Introduction of Cell Death--Autophagy

Apoptosis is a "self-killing" phenomenon in cells, while autophagy is a "self-eating" phenomenon in cells. This article will introduce what autophagy is.

1. Definition of autophagy

Autophagy refers to the transport of damaged, denatured or aged proteins and organelles in cells to lysosomes under the regulation of autophagy associated genes (Atg), where they are digested and degraded by lysosomes. process. In order to maintain the dynamic balance of the intracellular environment, normal animal cells need to continuously degrade dysfunctional or unnecessary cellular structures. Usually, short-lived proteins are degraded through the ubiquitin -proteasome pathway, while longer-lived proteins and cellular structures It is degraded by lysosomes through the autophagy pathway.

Figure 1. Autophagy process (Source: nobelprize)

2. Characteristics of autophagy

Current autophagy can be divided into three categories, including macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), of which macroautophagy is the most important and the most studied form. The process of autophagy mainly includes the formation and expansion of the isolation membrane (Phagophore), the formation of the autophagosome, and the fusion of the autophagosome and the lysosome to form the autophagolysosome (Autophagolysosome) . After the formation of autophagolysosomes, the contents are digested by hydrolases in the lysosomes.

Figure 2. Three types of autophagy in mammals

3. The process of autophagy

The process of autophagy is not a completely passive cellular process, but an active biological process that the cells themselves trigger through a series of intracellular signal transductions to maintain internal environment stability when they are stimulated by the outside world. The complete process of autophagy can be roughly divided into the following four stages: initiation of autophagy → formation of isolation membrane and autophagosome → fusion of autophagosome and lysosome → cleavage of autophagosome.

Figure 3. The process of autophagy (Li et al. Molecular Cancer, 2020)

4. Common detection methods of autophagy

There are three main methods to detect autophagy levels: electron microscopy, Western Blot detection and fluorescent protein labeling detection.

① Electron microscope observation

Detection of autophagy by transmission electron microscopy is based on identifying the structure of autophagosomes and is the most direct and classic method to observe the phenomenon of autophagy.

Table 1. Autophagy markers and their characteristics under transmission electron microscopy

Figure 4. Morphology of autophagosomes (single arrow) and autolysosomes (double arrow) under transmission electron microscope.

② Western Blot

1) During the process of autophagy, the C-terminus of LC3 is cleaved into LC3-I after synthesis, and is scattered in the cytoplasm; when the autophagosome forms LC3-I, it is coupled with phosphatidyl ethanolamine to form LC3 -II, localized on the inner and outer membranes of autophagosomes, and remains stably until fusion with lysosomes. Therefore, WB can be used to detect changes in the LC3-Ⅱ/Ⅰ ratio to evaluate the strength of autophagy.

2) As an important receptor for autophagy, p62 mainly relies on the autophagy pathway for degradation. Therefore, p62 protein levels are often used to indicate autophagy degradation.

Figure 5. WB detection of LC3 and p62 protein expression

③ Fluorescent protein labeling

1) GFP-LC3

When there is no autophagy, GFP-LC3 is diffusely distributed in the cytoplasm; when autophagy is formed, GFP-LC3 accumulates on the autophagosome membrane. Multiple bright green fluorescent spots are formed under a fluorescence microscope, and the number of observed point-like aggregations is the number of autophagosomes.

LC3 on the inner membrane of autophagosomes is degraded in lysosomes. Therefore, the overall degradation amount of LC3 can be quantitatively detected by flow cytometry through the decrease in GFP-LC3 fluorescence.

Figure 6. GFP intensity is reduced by nutrient starvation, while autophagy or lysosome inhibitors enhance GFP intensity.

2) RFP -GFP-LC3 dual fluorescence indicator system

The acidic environment within lysosomes causes quenching of the GFP fluorescence signal, and red fluorescent protein has good tolerance to the acidic environment, allowing the construction of RFP-GFP-LC3 concatemers. When autophagy is formed, autophagosomes are marked in yellow (green and red images overlap), and autophagolysosomes are marked in red.

Figure 7. RFP-GFP-LC3 dual fluorescent indicator system

5. What autophagy detection related products does Elabscience have?

The above is an introduction of autophagy. For more content on cell death, please continue to pay attention to the cell death inventory series.