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 prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for 
CMA, and by treatments that inactivate CMA. These results suggest that lysosomal 
accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed 
that mutant γPKC, which causes spinocerebellar ataxia type 14, decreased CMA 
activity in cultured PCs.
CONCLUSION/SIGNIFICANCE: In the present study, we established a novel 
fluorescent-based method to evaluate CMA activity in a single neuron. This novel 
method should be useful and valuable for evaluating the role of CMA in various 
neuronal functions and neural disease pathogenesis.