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

Abstract

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.

Keywords: ATG9; Ca(2+) transient; EI24; ER; FIP200; autophagosome formation; liquid-liquid phase separation; lysosome.