Fluorescence anisotropy reveals order and disorder of protein domains in the nuclear pore complex.
We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport.
Mattheyses
Alexa L
AL
Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York, USA.
Kampmann
Martin
M
Atkinson
Claire E
CE
Simon
Sanford M
SM
D021581:Active Transport, Cell Nucleus; D003593:Cytoplasm / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D004718:Saccharomycetales / Q000166:cytology; D013382:Subtraction Technique
D005656:Fungal Proteins; D028861:Nuclear Pore Complex Proteins