Live-cell 3D mapping reveals the organized architecture of FG-nucleoporins within the nuclear pore complex

This is the authors' abstract. We don't add key points for this paper.

The nuclear pore complex (NPC) governs selective nucleocytoplasmic exchange, yet the spatial organization of its intrinsically disordered FG nucleoporins (FG-Nups) in living cells has remained unresolved. Here we reconstruct the three-dimensional distributions of all eleven mammalian FG-Nups within individual NPCs in live cells by combining high-speed 2D single-molecule tracking with post-localization 2D-to-3D transformation, validated by Monte Carlo simulations and independent 3D MINFLUX measurements. The resulting map reveals that the FG barrier is not a uniform disordered meshwork but a stratified architecture. Five centrally anchored FG-Nups form an annular structure surrounding a sparsely occupied axial corridor, whereas six asymmetrically positioned FG-Nups extend flexibly beyond the scaffold to generate dynamic caps at the cytoplasmic and nucleoplasmic faces. Nup214 and Nup153 switch between open and closed axial conformations, whereas Nup98 spans nearly the full pore length and overlaps with most other FG-Nups, linking the major FG zones across the barrier. Together, these features define an adaptable capped channel extending up to ~270 nm across the nuclear envelope, with a ~ 40 nm annular central region and FG extensions reaching ~80–130 nm into the cytoplasm and ~60–100 nm into the nucleoplasm. Transport-route measurements in the same live-cell system show that passive diffusion preferentially traverses the relatively FG-poor central corridor, whereas facilitated transport follows peripheral FG-rich pathways. These findings provide a unified structural framework for NPC selectivity and support a model in which distributed, position-dependent FG architectures, rather than a single central barrier, organize transport. 3D imaging in living cells reveals how nuclear pore FG-Nups organize: five central FG-Nups form a ring around a sparse axial corridor, while six asymmetric FG-Nups form dynamic caps, together directing diffusion and selective transport.