A subcellular fractionation procedure was developed to isolate the different endosomal compartments present during reticulocyte maturation. After reticulocyte lysis and removal of excess haemoglobin by gel chromatography, membrane vesicles were separated over a discontinuous sucrose gradient (10-40%). Two fractions were isolated: P1 at the 25-35% sucrose interface and P2 at the 17-25% sucrose interface. These fractions were morphologically characterized by electron microscopy and the distribution of endocytic markers in the fractions was detected by Western blot. Moreover, this fractionation technique was used to study the effect of 3-methyladenine (3-MA), an autophagy inhibitor, on reticulocyte maturation. The presence of 3-MA during in vitro maturation of reticulocytes induced a decrease in exosome secretion, as measured by the amount of transferrin receptor (TfR) released in the extracellular medium. The subcellular fractionation results suggested that multivesicular endosome formation from early endosomes is the step affected by 3-MA.
Fractionation analysis of the endosomal compartment during rat reticulocyte maturation
Dardalhon, V.; Geminard, C.; Reggio, H.; Vidal, M.; Sainte-Marie, J.
Cell Biol Int
2002 / vol 26 / pages 669-78
Animals; Blotting, Western; Rats; Rats, Sprague-Dawley; Microscopy, Electron; *Cell Fractionation; Adenine/*analogs & derivatives/pharmacology; Autophagy/drug effects/physiology; Cell Compartmentation/drug effects/*physiology; Cell Differentiation/drug effects/*physiology; Endosomes/drug effects/*metabolism/ultrastructure; Extracellular Space/drug effects/metabolism; HSP70 Heat-Shock Proteins/drug effects/metabolism; rab5 GTP-Binding Proteins/drug effects/metabolism; Receptors, Transferrin/drug effects/metabolism; Reticulocytes/drug effects/*metabolism/ultrastructure; Secretory Vesicles/drug effects/metabolism/ultrastructure; Subcellular Fractions/metabolism/ultrastructure