Fig. 10. Behavior of multiple NFs in a single axon. A 146-μm-long segment of an axon was imaged for 5 min. This axon segment contains several gaps in the GFP–NFH fluorescence, thereby permitting detection of the ends of several NFs. A shows selected frames from the sequence in which the ends of eight different NFs are indicated. Six of these (1–4, 6, and7) moved very little or not at all and are highlighted with single arrowheads. One (5) moved anterogradely; its trailing end is indicated with a double arrowhead. The remaining NF (8) moved retrogradely, and its leading edge is indicated with an arrow. See supplementary information for a video of this sequence. B shows cumulative distance plots for five of these NFs, specifically those labeled1, 3, 4, 5, and 8. Note that NFs1, 3, and 4 undergo very little net movement during the sequence and, in fact, spend most of the total time paused, undergoing no detectable movement (to minimize overlap of the data points for the plots for NFs 1, 3, and4, we have shifted them on the y-axis). The anterogradely moving NF (5) shows clear intervals of movement but spends most of the time paused. The retrogradely moving NF (8) first appears in the sequence at 205 sec and moves in a relatively sustained manner until the end of the sequence. The asterisk (inA) identifies an ovoid structure that contains GFP–NFH that moves in a net retrograde direction. This object is much brighter than individual NFs that contain GFP–NFH and is distinctly different in shape. Similar structures have been seen by Hollenbeck and Bray (1987) and Chang et al. (1999) and probably are associated with the endosomal/lysosomal pathway.