Human Reticulocytes Extrude Inside-Out, Phosphatidylserine-Exposed, Autophagic Vesicles During The Final Step In The Formation Of Erythrocytes

During maturation to an erythrocyte, a reticulocyte must degrade or eliminate its residual cytosolic organelles and reduce its surface area. Using confocal microscopy, we show that in late reticulocytes produced from an in vitro culture system, this is achieved through a novel form of exocytosis whereby large (∼1.4um) intact, inside-out phosphatidylserine-exposed vesicles are expelled from the maturing reticulocyte. The vesicles contain organelle marker proteins and numerous erythroid membrane proteins, notably CD71, CD147 and stomatin. The presence of ubiquitin within these vesicles suggests a recognised mechanism for the targeting of proteins for extracellular export or degradation. The exocytosed vesicles are identical to intracellular GPA-decorated autophagic vesicles previously identified in cultured reticulocytes (Griffiths et al 2012). GPA-decorated vesicles are also seen in some cells in peripheral blood. Proteins involved in vesicle trafficking, SNARE (VAMP7), ESCRT (CHMP4B) and myosin, locate to defined positions at the point of vesicle extrusion. We hypothesize that the exposed “eat me” phosphatidylserine signal ensures that released autophagic vesicles are rapidly removed from circulation by professional phagocytic cells within the spleen thus ensuring maturation to erythrocytes without the release of potentially toxic material into circulation. Our results describe a previously unrecognised mode of exocytosis which may have significance beyond erythropoiesis particularly with respect to apoptosis and autophagy and reveal the final step in the formation of human erythrocytes.