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MIRAVA POLYSCOPE – All in one and on for all: the perfect image
Science beyond Barriers

abberior instruments

Cell Biology

2023
bioRxiv

Nucleoporin-binding nanobodies that either track or inhibit nuclear pore complex assembly

Authors:

Mireia Solà Colom, Zhenglin Fu, Thomas Güttler, Sergei Trakhanov, Vasundara Srinivasan, Kathrin Gregor, Tino Pleiner, Dirk Görlich

Keywords:

nuclear pore complex, NPC, nanobodies, NPC biogenesis, nucleoporin, Nup

Abstract:

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact NPCs from frog to human. We further report a simplified assay that directly tracks postmitotic NPC assembly by added labeled anti-Nup nanobodies. In interphase, NPCs are inserted into a pre-existing nuclear envelope. This makes it difficult to monitor this process as newly-assembled NPCs must be distinguished from pre-existing ones. We solved this problem by inserting Xenopus NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We also asked whether anti-nucleoporin (Nup) nanobodies could serve as NPC assembly inhibitors. A first generation, selected from immune libraries against Xenopus Nups, comprised only bright stainers of intact NPCs but no inhibitors, perhaps because the immune response was biased towards non-conserved and, thus, functionally-irrelevant epitopes. To overcome this bias, we selected for crossreactivity between Xenopus and human Nups and obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93-α-solenoid in recruiting Nup358 and the Nup214·88·62 complex, and for Nup155 and the Nup98 autoproteolytic domain in NPC-scaffold assembly. The latter suggests an assembly checkpoint linking pore formation to permeability barrier assembly.

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