Pi3K68D localizes at endolysosomes and cell periphery with microtubule-dependent motility. (A–A”) mCherry:Pi3K68D localized to dots (A and A’), rings (A and A”), and cell periphery (arrows) in hemocytes. (B and B’) Pi3K68D:GFP surrounding acidified endolysosomes. Red, LysoTracker. (C) Mtm (red) and Pi3K68D (green) did not colocalize when coexpressed. (D–I) Pi3K68D microtubule-dependent motility and localization. (D and D’) mCherry:Pi3K68D (red) along microtubules (green). (E and E’) Localization persisted with latrunculin B (+LatB). (F) Pi3K68D:GFP colocalized with microtubules (red) in latrunculin B–treated Kc₁₆₇ cells. (G–I) Pi3K68D:GFP time-lapse imaging in live Kc₁₆₇ cells. (G) Pi3K68D on oscillating puncta. (H) Disruption of F-actin (+LatB) revealed extensive Pi3K68D:GFP particle motility (arrowheads). (I) Disruption of microtubules (+nocod) arrested all Pi3K68D:GFP motility. Arrowheads, examples of stationary Pi3K68D:GFP particles. (J) Pi3K68D:GFP dynamic colocalization relating to LysoTracker (red) compartments in single z sections from time-lapse microscopy of hemocytes (the following show frames from Video 4: PI3K68D:GFP movement toward [i] and away [ii and iii] from endolysosomes). Examples of persistent (arrowheads) and transient colocalization between motile Pi3K68D:GFP (arrows) and Lysotracker. Blue, DAPI. Boxed areas are shown in higher magnification on the right.