We have applied an optical super-resolution technique based on single-molecule localization to examine the peripheral distribution of a cardiac signaling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. RyRs form clusters with a mean size of approximately 14 RyRs per cluster, which is almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often <50 nm, suggesting that peripheral RyR clusters may exhibit strong intercluster signaling. The wide variation of cluster size, which follows a near-exponential distribution, is compatible with a stochastic cluster assembly process. We suggest that calcium sparks may be the result of the concerted activation of several RyR clusters forming a functional ''supercluster'' whose gating is controlled by both cytosolic and sarcoplasmic reticulum luminal calcium levels.excitation-contraction coupling ͉ fluorescence ͉ heart ͉ single molecule ͉ super-resolution

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.

BackgroundOptical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.Methodology/Principal FindingsWe show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk.Conclusions/SignificanceOur approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.

Confocal and total internal reflection fluorescence imaging was used to examine the distribution of caveolin-3, sodium-calcium exchange (NCX) and ryanodine receptors (RyRs) in rat ventricular myocytes. Transverse and longitudinal optical sectioning shows that NCX is distributed widely along the transverse and longitudinal tubular system (t-system). The NCX labeling consisted of both punctate and distributed components, which partially colocalize with RyRs (27%). Surface membrane labeling showed a similar pattern but the fraction of RyR clusters containing NCX label was decreased and no nonpunctate labeling was observed. Sixteen percent of RyRs were not colocalized with the t-system and 1.6% of RyRs were found on longitudinal elements of the t-system. The surface distribution of RyR labeling was not generally consistent with circular patches of RyRs. This suggests that previous estimates for the number of RyRs in a junction (based on circular close-packed arrays) need to be revised. The observed distribution of caveolin-3 labeling was consistent with its exclusion from RyR clusters. Distance maps for all colocalization pairs were calculated to give the distance between centroids of punctate labeling and edges for distributed components. The possible roles for punctate NCX labeling are discussed.

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Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a β-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.

We show that high quantum efficiency fluorophores can exhibit reversible photobleaching. This observation provides the basis for an imaging technique we call reversible photobleaching microscopy. We demonstrate applicability of this technique using antibody labeled biological samples in standard aqueous (or glycerol based) media to produce far-field images at approximately 30 nm resolution. Our novel method relies on intense illumination to reversibly induce a very long-lived (>10 s) dark state from which single fluorochromes slowly return stochastically. As in other localization microscopy methods, reversible photobleaching microscopy localizes single fluorochromes, but has the advantage that specialized photoactivatible and photoswitchable molecules or special immersion/embedding media are not required.

Signalling nanodomains requiring close contact between the plasma membrane and internal compartments, known as ‘junctions’, are fast communication hubs within excitable cells such as neurones and muscle. Here we have examined two transgenic murine models probing the role of junctophilin-2, a membrane tethering protein crucial for the formation and molecular organisation of sub-microscopic junctions in ventricular muscle cells of the heart. Quantitative single molecule localisation microscopy showed that junctions in animals producing above-normal levels of junctophilin-2 were enlarged, allowing the re-organisation of the primary functional protein within it, the ryanodine receptor (RyR). Although this change was associated with much enlarged RyR clusters that due to their size should be more excitable, functionally it caused a mild inhibition in the calcium signalling output of the junctions (calcium sparks). Analysis of the single molecule densities of both RyR and junctophilin-2 revealed an ∼3-fold increase in the junctophilin-2 to RyR ratio. This molecular rearrangement is compatible with direct inhibition of RyR opening by junctophilin-2 to intrinsically stabilise the calcium signalling properties of the junction and thus the contractile function of the cell.