Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein, Tandem Fluorescent-Tagged LC3

Kimura, Shunsuke; Noda, Tetsuji; Yoshimori, Tamotsu

doi:10.4161/auto.4451

Cited by 1,989 publications

(2,050 citation statements)

References 40 publications

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“…This step can be monitored using a transgene encoding Atg8a fused to tandem GFP-mCherry (GFP-mCherry-Atg8a). Fusion of the relatively neutral autophagosomes to acidic degradative LEs and lysosomes selectively quenches the more pH-sensitive and proteolytically labile GFP fluorescence, shifting the emitted fluorescence ratio from green/yellow towards red [52]. Autophagy is active in Drosophila photoreceptor cells, as evidenced by massive accumulation of autophagosomes in the eyes of flies with loss-of-function mutations in the HOPS complex components car/Vps33A and dor/Vps18 , due to a failure of HOPS-dependent autophagosome-lysosome fusion [28,49].…”

Section: Resultsmentioning

confidence: 99%

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

2018

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Rab2 is a conserved Rab GTPase with a well-established role in secretory pathway function and phagocytosis. Here we demonstrate that Drosophila Rab2 is recruited to late endosomal membranes, where it controls the fusion of LAMP-containing biosynthetic carriers and lysosomes to late endosomes. In contrast, the lysosomal GTPase Gie/Arl8 is only required for late endosome-lysosome fusion, but not for the delivery of LAMP to the endocytic pathway. We also find that Rab2 is required for the fusion of autophagosomes to the endolysosomal pathway, but not for the biogenesis of lysosome-related organelles. Surprisingly, Rab2 does not rely on HOPS-mediated vesicular fusion for recruitment to late endosomal membranes. Our work suggests that Drosophila Rab2 is a central regulator of the endolysosomal and macroautophagic/autophagic pathways by controlling the major heterotypic fusion processes at the late endosome.

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Section: Resultsmentioning

confidence: 99%

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

2018

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“…We isolated APG (enriched in LC3‐II and p62; note that the low amounts of LAMP1 and cathepsin observed in this fraction could represent amphisomes [resulting from fusion of APG and late endosomes]) and AUT (also enriched in both markers but with higher abundance of lysosomal markers (LAMP1, CathB, and mucolipin; Figure 4a ). To determine whether our isolation procedure preserved vesicle‐associated motor proteins, we performed immunoblots for the minus‐end‐directed motor dynein (Jahreiss et al., 2008; Katsumata et al., 2010; Kimura, Noda & Yoshimori, 2007; Kimura et al., 2008) and the plus‐end‐directed motor KIF5B (Cardoso et al., 2009; Geeraert et al., 2010) previously described to contribute to trafficking of autophagic compartments. Immunofluorescence and immunoblot for motor proteins (dynein shown in Figure 4a,b and S4a–c) in the isolated fractions confirmed that motors were indeed present on the surface of the isolated APG and were not due to contamination of the preparation with cytosol.…”

Section: Resultsmentioning

confidence: 99%

“…Independent of these possible mechanisms, in this work we have identified a defect at the level of vesicle‐associated molecular motors that can account for the differences in motility with age. Dynein dysfunction with age, manifested as defective interaction with dynactin, has been previously described and shown sufficient to reproduce age‐dependent retromer deficiency (Kimura et al., 2007, 2016). Our analysis of the molecular motors associated with APG and lysosomes in liver confirmed the previously reported presence of the plus‐end‐directed motor KIF5B (member of kinesin‐1 family) in APG (Toda et al., 2008).…”

Section: Discussionmentioning

confidence: 99%

Defective recruitment of motor proteins to autophagic compartments contributes to autophagic failure in aging

et al. 2018

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SummaryInability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule‐based minus‐end‐directed motor proteins, the well‐characterized dynein, and the less‐studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti‐aging purposes.

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“…Autophagosome clearance, which begins from the autolysosome formation by fusion of autophagosome with lysosome, is the second step for the complement of autophagic process 27, 30. As shown in Figure 2A, significantly more autolysosomes (red) were observed in the cells treated with tetracaine (1345.8%), bupivacaine (1169.5%), ropivacaine (1571.1%), procaine (1964.0%) and lidocaine (1637.0%), respectively, compared with untreated controls ( P < 0.01).…”

Section: Resultsmentioning

confidence: 99%

Autophagy activated by tuberin/mTOR/p70S6K suppression is a protective mechanism against local anaesthetics neurotoxicity

Xiong

Kong

Dai

et al. 2016

J Cellular Molecular Medi

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The local anaesthetics (LAs) are widely used for peripheral nerve blocks, epidural anaesthesia, spinal anaesthesia and pain management. However, exposure to LAs for long duration or at high dosage can provoke potential neuronal damages. Autophagy is an intracellular bulk degradation process for proteins and organelles. However, both the effects of LAs on autophagy in neuronal cells and the effects of autophagy on LAs neurotoxicity are not clear. To answer these questions, both lipid LAs (procaine and tetracaine) and amide LAs (bupivacaine, lidocaine and ropivacaine) were administrated to human neuroblastoma SH‐SY5Y cells. Neurotoxicity was evaluated by MTT assay, morphological alterations and median death dosage. Autophagic flux was estimated by autolysosome formation (dual fluorescence LC3 assay), LC3‐II generation and p62 protein degradation (immunoblotting). Signalling alterations were examined by immunoblotting analysis. Inhibition of autophagy was achieved by transfection with beclin‐1 siRNA. We observed that LAs decreased cell viability in a dose‐dependent manner. The neurotoxicity of LAs was tetracaine > bupivacaine > ropivacaine > procaine > lidocaine. LAs increased autophagic flux, as reflected by increases in autolysosome formation and LC3‐II generation, and decrease in p62 levels. Moreover, LAs inhibited tuberin/mTOR/p70S6K signalling, a negative regulator of autophagy activation. Most importantly, autophagy inhibition by beclin‐1 knockdown exacerbated the LAs‐provoked cell damage. Our data suggest that autophagic flux was up‐regulated by LAs through inhibition of tuberin/mTOR/p70S6K signalling, and autophagy activation served as a protective mechanism against LAs neurotoxicity. Therefore, autophagy manipulation could be an alternative therapeutic intervention to prevent LAs‐induced neuronal damage.

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Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein, Tandem Fluorescent-Tagged LC3

Cited by 1,989 publications

References 40 publications

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

Defective recruitment of motor proteins to autophagic compartments contributes to autophagic failure in aging

Autophagy activated by tuberin/mTOR/p70S6K suppression is a protective mechanism against local anaesthetics neurotoxicity

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