Fig. 7. Effect of activation of exocytosis on secretory granules labelled with EGFP-ppANF, EGFP-Rab3A or EGFP-Rab27A in live cells. Cells were transfected to express EGFP-ppANF, EGFP-Rab3A or EGFP-Rab27A and imaged 18 hours after transfection. Cells showing low levels of fusion protein expression were selected for observation and stimulated by perfusion with 300 µM ATP. (A) Example of the time course of Ca²⁺ changes from monitoring of X-Rhod fluorescence following ATP stimulation (n=11 cells). (B) Disappearance of a ppANP-EGFP-labelled granule during stimulation. The images shown are sequential frames in which the sudden disappearance of a granule (arrow) can be observed. (C) Effect of stimulation with 300 µM ATP on secretory granules labelled with EGFP-ppANF, EGFP-Rab3A or EGFP-Rab27A as indicated. Images were taken at 19 seconds and 76 seconds (after the elevation in cytosolic Ca²⁺). Overlay images are shown with t=19 seconds in green and t=76 seconds in red so that granules that disappear between the images appear green. Granules that disappeared in the ppANF-EGFP cell are indicated with arrowheads. Some granules did not disappear but moved during the recording and so appear green in the overlay; an example is indicated by the arrow in the EGFP-Rab3A-transacted cell. Bars, 4 µm. (D) The number of granules that disappeared between the before and after images was identified by the comparison of images at 19 and 76 seconds, quantified and shown as mean ± s.e.m. for eight cells expressing each construct.