Extended synaptotagmin 2 helps regulate the number of ER–PM Sac1 puncta to control PM phosphoinositide metabolism. (A, left) Superresolution_EPI localization maps of E-Syt2-mCherry, GFP-Sac1, and merge. Bar, 5 µm. (right) Intensity profile of GFP-Sac1 (green line) and E-Syt2-mCherry (red line) in the region between the white arrows. (B) Superresolution_TIRF localization maps from a tsA201 cell expressing GFP-Sac1 (left) and E-Syt2-mCherry (right), and merge. Insets: enlarged images from the region within the white box. Bar, 5 µm. (C, left) Summary of the overlap coefficent between Sac1 and E-Syt2 (mean ± SEM, n = 6) and (right) histogram of relative distance of GFP-Sac1 to E-Syt2-mCherry (means ± SEM, n = 6). (D, left) Superresolution_TIRF localization map of GFP-Sac1 from a single, fixed tsA201 cell after treatment with siRNA against E-Syt2. Bar, 5 µm. Inset is magnified 4×. (middle) Superresolution_TIRF localization map of a tsA201 cell stained for anti-Sac1. Inset is magnified 3×. (right) Superresolution_TIRF localization map of a tsA201 cell treated with siRNA against E-Syt2 and stained for anti-Sac1. Inset is magnified 3× (E) Histograms summarizing the effect of siRNA treatment against E-Syt2 on total PIP (left) and PIP₂ (right; means ± SEM, n = 6, t test, *, P < 0.05). (F, left) Representative Inverted TIRF images from a tsA201 cell expressing GFP-Sac1 (left) and E-Syt2-mCherry (right) before (top row) and after (bottom row) the activation of the M₁R. (right, top) Kymographs taken from the red dashed line in left panel. Bar, 5 µm. (right, bottom) Normalized time series kinetics of GFP-Sac1 (green line) and E-Syt2-mCherry (red line) after the addition of Oxo-M (10 µM). (G) Representative kymograph (top) and normalized time series kinetics (bottom) of GFP-Sac1 (green line) and E-Syn2 C2CΔ-mCherry (red line). Bars, 4 µm.