In vivo imaging of single-molecule translocation through nuclear pore complexes by pair correlation functions.
BACKGROUND
Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. Although there are many studies that look at the traffic in the nucleus and through the nuclear envelope we propose a method to detect the nucleocytoplasmic transport kinetics in an unperturbed cell, with no requirement for specific labeling of isolated molecules and, most important, in the presence of the cell milieu.


METHODS
The pair correlation function method (pCF) measures the time a molecule takes to migrate from one location to another within the cell in the presence of many molecules of the same kind. The spatial and temporal correlation among two arbitrary points in the cell provides a local map of molecular transport, and also highlights the presence of barriers to diffusion with millisecond time resolution and spatial resolution limited by diffraction. We use the pair correlation method to monitor a model protein substrate undergoing transport through NPCs in living cells, a biological problem in which single particle tracking (SPT) has given results that cannot be confirmed by traditional single-point FCS measurements because of the lack of spatial resolution.


CONCLUSIONS
We show that obstacles to molecular flow can be detected and that the pCF algorithm can recognize the heterogeneity of protein intra-compartment diffusion as well as the presence of barriers to transport across NE.
Cardarelli
Francesco
F
Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California Irvine, Irvine, California, United States of America.
Gratton
Enrico
E
D000818:Animals; D001693:Biological Transport, Active; D016466:CHO Cells; D002451:Cell Compartmentation; D006224:Cricetinae; D003412:Cricetulus; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008954:Models, Biological; D057054:Molecular Imaging / Q000379:methods*; D019913:Nuclear Localization Signals / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
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