mtm controls PI(3)P turnover, membrane compartment identity, and flux. (A) Mtm-dephosphorylated PI(3)P and PI(3,5)P2. Mutant Mtm-R403C was inactive for both substrates (released phosphate/reaction). (B–E’) PI(3)P distribution detected by GFP:2xFYVE. PI(3)P expanded distribution upon mtm RNAi in Kc167 cells (B’) and abnormal association with LysoTracker-positive organelles upon mtm depletion in hemocytes (C’). (D) PI(3)P pools detected within small dots with rapid, directional movement (arrowhead; motile particle imaged as line) by time-lapse microscopy in control Kc167 cells. (D’) PI(3)P pools detected on expanded, tethered rings and lack of particle motility upon mtm RNAi. (E) Time-lapse microscopy of PI(3)P pools detected by mCherry:2xFYVE on rings with obvious tubulation (arrowheads) in hemocytes. (E’) PI(3)P pools detected on expanded, tethered rings that underwent fusion (arrows) without detectable tubulation in mtm-depleted hemocytes; still images from Video 2 are shown. (F and F’) GFP:Rab7 motility (F, arrowheads) and dynamic extension and retraction of tubules (F’, arrows) in control hemocytes (Video 3). LysoTracker, red; DAPI, blue. (G and G’) Tethered motion and fusion (arrows) between enlarged GFP:Rab7 compartments in mtm-depleted hemocytes. (H–H″) GFP:Mtm localized to dots (H) and big (H’) and small rings (H”, arrowheads). (I and I’) mCherry:Mtm on small rings detected within GFP:Rab7 compartments (GFP:Rab7 ring, arrowheads). (J and J’) Mtm at periphery of acidified endolysosomes. Red, LysoTracker. Boxed areas are shown in higher magnification on the right. Error bars indicate SEM.
