Nomethods proposed thus far have the sensitivity tomeasure the transport of singlemolecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with realtime 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 systemof a pore with high temporal (millisecond) andspatial (limited by diffraction) resolution.Wefindthat the transport of the classical receptor karyopherin-b1 (Kapb1) 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 Kapb1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions.
Fluorescence correlation spectroscopy of intact nuclear pore complexes
LUCA LANZANO';
2011-01-01
Abstract
Nomethods proposed thus far have the sensitivity tomeasure the transport of singlemolecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with realtime 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 systemof a pore with high temporal (millisecond) andspatial (limited by diffraction) resolution.Wefindthat the transport of the classical receptor karyopherin-b1 (Kapb1) 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 Kapb1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions.File | Dimensione | Formato | |
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