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Autophagy is a cellular process in which intracellular components are degraded and recycled. It is broadly categorized into macroautophagy, which involves the formation of autophagosomes, and microautophagy, where lysosomes directly engulf target materials without autophagosome intermediates1. While mitophagy is typically mediated by macroautophagy, recent studies have uncovered a microautophagy-like mechanism by which lysosomes selectively remove damaged portions of mitochondria rather than the entire organelle2. |
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1. Autophagy genes in biology and disease (Nature Reviews Genetics, 2024) Related techniques Autophagic Flux Detection |
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2. Lysosomes drive the piecemeal removal of mitochondrial inner membrane (Nature, 2024) Highlighted technique: This study uses a combination of super-resolution microscopy, live-cell imaging, and specific fluorescent probes and proteins to visualize and track in real time the dynamic process of IMM protruding through the OMM and their subsequent uptake by lysosomes. Related techniques Lysosomal Function Analysis, Mitophagy detection |
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Related Techniques (click to open/close)
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Application Note I (click to open/close)
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We determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification. Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD+) biosynthetic enzyme, Nampt induces lysosomal deacidification1). In this section, we tried to determine how NAD+ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway. 1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021) |
Application Note II (click to open/close)
> Induction of Mitophagy in Parkin Expressed HeLa cells
| Induction of mitophagy by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a mitochondrial-uncoupling reagent with Parkin expressed HeLa cells. | |
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HeLa cells were seeded on μ-slide 8 well (Ibidi) and cultured at 37oC overnight in a 5%-CO2 incubator. The cells were transfected with Parkin plasmid vector by HilyMax transfection reagent (Code#:H357), and incubated at 37oC overnight. The Parkin expressed HeLa cells were washed with Hanks’ HEPES buffer twice and then incubated at 37oC for 30 minutes with 250 μl of 100 nmol/l Mtphagy Dye working solution containing 100 nmol/l MitoBright LT Deep Red (※). After the washing of the cells with Hanks’ HEPES buffer twice, the culture medium containing 10 μmol/l CCCP was added to the well. After 24 hours incubation, mitophagy was observed by fluorescence microscopy. After removing the supernatant, 250 μl of 1 μmol/l Lyso Dye working solution were added to the cells and incubated at 37oC for 30 minutes. The cells were washed with Hanks’ HEPES buffer twice and then co-localization of Mtphagy, Lyso Dye and MitoBright Deep Red was observed by confocal fluorescence microscopy. Observation of mitophagy using Parkin ※Our sincere apology that we discontinued distributing MitoBright Deep Red.
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