Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron.

ABSTRACT:

Background

Chaperone-mediated autophagy (CMA) is a selective autophagy-lysosome protein degradation pathway. The role of CMA in normal neuronal functions and in neural disease pathogenesis remains unclear, in part because there is no available method to monitor CMA activity at the single-cell level.

Methodology/principal findings

We sought to establish a single-cell monitoring method by visualizing translocation of CMA substrates from the cytosol to lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT (GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA and prevent

SUBMITTER: Seki T 

PROVIDER: S-EPMC3280339 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature