Figure 6.

Slow kinesin-1 and fast kinesin-3 motors in complex do not coordinate but alternate their activities in vitro. (A) Schematic of experimental setup. COS7 cells were transfected with plasmids for self-assembly of a slow kin1-mNeGr motor and a fast kin3-2×mCh motor on a 20-nm scaffold in cells. (B–E) Analysis of kin1+kin3 motility in vitro. (B) Representative kymographs. In the presence of scaffold, a subset of the motility events show colocalized green and red trajectories (yellow in merge). In the absence of scaffold, kin1-mNeGr (green) and kin3-2×mCh (red) walk independently with characteristic speeds and processivities. Time is on the x axis (bar, 1 s) and distance is on the y axis (bar, 1 µm). (C) The velocities were determined for single kin1-mNeGr motors (green bars, n = 207 events) and single kin3-2×mCh motors in the absence of scaffold (red bars, n = 211 events), and for two-color two-motor events in the presence of scaffold (yellow bars, n = 203 events) in three independent experiments. The mean ± SE is indicated. (D) Four types of behavior were observed for kin1+kin3 complexes: slow, fast, intermediate, and speed change events. Representative kymographs are shown for each. Bars: 1 µm vertical, 1 s horizontal. See Fig. S4 C for additional examples. Slow, n = 103 events; fast, n = 23 events; intermediate, n = 17 events; speed change, n = 60 events. (E) Representative kymographs of merging and splitting behaviors observed for kin1+kin3 complexes. In the presence of scaffold, kin1-mNeGr (green) and kin3-2×mCh (red) traces were observed to merge together (11% of all two-color events) and to split apart (21% of all two-color events). Bars: 1 µm vertical, 1 s horizontal.