Super-Resolution Analysis of the Distribution of RyR, Cav1.2 and NCX within the Mammalian Couplon

We have developed a dynamically stable 3D dSTORM system, modified to give highly accurate blink positioning deep within a thick sample, to investigate the distribution and localization of the ryanodine receptor (RyR), the L-type calcium channel (Cav1.2) and the sodium-calcium exchanger (NCX) both on the surface and in the interior of the rat cardiomyocyte. The images have a resolution of 10nm in X and Y and 40 nm in Z, and cover areas of up to 1000 µm2 in XY with depths of up to 700nm in Z.We have been able to identify the position of individual RyR tetramers and have confirmed the recently published tomographic finding that in the resting state most RyR clusters are neither homogenous nor well-ordered. In addition, we found that surface RyR clusters are smaller both in extent and membership than those in the cell interior.Cav1.2 was commonly found in small spherical clusters of between 30-100 nm in diameter and were smaller and far denser than their RyR counterparts, with their centers appearing to be tightly packed. Cav1.2 clusters on the surface of the cell seemed little different from those in the interior, although the surface density was much higher. Super-clusters, made of 3 or more individual clusters were observed throughout the cell.NCX was more widely distributed and than either RyR or Cav1.2 and often formed a dense carpet on the cell surface made up of many small spherical clusters (∼50 nm dia). The mouth of the t-tubules were often very heavily labelled for 2-3 µm into the cell.