Using Multi-Color Super-Resolution Microscopy to Probe the Organization of Dyadic Proteins within Rat Cardiac Myocytes

Abstract

Cardiac excitation-contraction coupling relies on calcium release from internal stores (the sarcoplasmic reticulum -SR) via ryanodine receptors (RyRs), which are intracellular calcium channels. Most RyRs are organized into clusters within narrow dyadic junctions between sarcolemmal and sarcoplasmic reticulum (SR) membranes. It is still unclear how junctions are formed and maintained. One protein that has been implicated in dyadic junction formation is the cardiac isoform of the junctophilin family, junctophilin-2 (JPH2). With the increased resolution available in multi-color super-resolution microscopy it is now possible to investigate the nanoscale relationship between proteins such as RyRs and JPH2 which are found within the junction. Using single fluorophore localization based super-resolution microscopy we have studied the distribution of a number of such proteins in rat ventricular myocytes.Analysing the peripheral distributions of the cardiac ryanodine receptor (RyR) and a junctional protein, junctophilin-2 (JPH2), we have found that JPH2 was almost completely associated with RyR clusters. Estimates of co-localization of ∼89% between JPH2 and RyRs confirmed near complete colocalization within peripheral couplons. The shape of associated RyR clusters and JPH2 clusters were very similar, suggesting that JPH2 is dispersed throughout RyR clusters and that the packing of JPH2 into junctions and the assembly of RyR clusters are tightly linked. Our data shows, with nanoscale resolution, that junctophilin is not constrained to specific anchoring domains within the junction, but is interspersed throughout the entire area occupied by RyRs.These results demonstrate the utility of fluorescent multi-colour super-resolution immuno-labeling to investigate protein proximity at the nanometer scale. The ability to resolve the association of different molecules into the same biological structure at the nanoscale (here the dyad junction) should give new insight into the assembly and function of macromolecular signaling complexes.