Mitochondrial degradation by autophagy (mitophagy) in GFP-LC3 transgenic hepatocytes during nutrient deprivation

Abstract

Fasting in vivo and nutrient deprivation in vitro enhance sequestration of mitochondria and other organelles by autophagy for recycling of essential nutrients. Here our goal was to use a transgenic mouse strain expressing green fluorescent protein (GFP) fused to rat microtubule-associated protein-1 light chain 3 (LC3), a marker protein for autophagy, to characterize the dynamics of mitochondrial turnover by autophagy (mitophagy) in hepatocytes during nutrient deprivation. In complete growth medium, GFP-LC3 fluorescence was distributed diffusely in the cytosol and incorporated in mostly small (0.2-0.3 μm) patches in proximity to mitochondria, which likely represent preautophagic structures (PAS). After nutrient deprivation plus 1 μM glucagon to simulate fasting, PAS grew into green cups (phagophores) and then rings (autophagosomes) that enveloped individual mitochondria, a process that was blocked by 3-methyladenine. Autophagic sequestration of mitochondria took place in 6.5 ± 0.4 min and often occurred coordinately with mitochondrial fission. After ring formation and apparent sequestration, mitochondria depolarized in 11.8 ± 1.4 min, as indicated by loss of tetramethylrhodamine methylester fluorescence. After ring formation, LysoTracker Red uptake, a marker of acidification, occurred gradually, becoming fully evident at 9.9 ± 1.9 min of ring formation. After acidification, GFP-LC3 fluorescence dispersed. PicoGreen labeling of mitochondrial DNA (mtDNA) showed that mtDNA was also sequestered and degraded in autophagosomes. Overall, the results indicate that PAS serve as nucleation sites for mitophagy in hepatocytes during nutrient deprivation. After autophagosome formation, mitochondrial depolarization and vesicular acidification occur, and mitochondrial contents, including mtDNA, are degraded.

Fig. 1.

Induction of autophagosomes during nutrient deprivation plus glucagon in GFP-LC3 transgenic mouse hepatocytes. A: hepatocytes from green fluorescent protein (GFP)-microtubule-associated protein-1 light chain 3 (LC3) mice were incubated either in Waymouth's growth medium (WM) or in nutrient-free Krebs-Ringer-HEPES buffer plus glucagon (KRH/G) to stimulate nutrient deprivation. Images were collected after 90 min by laser-scanning confocal microscopy. In WM, the majority of GFP-LC3 fluorescence was diffuse in the cytosol and nucleus or incorporated into green patches [preautophagic structures (PAS), arrowheads] and rarely in disks (autophagosomes, double arrows). In KRH/G, GFP-LC3 fluorescence appeared in numerous rings and disks (autophagosomes, double arrows) and cup-shaped structures (phagophores, arrows). Insets: magnified images of autophagic structures. B: hepatocytes from wild-type mice were incubated in WM or KRH/G for 0 to 90 min, and LC3 I and LC3 II protein expression was assessed in cell extracts by Western blotting, as described in materials and methods.

Fig. 2.

Confocal microscopy of mitophagy during nutrient deprivation plus glucagon in GFP-LC3 hepatocytes. GFP-LC3 hepatocytes were loaded with tetramethylrhodamine methylester (TMRM) to label mitochondria, as described in materials and methods. A: confocal images of TMRM-loaded GFP-LC3 hepatocytes incubated in WM (left), KRH/G (middle), or KRH/G plus 10 mM 3-methyladenine (3MA; right) for 90 min. In WM, green-fluorescing GFP-LC3 patches were present that sometimes resided adjacent to red-fluorescing mitochondria. Autophagosomes (green rings and disks) were rare but increased greatly in KRH/G and often enclosed TMRM-labeled mitochondria. 3MA blocked autophagosome formation in WM. B: the numbers of PAS (green patches), phagophores (green cup-shaped structures), polarized mitophagosomes (green rings or disks containing TMRM), and depolarized autophagosomes/autolysosomes (green rings or disks not containing TMRM) per cellular confocal image were quantified for GFP-LC3 hepatocytes incubated as described in A from three different hepatocyte isolations per treatment group. Differences in numbers of PAS, mitophagosomes, and autolysosomes in KRH/G compared with WM and 3MA were statistically significant (P < 0.05, n = 5 cells/group). C: representative structures of a PAS (arrowhead), phagophore (arrow), polarized mitophagosome (double arrow), and autolysosome (double arrowhead).

Fig. 3.

