Detecting Nuclear Pore Complex assembly in living cells

The formation of nuclear pore complexes (NPCs)--vital gateways regulating nuclear-cytoplasmic transport--is a highly orchestrated process requiring the integration of hundreds of nucleoporins into the nuclear envelope. A major challenge in studying this dynamic assembly process has been the difficult to distinguish newly forming NPCs from their mature counterparts. Here, we present a powerful nanobody-based approach that overcomes this limitation. Using pulsed-labelling mass spectrometry, conventional and super resolution live-cell imaging, and correlative light and electron microscopy, we demonstrate that a nanobody targeting the nucleoporin Nic96 from Saccharomyces cerevisiae selectively binds newly synthesized Nic96 subcomplexes prior to their incorporation into NPCs. Importantly, the nanobody does not disrupt NPC assembly, nuclear transport, cell growth, or lifespan, and shows no genetic interactions with known NPC assembly surveillance pathways--making it an ideal, non-perturbing tool to study NPC biogenesis in yeast. We use the Nic96 nanobody to reveal distinct assembly rates between haploid and diploid cells and to visualize early stages of assembly. We show that newly synthesized nucleoporins accumulate on Pdr16- and Ldo16-positive lipid droplets near the nucleus-vacuole junction. This suggests a regulatory balance between NPC incorporation and nucleoporin sequestration, potentially buffering assembly kinetics or targeting unassembled subunits for degradation. Our nanobody-based pulsed-labelling strategy opens new avenues for dissecting the spatiotemporal regulation of NPC assembly, with implications for understanding aging and diseases linked to NPC dysfunction.