Three-dimensional structure and flexibility of a membrane-coating module of the nuclear pore complex.

The nuclear pore complex mediates nucleocytoplasmic transport in all eukaryotes and is among the largest cellular assemblies of proteins, collectively known as nucleoporins. Nucleoporins are organized into distinct subcomplexes. We optimized the isolation of a putative membrane-coating subcomplex of the nuclear pore complex, the heptameric Nup84 complex, and analyzed its structure by EM. Our data confirmed the previously reported 'Y' shape. We discerned additional structural details, including specific hinge regions at which the particle shows great flexibility. We determined the three-dimensional structures of two conformers, mapped the localization of two nucleoporins within the subcomplex and docked known crystal structures into the EM maps. The free ends of the Y-shaped particle are formed by beta-propellers; the connecting segments consist of alpha-solenoids. Notably, the same organizational principle is found in the clathrin triskelion, which may share a common evolutionary origin with the heptameric complex

Biophysical Characterization of Structure and Dynamics of Nuclear Pore Complex Components

The Nuclear Pore Complex (NPC) mediates nucleo-cytoplasmic transport in all eukaryotes and is among the largest cellular assemblies of proteins, called nucleoporins (nups). The details of NPC architecture, dynamics, and mechanism are still unknown. NPCs can be dissected biochemically into distinct subcomplexes. One of the best-characterized subcomplexes, the Nup84 complex, consists of seven nups and was proposed to form a membrane-coating module of the NPC. I optimized the isolation of the heptameric complex from budding yeast and analyzed its structure by negative-stain electron microscopy (EM). My data confirm the previously reported flexible Y-shape. I solved the three-dimensional structures of two conformers of the heptamer and discerned additional details, including specific hinge regions. Tagged versions of two nups were localized within the heptamer and known crystal structures were docked into the EM map. The globular ends of the arms and the stem are formed by β-propeller domains; thinner connecting segments are formed by α-solenoids. Strikingly, the same organizational principle is found in the clathrin triskelion, which was proposed to share a common evolutionary origin with the heptameric complex. A second focus of this thesis is the investigation of NPC dynamics in live cells, using polarized fluorescence microscopy. Two types of NPC dynamics have been suggested to play important functional roles: the dilation of the NPC to accommodate the transport of large cargoes, and the movement of disordered FG domains of nups to gate the NPC via entropic exclusion. An alternative model envisages a static FG domain meshwork that operates via hydrophobic exclusion. I analyzed theoretically how anisotropy measurements of GFP-tagged nups can be used to monitor nup orientation and dynamics. In a collaboration with the Simon lab (The Rockefeller University), we established techniques to analyze GFP anisotropy in live yeast cells. GFP attached to ordered nup domains displayed defined orientations with respect to the NPC, whereas GFP attached to the FG domains is randomly oriented. Homo-FRET between GFP-tags was observed in two cases. Future experiments should enable us to distinguish between different models for the role of FG domains in NPC gating, and to investigate NPC dilation during transport

Neurodegeneration enters the era of functional genomics

There are no cures for the most common neurodegenerative diseases. None of the currently approved treatments cure or halt these conditions; rather, they address symptoms or slow disease progression. A focus on protein deposits in the brain—a hallmark of Alzheimer’s disease (AD) and Parkinson’s disease (PD)—has led to the development of immunotherapy drugs. Other promising avenues of investigation include the roles of neuroinflammation in neurodegeneration. However, the clinical impact of these approaches is still uncertain. What about exploiting our knowledge of the human genome and the ability to modify it with surgically precise tools? Can functional genomics approaches in neurodegenerative disease research provide the breakthroughs we need

