How to detect autophagosome?

Western Blotting. The most widely used antibodies to detect autophagy by western blotting are those that detect LC3. LC3 lipidation (LC3-I to LC3-II) results in an increase in molecular weight that can be detected by western blot (Figure 1).

How do you induce autophagy in a cell line?

Serum starvation for 12–24 h can also be used to induce autophagic flux in some cell types. Inhibition of glycolysis with 2-deoxyglucose (5 mM for 12–24 h) will also induce energetic stress and autophagy in cells.

What is the molecular mechanism of autophagosome formation?

The molecular mechanism of autophagy has been extensively studied using the yeast, Saccharomyces cerevisiae, during these past 10 years. These studies suggested that the molecular machinery of autophagosome formation is well conserved from yeast to higher eukaryotes.

How are autophagosomes generated in yeast and mammals?

Autophagosome formation. For example, LC3 in mammals is Atg8 in yeast and autophagosomes are generated from Pre-Autophagosomal Structure (PAS) which is distinct from the precursor structures in mammalian cells. The pre-autophagosomal structure in yeast is described as a complex localized near the vacuole.

How does ATG12-ATG5 dissociate from the autophagosome?

After the formation of the spherical structure, the complex of ATG12-ATG5:ATG16L1 dissociates from the autophagosome. LC3 is cleaved by ATG4 protease to generate cytosolic LC3. LC3 cleavage is required for the terminal fusion of an autophagosome with its target membrane.

How are endosomes and autophagosomes related to each other?

At first, autophagosomes fuse with endosomes or endosome-derived vesicles. These structures are then called amphisomes or intermediate autophagic vacuoles. Nonetheless, these structures contain endocytic markers even small lysosomal proteins such as cathepsin D .