Fig. 3: Simultaneous dual-channel single-molecule tracking of both termini of LBR.

a Transport route of LBR tagged with EGFP on the N terminus and RFP on the C terminus (GFP-LBR-RFP). (i) Schematic cartoon representation of each INM protein (orientation N-terminus to the left and C-terminus to the right). The depicted membrane represents a single bilayer of the NE. (ii) 2D spatial locations (projected) of each INM protein candidate. In the X dimension of the Cartesian graph, 0 represents the axial center of the NPC, and in the Y dimension, +20 represents the ONM, and −20 represents the INM. (iii) A normalized 2D probability density map showing the regions where the projected single-molecule localizations had the highest and lowest density. (iv) 3D probability density map generated by using the 2D to 3D transformation algorithm⁵², with regions of highest to lowest density (each color bar shows the gradient color change between regions of high and low density). b Typical co-tracking trajectories of GFP-LBR-RFP in the NPC show that the EGFP and RFP tagged termini move in the same direction with close proximity to each other. Green dots represent EGFP, red dots represent RFP. These trajectories have been overlaid on top of the 2D probability density map of GFP-LBR-RFP. c Histogram of ~300 different dual tracked trajectories shows that the average distance between EGFP tagged N terminus and RFP tagged C terminus is ~18 nm during transit through the NPC. No FRET was observed during these experiments further confirming that the N and C termini do not transit in the same channel. d Cryo-EM image of the NPC with a cartoon representation of how LBR transits from the ONM to INM (figure modified from ref. ⁵⁵ with permission).