title: A photoconvertible fluorescent reporter to track chaperone-mediated autophagy.
abstract: Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble proteins in lysosomes. CMA contributes to cellular quality control and is activated as part of the cellular response to different stressors. Defective CMA has been identified in ageing and different age-related diseases. Until now, CMA activity could only be measured in vitro using isolated lysosomes. Here we report the development of a photoconvertible fluorescent reporter that allows monitoring of CMA activity in living cells. Activation of CMA increases the association of the reporter with lysosomes which can be visualized as a change in the intracellular fluorescence. The CMA reporter can be utilized in a broad variety of cells and is suitable for high-content microscopy. Using this reporter, we find that levels of basal and inducible CMA activity are cell-type dependent, and we have identified an upregulation of this pathway in response to the catalytic inhibition of the proteasome.
authors.affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Koga
authors.forename: Marta
authors.initials: M
authors.lastname: Martinez-Vicente
authors.forename: Fernando
authors.initials: F
authors.lastname: Macian
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Cuervo
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001343:Autophagy / Q000502:physiology*; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D008247:Lysosomes / Q000502:physiology*; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism / Q000502:physiology*; D015336:Molecular Probe Techniques; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics
chemical_list: D018832:Molecular Chaperones