Imaging interorganelle membrane contact sites using dimerization-dependent fluorescent proteins

Membrane contact sites (MCSs), sites of close apposition between membrane-bound organelles, mediate key intracellular processes to coordinate organelle function and are implicated in a wide range of human diseases. Because MCSs can span as little as 10 nm of distance, the limited resolution of light microscopy can hamper the ability to study these structures in live cells. Dimerization-dependent fluorescent proteins consist of a weakly fluorescent and non-fluorescent monomer that produce greater signal when the two monomers interact, thereby allowing the user to identify sites of proximity through protein-protein interaction. Here, we describe a protocol using Contact-FP - our suite of organelle-targeted dimerization-dependent fluorescent proteins - to study MCSs using confocal or Airyscan microscopy in live cells. The protocol includes guidance for transfection, imaging, and analysis of Contact-FP biosensors. It includes instructions on how to leverage this tool to study a single MCS type, identify two MCSs involving the same organelle, or induce MCSs using high levels of transfection of Contact-FP probes. We also suggest troubleshooting steps for transfection, imaging, and analysis. This protocol provides a specific example for using this tool in U-2 OS osteosarcoma cells but is amenable to adjustment for other cell types.