Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and DNA PAINT single molecule localisation microscopy

Abstract

Aberrant Ryanodine receptor behaviour is highly implicated in cardiovascular disease. Post-translational modifications are used widely in the body to control the dynamics of proteins to respond to acute and chronic demands. Phosphorylation is a key, highly tuneable modification used by cells by the reversible enzymatic addition of a phosphate group to single amino acids within protein structures. Common cardiac diseases such as arrhythmia, hypertension, and heart failure have been linked to excessive ryanodine receptor phosphorylation and transgenic constitutive phosphorylation has shown disease aetiology in disease models. Phosphorylation is typically measured en masse by use of Western blots, or more recently phosphoproteomics, however the spatial distribution has remained a mystery. Ryanodine receptors are found in clusters and their influence tightly controlled spatially. As phosphorylation increases their range of influence, it is of great interest to observe the pattern of phosphorylation within and between ryanodine receptor clusters. Ryanodine receptor clusters have been well characterised in electron microscopy and the fluorescence based super resolution microscopy, achieving single receptor resolution. This thesis details the validation pipeline for translation phosphorylation-state specific antibodies from Western blot through to super resolution microscopy. The phosphorylation distribution was compared between isolated ventricular cardiomyocytes and ventricular tissue sections for Ser 2808 and Ser 2814 phosphorylation. Strong reductions in basal phosphorylation caused by the isolation procedure were observed for Ser 2808 but not Ser 2814. These differences in Ser¬ 2808 phosphorylation were then investigated in dual channel STORM super resolution microscopy, highlighting stark contracts in colocalisation between the confocal and STORM techniques. This population and sub population experiment was then translated into the new DNA PAINT technology and a direct comparison of performance between STORM and DNA PAINT was discussed. The data described in this thesis shows a methodological approach to enabling other biophysicists to perform quantitative super resolution microscopy to determine the extent and spatial distribution pattern of phosphorylation of a protein of interest at the nanoscale. Important differences were observed in the phosphorylation state due to cardiomyocyte isolation procedures that are of interest to a wide audience of cardiovascular researchers. DNA-PAINT is emerging as the progression of SMLM from STORM microscopy due to the greater control of imaging parameters it affords. Parallel experiments of Ryanodine receptor Ser-2808 phosphorylation were performed in tissue sections. Comparisons between dual channel STORM and DNA-PAINT were evaluated. Open questions about DNA PAINT are also highlighted and discussed.