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. Author manuscript; available in PMC: 2014 Jun 20.
Published in final edited form as: Cell. 2013 Jun 20;153(7):1494–1509. doi: 10.1016/j.cell.2013.05.026

Figure 4. Interaction of the C2C domains of E-Syt2/3 with PM PI(4,5)P2 is required for the ER-PM tethering function of E-Syt2/3.

Figure 4

(A) E-Syt2 constructs examined and their localizations. (B) Confocal images of HeLa cells of constructs lacking the C2C domain or mutated in the basic patch in this domain. The C2C domain alone is targeted to the cell cortex. Scale bar, 10μm. (C) Confocal images of a COS-7 cell expressing E-Syt3-mCherry, the PI(4,5)P2 reporter iRFP-PH-PLCδ1, and the two components of blue light-dependent PI(4,5)P2 depletion system at the end of 10 min blue light illumination (arrow indicates cortical ER). Fluorescence is shown in black. N = nucleus. (see also movie S3). Scale bar, 5μm. (D–F) Immuno-EM micrograph (E-Syt3-EGFP, 10nm gold and endogenous PDI, 5nm gold) of cells processed as above and fixed at the end of the illumination. Quantification of the results is shown in E and F. Note that the redistribution of E-Syt3-EGFP from cell periphery (cortical ER, cER) (compare with Figure 1J) into the non-cortical ER (arrows) is accompanied by loss of cER (mean±SD) (F). * P<0.001. (G – J) Time-course of normalized mCherry fluorescence, as assessed by TIRF microscopy, from COS-7 cells expressing the indicated E-Syt fusion proteins together with EGFP-PH-PLCδ1 and the two components of blue light-dependent PI(4,5)P2 depletion system (G – I) or the same two components but with a catalytically inactive phosphatase domain (DEAD 5-ptaseOCRL) (J). (Top) kymographs of representative cells, and (bottom) average traces (n=5). Data are represented as mean±SEM. The bar graphs on the right show levels of PI(4,5)P2, as assessed by EGFP-PH-PLCδ1 fluorescence at time zero and at the end of the 3 min illumination. * P<0.001. See also Figure S3.