Key challenges and recommendations for defining organelle membrane contact sites

Intracellular membrane contact sites (MCSs) between organelles have crucial roles in cellular signalling and homeostasis. These sites, which are often disrupted in pathological conditions, enable the exchange of ions, lipids and metabolites between membrane-bound compartments, helping cells adapt to varying physiological conditions. Specific tether proteins and complexes stabilize these interactions and mediate responses to different intracellular or extracellular stimuli. The study of MCSs has progressed in recent years, owing to the development of new methods such as genetically encoded reporter constructs, advanced imaging techniques, including super-resolution microscopy and electron tomography, and proteomic approaches based on mass spectrometry. These tools have enabled unprecedented visualization and quantification of organelle interactions, as well as identification of the molecular players involved. This Expert Recommendation aims to define and map the 'organelle contactome', describing key proteins involved in contact site formation and the roles of MCSs in cellular function. We also explore contact site dynamics and detail advantages and disadvantages of the methodologies for studying them. Importantly, we consolidate open questions in contact site research and discuss challenges and limitations of the current experimental approaches.