Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S.

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.

A nationwide observational study of locomotive syndrome in Japan using the ResearchKit: The Locomonitor study.

BACKGROUND: We developed the Locomonitor application (app), the world's first iOS app to study locomotive syndrome, using the ResearchKit and examined the prevalence and risk factors for locomotive syndrome in Japanese general individuals 20-69 years old in a nationwide cross-sectional observational study. METHODS: The participants were recruited from February to August 2016. The outcome measures for the locomotive function were evaluated by locomotive syndrome risk tests (LSRTs) using the Locomonitor app. The chi-squared test, a linear-by-linear association trend analysis, and Spearman's correlation test were performed as statistical analyses. RESULTS: A total of 2177 subjects from all prefectures in Japan were included (average 42.2 years old). The Locomo25 and Stand-Up test scores in female participants and the Two-Step test scores in male participants showed age-dependent deterioration. In the overall population, the incidence of Locomo stage 1 and 2, as evaluated by the Locomo25, Stand-Up test or Two-Step test, was 30.2% and 29.2%, respectively. In subjects without locomotive syndrome (40.5%), LSRT scores showed age-dependent deterioration in both sexes. Locomotive syndrome in participants with a body mass index (BMI) of ≥25 kg/m2 was more frequent than in those with a BMI of <25 kg/m2 (age- and gender-adjusted odds ratio [OR] 1.344 [95% confidence interval {CI} 1.03-1.75, p = 0.027]). Locomotive syndrome in participants with an exercise habit was less frequent than in those without an exercise habit (age- and gender-adjusted OR 0.499 [95% CI 0.33-0.755, p < 0.0001]). CONCLUSIONS: The Locomonitor app, a newly developed remote platform, revealed that approximately 20%-30% of Japanese individuals 20-69 years old in the general population met the definition of locomotive syndrome. Locomotive syndrome in participants with obesity was more frequent than those without obesity, while locomotive syndrome in participants with an exercise habit was less frequent than those without an exercise habit.

Metabolic contribution of hepatic autophagic proteolysis: old wine in new bottles.

Pioneering work on autophagy was achieved soon after the discovery of lysosomes more than 50 years ago. Due to its prominent lysosomal activity and technical ease of handling, the liver has been at the center of continuous and vigorous investigations into autophagy. Many important discoveries, including suppression by insulin and plasma amino acids and stimulation by glucagon, have been made through in vivo and in vitro studies using perfused liver and cultured hepatocytes. The long-term controversy about the origin and nature of the autophagosome membrane has finally led to the conclusion of "phagophore," through extensive molecular cell biological approaches enlightened by the discovery of autophagy-essential ATG genes. Furthermore, recent studies using liver-specific autophagy-deficient mice have thrown light on the unique role of a selective substrate of autophagy, p62. The stabilized p62 accumulating in autophagy-deficient liver manipulates Nrf2-dependent transcription activation through specific binding to Keap1, which results in the elevated gene expression involved in detoxification. This is the first example of the dysregulation of gene expression under autophagy deficiency. Thus, basal liver autophagy makes a large contribution to the maintenance of cell homeostasis and health. Meanwhile, precise comparisons of wild-type and liver-specific autophagy-deficient mice under starvation conditions have revealed that amino acids released by autophagic degradation can be metabolized to produce glucose via gluconeogenesis for the maintenance of blood glucose, and can also be excreted to the circulation to supply serum amino acids. These results strongly confirm that induced liver autophagy plays a pivotal role in metabolic compensation. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Differences in expression patterns of cathepsin C/dipeptidyl peptidase I in normal, pathological and aged mouse central nervous system.

