Figure 5. E-Syt1 makes dynamic contacts with the PM in a Ca²⁺- and PI(4,5)P₂-dependent manner.

(A) TIRF microscopy images of a HeLa cell expressing EGFP-E-Syt1 before and after stimulation with 2μM thapsigargin (TG) at the indicated times. Scale Bar, 10 μm. (B) Time-course of TG-induced recruitment of EGFP-E-Syt1 to the PM, as shown in A (mean±SEM, n=5). (C) Immuno-EM micrographs of HeLa cells expressing E-Syt1-EGFP, untreated or treated with TG (2 μM, 10 s). E-Syt1-EGFP (10nm gold) and endogenous PDI (5nm gold). Arrows indicate E-Syt1-positive non-cortical ER and arrowheads indicate E-Syt1-positive cortical ER (magnified in the inset). Scale bar, 200 nm. Quantification of the results is shown in D. (means±SD). (E) Quantification of the response of wild-type (WT) or Ca²⁺-binding deficient (Mut) mCherry-E-Syt1 to TG (2 μM, peak response) in HeLa cells, as assessed by TIRF microscopy, with or without optogenetic depletion of PM PI(4,5)P₂ (mean±SEM, n=3 for each, * P<0.001 for comparison to WT [TG]). (F) Confocal images of the ventral region of COS-7 cells expressing WT or Mut mCherry-E-Syt1 before and 10 s after UV photolysis of caged Ca²⁺ with or without optogenetic depletion of PM PI(4,5)P₂. Hot spots of fluorescence represent focal accumulation of E-Syt1 at the PM. (G) Time-course of results shown in F (mean±SEM, n=7–15, * P<0.01 compared to no PI[4,5]P₂). (H) TIRF microscopy images of a HeLa cell expressing E-Syt1-EGFP and the M1 muscarinic receptor (M1R) before and after stimulation with 10 μM oxotremorine-M (Oxo-M). (Bottom) Kymograph of EGFP fluorescence of a representative cell. Fluorescence in A, F and H is shown in black. See also Figure S4.