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 OยฐC and resealed by a brief incubation at 37OC. 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 lysosomes. 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 20ยฐC. The sequestration step is energy-dependent, and can probably account for the known energy requirement of autophagic-lysosomal protein degradation.
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Proteins are continuously synthesized and degraded in living cells, and a balance between their synthesis and degradation is vital for cell homeostasis. While considerable information has been generated regarding the HAROLD 0. CONN, EDITOR