Main function of lysosomes

Endocytosis is a dynamic cellular process that actively transports particles into a cell. Late endosome fusion with the lysosome is a crucial step in the delivery of newly synthesized lysosomal proteins and endocytosed cargo for degradation. Disturbing this step in neurons is associated with neurological disorders. Thus, studying endosome-lysosome fusion in neurons will provide new insight into the mechanisms of these diseases and open new possibilities for therapeutic treatment. However, measuring endosome-lysosome fusion is challenging and time consuming, which limits the research in this area. Here we developed a high throughput method using pH-insensitive dye-conjugated dextrans and the Opera Phenix® High Content Screening System. By using this method, we successfully separated endosomes and lysosomes in neurons, and time-lapse images were collected to capture endosome-lysosome fusion events in hundreds of cells. Both assay set-up and analysis can be completed in an expeditious and efficient manner. Key words • Endosome-lysosome fusion • Opera Phenix® • High throughput assay • Time-lapse images • Dye-conjugated dextran • Neurological disorders • Goldstein JL, Brown MS, Anderson RGW et al (1985) Receptor mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol 1:1–39 • Gruenberg JE, Howell KE (1989) Membrane traffic in endocytosis: insights from cell-free assays. Annu Rev Cell Biol 5:453–481 • Kornfeld S, Mellman I (1989) The biogenesis o...

• Lysosomes are cellular organelles involved in the degradation and recycling of cellular waste. Extracellular and intracellular materials to be degraded reach the lysosome via endocytosis and autophagy, respectively. Lysosomes are also involved in secretion and plasma membrane repair, by fusing to the plasma membrane in a process termed lysosomal exocytosis. • Lysosomal function is performed by lumenal hydrolases that are responsible for substrate digestion and by membrane-associated proteins that handle trafficking of materials into and out of the lysosome. • A complex machinery, which includes the kinase complex mammalian target of rapamycin complex 1 (mTORC1, a major regulator of cell growth), the vesicular ATPase complex and additional complexes, is located on the lysosomal surface and is devoted to sensing the nutrient content of the lysosome. This complex is called the lysosomal nutrient sensing (LYNUS) machinery. • Most genes encoding lysosomal proteins belong to a gene network termed CLEAR (coordinated lysosomal expression and regulation), and they are transcriptionally regulated by transcription factor EB (TFEB), the master regulator for lysosomal biogenesis. Using this regulatory mechanism, cells can adapt lysosomal function to respond to environmental cues. • The activity of TFEB is induced following starvation, by both transcriptional autoregulation and a phosphorylation-dependent mechanism. Once activated, TFEB mediates the starvation response by activating l...