Figure 3

STVs and PTVs pause at the same sites. (A) Time-lapse images of a segment of axon expressing both synaptophysin-mcherry (magenta) and GFP-bassoon (green). In each panel, fluorescence signals from the two channels are overlaid, and co-localization is indicated by white. The orange box outlines a site where a PTV is paused. The arrow tracks a STV that then pauses at that site. Scale bars: 5 μm. (B) Kymographs showing two examples of STVs and PTVs that pause at the same sites. On the ordinate axis, one pixel corresponds to 10 s. Bottom panel: overlay of STV and PTV fluorescence. Orange boxes outline three shared pause sites. (C) Simulations of model axons were performed by randomizing initial positions of vesicles while maintaining movement and pausing characteristics observed for the original experimental vesicles. Diagrams show the superimposed locations of all pause sites for individual STVs and PTVs in model axons. Three simulations of the same experimental data are shown. STV pause sites are indicated with a dot while PTV pause sites are indicated with a plus sign. Each color represents an individual vesicle. Cyan and magenta boxes outline the pause sites of the same PTV and STV, respectively, in each model axon. For each axon imaged, 100 such simulations were performed, allowing estimation of the degree of co-transport and co-pausing expected from chance alone. (D) Plot illustrating the percentage of PTV (green) and STV (magenta) pause sites that are shared with STVs and PTVs, respectively. The fraction of shared sites is much higher than predicted by chance (via simulations, light green and light magenta). (E) A large majority of PTVs that encountered STV pause sites paused at those sites. Similarly, most STVs that encountered PTV pause sites then paused at those sites. Error bars display the 95% confidence interval.