P‐Selectin and CD63 Use Different Mechanisms for Delivery to Weibel–Palade Bodies

Abstract

The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the transGolgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC). Weibel-Palade bodies (WPB) are endothelial-specific cigarshaped secretory organelles (1, reviewed in 2) which store the haemostatic protein von Willebrand factor (VWF) (3) within a limiting membrane that includes the leukocyte receptor P-selectin (4,5) and the tetraspanin CD63 (6). Their composition and tissue distribution thus place them at the centre of both haemostasis and inflammation. Despite this, WPB biogenesis is not well understood. The little that is known suggests that they share at least some characteristics with lysosome-related organelles (LRO).LRO are cell-type-specific structures with secretory functions, including melanosomes, cytotoxic T-lymphocyte lytic granules, platelet dense granules, the MHC class II compartment, basophil and azurophil granules, and lamellar bodies. They are thus often seen in cells with a haemopoietic lineage. All share some characteristics with endosomes and lysosomes (and some are called secretory lysosomes), including protein composition, low intraorganellar pH and access from the endocytic pathway without re-entering the biosynthetic pathway at the level of the Golgi (7-10). Their biogenesis is often marked by the delivery of specialized components to endocytic organelles, thereby transforming them into LRO. One common LRO characteristic is a role for the adaptor protein complex 3 (AP-3) in this process. In endothelial cells, AP-3 has been reported as being required for P-selectin recruitment to WPB (11); it is also reported to affect the trafficking of CD63 in NRK and NIH3T3 cells (12).However, we also know that WPB formation is driven by VWF (13), whose heterologous expression in HEK293 cells can cause the appearance of cigar-shaped, ultrastructurally correct 'WPB' that recruit appropriate membrane proteins and respond to secretagogue (14). Moreover, WPB are thought to form at the trans Golgi network (TGN), and heterologously expressed P-selectin is recruited to forming secretory granul...

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

“…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 .…”

“…However, differences appear to exist between the transport of endosomal CD63 and P-selectin to WPB. P-selectin is delivered to newly forming WPB already at the level of the trans-Golgi network, whereas CD63 is only recruited to the secretagogue-responsive, mature WPB 10,14 . This is in line with our antibody uptake experiments in anxA8-depleted cells that revealed a defect in the transport of CD63, leading to diminished CD63 levels in WPB, while transfer of P-selectin to WPB remained unaltered.…”

“…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 .…”

Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells

“…P-selectin is also recruited to the WPB membrane at the TGN (8) and functions in leukocyte recruitment after delivery to the endothelial cell surface by WPB exocytosis. Maturation of WPBs is marked by the recruitment of the membrane-associated GTPases Rab27a (9) and Rab3D (10) and the membrane tetraspanin CD63 (8,11). Exogenous expression of VWF leads to the de novo formation of WPBs in a number of non-endothelial mammalian cell lines (12).…”

A role for Rab10 in von Willebrand factor release discovered by an AP‐1 interactor screen in C. elegans

“…by immunofluorescent microscopy of the Golgi association of AP-1 and the recruitment of CD63 (lost in AP-3 deficient cells [40]) or of MyRIP (recruited late in biogenesis and affecting exocytosis by anchoring the WPB to actin [41]) all appear to be normal (Fig. S1).…”