The Tetraspanin CD63/lamp3 Cycles between Endocytic and Secretory Compartments in Human Endothelial Cells

Abstract

In the present study, we show that in human endothelial cells the tetraspanin CD63/lamp3 distributes predominantly to the internal membranes of multivesicular-multilamellar late endosomes, which contain the unique lipid lysobisphosphatidic acid. Some CD63/lamp3 is also present in Weibel-Palade bodies, the characteristic secretory organelle of these cells. We find that CD63/lamp3 molecules can be transported from late endosomes to Weibel-Palade bodies and thus that CD63/lamp3 cycles between endocytic and biosynthetic compartments; however, movement of CD63/lamp3 is much slower than that of P-selectin, which is known to cycle between plasma membrane and Weibel-Palade bodies. When cells are treated with U18666A, a drug that mimics the Niemann-Pick type C syndrome, both proteins accumulate in late endosomes and fail to reach Weibel-Palade bodies efficiently, suggesting that P-selectin, like CD63/lamp3, cycles via late endosomes. Our data suggest that CD63/lamp3 partitions preferentially within late endosome internal membranes, thus causing its accumulation, and that this mechanism contributes to CD63/lamp3 retention in late endosomes; however, our data also indicate that the protein can eventually escape from these internal membranes and recycle toward Weibel-Palade bodies to be reused. Our observations thus uncover the existence of a selective trafficking route from late endosomes to Weibel-Palade bodies.

Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

“…5a and Supplementary Movie 4). It was previously shown that the highest amount of internalized anti-CD63 antibodies is found in WPB 24 h post antibody addition 10 . In accordance, at this time point we detected a considerable fraction of the internalized antibodies in the WPB of control cells, indicative of an efficient transfer of the internalized CD63 to WPB.…”

“…The trafficking pathways of CD63 in endothelial cells appear to be rather complex, with CD63 cycling between the plasma membrane and WPB via endosomal compartments 10 . Experimental evidence indicates that the CD63 present in WPB originates from late endosomes that are typically multivesicular and contain luminal vesicles surrounded by the limiting membrane 10,14,18 .…”

“…Experimental evidence indicates that the CD63 present in WPB originates from late endosomes that are typically multivesicular and contain luminal vesicles surrounded by the limiting membrane 10,14,18 . CD63-specific antibodies are internalized and travel through the early and then the late endosomal compartments before eventually reaching the WPB 10 . Likewise, internalized anti-P-selectin antibodies can be found in WPB following transport through endosomes 9 .…”

“…Antibody uptake experiments revealed that plasma membrane CD63 is internalized to late endosomes and a fraction is then transported to WPB. Endothelial activation, in turn, leads to WPB exocytosis and cell surface presentation of CD63, indicating that this tetraspanin cycles between late endosomes and the plasma membrane via WPB 9,10 .…”

“…Although lysosomal targeting signals were identified in the C-terminal cytoplasmic tail of CD63 and its endocytic transport to late endosomes/lysosomes is well characterized [11][12][13] , the trafficking route of endosomal CD63 to WPB is not clearly resolved, for example, with respect to the contribution of the trans-Golgi network or other intermediate compartments 10,14 . Considering the role of WPB-associated CD63 in P-selectinmediated leukocyte adhesion, this transport process is, however, of prime importance, as it delivers CD63 to WPB and thus guarantees the simultaneous cell surface presentation of CD63 and P-selectin and their function in leukocyte recruitment 6 .…”