Arg206 of SNAP-25 is essential for neuroexocytosis at the Drosophila melanogaster neuromuscular junction

An analysis of SNAP-25 isoform sequences indicates that there is a highly conserved arginine residue (198 in vertebrates, 206 in the genus Drosophila ) within the C-terminal region, which is cleaved by botulinum neurotoxin A, with consequent blockade of neuroexocytosis. The possibility that it may play an important role in the function of the neuroexocytosis machinery was tested at neuromuscular junctions of Drosophila melanogaster larvae expressing SNAP-25 in which Arg206 had been replaced by alanine. Electrophysiological recordings of spontaneous and evoked neurotransmitter release under different conditions as well as testing for the assembly of the SNARE complex indicate that this residue, which is at the P 1 ′ position of the botulinum neurotoxin A cleavage site, plays an essential role in neuroexocytosis. Computer graphic modelling suggests that this arginine residue mediates protein–protein contacts within a rosette of SNARE complexes that assembles to mediate the fusion of synaptic vesicles with the presynaptic plasma membrane

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

SNARE complexes associate in a rosette-like structure at the Drosophila neuromuscular junction

Secretion in all eukaryotic cells involves a family of proteins known as SNAREs (soluble Nethylmaleimide‐sensitive factor attachment protein receptors). In the nervous system, these proteins are the main molecular constituents of the synaptic vesicle fusion machinery. The release of molecules contained inside exocytic granules and synaptic vesicles is mediated by the assembly of a four helix bundle complex: the SNARE complex. It is formed by the coilcoiling of three proteins: the v‐SNARE VAMP/Synaptobrevin and the t‐SNAREs, Syntaxin and SNAP‐25 (synaptosome‐associated protein of 25 kDa). A cooperative interaction between SNARE complexes has been suggested to be necessary for the fusion of a synaptic vesicle with the presynaptic membrane to occur. Previous experiments, made with botulinum neurotoxins type A and E on mouse neuromuscular junction, led to predict a central role of the SNAP‐25 C‐terminus in protein‐protein contacts between SNARE complexes. Previously conducted sequence comparisons and molecular dynamics simulations, led to the finding that this central role in such interactions could be played by a key Arginine residue at the SNAP‐25 C‐terminus. In particular, the SNAP‐25 Arg206 residue possibly forms an ionic bond with a negative charge amino acid of Syntaxin from an adjacent SNARE complex. A computational model, based on such interactions, proposes that the formation of the vesicle/presynaptic membrane fusion pore is catalyzed by a SNARE supercomplex consisting in a "ten petal" rosette. This prediction was tested in Drosophila melanogaster by altering the putative key amino acids, thought to be involved in rosette formation, by site‐directed mutagenesis. Two transgenic lines (harboring the mutation bearing constructs in a SNAP‐25 wild‐type background) were thus generated: UAS‐SNAP‐25WT (control) and UAS‐SNAP‐ 25R206A. Using the UAS/GAL4 system the expression of these constructs was directed specifically in the nervous system. The mRNA expression profiles, the locomotor behaviour and the morphology of the neuromuscular junction of these mutants were characterized in third instar larvae. Electrophysiological recordings performed at the larval neuromuscular junction showed that the ElavALl4/UAS‐SNAP‐25R206A mutants present a decrease in the probability of vesicle fusion as compared to ElavGAL4/UAS‐SNAP‐25WT, our control, in both spontaneous and evoked exocytosis. These findings highlight the key role of Arg206 and support the model entailing the involvement of a rosette of SNARE complexes in the process of neuroexocytosis.L’esocitosi regolata è un processo essenziale in tutte le cellule eucariotiche. A livello della sinapsi, il rilascio di molecule contenute all’interno delle vescicole è mediato dalla formazione del complesso SNARE, costituito dall’interazione di tre proteine: sintassina, SNAP‐25 e VAMP/sinaptobrevina. Numerose evidenze sperimentali suggeriscono che la cooperazione tra complessi SNARE adiacenti sia necessaria affinché una vescicola sinaptica si fonda con la membrane plasmatica. Da un’analisi della sequenza amminoacidica dell’isoforma neuronale di questa proteina è emerso che, nella porzione C‐terminale, un residuo aminoacidico è altamente conservato anche se non è situate nella regione di interazione tra SNAP‐25 e le altre componenti molecolari del complesso. In mammifero, tale residuo è l’arginina in posizione 198. Esperimenti condotti incubando la giunzione neuromuscolare di topo in presenza della tossina botulinica di tipo A hanno evidenziato il ruolo chiave della porzione C‐terminale della proteina SNAP‐25 nel processo di fusion vescicolare e, in particolare, dell’Arg198, poiché la tossina taglia esattamente la proteina a monte di questo residuo, determinando il blocco dell’esocitosi. Inoltre, simulazione di modeling effettuate al computer hanno suggerito che tale arginine interagisce con un amminoacido carico negativamente posizionato sulla sintassina del complesso SNARE adiacente, formando di un legame ionico. Questa ipotesi è stata verificata in vivo usando Drosophila melanogaster come organismo modello. Poiché nel moscerino della frutta la sequenza di SNAP‐25 è più lunga di quella in mammifero, l’arginina conservata risulta essere in posizione 206. Tale Arg206 è stata sostituita con un’alanina mediante mutagenesi sitospecifica. Quindi, sono state generate due linee transgeniche in cui i costrutti pUAST‐SNAP‐25WT e pUAST‐SNAP‐25R206A sono stati inseriti in background SNAP‐25 wild‐type. Sfruttando il sistema GAL4/UAS, i transgeni sono stati espressi nel sistema nervoso utilizzando il driver pan‐neuronale ElavGAL4. In entrambe le linee ElavGAL4/UAS‐SNAP‐25WT (controllo) (controllo) e ELAV‐GAL4/UAS‐SNAP‐25R206A (mutante) sono stati quantificati i profili di espressione dell’mRNA dei trascritti mutante e wild‐type; inoltre, è stata caratterizzata la giunzione neuromuscolare a livello larvale, musurandone la morfologia e la morfometria. L’analisi elettrofisiologica del rilascio spontaneo ed evocato ha mostrato una significativa riduzione della probabilità di fusione tra vescicola e membrana plasmatica. Questo risultato conferma il ruolo chiave ricoperto dall’Arg206 durante il processo di esocitosi a livello del terminale sinaptico e supporta l’ipotesi secondo cui i complessi SNARE cooperano tra loro, formando un supercomplesso necessario alla formazione del poro di fusione

