Bipartite Genetically Encoded Biosensors to Sense Calcium Ion Dynamics at Membrane–Membrane Contact Sites

Self-complementing bipartite fluorescent proteins (FPs) are useful tools for the detection of protein–protein proximity and for localizing fluorophores to membrane–membrane contact sites. Here, we report versions of circularly permuted green FP (GFP), red FP (RFP), and mNeonGreen (NG), which are split into a large fragment composed of nine β-strands and a small fragment composed of two β-strands. In each case, the large and small fragments can associate in live cells to form the complete 11-stranded FP β-barrel. We further converted each of these three self-complementing FPs into bipartite calcium ion (Ca2+) biosensors. We demonstrate that appropriately targeted versions of these split FPs, and split FP-based biosensors, can be functionally assembled at membrane–membrane contact sites. We employ the bipartite NG-based Ca2+biosensor for visualization of pharmacologically induced Ca2+release at mitochondria-endoplasmic reticulum contact sites (MERCs).