Charge Effects on Nucleocytoplamsic Transport Studied by Single-Molecule Microscopy

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In eukaryotic cells, the transport of genetic material and proteins between nucleus and cytoplasm is mediated by the nuclear pore complexes (NPCs) embedded in the double-membrane nuclear envelope. A highly selective barrier formed by phenylalanine-glycine (FG)-nucleoporin (Nup) with net positive charge in the NPC allows for passive diffusion of signal-independent small molecules (< 40 kDa) and transportin-facilitated translocation of signal-dependent large molecules (up to 50 MDa). Previously it was suggested that the positively charged FG-barrier would inhibit the transport of positively charged transiting molecules. However, the fundamental inhibition mechanism and the detailed pathways for both transport modes remain poorly understood. Here, we employ an innovative single-molecule technique, single-point edge-excitation subdiffraction (SPEED) microscopy, to track fluorescent single molecules with different charges transiting through single native NPCs. We have obtained the transport kinetics and the 3D transport pathways for both passive diffusion of variously charged GFPs (27 kDa) and facilitated translocation of transportin-cargo complexes with different charges. Our results indicate that (i): the positively charged selective barrier in the NPC possesses an axial central conduit for passive diffusion of small molecules with different charges, and allows for transportin-facilitated translocation of differently charged cargos via the peripheral regions around the central conduit; and (ii) the positively charged environment in the NPC significantly affects the transport pathway, transport time and efficiency for both passive and facilitated transport.