Zhang Hong's team revealed that calcium transients on the surface of the endoplasmic reticulum are the key signals that initiate the formation of autophagosomes in the endoplasmic reticulum

Autophagy refers to the process of forming a double-layered membrane structure of autophagosomes, enveloping part of the cytoplasm and transporting them to lysosomes for degradation and recovery, which is essential for resisting various stresses and maintaining cell homeostasis
.
The key steps in autophagosome formation include the initiation, nucleation, extension, and closure
of the barrier membrane (autophagosome precursor).
Much of what is known about the molecular mechanisms of autophagosome formation comes from the study
of single-cell yeast autophagy.
The process of autophagosome formation in multicellular organisms is more complex, including multiple unique steps and the involvement
of autophagoblasts unique to multicellular organisms.
An important difference between autophagy in multicellular organisms and autophagy in yeast is the location of
autophagosome formation.
Yeast autophagosomes form on the vacuole membrane, while multicellular biological autophagosomes form
simultaneously at multiple sites in the endoplasmic reticulum.
Identifying the signals formed by autophagosomes on the endoplasmic reticulum is a long-standing scientific problem
in the field of autophagy.

On October 5, 2022, Zhang Hong's research group at the Institute of Biophysics of the Chinese Academy of Sciences published an online report in Cell magazine titled "Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation.
" sites" research paper.

This paper found that calcium transients on the surface of the endoplasmic reticulum caused by autophagy induction conditions are the key signals
that determine the formation of autophagosomes on the endoplasmic reticulum.
The calcium transient on the surface of the endoplasmic reticulum causes the liquid-liquid phase separation of the FIP200 complex involved in autophagy initiation, and the FIP200 condensate formed binds to the endoplasmic reticulum membrane proteins VAPs and ATLs to locate the endoplasmic reticulum and become the autophagosome starting site
.

The researchers first found that the calcium ion rapid chelating agent BAPTA-AM can inhibit the formation of condensate in the endoplasmic reticulum of FIP200 complexes involved in autophagy initiation, but this process cannot be blocked
by the slow calcium ion chelating agent EGTA-AM.
This suggests rapid local changes in calcium ions, not steady-state calcium concentrations, that may be involved in the autophagy initiation process
.
The researchers constructed a fusion protein of the endoplasmic reticulum transmembrane domain CYB5 and the rapid calcium ion probe GCaMP6f to detect the change in calcium ion concentration on the surface of the outer membrane of the
endoplasmic reticulum induced by autophagy.
Multimodal Superresolved Vivocell Imaging (Multi-SIM) shows calcium transients in the endoplasmic reticulum outer membrane under autophagy-inducing conditions such as starvation or Torin1 treatment, and these calcium signals can be blocked by BAPTA-AM but not by EGTA-AM
.

Further studies have shown that the calcium transient on the surface of the endoplasmic reticulum triggers the liquid-liquid phase separation of the FIP200 complex involved in autophagy initiation, and the FIP200 condensate formed is positioned on the endoplasmic reticulum by binding to the endoplasmic reticulum proteins VAPs and ATLs, which is the autophagosome initiation site
.
The study also found that the new autophagic protein EPG-4/EI24 localized by the endoplasmic reticulum identified by Zhang Hong's group in the early stage regulated the amplitude, frequency and duration
of calcium transients on the surface of the endoplasmic reticulum.
In EI24 knockout cells, the surface of the endoplasmic reticulum exhibits continuous calcium transients, resulting in abnormal accumulation
of FIP200 complexes in the endoplasmic reticulum.
Treatment by chemical reagents, or knocking down the activity of calcium channels on the surface of the endoplasmic reticulum, can reduce the calcium transients caused by EI24 knockout and save the phenotype
of its autophagy defects.

In summary, this study found that calcium transients occurring on the surface of the endoplasmic reticulum induced liquid-liquid phase separation of the starting FIP200 complex of autophagy under autophagy induction
conditions.
The FIP200 condensate then binds to the endoplasmic reticulum proteins VAPs and ATLs and is stably localized on the endoplasmic reticulum as the starting site
of autophagy.
This work revealed that calcium transients on the surface of the endoplasmic reticulum are the key signals that determine the formation of autophagosomes on the endoplasmic reticulum, greatly advancing our understanding of the molecular mechanisms of
autophagy in multicellular organisms.

Figure.
Pattern diagram of the endoplasmic reticulum autophagy start site by the calcium transient of the outer membrane of the endoplasmic reticulum triggers the liquid-liquid phase separation of the FIP200 complex and forms the starting point of autophagy on the endoplasmic reticulum

Professor Zhang Hong, Institute of Biophysics, Chinese Academy of Sciences, is the corresponding author
of this paper.
Zheng Qiaoxia, assistant researcher of Zhang Hong's research group, and Chen Yong, assistant researcher, are co-first authors
of this paper.
The research was funded
by the National Natural Science Foundation of China and the National Key Research and Development Program.
The Biophysical Institute imaging platform provided important technical support
for the study.

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(Contributed by: Zhang Hong Research Group)