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Simple rules for passive diffusion through the nuclear pore complex.

ABSTRACT: 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.

SUBMITTER: Timney BL

PROVIDER: S-EPMC5057280 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex.

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Project description:The nuclei were isolated from HeLa cells with nuclear extraction reagents (Thermo, NE-PER™) and suspended in 1 X PBS buffer (pH 7.4). The suspension of intact nuclei was crosslinked by adding 5 mM BSP (chemical structure shown in Figure S1, with the maximum Cα-Cα distance restraints of 28 Å ) every 20 min for 3 times at room temperature and quenched with 50 mM ammonium bicarbonate. To further improve the identification coverage of NRPC, the cross-linker of 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride (DMTMM) was used, combining with the amino-reactive BSP for the crosslinking of nuclear envelopes (NEs) isolated from HeLa cells with commercial kit (invent, NE-013™), respectively.

2021-09-14 | PXD028457 |

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