Role of Ankyrin-G in the Preservation of Intercellular Junctions. Visualization in the Nanoscale Using Direct Stochastic Optical Reconstruction Microscopy

Abstract

Background Despite advances in technology, resolution of fluorescent microscopy images (including confocal, deconvolution, or TIRF) remains limited to 210–250 nm. This prevents quantitative analysis of images to discern critical parameters such as protein cluster size, abundance, and separation from molecular partners. This limitation is apparent in the study of intercellular junction proteins. Gap junctions and desmosomes are defined molecular clusters, but their dimensions are in the nanoscale and, as such, escape quantitative analysis by immunofluorescence. To overcome this limitation, we implemented a novel method of imaging and analysis called direct stochastic reconstruction microscopy (dSTORM) and analyzed the effect that loss of Ankyrin-G (AnkG) expression exerts on connexin43 (Cx43) and plakophilin-2 (PKP2) plaques at the site of cell apposition. Methods Neonatal rat ventricular myocytes were labeled with antibodies to Cx43 and PKP2. Cells were treated with siRNA for AnkG, or non-targeted constructs. A total of 2.000 images were obtained to detect on–off cycles of emission, and reconstructed images were analyzed using custom-made software. Results Images yielded resolutions of 10–20 nm. We measured size, shape, fluorescence intensity profile, and abundance of clusters of Cx43 and PKP2, as well as the distance separating them. Loss of AnkG increased Cx43 cluster numbers from 16 ± 1 to 29 ± 8 clusters per region of interest. This seemed concurrent with a decrease in size of individual clusters. Moreover, in control, 16.6% ± 1.4 (34/204) clusters were within 100 nm from each other; the number increased to 32.8% ± 5.5 (145/433) in cells lacking AnkG. Similar results were observed for PKP2 plaques. Finally, 45% (168/334) of all Cx43 plaques were within 20 nm of a PKP2 plaque, and the number was unaffected by AnkG expression (274/580). Conclusions Our data show that a large fraction of Cx43–PKP2 molecules are within distances compatible with direct intermolecular interaction. Moreover, the separation between Cx43 and PKP2 is unaffected by AnkG expression, whereas loss of AnkG leads to fragmented Cx43 and PKP2 plaques. We demonstrate the applicability of dSTORM to study cardiac proteins within a resolution previously reserved for electron microscopy.