Chapter 13 Cytosolic LC3 Ratio as a Quantitative Index of Macroautophagy
Introduction
Macroautophagy constitutes a cellular self‐restructuring system through degradation of intracellular components (e.g., organelles, proteins, RNA) and is actively controlled by nutritional, physiological, and pathological processes (Mortimore and Kadowaki, 2001, Meijer and Codogno, 2004, Cuervo, 2004, Yorimitsu and Klionsky, 2005, Kadowaki et al., 2006). Although several biochemical and morphological methods have been developed for autophagy assessment, they are associated with limited and somewhat unsatisfactory results in mammalian cells (Mizushima, 2004, Klionsky et al., 2007). In general, biochemical methods are quantitative. However, it is difficult to distinguish autophagy from other intracellular degradation pathways such as the ubiquitin‐proteasome system or endocytosis, although autophagy is believed to account for the majority of long‐lived proteolysis (Mortimore and Kadowaki, 2001). Until recently, morphological and morphometric methods utilizing electron microscopy have been the most reliable methods for monitoring autophagy, but they are time‐consuming and require advanced skills (Schwörer et al., 1981, Mizushima, 2004, Kawai et al., 2006, Klionsky et al., 2007). A fluorescence method based on overexpression of GFP‐LC3 was recently introduced as a relatively simple and specific marker, but it does not provide a convenient measure for assessing autophagic flux (Mizushima, 2004).
Although LC3‐II is a very specific marker of autophagosomes/autolysosomes, detection of its steady‐state levels is not appropriate for estimating cellular autophagic flux (Tanida et al., 2005) (see also the chapter by Kimura et al. in this volume). For this reason, we calculated the ratio between LC3‐II and LC3‐I to estimate the activation step of autophagy formation that could be compared with the bulk proteolytic flux in isolated rat hepatocytes and rat hepatoma H4‐II‐E cells. During our analysis of the subcellular localization of LC3, we discovered a soluble delipidated form of LC3‐II that was localized in the cytosolic fraction. This soluble form, termed LC3‐IIs, was different from the typical form found in membrane fractions (LC3‐IIm). When the ratio between LC3‐IIs and LC3‐I in the cytosolic fraction was assessed, it was found to reflect quantitative changes in the proteolytic flux much better than that of the total homogenate. Thus, in this chapter, we describe the cytosolic LC3 ratio (LC3‐IIs/LC3‐I) as a novel sensitive index of macroautophagy in hepatocytes and H4‐II‐E cells.
Section snippets
Measurement of Proteolysis
We describe proteolysis measurement techniques for comparison with the LC3 method because it is still a reliable method for quantifying bulk proteolysis including autophagy. Here, two different proteolytic methods are described. For the study of fresh hepatocytes, we employ the combination of monitoring valine (Val) release from the hepatocytes (within an hour) in the presence of cycloheximide, relying on the fluorescent detection of Val by HPLC, because this method is more sensitive to
Subcellular fractionation
Because this method is rather simple but requires obtaining the cytosolic fraction, we describe the subcellular fractionation technique (Furuya et al., 2001).
- 1
To collect hepatocytes, 1.2 mL of cell suspensions (2 × 106 cells/mL) in the KRB buffer are centrifuged at 1,000 rpm for 50 s at 4 °C in a Kubota 6930 centrifuge with an RA‐300F rotor.
- 2
The supernatant fraction is discarded and the cell pellet is resuspended in 800 μL of 0.25 M sucrose/1 mM EDTA, pH 7.4, and homogenized by 120 strokes with a
Concluding Remarks
In conclusion, we identified a cytosolic subpopulation of LC3‐II, designated LC3‐IIs, which is hydrophilic and not lipidated, in fresh rat hepatocytes and the H‐4‐II‐E cell line. The cytosolic LC3 ratio derived from LC3‐IIs was found to represent a reliable index of autophagy formation and was much more sensitive and proportional to changes in the proteolytic rates than the ratio from the total homogenate. The use of this index should lead to a better understanding of the mechanisms of
References (31)
- et al.
Dominant negative Rab3D mutants reduce GTP‐bound endogenous Rab3D in pancreatic acini
J. Biol. Chem.
(2003) - et al.
Nutrient control of macroautophagy in mammalian cells
Mol. Aspects Med.
(2006) - et al.
Amino acids and insulin control autophagic proteolysis through different signaling pathways in relation to mTOR in isolated rat hepatocytes
J. Biol. Chem.
(2004) - et al.
Quantitative monitoring of autophagic degradation. Biochem. Biophys
Res. Commun.
(2006) - et al.
Assessment of protein turnover in perfumed rat liver: evidence for amino acid compartmentation from differential labeling of free and tRNA‐bound valine
J. Biol.Chem.
(1976) - et al.
Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation
J. Biol. Chem.
(2006) - et al.
Protein measurement with the folin phenol reagent
J. Biol. Chem.
(1951) - et al.
Regulation and role of autophagy in mammalian cells
Int. J. Biochem. Cell Biol.
(2004) Methods for monitoring autophagy
Int. J. Biochem. Cell Biol.
(2004)- et al.
Quantitative relationship between autophagy and proteolysis during graded amino acid deprivation in perfused rat liver
J. Biol. Chem.
(1981)
Phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian Atg8 modifiers, LC3, GABARAP, and GATE‐16
J. Biol. Chem.
HsAtg4B/HsApg4B/autophagin‐1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule‐associated protein light chain 3‐ and GABA receptor‐associated protein‐phospholipid conjugates
J. Biol. Chem.
LC3 conjugation system in mammalian autophagy
Int. J. Biochem. Cell Biol.
Dissection of autophagosome biogenesis into distinct nucleation and expansion steps
J. Cell Biol.
Cited by (124)
DIRAS3 regulates autophagy in an endometriosis epithelial cell line
2023, Reproductive BioMedicine OnlineThe anti-apoptotic and anti-autophagic effects of EPO through PI3K/Akt/mTOR signaling pathway in MAC-T cells
2022, Research in Veterinary ScienceMiR-125b enhances autophagic flux to improve septic cardiomyopathy via targeting STAT3/HMGB1
2021, Experimental Cell ResearchCitation Excerpt :Autophagy is a highly conserved regulated intracellular process in which unnecessary or dysfunctional proteins or organelles are engulfed by autophagosomes and transported to lysosomes for degradation [10]. Microtubule-associated protein 1 light chain 3 (LC3) plays a key role in macroautophagy formation and has been widely used as a marker for autophagy [11]. The ratio between the precursor form (LC3-I) and the active form (LC3-II) reflects the level of autophagic flux.