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ACS Nano. 2019 Feb 26;13(2):2143-2157. doi: 10.1021/acsnano.8b08742. Epub 2019 Feb 8.

Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy.

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School of Biomedical Sciences, Faculty of Biological Sciences , University of Leeds , Leeds LS2 9JT , United Kingdom.
Institute of Experimental Medical Research , Oslo University Hospital Ullevål , Oslo 0407 , Norway.
School of Physics and Astronomy, Faculty of Mathematics and Physical Sciences , University of Leeds , Leeds LS2 9JT , United Kingdom.
Living Systems Institute , University of Exeter , Devon EX4 4QL , United Kingdom.
Auckland Bioengineering Institute , University of Auckland , UniServices House, Level, 6/70 Symonds St , Grafton, Auckland 1010 , New Zealand.


Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a β-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.


computational modeling of intracellular calcium; expansion microscopy; ryanodine receptor; signaling nanodomains; site-specific phosphorylation

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