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Review
. 2010 Jun;221(2):117-24.
doi: 10.1002/path.2694.

Autophagy: assays and artifacts

Sandra Barth  1 , Danielle Glick, Kay F Macleod
Affiliations
Review

Autophagy: assays and artifacts

Sandra Barth et al. J Pathol. 2010 Jun.

Abstract

Autophagy is a fundamental and phylogenetically conserved self-degradation process that is characterized by the formation of double-layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation. The increasing significance attached to autophagy in development and disease in higher eukaryotes has placed greater importance on the validation of reliable, meaningful and quantitative assays to monitor autophagy in live cells and in vivo in the animal. To date, the detection of processed LC3B-II by western blot or fluorescence studies, together with electron microscopy for autophagosome formation, have been the mainstays for autophagy detection. However, LC3 expression levels can vary markedly between different cell types and in response to different stresses, and there is also concern that over-expression of tagged versions of LC3 to facilitate imaging and detection of autophagy interferes with the process itself. In addition, the realization that it is not sufficient to monitor static levels of autophagy but to measure 'autophagic flux' has driven the development of new or modified approaches to detecting autophagy. Here, we present a critical overview of current methodologies to measure autophagy in cells and in animals.

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Figures

Figure 1
Figure 1
Technical approaches for measuring autophagy. (A) An example of TEM to identify autophagosomes. TEM remains an important tool for detecting autophagosomes and, while not readily quantifiable, can provide significant insight to the extent of on-going autophagy in cells. This image shows human HCC-38 breast tumour cells that have been exposed to hypoxia (1% oxygen) for 24 h, undergoing selective autophagy of mitochondria, a process referred to as mitophagy. Mitochondria are seen inside double-membrane vesicles (black arrows). (B) The use of LC3B processing to measure autophagy. Measuring processing of endogenous LC3B by western blot is one of the most commonly used approaches to detecting increased autophagy in cells. In the example shown, HCC38 breast tumour cells are starved of nutrients by growth for several hours in Earle’s balanced salt solution (EBSS) in the presence or absence of Bafilomycin A1 (lanes 3, 5). Starvation induces LC3B processing (lane 4) compared to growth in regular DMEM medium (lanes 1, 4) and treatment with Bafilomycin A1 increases the amount of processed LC3B-II detected (lanes 3, 5), indicating that these cells have a high basal rate of autophagy (lane 3) that is further increased by starvation (lane 5). (C) Formation of LC3-positive puncta as evidence of autophagy. Processed LC3 can also be detected by immunofluorescence for LC3B-II, using epitope-specific antibodies on methanol-fixed cells. In the example shown, HCC38 cells were starved by growth in EBSS in the presence or absence of Bafilomycin A1. Starvation induced increased punctate staining for LC3 that was further increased by the addition of Bafilomycin A to block turnover at the lysosome.
Figure 2
Figure 2
Diagram summarizing approaches to measure autophagy at different points during the autophagic process. Different agents and approaches can be used to induce or to block different steps in the autophagic process, from phagophore formation to lysosomal protease activity. (A) The activity of the Vps34-Beclin-1 complex at the ER is a focal point for artificial modulation of autophagy, with knockdown or knockout of Beclin1 being one of the most common genetic approaches to inhibiting autophagy. Treatment of cells with 3-methyl adenine or wortmannin, which inhibits the activity of Class III PI3 kinases such as Vps34, is a common chemical approach to inhibiting autophagy, although these drugs also likely have other off-target effects in the cell that can make interpretation of data difficult at times. Autophagy can be artificially induced in autophagy-competent cells by treatment with agents such as lithium chloride (which inhibits inositol phosphatase) that promote the levels of PI3P, the product of Vps34 activity and which is required for the recruitment of key factors to the expanding phagophore. Other agents, such as thapsigargin, act by inducing ER stress and mimicking physiological stress. Targeted peptides, such as ABT737, which block the interaction of Beclin-1 with Bcl-2 and other BH3-containing molecules, have also been validated as promoting autophagy [63]. (B) Akin to knockdown of Beclin-1, effective inhibition of autophagy may be achieved through knockdown of Atg5 [64]. With the discovery that there are Atg5-independent forms of autophagy, however, knockdown of Beclin-1 may be the preferred approach. (C) Rapamycin has been widely used to experimentally induce autophagy through its ability to block the inhibitory action of mTOR on Atg1 and autophagy. More efficacious derivatives of rapamycin, known as ‘rapalogues’ are in clinical trials for cancer therapy. Such rapalogues include temsirolimus and everolimus. (D) Various agents are commonly used to inhibit lysosomal turnover of autophagosome content, such as Bafilomycin A1, which inhibits the lysosomal Na+H+ ATPase, and chloroquine, which increases the pH of the lysosome, thereby preventing the activity of lysosomal acid proteases and causing autophagosomes to accumulate. Similar effects are induced by treatment with specific inhibitors of lysosomal proteases, such as pepstatin A or E64d. The increased accumulation of autophagosomes under conditions in which lysosomal proteases are inhibited is then used to assess the rate of autophagic flux in response to specific stresses.
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