# In Situ Differential Analysis of α- and β-Glycosidase Activities in Lysosomes After Internalization Using Glucosylcerebroside-Based Liposomes

## TL;DR

This paper introduces a method to study glycosidase activities in living cells using liposomes, enabling detailed imaging of enzyme activity within lysosomes.

## Contribution

The study introduces GlcCer liposomes as a novel platform for in situ differential analysis of α- and β-glycosidase activities in live cells.

## Key findings

- GlcCer liposomes successfully deliver fluorogenic substrates into PC12 cells for confocal imaging.
- Dual-channel imaging revealed variable relative intensities of α- and β-glycosidase activities in lysosomes.
- The method allows long-term and differential analysis of enzyme activities in living cells.

## Abstract

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.