MODIFIED ABSTRACT The enclosure of genetic information in the nucleus is one of the great hallmarks of evolution, but creates the necessity for dedicated portals through which folded proteins and protein/nucleic acid complexes can cross the nuclear envelope (NE). The nuclear pore complex (NPC), a cylindrical supramolecular structure embedded in circular pores permeating the NE, is the sole gateway for passage through the NE and can accomplish the selective bidirectional transport of macromolecules of up to ~40 nm in diameter at a rate of several hundred events per second. Beyond its primary role in nucleocytoplasmic transport, the NPC also contributes to additional modes of gene regulation for example through direct interaction with the transcription and mRNA export machineries. The NPC thus represents an essential organelle for all eukaryotic life and, accordingly, NPC dysfunction has been associated with various forms of human disease. Architecturally, the NPC consists of a central symmetric core to which asymmetric components called cytoplasmic filaments and nuclear basket are attached. The NPC is built from ~34 different proteins termed nucleoporins that are each present in multiple copies such that the entire assembly reaches the extraordinary mass of ~110 MDa in humans. Nucleoporins are organized into distinct subcomplexes which constitute physiological building blocks of the intact NPC in vivo. To determine the atomic architecture of the NPC, my group has been pursuing a divide-and-conquer approach, in which we have mapped nucleoporin interactions, reconstituted recombinant nucleoporin complexes and determined their crystal structures to be fit into cryo-electron tomographic reconstructions of the intact NPC. In this way, we achieved a near-atomic composite structure of the ~60MDa human NPC symmetric core in the previous grant period. Building on this progress, we now propose to expand our structural characterization to still unresolved parts of the NPC and to use our already gained knowledge to address fundamental NPC-associated cell biological questions. Specifically, we plan to elucidate the molecular interactions in the NPC?s inner ring that are essential for the formation of its central transport channel, and between the symmetric core and transmembrane NPC components that are essential for NPC anchoring in the NE pores. The outcome of the proposed research is expected to greatly increase our understanding of the molecular mechanisms by which the NPC regulates nucleocytoplasmic transport and associated cellular processes, while simultaneously creating a mechanistic basis for currently untreatable ?nup diseases.? Furthermore, the methodologies developed herein will serve as a paradigm for the characterization of other essential cellular mega-assemblies as large, flexible and complex as the NPC whose functional mechanisms have remained elusive due to lack of structural insight.
In human cells, the hereditary material is sequestered in a separate compartment, which is accessible only through so-called nuclear pore complexes (NPCs). NPCs constitute massive transport channels through which traffic is tightly regulated and changes to NPC components have been linked to a diverse set of human diseases, such as aggressive forms of leukemia. Our research seeks to enhance our understanding of the NPC in health and disease by creating a molecular snapshot of the NPC that depicts its atomic architecture.
| Hoelz, André; Glavy, Joseph S; Beck, Martin (2016) Toward the atomic structure of the nuclear pore complex: when top down meets bottom up. Nat Struct Mol Biol 23:624-30 |
| Lin, Daniel H; Stuwe, Tobias; Schilbach, Sandra et al. (2016) Architecture of the symmetric core of the nuclear pore. Science 352:aaf1015 |
| Stuwe, Tobias; Bley, Christopher J; Thierbach, Karsten et al. (2015) Architecture of the fungal nuclear pore inner ring complex. Science 350:56-64 |
| Stuwe, Tobias; Lin, Daniel H; Collins, Leslie N et al. (2014) Evidence for an evolutionary relationship between the large adaptor nucleoporin Nup192 and karyopherins. Proc Natl Acad Sci U S A 111:2530-5 |
