Figure 10.

PtdIns(3)P on endosomes is controlled by pH. (a) The cytoplasmic pH of pHrodo-AM–loaded RAW macrophages over time. RAW macrophages were incubated in either culture media (vehicle) or acidic media (pH 4.0), with or without 1 µM ConA. pHrodo-AM fluorescence intensity is shown in rainbow palette. Bars, 5 µm. Graph illustrates the changes in fluorescence intensity over time. Each time point was normalized to 0 mins, represented as relative units (R.U). Data shown are means ± SEMs from three independent experiments (n > 50 cells for each condition). ANOVA test was used to compare each treatment condition to control; ***, P < 0.001. (b) RAW macrophages expressing 2FYVE-GFP (white) show PtdIns(3)P containing endosomes (arrows). 2FYVE-GFP–positive endosomes disappeared when macrophages were exposed to acidic cell-culture medium (pH 4.0) for the time points indicated (representative of the independent experiments). Bars, 5 µm. (c) Determination of PtdIns(3)P level in RAW macrophages. Cells were incubated with ³H-myo-inositol overnight and subjected to the conditions indicated for 20 min. After inositol isolation and separation, levels of PtdIns(3)P and PtdIns(4,5) were determined. For each treatment, PtdInsP levels were normalized to the parent PtdIns peak and compared with control. Data shown are means ± SEMs from four independent experiments. *, P < 0.05.