Molecular-scale 3D visualisation of the cardiac ryanodine receptor clusters and the molecular-scale fraying of dyads

Abstract

Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery in cardiomyocytes. Various adaptations of super-resolution microscopy have revealed intricate details of the structure, molecular composition and locations of these couplons. However, most optical super-resolution techniques lack the capacity for three-dimensional (3D) visualisation. Enhanced Expansion Microscopy (EExM) offers resolution (in-plane and axially) sufficient to spatially resolve individual proteins within peripheral couplons and within dyads located in the interior. We have combined immunocytochemistry and immunohistochemistry variations of EExM with 3D visualisation to examine the complex topologies, geometries and molecular sub-domains within RyR clusters. We observed that peripheral couplons exhibit variable co-clustering ratios and patterns between RyR and the structural protein, junctophilin-2 (JPH2). Dyads possessed sub-domains of JPH2 which occupied the central regions of the RyR cluster, whilst the poles were typically devoid of JPH2 and broader, and likely specialise in turnover and remodelling of the cluster. In right ventricular myocytes from rats with monocrotaline-induced right ventricular failure, we observed hallmarks of RyR cluster fragmentation accompanied by similar fragmentations of the JPH2 sub-domains. We hypothesise that the frayed morphology of RyRs in close proximity to fragmented JPH2 structural sub-domains may form the primordial foci of RyR mobilisation and dyad remodelling.

Three-dimensional visualization of the cardiac ryanodine receptor clusters and the molecular-scale fraying of dyads

“…All data included in the paper are processed data. In the electronic supplementary material, data (zip) file [37], we have attached exemplar datasets and exemplar Python and IDL scripts used for the quantitative image analysis.…”

Expansion microscopy reveals subdomains inC. elegansgerm granules

“…Although previous EM studies show that the P granule consists of a crest and a base ( Sheth et al, 2010 ), we were not able to observe these features using pan-protein staining. It will be interesting to examine the P granule at a higher resolution through higher orders of EExM, which might allow us to observe these features; however, the risks of spatial distortions that accompany the combination of multicellular tissue imaging with 10× or greater EExM need to be carefully managed ( Sheard et al, 2021 Preprint ). It is also possible that the crest is composed of other materials, for example, RNA, which would not be stained by the pan-protein stain but could be labelled by nucleic acid–based approach.…”

Expansion microscopy reveals subdomains in C. elegans germ granules

“…Whilst previous EM studies show that the P granule consists of a crest and a base (Sheth et al, 2010), we were not able to observe these features using pan-protein staining. It will be interesting to examine the P granule at a higher resolution through higher orders of EExM, which might allow us to observe these features, however the risks of spatial distortions that accompany the combination of multicellular tissue imaging with 10x or greater EExM need to be carefully managed (Sheard et al, 2021). It is also possible that the crest is composed of other materials e.g.…”