The Merlin OMS Benchmark - Definition, Implementations and Results

The bottleneck of highly integrated software development environments (SDEs) could still be the use of an underlying central data store. An environment is called highly integrated, if the included tools support most of the life-cycle phases and especially inform, analyse and check document interdependencies and even propagate changes on basically any level of granularity. In that case a central data store for documents and document interdependencies is the necessary key information base. A number of so-called object management systems (OMSs) have recently been developed to address the needs of SDEs. In order to support an environment builder to select the most appropriate one, this paper presents a new benchmark for such databases. We precisely define that benchmark in an abstract way in terms of its conceptual schema, possible initial database states and the benchmark operations. The benchmark was implemented twice to allow for a comparison of the OMSs GRAS and GemStone. We describe these two implementations as well as the results we gained when we performed the benchmark

Zivil- und zivilprozessrechtliche Aspekte der großen Justizreform in Frankreich (Justice du 21ème siècle)

By two laws issued on 8 August 2016 and 18 November 2016, the French legislator has under-taken extensive reforms of the civil and the civil procedure law, called “Justice of the 21st cen-tury” (Justice du 21ème siècle). These laws aim at setting up the judiciary for the 21st century by making it more efficient, transparent and easily accessible. The propositions have been dis-cussed since 2014 and include several sets of measures. With the key issue of an efficient judi-cial system, justice shall be relieved by transferring several competences to other public author-ities. The judicial organization shall be simplified and more easily accessible. For this reason, the laws “for a justice of the 21st century” contain instruments for procedural simplification and facilitating access to the courts. Finally, the independence of judiciary shall be guaranteed. By different measures, the judicial independence is being strengthened.Durch zwei Gesetze vom 8. August 2016 und vom 18. November 2016, betreffend „Die Justiz des 21. Jahrhunderts“ (Justice du 21ème siècle), hat der französische Gesetzgeber umfassende Re-formen des Zivil- und Zivilprozessrechts vorgenommen. Ziel dieses Gesetzes ist es, die Justiz auf das 21. Jahrhundert vorzubereiten und die Justiz effizienter, transparenter und leichter zu-gänglich zu machen. Die seit 2014 diskutierten Vorschläge zur Ausgestaltung der Justiz des 21. Jahrhunderts umfassen mehrere Maßnahmenpakete. Unter dem Stichwort einer effizienteren Justiz soll zunächst eine Justizentlastung durch Verlagerung bestimmter bisher von der Justiz erledigter Aufgaben auf andere staatliche Stellen erreicht werden. Die Justiz soll zudem einfa-cher ausgestaltet werden und leichter zugänglich sein. Die Gesetze zur Justiz des 21. Jahrhun-derts enthalten daher auch Instrumente der Verfahrensvereinfachung und zur Erleichterung des Zugangs zu Gericht. Diskutiert werden diese Maßnahmen unter dem Stichwort einer leichter zugänglichen Justiz. Schließlich soll die Justiz auch unabhängiger werden. Durch verschiedene, in den Gesetzen enthaltene Maßnahmen soll die richterliche Unabhängigkeit gestärkt werden

Extending Chemical Perturbations Of The Ubiquitin Fitness Landscape In A Classroom Setting [preprint]

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al. 2016), we used deep mutational scanning to determine how twelve new chemicals reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a highly sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales

Der Tod des Herrschers. Aspekte der zeremoniellen und literarischen Verarbeitung des Todes politischer Führungsfiguren

Die vier Beiträge des Bandes untersuchen, wie der Tod des Herrschers sich auf die Stabilität der Herrschaft selbst auswirkt - ein Problem, welches historischen Wandlungen unterliegt. Im Tod des Fürsten werden die Kontingenz menschlicher Existenz und irdischer Ordnung zugleich sichtbar. Deren Interdependenz wird hinterfragt. Wie Gesellschaften den Tod ihrer politischen Führer verarbeiteten - vor allem, wenn ihre Existenz semantisch an den Fortbestand der Einherrschaft gebunden ist, ist ein dringendes Problem bis in die europäische Frühe Neuzeit hinein. Gefragt wird auch, ob dieses Problem im Übergang zur repräsentativen Verfassung verschwand, oder ob es in transformierter Form weiterlebte

Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales