VAMP3 is associated with endothelial weibel-palade bodies and participates in their Ca(2+)-dependent exocytosis

Weibel-Palade bodies (WPBs) are secretory organelles of endothelial cells that store the thrombogenic glycoprotein von Willebrand factor (vWF). Endothelial activation, e.g. by histamine and thrombin, triggers the Ca(2+)-dependent exocytosis of WPB that releases vWF into the vasculature and thereby initiates platelet capture and thrombus formation. Towards understanding the molecular mechanisms underlying this regulated WPB exocytosis, we here identify components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery associated with WPB. We show that vesicle-associated membrane protein (VAMP) 3 and VAMP8 are present on WPB and that VAMP3, but not VAMP8 forms a stable complex with syntaxin 4 and SNAP23, two plasma membrane-associated SNAREs in endothelial cells. By introducing mutant SNARE proteins into permeabilized endothelial cells we also show that soluble VAMP3 but not VAMP8 mutants comprising the cytoplasmic domain interfere with efficient vWF secretion. This indicates that endothelial cells specifically select VAMP 3 over VAMP8 to cooperate with syntaxin 4 and SNAP23 in the Ca(2+)-triggered fusion of WPB with the plasma membrane. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

VAMP3 is associated with endothelial Weibel-Palade bodies and participates in their Ca-dependent exocytosis

Weibel-Palade bodies (WPBs) are secretory organelles of endothelial cells that store the thrombogenic glycoprotein von Willebrand factor (vWF). Endothelial activation, e.g. by histamine and thrombin, triggers the Ca²⁺-dependent exocytosis of WPB that releases vWF into the vasculature and thereby initiates platelet capture and thrombus formation. Towards understanding the molecular mechanisms underlying this regulated WPB exocytosis, we here identify components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery associated with WPB. We show that vesicle-associated membrane protein (VAMP) 3 and VAMP8 are present on WPB and that VAMP3, but not VAMP8 forms a stable complex with syntaxin 4 and SNAP23, two plasma membrane-associated SNAREs in endothelial cells. By introducing mutant SNARE proteins into permeabilized endothelial cells we also show that soluble VAMP3 but not VAMP8 mutants comprising the cytoplasmic domain interfere with efficient vWF secretion. This indicates that endothelial cells specifically select VAMP 3 over VAMP8 to cooperate with syntaxin 4 and SNAP23 in the Ca²⁺-triggered fusion of WPB with the plasma membrane. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Myosin Va acts in concert with Rab27a and MyRIP to regulate acute von-Willebrand factor release from endothelial cells

Von-Willebrand factor (vWF) is a highly multimerized hemostatic glycoprotein that is stored in endothelial Weibel-Palade bodies (WPB) and secreted upon cell stimulation to act in recruiting platelets to sites of vessel injury. Only fully matured multimeric vWF represents an efficient anchor for platelets, and endothelial cells have developed mechanisms to prevent release of immature vWF. Full maturation of vWF occurs within WPB following their translocation from a perinuclear site of emergence at the trans-Golgi network (TGN) to the cell periphery. The WPB-associated small GTPase Rab27a is involved in restricting immature WPB exocytosis and we searched for links between Rab27a and the actin cytoskeleton that could anchor WPB inside endothelial cells until they are fully matured. We here identify myosin Va as such link. Myosin Va forms a tripartite complex with Rab27a and its effector MyRIP and depletion of or dominant-negative interference with myosin Va leads to an increase in the ratio of perinuclear to more peripheral WPB. Concomitantly, myosin Va depletion results in an elevated secretion of less-oligomeric vWF from histamine-stimulated endothelial cells. These results indicate that a Rab27a/MyRIP/myosin Va complex is involved in linking WPB to the peripheral actin cytoskeleton of endothelial cells to allow full maturation and prevent premature secretion of vWF.     

