Small molecule modulation of the Drosophila Slo channel elucidated by cryo-EM.

Slowpoke (Slo) potassium channels display extraordinarily high conductance, are synergistically activated by a positive transmembrane potential and high intracellular Ca2+ concentrations and are important targets for insecticides and antiparasitic drugs. However, it is unknown how these compounds modulate ion translocation and whether there are insect-specific binding pockets. Here, we report structures of Drosophila Slo in the Ca2+-bound and Ca2+-free form and in complex with the fungal neurotoxin verruculogen and the anthelmintic drug emodepside. Whereas the architecture and gating mechanism of Slo channels are conserved, potential insect-specific binding pockets exist. Verruculogen inhibits K+ transport by blocking the Ca2+-induced activation signal and precludes K+ from entering the selectivity filter. Emodepside decreases the conductance by suboptimal K+ coordination and uncouples ion gating from Ca2+ and voltage sensing. Our results expand the mechanistic understanding of Slo regulation and lay the foundation for the rational design of regulators of Slo and other voltage-gated ion channels.

The Cellular Innate Immune Response of the Invasive Pest Insect Drosophila suzukii against Pseudomonas entomophila Involves the Release of Extracellular Traps.

Drosophila suzukii is a neobiotic invasive pest that causes extensive damage to fruit crops worldwide. The biological control of this species has been unsuccessful thus far, in part because of its robust cellular innate immune system, including the activity of professional phagocytes known as hemocytes and plasmatocytes. The in vitro cultivation of primary hemocytes isolated from D. suzukii third-instar larvae is a valuable tool for the investigation of hemocyte-derived effector mechanisms against pathogens such as wasp parasitoid larvae, bacteria, fungi and viruses. Here, we describe the morphological characteristics of D. suzukii hemocytes and evaluate early innate immune responses, including extracellular traps released against the entomopathogen Pseudomonas entomophila and lipopolysaccharides. We show for the first time that D. suzukii plasmatocytes cast extracellular traps to combat P. entomophila, along with other cell-mediated reactions, such as phagocytosis and the formation of filopodia.

Drosophila Models Rediscovered with Super-Resolution Microscopy.

With the advent of super-resolution microscopy, we gained a powerful toolbox to bridge the gap between the cellular- and molecular-level analysis of living organisms. Although nanoscopy is broadly applicable, classical model organisms, such as fruit flies, worms and mice, remained the leading subjects because combining the strength of sophisticated genetics, biochemistry and electrophysiology with the unparalleled resolution provided by super-resolution imaging appears as one of the most efficient approaches to understanding the basic cell biological questions and the molecular complexity of life. Here, we summarize the major nanoscopic techniques and illustrate how these approaches were used in Drosophila model systems to revisit a series of well-known cell biological phenomena. These investigations clearly demonstrate that instead of simply achieving an improvement in image quality, nanoscopy goes far beyond with its immense potential to discover novel structural and mechanistic aspects. With the examples of synaptic active zones, centrosomes and sarcomeres, we will explain the instrumental role of super-resolution imaging pioneered in Drosophila in understanding fundamental subcellular constituents.

Mechanical Permeabilization as a New Method for Assessment of Mitochondrial Function in Insect Tissues.

Respirometry analysis is an effective technique to assess mitochondrial physiology. Insects are valuable biochemical models to understand metabolism and human diseases. Insect flight muscle and brain have been extensively used to explore mitochondrial function due to dissection feasibility and the low sample effort to allow oxygen consumption measurements. However, adequate plasma membrane permeabilization is required for substrates/modulators to reach mitochondria. Here, we describe a new method for study of mitochondrial physiology in insect tissues based on mechanical permeabilization as a fast and reliable method that do not require the use of detergents for chemical permeabilization of plasma membrane, while preserves mitochondrial integrity.

Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes.

BACKGROUND: ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans. METHODS: To expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, we performed exome sequencing for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants. RESULTS: We report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, we showed that Phe50Leu, Gly236Val, and Arg327Pro cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles. CONCLUSION: Our findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation.

