Reference - Detail
|Seki T, Yoshino KI, Tanaka S, Dohi E, Onji T, Yamamoto K, Hide I, Paulson HL, Saito N, Sakai N.
|Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron.
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.
|Animals Autophagy* Cytosol / metabolism Fluorescence Fluorescent Dyes / metabolism* Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / metabolism HSC70 Heat-Shock Proteins / metabolism HeLa Cells Humans Lysosomes / metabolism Mice Mice, Inbred ICR Molecular Chaperones / metabolism* Mutation / genetics Neurons / metabolism* Neurons / pathology* Protein Binding Protein Kinase C / genetics Protein Transport Purkinje Cells / enzymology Purkinje Cells / pathology Single-Cell Analysis / methods* Spinocerebellar Ataxias / enzymology Spinocerebellar Ataxias / genetics
|Human and Animal Cells