Sac1 at ER–PM junctions reduces PM PI(4)P. (A, left) Inverted TIRF footprint from a single tsA201 cell expressing GFP-STIM1 (top row) and Sac1-mCherry (bottom row), before (left) and after (right) the addition of thapsigargin (500 nM). (second panel) Normalized kinetics of GFP-STIM1 and Sac1-mCherry changes after the addition of thapsigargin. (third panel) Temporal relationship between STIM1 and Sac1 recruitment to the PM (same cell as left panel). (right) Summary of the change in intensity and kinetics of recruitment to the PM of STIM1 and Sac1 (means ± SEM, n = 11). (B) Similar experiments as A, except P4M-mCherry is expressed and monitored (means ± SEM, n = 9). (C) and D) Same experimental design as before, except 100 µM UTP is used to initiate STIM1–Orai1 interactions (means ± SEM, n = 8–13). (E) Same experiment as D, except RFP-PH_PLCδ1 is expressed. (F, left) Confocal micrographs of two tsA201 cells expressing Sac1 (top) and P4M-mCherry (bottom), before (left) and after (right) the addition of rapamycin (5 µM) to recruit the ER to the mitochondria. (middle) Higher magnification confocal images from the regions labeled in A, before (left) and after (right) the addition of rapamycin. (right) (top) Normalized kinetics. (bottom) Summary histograms (means ± SEM, n = 4). All changes in P4M intensity are significantly different compared with no application of rapamycin. Bars, 5 µm. N.S., P > 0.05; *, P < 0.05.