Autophagy is a fundamental process that degrades various components within the cell. Autophagy can be non-selective, where cytoplasmic materials are sequestered into autophagosomes for engulfment, typically occurring during nutrient deprivation. In contrast, selective autophagy degrades specific targets, such as damaged organelles: mitophagy, lysophagy, and ER-phagy, playing a crucial role in cellular quality control (Fig. 1)^[1][2].

Autophagy is a highly integrated process that maintains cellular homeostasis by promoting cell survival or leading to cell death^[3][4].

2.1 Autophagy-Mediated Cytoprotection

The primary function of autophagy is to promote cell survival following stress or nutrient deprivation by recycling essential cellular components. Autophagy is induced by various stimuli, including nutrient and energy stress, hypoxia, oxidative stress, and mitochondrial damage^[4].

For example, when cells are cultured under conditions of simultaneous nutrient and growth factor deprivation, autophagy reaches its highest level^[6]. Additionally, in mice, after 24-48 hours of starvation, most cells in various tissues exhibit an increase in autophagosome numbers^[7]. Furthermore, cells must clear damaged mitochondria to prevent the accumulation of reactive oxygen species (ROS)^[5]. Certain stress pathways, such as moderate hypoxia, also induce autophagy to prevent cell death (Fig. 2)^[5][8].

2.2 Autophagy-Induced Cell Death

Autophagy is often observed in the context of cell death, and in some cases, inhibiting autophagy can prevent cell death^[9]. Due to extensive crosstalk between different signaling pathways, the pro-death effects of autophagy are very complex^[10].

Case 1: Autophagy-Dependent Ferroptosis

Recently, Jiao Liu and colleagues reported the critical role of TMEM164 in selectively mediating ATG5-dependent autophagosome formation during ferroptosis (rather than during starvation). TMEM164 promotes the death of iron-dependent cells by activating autophagy to degrade ferritin, GPX4, and lipid droplets, thereby increasing iron accumulation and lipid peroxidation. The loss of TMEM164 limits the anticancer activity of ferroptosis-mediated cytotoxicity in mice, establishing a new paradigm for autophagy-dependent ferroptosis^[11].

Case 2: Autophagy-Triggers Necroptotic Apoptosis

Research indicates that GX15-070 can induce autophagy by increasing the accumulation of autophagosomes and promoting the interaction of Atg5 (a component of the autophagosome membrane) with key components of the necrosome, namely FADD, RIP1, and RIP3, triggering the assembly of the necrosome on autophagosomes. This leads to the formation of cytoplasmic cell death signaling complexes, initiating necrotic cell death^[12].

Autophagy plays a dual role in cancer, depending on the type and stage of the cancer^[10][13]. On one hand, autophagy can promote tumorigenesis and metastasis; currently, the only FDA-approved autophagy inhibitors are Chloroquine and Hydroxychloroquine^[14]. On the other hand, autophagy can enhance the efficacy of cancer treatments by promoting cell death either independently or in conjunction with other cell death pathways (Table 1)^[10].

With the increasing interest in autophagy research, related detection methods have also become a focal point for researchers. The number of autophagosomes and autophagic flux are often used as indicators of cellular autophagic activity levels.

3.1 Monitoring the Number of Autophagosomes

Currently, there are three main methods used to monitor the number of autophagosomes: electron microscopy, optical microscopy for detecting the subcellular localization of LC3, and biochemical assays for the membrane-associated form of LC3^[15].

The most traditional method is electron microscopy, which allows for the observation of autophagic vacuole-like structures in samples. Under electron microscopy, cells undergoing autophagy display damaged organelles, such as swollen mitochondria, surrounded by vacuolar double-membrane-like structures, or double-membranes encircling mitochondria to form autophagosomes. Residual bodies that cannot be degraded are also visible within autolysosomes (Fig. 5 and 6A)^[15][16].

Secondly, the mammalian autophagy protein LC3 is a marker for autophagosomes and can be detected using more widely used optical microscopy and biochemical methods. Endogenous LC3 or GFP-LC3 can be observed via fluorescence microscopy as a diffuse cytoplasmic pool or as punctate structures primarily representing autophagosomes (Fig. 6B-C).

3.2 Monitoring Autophagic Flux

One of the main methods for measuring autophagic flux is monitoring the turnover of LC3. When cells are treated with lysosomal agents (such as ammonium chloride, Chloroquine, or Bafilomycin A1) or lysosomal protease inhibitors (such as E64d), the degradation of LC3-II is blocked, leading to its accumulation. Therefore, the differences in LC3-II levels between samples represent the amount of LC3 delivered to lysosomes for degradation (Fig. 6D)^[15].

Additionally, the total cellular LC3 levels can be quantified through immunoblotting analysis or flow cytometry, or qualitatively observed via fluorescence microscopy, which inversely correlates with autophagic flux. Besides LC3, the levels of other autophagic substrates can also be used to monitor autophagic flux (Fig. 6).

In this issue, our little M has summarized the types of cellular autophagy, related mechanisms, and characteristics, and introduced the dual functions of autophagy in cell protection and promotion of cell death. Finally, we have organized the relevant detection methods for cellular autophagy!