KIF1A–cargo interactions in the engineered motor assay. Related to Fig. 1. Images validating the engineered motor assay and showing lysosome, SV, and endosomal vesicle localization and motility in the assay. (A) Representative images of hippocampal neurons coexpressing FRB-3myc-KIF1Atd and FKBP-mRFP-KIF5Cmd without (upper panels) or with (lower panels) addition of 1 µM rapalog. Pink lines mark the cell soma. Blue dotted circles indicate examples of axonal tips. Zooms of the boxed regions are shown in the top right of each image. (B, D, and F) Representative images of hippocampal neurons coexpressing FRB-3myc-KIF1Atd, FKBP-mRFP-KIF5Cmd, and LAMP1-GFP (B), Rab3-GFP (D), or TfR-GFP (F) without (upper panels) or with (lower panels) addition of 1 µM rapalog. Pink lines mark the cell soma. Blue dotted circles indicate examples of axonal tips. Zooms of the boxed regions are shown as a merge on the right. (C, E, and G) Representative kymographs showing movement of LAMP1 (C), Rab3 (E), or TfR (G) vesicles in the AIS before (left) and 15 min after (right) addition of 1 µM rapalog. Scale bars, 20 µm.