Cathepsin C (CC) (EC 3.4.14.1, dipeptidyl peptidase I) is a lysosomal cysteine protease that is required for the activation of several granule-associated serine proteases in vivo. CC has been shown to be constitutively expressed in various tissues, but the enzyme is hardly detectable in central nervous system (CNS) tissues. In the present study, we investigated the regional and cellular distribution of CC in normal, aging and pathological mouse brains. Immunoblotting failed to detect CC protein in whole brain tissues of normal mice, as previously described. However, low proteolytic activity of CC was detected in a brain region-dependent manner, and granular immunohistochemical signals were found in neuronal perikarya of particular brain regions, including the accessory olfactory bulb, the septum, CA2 of the hippocampus, a part of the cerebral cortex, the medial geniculate, and the inferior colliculus. In aged mice, the number of CC-positive neurons increased to some extent. The protein level of CC and its proteolytic activity showed significant increases in particular brain regions of mouse models with pathological conditions--the thalamus in cathepsin D-deficient mice, the hippocampus of ipsilateral brain hemispheres after hypoxic-ischemic brain injury, and peri-damaged portions of brains after penetrating injury. In such pathological conditions, the majority of the cells that were strongly immunopositive for CC were activated microglia. These lines of evidence suggest that CC is involved in normal neuronal function in certain brain regions, and also participates in inflammatory processes accompanying pathogenesis in the CNS.

Doxorubicin-induced glomerulosclerosis with proteinuria in GFP-GABARAP transgenic mice.

Autophagy is a process of cellular degradation, and its dysfunction elicits many pathological symptoms. However, the contribution of autophagy to kidney glomerular function has not been fully clarified. We previously reported that LC3, a promising executor of autophagy, played an important role in recovery from podocyte damage in an experimental nephrosis model (Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T, Kominami E, Tomino Y. FASEB J 17: 1165-1167, 2003). γ-Aminobutyric acid A receptor-associated protein (GABARAP), has recently been characterized as another homolog of LC3, although its precise role in autophagy remains unclear. We recently generated green fluorescent protein (GFP)-GABARAP transgenic mice, in which GFP-GABARAP is abundantly expressed in glomerular podocytes. We found that the transgenic mice showed no obvious phenotype, and podocytes isolated from these mice manifested autophagic activity almost equivalent to that of wild-type mice when measured in vitro. Surprisingly, a single injection of doxorubicin caused a greater increase in proteinuria and sclerotic glomeruli in transgenic mice compared with wild-type mice. Under these conditions, neither GFP-GABARAP nor endogenous GABARAP appeared to be recruited to autophagosomes, and both remained in the cytosol. Moreover, the cytosolic GFP-GABARAP was significantly colocalized with p62 to form aggregates. These results indicate that the GFP-GABARAP/p62 complex is responsible for impairment of glomerular function and that it retards recovery from the effects of doxorubicin.

Loss of autophagy in the central nervous system causes neurodegeneration in mice.

Protein quality-control, especially the removal of proteins with aberrant structures, has an important role in maintaining the homeostasis of non-dividing neural cells. In addition to the ubiquitin-proteasome system, emerging evidence points to the importance of autophagy--the bulk protein degradation pathway involved in starvation-induced and constitutive protein turnover--in the protein quality-control process. However, little is known about the precise roles of autophagy in neurons. Here we report that loss of Atg7 (autophagy-related 7), a gene essential for autophagy, leads to neurodegeneration. We found that mice lacking Atg7 specifically in the central nervous system showed behavioural defects, including abnormal limb-clasping reflexes and a reduction in coordinated movement, and died within 28 weeks of birth. Atg7 deficiency caused massive neuronal loss in the cerebral and cerebellar cortices. Notably, polyubiquitinated proteins accumulated in autophagy-deficient neurons as inclusion bodies, which increased in size and number with ageing. There was, however, no obvious alteration in proteasome function. Our results indicate that autophagy is essential for the survival of neural cells, and that impairment of autophagy is implicated in the pathogenesis of neurodegenerative disorders involving ubiquitin-containing inclusion bodies.

Crucial role for autophagy in degranulation of mast cells.

