PMID- 22363588
OWN - NLM
STAT- MEDLINE
DCOM- 20120629
LR  - 20211021
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 7
IP  - 2
DP  - 2012
TI  - Establishment of a novel fluorescence-based method to evaluate chaperone-mediated 
      autophagy in a single neuron.
PG  - e31232
LID - 10.1371/journal.pone.0031232 [doi]
LID - e31232
AB  - 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 gammaPKC, 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.
FAU - Seki, Takahiro
AU  - Seki T
AD  - Department of Molecular and Pharmacological Neuroscience, Graduate School of 
      Biomedical Sciences, Hiroshima University, Hiroshima, Japan. 
      tseki@hiroshima-u.ac.jp
FAU - Yoshino, Ken-ich
AU  - Yoshino KI
FAU - Tanaka, Shigeru
AU  - Tanaka S
FAU - Dohi, Eisuke
AU  - Dohi E
FAU - Onji, Tomoya
AU  - Onji T
FAU - Yamamoto, Kazuhiro
AU  - Yamamoto K
FAU - Hide, Izumi
AU  - Hide I
FAU - Paulson, Henry L
AU  - Paulson HL
FAU - Saito, Naoaki
AU  - Saito N
FAU - Sakai, Norio
AU  - Sakai N
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20120207
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Fluorescent Dyes)
RN  - 0 (HSC70 Heat-Shock Proteins)
RN  - 0 (Molecular Chaperones)
RN  - EC 1.2.1.12 (Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating))
RN  - EC 2.7.1.- (protein kinase C gamma)
RN  - EC 2.7.11.13 (Protein Kinase C)
SB  - IM
EIN - PLoS One. 2014;9(9):e109006
MH  - Animals
MH  - *Autophagy
MH  - Cytosol/metabolism
MH  - Fluorescence
MH  - Fluorescent Dyes/*metabolism
MH  - Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism
MH  - HSC70 Heat-Shock Proteins/metabolism
MH  - HeLa Cells
MH  - Humans
MH  - Lysosomes/metabolism
MH  - Mice
MH  - Mice, Inbred ICR
MH  - Molecular Chaperones/*metabolism
MH  - Mutation/genetics
MH  - Neurons/*metabolism/*pathology
MH  - Protein Binding
MH  - Protein Kinase C/genetics
MH  - Protein Transport
MH  - Purkinje Cells/enzymology/pathology
MH  - Single-Cell Analysis/*methods
MH  - Spinocerebellar Ataxias/enzymology/genetics
PMC - PMC3280339
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2012/03/01 06:00
MHDA- 2012/06/30 06:00
PMCR- 2012/02/07
CRDT- 2012/02/25 06:00
PHST- 2011/05/28 00:00 [received]
PHST- 2012/01/04 00:00 [accepted]
PHST- 2012/02/25 06:00 [entrez]
PHST- 2012/03/01 06:00 [pubmed]
PHST- 2012/06/30 06:00 [medline]
PHST- 2012/02/07 00:00 [pmc-release]
AID - PONE-D-11-09710 [pii]
AID - 10.1371/journal.pone.0031232 [doi]
PST - ppublish
SO  - PLoS One. 2012;7(2):e31232. doi: 10.1371/journal.pone.0031232. Epub 2012 Feb 7.