Structure and Organization of Nuclear Pore Proteins

The nuclear pore complex (NPC) is a large macromolecular assembly that controls the flow of molecules between the nucleus and the cytoplasm. Elucidating the structure and organization of this complex will be crucial to understanding the mechanism of nucleocytoplasmic transport, but due to its size, the NPC presents a challenge to structure determination. There are electron microscopy (EM) structures of the entire NPC, and high resolution crystal structures of some of the individual components have been solved, but the arrangement of proteins within the overall complex remains unknown. An approach to solving structures of macromolecular complexes is to fit high resolution structures into an EM map, but the resolution of the EM structures of the NPC is too low to do so accurately. Additionally, approximately one third of the proteins making up the NPC contain unstructured regions that are not amenable to traditional structural techniques. These proteins (the FG nups) contain multiple phenylanine-glycine repeats, which bind cargo and transport it through the pore. Although many conflicting models for transport have been proposed, the mechanism still remains unclear. Polarization fluorescence microscopy can be used to determine the orientation, mobility, and proximity of molecules. In this work, polarization microscopy techniques were developed to study aspects of the structure and organization of the NPC in live cells. In the first part of this project, structural proteins were tagged with GFP in a defined position and their orientation within the NPC was determined. This showed that Nic96 is arranged in a head-to-tail ring around the perimeter of the NPC lumen and that the Yshaped Nup107/160 complex is oriented with its long axis parallel to the plane of the nuclear envelope. This orientation technique can be used to help bridge the gap between EM and crystal structures of the NPC and its constituent parts. In the second part of this work, the FG nups were tagged with GFP and polarization microscopy was used to determine their organization within the NPC. This showed that some of the FG nups adopted an ordered conformation which was unpredicted in any models for transport. FG nups which are located in center of the NPC lumen were more ordered than those at the periphery. This order was conserved between mammalian and yeast homologues of the same protein, suggesting functional relevance. Factors which affect the ordering of the FG domains were investigated. Domain swapping experiments showed that relocating the FG domains of the central nups to the cytoplasmic side caused them to become disordered, suggesting a role for local interactions in their organization. Removal of the FG repeats had no effect on the behavior of Nup62, only a limited effect on Nup54, and prevented Nup98 from localizing to the pore, indicating that FG-FG interactions are not dominant in determining order. Ameliorating active transport, and disassociation of cargo from the NPC had no effect on the behavior of the FG domains. Taken together, these data led to development of a model for nematic ordering of the FG domains, in which dense packing and alignment of these domains within the NPC lumen leads to organizational, but not positional, order