The actin polymerization factor Diaphanous and the actin severing protein Flightless I collaborate to regulate sarcomere size.

The sarcomere is the basic contractile unit of muscle, composed of repeated sets of actin thin filaments and myosin thick filaments. During muscle development, sarcomeres grow in size to accommodate the growth and function of muscle fibers. Failure in regulating sarcomere size results in muscle dysfunction; yet, it is unclear how the size and uniformity of sarcomeres are controlled. Here we show that the formin Diaphanous is critical for the growth and maintenance of sarcomere size: Dia sets sarcomere length and width through regulation of the number and length of the actin thin filaments in the Drosophila flight muscle. To regulate thin filament length and sarcomere size, Dia interacts with the Gelsolin superfamily member Flightless I (FliI). We suggest that these actin regulators, by controlling actin dynamics and turnover, generate uniformly sized sarcomeres tuned for the muscle contractions required for flight.

Microtubules provide guidance cues for myofibril and sarcomere assembly and growth.

BACKGROUND: Muscle myofibrils and sarcomeres present exceptional examples of highly ordered cytoskeletal filament arrays, whose distinct spatial organization is an essential aspect of muscle cell functionality. We utilized ultra-structural analysis to investigate the assembly of myofibrils and sarcomeres within developing myotubes of the indirect flight musculature of Drosophila. RESULTS: A temporal sequence composed of three major processes was identified: subdivision of the unorganized cytoplasm of nascent, multi-nucleated myotubes into distinct organelle-rich and filament-rich domains; initial organization of the filament-rich domains into myofibrils harboring nascent sarcomeric units; and finally, maturation of the highly-ordered pattern of sarcomeric thick (myosin-based) and thin (microfilament-based) filament arrays in parallel to myofibril radial growth. Significantly, organized microtubule arrays were present throughout these stages and exhibited dynamic changes in their spatial patterns consistent with instructive roles. Genetic manipulations confirm these notions, and imply specific and critical guidance activities of the microtubule-based cytoskeleton, as well as structural interdependence between the myosin- and actin-based filament arrays. CONCLUSIONS: Our observations highlight a surprisingly significant, behind-the-scenes role for microtubules in establishment of myofibril and sarcomere spatial patterns and size, and provide a detailed account of the interplay between major cytoskeletal elements in generating these essential contractile myogenic units.

Structural and transcriptional evidence of mechanotransduction in the Drosophila suzukii ovipositor.

Drosophila suzukii is an invasive pest that prefers to lay eggs in ripening fruits, whereas most closely related Drosophila species exclusively use rotten fruit as oviposition site. This behaviour is allowed by an enlarged and serrated ovipositor that can pierce intact fruit skin, and by multiple contact sensory systems (mechanosensation and taste) that detect the optimal egg-laying substrates. Here, we tested the hypothesis that bristles present in the D. suzukii ovipositor tip contribute to these sensory modalities. Analysis of the bristle ultrastructure revealed that four different types of cuticular elements (conical pegs type 1 and 2, chaetic and trichoid sensilla) are present on the tip of each ovipositor plate. All of them have a poreless shaft and are innervated at their base by a single neuron that ends in a distal tubular body, thus resembling mechanosensitive structures. Fluorescent labelling in D. suzukii and D. melanogaster revealed that pegs located on the ventral side of the ovipositor tip are innervated by a single neuron in both species. RNA-sequencing profiled gene expression, notably sensory receptor genes of the terminalia of D. suzukii and of three other Drosophila species with changes in their ovipositor structure (from serrated to blunt ovipositor: Drosophila subpulchrella, Drosophila biarmipes and D. melanogaster). Our results revealed few species-specific transcripts and an overlapping expression of candidate mechanosensitive genes as well as the presence of some chemoreceptor transcripts. These experimental evidences suggest a mechanosensitive function for the D. suzukii ovipositor, which might be crucial across Drosophila species independently from ovipositor shape.

