Neuronal PIK3C3 and PtdIns(3)P lipids regulate the transport dynamics of multiple axonal cargoes. (A–C) Distribution of synaptophysin-YFP (A), LAMP1-GFP (B), and Rab5-RFP (C) in Pik3c3^flox/flox axons expressing BFP or Cre-BFP proteins. Arrowheads indicate large axonal aggregates. Kymographs illustrate synaptophysin-YFP, LAMP1-GFP, and Rab5-RFP motility in axons. For each kymograph, both grayscale images showing the motility of all particles and color-coded trajectories for a small subset of particles are provided. (D and E) Axonal velocity (D) and run length (E) for synaptophysin-YFP, LAMP1-GFP, and Rab5-RFP cargoes. (F) Relative motility of axonal synaptophysin-YFP, LAMP1-GFP, and Rab5-RFP in Pik3c3^flox/flox neurons expressing BFP or Cre-BFP proteins. (G) Quantification of axonal swellings. For all graphs, the data shown are for one experiment out of three repeats. For the experiment shown, motility parameters were quantified from a total of 10 axons, 5 independent preparations, and 250 total particles for each organelle per genotype tested. Data represent means ± SEM. *, P < 0.05; ***, P < 0.001, two-tailed t test.