Acidification of autophagosomes during nutrient deprivation plus glucagon in GFP-LC3 hepatocytes. GFP-LC3 hepatocytes were loaded with LysoTracker Red (LTR) to label acidified vesicles, as described in materials and methods. A: GFP-LC3 hepatocytes were incubated in WM (left), KRH/G (middle), or KRH/G plus 10 mM 3MA (right) for 90 min. After incubation in WM, individual PAS were distributed throughout the cytosol, and LTR staining was mostly confined to small primary lysosomes/late endosomes. In KRH/G, LTR-labeled vesicles proliferated, often in association with GFP-LC3 fluorescence. Cells incubated in KRH/G with 3MA were indistinguishable from cells in WM. B: the numbers of primary lysosomes/acidified late endosomes, acidified autophagosomes, and autolysosomes were quantified from confocal images of GFP-LC3 hepatocytes incubated as described in A from three different hepatocyte isolations per treatment group. Differences in numbers of primary lysosomes, autophagosomes, and autolysosomes in KRH/G compared with WM and 3MA were statistically significant (P < 0.05, n = 5 cells/group). C: representative structures of a primary lysosome (arrowhead), autophagosome (double arrow), and autolysosome (double arrowhead).

Fig. 4.

Progression of autophagic sequestration of polarized mitochondria. A and B: TMRM-loaded GFP-LC3 hepatocytes were incubated in KRH/G, and time-lapse images of TMRM-labeled mitochondria were collected every minute. Representative images are shown. In favorable views along the long axis of forming phagophores, note progression from a GFP-LC3 (green)-labeled PAS patch (arrowheads) to a cup-shaped phagophore (arrows) to a ring-shaped mitophagosome (double arrows). Loss of red TMRM fluorescence (mitochondrial depolarization) occurred at or after ring closure (double arrowheads). C: TMRM fluorescence intensity of mitochondria during the progression of mitophagy is plotted. Baseline is background-subtracted TMRM fluorescence of cellular mitochondria not in association with GFP-LC3 normalized to 100%. Other fluorescence values are for same cell mitochondria in association with PAS, phagophores, and GFP-LC3 rings before and after the beginning of mitochondrial depolarization. *P < 0.05 compared with other groups; n ≥ 4 per group.

Fig. 5.

Acidification of autophagosomes. A: LTR-loaded GFP-LC3 hepatocytes were incubated in KRH/G, and time-lapse images were collected every minute. Double arrows indicate the formation and maturation of an autophagosome. In this example, autophagosome formation (GFP-LC3 ring) was complete at 29 min and was followed by vesicular acidification (LTR uptake) beginning between 35 and 37 min. Acidification (LTR uptake) become maximal at ∼42 min. B: GFP-LC3 hepatocytes were loaded with LTR and MitoFluor Far Red (MFFR) and incubated 90 min in KRH/G. Arrows, double arrows, arrowheads, and double arrowheads indicate a polarized mitophagosome, a later depolarizing mitophagosome, an autolysosome still containing GFP-LC3 fluorescence, and a later autolysosome that has lost remnants of GFP-LC3, respectively.

Fig. 6.

Mitochondrial fission during nutrient deprivation-induced mitophagy. GFP-LC3 hepatocytes were loaded with TMRM and incubated in KRH/G. Images were collected every minute. A: a PAS became associated with the middle part of a TMRM-labeled mitochondrion and elongated to form a phagophore (arrows). The middle part of the mitochondria was then enveloped, and completion of sequestration occurred coordinately with mitochondrial fission. Afterward, the sequestered mitochondrial fragment depolarized, as shown by loss of red TMRM fluorescence. B: a PAS localized to the end of a TMRM-labeled mitochondrion and elongated into a phagophore (arrows). Again, completion of sequestration occurred coordinately with mitochondrial fission and was followed by depolarization of the sequestered mitochondrion.

Fig. 7.

Mitochondrial DNA sequestration and degradation by mitophagy. Hepatocytes from wild-type mice were coloaded with green-fluorescing PicoGreen to label DNA and red-fluorescing TMRM or LTR, as described in materials and methods. A: PicoGreen- and TMRM-loaded hepatocytes were incubated in WM. Note one to several mtDNA nucleoids inside individual mitochondria that appear yellow in the overlay. B: PicoGreen and LTR-colabeled hepatocytes were incubated in KRH/G, and images were collected at 1 and 120 min. Initially, PicoGreen-labeled mtDNA rarely localized with LTR. After 120 min incubation with KRH/G, some LTR-labeled autolysosomes contained PicoGreen staining (arrows). In the top left corner is a portion of a nucleus, which also strongly labeled with PicoGreen. C: time-lapse images of PicoGreen and LTR-colabeled hepatocytes in KRH/G were collected. Arrows identify mtDNA nucleoids of a single mitochondrion that was subsequently sequestered by mitophagy. Afterward, PicoGreen fluorescence of the sequestered mtDNA decreased and virtually disappeared.