Fig. 2. KRAP tethers immobile IP₃Rs to actin near the PM.

a, b, STORM images of KRAP overlying TIRF images of IP₃R (a), and STORM images of IP₃R overlying TIRF images of KRAP (b). Scale bars, 5 µm in first image, 2 µm in enlargements of boxed areas, 500 nm in images of individual puncta. Typical of at least 3 experiments. The resolution of our STORM images (FWHM 20–25 nm) is too low to confidently distinguish single IP₃R tetramers (~20 nm across) from tightly packed small clusters. Pixels evident in the enlarged overlays are from the TIRF, rather than STORM, images (pixel sizes 100 nm and 10 nm, respectively). c, d, Fluorescence intensity profiles across puncta (dashed lines in (a) and (b)). e, f, Summary shows distances between centroids of KRAP and IP₃R within each punctum (e, 190 puncta, 3 cells) and distribution of numbers of resolved foci within each punctum (f, 118 IP₃R puncta from 4 cells, 150 KRAP puncta from 3 cells). g, h, TIRF images of EGFP-IP₃R1 HeLa cells transfected with non-silencing (NS) (g) or KRAP siRNA (h) and time overlays (30-s interval) showing mobile and immobile IP₃R puncta. Scale bars, 10 μm (5 µm for enlargements). i, Summary (mean ± s.e.m., 10 cells from 5 independent dishes, with 41–253 puncta analysed in each) shows numbers of immobile IP₃R puncta per cell. ^****P < 0.0001, Student’s t test. j Effects of siRNA on mobile fractions (M_f) determined by FRAP for peripheral and perinuclear IP₃R puncta (Supplementary Fig. 4i, j). Mean ± s.d., n = 7 cells (peripheral, NS siRNA), 12 cells (peripheral and perinuclear, KRAP siRNA), and 13 cells (perinuclear, NS siRNA). ^**P < 0.01 Student’s t test. k–m, Effects of siRNA on the sum of the fluorescence intensities of all IP₃R puncta in the TIRF field (k), the average intensities of individual puncta (l), and numbers of puncta (m). Mean ± s.d., n = 22 cells for each, ^****P < 0.0001, ^**P < 0.01, ^*P < 0.05, Student’s t test (k–m). n KRAP, via its interaction with IP₃Rs or scaffold molecules (pale blue), tethers pre-assembled clusters of sparsely distributed IP₃Rs to actin. o Effects of siRNA against the three IP₃R subtypes (10 cells) or NS siRNA (9 cells) on distribution of distances between the centroids of each STIM1 punctum and the nearest actin-associated KRAP punctum in cells treated with thapsigargin (P < 0.05, for both distributions relative to distances after randomization of KRAP distribution; Costes randomization test for each cell).