Fig. 2
SR Ca2+ flux within a cell-wide circuit of cytoplasmic nanocourses of arterial myocytes. a (left to right), confocal z sections through acutely isolated arterial myocyte loaded with Fluo-4 (green, calcium indicator) and Draq 5 (blue, nucleus), then deconvolved, and pseudocolor applied to show relative Fluo-4 intensity. Regions of interest identify exemplar subplaslemmal (white), extraperinuclear (blue), perinuclear (green) and nuclear (yellow) nanocourses. b (left to right), nanocourses in (a) at higher magnification and different time points; note, thresholds set independently to visualise hotspots. Grey circles identify hotspots (H1, black; H2, orange) of Ca2+ flux in exemplar nanocourses. c Fluo-4 fluorescence ratio (Fx/F0; where F0 = fluorescence at time 0 and Fx = fluorescence at time = x) versus time (sampling frequency = 0.5 Hz) for H1 and H2 (upper panels, left to right) and the average for the whole nanocourse (lower panels, left to right). d Scatter plot shows distances separating hotspots (mean ± SEM; ≥36 hotspots, n = 7 cells from 7 rats) within subplasmalemmal (white), extraperinuclear (blue), perinuclear (green) and nuclear (yellow) nanocourses. e Dot plots show the effect of thapsigargin (1 µM; 30 min pre-incubation; n = 3 cells from 3 rats) and tetracaine (1 mM; 4 h pre-incubation; n = 5 cells from 4 rats) on the amplitude (mean ± SEM) of Fluo-4 fluorescence ratio change (ΔFX/F0); t-test with Welch’s correction: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. f Image time series highlights (white rectangle) time-dependent intensity fluctuation of one hotspot in a different subplasmalemmal nanocourse (arrow in (a), upper panel, right most image), with a record of fluorescence intensity against time (ΔFH/FN0; H = hotspot, N0 = nanocourse at time = 0) from basal (B) to high intensity (H) states; note prolonged sub-state. g Deconvolved time series of z sections (0.25 Hz) show LysoTracker Red labelled endolysosomes in cytoplasmic nanocourses identified by Fluo-4 (confirmed in 3 cells from 3 different animals). h As for (g), but for mitochondria labelled with MitoTracker Red (confirmed in 3 cells from 3 different animals). Pseudocolour look up tables in (a) and (b) indicate relative fluorescence intensity in arbitrary units
