FlyBase: updates to the Drosophila genes and genomes database.

FlyBase (flybase.org) is a model organism database and knowledge base about Drosophila melanogaster, commonly known as the fruit fly. Researchers from around the world rely on the genetic, genomic, and functional information available in FlyBase, as well as its tools to view and interrogate these data. In this article, we describe the latest developments and updates to FlyBase. These include the introduction of single-cell RNA sequencing data, improved content and display of functional information, updated orthology pipelines, new chemical reports, and enhancements to our outreach resources.

Direct real-time measurements of superoxide release from skeletal muscles in rat limbs and human blood platelets using an implantable Cytochrome C microbiosensor.

Oxidative stress and excessive accumulation of the superoxide (O2.-) anion are at the genesis of many pathological conditions and the onset of several diseases. The real time monitoring of (O2.-) release is important to assess the extent of oxidative stress in these conditions. Herein, we present the design, fabrication and characterization of a robust (O2.-) biosensor using a simple and straightforward procedure involving deposition of a uniform layer of L-Cysteine on a gold wire electrode to which Cytochrome C (Cyt c) was conjugated. The immobilized layers, studied using conductive Atomic Force Microscopy (c-AFM) revealed a stable and uniformly distributed redox protein on the gold surface, visualized as conductivity and surface topographical plots. The biosensor enabled detection of (O2.-) at an applied potential of 0.15 V with a sensitivity of 42.4 nA/µM and a detection limit of 2.4 nM. Utility of the biosensor was demonstrated in measurements of real time (O2.-) release in activated human blood platelets and skeletal rat limb muscles following ischemia reperfusion injury (IRI), confirming the biosensor's stability and robustness for measurements in complex biological systems. The results demonstrate the ability of these biosensors to monitor real time release of (O2.-) and estimate the extent of oxidative injury in models that could easily be translated to human pathologies.

Molecular stiffness cues of an interpenetrating network hydrogel for cell adhesion.

Understanding cells' response to the macroscopic and nanoscale properties of biomaterials requires studies in model systems with the possibility to tailor their mechanical properties and different length scales. Here, we describe an interpenetrating network (IPN) design based on a stiff PEGDA host network interlaced within a soft 4-arm PEG-Maleimide/thiol (guest) network. We quantify the nano- and bulk mechanical behavior of the IPN and the single network hydrogels by single-molecule force spectroscopy and rheological measurements. The IPN presents different mechanical cues at the molecular scale, depending on which network is linked to the probe, but the same mechanical properties at the macroscopic length scale as the individual host network. Cells attached to the interpenetrating (guest) network of the IPN or to the single network (SN) PEGDA hydrogel modified with RGD adhesive ligands showed comparable attachment and spreading areas, but cells attached to the guest network of the IPN, with lower molecular stiffness, showed a larger number and size of focal adhesion complexes and a higher concentration of the Hippo pathway effector Yes-associated protein (YAP) than cells linked to the PEGDA single network. The observations indicate that cell adhesion to the IPN hydrogel through the network with lower molecular stiffness proceeds effectively as if a higher ligand density is offered. We claim that IPNs can be used to decipher how changes in ECM design and connectivity at the local scale affect the fate of cells cultured on biomaterials.

Optoregulated force application to cellular receptors using molecular motors.

Progress in our understanding of mechanotransduction events requires noninvasive methods for the manipulation of forces at molecular scale in physiological environments. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the entangled polymer chains, which will in turn effectively "pull" on engaged cell membrane receptors (e.g., integrins, T cell receptors) within the illuminated area. Applied forces have physiologically-relevant magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in force-dependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate a functionality which at the moment cannot be achieved by other technologies for force application.

Sleep Induction by Mechanosensory Stimulation in Drosophila.

People tend to fall asleep when gently rocked or vibrated. Experimental studies have shown that rocking promotes sleep in humans and mice. However, the mechanisms underlying the phenomenon are not well understood. A habituation model proposes that habituation, a form of non-associative learning, mediates sleep induction by monotonous stimulation. Here, we show that gentle vibration promotes sleep in Drosophila in part through habituation. Vibration-induced sleep (VIS) leads to increased homeostatic sleep credit and reduced arousability, and can be suppressed by heightened arousal or reduced GABA signaling. Multiple mechanosensory organs mediate VIS, and the magnitude of VIS depends on vibration frequency and genetic background. Sleep induction improves over successive blocks of vibration. Furthermore, training with continuous vibration does not generalize to intermittent vibration, demonstrating stimulus specificity, a characteristic of habituation. Our findings suggest that habituation plays a significant role in sleep induction by vibration.

In Situ Imaging of Candida albicans Hyphal Growth via Atomic Force Microscopy.

