A postsynaptic Spectrin scaffold defines active zone size, spacing, and efficacy at the Drosophila neuromuscular junction

Synaptic connections are established with characteristic, cell type–specific size and spacing. In this study, we document a role for the postsynaptic Spectrin skeleton in this process. We use transgenic double-stranded RNA to selectively eliminate α-Spectrin, β-Spectrin, or Ankyrin. In the absence of postsynaptic α- or β-Spectrin, active zone size is increased and spacing is perturbed. In addition, subsynaptic muscle membranes are significantly altered. However, despite these changes, the subdivision of the synapse into active zone and periactive zone domains remains intact, both pre- and postsynaptically. Functionally, altered active zone dimensions correlate with an increase in quantal size without a change in presynaptic vesicle size. Mechanistically, β-Spectrin is required for the localization of α-Spectrin and Ankyrin to the postsynaptic membrane. Although Ankyrin is not required for the localization of the Spectrin skeleton to the neuromuscular junction, it contributes to Spectrin-mediated synapse development. We propose a model in which a postsynaptic Spectrin–actin lattice acts as an organizing scaffold upon which pre- and postsynaptic development are arranged

Analyse von Neuron-Glia Interaktionen im embryonalen Nervensystem von Drosophila

Wichtige Aspekte der Entwicklung und Funktion eines Nervensystems werden durch Neuron-Glia Interaktionen kontrolliert. Im Rahmen der Dissertation wurde die Interaktion zwischen Mittellinienneuronen und Mittelliniengliazellen während der Embryonalentwicklung von Drosophila analysiert. Neben der konfokalen Analyse der wildtypischen Interaktionen zwischen den beiden Zelltypen wurden die drei Mutationen klötzchen, kästchen und schmalspur phänotypisch und molekular charakterisiert. Die drei Gene werden für unterschiedliche Aspekte der Neuron-Glia Interaktion benötigt. kästchen kodiert für ein konserviertes Transmembran-Protein, das die Kommunikation zwischen den Zellen kontrolliert. klötzchen ist an der Regulation des Spektrin-Zytoskeletts während der Migration der Gliazellen entlang der axonalen Ausläufer der Mittellinienneurone beteiligt. schmalspur kodiert für einen transkriptionellen Regulator, der für die Entwicklung der Mittelliniengliazellen benötigt wird

Review of underground logistic systems in the Netherlands: an ex-post evaluation of barriers, enablers and spin-offs

Now, 10 years after the first plans, we analyse in this paper what has happened with Underground Logistic Systems (ULS). The major question in this paper is: Which barriers and enablers led to the failure of ULS and what ULS spin-offs can be found nowadays? Several factors can be classified as barriers or enablers. The main conclusions that can be drawn are that the opportunities for try-out were too limited; political support could have been gained on higher levels; the costs were too high, the catchment area was too limited; ULS in itself is a very promising system, but there was no one clear goal. In particular, the lack of a thorough and positive business model in combination with a lack of sufficient freight volumes almost immediately guaranteed the failure of the initiative. The spin-offs seem to have taken place in different directions: ranging from rather soft impacts (e.g. scientific knowledge) to more hard developments (adopting and developing transport and tunnelling technologies), and, although difficult to quantify, they are of great value

The Ankyrin Repeat Domain Controls Presynaptic Localization of Drosophila Ankyrin2 and Is Essential for Synaptic Stability

The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. Here, we address the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. We used a P[acman] based rescue approach to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. We show that specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and we demonstrate that these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, our data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance

RNAi Screen Reveals an Abl Kinase-Dependent Host Cell Pathway Involved in Pseudomonas aeruginosa Internalization

Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of ∼80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa–induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections

Motor control of Drosophila feeding behavior

The precise coordination of body parts is essential for survival and behavior of higher organisms. While progress has been made towards the identification of central mechanisms coordinating limb movement, only limited knowledge exists regarding the generation and execution of sequential motor action patterns at the level of individual motoneurons. Here we use Drosophila proboscis extension as a model system for a reaching-like behavior. We first provide a neuroanatomical description of the motoneurons and muscles contributing to proboscis motion. Using genetic targeting in combination with artificial activation and silencing assays we identify the individual motoneurons controlling the five major sequential steps of proboscis extension and retraction. Activity-manipulations during naturally evoked proboscis extension show that orchestration of serial motoneuron activation does not rely on feed-forward mechanisms. Our data support a model in which central command circuits recruit individual motoneurons to generate task-specific proboscis extension sequences

The pupil near response is short lasting and intact in virtual reality head mounted displays

The pupil of the eye constricts when moving focus from an object further away to an object closer by. This is called the pupil near response, which typically occurs together with accommodation and vergence responses. When immersed in virtual reality mediated through a head-mounted display, this triad is disrupted by the vergence-accommodation conflict. However, it is not yet clear if the disruption also affects the pupil near response. Two experiments were performed to assess this. The first experiment had participants follow a target that first appeared at a far position and then moved to either a near position (far-to-near; FN) or to another far position (far-to-far; FF). The second experiment had participants follow a target that jumped between five positions, which was repeated at several distances. Experiment 1 showed a greater pupil constriction amplitude for FN trials, compared to FF trials, suggesting that the pupil near response is intact in head-mounted display mediated virtual reality. Experiment 2 did not find that average pupil dilation differed when fixating targets at different distances, suggesting that the pupil near response is transient and does not result in sustained pupil size changes

Novel Behavioral and Developmental Defects Associated with Drosophila single-minded

In Drosophila, the development of the midline cells of the embryonic ventral nerve cord depends on the function of the bHLH-PAS transcription factor Single-minded (Sim). The expression domain of sim, however, is also found anterior and posterior to the developing ventral cord throughout the germ band. Indeed, mutations in sim were identified based on their characteristic cuticle phenotype. Eight abdominal segments (A1–A8) can be easily seen in the larval cuticle, while three more can be identified during embryogenesis. Cells located in A8–A10 give rise to the formation of the genital imaginal discs, and a highly modified A11 segment gives rise to the anal pads that flank the anus. sim is expressed in all these segments and is required for the formation of both the anal pads and the genital imaginal discs. A new temperature-sensitive sim allele allowed an assessment of possible postembryonic function(s) of sim. Reduction of sim function below a 50% threshold leads to sterile flies with marked behavioral deficits. Most mutant sim flies were only able to walk in circles. Further analyses indicated that this phenotype is likely due to defects in the brain central complex. This brain region, which has previously been implicated in the control of walking behavior, expresses high levels of nuclear Sim protein in three clusters of neurons in each central brain hemisphere. Additional Sim localization in the medullary and laminar neurons of the optic lobes may correlate with the presence of ectopic axon bundles observed in the optic lobes of sim mutant flies