Shining Light on the ‘Dark Matter’ of the Nuclear Pore Complex

Abstract

The nuclear pore complex (NPC) is the gatekeeper for the nucleocytoplasmic transport of macromolecules in eukaryotic cells. The centralchannel of the NPC is filled with intrinsically disordered proteins (IDPs) enriched in phenylalanine glycine (FG) residues, known as FG-Nups. Due to current technical limitations, how the FG-Nups line the NPC channel and form a selective permeability barrier for transport, still remains elusive. Here we develop a novel high-resolution imaging tool that allows direct visualization of FG-Nups in situ. We generate artificial organelles in the cell that allow to exclusively expand the genetic code for targeted FG-Nups. We use them to incorporate non-canonical amino acids into FG-Nups with single-residue precision, which we subsequently label with small, photostable fluorescent dyes via click-chemistry. This labeling method ensures minimal interference with the structures and biological activities of the labeled FG-Nups. By synergistically combining this labeling method with a double strategy for imaging (i.e. super-resolution microscopy and fluorescence lifetime imaging microscopy), we can bridge the resolution boundary between single-molecule and super-resolution techniques, and acquire complementary spatial information about FG-Nups in situ. This work paves the way for better understanding the conformations, dynamics, and functions of FG-Nups in the cell. The developed novel tool is generally applicable to study the plasticity and functions of other IDPs, filling the current technology gap in the field.