Autophagy is a degradation process of cytoplasmic dysfunctional proteins and organelles. In this process, an isolation membrane composed of double membrane appear in cytosol, expands gradually, enfold with the aggregated proteins and damaged organelles, and close to form autophagosomes. The autophagosomes are fused with lysosomes to form autolysosomes in which are acidic environment. The contents in autolysosomes are decomposed by digestive enzymes in lysosomes. Since this cellular function is said to be related to aging as well as neurodegenerative diseases such as Parkinson’s disease, a simple autophagy detection method is being required.
New Insights into Autophagic and Endocytic Pathways
Autophagy is a cellular process that involves the degradation and recycling of cellular components such as damaged organelles, misfolded proteins, and intracellular pathogens. Autophagy is regulated by a complex network of several pathways, including the endocytic pathway. The endocytic pathway is responsible for the trafficking and sorting of proteins and lipids between different cellular compartments, including the plasma membrane, endosomes, lysosomes, and the trans-Golgi network. These pathways sometimes cooperate and sometimes independently contribute to cellular homeostasis. Today, we introduce you to three highlighted articles related to Autophagic and Endocytic pathways focusing on Nucleophay and aging, Endosomal lipid signaling, and Autophagy and the circadian clock.
- The nematode Caenorhabditis elegans nuclear envelope anchor protein ANC-1 and its m ammlian ortholog nesprin-2 are cleared out by autophagy and restrict the nucleolar size, a biomarker of aging.
- Nucleolar degradation at the most proximal oocyte by ANC-1 and key autophagic components relates to a germline immortality.
- Perturbation of this clearance pathway causes tumor-like structures in C. elegans, and genetic ablation of nesprin-2 causes ovarian carcinomas in mice.
- Nutrient starvation triggers changes in metabolism that are coordinated across the cell and its organelles.
- Jang et al. studied how endosomal signaling lipid turnover through MTM1 reshapes the endoplasmic reticulum to control mitochondrial morphology and oxidative metabolism.
- A lipid-controlled organellar relay transmits nutrient-triggered changes in endosomal signaling lipid levels to mitochondria to enable metabolic rewiring.
- Chaperone-mediated autophagy (CMA) contributes to the rhythmic removal of clock machinery proteins and the circadian remodeling.
- Disruption of this autophagic pathway in vivo leads to fragmented circadian patterns, resembling those in sleep disorders and aging.
- Conversely, loss of the circadian clock abolishes the rhythmicity of CMA, leading to pronounced changes in the CMA-dependent cellular proteome.
Tracing autophagosome to autolysosome in live cells
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.
To confirm the effect of the Nampt inhibitor, FK866, on lysosomal acidification, HeLa cells were first labeled by the lysosomal pH detection dye pHLys Red. The cells were then treated with FK866, and lysosomal acidification inhibitor Bafilomycin A1 was used as a positive control. FK866 and Bafilomycin A1-treatment each decreased the fluorescent pHLys Red signal, indicating lysosome neutralization.
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.
We next determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with 
