Reconstructing Spatial Transport Distributions In The Nuclear Pore Complex From 2D Images -- How Reliable Is It?
Imaging single molecules in living cells and reconstituted cell systems has resulted in a new understanding of the dynamics of nuclear pore complex functions over the last decade. It does, however, fall short on providing insights into the functional relationships between the pore and nucleocytoplasmic cargo in three-dimensional space. This limited ability is the result of insufficient resolution of optical microscopes along the optical axis and limited fluorescent signal due to the short timescales involved in nuclear transport (fractions of a second). To bypass current technological limitations, it was suggested that highly time-resolved 2D single molecule data could be interpreted as projected cargo locations and could subsequently be transformed into a spatial cargo distribution by assuming cylindrical symmetry 1. Such cargo distributions would provide valuable insights into the NPC-mediated transport in cells. This method, termed 3D-SPEED, has attracted large interest inside and beyond the nuclear pore field, but has also been sharply critiqued for a lack of critical evaluation. Here we present such an evaluation, testing the robustness, reconstruction quality and model-dependency.