Weibel-Palade bodies (WPBs) are elongated secretory organelles specific to endothelial cells that contain von Willebrand factor (VWF) and a variety of other proteins that contribute to inflammation, angiogenesis, and tissue repair. The remarkable architecture of WPBs is because of the unique properties of their major constituent VWF. VWF is stored inside WPBs as tubules, but on its release, forms strikingly long strings that arrest bleeding by recruiting blood platelets to sites of vascular injury. In recent years considerable progress has been made regarding the molecular events that underlie the packaging of VWF multimers into tubules and the processes leading to the formation of elongated WPBs. Mechanisms directing the conversion of tightly packaged VWF tubules into VWF strings on the surface of endothelial cells are starting to be unraveled. Several modes of exocytosis have now been described for WPBs, emphasizing the plasticity of IntroductionIn 1964 Ewald Weibel and George Palade used transmission electron microscopy (EM) to discover that "a hitherto unknown rod-shaped cytoplasmic component which consists of a bundle of fine tubules, enveloped by a tightly fitted membrane, was regularly found in endothelial cells of small arteries in various organs in rat and man." 1 Subsequent studies confirmed the presence of those organelles in a variety of vertebrates, including hagfish, 2 which suggests they were present at least 500 million years ago. These organelles have a diameter of 0.1-0.3 m, length of 1-5 m, and characteristic longitudinal striations (Figure 1). 3 In cross section they consist of electron dense tubules with an inside diameter of 12 nm, surrounded by a less dense matrix, and packed in parallel bundles that are surrounded by a lipid bilayer. We now refer to these organelles as Weibel-Palade bodies (WPBs).At the time of their discovery, the biologic function of WPBs was unknown, although their conserved ultrastructure and wide distribution suggested that they must play a significant role in vertebrate endothelium. Almost 20 years later WPBs were shown to contain von Willebrand factor (VWF), 4 a multimeric hemostatic protein that is secreted into the blood in response to a variety of agonists and mediates platelet adhesion at sites of vascular injury. Inherited defects in VWF cause von Willebrand disease (VWD), the most common inherited bleeding disorder. 5 Interestingly, VWF is also produced by megakaryocytes and platelets, 6 the latter containing tubular structures similar to those found in WPBs but eccentrically localized in the ␣-granule, and shown to be positive for VWF by immunogold EM techniques. 7 In fact, VWF is the only protein readily detected in WPBs after biosynthetic labeling of endothelial cells. 8 Several other proteins have been identified as constituents of WPBs including tissue-type plasminogen activator (tPA), P-selectin, interleukin-8 (IL-8), eotaxin-3, angiopoietin-2, osteoprotegerin, endothelin-1, endothelin-converting enzyme, and calcitonin generelated peptide. 9,10 In addi...

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IntroductionRegulated exocytosis from vascular endothelial cells forms the first line of repair following tissue damage and inflammation. 1 Endothelial-specific secretory granules, the so-called Weibel-Palade bodies (WPBs), 2 release their contents in response to various physiologic stimuli such as physical trauma, mediators of inflammation, and hypoxia. The major secretory product of WPBs, von Willebrand Factor (VWF), assembles into remarkably long strings (up to several millimeters long) that capture flowing platelets and bind to connective tissue at the site of vascular injury to form a hemostatic plug. [3][4][5] WPBs have a distinctive elongated shape of 0.1 to 0.2 m wide and up to 5 m long, with a uniform pattern of striations running along the longitudinal axis. [6][7][8] These striations represent VWF filaments that have assembled into helical tubules. 9 Packing of VWF multimers into tubules requires both the N-terminal domains of mature VWF and the cleaved VWF propeptide, while the maintenance of the tubules in WPBs depends on a pH-sensitive interaction between mature VWF and the propeptide. 4,10,11 Microscopic imaging techniques have been instrumental in advancing our knowledge of WPB biogenesis and exocytosis. In particular, live-cell imaging studies using genetically labeled WPB cargo proteins have stressed the extraordinary plasticity of the regulated secretory pathway leading to WPB exocytosis. [12][13][14][15][16] Thus secretagogues that elevate intracellular cAMP levels cause a subset of WPBs to cluster at the level of the microtubuleorganizing center so that they do not partake in exocytosis. 12 Secretagogues that elevate intracellular Ca 2ϩ levels, on the other hand, do not elicit this effect. On the basis of these findings, and taking into account evidence for the existence of WPB subpopulations that except for VWF differ in their content of cargo molecules, it has been suggested that WPB clustering allows for the differential release of bioactive molecules from WPBs. 17 Further modulation of the release of WPB constituents is possible during the exocytosis process itself, as it has been shown that WPBs can engage in 2 modes of exocytosis, full-collapse and a slow form of kiss-and-run (lingering kiss). 14 In the latter mode, a 10-to 12-nm fusion pore is formed that acts as a molecular sieve allowing for the selective release of smaller molecules (interleukin-8, CD63) while larger molecules such as VWF are retained.In the present report, we expand the palette of exocytosis modes of WPBs by providing evidence for multigranular exocytosis, that is, the homotypic fusion of secretory granules prior to exocytosis. Using confocal, live-cell, correlative, scanning electron, and electron tomographic imaging techniques applied to human umbilical vein endothelial cells (HUVECs), we identified a novel structure, which we termed secretory pod, and which represents a secretory intermediate resulting from the coalescence of WPBs. In addition, our data suggest that fusion of WPBs with secretory pods is mediate...

The present study describes a novel phenomenon in pancreatic acinar cells undergoing regulated exocytosis. When acinar cell preparations were challenged with the secretagogue carbamylcholine, a subpopulation of zymogen granules became coated with filamentous actin. These zymogen granules were always in proximity of the acinar cell apical membrane (the site of exocytosis) but did not appear to have fused yet. They were distinct from regular zymogen granules not only because of their association with filamentous actin, but also because the majority of them lacked the zymogen granule marker rab3D, a small GTPase implicated in regulated exocytosis. The apparent loss of rab3D, presumed to result from the release of rab3D from the granule membranes, could be prevented by agents that modulate the actomyosin system as well as by GTP [␥S]. These data suggest that zymogen granules engaging in exocytosis become coated with actin before fusion and that this actin coating is tightly coupled to the release of rab3D. We propose that rab3D is involved in the regulation of actin polymerization around secretory granules and that actin coating might facilitate the movement of granules across the subapical actin network and toward their fusion site.

The epithelial sodium channel (ENaC) provides the rate-limiting step in the reabsorption of sodium by many epithelia. The number of channels at the cell surface is tightly regulated; most cells express only a few channels. We have examined the biosynthesis and cell surface expression of ENaC in Xenopus oocytes. The subunits of ENaC are readily synthesized in the endoplasmic reticulum, but most of them remain as immature proteins in pre-Golgi compartments, where they are degraded by the proteasomal pathway without apparent ubiquitination. Even when the three subunits, ␣, ␤, and ␥, are expressed in the same cell, only a very small fraction of the total channel population leave the endoplasmic reticulum, acquire complex oligosaccharides, and reach the plasma membrane. Overexpression of subunits does not increase the number of channels in the plasma membrane but results in the appearance of cytoplasmic subunits in a form not membrane bound. The data indicate that maturation and assembly of the subunits are slow and inefficient processes, and constitute limiting steps for the expression of functional ENaC channels in the plasma membrane.The amiloride-sensitive epithelial sodium channel (ENaC) 1 is a heteromultimeric protein formed by three homologous subunits, ␣, ␤, and ␥, that associate in a protein complex, whose number of subunits and stoichiometry have not been determined definitively yet (1, 2). Single subunits induce small amiloride-sensitive current and are not detected at the cell surface (3, 4). Even when the three subunits are present, the expression of ENaC at the plasma membrane is of small magnitude; in most tissues only a few copies are found per cell (5, 6), suggesting the presence of highly regulated mechanisms that control its surface expression. Since ENaC is a constitutively active channel with high open probability (7), control of the number of channels at the cell surface is crucial for the regulation of sodium reabsorption. In addition to turnover of channels by endocytosis (8), another mechanism that controls the number of channels in the plasma membrane is the rate of delivery of newly synthesized channels.Xenopus oocytes have become the most frequently used expression system to study structure, function, and regulation of ENaC, and where most of the recent characterizations of wildtype and mutant channels have been performed. Here, we have examined the biosynthesis of wild-type ENaC and of channels with deletions of the carboxyl termini (the latter known to increase the activity of ENaC by 3-5-fold) in Xenopus oocytes (9). Our results show that subunits are readily synthesized in the endoplasmic reticulum (ER) but they remain as core-glycosylated, immature proteins that are degraded in pre-Golgi compartments by a process that requires the activity of the proteasome but without the need for ubiquitination. Co-expression of all three subunits significantly stabilizes the subunits in the ER, but still, only a very small fraction is converted into a mature form that bears complex oligosaccharides...