Reconstructing Spatial Features of Nucleocytoplasmic Transport using Projected Cargo Localizations

Time-resolved single molecule experiments have provided tremendous information pertaining to molecular dynamics and localization over the last decade. Biological processes take place in a light sensitive environment, on time scales from sub-millisecond (ms) to hours and length scales from nanometer to millimeter, presenting a number of experimental challenges. One such challenge is the need for speed in image acquisition to correctly follow single molecule movements during translocation through the nuclear pore complex (NPC). Translocation through the central channel has repeatedly been reported to be faster than 20 ms.Biochemical and structural data of the components that make up the NPC have led to the question if specific spatial transport routes exist within the NPC in vivo. Millisecond time resolutions and three-dimensional spatial resolution in the range of only a few nm are needed in order to resolve the path traveled by transport receptors and cargos by single molecule real-time microscopy. These imaging requirements are challenging and are not met by current technology; however, it was suggested by Ma & Yang that highly time-resolved 2D tracking data can be interpreted as projected cargo densities and subsequently transformed into a 3D cargo distribution. Such distributions would provide valuable insights into the function of NPC mediate transport in cells. Here we present a thorough analysis of the conditions needed for this method to work for the nuclear pore complex and the limits to which the results may be interpreted.