Quantitative FRET FLIM imaging reveals multiple interactions between proteins of the ESYT, ORP and VAP families at the endoplasmic reticulum membrane

concentration, as observed for ESYT1. We also identify novel inter-family organization, including clustering between VAPB and ORP8, and between ESYT2 and ORP5/8. Finally, our approach highlights the broad interaction network (interactome) of VAPA/B, and shows how various potential binding partners can influence FRET efficiency.