A toolkit to study nuclear transport pathways: nanobody-mediated inhibition of importin β-related nuclear transport receptors (NTRs)

Perera, Dilantha

• Publisher: Cuvillier Verlag
• Year: 2023
• eBook ISBN:
  9783736968479
• Print ISBN:
  9783736978478
• Edition: 1

Eukaryotic cells are divided into a nuclear and a cytoplasmic compartment. This separates transcription from translation and makes gene expression dependent on nucleocytoplasmic transport. The members of the importin ? superfamily function as shuttling nuclear transport receptors (NTRs) that recognize and actively transport cargoes through nuclear pores. An estimated 5 000 to 10 000 different human proteins are subject to active nuclear transport. Numerous cargo/NTR pairs have been identified, however, we are still far from a complete understanding as it has been very challenging to setup a systematic in vivo analysis that integrates the impact of all transport pathways.In this study, we obtained anti-NTR nanobodies against TRN1, Xpo4, Xpo7, and CAS. Our aim was to identify nanobodies, and prepare nanobody fusions, that impede nuclear pore-passage of the targeted NTR and thus, interrupt a given transport cycle. These nanobody fusions were observed to inhibit the partition of NTR/cargo complexes into a reconstituted FG phase. We also observed that the nanobodies and nanobody fusions inhibit NTR transport in permeabilized cells.

Table of contents

• Thesis Committee and Examination Board Members
• Epigraph
• List of Figures
• List of Tables
• Summary
• 1 Introduction
• 1.1 Compartmentalization of eukaryotic cells
• 1.2 Nuclear pore complex
• 1.3 Permeability barrier of the NPC
• 1.4 Nuclear transport receptors
• 1.5 Overview of selected NTRs
• 1.6 Tools to investigate NTR mediated transport pathways
• 1.7 Nanobodies
• 1.8 Aim of this study
• 2 Results
• 2.1 Biochemical characterization of anti-NTR nanobodies
• 2.2 In vivo validation of Nb-mediated NTR pathway inhibition
• 2.3 Inhibition of NTR-facilitated transport through NPCs inpermeabilized cells
• 2.4 Tag-Nbs to interrupt NTR-facilitated transport through NPCsin permeabilized cells
• 2.5 Impeding NTR-facilitated transport into reconstituted FGhydrogels
• 3􀀁Discussion
• 3.1 Functional classification of the selected anti-NTR Nbs
• 3.2 Tag-Nbs: the good, the bad, and the redundant
• 3.3 Future Outlook
• 4 Materials and Methods
• 4.1 Chemicals, proteins, and reagents
• 4.2 Molecular cloning
• 4.3 Protein purification
• 4.4 Binding assays
• 4.5 In vivo experiments
• 4.6 Permeabilized cell assays
• 4.7 Experiment using FG phase hydrogels
• References
• Acknowledgements