Fig. 3.

IP₃Rs within mobile puncta do not exchange with immobile puncta. a TIRFM images of FRAP experiment show images before, immediately after and 60 min after bleaching. Bleached area shown by white rectangle. Times shown as h:min:s. b, c Enlarged images of yellow boxed area in a. Immobile puncta were identified by overlaying two frames (30 s apart) using pseudocolours for each (green and magenta), such that immobile puncta appear white in the overlay (iii, iv) (Supplementary Fig. 7). Images were captured before photobleaching (b) and after recovery for 15 min (c). Enlarged areas (red boxes in iii) are shown in (iv). Scale bars (a–c) = 10 µm. d Enlargements (blue boxes in b iv and c iv) show images before and 15 min after bleaching. Scale bar = 2 µm. Abundant green and magenta puncta in the post-bleach images (c, and lower in d) indicate rapid exchange of mobile EGFP-IP₃R, while the scarcity of white puncta suggests very slow exchange of immobile EGFP-IP₃R1. e Summary results show fluorescence recovery (F/F ₀, %, mean ± SEM) from three cells (26 ± 15 immobile puncta per cell were identified in the initial images). f Example trace from a region centred on an immobile punctum (circled in first image) shows stepwise increase and decrease in fluorescence intensity as a mobile punctum (arrows) moves through the immobile punctum (Supplementary Movie 2). Scale bar = 1 μm