Candida albicans is an opportunistic fungal pathogen of humans known for its ability to cause a wide range of infections. One major virulence factor of C. albicans is its ability to form hyphae that can invade host tissues and cause disseminated infections. Here, we introduce a method based on atomic force microscopy to investigate C. albicans hyphae in situ on silicone elastomer substrates, focusing on the effects of temperature and antifungal drugs. Hyphal growth rates differ significantly for measurements performed at different physiologically relevant temperatures. Furthermore, it is found that fluconazole is more effective than caspofungin in suppressing hyphal growth. We also investigate the effects of antifungal drugs on the mechanical properties of hyphal cells. An increase in Young's modulus and a decrease in adhesion force are observed in hyphal cells subjected to caspofungin treatment. Young's moduli are not significantly affected following treatment with fluconazole; the adhesion force, however, increases. Overall, our results provide a direct means of observing the effects of environmental factors and antifungal drugs on C. albicans hyphal growth and mechanics with high spatial resolution.IMPORTANCECandida albicans is one of the most common pathogens of humans. One important virulence factor of C. albicans is its ability to form elongated hyphae that can invade host tissues and cause disseminated infections. Here, we show the effect of different physiologically relevant temperatures and common antifungal drugs on the growth and mechanical properties of C. albicans hyphae using atomic force microscopy. We demonstrate that minor temperature fluctuations within the normal range can have profound effects on hyphal cell growth and that different antifungal drugs impact hyphal cell stiffness and adhesion in different ways.

Generation and characterization of inner photoreceptor-specific enhancer-trap lines using a novel piggyBac-Gal4 element in Drosophila.

The Drosophila inner photoreceptors R7 and R8 are responsible for color vision and their differentiation starts at the third instar larval stage. Only a handful of genes with R7 or R8-cell-specific expression are known. We performed an enhancer-trap screen using a novel piggyBac transposable element, pBGay, carrying a Gal4 sequence under the control of the P promoter to identify novel genes expressed specifically in R7 or R8 cells. From this screen, three lines were analyzed in detail: piggyBacAC109 and piggyBacAC783 are expressed in R8 cells and piggyBacAC887 is expressed in R7 cells at the third instar larval stage and pupal stages. Molecular analysis showed that the piggyBac elements were inserted into the first intron of CG14160 and CG7985 genes and the second intron of unzipped. We show the expression pattern in the developing eye imaginal disc, pupal retina as well as the adult retina. The photoreceptor-specific expression of these genes is reported for the first time and we propose that these lines are useful tools for studying the development of the visual system.

The mechanics of single cross-links which mediate cell attachment at a hydrogel surface.

The response of cultured cells to the mechanical properties of hydrogel substrates depends ultimately on the response of single crosslinks to external forces exerted at cell attachment points. We prepared hydrogels by co-polymerization of poly(ethylene glycol diacrylate) (PEGDA) and carboxy poly(ethylene glycol) acrylate (ACPEG-COOH) and confirmed fibroblast spreading on the hydrogel after the ACPEG linker was functionalized with the RGD cell adhesive motif. We performed specific force spectroscopy experiments on the same ACPEG linkers in order to probe the mechanics of single cross-links which mediate the cell attachment and spreading. Measurements were performed with tips of an atomic force microscope (AFM) functionalized with streptavidin and ACPEG linkers functionalized with biotin. We compared hydrogels of varying elastic modulus between 4 and 41 kPa which exhibited significant differences in cell spreading. An effective spring constant for the displacement of single cross-links at the hydrogel surface was derived from the distributions of rupture force and molecular stiffness. A factor of ten in the elastic modulus E of the hydrogel corresponded to a factor of five in the effective spring constant k of single crosslinks, indicating a transition in scaling with the mesh size ξ from the macroscopic E∝ξ-3 to the molecular k∝ξ-2. The quantification of stiffness and deformation at the molecular length scale contributes to the discussion of mechanisms in force-regulated phenomena in cell biology.

Blimp-1 Mediates Tracheal Lumen Maturation in Drosophila melanogaster.

The specification of tissue identity during embryonic development requires precise spatio-temporal coordination of gene expression. Many transcription factors required for the development of organs have been identified and their expression patterns are known; however, the mechanisms through which they coordinate gene expression in time remain poorly understood. Here, we show that hormone-induced transcription factor Blimp-1 participates in the temporal coordination of tubulogenesis in Drosophila melanogaster by regulating the expression of many genes involved in tube maturation. In particular, we demonstrate that Blimp-1 regulates the expression of genes involved in chitin deposition and F-actin organization. We show that Blimp-1 is involved in the temporal control of lumen maturation by regulating the beginning of chitin deposition. We also report that Blimp-1 represses a variety of genes involved in tracheal maturation. Finally, we reveal that the kinase Btk29A serves as a link between Blimp-1 transcriptional repression and apical extracellular matrix organization.

