RRC ID 63488
Author Ishii S, Matsuura A, Itakura E.
Title Identification of a factor controlling lysosomal homeostasis using a novel lysosomal trafficking probe.
Journal Sci Rep
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.
Volume 9(1)
Pages 11635
Published 2019-8-12
DOI 10.1038/s41598-019-48131-2
PII 10.1038/s41598-019-48131-2
PMID 31406169
PMC PMC6690932
MeSH Benzazepines / pharmacology Cyclin-Dependent Kinase 5 / antagonists & inhibitors Cyclin-Dependent Kinase 5 / genetics Cyclin-Dependent Kinase 5 / metabolism* Endocytosis / drug effects Endocytosis / physiology* Flow Cytometry Gene Knockdown Techniques HEK293 Cells HeLa Cells Humans Indoles / pharmacology Luminescent Agents / chemistry Luminescent Proteins / chemistry* Luminescent Proteins / genetics Lysosomes / drug effects Lysosomes / metabolism* Microscopy, Confocal / methods Microscopy, Fluorescence / methods Molecular Probes / chemistry* Molecular Probes / genetics Plasmids / genetics Purines / pharmacology
IF 3.998
DNA material pMRX-IP TFEB-sfGFP (RDB18607) pCW DNAseIIα-sfGFP-T2A-mCherry (RDB18608)