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Precise mapping of disordered regions of nucleoporins in situ

Kang, Jun Hee

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Abstract

The nuclear pore complex (NPC) is an immense structure which functions as a primary gateway for transport of biomolecules in and out of the nucleus. The NPC allows passive diffusion of small molecules (< 40 kDa, < 4 nm) while blocking large cargo molecules unless they are bound to nuclear transport receptors (NTRs). It is composed of roughly 30 different types of proteins called nucleoporins (Nups). The central transport channel is filled with Nups containing numerous phenylalanine-glycine (FG) repeats which play a major role in forming the permeability barrier. Recent advancements in structural biology approaches have greatly contributed to deciphering the structure of the ring-like NPC scaffold. However, insights about the fundamental nature of the FG Nups such as their spatial organization and dynamics in the central transport channel of the NPC still remains challenging. This is mainly due to technical limitations when aiming to study the flexible intrinsically disordered regions of FG Nups. The goal of my thesis was to devise an experimental strategy to study the spatial organization of FG Nups with residue precision in cells using super-resolution microscopy (SRM). One main bottleneck to achieve this goal was to develop a tool to site-specifically label intracellular proteins with synthetic fluorophores (SFs). In this thesis, I developed and established a labeling strategy implementing genetic code expansion (GCE) technology and click-chemistry reaction in order to label FG Nups in situ in mammalian cells with residue specificity. Moreover, I designed a dual-color imaging strategy using spectral demixing coupled to a robust automated custom-written analysis to quantitatively assess and compare different labeling positions along the disordered regions of FG Nups. I validated the developed labeling and analysis strategy by determining the localization of Nup153, which is one of the largest and in terms of function, one of the most debated FG Nups in the NPC of mammalian cells. Ultimately, I fully utilized the new approach to map the spatial organization of distinct Nup98 regions in situ in mammalian cells. I will discuss possible interpretations and pitfalls of the acquired results and also the steps that are needed to make the labeling strategy live-cell compatible. Further application of my developed approach to other FG Nups in the central channel of the NPC will contribute in acquiring a deeper understanding of the molecular mechanisms behind nucleocytoplasmic transport. In general, my work presents a complementary approach to study the structure and dynamics of flexible proteins inside cells and paves the way towards precise in-cell structural biology.

Item Type: Dissertation
Supervisor: Melchior, Prof. Dr. Frauke
Date of thesis defense: 11 October 2016
Date Deposited: 02 Nov 2016 10:11
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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