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No methods proposed thus far have the sensitivity to measure the transport of single molecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with real-time tracking of the center of mass of single NPCs in live, unperturbed cells allows us to detect the transport of single molecules in a reference system of a pore with high temporal (millisecond) and spatial (limited by diffraction) resolution. We find that the transport of the classical receptor karyopherin-β1 (Kapβ1) is regulated so as to produce a peculiar distribution of characteristic times at the NPC. This regulation, which is spatially restricted to the pore, depends on the properties and metabolic energy of Kapβ1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Nuclear pore complexes (NPCs) are the gateways for nucleocytoplasmic exchange. Because single molecules undergo rapid transport, it is challenging to follow their motion in live cells. Hence fundamental questions remain in regard to the nanomechanical basis of selective gating of molecules through the NPC in live cells.We set out to address these issues by a combination of fluorescence correlation spectroscopy (FCS) and real-time tracking of the center of mass of single NPCs in live cells. The center of mass tracking allows us to create an “Einstein trap” in which the thermal motion of the entire pore is compensated so that we observe the shuttling of single molecules in the reference frame of the pore. Using this setup we demonstrate that the transport of Karyopherin-β1 (Kapβ1) receptor is regulated to produce a characteristic narrow correlation time within the NPC, which is the signature of directed-motion events. We also show that the back and forth components of Kapβ1 transport are coupled by energy-consuming processes. Analogously, the dynamics of nucleoporin-153 (Nup153) at the nanoscale is characterized by a similar correlation pattern between two separate positions within the NPC. By means of the pair correlation function (pCF) analysis we separate the two components of Nup153 exchange: a fast collapse into compact conformations (cytoplasm-to-nucleus) and a slightly slower release into extended conformations (nucleus-to-cytoplasm). We demonstrate that this signature activity is directly linked to the functional import of classical transport receptors and cargoes.Thus, we propose that the selective gating through intact NPCs may be powered by spring-like molecular engines As far as we know, this is the first time that a selective gating model is proposed on the basis of experimental evidences obtained in live, unperturbed cells.Work supported by NIH-P41 P41-RRO3155,P50-GM076516.

Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleocytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to tackle this issue in intact cells by an established combination of fluorescence correlation spectroscopy and real-time tracking of the center of mass of single NPCs. We find the dynamics of nucleoporin Nup153 to be regulated so as to produce rapid, discrete exchange between two separate positions within the NPC. A similar behavior is also observed for both karyopherinβ1 transport-receptor and cargoes destined to nuclear import. Thus, we argue that directed Nup-mediated molecular motion may represent an intrinsic feature of the overall selective gating through intact NPCs.