A perinuclear microtubule-organizing centre controls nuclear positioning and basement membrane secretion.

Non-centrosomal microtubule-organizing centres (ncMTOCs) have a variety of roles that are presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization and function. Here we report a perinuclear MTOC in Drosophila fat body cells that is anchored by the Nesprin homologue Msp300 at the cytoplasmic surface of the nucleus. Msp300 recruits the microtubule minus-end protein Patronin, a calmodulin-regulated spectrin-associated protein (CAMSAP) homologue, which functions redundantly with Ninein to further recruit the microtubule polymerase Msps-a member of the XMAP215 family-to assemble non-centrosomal microtubules and does so independently of the widespread microtubule nucleation factor γ-Tubulin. Functionally, the fat body ncMTOC and the radial microtubule arrays that it organizes are essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture that regulates microtubules, serving vital functions.

Analysis of a cellular structure observed in the compound eyes of Drosophila white; yata mutants and white mutants.

We previously identified the Drosophila yata mutant, which showed phenotypes including progressive vacuolization of the white-coloured compound eye, progressive shrinkage of the brain and a shortened lifespan. The yata gene was shown to be involved in controlling intracellular trafficking of the Amyloid precursor protein-like protein, which is an orthologue of Amyloid precursor protein, which is a causative molecule of Alzheimer's disease. In this study, we examined the phenotype of the compound eye of the yata mutant using electron microscopy and confocal microscopy. We found that abnormal cellular structures that seemed to originate from bleb-like structures and contained vesicles and organelles, such as multivesicular bodies and autophagosomes, were observed in aged white; yata mutants and aged white mutants. These structures were not observed in newly eclosed flies and the presence of the structures was suppressed in flies grown under constant dark conditions after eclosion. The structures were not observed in newly eclosed red-eyed yata mutants or wild-type flies, but were observed in very aged red-eyed wild-type flies. Thus, our data suggest that the observed structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies.

Inter-axonal recognition organizes Drosophila olfactory map formation.

Olfactory systems across the animal kingdom show astonishing similarities in their morphological and functional organization. In mouse and Drosophila, olfactory sensory neurons are characterized by the selective expression of a single odorant receptor (OR) type and by the OR class-specific connection in the olfactory brain center. Monospecific OR expression in mouse provides each sensory neuron with a unique recognition identity underlying class-specific axon sorting into synaptic glomeruli. Here we show that in Drosophila, although OR genes are not involved in sensory neuron connectivity, afferent sorting via OR class-specific recognition defines a central mechanism of odortopic map formation. Sensory neurons mutant for the Ig-domain receptor Dscam converge into ectopic glomeruli with single OR class identity independent of their target cells. Mosaic analysis showed that Dscam prevents premature recognition among sensory axons of the same OR class. Single Dscam isoform expression in projecting axons revealed the importance of Dscam diversity for spatially restricted glomerular convergence. These data support a model in which the precise temporal-spatial regulation of Dscam activity controls class-specific axon sorting thereby indicating convergent evolution of olfactory map formation via self-patterning of sensory neurons.

Syndapin constricts microvillar necks to form a united rhabdomere in Drosophila photoreceptors.

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, we 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.

Imaging neuropeptide release at synapses with a genetically engineered reporter.

Research on neuropeptide function has advanced rapidly, yet there is still no spatio-temporally resolved method to measure the release of neuropeptides in vivo. Here we introduce Neuropeptide Release Reporters (NPRRs): novel genetically-encoded sensors with high temporal resolution and genetic specificity. Using the Drosophila larval neuromuscular junction (NMJ) as a model, we provide evidence that NPRRs recapitulate the trafficking and packaging of native neuropeptides, and report stimulation-evoked neuropeptide release events as real-time changes in fluorescence intensity, with sub-second temporal resolution.

