Cardiomyocyte ryanodine receptor clusters expand and coalesce after application of isoproterenol

Figures

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  Figure S1.. Imaging system parameters. (A) Stability of the x, y, and z axes of the piezoelectric stage for the duration of a single image acquisition. (B) Estimated errors in the positioning of the x, y, and z values for 2.6 × 106 control blinks.

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  Figure S2.. Calculating the blink density. (A) An arbitrary set of points with a Delaunay triangulation (blue). The density of the black point is given by the reciprocal of the sum of the areas in red. If there is more than one blink at a point, the density is multiplied by the number of blinks. (B i and ii) Histogram of blink density plotted on a linear (i) and logarithmic (ii) scale. The data is from a cell, a surface segment of which is displayed in Fig. 1 A.

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  Figure S3.. Examples of α-shapes and minimum ellipses. (A) Control cell (Fig. 1 B) showing the α-shape generated boundaries (vermillion) of each cluster. Scale bar, 500 nm. (B) Control cell (Fig. 1 B) showing fitted ellipses (sky blue). Neighborhood limit was 50 nm. Scale bar, 500 nm.

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  Figure 1.. Blink distribution on the surface of a cardiomyocyte. (A) Blink data showing tetramer clusters on the surface of a cardiomyocyte. Area is 13.0 × 11.8 µm. Z lines run left to right and are about 1.75 µm apart. Scale bar: 1 µm. The cluster structure and, in some cases, individual tetramers are clearly visible with a twofold zoom. (B) Enlargement of the section in A outlined in red. Densities and the size of tetramers can be seen throughout the image. Scale bar: 1 µm.

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  Figure 2.. Tetramer distributions in RyR2 clusters. (A and B) Analysis of the tetramer distribution of two clusters (A and B) marked in red in Fig. 1 B. (i–iv) Descriptions apply to both clusters: (i) blinks before any size filters were applied; (ii) blink density coded so that those blinks above the threshold are in vermillion while those below are in sky blue; (iii) preliminary fit of single tetramers (bluish green); (iv) proposed tetramer distributions. Density threshold was −1.6. Each blink is represented by a 2 × 2 pixel for visibility. Scale bars: 50 nm.

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  Figure S4.. Control cell blink distribution. (A–C) Clusters showing tetramers on the surface of three different control cells. Note the large variation in size, shape, and density of the clusters. Scale bars, 500 nm.

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  Figure S5.. Blink distribution in phosphorylated cells. (A and B) Blink data showing the effect of 300 nmol/liter ISO on the surface distribution of clusters in two different cells. Scale bar, 1 μm.

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  Figure 3.. Blink and tetramer dsitribution in phosphorylated cells. (A) Blink data showing clusters on the surface of a cardiomyocyte treated with 300 nmol/liter ISO. Area is 12.3 × 4.1 µm. Z lines are parallel to the x axis and 1.7 µm apart. Scale bar: 1 µm. (B–D) Three clusters taken from image A. (i) Blinks as they appear in image A; (ii) blink density coded so that those blinks above threshold (−1.6) are in vermillion while those below are in sky blue; (iii) proposed tetramer distributions (bluish green, B and C only). Each blink is represented by a 2 × 2 pixel for visibility. Scale bars: 100 nm.

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  Figure 4.. Neighborhood limit and cluster grouping. (A) A group of clusters with their α-shape boundaries in vermillion. (B–D) Effects of changing the neighborhood limit on the group in A. In each image, the clusters are marked by a convex hull (sky blue). B, 30 nm; C, 50 nm; and D, 80 nm. Increasing the neighborhood limit decreases the number of clusters from four (B) to one (D). Each blink is represented by a 3 × 3 pixel for visibility. Scale bar: 100 nm.

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  Figure 5.. Cluster area distributions. (A) Control cells: (i) Cluster area distribution. Each bar is 104 nm2 wide and the y axis is logarithmic. (ii) Total area contribution from the summed areas of the clusters defined by each bar. (B) Cells treated with 300 nmol/liter ISO. (i) Cluster area distribution; bar size and y axis is the same as control. Compared with control cells (A i), the distribution is flatter for the lower value areas. (ii) Total area contributions. Changes in B i are reflected in a reduction in the contribution from small areas and a shift in the sum median to the right. (C) CDF of the cluster areas of control and ISO-treated myocytes. The distribution of the ISO-treated cells is significantly shifted to the right (p< 10−6).

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  Figure 6.. Summed area distributions and the neighborhood limit. Summed area distributions for (A) control cells and (B) cells treated with ISO. (i–iv) For both A and B, the NLs are: (i) 40 nm; (ii) 60 nm; (iii) 100 nm; and (iv) 150 nm. The dark areas represent the proportion of the areas that are due to multiclusters. All axes are to the same scale including B iv, where the x axis was expanded 60%. The values of mean, median, and median sum are listed in Table 1.

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  Figure 7.. Subcluster distributions at a 150 nm NL. (A) Plot of the number of subclusters versus cluster area. Control cell values (vermillion) are offset from ISO values (bluish-green). (B i and ii) Plot of largest subcluster versus second largest subcluster (i) control cells (ii) ISO-treated cells.