Identification of a factor controlling lysosomal homeostasis using a novel lysosomal trafficking probe

Abstract

Lysosomes are largely responsible for significant degradation of intracellular and extracellular proteins via the secretory pathway, autophagy, and endocytosis. Therefore, dysregulation of lysosomal homeostasis influences diverse cellular functions. However, a straightforward and quantitative method to measure the integrity of the lysosomal pathway has not been developed. Here, we report the plasmid-based lysosomal-METRIQ (MEasurement of protein Transporting integrity by RatIo Quantification) probe that enables simple quantification of lysosomal integrity by lysosomal green and cytosolic red fluorescent proteins using a flow cytometer. In cultured cells, the lysosomal-METRIQ probe detected not only suppression of the lysosomal pathway but also upregulation of lysosomal activity such as lysosomal biogenesis. To identify factors involved in lysosomal homeostasis, we carried out compound screening and found that the cyclin-dependent kinase (CDK) inhibitors kenpaullone and purvalanol A induce synthesis of cathepsin D and an increase in the number of lysosomes. Subsequent studies revealed that CDK5 maintains lysosomal homeostasis independently of cell cycle arrest. Our results suggest that the lysosomal-METRIQ probe is an effective and efficient tool for measuring lysosomal activity in mammalian cells.

Regulatory Function of Sympathetic Innervation on the Endo/Lysosomal Trafficking of Acetylcholine Receptor

“…Particularly, overexpression of a dominant-negative mutant of Cdk5 (DN-Cdk5) caused a reduction of denervation-induced endocytic AChR-containing vesicles ( Wild et al, 2016 ). These data are consistent with the observation of a cooperative function of Cdk5, Endophilin B1, and Beclin1 in autophagy ( Wong et al, 2011 ; Ishii et al, 2019 ). At rodent NMJs, degradation of endocytosed AChR vesicles has been shown to occur via autophagy in an Endophilin B1 and MuRF1-dependent manner ( Khan et al, 2014 ).…”

Lysosomes and signaling pathways for maintenance of quiescence in adult neural stem cells

“…Further investigations, using both proteomic approaches and protein analysis at the molecular level, are required to reveal the role of lysosomes in quiescence. Imaging in live cells with fluorescent probes for lysosomal activity could also be used to monitor lysosomal function and dynamics [51,[85][86][87]. Because the lysosome is a multifunctional organelle, the inhibition or activation of lysosomes alters multiple aspects of cellular functions, including metabolic changes and environmental stresses associated with aging.…”

The Autophagy Machinery in Human-Parasitic Protists; Diverse Functions for Universally Conserved Proteins

“…However, it should be noted that the degradation of the autophagocytosed material is often not demonstrated in parasitic protists. It would thus be particularly important to develop and adapt autophagy probes and monitoring assays that are useful in other eukaryotic systems to not only detect autophagy, but also to measure the autophagic flux [39][40][41] and lysosomal activity [124].…”