Copy Number and Function of Individual Disordered Proteins in the Nuclear Pore Complex Revealed by Combining Auxin-Inducible Degron Strategy and High-Speed Single -Molecule Microscopy

Abstract

Intrinsically disordered nucleoporins (Nup) rich in phenylalanine-glycine (FG) repeats form the selectivity barrier in nuclear pore complexes to mediate nucleocytoplasmic transport in eukaryotic cells. However, the definite copy number of an individual FG Nup and its specific roles in mediating nucleocytoplasmic transport remain controversial. Here we demonstrated an approach to count the copy number of individual FG Nup and determine the consequences of the absence of each FG Nup on the nucleocytoplasmic transport kinetics in live cells. First, with CRISPR/Cas9 technique, we generated stable human cell lines expressing FG Nups fused with fluorescent proteins and degron tags, and then utilized auxin-inducible degron (AID) strategies for selective and rapid degradation of individual FG-Nups. Then we used high-speed single-molecule microscopy to directly quantify the copy number for each FG Nup in individual NPCs, as well as track nuclear export of firefly luciferase mRNPs at the presence or absence of a specific FG Nup.Our results revealed significant deviations on the copy number and facilitating roles for some of the FG Nups from previous measurements. For example, we found that in DLD-1 cells, all of the three nuclear basket FG Nups, Nup153, Nup50 and Tpr, have consistently shown an average of 8 copies in over 300 NPCs from 50 live cells, in contrast with previous studies, where 16-32 copies were estimated. Moreover, high-speed single-particle tracking of nuclear export of mRNAs in live cells in the absence of Nup153 at the NPCs, showed approximately a 4-fold reduction in their nuclear export efficiency, while the nuclear export times of successful mRNPs export events with and without the presence of Nup153 in the NPCs showed almost no differences.