Single-Molecule Studies on Nuclear Pore Complex Structure and Function

Abstract

Nuclear pore complexes (NPCs) are large macromolecular gateways embedded in the nuclear envelope of Eukaryotic cells that serve to regulate bi-directional trafficking of particles to and from the nucleus. NPCs have been described as creating a selectively permeable barrier mediating the nuclear export of key endogenous cargoes such as mRNA, and pre-ribosomal subunits as well as allow for the nuclear import of nuclear proteins and some viral particles. Remarkably, other particles that are not qualified for nucleocytoplasmic transport are repelled from the NPC, unable to translocate. The NPC is made up of over 30 unique proteins, each present in multiples of eight copies. The two primary protein components of the NPC can be simplified as scaffold nucleoporins which form the main structure of the NPC and the phenylalanine-glycine (FG) motif containing nucleoporins (FG-Nups) which anchor to the scaffold and together create the permeability barrier within the pore. Advances in fluorescence microscopy techniques including single-molecule and super-resolution microscopy have made it possible to label and visualize the dynamic components of the NPC as well as track the rapid nucleocytoplasmic transport process of importing and exporting cargoes. The focus of this dissertation will be on live cell fluorescence microscopy application in probing the dynamic components of the NPC as well as tracking the processes of nucleocytoplasmic transport.