Streptococcus pneumoniae induces exocytosis of Weibel-Palade bodies in pulmonary endothelial cells

Invasive pneumococcal infections due to Streptococcus pneumoniae lead to inflammatory infiltration of leucocytes into lung alveolus, meninges and to septic dissemination within the vascular system. The lung microvasculature is covered by pulmonary endothelial cells containing Weibel‐Palade bodies (WPB) releasing procoagulant von Willebrand factor (vWF) and other proteins in response to inflammatory stimuli. The influence of pathogenic pneumococci on secretion of WPB proteins is unknown. Here, we report that adherence of S. pneumoniae to primary human pulmonary microvascular endothelial cells (HPMEC) stimulates the WPB exocytosis and the secretion of vWF and interleukin 8 (IL‐8). Moreover, infection analyses performed with pneumococcal mutants deficient in the expression of cytotoxic pneumolysin demonstrated that, in addition to direct bacterial adherence, sublytic concentrations of pneumolysin stimulated vWF secretion. The release of vWF was induced after infection with pneumococci from both the apical and the basal cell surfaces, which implies a stimulation of WPB exocytosis in both directions: from inside the vasculature and also following invasive pneumococcal transmigration from pulmonary tissue into the bloodstream. In conclusion, this study demonstrates that the most relevant pulmonary pathogen S. pneumoniae induces release of proinflammatory and procoagulative components directly contributing to pathophysiological processes leading to fatal tissue injury during course of infection.     

Differential Cargo Mobilisation within Weibel-Palade Bodies after Transient Fusion with the Plasma Membrane

Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH₄Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼2.4 µm²/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis.     

Differential kinetics of cell surface loss of von Willebrand factor and its propolypeptide after secretion from Weibel-Palade bodies in living human endothelial cells

The time course for cell surface loss of von Willebrand factor (VWF) and the propolypeptide of VWF (proregion) following exocytosis of individual Weibel-Palade bodies (WPBs) from single human endothelial cells was analyzed. Chimeras of enhanced green fluorescent protein (EGFP) and full-length pre-pro-VWF (VWF-EGFP) or the VWF propolypeptide (proregion-EGFP) were made and expressed in human umbilical vein endothelial cells. Expression of VWF-EGFP or proregion-EGFP resulted in fluorescent rod-shaped organelles that recruited the WPB membrane markers P-selectin and CD63. The WPB secretagogue histamine evoked exocytosis of these fluorescent WPBs and extracellular release of VWF-EGFP or proregion-EGFP. Secreted VWF-EGFP formed distinctive extracellular patches of fluorescence that were labeled with an extracellular antibody to VWF. The half-time for dispersal of VWF-EGFP from extracellular patches was 323.5 +/- 146.2 s (+/-S.D., n = 20 WPBs). In contrast, secreted proregion-EGFP did not form extracellular patches but dispersed rapidly from its site of release. The half-time for dispersal of proregion-EGFP following WPB exocytosis was 2.98 +/- 1.88 s (+/-S.D., n = 32 WPBs). The slow rate of loss of VWF-EGFP is consistent with the adhesive nature of this protein for the endothelial membrane. The much faster rate of loss of proregion-EGFP indicates that this protein does not interact strongly with extracellular VWF or the endothelial membrane and consequently may not play an adhesive role at the endothelial cell surface.     

Osteoprotegerin (OPG) is localized to the Weibel-Palade bodies of human vascular endothelial cells and is physically associated with von Willebrand factor

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra-skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co-localizes with P-selectin and von Willebrand factor (vWF), within the Weibel-Palade bodies (WPB). Treatment of HUVEC with the pro-inflammatory cytokines, tumor necrosis factor (TNF)-alpha and IL-1beta, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF-alpha treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24-h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P-Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP-1), raising the intriguing possibility that OPG may provide a link between TSP-1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.     

Ca2+ -regulated secretion of tissue-type plasminogen activator and von Willebrand factor in human endothelial cells

von Willebrand factor (vWF) and tissue-type plasminogen activator (tPA) are products of endothelial cells which are secreted into the bloodstream upon a stimulus-induced rise in intracellular Ca(2+). Although the release of both factors appears to be regulated similarly, they exhibit opposing physiological effects in the vasculature with vWF inducing coagulation and platelet aggregation and tPA triggering fibrinolysis and thrombolysis. To analyze possible differences in the regulated secretion of vWF and tPA in more detail, we recorded the Ca(2+)-triggered exocytosis of both factors in cultured human endothelial cells. We demonstrate that vWF and tPA which are stored in different granules within endothelial cells are released with different kinetics following endothelial stimulation with histamine or the Ca(2+) ionophore A23187. While the stimulus-induced release of vWF increases with time over a course of 30 min, maximal acute secretion of tPA is observed 5 min following stimulation and subsequently drops to background levels. In the case of vWF, secretion can also be monitored indirectly through an antibody-reinternalization assay which indicates an incomplete release of vWF during single exocytotic fusion events. Our data thus point to differences in the Ca(2+)-triggered secretion of vWF and tPA which could allow a fine-tuning of their release thereby ensuring a balanced physiological action.     

Rab3D and annexin A2 play a role in regulated secretion of vWF, but not tPA, from endothelial cells

von-Willebrand factor (vWF) and tissue-type plasminogen activator (tPA) are products of endothelial cells acutely released into the vasculature following cell activation. Both factors are secreted after intraendothelial Ca2+ mobilization, but exhibit opposing physiological effects with vWF inducing coagulation and tPA triggering fibrinolysis. To identify components that could regulate differentially the release of pro- and antithrombogenic factors, we analyzed the contribution of Rab3D and the annexin A2/S100A10 complex, proteins implicated in exocytotic events in other systems. We show that mutant Rab3D proteins interfere with the formation of bona fide Weibel-Palade bodies (WPbs), the principal storage granules of multimeric vWF, and consequently the acute, histamine-induced release of vWF. In contrast, neither appearance nor exocytosis of tPA storage granules is affected. siRNA-mediated downregulation of annexin A2/S100A10 and disruption of the complex by microinjection of peptide competitors result in a marked reduction in vWF but not tPA secretion, without affecting the appearance of WPbs. This indicates that distinct mechanisms underlie the acute secretion of vWF and tPA, enabling endothelial cells to fine-regulate the release of thrombogenic and fibrinolytic factors.     

The Epac-Rap1 signaling pathway controls cAMP-mediated exocytosis of Weibel-Palade bodies in endothelial cells

Endothelial cells contain specialized storage organelles called Weibel-Palade bodies (WPBs) that release their content into the vascular lumen in response to specific agonists that raise intracellular Ca(2+) or cAMP. We have previously shown that cAMP-mediated WPB release is dependent on protein kinase A (PKA) and involves activation of the small GTPase RalA. Here, we have investigated a possible role for another PKA-independent cAMP-mediated signaling pathway in the regulation of WPB exocytosis, namely the guanine nucleotide exchange factor Epac1 and its substrate, the small GTPase Rap1. Epinephrine stimulation of endothelial cells leads to Rap1 activation in a PKA-independent fashion. siRNA-mediated knockdown of Epac1 abolished epinephrine-induced activation of Rap1 and resulted in decreased epinephrine-induced WPB exocytosis. Down-regulation of Rap1 expression and prevention of Rap1 activation through overexpression of Rap1GAP effectively reduced epinephrine- but not thrombin-induced WPB exocytosis. Taken together, these data uncover a new Epac-Rap1-dependent pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP.     

The number of secretory vesicles remains unchanged following exocytosis.

Earlier studies using electron microscopy demonstrate that there is no loss of secretory vesicles following exocytosis. Depletion however, of vesicular contents resulting in the formation of empty or partially empty vesicles is seen in electron micrographs, post exocytosis, in a variety of cells. Our studies using atomic force microscopy (AFM) reveal that following stimulation of secretion, live pancreatic acinar cells having 100-180 nm in diameter fusion pores located at the apical plasma membrane, dilate only 25-35% during exocytosis. Since secretory vesicles in pancreatic acinar cells range in size from 200 nm to 1200 nm in diameter, their total incorporation at the fusion pore, would distend the structure much more then what is observed. These earlier results prompted the current study to determine secretory vesicle dynamics in live pancreatic acinar cells following exocytosis. AFM studies on live acinar cells reveal no loss of secretory vesicle number following exocytosis. Parallel studies using electron microscopy, further confirmed our AFM results. These studies demonstrate that following stimulation of secretion, membrane-bound secretory vesicles transiently dock and fuse to release vesicular contents.     

