FIGURE 7:

p150 activates DDB in kinesin–DDB bidirectional transport. (A) Diagram of the reconstituted bidirectional transport system. Single kinesin-1 and DDB are connected through single-stranded DNA-functionalized GBP1 and GBP2 adapters to a double-stranded DNA scaffold, linked at its biotinylated 5′ end to a streptavidin-coated Qdot. (B) Kymographs Qdot-labeled DDB–kinesin-1 (top) in the 647 nm channel, and the excess kinesin-1 motors streaming to the plus end in the GFP channel (bottom), used to identify the polarity of the microtubule. See also Supplemental Video S2. (C) Sample traces of DDB–kinesin-1 for control (top) and the Ab_p150 group (bottom). (D) Velocities of the control group (orange; −9.1 ± 9.2 nm/s; mean ± SEM, n = 33) and the Ab_p150 group (blue: 62 ± 17 nm/s; mean ± SEM, n = 32) calculated from linear regression to entire traces. The two groups were significantly different by two-tailed t test, ***, p < 0.0005. (E) Percent of plus-end directed cargoes (yellow) and minus-end directed cargoes (gray) for the control kinesin–DDB group (left) and the Ab_p150 group (right). (F) Graphical explanation of velocity shift. Blocking p150 increased the fraction of time in the diffusive state by 12%. If it is assumed that the complexes move at the unloaded kinesin-1 velocity during this time, then the expected shift in mean velocity is 0.12 * 600 nm/s = 72 nm/s toward the plus end, in agreement with measurements.