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

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

SketchyTuna:Exploring A Design For Screenless Creativity

(Abstract to follow

Dual gene activation and knockout screen reveals directional dependencies in genetic networks.

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention