Linking a kinesin tail–vesicle complex to an active axon-selective motor domain causes a distinctive increase in axonal vesicle transport. (A) Hippocampal neurons were transfected with a split kinesin consisting of an FRB-GFP-KIF1A tail, which interacts with endogenous vesicles, and the KIFC⁵⁵⁹-tdTM-FKBP motor domain. The constructs were expressed for 18 h before live imaging to evaluate the transport of KIF1A-labeled vesicles. (B) Before addition of linker drug, the FRB-GFP-KIF1A–labeled vesicles were present in both the axon and the dendrites. In this and all subsequent figures, the contrast was inverted so that brightly labeled vesicles appear dark. Bars: (top panels) 20 µm; (high magnification panels) 5 µm. (C and D) Kymographs illustrate the transport of KIF1A-labeled vesicles before (0 min) and 25 min after addition of 1 µM AP 21967. Before addition of the linker, labeled vesicles were transported bi-directionally in the axon and the dendrites. After addition of the linker drug, there was a pronounced increase in long-range anterograde events in the axon. Graphs with red lines illustrate all anterograde events visible on the corresponding kymographs. In the kymographs, time is shown on the x axis and position along the neurite on the y axis. Diagonal lines with positive slope represent movements away from the cell body. Time and distance calibration are the same for all kymographs. See also Video 1.