MINFLUX microscopy resolves subunits of the cardiac ryanodine receptor and its 3D orientation in cells

Authors:

Alexander H. Clowsley, Anna Meletiou, Radoslav Janicek, Alexandre F. E. Bokhobza, Evelina Lučinskaitė, Gabriela Bleuer, Isabelle Jansen, Peter P. Jones, William E. Louch, Christian Soeller

Keywords:

cardiac ryanodine receptor; RyR2; 3D MINFLUX; multi-subunit complexes

Abstract:

The cardiac ryanodine receptor (RyR2) constitutes the molecular basis of the process of calcium-induced calcium release where activation of RyR2s can be locally regenerative. Here, we present purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX microscopy. Using single-domain antibodies and DNA-PAINT we determine the location of individual RyR2 subunits with high precision (~3 nm) and resolve the 3D orientations of RyR2s in-situ. We measured labeling efficiencies of ~50%, implying RyR2 tetramer detection probability approaching 95%. In HEK293 cells, RyR2 expression was dense, with some clusters containing several hundred RyR2s. Ventricular myocytes from mice contained large clusters containing many tens of close-packed RyR2s, resolving apparent discrepancies between electron microscopy and previous super-resolution microscopy data. The methodology developed here reveals the full 3D morphological complexity of RyR2 channels and is applicable to other multi-subunit complexes in a variety of cell types.

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