PtdIns(3)P lipids are required for AnkB association with membrane cargo in axons. (A) Images show loss of PIK3C3 expression and diffuse staining for the PtdIns(3)P biosensor 2×FYVE in Pik3c3^flox/flox neurons expressing Cre-BFP (dotted lines, asterisks) when compared with untransfected cells (arrowheads). (B) Detection of PtdIns(3)P distribution using the 2×FYVE-GFP probe in Pik3c3^flox/flox neurons expressing BFP or Cre-BFP proteins. Insets are enlarged views of boxed axonal regions. Arrowheads indicate areas of localization of GFP signal to vesicular structures. (C) Time-lapse image and kymographs of synaptophysin-YFP and WT 220-kD AnkB-mCherry localization and dynamics in Pik3c3^flox/flox axons transfected with BFP or Cre-BFP. Arrowheads indicate large axonal swellings. (D) Quantification of AnkB-mCherry localization to YFP-positive synaptic vesicles (n = 10 axons, 5 independent preparations; n = 100 YFP vesicles). Data represent means ± SEM and correspond to one experiment out of three repeats. ***, P < 0.001, two-tailed t test.