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.
Highlights
The advent of single-molecule switching and localization-based PALM, STORM, and related super-resolution microscopies (Betzig et al, 2006; Hess et al, 2006; Rust et al, 2006) has greatly advanced insight in cell biology over the last decade
This includes the visualization of the clusters of the giant (2 MDa) ryanodine receptor-2 (RyR) Ca2+ release channels in cardiomyocytes to characterize the calcium signaling nanodomains, which are the structural units of calcium signaling in cardiac myocytes (Baddeley et al, 2009) using dSTORM (Heilemann et al, 2008)
The cardiac ryanodine receptor, RyR2, is strongly expressed in the heart and brain and provides the molecular basis of a mechanism known as calcium-induced calcium release (CICR), in which RyRs are transiently opened via calcium from adjacent calcium channels (Fabiato, 1985; Franzini-Armstrong and Protasi, 1997)
Summary
The advent of single-molecule switching and localization-based PALM, STORM, and related super-resolution microscopies (Betzig et al, 2006; Hess et al, 2006; Rust et al, 2006) has greatly advanced insight in cell biology over the last decade. Significant breakthroughs in visualizing nanostructures within cells include optically resolved nuclear pore complexes (Szymborska et al, 2013), microtubules (Mikhaylova et al, 2015), actin-spectrin scaffolds for membranes (Xu et al, 2013), membrane compartments (Shim et al, 2012), and protein ensembles in signaling nanodomains (Gambin et al, 2013). The cardiac ryanodine receptor, RyR2, is strongly expressed in the heart and brain and provides the molecular basis of a mechanism known as calcium-induced calcium release (CICR), in which RyRs are transiently opened via calcium from adjacent calcium channels (Fabiato, 1985; Franzini-Armstrong and Protasi, 1997). At the supra-molecular level the clustering of RyRs is of major interest, both because it can dramatically modulate the excitability of RyRs (Walker et al, 2014) and due to their general role in calcium signaling in muscle (Allen et al, 2008; Cannell and Kong, 2012), neurons (Manita and Ross, 2009), and secretory cells like pancreatic beta cells
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