title: 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-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.
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California, USA.
authors.forename: Francesco
authors.initials: F
authors.lastname: Cardarelli
authors.forename: Luca
authors.initials: L
authors.lastname: Lanzano
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D028961:beta Karyopherins / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins