Activity-dependence of synaptic vesicle dynamics

The proper function of synapses relies on efficient recycling of synaptic vesicles. The small size of synaptic boutons has hampered efforts to define the dynamical states of vesicles during recycling. Moreover, whether vesicle motion during recycling is regulated by neural activity remains largely unknown. We combined nanoscale-resolution tracking of individual synaptic vesicles in cultured hippocampal neurons from rats of both sexes with advanced motion analyses to demonstrate that the majority of recently endocytosed vesicles undergo sequences of transient dynamical states including epochs of directed, diffusional, and stalled motion. We observed that vesicle motion is modulated in an activity-dependent manner, with dynamical changes apparent in ∼20% of observed boutons. Within this subpopulation of boutons, 35% of observed vesicles exhibited acceleration and 65% exhibited deceleration, accompanied by corresponding changes in directed motion. Individual vesicles observed in the remaining ∼80% of boutons did not exhibit apparent dynamical changes in response to stimulation. More quantitative transient motion analyses revealed that the overall reduction of vesicle mobility, and specifically of the directed motion component, is the predominant activity-evoked change across the entire bouton population. Activity-dependent modulation of vesicle mobility may represent an important mechanism controlling vesicle availability and neurotransmitter release.SIGNIFICANCE STATEMENTMechanisms governing synaptic vesicle dynamics during recycling remain poorly understood. Using nanoscale resolution tracking of individual synaptic vesicles in hippocampal synapses and advanced motion analysis tools we demonstrate that synaptic vesicles undergo complex sets of dynamical states that include epochs of directed, diffusive, and stalled motion. Most importantly, our analyses revealed that vesicle motion is modulated in an activity-dependent manner apparent as the reduction in overall vesicle mobility in response to stimulation. These results define the vesicle dynamical states during recycling and reveal their activity-dependent modulation. Our study thus provides fundamental new insights into the principles governing synaptic function

Myosin V regulates spatial localization of different forms of neurotransmitter release in central synapses

Synaptic active zone (AZ) contains multiple specialized release sites for vesicle fusion. The utilization of release sites is regulated to determine spatiotemporal organization of the two main forms of synchronous release, uni-vesicular (UVR) and multi-vesicular (MVR). We previously found that the vesicle-associated molecular motor myosin V regulates temporal utilization of release sites by controlling vesicle anchoring at release sites in an activity-dependent manner. Here we show that acute inhibition of myosin V shifts preferential location of vesicle docking away from AZ center toward periphery, and results in a corresponding spatial shift in utilization of release sites during UVR. Similarly, inhibition of myosin V also reduces preferential utilization of central release sites during MVR, leading to more spatially distributed and temporally uniform MVR that occurs farther away from the AZ center. Using a modeling approach, we provide a conceptual framework that unites spatial and temporal functions of myosin V in vesicle release by controlling the gradient of release site release probability across the AZ, which in turn determines the spatiotemporal organization of both UVR and MVR. Thus myosin V regulates both temporal and spatial utilization of release sites during two main forms of synchronous release

Safe liquid scintillators for large scale detectors

Many experiments in particle physics, in particular in the field of neutrino searches, rely on organic liquid scintillators as target and detection material. The size of these detectors was continously growing in the last decades, up to the kiloton scale. In several cases these detectors are located at sites with enhanced safety requirements as underground laboratories or in the vicinity of nuclear reactors. Therefore, there is strong demand in liquids which are safe with respect to aspects as fire protection, human health or environmental pollution. In particular, properties as the flash point, the vapor pressure or the toxicity need to be significantly improved as compared to classical solvents such as xylene or pseudocumene. We present and compare the performance and optical properties of scintillators based on high flash point solvents. In particular polysiloxane based scintillators are characterized by outstanding properties in terms of safety.Comment: 13 pages, 4 figure

