Immunofluorescence of Autophagic Cargo Receptors and P-Tbk1 at LAMP1 Lysosomes During Lysophagy V1

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 method for monitoring protein recruitment to damaged lysosomes via immunofluorescence and confocal imaging.