Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome...

The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca²⁺ transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca²⁺ transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca²⁺ transients. Multi-modal SIM analysis revealed that Ca²⁺ transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca²⁺ transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca²⁺ transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.