RpH-ILV: Probe for lysosomal pH and acute LLOMe-induced membrane permeabilization in cell lines and Drosophila

Authors:

Izaak J. Cheetham-Wilkinson, Bhavya Sivalingam, Chloe Flitton, Franziska Flottmann, Luisa Vehling, Maik Drechsler, Marija Stojchevska, Andrea Raimondi, Achim Paululat, Florian Fröhlich, Laura E. Swan, Massimiliano Stagi

Keywords:

RpH-ILV; Drosophila; pathophysiology; lysosome; pH dysregulation; LAMP1; vesicles; LLOMe; lysosomal pH sensor

Abstract:

Lysosomal pH dysregulation is a critical element of the pathophysiology of neurodegenerative diseases, cancers, and lysosomal storage disorders (LSDs). To study the role of lysosomes in pathophysiology, probes to analyze lysosomal size, positioning, and pH are indispensable tools. Here, we developed and characterized a ratiometric genetically encoded lysosomal pH probe, RpH-ILV, targeted to a subpopulation of lysosomal intraluminal vesicles. This subpopulation behaves similarly to the general population of LAMP1-positive vesicles in terms of pH response to pharmacological stresses. In addition, RpH-ILV, which is trafficked to the lysosome via a different cytosolic motif than our previous ratiometric sensor, RpH-LAMP1, is well tolerated by the model organism Drosophila melanogaster, exhibits minimal plasma membrane fluorescence, and reveals sensitivity to the lysosomal damaging agent LLOMe, adding a valuable tool to our repertoire of lysosomal pH sensors.

< Back to publications

Full article >