We have applied an optical super-resolution technique based on single-molecule localization to examine the peripheral distribution of a cardiac signaling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. RyRs form clusters with a mean size of approximately 14 RyRs per cluster, which is almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often <50 nm, suggesting that peripheral RyR clusters may exhibit strong intercluster signaling. The wide variation of cluster size, which follows a near-exponential distribution, is compatible with a stochastic cluster assembly process. We suggest that calcium sparks may be the result of the concerted activation of several RyR clusters forming a functional ''supercluster'' whose gating is controlled by both cytosolic and sarcoplasmic reticulum luminal calcium levels.excitation-contraction coupling ͉ fluorescence ͉ heart ͉ single molecule ͉ super-resolution

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Our results show that the WNK 1, 3, 4, OSR1, and SPAK signaling system known to play a role in regulating the phosphorylation status, and hence activity of the CCCs in other tissues, is also present in the rat and human lenses. The increased susceptibility of SPAK lenses to opacification suggests that disruption of this signaling pathway may compromise the ability of the lens to control its volume, and its ability to maintain its transparency.