Mechanosensory channels mediate ER Ca2+ transients to trigger assembly of autophagosome initiation sites for degradation of ER subdomains

ER-phagy involves the selective autophagosomal engulfment of ER fragments, but the signaling events, selection mechanisms, and membrane source of ER-phagic autophagosomes remain elusive. Here, using state-of-the-art super-resolution multi-SIM imaging, we reveal that stresses (prolonged starvation, Cholesterol dyshomeostasis, and high-Ca²⁺ insults) trigger the expansion of sheet ER subdomains containing high levels of luminal CA²⁺ in mammalian cells, which are subsequently degraded by ER-phagy. Autophagosome formation and sequestration of ER sheets require the concerted actions of FAM134B and lipidated LC3, whereas the Autophagy proteins ATG14 and ATG9 are partially dispensable. Electron microscopy and cryo-electron tomography show that the membranes of autophagosomes enclosing high-Ca²⁺-containing ER sheets are directly remodeled from the ER. The ER-localized cation channels PIEZO1 and TRPV1 are enriched at and mediate CA²⁺ transients from high-Ca²⁺-containing ER sheets, triggering liquid-liquid phase separation of the autophagosome-initiating FIP200 complex to initiate ER-phagy. Thus, distinct mechanisms are employed for the formation of high-Ca²⁺-containing ER-enclosing autophagosomes and non-selective autophagosomes.