Using nanobodies to dissect the role of membrane proteins in nuclear assembly

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• Publisher: Cuvillier Verlag
• Year: 2024
• eBook ISBN:
  9783736969322
• Print ISBN:
  9783736979321
• Edition: 1

Organisms undergoing open mitosis disassemble their nuclei during cell division. Upon returning to interphase, the nuclear en-velope (NE) and nuclear pore complexes (NPCs) must reassemble to reestablish cell compartmentalization and nucleocytoplasmic icles, ER-embedded membrane proteins, membrane fusion, and NPC assembly from soluble nucleoporin complexes. This complex process involves numerous players that need to coordinate in space and time, and is so far little understood.To explore NE and NPC assembly mechanisms and the roles ofransmembrane Protein 209), we generated target-specific nanobodies (Nbs). Some Nbs allowed protein visualization via confocal and super-resolution microscopy, while others disrupted essential protein-protein interactions, hindering functional nuclei assembly. Nbs targeting the GTPase and ER integral membrane protein Atlastin impeded import-competent nuclei assembly in vitro, illustrating the Nbs' utility in disrupting membrane protein functions. Conversely, Nbs against the Lamin B Receptor facilitated the tracking of the proteins' recruitment during NE assembly via immunofluorescence microscopy.Orthogonal Nbs, targeting the Transmembrane Protein 209 (TMEM209), allowed us to follow its recruitment during NE assembly, and to pinpoint its localization by super-resolution microscopy. We showed that TMEM209 co-localizes with NPC proteins and established its relative position in the NPC with regard to some of the NUPs, and thus identified TMEM209 as a novel transmembrane NUP.

Table of contents

• Epigraph
• Contents
• List of Figures
• List of Tables
• Summary
• Introduction
• 1.1 The architecture of the endoplasmic reticulum
• 1.2 The nuclear envelope
• 1.3 Nuclear Pore Complex
• 1.4 NE and NPC breakdown and reassembly
• 1.5 Atlastin
• 1.6 Lamin B Receptor
• 1.7 Transmembrane Protein 209
• 1.8 Single domain antibodies (Nanobodies)
• 1.8.1 Antibodies and derived fragments
• 1.9 Aim of this study
• Results
• 2.1 Nanobodies to study Atlastin
• 2.2 Nanobodies to study the Lamin B Receptor
• 2.3 Characterization of Transmembrane Protein 209
• Discussion
• 3.1 Anti-ATL nanobody as a valuable tool to study ATLmediatedmembrane fusion
• 3.2 Anti-LBR tudor domain nanobodies for the visualizationof the NE and LBR
• 3.3 TMEM209 is associated with the NPC
• Materials and Methods
• 4.1 Molecular cloning
• 4.2 Protein expression and purification
• 4.3 Genome editing in human cell lines
• 4.4 Nanobody library construction and selection
• 4.5 Nanobody preparation and characterization
• 4.6 Xenopus egg extract preparation
• 4.7 Reconstituting postmitotic NE/NPC assembly
• 4.8 Reconstituting interphasic NPC assembly
• 4.9 Characterization of proteins by immunofluorescence microscopy(IFM)
• 4.10 Protein crystallography
• References
• Abbreviations
• Acknowledgements