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. 2013 Aug 28;32(17):2336-47.
doi: 10.1038/emboj.2013.171. Epub 2013 Aug 6.

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

Ikuko Maejima  1 , Atsushi Takahashi, Hiroko Omori, Tomonori Kimura, Yoshitsugu Takabatake, Tatsuya Saitoh, Akitsugu Yamamoto, Maho Hamasaki, Takeshi Noda, Yoshitaka Isaka, Tamotsu Yoshimori
Affiliations

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

Ikuko Maejima et al. EMBO J. .

Abstract

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The recruitment of ubiquitin and LC3 to GFP-Gal3-positive damaged lysosomes. (A–C) Atg7+/+ and Atg7–/– MEFs stably expressing GFP-Gal3 (A, C) or GFP-Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti-LC3 and anti-Lamp1 (A), anti-p62 and anti-Lamp1 (B), or anti-FK2 and anti-p62 (C). Bars: 10 μm.
Figure 2
Figure 2
Decrease in the number of GFP-Gal3 puncta is dependent on time and autophagy. (A, B) NIH3T3 cells stably expressing GFP-Gal3 and either empty vector (control) or mStrawberry-Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP-Gal3 or Lamp1 puncta per cell was quantified using G-Count (see also Supplementary Figure S4A and B). Then, the percent of GFP-Gal3-positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm. Source data for this figure is available on the online supplementary information page.
Figure 3
Figure 3
tfGal3 GFP signal in puncta attenuates in an autophagy-dependent manner. (A) Diagram of the primary structure of tandem fluorescence-tagged Galectin-3 (tfGal3). (B) Schematic diagram of the fate of tfGal3 recruited to damaged lysosomes. (C–E) HeLa cells transfected with tfGal3 and either One-STrEP-FLAG-tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G-Count (D). Then, the percent of GFP+RFP+ tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm. (F–H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry-Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B-mutant (H) cells was quantified by G-Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm. Source data for this figure is available on the online supplementary information page.
Figure 4
Figure 4
CLEM analysis of mSt-Gal3- and GFP-LC3-associated membranes. (A–F) HeLa cells stably expressing GFP-LC3 were transfected with mStrawberry-Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B–F). Green: LC3; magenta: Gal3; blue: DAPI. (G–I) NIH3T3 cells stably expressing CFP-Gal3 and YFP-LC3, and either empty vector (control) (G, H) or mStrawberry-Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane.
Figure 5
Figure 5
Autophagy-deficient renal tubules exhibit severe injury and dysfunction under hyperuricaemia. (A) Plasma creatinine and urea nitrogen in Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P<0.05) are indicated. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. (B, C) PAS-stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4–7). Bars: 40 μm (B). Kidney injury score of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (C). (D) Kidney cortexes from Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA were subjected to immunohistocheminal analysis of Lamp1 and LC3. Green: LC3; magenta: Lamp1; blue: DAPI. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. Bar: 5 μm. (E) Electron micrographs of kidney proximal tubules in Atg5 F/F and Atg5F/F;KAP mice treated with UA+OA. Yellow arrowhead: autophagosome; asterisk: lysosome; Mt: mitochondrion. Bar: 1 μm. (F) Model of autophagic clearance of damaged lysosomes. Source data for this figure is available on the online supplementary information page.
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