Nucleoporin-binding nanobodies that either track or inhibit nuclear pore complex assembly
Mireia Solà Colom, Zhenglin Fu, Thomas Güttler, Sergei Trakhanov, Vasundara Srinivasan, Kathrin Gregor, Tino Pleiner, Dirk Görlich
nuclear pore complex, NPC, nanobodies, NPC biogenesis, nucleoporin, Nup
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.