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Abstract

The nuclear pore complex (NPC) is the largest protein complex in the nuclear envelope of eukaryotic cells. It provides a permeability barrier for rapid and selective transport of biomolecules in and out of the nucleus. While the scaffold structure and composition of the NPC are well understood, little is known about the permeability barrier, which is formed of multiple copies of about ten different intrinsically disordered nucleoporins. These proteins characteristically contain multiple phenylglycine repeats in their sequence and are referred to as FG-Nups. Due to technical limitations in the study of these highly flexible and dynamic proteins, the conformational dynamics and spatial arrangement of FG-Nups in the permeability barrier of the NPC remain elusive. In this thesis I developed and established two complementary imaging techniques to visualise FG-Nups in the NPC in mammalian cells. To achieve this, I further developed a combination of genetic code expansion using unnatural amino acids and click-chemistry technologies suitable for high resolution fluorescence imaging. This method allows for efficient site-specific labelling of two sites in FG-Nups with small, photostable organic fluorophores with residue precision in situ. The first imaging approach used super-resolution localisation microscopy to precisely map the labelled site of the FG-Nups to a reference in the NPC. This resulted in images showing two-dimensional projection of FG-Nups distribution in NPCs, resolving distances down to 10nm. The second approach involved a complimentary technique based on confocal scanning microscopy: using fluorescence lifetime imaging microscopy (FLIM) to directly measure end-to-end distances between two sites in an FG-Nup less than 10nm apart using Förster resonance energy transfer (FRET). The ultimate goal of my work was to use the approaches to determine the structural arrangement of FG-Nups in the permeability barrier of the NPC. I further applied polymer physics concepts of scaling laws to my results from FLIM-FRET studies. I was able to show that the two major players in the permeability barrier FG-Nups, Nup62 and Nup98, tend to resemble a state between a collapsed coil and an ideal chain. This finding demonstrates the first experimental evidence of the actual scaling of FG-Nups in vivo, which has implications for existing transport models. Aside from its contribution to understanding the NPC permeability barrier function, the developed approach lays the groundwork for a more detailed understanding of disordered protein dynamics, dimensions and functions inside the cell.