drosophila. J Neurosci. PubMed ID: 31235647
Summary:
Normal brain function requires proper targeting of synaptic-vesicle (SV) and active-zone (AZ) components for presynaptic assembly and function. Whether and how synaptogenic signals (e.g. adhesion) at axo-dendritic contact sites promote axonal transport of presynaptic components for synapse formation, however, remain unclear. This study shows that Borderless (Bdl), a member of the conserved IgSF9-family trans-synaptic cell adhesion molecules, plays a novel and specific role in regulating axonal transport of SV components. Loss of bdl disrupts axonal transport of SV components in photoreceptor R8 axons, but does not affect the transport of mitochondria. Genetic mosaic analysis, transgene rescue and cell-type-specific knockdown indicate that Bdl is required both pre- and postsynaptically for delivering SV components in R8 axons. Consistent with a role for Bdl in R8 axons, loss of bdl causes a failure of R8-dependent phototaxis response to green light. bdl interacts genetically with imac encoding for a member of the UNC-104/Imac/KIF1A-family motor proteins, and is required for proper localization of Imac in R8 presynaptic terminals. These results support a model in which Bdl mediates specific axo-dendritic interactions in a homophilic manner, which up-regulates the Imac motor in promoting axonal transport of SV components for R8 presynaptic assembly and function.
Shimozono, M., Osaka, J., Kato, Y., Araki, T., Kawamura, H., Takechi, H., Hakeda-Suzuki, S. and Suzuki, T. (2019). Cell surface molecule, Klingon, mediates the refinement of synaptic specificity in the Drosophila visual system. Genes Cells. PubMed ID: 31124270
Summary:
In the Drosophila brain, neurons form genetically specified synaptic connections with defined neuronal targets. It is proposed that each central nervous system neuron expresses specific cell surface proteins, which act as identification tags. Through an RNAi screen of cell surface molecules in the Drosophila visual system, this study found that the cell adhesion molecule Klingon (Klg) plays an important role in repressing the ectopic formation of extended axons, preventing the formation of excessive synapses. Cell-specific manipulation of klg showed that Klg is required in both photoreceptors and the glia, suggesting that the balanced homophilic interaction between photoreceptor axons and the glia is required for normal synapse formation. Previous studies suggested that Klg binds to cDIP and genetic analyses indicate that cDIP is required in glia for ectopic synaptic repression. These data suggest that Klg play a critical role together with cDIP in refining synaptic specificity and preventing unnecessary connections in the brain.
Lorincz, P., Kenez, L. A., Toth, S., Kiss, V., Varga, A., Csizmadia, T., Simon-Vecsei, Z. and Juhasz, G. (2019). Vps8 overexpression inhibits HOPS-dependent trafficking routes by outcompeting Vps41/Lt. Elife 8. PubMed ID: 31194677
Summary:
Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding CORVET that was suggested to transform into Rab7-binding HOPS. Previous work has identified miniCORVET, containing Drosophila Vps8 and three shared core proteins, which are required for endosome maturation upstream of HOPS in highly endocytic cells. This study shows that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Light and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation.
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