Mitophagy Diversified: Discovery of a Microautophagy-like Mechanism [Jul. 15, 2025]

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)

HeLa cells were seeded on μ-slide 8 well (Ibidi) and cultured at 37^oC overnight in a 5%-CO₂ incubator. The cells were transfected with Parkin plasmid vector by HilyMax transfection reagent (Code#:H357), and incubated at 37^oC overnight. The Parkin expressed HeLa cells were washed with Hanks’ HEPES buffer twice and then incubated at 37^oC 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 37^oC 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
expressed HeLa cells (upper panel) and normal HeLa cells (Lower)
A, E) Fluorescent images of Mtphagy Dye; B, F) Fluorescent images of Lyso Dye;
C, G) Fluorescent images of MitoBright Deep Red (※); D, H) Merged images

※Our sincere apology that we discontinued distributing MitoBright Deep Red.
     Please refer to MitoBright LT Deep Red (Code#:MT12) which improved retention ability.