Plasma Membrane Loss in Maturing Human Reticulocytes Occurs Through Endocytosis of Large Glycophorin A- Containing Vacuoles Which Fuse with Autophagosomes Before Exocytosis

Abstract 177

The erythrocyte is one of the best characterized human cells. However, studies of the process whereby human reticulocytes mature to erythrocytes have been hampered by the difficulty of obtaining sufficient numbers of cells for analysis. We have developed an in vitro culture system permitting the production of millilitre quantities of functional mature human adult reticulocytes from peripheral blood CD34+ cells. Analysis of reticulocytes obtained from culture using confocal microscopy on permeabilized cells reveals that the final stage of reticulocyte maturation occurs by a previously undescribed mechanism in which large Glycophorin A-containing vacuoles forming at the cytosolic face of the plasma membrane are internalised and fuse with autophagosomes (defined by reactivity with anti-LC3) before expulsion from the cell. Our results constitute the first demonstration of linkage between plasma membrane remodelling and autophagy, and suggest reticulocyte plasma membrane remodelling occurs in two stages. Early maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, β1 integrin) through the endosome-exosome pathway. In contrast, final maturation is characterized by the generation and expulsion of large Glycophorin A-decorated vacuoles of autophagic origin, thereby simultaneously permitting optimisation of membrane:skeleton interactions to yield the more stable and deformable membrane of mature erythrocytes and eliminating unwanted residual organelles.