"The found in neurons" mutation reduces courtship levels and causes B-lobe fusion in adult Drosophila mushroom bodies with no impact on courtship memory.

International audienc

A Multilayered Imaging and Microfluidics Approach for Evaluating the Effect of Fibrinolysis in <i>Staphylococcus aureus</i> Biofilm Formation

The recognition of microbe and extracellular matrix (ECM) is a recurring theme in the humoral innate immune system. Fluid-phase molecules of innate immunity share regulatory roles in ECM. On the other hand, ECM elements have immunological functions. Innate immunity is evolutionary and functionally connected to hemostasis. Staphylococcus aureus (S. aureus) is a major cause of hospital-associated bloodstream infections and the most common cause of several life-threatening conditions such as endocarditis and sepsis through its ability to manipulate hemostasis. Biofilm-related infection and sepsis represent a medical need due to the lack of treatments and the high resistance to antibiotics. We designed a method combining imaging and microfluidics to dissect the role of elements of the ECM and hemostasis in triggering S. aureus biofilm by highlighting an essential role of fibrinogen (FG) in adhesion and formation. Furthermore, we ascertained an important role of the fluid-phase activation of fibrinolysis in inhibiting biofilm of S. aureus and facilitating an antibody-mediated response aimed at pathogen killing. The results define FG as an essential element of hemostasis in the S. aureus biofilm formation and a role of fibrinolysis in its inhibition, while promoting an antibody-mediated response. Understanding host molecular mechanisms influencing biofilm formation and degradation is instrumental for the development of new combined therapeutic approaches to prevent the risk of S. aureus biofilm-associated diseases

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

Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion.

Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, we found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs