Autophagy : Reagent Selection Guide

Recent research reveals mechanisms that some autophagy receptors and liquid-liquid phase separation, which are triggers to induce selective autophagy. Autophagy plays a critical role in maintaining homeostasis and dysregulated autophagy contributes to diseases such as neurodegeneration and cancer. Here are key papers exploring selective autophagy mechanisms and disease associations.

Selective autophagy is a critical cellular process that targets and degrades specific damaged or unnecessary components, such as protein aggregates, organelles, or invading pathogens. This selective clearance prevents the accumulation of toxic materials, thereby maintaining cellular homeostasis and promoting cell survival under stress conditions. For example, by removing dysfunctional mitochondria through mitophagy, selective autophagy minimizes the production of reactive oxygen species, thereby preserving energy balance and genomic integrity. Dysregulation of selective autophagy is associated with several diseases, including neurodegeneration, cancer and metabolic disorders, highlighting its essential role in cellular health.

Point of Interest
- Phase separation forms initiation hubs on cargo surfaces, enabling phagophore nucleation for selective autophagy in yeast and human cells.

- Multivalent and low-affinity receptor-cargo interactions drive hub formation, while high-affinity binding blocks autophagy progression.

- Phase separation regulates mass and selective autophagy initiation in diverse organisms, linking cargo to the ER for phagophore assembly.

Point of Interest
- Autophagy selectively degrades topoisomerase 1 cleavage complexes (TOP1cc) DNA lesions, thereby maintaining genome stability.

- TOP1cc are targeted to lysosomes via nuclear envelope modifications, independent of the proteasome.

- The autophagy receptor TEX264 recognizes TOP1cc at replication forks and directs lysosomal processing via p97 ATPase, MRE11 nuclease and ATR kinase.

Point of Interest
- Mitochondrial transcription factor A (TFAM) uses autophagy to remove leaked mitochondrial DNA (mtDNA), thereby preventing inflammation.

- TFAM’s LC3 interacting region (LIR) motif allows binding to autophagy proteins that mediate mtDNA degradation through nucleoid-phagy.

- Mutation of TFAM's LIR motif leads to accumulation of cytoplasmic mtDNA and activation of inflammatory pathways despite normal mitochondrial function.

NAD⁺ Depletion and Autophagy-Lysosomal Pathway Response

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)

We next 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.

Products in Use
   - Autophagic Flux Assay Kit 
     (combination kit of DAPRed / DALGreen)
   - DAPGreen

Simultaneous Detection of Lysosomal and Mitochondrial Dysfunction

We tried the simultaneous detection of lysosomal and mitochondrial dysfunction in Hela cells treated with CCCP or Antimycin (AN). CCCP and AN are well-known inducers of mitochondrial ROS regarding loss of mitochondrial membrane potential. Recent research showed the result that CCCP induces not only mitochondrial ROS but also lysosomal neutralization. To detect mitochondrial ROS, HeLa cells were labeled by MitoBright ROS Deep Red - Mitochondrial Superoxide Detection, and the lysosomal mass and pH were detected separately with LysoPrime Green and pHLys Red. Co-staining with MitoBright ROS and Lysosomal dyes demonstrated that CCCP causes lysosomal neutralization and mitochondrial ROS induction at the same time.

Products in Use
   - Lysosomal Acidic pH Detection Kit
     (combination kit of LysoPrime Green / pHLys Red)
   - MitoBright ROS Deep Red - Mitochondrial Superoxide Detection