Single Molecule Fluorescence Studies of Nucleocytoplasmic Transport

Single Molecule Fluorescence Studies of Nucleocytoplasmic Transport

Cellular membranes serve to compartmentalize biochemical reactions to specific microenvironments. However, communication and exchange of molecules between the various cellular compartments is essential for cell vitality. The transport of various molecules across membranes requires elaborate molecular machines to prevent compromising the membranes' role as a permeability barrier. The nuclear pore complex (NPC), which spans the double-membrane nuclear envelope, fulfills the essential physiological role of facilitating and regulating the extensive trafficking of proteins and RNAs between the cytoplasm and nucleoplasm in eukaryotic cells. It is unique among protein transporters because it regulates the trafficking of diverse substrates in both directions and it can accommodate the passage of molecules as large as ribosomal subunits. We have pioneered the application of single molecule fluorescence and particle tracking techniques to visualize nuclear transport processes occurring in real time in permeabilized and live cells. Diffusing molecules are directly observed in twodimensions with 2 ms and ~40 nm resolution. NPC translocation time and transport efficiency are strongly dependent on conditions, potentially indicating that these parameters can be regulated in response to cellular transport needs. Our results are consistent with a model in which the basic mechanism of cargo migration through and within the NPC is unbiased diffusion, but that bias can be introduced by changes in pore structure or the location at which transport complexes are assembled and disassembled.