PM PI(4,5)P₂ is necessary for ER–PM Sac1 localization. (A, left) Inverted TIRF footprints from living tsA201 cells before, during, and after the addition of Oxo-M (10 µM). (middle, top) Kymograph of GFP-Sac1 taken from the red line in A. Bar, 5 µm. (middle, bottom) Normalized time course of GFP-Sac1 after the addition of Oxo-M. (right) Summary histograms (means ± SEM, n = 15, t test; *, P < 0.05). (B, left) TIRF footprint from a single tsA201 cell expressing GFP-Sac1 (left column), RFP-PH_PLCδ1 (middle column), and merge, before (top row), during (middle row), and after (bottom row) M₁R activation. (middle, top) Kymographs from the cell in left panel. Bars, 3 µm. (middle, bottom) Normalized kinetics of GFP-Sac1 and RFP-PH_PLCδ1 from cell in left panel. (right) Summary (means ± SEM, n = 12, t test; *, P < 0.05) of the time constants of GFP-Sac1 (green bars) and RFP-PH_PLCδ1 (red bars). (C, left) Experimental design: recruited PI(4)P 5-kinase phosphorylates PM PI(4)P into PI(4,5)P₂. (right) Representative TIRF footprint kinetics of GFP-Sac1 and RFP-PH_PLCδ1 after the addition of rapamycin to recruit PI(4)P 5-kinase to the PM. (D, left) Experimental design: VSP rapidly dephosphorylates PM PI(4,5)P₂ into PI(4)P. (middle) Representative TIRF images from a patch-clamped tsA201 cell expressing GFP-Sac1 and VSP and held at −60 mV (top) and +100 mV to activate VSP (bottom). (right) Normalized kinetics of GFP-Sac1 from the same cell after two sequential steps to +100 mV for 4 s to activate VSP. Bar, 5 µm.