Mapping Transport Pathways through Nuclear Pores using 3D Super-Resolution Microscopy

PROJECT SUMMARY Nuclear pore complexes (NPCs) mediate the bi-directional transport of proteins, RNAs and ribonucleoprotein complexes across the double-membrane nuclear envelope of eukaryotic cells. Consequently, NPCs are essential for the ability of many biosynthetic, signaling and gene regulatory processes to maintain cellular health and viability. Protein mis-localization due to recognition defects or altered NPC structure and function is linked to diseases as diverse as primary biliary cirrhosis, amyotrophic lateral sclerosis (ALS), leukemias and cancers, and Alzheimer's and Huntington's diseases. While the protein components of the NPC and many soluble nuclear transport factors have been identified and extensively studied, the mechanism(s) by which bi-directional transport occurs without clogging the pore remains unknown. The NPC has an octagonally symmetric approximately cylindrical structure with an hourglass-shaped central pore that has an internal minimal diameter of ~50-60 nm in humans. Occluding this pore and decorating its exits is a network of > 200 mobile intrinsically disordered polypeptides with thousands of phenylalanine-glycine (FG) repeat motifs that provide binding sites for the nuclear transport receptors (NTRs) that carry cargos through the NPC. At steady-state, up to ~100 NTRs are asymmetrically distributed throughout this FG-network. The heterogeneous and dynamic NTR/FG-network establishes a permeability barrier while simultaneously providing pathways for the translocation of import and export complexes of a wide range of sizes, affinities and surface properties. Multiple preferred paths through the central pore exist. However, the overlap, selectivity, and geometric and functional properties of these translocation conduits are largely unexplored due to the historical absence of technological tools to dissect these pathways with the necessary spatial and temporal resolution. To address this deficiency, a multi-color three- dimensional (3D) super-resolution fluorescence microscopy approach was developed in the last grant period that is capable of determining the position and orientation of individual functional NPCs combined with single particle trajectories of transiting cargo. This approach will be used to determine the structural and functional properties of multiple translocation conduits and the FG-network barrier. The Specific Aims are: 1) to determine the number and nature of protein translocation conduits; and 2) to determine the FG-polypeptide and NTR distributions within the FG-network. Aim 1 seeks to explore the possibility of at least three distinct translocation conduits, whether some of these consist of 8 distinct channels, and whether any are dedicated to either import or export. Aim 2 seeks to determine how the physical arrangement and properties of components of the FG- network are linked to promoting the identified translocation conduits. This work will directly address whether preferred routes through the permeability barrier provide a means to avoid the competition and clogging expected for two-way traffic through the same channel. In addition, improved super-resolution microscopy technologies and algorithms are expected to comprise a necessary toolkit for the field as well as to have broad applicability.