Huygens STED Deconvolution Increases Signal-to-Noise and Image Resolution towards 22 nm

Image restoration aims to make optimal use of the data provided by the light microscope to recover the object being imaged; in other words, to "figure out what the microscope is actually trying to tell us.” To achieve this, the distortions introduced by the imaging process need to be undone insofar possibl

The effectiveness of conventional trickling filter treatment plants at reducing concentrations of copper in wastewaters

This is the post-print version of the final paper published in Science of the Total Environment. The published article is available from the link below. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. Copyright @ 2009 Elsevier B.V.Eight different sewage treatment works were sampled in the North West of England. The effectiveness of the conventional treatment processes (primary sedimentation and biological trickling filters) as well as various tertiary treatment units in terms of both total and dissolved copper removal was evaluated. The removal of total copper across primary sedimentation averaged 53% and were relatively consistent at all sites, however, at three sites the removal of dissolved copper also occurred at this stage of treatment. Removal of total copper by the biological trickling filters averaged 49%, however, substantial dissolution of copper occurred at two sites, which highlighted the unpredictability of this treatment process in the removal of dissolved copper. Copper removal during tertiary treatment varied considerably even for the same treatment processes installed at different sites, primarily due to the variability of insoluble copper removal, with little effect on copper in the dissolved form being observed. The proportion of dissolved copper increased significantly during treatment, from an average of 22% in crude sewages to 55% in the final effluents. There may be the potential to optimise existing, conventional treatment processes (primary or biological treatment) to enhance dissolved copper removal, possibly reducing the requirement for installing any tertiary processes specifically for the removal of copper.United Utilities PL

Functional architecture of reward learning in mushroom body extrinsic neurons of larval Drosophila.

The brain adaptively integrates present sensory input, past experience, and options for future action. The insect mushroom body exemplifies how a central brain structure brings about such integration. Here we use a combination of systematic single-cell labeling, connectomics, transgenic silencing, and activation experiments to study the mushroom body at single-cell resolution, focusing on the behavioral architecture of its input and output neurons (MBINs and MBONs), and of the mushroom body intrinsic APL neuron. Our results reveal the identity and morphology of almost all of these 44 neurons in stage 3 Drosophila larvae. Upon an initial screen, functional analyses focusing on the mushroom body medial lobe uncover sparse and specific functions of its dopaminergic MBINs, its MBONs, and of the GABAergic APL neuron across three behavioral tasks, namely odor preference, taste preference, and associative learning between odor and taste. Our results thus provide a cellular-resolution study case of how brains organize behavior

Struktur und Dynamik der neuromuskulären Synapse von Drosophila melanogaster : Untersuchungen am Dlg-Gerüstkomplex mittels hochauflösender Lichtmikroskopie und 3D/4D-Bildanalysen

