A genetically-encoded fluorescence-based reporter to spatiotemporally investigate mannose-6-phosphate pathway

Maintenance of a pool of active lysosomes with acidic pH and degradative hydrolases is crucial for cell health. Abnormalities in lysosomal function are closely linked to diseases, such as lysosomal storage disorders, neurodegeneration, intracellular infections, and cancer among others. Emerging body of research suggests the malfunction of lysosomal hydrolase trafficking pathway to be a common denominator of several disease pathologies. However, available conventional tools to assess lysosomal hydrolase trafficking are insufficient and fail to provide a comprehensive picture about the trafficking flux and location of lysosomal hydrolases. To address some of the shortcomings, we designed a genetically-encoded fluorescent reporter containing a lysosomal hydrolase tandemly tagged with pH sensitive and insensitive fluorescent proteins, which can spatiotemporally trace the trafficking of lysosomal hydrolases. As a proof of principle, we demonstrate that the reporter can detect perturbations in hydrolase trafficking, that are induced by pharmacological manipulations and pathophysiological conditions like intracellular protein aggregates. This reporter can effectively serve as a probe for mapping the mechanistic intricacies of hydrolase trafficking pathway in health and disease and is a utilitarian tool to identify genetic and pharmacological modulators of this pathway, with potential therapeutic implications.