True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors

Abstract

SummarySignaling nanodomains rely on spatial organization of proteins to allow controlled intracellular signaling. Examples include calcium release sites of cardiomyocytes where ryanodine receptors (RyRs) are clustered with their molecular partners. Localization microscopy has been crucial to visualizing these nanodomains but has been limited by brightness of markers, restricting the resolution and quantification of individual proteins clustered within. Harnessing the remarkable localization precision of DNA-PAINT (<10 nm), we visualized punctate labeling within these nanodomains, confirmed as single RyRs. RyR positions within sub-plasmalemmal nanodomains revealed how they are organized randomly into irregular clustering patterns leaving significant gaps occupied by accessory or regulatory proteins. RyR-inhibiting protein junctophilin-2 appeared highly concentrated adjacent to RyR channels. Analyzing these molecular maps showed significant variations in the co-clustering stoichiometry between junctophilin-2 and RyR, even between nearby nanodomains. This constitutes an additional level of complexity in RyR arrangement and regulation of calcium signaling, intrinsically built into the nanodomains.

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes

“…; Jayasinghe et al . ). In the present work we developed techniques for 3D quantification of RyR localization, allowing axial separation of neighbouring clusters.…”

“…Such assumptions are more problematic for the tetrameric RyR, as several subunits are simultaneously labelled by antibodies (Jayasinghe et al . ). On the other hand, RyR tallying on the cell surface is abetted by the fact that the protein is of similar size to the presently attained xy resolution.…”

“…; Jayasinghe et al . ; Kolstad et al . 2018) [Correction made on 14 December 2018, after first online publication: Kolstad et al .…”

“…In cardiomyocytes, several other dyadic proteins have been demonstrated to display clustering properties, including junctophilin‐2 (Jayasinghe et al . ), L‐type Ca 2+ channels (Scriven et al . ) and the sodium–calcium exchanger (Wang et al .…”

“…; Jayasinghe et al . ). Moreover, these studies have indicated that Ca 2+ diffusion distances between neighbouring RyR clusters are often sufficiently short (<100 nm) to support their concerted generation of Ca 2+ sparks (Sobie et al .…”