Abstract 14385: Novel STORM-based Quantitative Nanoscale Assessment Reveals Two Pools of Sodium Channels Within the Cardiac Intercalated Disk
Background: The spatial association between proteins is crucial to understanding how proteins function in biological systems. Colocalization assessed from diffraction-limited fluorescence microscopy images merely indicates that the proteins are within 200-300 nm of each other in the xy plane and within 500-700 nm of each other along the z-axis. We sought to develop a method applicable to single molecule positional data from super-resolution STochastic Optical Reconstruction Microscopy (STORM) which achieves resolutions of 20 nm laterally and <50 nm in the z-dimension.
Methods and Results: To this end, we developed STORM-based Relative Localization Analysis (STORM-RLA) which quantifies the spatial relationship between clusters of co-labeled proteins: 1) the instance and degree of overlap between clusters, and, 2) the distance from each cluster to the nearest cluster of the co-labeled protein. Here, we apply STORM-RLA, a novel, 3-dimensional quantitative analysis, to analyze sodium channel (NaV1.5) localization relative to connexin43 (Cx43) and N-cadherin (N-Cad) within the intercalated disk (ID) in guinea pig ventricular myocardium. Examination of 2190 NaV1.5 clusters and 1767 Cx43 clusters revealed that 17% of NaV1.5 clusters overlapped a Cx43 clusters; however, 31% was located less than 200 nm from a Cx43 cluster. Overlap, where it occurred, involved 22 ± 4% of the NaV1.5 and 35 ± 7% of the Cx43 clusters. Overall, 61% of Cx43 clusters had NaV1.5 less than 200 nm away, with a median distance of 75 ± 25 nm. In contrast, 30% of NaV1.5 clusters and 29% of N-Cad clusters overlapped with each other across 2030 NaV1.5 clusters and 1768 N-Cad clusters examined with a median distance of 77 ± 19 nm. Overlap, where it occurred, involved 48 ± 7% of the NaV1.5 and 43 ± 5% of the N-Cad clusters. Immuno-electron microscopy quantitatively confirmed segregation of NaV1.5 between these two junction-associated ID nanodomains.
Conclusions: Two pools of NaV1.5 may exist within the ID: one adjacent Cx43 GJ located in interplicate ID regions and another co-distributed with N-cadherin within plicate ID regions. Overall, STORM-RLA represents a significant advance in the high throughput quantitative assessment of the spatial organization of proteins.
Author Disclosures: R. Veeraraghavan: None. R.G. Gourdie: None.
- © 2016 by American Heart Association, Inc.