BACKGROUND: Autophagy plays a crucial role in controlling various biological responses including starvation, homeostatic turnover of long-lived proteins, and invasion of bacteria. However, a role for autophagy in development and/or function of mast cells is unknown. OBJECTIVE: To investigate a role for autophagy in mast cells, we generated bone marrow-derived mast cells (BMMCs) from mice lacking autophagy related gene (Atg) 7, an essential enzyme for autophagy induction. METHODS: Bone marrow-derived mast cells were generated from bone marrow cells of control and IFN-inducible Atg7-deficient mice, and morphologic and functional analyses were performed. RESULTS: We found that conversion of type I to type II light chain (LC3)-II, a hallmark of autophagy, was constitutively induced in mast cells under full nutrient conditions, and LC3-II localized in secretory granules of mast cells. Although deletion of Atg7 did not impair the development of BMMCs, Atg7(-/-) BMMCs showed severe impairment of degranulation, but not cytokine production on FcεRI cross-linking. Intriguingly, LC3-II but not LC3-I was co-localized with CD63, a secretory lysosomal marker, and was released extracellularly along with degranulation in Atg7(+/+) but not Atg7(-/-) BMMCs. Moreover, passive cutaneous anaphylaxis reactions were severely impaired in mast cell-deficient WBB6F1-W/W(V) mice reconstituted with Atg7(-/-) BMMCs compared with Atg7(+/+) BMMCs. CONCLUSION: These results suggest that autophagy is not essential for the development but plays a crucial role in degranulation of mast cells. Thus, autophagy might be a potential target to treat allergic diseases in which mast cells are critically involved.

COVID-19 Severity and Thrombo-Inflammatory Response Linked to Ethnicity.

Although there is strong evidence that SARS-CoV-2 infection is associated with adverse outcomes in certain ethnic groups, the association of disease severity and risk factors such as comorbidities and biomarkers with racial disparities remains undefined. This retrospective study between March 2020 and February 2021 explores COVID-19 risk factors as predictors for patients' disease progression through country comparison. Disease severity predictors in Germany and Japan were cardiovascular-associated comorbidities, dementia, and age. We adjusted age, sex, body mass index, and history of cardiovascular disease comorbidity in the country cohorts using a propensity score matching (PSM) technique to reduce the influence of differences in sample size and the surprisingly young, lean Japanese cohort. Analysis of the 170 PSM pairs confirmed that 65.29% of German and 85.29% of Japanese patients were in the uncomplicated phase. More German than Japanese patients were admitted in the complicated and critical phase. Ethnic differences were identified in patients without cardiovascular comorbidities. Japanese patients in the uncomplicated phase presented a suppressed inflammatory response and coagulopathy with hypocoagulation. In contrast, German patients exhibited a hyperactive inflammatory response and coagulopathy with hypercoagulation. These differences were less pronounced in patients in the complicated phase or with cardiovascular diseases. Coagulation/fibrinolysis-associated biomarkers rather than inflammatory-related biomarkers predicted disease severity in patients with cardiovascular comorbidities: platelet counts were associated with severe illness in German patients. In contrast, high D-dimer and fibrinogen levels predicted disease severity in Japanese patients. Our comparative study indicates that ethnicity influences COVID-19-associated biomarker expression linked to the inflammatory and coagulation (thrombo-inflammatory) response. Future studies will be necessary to determine whether these differences contributed to the less severe disease progression observed in Japanese COVID-19 patients compared with those in Germany.

A novel type of E3 ligase for the Ufm1 conjugation system.

The ubiquitin fold modifier 1 (Ufm1) is the most recently discovered ubiquitin-like modifier whose conjugation (ufmylation) system is conserved in multicellular organisms. Ufm1 is known to covalently attach with cellular protein(s) via a specific E1-activating enzyme (Uba5) and an E2-conjugating enzyme (Ufc1), but its E3-ligating enzyme(s) as well as the target protein(s) remain unknown. Herein, we report both a novel E3 ligase for Ufm1, designated Ufl1, and an Ufm1-specific substrate ligated by Ufl1, C20orf116. Ufm1 was covalently conjugated with C20orf116. Although Ufl1 has no obvious sequence homology to any other known E3s for ubiquitin and ubiquitin-like modifiers, the C20orf116 x Ufm1 formation was greatly accelerated by Ufl1. The C20orf116 x Ufm1 conjugate was cleaved by Ufm1-specific proteases, implying the reversibility of ufmylation. The conjugation was abundant in the liver and lungs of Ufm1-transgenic mice, fractionated into membrane fraction, and impaired in Uba5 knock-out cells. Intriguingly, immunological analysis revealed localizations of Ufl1 and C20orf116 mainly to the endoplasmic reticulum. Our results provide novel insights into the Ufm1 system involved in cellular regulation of multicellular organisms.

Peroxisome degradation in mammals.

This review summarizes the historical aspects of the study of peroxisome degradation in mammalian cells. Peroxisomes have diverse metabolic roles in response to environmental changes and are degraded in a preferential manner, by comparison with cytosolic proteins. This review introduces three hypotheses on the degradation mechanisms: (a) the action of the peroxisome-specific Lon protease; (b) the membrane disruption effect of 15-lipoxygenase; and (c) autophagy that sequesters and degrades the organelles by lysosomal enzymes. Among these hypotheses, autophagy is now recognized as the most important mechanism for excess peroxisome degradation. One of the most striking characteristics of peroxisomes is that they are markedly proliferated in the liver by the administration of hypolipidemic drugs and industrial plasticizers. The effects of these substances were fully reversed after withdrawal of the substances, and most of the excess peroxisomes were selectively degraded and recovered to a normal number and size. Autophagic degradation of peroxisomes has been examined using this characteristic phenomenon. Excessive peroxisome degradation that occurs after cessation of hypolipidemic drugs has been extensively investigated biochemically and morphologically. The evidence shows that the degradation of excess peroxisomes and peroxisomal enzymes is inhibited by 3-methyladenine (3-MA), a specific inhibitor of autophagy. Furthermore, in liver-specific autophagy-deficient mice, rapid removal of peroxisomes was exclusively impaired, and degradation of peroxisomal enzymes was not detected. Thus, the significant contribution of autophagic machinery to peroxisomal degradation in mammals was confirmed. However, the important question of the mechanism for the selective recognition of peroxisomes by autophagosomes remains to be fully elucidated.

Dendrin location in podocytes is associated with disease progression in animal and human glomerulopathy.

BACKGROUND: Adriamycin (ADR) nephrosis in mice has been extensively studied and has enabled a greater understanding of the processes underlying the progression of renal injury. Dendrin is a novel component of the slit diaphragm with proapoptotic signaling properties, and it accumulates in the podocyte nucleus in response to glomerular injury in mice. The present study re-evaluated chronic progressive nephropathy in ADR mice and the localization of dendrin in mice and in human glomerulopathy. METHODS: To investigate the localization of dendrin, a mouse model of nephrosis and glomerulosclerosis was used, in which ADR was injected once. WT-1-positive cells and apoptotic cells were counted in vivo and in vitro. To check the expression of dendrin in ADR mice, immunostaining and Western blot were performed. A survey of dendrin staining was performed on human kidney biopsy specimens. RESULTS: The injection of ADR induced proteinuria, podocyte loss and glomerulosclerosis. It also caused the relocation of dendrin from the slit diaphragm to the podocyte nucleus. We demonstrated the location of dendrin to podocyte nuclei in several cases of human glomerulopathy. The mean occurrence of dendrin-positive nucleus per glomerulus increased in several cases of human glomerulopathy. CONCLUSIONS: These findings suggest that the relocation of dendrin to the podocyte nuclei is useful as a novel marker of podocyte injury in human glomerulopathy.

Autophagic compartments gain access to the MHC class II compartments in thymic epithelium.

The presentation of self-peptides in the context of MHC molecules by thymic epithelial cells (TECs) is essential for T cell repertoire selection in the thymus. However, the underlying mechanisms of this process have not been fully elucidated. To address whether autophagy, a catabolic process involving the degradation of a cell's components through the lysosomal machinery, intersects the MHC class II-restricted Ag presentation pathway in TECs, we investigated the colocalization of LC3, a peculiar autophagy marker molecule, with MHC class II compartments in in vitro-established TEC lines by immunofluorescence microscopy and Western blotting analyses. We found that in both cortical and medullary TEC lines, LC3 was colocalized with the H2-DM-positive lysosomal compartments, in which MHC class II plus class II-associated invariant chain peptides complexes are formed. Furthermore, our analysis of thymic cryosections from 1-day-old mice revealed that LC3 colocalizes with the H2-DM-positive compartments in TECs. These results strongly suggest that the cytoplasmic self-Ags gain access to the H2-DM-positive compartments via the autophagic process in the thymus.

