Snx10 controls levels of PI(3,5)P ₂ on late endolysosomes. (A) HeLa cells were transiently transfected with mRFP-FKBP-PIKfyve^CC, iRFP-FRB-Rab7, and NES-mNG-NES-(2x)DdSnxA^PX 18 h prior to imaging. Representative fluorescence images of cells prior to (top 3 panels) and 1 min after the addition of 100 nM rapamycin (bottom 3 panels). (B and C) HeLa cells transiently transfected with Snx10-GFP, iRFP-FRB-Rab7, and either (B) mRFP-FKBP-PIKfyve^CC or (C) mRFP-FKBP-PIKfyve^CC K1877A for 18 h prior to imaging. Representative fluorescence images of cells before (top 3 panels), 1 min (middle 3 panels) or 60 min after the addition of 100 nM rapamycin (bottom 3 panels). (D) Mean volume of Rab7-positive vacuoles from experiments like those represented in B and C. n = 3, each point representing the average volume of >30 vacuoles. (E and F) WT (grey) and Snx10 KO RAW 264.7 cells (magenta) were transiently transfected with NES-mNG-NES-(2x)DdSnxA^PX, mRFP-FKBP-PIKfyve^CC, and either (E) iRFP-FRB-Rab7 or (F) iRFP-FRB-Rab5 for 12 h prior to imaging. The ratio of NES-mNG-NES-(2x)DdSnxA^PX–positive compartments to either (E) iRFP-FRB-Rab7 or (F) iRFP-FRB-Rab5 before 1 min and 30 min after addition of 100 nM rapamycin is shown. n = 3. (G) WT and Snx10 KO RAW 264.7 macrophages were co-transfected with PX-GFP and mRFP-Rab7. The percentage of organelles found to be positive for both markers is indicated. n = 3. (H) HeLa cells were co-transfected with both p40_phox^PX-RFP and iRFP-FRB-Rab7 (control), and Snx10-GFP where indicated, then subject to treatment with either vehicle control or 100 nM YM201636 for 30 min prior to imaging. The percentage of organelles found to be positive for both p40_phox^PX and Rab7 is indicated. n = 3.