Mechanism of Autophagy in Permeabilized Hepatocytes
Autophagic vacuoles in hepatocytes are formed from membranes of rough and smooth endoplasmic reticulum (ER) by processes that are under immediate physiologic control by amino acids, insulin, and glucagon (reviewed in 1). Little, though, is known of the molecular steps involved. Microinjection (2) and electropermeabilization (3) have been used to introduce markers into cells and newly formed vacuoles. But because the pores are transient, observations are restricted to events that occur after membrane resealing. In order to gain access to autophagy under steady state conditions, we permeabilized hepatocytes with α-toxin from Staphylococcus aureus, an agent which forms stable ≈1.5 nm channels that limit exchange to molecules of approximately 1000 Da (4). Such pores will admit nucleotides and labeled residualizing probes without loss of cell proteins, a desirable, possibly necessary, condition for evaluating autophagically-mediated proteolysis.
KeywordsVolume Density Autophagic Vacuole Cytoplasmic Sequestration Membrane Reseal Permeabilized Hepatocyte
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