Free fatty acids stimulate autophagy in pancreatic ß-cells via JNK pathway.

Recent studies have suggested that free fatty acids stimulate autophagy of pancreatic beta cells. The aim of this study was to verify the free fatty acids (FFA)-induced autophagy and investigate its molecular mechanism. As reported previously, palmitate strongly enhanced the conversion of light chain (LC)3-I to LC3-II, a marker of activation of autophagy in INS-1 beta cells. Palmitate-induced conversion of LC3-I to LC3-II was also observed in neuron-, muscle-, and liver-derived cells. In addition, palmitate induced the formation of typical autophagosomes and autolysosomes and enhanced the degradation rate of long-lived proteins. These results confirmed that palmitate activates autophagic flux in most of the cells. While FFAs reportedly activate several signal transduction pathways in beta cells, palmitate-induced autophagy was blocked by a JNK inhibitor. Although enhanced oxidative stress and endoplasmic reticulum (ER) stress are suspected to mediate FFA-induced activation of JNK1, the induction of autophagy was not associated with changes in molecular markers related to oxidative and endoplasmic reticulum stresses. On the other hand, phosphorylation of double stranded RNA-dependent protein kinase (PKR) paralleled JNK1 activation. Considered together, our study suggested that FFA stimulated functional autophagy possibly through the PKR-JNK1 pathway independent of ER or oxidative stress.

LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation.

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism.

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

The MAP1-LC3 conjugation system is involved in lipid droplet formation.

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.

Phospholipase C-related inactive protein is involved in trafficking of gamma2 subunit-containing GABA(A) receptors to the cell surface.

The subunit composition of GABA(A) receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of gamma2 subunit-containing GABA(A) receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABA(A) receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of gamma2 subunit-containing GABA(A) receptors. The surface numbers of diazepam binding sites (alpha/gamma2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (alpha/beta subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABA(A) receptors and GABA(A) receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABA(A) receptor beta subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of gamma2 subunit-containing GABA(A) receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of gamma2 subunit-containing GABA(A) receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.

Comprehensive proteomics analysis of autophagy-deficient mouse liver.

Autophagy is a bulk protein degradation system for the entire organelles and cytoplasmic proteins. Previously, we have shown the liver dysfunction by autophagy deficiency. To examine the pathological effect of autophagy deficiency, we examined protein composition and their levels in autophagy-deficient liver by the proteomic analysis. While impaired autophagy led to an increase in total protein mass, the protein composition was largely unchanged, consistent with non-selective proteins/organelles degradation of autophagy. However, a series of oxidative stress-inducible proteins, including glutathione S-transferase families, protein disulfide isomerase and glucose-regulated proteins were specifically increased in autophagy-deficient liver, probably due to enhanced gene expression, which is induced by accumulation of Nrf2 in the nuclei of mutant hepatocytes. Our results suggest that autophagy deficiency causes oxidative stress, and such stress might be the main cause of liver injury in autophagy-deficient liver.

LC3 and Autophagy.

Microtubule-associated protein 1A/1B-light chain 3 (LC3) is a soluble protein with a molecular mass of approximately 17 kDa that is distributed ubiquitously in mammalian tissues and cultured cells. During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. Autophagosomes fuse with lysosomes to form autolysosomes, and intra-autophagosomal components are degraded by lysosomal hydrolases. At the same time, LC3-II in autolysosomal lumen is degraded. Thus, lysosomal turnover of the autophagosomal marker LC3-II reflects starvation-induced autophagic activity, and detecting LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy and autophagy-related processes, including autophagic cell death. Here we describe basic protocols to assay for endogenous LC3-II by immunoblotting, immunoprecipitation, and immunofluorescence.