Coating agent-induced mechanical behavior of 3D self-assembled nanocrystals.

The Young's modulus of three-dimensional self-assembled Ag nanocrystals, as so-called supracrystals, is correlated with the type of coating agent as well as the nanocrystal morphology. The Young's moduli of supracrystals of icosahedral Ag nanocrystals are measured in the range of tens to hundreds of megapascals revealing an extremely soft mechanical behavior. The alkylamine molecules used as coating agents weakly interact with the Ag nanocrystal surface favoring translational and orientational ordering of atomic lattice planes of nanocrystals. Under such experimental conditions, both the average distance between nanocrystals and the increase of the nanocrystal diameter control the measured Young's modulus: it increases with decreasing interparticle distance and increasing nanocrystal diameter. When dodecylamine (C12NH2) molecules are replaced by dodecanethiol (C12SH), the orientational ordering between nanocrystals, produced from the same batch as C12NH2, disappears by inducing a drop in the Young's modulus. This is attributed to the formation of a "skin" at the nanocrystal surface causing a transition from shaped to spherical nanocrystals. Finally, by comparing with various studies performed in our group with Co and Au nanocrystals, we explain the formation of such extremely soft materials with Ag nanocrystals by both the strength of the binding between nanocrystals and coating agents and the ligand-ligand interactions.

Identification of octopaminergic neurons that modulate sleep suppression by male sex drive.

Molecular and circuit mechanisms for balancing competing drives are not well understood. While circadian and homeostatic mechanisms generally ensure sufficient sleep at night, other pressing needs can overcome sleep drive. Here, we demonstrate that the balance between sleep and sex drives determines whether male flies sleep or court, and identify a subset of octopaminergic neurons (MS1) that regulate sleep specifically in males. When MS1 neurons are activated, isolated males sleep less, and when MS1 neurons are silenced, the normal male sleep suppression in female presence is attenuated and mating behavior is impaired. MS1 neurons do not express the sexually dimorphic FRUITLESS (FRU) transcription factor, but form male-specific contacts with FRU-expressing neurons; calcium imaging experiments reveal bidirectional functional connectivity between MS1 and FRU neurons. We propose octopaminergic MS1 neurons interact with the FRU network to mediate sleep suppression by male sex drive.

Regulation of sleep plasticity by a thermo-sensitive circuit in Drosophila.

Sleep is a highly conserved and essential behaviour in many species, including the fruit fly Drosophila melanogaster. In the wild, sensory signalling encoding environmental information must be integrated with sleep drive to ensure that sleep is not initiated during detrimental conditions. However, the molecular and circuit mechanisms by which sleep timing is modulated by the environment are unclear. Here we introduce a novel behavioural paradigm to study this issue. We show that in male fruit flies, onset of the daytime siesta is delayed by ambient temperatures above 29 °C. We term this effect Prolonged Morning Wakefulness (PMW). We show that signalling through the TrpA1 thermo-sensor is required for PMW, and that TrpA1 specifically impacts siesta onset, but not night sleep onset, in response to elevated temperatures. We identify two critical TrpA1-expressing circuits and show that both contact DN1p clock neurons, the output of which is also required for PMW. Finally, we identify the circadian blue-light photoreceptor CRYPTOCHROME as a molecular regulator of PMW, and propose a model in which the Drosophila nervous system integrates information encoding temperature, light, and time to dynamically control when sleep is initiated. Our results provide a platform to investigate how environmental inputs co-ordinately regulate sleep plasticity.

A feedback mechanism converts individual cell features into a supracellular ECM structure in Drosophila trachea.

The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. Here, we used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. We show that there is an active feedback mechanism between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, we reveal that cell-cell junctions are key players in this aECM patterning and organisation and that individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, we propose that Src42A phosphorylation levels provide a link for the ECM environment to ensure proper cytoskeletal organisation.

Drosophila chitinous aECM and its cellular interactions during tracheal development.

The morphology of organs, and hence their proper physiology, relies to a considerable extent on the extracellular matrix (ECM) secreted by their cells. The ECM is a structure contributed to and commonly shared by many cells in an organism that plays an active role in morphogenesis. Increasing evidence indicates that the ECM not only provides a passive contribution to organ shape but also impinges on cell behaviour and genetic programmes. The ECM is emerging as a direct modulator of many aspects of cell biology, rather than as a mere physical network that supports cells. Here, we review how the apical chitinous ECM is generated in Drosophila trachea and how cells participate in the formation of this supracellular structure. We discuss recent findings on the molecular and cellular events that lead to the formation of this apical ECM (aECM) and how it is influenced and affects tracheal cell biology.