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Title: Probing the transport barrier of the nuclear pore complex
Author: Stanley, George
ISNI:       0000 0004 7661 1983
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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The nuclear pore complex (NPC) is the selective gateway through which macromolecules must pass when entering or exiting the nucleus. It is a cog in the gene expression pathway, an entrance to the nucleus exploited by viruses, and a highly-tuned nanoscale filter. The NPC is a large proteinaceous assembly with a central channel occluded by natively disordered proteins, known as FG-nucleoporins (or FG-nups). These FG-nups, along with a family of soluble proteins (known as nuclear transport receptors, or NTRs), form the selective transport barrier. Although much is known about the transport cycle and the necessity of NTRs for chaperoning cargo molecules through the NPC, the mechanism by which NTRs and NTR•cargo complexes translocate the selective transport barrier is not well understood. How can intrinsically disordered FG-nups and soluble NTRs form a transport barrier that is selective, ATP-free, and fast? In this thesis, high-resolution atomic force microscopy (AFM) and a new, fast force-spectroscopy technique (PeakForce QNM) are used to provide a structural and nanomechanical analysis of individual NPCs. This data highlights the structural diversity and complexity at the nuclear envelope, showing the interplay between the lamina network, actin filaments, and the NPCs. It reveals the dynamic behaviour of NPC scaffolds and displays pores of varying sizes. Of functional importance, the NPC central channel shows large structural diversity (in both its mechanical properties and topography), supporting the notion that FG-nup cohesiveness is in a range that facilitates collective rearrangements at little energetic cost. Furthermore, various NTRs are shown to interact in qualitatively different ways with the FG-nups, with particularly strong binding of importin-β. Next, a method for analysing the dynamics of reconstituted FG-nups inside mimetic NPCs is presented - with the results highlighting the surprisingly slow time-scale for collective rearrangement of FG-nup morphologies in the pore geometry. When this analysis is applied to the real NPC, however, no dynamic movement of FG-nups is detected. Finally, preliminary results from AFM imaging experiments of large cargoes (in this case, the hepatitis B virus capsid) translocating the NPC, are presented. This thesis supports the notion that FG-nup cohesiveness is tuned such that the energetics of stable FG-nup morphologies lie near transition states, thereby allowing the collective rearrangement of FG-nups at little energetic cost. Furthermore, it suggests that NTRs with several FG-nup binding sites (such as importin-β) are an intrinsic component of the transport barrier.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available