In Situ Differential Analysis of α- and β-Glycosidase Activities in Lysosomes After Internalization Using...

Fluorogenic glycosides are widely used substrates for assaying lysosomal glycosidase activities in vitro, but they do not provide subcellular information in living cells. In this study, we used glucosylceramide (GlcCer) liposomes as carriers to deliver fluorogenic substrates into live PC12 cells for confocal imaging. The α-4-methylumbelliferyl glucoside (α-4MUG) and β-glucosidase substrate β-4-(trifluoromethyl)umbelliferyl glucoside (β-4FMUG) were co-encapsulated in liposomes. The liposomes (approximately 100 nm in diameter) were taken up by PC12 cells after pulse exposure. Punctate fluorescence signals from both hydrolyzed substrates were observed. The relative intensity of two signals varied among puncta, as assessed by dual-channel imaging and line-scan analysis. These results show that GlcCer liposomes provide a practical platform for long-term and differential analyses of relative α- and β-glucosidase activities in living cells.