ER membranes exhibit phase behavior at sites of organelle contact

Significance Membrane phase behavior in cells permits transient concentration of specific proteins and lipids into dynamic nanoscopic domains. Here, we tested the existence and role of such phase behavior in endoplasmic reticulum (ER) membranes. Employing hypotonic cell swelling, we created large intracellular vesicles (LICVs) from internal organelles. ER LICVs maintained stable interorganelle contacts, with known protein tethers concentrated at the contact sites. Cooled ER LICVs underwent reversible phase separation into microscopically visible domains with different lipid order and membrane fluidity. The phase-separated domains specified sites of contact between the ER and different organelles.