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

Statistics In Diffractive Imaging

This dissertation describes reconstruction techniques in diffractive imaging when the data is exceptionally noisy and when crucial experimental parameters are unmeasured. In particular, this work focuses on two applications of diffractive imaging, single particle imaging with unoriented data and ultrafast magnetic imaging with unmeasured charge distribution, both of which are exciting experiments planned for free electron laser facilities. Concerning single particle imaging, in chapter 2 we introduce the EMC algorithm for reconstructing a particle's 3D diffraction intensity from very many photon-shot-noise limited 2D measurements, when the particle orientation in each measurement is unknown. We coin such an imaging technique cryptotomography. In this chapter, we also study the noise limits beyond which cryptotomography is impossible. This is followed by an experimental demonstration of EMC in chapter 3, where we reconstruct the 3D Fourier intensity distribution of mono-disperse prolate nano-particles using single-shot 2D diffraction patterns collected at DESYs FLASH facility when a bright, coherent, ultrafast X-ray pulse intercepted individual particles of random, unmeasured orientations. This experimental demonstration of cryptotomography extended the Expansion-Maximization-Compression (EMC) framework to accommodate unmeasured fluctuations in photon fluence and loss of data due to saturation or background scatter. In chapter 4 we discuss magnetic imaging. We study, using simulated experiments, the feasibility of phase retrieval in X-ray diffractive imaging of thin-film magnetic domains in the presence of intrinsic charge scattering given only photon-shot-noise limited diffraction data. We also chart out the limits of diffractive imaging when we vary both photon-shot-noise and the intensity of charge-scattering noise. This work is directly relevant to the time-resolved imaging of magnetic dynamics using coherent and ultrafast radiation from Xray free electron lasers

52nd Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 52nd annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-endorsed by the Colorado Section of the American Chemical Society and the Society for Applied Spectroscopy. Held in Snowmass, Colorado, August 1-5, 2010