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.

Glycosome turnover inLeishmania majoris mediated by autophagy

“…2C), and we observed an increase in the percentage of total glycosomes colocalizing with autophagosomes from 1% during log phase growth to 3% during differentiation (Table S3). Therefore, depending on the time it takes for an autophagosome to sequester and traffic its cargo to the lysosome (observed to be »16 min during mitophagy in mammals), 48 it is possible that the whole population of glycosomes could be turned over by autophagy within »8 h; this is consistent with the timescale of differentiation. This would result in a gradual degradation of glycosomes while new organelles could be synthesized, a process which would require selective turnover of old vs. new glycosomes.…”

“…The single Leishmania lysosome had a mean diameter of 0.5 to 0.6 mm; in contrast, yeast typically contain 1 to 3 large vacuoles 1 to 5 mm in diameter 53 and mammalian cells contain multiple small lysosomes 0.2 to 1.5 mm in size. 48 These data provide a useful reference for recognition of Leishmania compartments based on their size and shape, as well as comparison with those of other organisms. Autophagy plays an important role in mitochondrial maintenance in mammalian cells.…”

“…Autophagosomes under normal growth conditions had an average of 0.30 to 0.36 mm in diameter, making them smaller than those of yeast, which were measured by electron microscopy to have a diameter in the range 0.3 to 0.9 mm, 52,53 and mammalian cells, which measure 0.5 to 1.0 mm. 48,54 The autophagosomes of L. major were relatively uniform in size (Fig. 6), both during normal growth and starvation conditions, suggesting that their cargo normally was no larger than »0.3 mm wide.…”

“…Our results could be consistent with the presence of a specific set of autophagosomes that target glycosomes, supporting the idea that glycosomes are selective cargo. In starved rat hepatocytes, 85% of autophagosomes were observed to contain mitochondria 48 and it was concluded that under these conditions these organelles were being nonselectively sequestered by the majority of autophagosomes. Our data for glycosomes in starving promastigotes suggest that they are not the major source of nutrients targeted via nonselective autophagy to ensure survival.…”

Autophagy in adipose tissue and the beta cell: implications for obesity and diabetes

“…Stimulating autophagy during fasting may prevent oxidative injury by enhancing the clearance of damaged and/or depolarised mitochondria, which have accumulated during 'hyperactive' (postprandial) periods. This is largely reminiscent of the proposed role of augmented mitophagy during fasting or in response to glucagon in the liver [66].…”