Nanodomain clustering mechanisms of Junctophilin-2 in human kidney, cardiac and skeletal muscle cells

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Junctophilin-2 (JPH2) –a tail-anchored protein located in the sarcoplasmic reticulum (SR) membrane– is known to stabilise Calcium Release Units (CRUs) in the ~12 nm wide junction between the sarcolemma (SL) and the SR. Recently, a decreased expression of the JPH2 protein was associated with a disruption of CRU function and distinct ultrastructural changes in cardiac dilation and hypertrophy. While an essential role of JPH2 for CRU stabilisation has emerged as a principle mechanism of junctional SR membrane contacts, the mechanisms behind JPH2 clustering and its organisation remain unknown. In this research work, I have studied the signal distribution based on recombinant JPH2 expression constructs in the endoplasmic or sarcoplasmic reticulum (ER/SR) in different cell models in order to investigate the subcellular mechanisms of JPH2 clustering. Expression of different JPH2 truncation constructs was studied in HEK293a cells, C2C12 myoblasts, C2C12 myotubes and neonatal rat cardiomyocytes (NRCM) using STimulated Emission Depletion (STED) superresolution microscopy and quantitative image analysis. N-terminal truncation of all cytosolic JPH2 domains resulted in a loss of local JPH2 clustering and pan-ER/SR distribution. Our data show a JPH2 construct-specific and cell type-dependent clustering behaviour. Specifically, the expression of recombinant full-length JPH2 and N-terminally truncated JPH2 (the latter including the divergent region and the TM domain while the alpha-helix and the MORN-motifs were deleted) led to a clustered signal pattern that was spatially associated with Ryanodine Receptor type 2 (RyR2) channels in NRCMs. In contrast, the signal distri-bution of truncated JPH2 devoid of all cytosolic domains resulted in a pan-ER/SR distribu-tion and altered spatial relations with RyR2 channels. In conclusion, the divergent region of JPH2 may promote at least in part the subcellular formation of JPH2 self-clustering and its local stabilisation in ER/SR nanodomains.