Passive macromolecular diffusion through nuclear pore complexes (NPCs) is thought to decrease dramatically beyond a 30-60-kD size threshold. Using thousands of independent time-resolved fluorescence microscopy measurements in vivo, we show that the NPC lacks such a firm size threshold; instead, it forms a soft barrier to passive diffusion that intensifies gradually with increasing molecular mass in both the wild-type and mutant strains with various subsets of phenylalanine-glycine (FG) domains and different levels of baseline passive permeability. Brownian dynamics simulations replicate these findings and indicate that the soft barrier results from the highly dynamic FG repeat domains and the diffusing macromolecules mutually constraining and competing for available volume in the interior of the NPC, setting up entropic repulsion forces. We found that FG domains with exceptionally high net charge and low hydropathy near the cytoplasmic end of the central channel contribute more strongly to obstruction of passive diffusion than to facilitated transport, revealing a compartmentalized functional arrangement within the NPC.
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Acknowledgments We thank Liesbeth Veenhoff and Patrick Onck for helpful discussion, sharing of unpublished data, and critical reading of the manuscript; and Laura Terry, who assisted in the assembly of strains for these studies. This work was supported by National Institutes of Health grants P41 GM109824 (to M.P. Rout and A. Sali), R01 GM112108 (to M.P. Rout and A. Sali), U01 GM098256 (to M.P. Rout and A. Sali), and R01 GM071329 (to M.P. Rout).
© 2016 Timney et al.