Cell Surface-expressed Phosphatidylserine and Annexin A5 Open a Novel Portal of Cell Entry

Abstract

Expression of phosphatidylserine (PtdSer) at the cell surface is part of the membrane dynamics of apoptosis. Expressed phosphatidylserine functions as an "eat me" flag toward phagocytes. Here, we report that the expressed phosphatidylserine forms part of a hitherto undescribed pinocytic pathway. Annexin A5, a phosphatidylserine-binding protein, binds to and polymerizes through protein-protein interactions on membrane patches expressing phosphatidylserine. The two-dimensional protein network of annexin A5 at the surface prevents apoptotic body formation without interfering with the progression of apoptosis as demonstrated by activation of caspase-3, PtdSer exposure, and DNA fragmentation. The annexin A5 protein network bends the membrane patch nanomechanically into the cell and elicits budding, endocytic vesicle formation, and cytoskeleton-dependent trafficking of the endocytic vesicle. Annexin A1, which binds to PtdSer without forming a two-dimensional protein network, does not induce the formation of endocytic vesicles. This novel pinocytic pathway differs from macropinocytosis, which is preceded by membrane ruffling and actin polymerization. We clearly showed that actin polymerization is not involved in budding and endocytic vesicle formation but is required for intracellular trafficking. The phosphatidylserine-annexin A5-mediated pinocytic pathway is not restricted to cells in apoptosis. We demonstrated that living tumor cells can take up substances through this novel portal of cell entry. This opens new avenues for targeted drug delivery and cell entry.

Knockout of caspase-like gene, YCA1 , abrogates apoptosis and elevates oxidized proteins in Saccharomyces cerevisiae

“…Nevertheless, oxidation of methionine residues in proteins has been shown to increase their susceptibility to proteolysis by the proteasome (19). In confirmation of the fact that apoptosis is associated with externalization of phosphatidylserine on the surface of the membrane (31,32) and the condensation of nucleic acid to form TUNEL-positive derivatives (33), we showed that upon exposure to H 2 O 2 , the ⌬yca1 strain failed to exhibit either of these characteristics. In contrast, exposure of the wild type to H 2 O 2 led to both phosphatidylserine externalization and formation of TUNEL-positive nuclei (Fig.…”

Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells

“…However, the studies of lactadherin binding have been very limited and the cell features recognized by the two proteins have not been compared. Our results of preferential internalization of annexin V by cells undergoing apoptosis agree with previous reports (3,29).…”

Death receptor ligation triggers membrane scrambling between Golgi and mitochondria

“…Thus, results obtained in different cell systems strongly supported the concept that Fas activation alters membrane traffic through the Golgi complex, possibly following the opening of a new portal of endocytosis propagating from the cell surface. 19,24 To investigate this possibility in detail, we expanded our studies on membrane traffic.…”

“…4,14 Consequently, the observed changes in intracellular organelles (Figures 1 and 2) would be entwined with events of membrane traffic, for instance enhanced pinocytosis. 19 To evaluate global aspects of endomembrane traffic, we exploited diverse fluorescent derivatives of Helix pomatia agglutinin (HPA), a lectin that recognizes membrane glycoproteins in the ER and proximal Golgi, 20 as well as at the surface of lymphoma cells. 21 The latter aspect reflects the traffic of glycoproteins that is modified during apoptosis, in part to stimulate binding of clearing cells.…”