Fig. 5: MINFLUX imaging of RyR2s in myocytes from PA-RFP RyR2 knock-in mice.

A RyR2 distribution in an isolated myocyte from a PA-RFP RyR2 mouse, stained with an anti-tagFP sdAB-Alexa647 and imaged in the confocal mode of the MINFLUX microscope. N > 5 independent repeats with similar results. B Enlargement of a region at the surface sarcolemma (green rectangle in A). C A MINFLUX DNA-PAINT 3D image, localizations are colored by the size of clusters as number of clustered subunits (SU) from DBSCAN cluster analysis. D Statistical analysis yields a mean cluster size of 9.0 ± 0.5 SUs or 16.5 ± 0.9 SUs when only looking at clusters with more than one SU – labeled “CS > 1 SU” (N = 22 MINFLUX data sets from n = 5 cell isolations). E A detail view containing 2 large clusters and a number of isolated SUs (arrows). F The majority of RyR2s reside in large clusters containing ≥50 SUs with on average 72.4 % ± 2.3 % of all RyR2s in these large clusters (N = 22 MINFLUX data sets from n = 5 cell isolations). G A large RyR2 cluster with a number of SUs in tretrad arrangements as indicated by RyR2-shaped overlays. H (i) Statistical analysis across datasets gives a mean RyR2 cluster size of 2.3 ± 0.1 RyR2s or 4.1 ± 0.2 RyR2s (CS > 1) when including only clusters which contain more than one SU. (ii) Fraction of large clusters (containing ≥50SUs, i.e. ≥12.5 RyR2s) with a mean of 4.7 ± 0.3% (N = 22 MINFLUX data sets from n = 5 cell isolations). I x-y (i) and x-z (ii) views of a peripheral coupling (PC) with the surface sarcolemma next to an internal coupling (IC) which is presumably arranged around a t-tubule invagination. Box plots show the median (center line), the 25th and 75th percentiles (bounds of the box), and the mean (large circular marker). Whiskers extend to the most extreme data points within 1.5x the interquartile range, and all individual data points are displayed as a swarm overlay. Scale bars B: 500 nm, C: 1 µm, E: 500 nm, G: 100 nm, I: 500 nm. Source data are provided with this paper.