Dissecting the dynamic turnover of GFP-LC3 in the autolysosome

Ni, Hong; Bockus, Abigail; Wozniak, Ann L.; Jones, Kellyann; Weinman, Steven A.; Yin, Xiao Ming; Ding, Wen

doi:10.4161/auto.7.2.14181

Cited by 295 publications

(261 citation statements)

References 36 publications

(57 reference statements)

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“…2D). These large LC3 structures are morphologically distinct from the autophagic LC3 puncta and are reminiscent of the LC3 aggregates observed when autophago-lysosomal protein degradation is blocked (13,14). Importantly, the pattern of these large-sized LC3 aggregates did not change upon nutrient starvation ( Fig.…”

Section: Resultsmentioning

confidence: 80%

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function

Jaber¹,

Dou²,

Chen³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

320

300

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A critical regulator of autophagy is the Class III PI3K Vps34 (also called PIK3C3). Although Vps34 is known to play an essential role in autophagy in yeast, its role in mammals remains elusive. To elucidate the physiological function of Vps34 and to determine its precise role in autophagy, we have generated Vps34 f/f mice, in which expression of Cre recombinase results in a deletion of exon 4 of Vps34 and a frame shift causing a deletion of 755 of the 887 amino acids of Vps34. Acute ablation of Vps34 in MEFs upon adenoviral Cre infection results in a diminishment of localized generation of phosphatidylinositol 3-phosphate and blockade of both endocytic and autophagic degradation. Starvation-induced autophagosome formation is blocked in both Vps34-null MEFs and liver. Liver-specific Albumin-Cre;Vps34 f/f mice developed hepatomegaly and hepatic steatosis, and impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34 f/f mice led to cardiomegaly and decreased contractility. In addition, while amino acid-stimulated mTOR activation was suppressed in the absence of Vps34, the steady-state level of mTOR signaling was not affected in Vps34-null MEFs, liver, or cardiomyocytes. Taken together, our results indicate that Vps34 plays an essential role in regulating functional autophagy and is indispensable for normal liver and heart function.LC3 | SQSTM1/p62 | 3-MA | epidermal growth factor receptor | transferrin M acroautophagy (referred to as autophagy hereafter) is a dynamic membrane trafficking process that involves the delivery of intracellular content to lysosomes for degradation. A fully executed autophagy includes the formation of doublemembraned autophagosomes, the fusion of autophagosomes to late endosomes/lysosomes, and the digestion of the enclosed content by lysosomal hydrolases. Autophagy is constantly maintained at the basal level and is up-regulated in response to stress conditions, such as nutrient and energy limitation, hypoxia, and DNA damage. Autophagy is necessary for cellular and tissue homeostasis, by eliminating damaged organelles and misfolded proteins, and its dysregulation is implicated in developmental defects and numerous diseases (1-5).

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Section: Resultsmentioning

confidence: 80%

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function

Jaber¹,

Dou²,

Chen³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

320

300

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“…This pattern was different from that of starvation-induced autophagy in which LC3-II level quickly rose in 0.5 to 2 h, followed by a decline, whereas p62 level changed conversely (14,24,25). p62 is an adaptor molecule for selective autophagy, but its level is often elevated in autophagy inhibition or deficiency (23,26).…”

Section: Resultsmentioning

confidence: 84%

Suppression of Lysosome Function Induces Autophagy via a Feedback Down-regulation of MTOR Complex 1 (MTORC1) Activity

Khambu

Zhang

et al. 2013

Journal of Biological Chemistry

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154

129

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Background: Lysosomes are required for autophagic degradation, which can be suppressed by lysosome inhibitors. Results: Inhibition of lysosome function resulted in autophagy activation via down-regulation of MTORC1. Conclusion: Lysosomes can affect autophagy initiation in addition to its role in autophagy degradation. Significance: The finding expands lysosome function to include regulation of autophagy activation and indicates a dual effect of lysosome inhibitors in autophagy.

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“…The total number of autophagic vacuoles, including autophagosomes and autolysosomes, was determined by counting all RFP fluorescent red puncta. The amount of autolysosomes, which represents the number of autophagosomes that were delivered to and degraded by lysosomes [36], was derived by subtracting the number of green puncta from the number of red puncta (red minus green). Consistent with our previous finding of increased LC3B-II protein levels in the mutants (Figure 5(A,B)), Gba L444P/WT neurons exhibited higher levels of total autophagic vacuoles than WT controls (Figure 5(D)), which is likely due to higher levels of autolysosomes.…”

Section: Resultsmentioning

confidence: 99%

Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations

et al. 2018

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Heterozygous mutations in GBA, the gene encoding the lysosomal enzyme glucosylceramidase beta/β-glucocerebrosidase, comprise the most common genetic risk factor for Parkinson disease (PD), but the mechanisms underlying this association remain unclear. Here, we show that in GbaL444P/WT knockin mice, the L444P heterozygous Gba mutation triggers mitochondrial dysfunction by inhibiting autophagy and mitochondrial priming, two steps critical for the selective removal of dysfunctional mitochondria by autophagy, a process known as mitophagy. In SHSY-5Y neuroblastoma cells, the overexpression of L444P GBA impeded mitochondrial priming and autophagy induction when endogenous lysosomal GBA activity remained intact. By contrast, genetic depletion of GBA inhibited lysosomal clearance of autophagic cargo. The link between heterozygous GBA mutations and impaired mitophagy was corroborated in postmortem brain tissue from PD patients carrying heterozygous GBA mutations, where we found increased mitochondrial content, mitochondria oxidative stress and impaired autophagy. Our findings thus suggest a mechanistic basis for mitochondrial dysfunction associated with GBA heterozygous mutations.Abbreviations: AMBRA1: autophagy/beclin 1 regulator 1; BECN1: beclin 1, autophagy related; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chloroyphenylhydrazone; CYCS: cytochrome c, somatic; DNM1L/DRP1: dynamin 1-like; ER: endoplasmic reticulum; GBA: glucosylceramidase beta; GBA-PD: Parkinson disease with heterozygous GBA mutations; GD: Gaucher disease; GFP: green fluorescent protein; LC3B: microtubule-associated protein 1 light chain 3 beta; LC3B-II: lipidated form of microtubule-associated protein 1 light chain 3 beta; MitoGreen: MitoTracker Green; MitoRed: MitoTracker Red; MMP: mitochondrial membrane potential; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH transcription factor; NBR1: NBR1, autophagy cargo receptor; Non-GBA-PD: Parkinson disease without GBA mutations; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; ROS: reactive oxygen species; SNCA: synuclein alpha; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1/Porin: voltage dependent anion channel 1; WT: wild type

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Dissecting the dynamic turnover of GFP-LC3 in the autolysosome

Cited by 295 publications

References 36 publications

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function

Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function

Suppression of Lysosome Function Induces Autophagy via a Feedback Down-regulation of MTOR Complex 1 (MTORC1) Activity

Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations

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