Mapping Transport Pathways Through Nuclear Pores Using 3D Super-Resolution Microscopy
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abstract
Nuclear pore complexes (NPCs) mediate the bi-directional transport of proteins, RNAs and ribonucleoproteincomplexes across the double-membrane nuclear envelope of eukaryotic cells. Consequently, NPCs areessential for the ability of many biosynthetic, signaling and gene regulatory processes to maintain cellular healthand viability. Protein mislocalization due to recognition defects or altered NPC structure and function is linkedto 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 nucleartransport factors have been identified and extensively studied, the mechanism(s) by which bi-directional transportoccurs without clogging the pore remains unknown. The NPC is an octagonally symmetric approximatelycylindrical structure with an hourglass-shaped central pore that has an internal minimal diameter of ~50 nm inhumans. Occluding this pore and decorating its exits is a network of > 200 mobile intrinsically disorderedpolypeptides. Thousands of phenylalanine-glycine (FG) repeat motifs within this disordered polypeptide network(FG-network) are binding sites for the nuclear transport receptors (NTRs) that carry cargos through the NPC. Atsteady-state, at least ~100 NTRs are asymmetrically distributed throughout the FG-network. The heterogeneousand dynamic NTR/FG-network establishes a permeability barrier while simultaneously providing pathways forthe translocation of import and export complexes of a wide range of sizes, affinities and surface properties.Multiple preferred paths through the ~50 nm diameter pore are predicted for typical cargo complexes of ~5-10nm. The extent of overlap in such pathways and the possibility of dynamic regulation remains largely unexploreddue to the absence of technological tools to dissect these pathways with the necessary spatial and temporalresolution. To address this deficiency, three-dimensional (3D) super-resolution single molecule fluorescencemicroscopy and single particle tracking techniques will be used to explore various transport pathways todetermine the extent of their structural and functional intersections. The Specific Aims are: 1) to develop a fastsuper-resolution 3D microscopy approach to characterize the translocation pathways through functional NPCs;and 2) to develop 3D distribution maps for NTRs and cargos undergoing transport. Aim 1 seeks to bring togethermultiple existing technologies to enable high precision 3D super-resolution microscopy on NPCs. Aim 2 seeksto then apply this technology to establishing a map of various transport pathways through the FG-network. Thiswork will establish whether discrete transport pathways within the FG-network exist, and thus, enhance transportefficiency by minimizing interactions between import and export cargos. While the primary goal of the proposedwork is to develop a comprehensive understanding of competing transport reactions and the potentialimplications for inhibition and regulation, the super-resolution microscopy technologies and algorithmsdeveloped are expected to comprise a necessary toolkit for the field as well as to have broad applicability.
