Autophagy

Autophagy is the process by which a cell breaks down old, damaged, abnormal components within the cytoplasm. In this way autophagy provides a conservative form of selective internal degradation that is a common attribute of an adaptive cellular response. It is one of the main three forms of cell death, with the other two being apoptosis and necrosis. Autophagy may be triggered or induced by a number of factors including starvation of essential gases, depletion of amino acids, or even insulin availability. The occurrence of autophagy is characterized by autophagosome formation, a typically double-membraned organelle, which occurs through the complete sequestration by the elongating phagophore in the cytoplasm.    



Autophagy can be divided into three subcategories, most identifiable by derogatory pathway.    

 

The most well-studied in mammals is macroautophagy, where an autophagosome fuses with a lysosome or vacuole in the cytoplasm, and gathers the cellular components in what is known as an autophagic body. This body then directly degrades or recycles the cargo it now holds via hydrolases.    

Microautophagy is lesser known, and unlike the other two processes it is mediated by direct engulfment of the cytoplasmic cargo, where material in the lysosome or vacuole is trapped by random membrane invagination.    

In chaperone-mediated autophagy, a chaperone complex in the cytosol identifies target proteins in the lysosome or vacuole, and pulls them to the surface of the organelle. These chaperones and proteins form complexes, then the chaperone unwinds and is pulled across the lysosomal membrane where it undergoes degradation inside the lumen.    

Utilization and recycling of degraded products gives autophagy many important physiological roles, not just as a survival response to starvation and hypoxia, but also in gluconeogenesis energy production, protein synthesis, and nutrient mobilization in development. Elimination of macromolecules and organelles enables autophagy to be a form of cellular quality control; such intracellular clearance may effectively rid the cytoplasm of surplus or hurtful proteins, organelles, bacteria, and/or pathogens. Additionally in immunity, autophagy may serve as a defense mechanism to allow the sequestration and packaging of viruses. Autophagy has also been observed to be used as a biosynthetic pathway, used to transport molecules from the cytoplasm to a lysosome or endosome.    

Detection and Measurement

---

Immunofluorescence

   
Immunofluorescence (IF) coupled with fluorescent microscopy may also be used to show cellular localization of protein. LC3 is the most common target, though other proteins namely p62, LAMP1/2, and cathepsin have been used. A typical IF protocol may be used, and staining should be done using an anti-LC3 antibody (or other antibody of choice) where transfected cells should be fluorescently tagged with LC3. Upon visualization, basal level autophagy will typically show a diffuse cytosolic LC3 expression pattern. After autophagy induction, LC3 becomes associated with the autophagosome membrane which will be seen as a dotted expression pattern.    
     

Electron Microscropy

   
Electron microscopy (EM) may be used to determine morphology and cellular position of autophagy samples. To quantify EM images, samples are scored for the presence and/or absence of autophagic structures in a section or entire cytoplasm of the cell. Alternatively, the entire cell volume occupied by autophagosomes within the cell may be estimated to determine autophagy activity and extent. Though this method offers high sensitivity, it is time consuming, low-throughput, and requires technical expertise for visualization and data analysis.    

   

Western Blot

   

One common semi-quantitative methodology of detecting autophagy is through Western blot (WB). Usually, antibodies that target LC3-II, a mammalian homolog of ATg8 and a major component of the autophagosomal inner membrane, are used for detection, though dozens of other pathway proteins may be utilized. Biologically, LC3-I is a protein found in the cytosol that becomes lipidated to form LC3-II. LC3-II will further become inserted into the membrane of autophagosomes, which make it an ideal target for the detection and analysis of autophagous samples. The key step in quantifying autophagy in WB lies in the analysis of the electrophoresis results, where LC3-I will migrate faster than LC3-II due to its hydrophobicity.    

One thing to consider during testing is that LC3-II is more sensitive to degradation than LC3-I, so use of fresh samples is highly advised compared to freeze-thawed samples. The loading control used should be considered carefully, and actin should not be used due to its inherent sensitivity to autophagy, though GAPDH and tubulin may provide better results.    

It is important to note that autophagy induction causes LC3-II levels to increase, but WB does not take into consideration LC3-II turnover so results may be misleading unless this assay is coupled with one that detects cellular autsophagy flux. Another consideration is that LC3 binds strongly to PVDF membranes, and the marker will be lost after the stripping step, so stripping and reprobing of the membrane should be avoided.    
   

Flow Cytometry

   
Flow cytometry can also be used to quantitate a large number of cells in a mixed cell population labeled with multiple antibodies simultaneously, meaning it can detect several components of the autophagy pathway simultaneously, too. LC3 is the most common target, though other proteins of the autophagic pathways including LAMP1/2 and Beclin 1 have been used. Effective experimental design must consider sample preparation, choice of fluorophore, positive controls, and autophagy flux. One common technique involved transfecting cells with fluorescently tagged LC3. Cytosolic LC3-I is then removed, normally through an incubation period with saponin, to leave behind membrane bound LC3-II. Additionally, endogenous levels of LC3 may also be measured.    

Experimentally, a few items must be considered before choosing flow cytometry to analyze autophagy. If using saponin to remove LC3-I, it is important to ensure careful titration and incubation so that excessive damage is not done to the cellular membrane. Where LC3 is bound to a reporter tag, cellular artifacts may result from overexpression. Endogenous LC3 may report levels lower than the level of detection, so this is not always a viable route. Primary cells in flow also may obscure positive signals hard to distinguish, making false positives more difficult to detect.

   

Immunohistochemistry

   
If immunohistochemistry (IHC) is preferred, LC3 (most commonly) or other proteins including  p62, ULK-1, LAMP1, Beclin 1, or ATG5/7/12 may be chosen as targets. IHC is not the most common method to determine autophagy, due to the lack of biomarkers in the autophagous pathway, but if chosen multiple targets should be chosen and tested together. IHC requires careful analysis as results are susceptible to misinterpretation, and high levels of cytosolic LC3 may make visualization and data interpretation difficult. False positives are common due to the nonspecific granular staining pattern of organelles within the cytosol, and cellular artifacts may obscure quantification of data.    

Distinguishing the characteristic double membrane of the autophagosome depends on the quality of the samples and/or fixative use, and paraformaldehyde fixation may present with lipid droplet formation so acetone fixation is recommended. Frozen samples exhibit poor morphology and resolution, so fresh samples are recommended. All-in-all, this assay must be coupled with another experiment to determine the true extent of autophagy within the sample. Frozen samples exhibit poor morphology and resolution, so fresh samples are recommended. All-in-all, this assay must be coupled with another experiment to determine the true extent of autophagy within the sample.    

       

   

Product Ordering Information

---