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. © 2011 Macmillan Publishers Limited. All rights reserved.