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Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy
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Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy
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Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy
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Journal Article
Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy
2011
Overview
Polarized fluorescence microscopy is used to investigate the orientation of nucleoporins tagged with GFP within the nuclear pore complex in live yeast and mammalian cells, by analyzing the anisotropy pattern of the fluorophore. This approach can be applied to study other protein assemblies.
The nuclear pore complex (NPC) perforates the nuclear envelope to facilitate selective transport between nucleus and cytoplasm. The NPC is composed of multiple copies of ∼30 different proteins, termed nucleoporins, whose arrangement within the NPC is an important unsolved puzzle in structural biology. Various alternative models for NPC architecture have been proposed but not tested experimentally in intact NPCs. We present a method using polarized fluorescence microscopy to investigate nucleoporin orientation in live yeast and mammalian cells. Our results support an arrangement of both yeast Nic96 and human Nup133–Nup107 in which their long axes are approximately parallel to the nuclear envelope plane. The method we developed can complement X-ray crystallography and electron microscopy to generate a high-resolution map of the entire NPC, and may be able to monitor nucleoporin rearrangements during nucleocytoplasmic transport and NPC assembly. This strategy can also be adapted for other macromolecular machines.
Publisher
Nature Publishing Group US,Nature Publishing Group
