- 136 pages
- English
- PDF
- Available on iOS & Android
About this book
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.
Frequently asked questions
- Essential is ideal for learners and professionals who enjoy exploring a wide range of subjects. Access the Essential Library with 800,000+ trusted titles and best-sellers across business, personal growth, and the humanities. Includes unlimited reading time and Standard Read Aloud voice.
- Complete: Perfect for advanced learners and researchers needing full, unrestricted access. Unlock 1.4M+ books across hundreds of subjects, including academic and specialized titles. The Complete Plan also includes advanced features like Premium Read Aloud and Research Assistant.
Please note we cannot support devices running on iOS 13 and Android 7 or earlier. Learn more about using the app.
Information
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
- 3Discussion
- 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