Date of Award
nuclear pore complex, electron microscopy, Nup84 complex, structural biology, fluorescence anisotropy, polarized fluorescence microscopy
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 Î²-propeller domains; thinner connecting segments are formed by Î±-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.
Kampmann, Martin, "Biophysical Characterization of Structure and Dynamics of Nuclear Pore Complex Components" (2009). Student Theses and Dissertations. 64.