Plasmanitrierung eines lufthärtenden Schmiedestahls mit mittlerem Mangangehalt

The impact of plasma nitriding on the microstructure and the hardness of a recently developed 4 wt.-% medium manganese steel are presented. In contrast to standard quench and tempering steels, the investigated material achieves its martensitic microstructure by air-cooling from the forging heat, which enables the reduction of the carbon footprint of the forged components. The influence of nitriding on this grade of steel has not been investigated so far, but fundamental differences in comparison to standard nitriding steels are expected due to the increased manganese concentration. To address this issue, nitriding treatments with different temperatures (350 °C, 580 °C and 650 °C) have been performed, followed by examinations of the microstructure, the phase composition, the obtained hardness profiles and the tensile properties of the bulk material after nitriding, accompanied by thermodynamic equilibrium calculations. It is demonstrated that after nitriding at 580 °C similar hardness profiles like standard nitriding steels are achieved, with a shorter process as austenitization and hardening were omitted, reaching a hardness of approximately 950 HV0.1. Furthermore, it was demonstrated that austenite can be stabilized by manganese and nitrogen partitioning to room temperature during nitriding in the intercritical phase region.Es wird der Einfluss des Plasmanitrierens auf das Gefüge und die Härte eines kürzlich entwickelten 4-Gew.-%-Mittelmanganstahls vorgestellt. Im Gegensatz zu Standard-Vergütungsstählen erreicht der untersuchte Werkstoff sein martensitisches Gefüge durch Luftabkühlung aus der Schmiedewärme, was die Reduzierung des Kohlenstoff-Fußabdrucks der Schmiedeteile ermöglicht. Der Einfluss des Nitrierens auf diese Stahlsorte wurde bisher noch nicht untersucht, doch werden aufgrund der erhöhten Mangankonzentration grundlegende Unterschiede im Vergleich zu Standard-Nitrierstählen erwartet. Um diese Frage zu klären, wurden Nitrierbehandlungen bei verschiedenen Temperaturen (350 °C, 580 °C und 650 °C) durchgeführt und anschließend die Mikrostruktur, die Phasenanteile, die erreichten Härteprofile und die Zugeigenschaften der Matrix nach dem Nitrieren untersucht, begleitet von thermodynamischen Gleichgewichtsberechnungen. Es wird gezeigt, dass nach dem Nitrieren bei 580 °C vergleichbare Härteprofile wie bei Standard-Nitrierstählen erzielt werden, wobei der Prozess deutlich verkürzt ist, da Austenitisierung und Härtung entfallen und eine Härte von etwa 950 HV0,1 erreicht wird. Darüber hinaus wurde gezeigt, dass Austenit durch Mangan- und Stickstoffpartitionierung bei Raumtemperatur während des Nitrierens im interkritischen Phasenbereich stabilisiert werden kann

Safe liquid scintillators for large scale detectors

Many experiments in particle physics, in particular in the field of neutrino searches, rely on organic liquid scintillators as target and detection material. The size of these detectors was continously growing in the last decades, up to the kiloton scale. In several cases these detectors are located at sites with enhanced safety requirements as underground laboratories or in the vicinity of nuclear reactors. Therefore, there is strong demand in liquids which are safe with respect to aspects as fire protection, human health or environmental pollution. In particular, properties as the flash point, the vapor pressure or the toxicity need to be significantly improved as compared to classical solvents such as xylene or pseudocumene. We present and compare the performance and optical properties of scintillators based on high flash point solvents. In particular polysiloxane based scintillators are characterized by outstanding properties in terms of safety

Design, Development, and Delivery of Novel Recombinant Proteins for Neuron-Targeted Therapy and Neuronal Differentiation

This thesis dissertation research has aimed to develop novel protein therapeutics with potential to treat neurological diseases for which there are currently limited available treatments and no cures including Parkinson’s disease and neuronopathic Gaucher’s disease. Although the individual chapters of this thesis had varied goals, all involved designing and expressing recombinant proteins, purifying the proteins, facilitating targeted and efficient protein delivery, and demonstrating in vitro activity. The effective targeting of neuronal cell types requires effective delivery strategies. As a result, initial thesis work focused on the evaluation of cell-penetrating peptides (CPPs) and receptor-dependent neuronal membrane binding proteins to improve protein delivery. This work led to the discovery of a novel protein delivery vector, Tat-Tetanus Toxin Fragment C (Tat-TTC), a highly neuron-specific domain capable of dramatically enhancing the binding and internalization of recombinant proteins. Both fluorescent microscopy and quantitative fluorimetry were utilized to demonstrate that, with Tat-TTC, model protein GFP was bound and internalized by neuronal cell types at least an order of magnitude better than Tat-GFP or TTC-GFP alone. Since the Tat-TTC protein delivery vector was endocytosed, this research project addressed the issue of endosomal entrapment of cargo protein. Photochemical internalization (PCI) was used to facilitate the endosomal release of Tat-TTC-linked cargo presenting the option of using the delivery vector to target endosomes/lysosomes or cytosolic or nuclear intracellular targets. Cell-penetrating peptide Tat and PCI were next utilized in the design, production, and delivery of recombinant transcription factors Mash1, Lmx1a, and Nurr1 intended for neuronal differentiation. These transcription factors had been shown to transdifferentiate human fibroblasts to dopaminergic neurons (Caiazzo et al., 2011), the cell type lost in Parkinson’s disease (PD) (Thomas, 2010). We hypothesized that direct protein delivery of Tat-linked transcription factors had the potential to generate dopaminergic neurons that would be safe for clinical cell-replacement therapy. Four transcription factors, Tat-Mash1, Tat-Mash1-GFP, Tat-Lmx1a, and Tat-Lmx1a-GFP were successfully expressed and purified from insoluble HEK293F cell lysate. The transcription factors were delivered to cells and endosomal escape was facilitated with PCI. Induction of promoter-reporter constructs with purified protein was limited, a finding which led us to question the bioactivity of the resolubilized factors. To address this issue, a cell-free mammalian expression system capable of producing high concentrations of soluble transcription factor was identified. In the future, this strategy could prove invaluable for transcription factor production. Research into novel PD therapies led to my strong interest in Gaucher’s disease (GD). Mutations in the GBA1 gene encoding lysosomal enzyme glucocerebrosidase (GCase), leading to GD, has recently been shown to represent the greatest genetic risk factor for PD (Siebert et al., 2014). There is currently no available treatment for the neuronopathic type 2 and type 3 forms of GD. I aimed to develop a neuron-targeted recombinant GCase with the potential to cross the blood-brain barrier (BBB). Since the target for delivered protein was the lysosome, protein delivery vectors taken up by endocytosis were predicted to work well for GCase delivery, without any need for PCI. Seventeen GCase variants were designed and expressed in combination with a variety of protein delivery vectors. Cell-based assays using a GCase-knockout neuronal cell line facilitated the identification of a rabies-derived peptide (RDP) linked GCase utilizing an IgA hinge linker region to remove steric hindrance by GCase on RDP. RDP-IgAh-GCase was shown to target GCase-knockout neurons ~2.5X better than Tat-GCase. Both RDP-IgAh-GCase and Tat-GCase reduced substrate glucosylsphingosine accumulation to wild-type levels with 72 hours of protein treatment, indicating the enzymes were appropriately trafficked to lysosomes and functional

Confluent Orthogonal Drawings of Syntax Diagrams

We provide a pipeline for generating syntax diagrams (also called railroad diagrams) from context free grammars. Syntax diagrams are a graphical representation of a context free language, which we formalize abstractly as a set of mutually recursive nondeterministic finite automata and draw by combining elements from the confluent drawing, layered drawing, and smooth orthogonal drawing styles. Within our pipeline we introduce several heuristics that modify the grammar but preserve the language, improving the aesthetics of the final drawing.Comment: GD 201

Electronic properties of alkali-metal loaded zeolites -- a "supercrystal" Mott insulator

First-principles band calculations are performed for the first time for an open-structured zeolite (LTA) with guest atoms (potassium) introduced in their cages. A surprisingly simple band structure emerges, which indicates that this system may be regarded as a "supercrystal", where each cluster of guest atoms with diameter $\sim$10\AA acts as a "superatom" with well-defined $s$- and $p$-like orbitals, which in turn form the bands around the Fermi energy. The calculated Coulomb and exchange energies for these states turn out to be in the strongly-correlated regime. With the dynamical mean-field theory we show the system should be on the Mott-insulator side, and, on a magnetic phase diagram for degenerate-orbital systems, around the ferromagnetic regime, in accord with experimental results. We envisage this class of systems can provide a new avenue for materials design.Comment: 4 pages, 4 figure

On the distortion of twin building lattices

We show that twin building lattices are undistorted in their ambient group; equivalently, the orbit map of the lattice to the product of the associated twin buildings is a quasi-isometric embedding. As a consequence, we provide an estimate of the quasi-flat rank of these lattices, which implies that there are infinitely many quasi-isometry classes of finitely presented simple groups. In an appendix, we describe how non-distortion of lattices is related to the integrability of the structural cocycle