Rare variants in MYH15 modify amyotrophic lateral sclerosis risk.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive muscular atrophy and respiratory failure. The G4C2 repeat expansion in the C9orf72 gene is the most prevalent genetic risk for ALS. Mutation carriers (C9ALS) display variability in phenotypes such as age-at-onset and duration, suggesting the existence of additional genetic factors. Here we introduce a three-step gene discovery strategy to identify genetic factors modifying the risk of both C9ALS and sporadic ALS (sALS) using limited samples. We first identified 135 candidate genetic modifiers of C9ALS using whole-genome sequencing (WGS) of extreme C9ALS cases diagnosed ~30 years apart. We then performed an unbiased genetic screen using a Drosophila model of the G4C2 repeat expansion with the genes identified from WGS analysis. This genetic screen identified the novel genetic interaction between G4C2 repeat-associated toxicity and 18 genetic factors, suggesting their potential association with C9ALS risk. We went on to test if 14 out of the 18 genes, those which were not known to be risk factors for ALS previously, are also associated with ALS risk in sALS cases. Gene-based-statistical analyses of targeted resequencing and WGS were performed. These analyses together reveal that rare variants in MYH15 represent a likely genetic risk factor for ALS. Furthermore, we show that MYH15 could modulate the toxicity of dipeptides produced from expanded G4C2 repeat. Our study presented here demonstrates the power of combining WGS with fly genetics to facilitate the discovery of fundamental genetic components of complex traits with a limited number of samples.

Electron microscopic observation of photoreceptor cells in directly inserted anesthetized Drosophila into a high-pressure freezing unit.

The high-pressure freezing (HPF) technique is known to cryofix water-containing materials with little ice-crystal formation in deep depths compared with other freezing techniques. In this study, HPF for anesthetized living Drosophila was performed by placing them directly on the carrier of the HPF unit and exposing them to light. Frozen Drosophila were freeze substituted, and their compound eyes were examined by transmission electron microscopy. The ultrastructures of ommatidia composed of photoreceptor cells were well preserved. The location of the cytoplasmic organelles inside the photoreceptor cells was observed. In some photoreceptor cells in ommatidia of the light-exposed Drosphila, the cytoplasmic small granules were localized nearer the base of rhabdomeres, compared with those of the nonlight-exposed Drosophila. Thus, HPF with the direct insertion of living Drosophila under light exposure into the HPF machine enabled us to examine changes to functional structures of photoreceptor cells that occur within seconds.

Ultrastructural variation and adaptive evolution of the ovipositor in the endemic Hawaiian Drosophilidae.

Ecological diversification of the endemic Hawaiian Drosophilidae has been accompanied by striking divergence in egg morphology, and ovarian structure and function. To determine how these flies successfully oviposit in a variety of breeding substrates, we used Scanning Electron Microscopy to examine the ultrastructure of the ovipositor of a sample of 65 Drosophila species and five Scaptomyza species of this hyperdiverse monophyletic group. The Drosophila species analyzed included representatives of the fungus-breeding haleakalae group, the leaf-breeding antopocerus and modified tarsus groups, the modified mouthparts species group, the nudidrosophila, and the picture wing clade; the latter sample of 41 species from four species groups included stem- and bark-breeders, as well as tree sap flux-breeders. Ovipositor length was found to vary more than 12-fold among Hawaiian drosophilids, with the longest ovipositors observed in the bark-breeding species and the shortest among the Scaptomyza and fungus-breeders. More noteworthy is the striking variation in overall shape and proportions of the ovipositor, in the shape of the apical region, and in the pattern of sensory structures or ovisensilla. Ultrastructural observations of the pair of long subapical sensilla on the ventral side identify these, for the first time, as taste bristles. Ovipositor form correlates strongly with the oviposition substrate used by the species, being of a distinctive shape and size in each case. We infer that the observed morphological divergence in the ovipositor is adaptive and the product of natural selection for successful reproduction in alternate microhabitats. The array of ovipositor forms that have evolved among the Hawaiian Drosophila species represent a series of ecomorphs that along with other divergent traits of the female reproductive system, have contributed to the successful adaptive radiation of this remarkable fauna.

High-precision automated reconstruction of neurons with flood-filling networks.