Brain functions, in particular learning and memory, are based on neuronal networks, which, once established are subject to experience-dependent adjustments. Neuronal networks in turn consist of neurons, which are connected with each other via synapses. The latter display plasticity, thereby being challenged by the need for both structural stability and adaptiveness in equal measure. In this regard, membrane-associated guanylate kinases (MAGuKs) are likely to play a pivotal role, as they act as scaffold proteins that determine the molecular architecture of synapses. The functional characterisation of these evolutionarily well-conserved proteins will thus add to a better understanding of brain functions at the cellular and molecular levels and of pathology- and age-related dysfunctions thereof. The larval neuromuscular junction (NMJ) of Drosophila as a widely used, genetically amenable model system for glutamatergic synapses is particularly useful to study the function of the prototypic MAGuK Dlg (Discs large). However, the number of known synaptic interacting partners of Dlg is limited. This work focusses on the interaction of Dlg with the scaffold and adaptor proteins Metro (Ménage à trois) and DLin-7, which are also conserved between vertebrates and invertebrates. Based on mutants and transgenic lines, it is shown that the three proteins are disproportionally interdependent regarding their localisation to NMJs. Postsynaptically, they localise peripheral to rather than within glutamate receptor fields. It is further shown that the complex restricts the size of these receptor fields. To determine the size of the receptor fields in 3D based on light microscopy, a protocol was established, which allows for measuring a large number of receptor fields in a reliable way. Moreover, it is documented that in the absence of Dlg, Metro or DLin-7, synaptic boutons are enlarged, irregularly shaped and often display features of incomplete differentiation, yet with no impact on the overall number of synaptic contacts. An elaborated live-Imaging analysis revealed that the dynamics of Dlg at the NMJ is regulated by its binding to Metro und DLin-7. The turnover rate of Dlg was found to be much lower than previously suggested and hence is likely comparable to the dynamics of glutamate receptors. Of note, it turned out that the expression level of fluorescently labelled Dlg is a critical analysis parameter. Finally, it is shown with unprecedented clarity that Dlg, Metro and DLin-7 are also enriched at the presynaptic membrane, thereby localising close to but not within synaptic release sites. In light of inconsistent findings in the literature, this result supports the assumption for the Dlg complex to play a presynaptic role in addition to the well-documented postsynaptic function.Hirnfunktionen, insbesondere Lernen und Gedächtnis, beruhen auf neuronalen Netzwerken, die nach ihrer Etablierung bedarfsgerechten Veränderungen unterliegen. Neuronale Netzwerke bestehen aus Neuronen, die über Synapsen untereinander verbunden sind. Letztere zeichnen sich durch eine Plastizität aus, die den Anforderungen an strukturelle Stabilität und funktionelle Anpassungsfähigkeit gleichermaßen genügen muss. Eine wichtige Rolle können hierbei sogenannte Membran-assoziierte Guanylatkinasen (MAGuKs) spielen, die als Gerüstproteine die molekulare Architektur von Synapsen maßgeblich mitbestimmen. Die funktionelle Charakterisierung dieser evolutionär gut konservierten Proteine trägt daher zu einem besseren Verständnis zellulärer Hirnfunktionen sowie deren krankheits- und altersbedingten Störungen bei. Die larvale neuromuskuläre Verbindung (NMJ) von Drosophila, als weithin genutztes, genetisch gut zugängliches Modellsystem für glutamaterge Synapsen, eignet sich gut dafür, da sich an ihr das prototypische MAGuK Dlg (Discs large) anreichert. Allerdings ist die Zahl der bekannten synaptischen Interaktionspartner von Dlg bisher gering. Im Fokus dieser Arbeit steht die Interaktion von Dlg mit den ebenfalls gut konservierten Gerüst- bzw. Adapterproteinen Metro (Ménage à trois) und DLin-7. Anhand von mutanten- und transgenen Tieren wird gezeigt, dass die drei Proteine in einem disproportionalen, wechselseitigen Abhängigkeitsverhältnis an der NMJ lokalisieren. Postsynaptisch lokalisiert der trimere Komplex peripher zu den Glutamatrezeptorfeldern. Es wird weiterhin gezeigt, dass der Komplex die Rezeptorfelder in ihrer Ausdehnung begrenzt. Dafür wurde in dieser Arbeit ein Protokoll ausgearbeitet, mit dem die Rezeptorfelder zuverlässig und in großer Anzahl lichtmikroskopisch in 3D vermessen wurden. Dokumentiert wird ferner, dass die Abwesenheit von Dlg, Metro oder DLin-7 zu strukturellen Abnormitäten und Differenzierungsdefiziten der synaptischen Boutons führt ohne die Zahl synaptischer Kontakte zu beeinträchtigen. Eine differenzierte live-Imaging-Analyse ergab, dass die Dynamik von Dlg an der NMJ durch die Bindung von Metro und DLin-7 reguliert wird. Ferner zeigte sich, dass der Umsatz des Dlg-Komplexes an der NMJ niedriger ausfällt als bislang angenommen und somit wohl der Dynamik von Glutamatrezeptoren vergleichbar ist. Das Expressionslevel des Fluoreszenz-markierten Dlg erwies sich dabei als kritischer Analyseparameter. Schließlich wird in dieser Arbeit erstmals deutlich gezeigt, dass Dlg, aber auch Metro und DLin-7, präsynaptisch angereichert sind und dabei peripher zu den synaptischen Zonen der Neurotransmitterfreisetzung lokalisiert sind. Mit Blick auf teils widersprüchliche Befunde in der Literatur, unterstützt dieser Ergebnisteil die Annahme, dass der Dlg-Komplex neben seiner Funktion in der Postsynapse auch in der Präsynapse eine Rolle spielt

Periodic F-actin structures shape the neck of dendritic spines

Most of the excitatory synapses on principal neurons of the forebrain are located on specialized structures called dendritic spines. Their morphology, comprising a spine head connected to the dendritic branch via a thin neck, provides biochemical and electrical compartmentalization during signal transmission. Spine shape is defined and tightly controlled by the organization of the actin cytoskeleton. Alterations in synaptic strength correlate with changes in the morphological appearance of the spine head and neck. Therefore, it is important to get a better understanding of the nanoscale organization of the actin cytoskeleton in dendritic spines. A periodic organization of the actin/spectrin lattice was recently discovered in axons and a small fraction of dendrites using super-resolution microscopy. Here we use a small probe phalloidin-Atto647N, to label F-actin in mature hippocampal primary neurons and in living hippocampal slices. STED nanoscopy reveals that in contrast to β-II spectrin antibody labelling, phalloidin-Atto647N stains periodic actin structures in all dendrites and the neck of nearly all dendritic spines, including filopodia-like spines. These findings extend the current view on F-actin organization in dendritic spines and may provide new avenues for understanding the structural changes in the spine neck during induction of synaptic plasticity, active organelle transport or tethering

