Analysis of Lysophagic Flux in Cultured Cells using Lyso-Keima v2

Vinay V. EapenSharan Swarup

Abstract

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a facile method for monitoring lysophagy using the acid sensitive fluorophore mKeima, affixed onto Galectin-3, which allows for the monitoring of lysophagic flux by Flow cytometry, Western blotting or Confocal imaging. This method, which we have termed Lyso-Keima, serves as a facile and quantitative assay for monitoring lysophagy in tissue culture cells.

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