Live imaging of intra-lysosome pH in cell lines and primary neuronal culture using a novel genetically encoded biosensor.

Autophagy
Amy H PonsfordMassimiliano Stagi

Abstract

Disorders of lysosomal physiology have increasingly been found to underlie the pathology of a rapidly growing cast of neurodevelopmental disorders and sporadic diseases of aging. One cardinal aspect of lysosomal (dys)function is lysosomal acidification in which defects trigger lysosomal stress signaling and defects in proteolytic capacity. We have developed a genetically encoded ratiometric probe to measure lysosomal pH coupled with a purification tag to efficiently purify lysosomes for both proteomic and in vitro evaluation of their function. Using our probe, we showed that lysosomal pH is remarkably stable over a period of days in a variety of cell types. Additionally, this probe can be used to determine that lysosomal stress signaling via TFEB is uncoupled from gross changes in lysosomal pH. Finally, we demonstrated that while overexpression of ARL8B GTPase causes striking alkalinization of peripheral lysosomes in HEK293 T cells, peripheral lysosomes per se are no less acidic than juxtanuclear lysosomes in our cell lines.Abbreviations: ARL8B: ADP ribosylation factor like GTPase 8B; ATP: adenosine triphosphate; ATP5F1B/ATPB: ATP synthase F1 subunit beta; ATP6V1A: ATPase H+ transporting V1 subunit A; Baf: bafilomycin A1; BLOC-...Continue Reading

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Citations

Nov 17, 2020·Chemistry : a European Journal·Matthieu StarckDavid Parker
Mar 18, 2021·Applied Microscopy·Yeseul JangSung Hoon Lee
Jun 9, 2021·ACS Sensors·Marcus Y ChinAimee W Kao
Aug 13, 2021·The Journal of Cell Biology·Ann-Christin BorchersChristian Ungermann
Sep 4, 2021·Molecular Omics·Pathma Muthukottiappan, Dominic Winter
Oct 16, 2021·Science Advances·Paula Gutiérrez-PérezLuisa Cochella

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Software Mentioned

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