Figure 1.

Imaging of cargos before, during, and after their interaction with NPCs. (A) Select video frames for two ICs that interacted with the same NPC in a permeabilized HeLa cell. The large image is a false-color bright-field image showing the nucleus (N) and the cytoplasm (C). The smaller images, corresponding to the boxed region in the large image (same scale), are 2-ms video frames for a cargo molecule that transported into the nucleus (top) and for a cargo molecule that underwent abortive transport, returning to the cytoplasm (bottom). These smaller images consist of SMF signals (green) overlaid onto a bright-field image (red) obtained with the same camera. The circular laser illumination area for SMF approximately filled the boxed region. The fluorescent cargo was NLS-2xGFP(4C) labeled with four Alexa 647 molecules. Numbers correspond to time in milliseconds. [fluorescent cargo] = 0.1 nM; [Imp α] = [Imp β] = 0.5 μM; [Ran] = 2 μM; [GTP] = 1 mM; [NTF2] = 1 μM. Bar, 5 μm. (B) Trajectories for the two interaction events shown in A with all frames included. The trajectory of the cargo molecule that transported into the nucleus (entry event) is shown in red, and the trajectory of the cargo molecule that underwent abortive transport (abortive event) is shown in blue. The beginning of each trajectory is identified by an asterisk. The red curve is the experimentally determined position of the NE from the bright-field image; the green and black curves are for reference at −100 and +100 nm from the NE, respectively.