Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside the sub-micrometer supramolecular complex. Here we combined super-resolution SPEED microscopy and nanobody specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveals both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provides evidence for their copy numbers of 8, 32, and 16 respectively per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes.