EXPOSURE OF PHOSPHATIDYLETHANOLAMINE ON THE SURFACE OF APOPTOTIC CELLS

In the early stages of apoptosis, phosphatidylserine (PS) is transloca ted from the inner side of the plasma membrane to the outer layer, whi ch allows phagocytes to recognize and engulf the apoptotic cells. In t his study we have analyzed the cell surface exposure of phosphatidylet hanolamine (PE) in apoptotic CTLL-2 cells, a cytotoxic T cell line, us ing a tetracyclic polypeptide of 19 amino acids (Ro09-0198) which spec ifically recognizes the structure of PE and forms a tight equimolar co mplex with the phospholipid. Fluorescence microscopic analysis showed that the peptide, conjugated with fluorescence-labeled streptavidin (F L-SA-Ro), bound effectively to the cell surface of cells undergoing ap optosis in response to withdrawal of interleukin-2 from the culture me dia, but not to non-apoptotic cells. The binding of FL-SA-Ro to apopto tic cells was not uniform and the intense staining was observed on sur face blebs of apoptotic cells. The FL-SA-Ro binding was inhibited spec ifically by liposomes containing PE, suggesting that PE is mainly expo sed on the surface blebs of apoptotic cells, The specific binding of F L-SA-Ro to the apoptotic cells was also confirmed using a fluorescence -activated cell sorter and the time-dependent cell surface exposure of PE correlated well with the exposure of PS, as detected by the bindin g of annexin V, This study provides the first direct evidence that PE as well as PS is exposed on the cell surface during the early stages o f apoptosis, resulting in the total loss of asymmetric distribution of aminophospholipids in the plasma membrane bilayer. (C) 1997 Academic Press.