Nucleoporin-binding nanobodies that either track or trap uclear pore complex assembly

Abstract

Nuclear pore complexes (NPCs) are large multi-protein assemblies that control the transport of macromolecules between the nucleus and the cytoplasm. Their formation from ~ 1,000 individual polypeptides is an impressive and still enigmatic example of self-assembly. NPCs are inserted during interphase into an intact nuclear envelope (NE), which requires a pore-forming fusion event between inner and outer nuclear membranes. In higher eukaryotes, NPCs also assemble upon mitotic exit concomitantly with the reformation of the NE. Either pathway is still poorly understood, foremost because it has been very di cult to identify assembly intermediates, put them into temporal order, and characterize them both biochemically and structurally. To solve this problem, we employed immune nanobody (Nb) libraries and a directed phage display strategy to generate Nbs that either trap or track the NPC assembly process. Trapping Nbs prevent essential Nup-Nup interactions, arrest NPC assembly at otherwise short-lived intermediate steps, and might therefore provide so far elusive snapshots of the assembly process. Since essential protein regions are likely preserved through evolution, trapping Nbs bind to conserved Nup epitopes, and allowed to unveil Nup regions that are critical for the NPC assembly process. In contrast, tracking Nbs do not interfere with NPC assembly but bind to epitopes that are exposed on intact NPCs. These Nbs yield bright and speci c uorescent signals when coupled to uorophores, and are thus useful for tracking NPC assembly by uorescence microscopy. Moreover, tracking Nbs are valuable tools to visualize the NPC structure by super-resolution microscopy. Next, we employed the generated Nbs for investigating NPC assembly at the end of mitosis and during interphase, respectively. We looked into postmitotic NPC assembly from Xenopus egg extracts, where trapping Nbs showed to be an attractive alternative to the conventional Nup depletions. To investigate NPC assembly during interphase, we established a novel in vitro assay that exploits the species-speci city of the tracking Nbs to distinguish newly inserted NPCs from `old', pre-existing ones. Combined with the trapping Nbs, this assay allowed to capture and characterize intermediates of interphase NPC assembly.