A correlative super-resolution protocol to map the local single-channel underpinnings of fast second-messenger signals in primary cell types

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Stream 6 (Frontiers): Development and Applications in Super Resolution Microscopy

Single molecule localisation microscopy, DNA-PAINT, correlative imaging, ryanodine receptor, calcium signalling

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Single molecule localisation microscopy (SMLM) is a common tool in resolving the clustering patterns of proteins. One such SMLM technique is DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT), which has enabled individual proteins within their clustered array to be localised and counted in situ at a spatial resolution ≤10 nm [1]However, nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated. The requirement for cells to undergo fixation and permeabilisation, alongside the incompatibility across image acquisition timescales, could partially explain the absent functional context to most SMLM data. We have developed a correlative imaging protocol [2] which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels in primary cell types. 


The RyR channel can be located across a range of vertebrate cell types and their role in mediating the release of fast Ca2+ signals, composed of Ca2+ sparks, is central to muscle force production and underpins intracellular homeostasis. The developed protocol can spatially overlay the two-dimensional (2D) total internal reflection fluorescence (TIRF) live-cell imaging of Ca2+ underneath a cell’s membrane, with the subsequent DNA-PAINT imaging of local RyR channels after cell fixation. To study the local regulation of RyR in regard to its functional release of Ca2+, we applied the experimental protocol to a drug-induced model of right ventricular (RV) heart failure. Enzymatically isolated RV cardiomyocytes were obtained from adult male Wistar rats injected with saline (Ctrl-RV) or monocrotaline (60 mg/kg) to induce pulmonary artery hypertension and compensated RV failure (MCT-RV). 


Using a semi-automated detection system [1], discretisation of individual RyR channels enabled spatial statistics of each channel to be extracted and their centroid to be detected. Within the same cellular region, Ca2+ sparks were characterised from the application of a 2D-Guassian filter detection protocol to identify the centroid of each spatial footprint (1-6 µm in width). Alignment of the respective RyR and Ca2+ spark localisations was by a two-step process, through the establishment of user-driven ‘primary alignment vectors’ followed by cross-correlation in Fourier space. When overlaid and aligned, spatial statistics could be undertaken in regard to the number of RyRs detected underneath the footprint of each Ca2+ spark. 


Within RV heart failure, remodelling of the sub-sarcolemmal RyR array patterns was observed, with a near-halving of MCT-RV RyR cluster size (MCT-RV 3.27±0.22 RyR/cluster; Ctrl-RV 5.85±0.51 RyR/cluster, mean±SEM; MCT-RV n=3; Ctrl-RV n=3; p<0.05, t-test). At a local level, when RyR pattern underneath each Ca2+ spark was examined, a steep correlation was revealed between the size of a Ca2+ spark and the spatial density of RyR, which ranged from 5-100 channels. A weaker correlation was observed in cardiomyocytes isolated from the model of RV heart failure, with a greater than halved reduction in Ca2+ spark mass (MCT-RV 20.32±2.93 AU; Ctrl-RV 51.36±11.81 AU, mean±SEM; MCT-RV n=8; Ctrl-RV n=5; p<0.05, t-test). A visible change in correlation upon the presentation of RV heart failure reflects the known dysfunction of local RyR regulation in such pathologies [3].  


It was observed that Ca2+ sparks were recorded in a spatially non-random pattern. These cellular regions where Ca2+ sparks spontaneously recurred over time were called ‘hot spots’. Using a Voronoi tessellation analysis, we spatially mapped these hot spots and correlated them with the DNA-PAINT maps of RyR channels. It was observed that these hot spots of regularly occurring Ca2+ sparks were in regions in between multiple RyR clusters. This observation is the first experimental confirmation that local recruitment of multiple clusters (typically 3-5 clusters in Ctrl-RV and 2-8 clusters in MCT-RV) is the key to the genesis of Ca2+ sparks in the heart. 


Here we have developed an experimental protocol for use in a primary cell type to probe the local structure-function relationship. This protocol is a useful blueprint for how imaging modalities can be combined to enable the correlative imaging of sub-plasmalemmal second-messenger signals (such as Ca2+ sparks), with the local spatial organisation of proteins at a nanometre-scale. 


[1] Jayasinghe, I. et al. (2018) True molecular scale visualization of variable clustering properties of Ryanodine Receptors. Cell Reports 22, 557-567. 

[2] Hurley, M.E. et al. (2020) A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types. Methods, DOI: 10.1016/j.ymeth.2020.10.005

[3] Sheard, T.M.D. et al. (2019) Three-dimensional and chemical mapping of intracellular signalling nanodomains in health and disease with enhanced expansion microscopy. ACS Nano, 13, 2143-2157.