Methods in mammalian autophagy research

Abstract

Autophagy has been implicated in many physiological and pathological processes. Accordingly, there is a growing scientific need to accurately identify, quantify, and manipulate the process of autophagy. However, as autophagy involves dynamic and complicated processes, it is often analyzed incorrectly. In this Primer, we discuss methods to monitor autophagy and to modulate autophagic activity, with a primary focus on mammalian macroautophagy.

Figure 1. The Process of Macroautophagy

A portion of cytoplasm, including organelles, is enclosed by a phagophore or isolation membrane to form an autophagosome. The outer membrane of the autophagosome subsequently fuses with the lysosome, and the internal material is degraded in the autolysosome. In yeast, autophagosomes are generated from the preautophagosomal structure (PAS), which has not yet been identified in mammalian cells. A partial list of treatments and reagents that modulate autophagy are indicated. Notably, lithium may also inhibit autophagy through mTOR activation. Atg proteins that have thus far been identified on isolation membranes include ULK1/2, Atg5, Beclin 1, LC3, Atg12, Atg13, Atg14, Atg16L1, FIP200, and Atg101.

Figure 2. Autophagosome and Autolysosome Morphology

Electron microscopic analysis of nutrient-starved mouse embryonic fibroblasts. Arrows indicate autophagosomes and double arrows indicate autolysosomes/amphisomes. Arrowheads indicate fragments of endoplasmic reticulum inside the autophagosome. (Image courtesy of Chieko Kishi.)

Figure 3. Dynamic Regulation of Autophagy and Cellular Accumulation of Different Autophagic Structures

Depicted are the relative amounts of isolation membrane (IM), autophagosomes (AP), and autolysosomes (AL). (A) Under normal conditions, basal autophagy occurs. (B) When autophagy is induced, for example by starvation, there is an increase in all types of autophagic structures. (C) When autophagy is suppressed at any step upstream, none of the autophagic structures are generated. (D) When autophagy is suppressed at any step after complete closure of the autophagosome, only autophagosomes accumulate. Autophagic flux increases in (B), but decreases in (C) and (D).

Figure 4. Methods for Monitoring Autophagosome Number and Autophagic Flux

(A) Detection of autophagosomes and autolysosomes by conventional electron microscopy. (B) The GFP-LC3 (or endogenous LC3) puncta formation assay counts the average number of punctate structures per cell by fuorescence microscopy. (C) Detection of the conversion of LC3-I (cytosolic form) to LC3-II (membrane-bound lipidated form) by immunoblotting. (D) In the LC3 turnover assay, degradation of LC3-II inside the autolysosome is estimated by the comparison of two samples with and without lysosomal inhibitor treatment. (E) Degradation of autophagy-selective substrates such as LC3 and p62 is detected by immunoblotting (LC3 is part of the autophagy machinery rather than a true substrate but is selectively degraded by autophagy). The degradation of GFP-LC3 can also be quantifed by flow cytometry. (F) Detection of autophagosomes labeled with a yellow signal (mRFP-GFP-LC3) and their maturation into autolysosomes labeled with a red signal (after quenching of GFP fuorescence in the lysosome). (G) Detection of the GFP fragment generated by the degradation of GFP-LC3 inside autolysosomes by immunoblotting with an anti-GFP antibody. (H) Measurement of long-lived protein degradation that is suppressed by autophagy inhibitors.

Figure 5. GFP-LC3 and mRFP-GFP-LC3 Puncta Formation Assays

(A) NIH 3T3 cells in culture stably expressing GFP-LC3 with (right) or without (left) 2 hr of starvation (depletion of both amino acids and serum). There is not only an increase in GFP-LC3 puncta number, but also a decrease in total GFP-LC3 fluorescent signals during the 2 hr incubation period. (B) Example of an analysis of GFP-LC3 transgenic mice. Skeletal muscle (extensor digitorum longus) and heart muscle samples were prepared from GFP-LC3 transgenic mice before (left) or after (right) 24 hr of starvation. (C) Mouse embryonic fibroblasts expressing mRFP-GFP-LC3 (left) were subjected to starvation treatment (2 hr) with (right) or without (middle) 100 nM bafilomycin A₁ to inhibit autophagosome/lysosome fusion. Note that both yellow (autophagosome) and red (autolysosome) puncta increase in the middle panel, whereas most puncta in the right panel are yellow (autophagosome). Scale bars represent 10 µm. (Images in A and C courtesy of Eisuke Itakura.)

Figure 6. LC3 Conversion and Turnover Assays

(A) Steady-state levels of LC3 expression. Mouse embryonic fibroblasts were cultured in regular Dulbecco’s modified Eagle’s medium (DMEM) culture medium (lane 1), DMEM without amino acids and serum (lane 2), and regular DMEM containing 20 µM chloroquine (lane 3) for 1 hr. Cell lysates were subjected to immunoblot analysis with an anti-LC3 antibody. The positions of LC3-I and LC3-II are indicated. (B) LC3 turnover assay. Cells were cultured as in (A), and the difference in LC3-II levels between samples with and without chloroquine was compared under nonstarvation and starvation conditions. (Data courtesy of Akiko Kuma.)