title: Fluorescent-based evaluation of chaperone-mediated autophagy and microautophagy activities in cultured cells.
abstract: The autophagy-lysosome protein degradation is further classified into macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). While MA is involved in various functions and disease pathogenesis, little is known about CMA and mA because of the absence of easy methods to assess their activities. We have recently established a method to assess CMA activity using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a CMA substrate, and HaloTag (HT) system. Another group has recently identified a mammalian mA pathway, in which substrates are delivered to late endosomes in an heat shock cognate protein (Hsc)70-dependent manner. Because Hsc70 is also involved in CMA, our method would detect both CMA and mA activities. In this study, we attempted to assess CMA and mA activities separately through the siRNA-mediated knockdown of CMA- and mA-related proteins. Knockdown of LAMP2A, a CMA-related protein, and TSG101, an mA-related protein, significantly but only partially decreased the punctate accumulation of GAPDH-HT in AD293 cells and primary cultured rat cortical neurons. Compounds that activate CMA significantly increased GAPDH-HT puncta in TSG101-knockdown cells, but not in LAMP2A-knockdown cells, suggesting that punctate accumulation of GAPDH-HT under LAMP2A- and TSG101-knockdown represents mA and CMA activities, respectively. We succeeded in establishing the method to separately evaluate CMA and mA activities by fluorescence observation.
authors.affiliation: Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.affiliation: Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Seki
authors.affiliation: Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Ayumu
authors.initials: A
authors.lastname: Konno
authors.affiliation: Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Hirai
authors.affiliation: Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kurauchi
authors.affiliation: Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto, Japan.|Program for Leading Graduate Schools "HIGO (Health life science: Interdisciplinary and Glocal Oriented) Program", Kumamoto University, Kumamoto, Japan.
authors.forename: Akinori
authors.initials: A
authors.lastname: Hisatsune
authors.affiliation: Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Katsuki
mesh_terms: D000818:Animals; D001343:Autophagy / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics*; D011992:Endosomes / Q000378:metabolism; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000096:biosynthesis* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000235:genetics*; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000302:isolation & purification; D009363:Neoplasm Proteins / Q000235:genetics*; D009474:Neurons / Q000378:metabolism; D059748:Proteolysis; D051381:Rats; D014157:Transcription Factors / Q000235:genetics*; D000099177:Tsg101 Protein; D059666:Lysosomal-Associated Membrane Protein 3
chemical_list: D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D051907:Lysosomal Membrane Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D014157:Transcription Factors; D000099177:Tsg101 Protein; C114233:LAMP3 protein, human; D059666:Lysosomal-Associated Membrane Protein 3