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

Abstract

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.

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy

“…In expansion microscopy of cultured cells, correctly estimating the dimensions of cell structures depends on precisely estimating the expansion factor at which these structures were pulled apart. The swelling of the gel block is commonly used as a prompt to estimate the expansion factor of the label and thus the "real" -pre-expansiondimension of the protein structures under study 15,16 . However, since the cell is a dense aggregate of proteins and these are cross-linked to the acrylamide polymer that is made within the cell, we speculated that the swelling ratios of this ionic polymer and those of the ionic polymer densely cross-linked to cellular proteins would differ.…”

“…In ExM, to extract quantitative information from expanded samples, it is imperative to have a correct estimation of the expansion factor at which the cellular components were pulled apart. So far, expansion factor estimations have been performed either by measuring the gel block before and after water dialysis-induced swelling 15,16 , by measuring the very same location before and after expansion 17,18 or by studying structures whose dimensions were already known by other techniques, such as fluorescence nanoscopy or electron microscopy. The values reported by these approaches vary significantly, with macroscopic measurement of the gel block seeming to underestimate the actual expansion of cellular proteins.…”

Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

“…7 In addition, various protocols have been introduced enabling subdiffraction-resolution imaging of proteins, RNA, and bacteria in cultured cells, neurons, and tissues by confocal fluorescence microscopy and in combination with superresolution microscopy. [5][6][7][8][9][10][11][12][13][14] In order to be usable for ExM, the molecule of interest has to exhibit amino groups that can react with glutaraldehyde (GA) 9 , MA-NHS 9 , AcX 10 , or Label-X 11 and be linked into the polyelectrolyte hydrogel. The plasma membrane of cells is mainly composed of glycerophospholipids, sphingolipids, and cholesterol.…”

Peptide ligands of the cardiac ryanodine receptor as super-resolution imaging probes

“…The molecular-level description of peripheral nanodomains was obtained using the sub-10 nm resolution technique DNA-PAINT, sufficient to localise individual RyRs 13 . More recently, the prevalence of RyR2 phosphorylation at the individual channel level has been studied with X10 expansion microscopy (ExM) 14 .…”