Reconstruction of neural circuits from volume electron microscopy data requires the tracing of cells in their entirety, including all their neurites. Automated approaches have been developed for tracing, but their error rates are too high to generate reliable circuit diagrams without extensive human proofreading. We present flood-filling networks, a method for automated segmentation that, similar to most previous efforts, uses convolutional neural networks, but contains in addition a recurrent pathway that allows the iterative optimization and extension of individual neuronal processes. We used flood-filling networks to trace neurons in a dataset obtained by serial block-face electron microscopy of a zebra finch brain. Using our method, we achieved a mean error-free neurite path length of 1.1 mm, and we observed only four mergers in a test set with a path length of 97 mm. The performance of flood-filling networks was an order of magnitude better than that of previous approaches applied to this dataset, although with substantially increased computational costs.

Comparative Antennal and Behavioral Responses of Summer and Winter Morph Drosophila suzukii (Diptera: Drosophilidae) to Ecologically Relevant Volatiles.

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a devastating global pest of berry crops and cherries. Little is understood about its biology during the winter in northern temperate regions, including potential resources that it may utilize during this period. In this study, olfactory and behavioral responses of female D. suzukii to six volatiles (methionol, acetic acid, linalool, bornyl acetate, isoamyl acetate, and geosmin) were evaluated separately for electroantennogram (EAG) and behavioral assays between summer and winter morphs. Results of EAG indicated that isoamyl acetate, acetic acid, and geosmin elicited significantly higher olfactory responses from the antennae of female summer morph D. suzukii compared with those of female winter morph D. suzukii. Winter morph D. suzukii showed reduced antennal response to the volatiles overall. Geosmin and bornyl acetate elicited significantly different behavioral responses from the two morphs in no-choice laboratory behavioral assays. T-maze behavioral assays with geosmin further revealed that summer morphs had a significant aversion, while winter morphs showed no significant aversion to geosmin. Overall, we demonstrate that responses of the two seasonally induced morphs to environmental stimuli are different, and future studies are justified to further understand how these physiological and behavioral differences may contribute to improved pest management of D. suzukii.

Epithelial cell shape change of Drosophila as a biomonitoring model for the dose assessment of environmental radiation.

Inevitable exposure to ionizing radiation from natural and human-made sources has been increasing over time. After nuclear disasters, such as the Fukushima accident, the public concerns on health risk of radiation exposure because of radioactive contamination of the environment have increased. However, it is very difficult to assess the biological effects of exposure caused by environmental radiation. A reliable and rapid bioassay to monitor the physiological effects of radiation exposure is therefore needed. Here, we quantitatively analyzed the changes in cell shape in Drosophila epidermis after irradiation as a model for biomonitoring of radiation. Interestingly, the number of irregularly shaped epithelial cells was increased by irradiation in a dose-dependent manner. A dose-response curve constructed with the obtained data suggests that the measurement of the number of irregular shaped cell in the epidermis is useful for the assessment of radiation dose. In addition, a comparison of the variation in the different samples and the data scored by different observers showed that our evaluation for cellular morphology was highly reliable and accurate and would, therefore, have immense practical application. Overall, our study suggests that detection of morphological changes in the epithelial cells is one of the efficient ways to quantify the levels of exposure to radioactive radiation from the environment.

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila.

In a previous paper, we showed a new giant lens called the Mesolens and presented performance data and images from whole fixed and intact fluorescently-stained 12.5-day old mouse embryos. Here, we show that using the Mesolens we can image an entire Drosophila larva or adult fly in confocal epifluorescence and show subcellular detail in all tissues. By taking several hundreds of optical sections through the entire volume of the specimen, we show cells and nuclear details within the gut, brain, salivary glands and reproductive system that normally require dissection for study. Organs are imaged in situ in correct 3D arrangement. Imaginal discs are imaged in mature larvae and it proved possible to image pachytene chromosomes in cells within ovarian follicles in intact female flies. Methods for fixing, staining and clearing are given.