A Dendritic Golgi Satellite between ERGIC and Retromer

The local synthesis of transmembrane proteins underlies functional specialization of dendritic microdomains in neuronal plasticity. It is unclear whether these proteins have access to the complete machinery of the secretory pathway following local synthesis. In this study, we describe a probe called pGolt that allows visualization of Golgi-related organelles for live imaging in neurons. We show that pGolt labels a widespread microsecretory Golgi satellite (GS) system that is, in contrast to Golgi outposts, present throughout basal and apical dendrites of all pyramidal neurons. The GS system contains glycosylation machinery and is localized between ERGIC and retromer. Synaptic activity restrains lateral movement of ERGIC, GS, and retromer close to one another, allowing confined processing of secretory cargo. Several synaptic transmembrane proteins pass through and recycle back to the GS system. Thus, the presence of an ER-ERGIC-GS-retromer microsecretory system in all neuronal dendrites enables autonomous local control of transmembrane protein synthesis and processing

F-actin patches associated with glutamatergic synapses control positioning of dendritic lysosomes

Organelle positioning within neurites is required for proper neuronal function. In dendrites, with their complex cytoskeletal organization, transport of organelles is guided by local specializations of the microtubule and actin cytoskeleton, and by coordinated activity of different motor proteins. Here, we focus on the actin cytoskeleton in the dendritic shaft and describe dense structures consisting of longitudinal and branched actin filaments. These actin patches are devoid of microtubules and are frequently located at the base of spines, or form an actin mesh around excitatory shaft synapses. Using lysosomes as an example, we demonstrate that the presence of actin patches has a strong impact on dendritic organelle transport, as lysosomes frequently stall at these locations. We provide mechanistic insights on this pausing behavior, demonstrating that actin patches form a physical barrier for kinesin-driven cargo. In addition, we identify myosin Va as an active tether which mediates long-term stalling. This correlation between the presence of actin meshes and halting of organelles could be a generalized principle by which synapses control organelle trafficking

A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-level analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins

Terminal Axonal Arborization and Synaptic Bouton Formation Critically Rely on Abp1 and the Arp2/3 Complex

<div><p>Neuronal network formation depends on properly timed and localized generation of presynaptic as well as postsynaptic structures. Although of utmost importance for understanding development and plasticity of the nervous system and neurodegenerative diseases, the molecular mechanisms that ensure the fine-control needed for coordinated establishment of pre- and postsynapses are still largely unknown. We show that the F-actin-binding protein Abp1 is prominently expressed in the <i>Drosophila</i> nervous system and reveal that Abp1 is an important regulator in shaping glutamatergic neuromuscular junctions (NMJs) of flies. STED microscopy shows that Abp1 accumulations can be found in close proximity of synaptic vesicles and at the cell cortex in nerve terminals. <i>Abp1</i> knock-out larvae have locomotion defects and underdeveloped NMJs that are characterized by a reduced number of both type Ib synaptic boutons and branches of motornerve terminals. Abp1 is able to indirectly trigger Arp2/3 complex-mediated actin nucleation and interacts with both WASP and Scar. Consistently, Arp2 and Arp3 loss-of-function also resulted in impairments of bouton formation and arborization at NMJs, i.e. fully phenocopied <i>abp1</i> knock-out. Interestingly, neuron- and muscle-specific rescue experiments revealed that synaptic bouton formation critically depends on presynaptic Abp1, whereas the NMJ branching defects can be compensated for by restoring Abp1 functions at either side. In line with this presynaptic importance of Abp1, also presynaptic Arp2 and Arp3 are crucial for the formation of type Ib synaptic boutons. Interestingly, presynaptic Abp1 functions in NMJ formation were fully dependent on the Arp2/3 complex, as revealed by suppression of Abp1-induced synaptic bouton formation and branching of axon terminals upon presynaptic Arp2 RNAi. These data reveal that Abp1 and Arp2/3 complex-mediated actin cytoskeletal dynamics drive both synaptic bouton formation and NMJ branching. Our data furthermore shed light on an intense bidirectional functional crosstalk between pre- and postsynapses during the development of synaptic contacts.</p></div

Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila

Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as ‘click chemistry’ serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in Drosophila melanogaster