Isolation of pancreatic autophagic vacuoles induced by vinblastine or neutral red. Separation of autophagosomes and autolysosomes by Percoll density gradient centrifugation.
Our purpose was to elaborate a cell fractionation method for the preparation and purification of macroautophagic vacuoles (AVs) and their subfractions: autophagosomes and autolysosomes. To overcome the difficulties caused in liver and some other cell types by the overlapping buoyant densities and sizes of different subclasses of lysosomes and other subcellular particles, we chose the murine pancreatic acinar cell as experimental system in which enormous numbers of large-sized AVs are readily accumulated upon certain treatments. As we measured by electron microscopic morphometry, cytoplasmic volume fraction of AVs was as small as 0.31% in the untreated cells, while it was elevated to 8.1% 4 h after the injection of 50 mg/kg body weight vinblastine sulfate (a widely used inducer of macroautophagy). From vinblastine-treated pancreas, a 5500g sediment containing AVs and mitochondria (AV-M fraction) was obtained by differential centrifugation. This fraction was resolved in a 50% Percoll gradient (15 min, 92,000g) into three distinct particle populations. Mitochondria were localized near the upper boundary of the gradient at a buoyant density of 1.075 to 1.08, whereas directly under them light AVs (1.085-1.09) were banded. Heavy AVs (1.13-1.14) formed a broad layer near the bottom of the tube. Electron microscopic comparison of the morphology of these fractions and AVs in situ showed that light AVs correspond to AVs in early, whereas heavy AVs to AVs in advanced and late stages of degradation of the segregated material. The activity of lysosomal enzymes were found low in both fractions, being several times higher in the heavy than in the light one.(ABSTRACT TRUNCATED AT 250 WORDS)
Fellinger
E
E
Department of General Zoology, Eötvös University, Budapest/Hungary.
Réz
G
G
D000818:Animals; D001343:Autophagy / Q000502:physiology*; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D008247:Lysosomes / Q000187:drug effects*; D008297:Male; D051379:Mice; D009499:Neutral Red / Q000494:pharmacology; D010179:Pancreas / Q000378:metabolism* / Q000648:ultrastructure; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000187:drug effects*; D011205:Povidone; D012822:Silicon Dioxide; D014747:Vinblastine / Q000494:pharmacology
D009499:Neutral Red; D014747:Vinblastine; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone