Responsabilidades en el proceso articulador

Endocytosis of ephrinB-EphB complexes can occur in a bidirectional fashion. The direction of endocytosis may depend on several parameters including ligand/receptor protein levels, experimental design and cellular context. NIH3T3 and HeLa cells ectopically expressing

EphB and ephrinB are capable of mediating ephrinB reverse and EphB forward endocytosis, which was further confirmed by time-lapse imaging of random encounters of mixed cultures of ephrinB and EphB expressing HeLa cells (Figure 41 and supplementary information on CD-Rom, movie 2). In a parallel study the authors showed that Swiss 3T3 and primary human endothelial (HUVECs) cells, when engaging in ephrinB-EphB trans-interactions primarily undergo forward endocytosis (Marston et al., 2003). However, in that case the experimental setup was different. Endocytosis and repulsion were studied several hours after microinjection of cells with expression constructs. Prolonged cell-cell contact may favor forward endocytosis by receptor expressing cells as was also observed with NIH3T3 cells (Figure 38), and therefore could also explain why reverse endocytosis was not observed in the accompanying study. In contrast, very immature primary neurons from mouse forebrain when engaged in growth cone-cell contact show little forward, but rather pronounced ephrinB reverse endocytosis (Figures 36, 42 and data not shown). Similar results were obtained with primary neurons from Xenopus retina when stimulated with soluble Fc fusion proteins (Mann, in press)

Other parameters that influence the direction of endocytosis are cell spreading and EphB forward signaling. One of the experimental setups in the presented work involved the stimulation with cells that had been removed from their substrate and therefore were deprived of a functional cytoskeleton. In this case, endocytosis was predominant in the pre- plated recipient cells independently whether or not they expressed ephrinB1 or EphB2. It is possible that the recipient cells have an advantage in their organization of endocytic and membrane trafficking machinery over the freshly seeded stimulator cells since the endocytic machinery might be linked to the actin cytoskeleton (Qualmann and Kessels, 2002). After the stimulator cells had spread out, endocytosis was favored in the EphB2 forward direction suggesting that in this tug-of-war EphB2 signaling is dominant over ephrinB signaling, at least in fibroblasts. Weakening the receptor’s ability to signal by C-terminal truncation or blocking its kinase activity shifted endocytosis towards ephrinB reverse signaling. On the ligand side, the cytoplasmic determinants that drive reverse endocytosis are not yet known. Neither signaling via tyrosine residues nor the PDZ domain seems to be required to mediate endocytosis. The tug-of-war between ephrinB and EphB molecules is illustrated in Figure 45.

Figure 45. EphB ephrinB bidirectional endocytosis: A Tug of war. Contact between ephrinB1 and EphB2 cells leads to bidirectional endocytosis (upper left panel). Forward endocytosis into the EphB2 cells is predominant when both ligand and receptor are wild-type (upper left panel). C-terminal truncation of the ligand completely abolishes endocytosis in reverse direction but leaving forward endocytosis intact (upper right panel). C-terminal truncation or inactivation of the kinase activity of the receptor shifts the predominant direction of endocytosis into the ligand cell (lower panels).

7.2.3. Endocytosis of full length transmembrane Eph/ephrin complexes

Independently of the cell type and the specific cellular response, the results of this study have shown that the endocytosed complex contains full length proteins indicating that one of the partners had to be transcytosed from one cell to its neighbor. In a parallel study the authors could demonstrate the concomitant transfer of a GFP marker hooked up to the inner leaflet of the plasma membrane. This suggests that internalization of the ephrinB-EphB complex includes pieces of the plasma membrane (Marston et al., 2003). No colocalization of ephrinB-EphB clusters with known markers of endocytic pathways including clathrin and caveolin and the early endosomal marker EEA1 could be seen (data not shown). Although clathrin mediated endocytosis has been shown to be capable to transcytose a transmembrane ligand in the case of boss/sevenless (Chang et al., 2002) it is unlikely to take place in Eph/ephrin bidirectional endocytosis. Clathrin coated pits have an average diameter of approximately 120nm. Caveolin and other clathrin and caveolin independent mechanisms even involve smaller membrane invaginations (Conner and Schmid, 2003). By contrast

surface and intracellular clusters of Eph/ephrin complexes appear at variant sizes of up to 1µm. Endocytosis is preceded by the formation of large membrane protrusions. Phagocytosis and macropinocytosis can lead to the invagination of particles, even of much larger size than 1µm, and involves actin driven membrane protrusions for initial engulfment. Actin protrusions required activity of the Rho family GTPases Rac and Cdc42 (Chimini and Chavrier, 2000; Nichols and Lippincott-Schwartz, 2001). The study of Marston et al. demonstrates the involvement of Rac in EphB mediated forward endocytosis. Thus, the underlying mechanism of Eph/ephrin endocytosis may resemble phagocytosis or macropinocytosis. Endocytosis of a transmembrane protein including plasma membrane may ultimately result in the formation of multivesicular bodies (Figure 46).

Figure 46. Model for bidirectional endocytosis of transmembrane receptor ligand complexes. (A) Contact between ephrinB (left) and EphB (right) expressing cells. (B) EphrinB reverse and EphB forward signaling leads to clustering in membrane protrusions followed by the invaginations of receptor/ligand complexes. (C) Entire transmembrane complexes are endocytosed either into the EphB or the ephrinB cell, including pieces of plasma membrane, leading to the formation of multivesicular bodies.