title: Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy.
abstract: 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.
authors.affiliation: Laboratory of Cell Biology, The Rockefeller University, New York, New York, USA. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Kampmann
authors.forename: Claire E
authors.initials: CE
authors.lastname: Atkinson
authors.forename: Alexa L
authors.initials: AL
authors.lastname: Mattheyses
authors.forename: Sanford M
authors.initials: SM
authors.lastname: Simon
mesh_terms: D000818:Animals; D005454:Fluorescence Polarization / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D022022:Nuclear Pore / Q000737:chemistry*; D028861:Nuclear Pore Complex Proteins / Q000032:analysis*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis
chemical_list: D028861:Nuclear Pore Complex Proteins; D029701:Saccharomyces cerevisiae Proteins