TY - JOUR
T1 - Electropermeabilization
T2 - Getting inside the cell to study autophagy
AU - Yamazaki, Kiyoshi
AU - Larusso, Nicholas F.
PY - 1988
Y1 - 1988
N2 - Electropermeabilisation—i.e. subjecting cells to a short series of high‐voltage pulses—has been used to make isolated rat hepatocytes transiently permeable to small molecules. The permeabilised cells can be loaded with [14C]sucrose at 0°C and resealed by a brief incubation at 37°C. The electrical treatment does not impair cellular function, since autophagic sequestration of sucrose occurs at the same rate as in untreated cells. The latter are permeable to sucrose immediately after isolation, due to the presence of hyperpermeable surface blebs similar to those formed in response to electrical treatment. In order to separate [14C]sucrose‐sequestering organelles from cytosol, the cells are broken up by electrodisruption—i.e. a single high‐voltage pulse given in a non‐ionic medium (isotonic sucrose)— and the resulting cell corpses are sedimented through a dense metrizamide/sucrose cushion to form a cytosol‐free pellet containing intact lyso‐somes. Since mitochondria also take up sucrose, the radioactivity in autophagosomes/lysosomes must be selectively extracted with a low concentration of digitonin. The resulting procedure provides a specific bioassay of autophagic sequestration. The sequestration is virtually completely inhibited by the autophagy inhibitor 3‐methyladenine, and at temperatures below 20°C. The sequestration step is energy‐dependent, and can probably account for the known energy requirement of autophagic‐lysosomal protein degradation.
AB - Electropermeabilisation—i.e. subjecting cells to a short series of high‐voltage pulses—has been used to make isolated rat hepatocytes transiently permeable to small molecules. The permeabilised cells can be loaded with [14C]sucrose at 0°C and resealed by a brief incubation at 37°C. The electrical treatment does not impair cellular function, since autophagic sequestration of sucrose occurs at the same rate as in untreated cells. The latter are permeable to sucrose immediately after isolation, due to the presence of hyperpermeable surface blebs similar to those formed in response to electrical treatment. In order to separate [14C]sucrose‐sequestering organelles from cytosol, the cells are broken up by electrodisruption—i.e. a single high‐voltage pulse given in a non‐ionic medium (isotonic sucrose)— and the resulting cell corpses are sedimented through a dense metrizamide/sucrose cushion to form a cytosol‐free pellet containing intact lyso‐somes. Since mitochondria also take up sucrose, the radioactivity in autophagosomes/lysosomes must be selectively extracted with a low concentration of digitonin. The resulting procedure provides a specific bioassay of autophagic sequestration. The sequestration is virtually completely inhibited by the autophagy inhibitor 3‐methyladenine, and at temperatures below 20°C. The sequestration step is energy‐dependent, and can probably account for the known energy requirement of autophagic‐lysosomal protein degradation.
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U2 - 10.1002/hep.1840080238
DO - 10.1002/hep.1840080238
M3 - Article
C2 - 3281890
AN - SCOPUS:0023974684
SN - 0270-9139
VL - 8
SP - 418
EP - 419
JO - Hepatology
JF - Hepatology
IS - 2
ER -