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Fluorescence correlation spectroscopy of intact nuclear pore complexes.

ABSTRACT: 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-?1 (Kap?1) 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 Kap?1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions.

SUBMITTER: Cardarelli F

PROVIDER: S-EPMC3175086 | biostudies-other | 2011 Aug

REPOSITORIES: biostudies-other

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Fluorescence correlation spectroscopy of intact nuclear pore complexes.

Cardarelli Francesco F Lanzano Luca L Gratton Enrico E

Biophysical journal 20110801 4

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 t ...[more]

PMID: 21843462

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