Roles of microfilaments in exocytosis: a new hypothesis

We observed the dynamic changes in the localization of microfilaments during the exocytic secretion of rat parotid and submandibular gland acinar cells, and obtained results which led us to propose a new concept of microfilament function in exocytosis. With the electron microscopy, NBD-Phallacidin (NBD-PL) fluorescence technique and immunohistochemistry for myosin, microfilaments consisting of F-actin and myosin were localized mainly underneath the luminal plasma membrane. Microfilaments were not detectable around the secretory granules which were stored in the cytoplasm, but were clearly observed around them whose membranes were continuous with the luminal plasma membrane. When viewed with NBD-PL and myosin fluorescence, the area of fused granule membranes revealed bright fluorescence in association with the luminal border, so that the luminal membrane undergoing exocytosis appeared like a 'bunch of grapes'. When excess exocytosis was stimulated by isoproterenol (IPR), the number of individual 'grapes' increased dramatically, indicating that the secretory granules are surrounded by microfilaments after the fusion with the luminal membrane. Microfilaments thus continuously undercoat the luminal membrane during exocytosis although the exocytic process involves the dilation and subsequent reduction of the luminal membrane due to the addition and removal of secretory granule membranes. This reduction of the dilated luminal membrane following exocytosis was, however, inhibited when the microfilaments were disrupted by cytochalasin D. Following this treatment, the lumina was expanded extraordinarily and the secretory products remained in the enlarged lumina, showing that the release of secretory products is inhibited when the microfilament function is disturbed. These results indicate that 1) microfilaments are localized mainly underneath the luminal plasma membrane and act as an obstacle to exocytosis when cells are at the resting phase and 2) at the secretory phase microfilaments allow exocytosis by disorganizing their barrier system and then, by encircling the discharged secretory granule membranes, provide forces for the extrusion of secretory products through the action of the acto-myosin contractile system.     

Characterization of a novel chemokine-containing storage granule in endothelial cells: evidence for preferential exocytosis mediated by protein kinase A and diacylglycerol

We have recently shown that several proinflammatory chemokines can be stored in secretory granules of endothelial cells (ECs). Subsequent regulated exocytosis of such chemokines may then enable rapid recruitment of leukocytes to inflammatory sites. Although IL-8/CXCL8 and eotaxin-3/CCL26 are sorted to the rod-shaped Weibel-Palade body (WPB), we found that GROalpha/CXCL1 and MCP-1/CCL2 reside in small granules that, similarly to the WPB, respond to secretagogue stimuli. In the present study, we report that GROalpha and MCP-1 colocalized in 50- to 100-nm granules, which occur throughout the cytoplasm and at the cell cortex. Immunofluorescence confocal microscopy revealed no colocalization with multimerin or tissue plasminogen activator, i.e., proteins that are released from small granules of ECs by regulated exocytosis. Moreover, the GROalpha/MCP-1-containing granules were Rab27-negative, contrasting the Rab27-positive, WPB. The secretagogues PMA, histamine, and forskolin triggered distinct dose and time-dependent responses of GROalpha release. Furthermore, GROalpha release was more sensitive than IL-8 release to inhibitors and activators of PKA and PKC but not to an activator of Epac, a cAMP-regulated GTPase exchange factor, indicating that GROalpha release is regulated by molecular adaptors different from those regulating exocytosis of the WPB. On the basis of these findings, we designated the GROalpha/MCP-1-containing compartment the type 2 granule of regulated secretion in ECs, considering the WPB the type 1 compartment. In conclusion, we propose that the GROalpha/MCP-1-containing type 2 granule shows preferential responsiveness to important mediators of EC activation, pointing to the existence of selective agonists that would allow differential release of selected chemokines.     

Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide

Background

Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca²⁺-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca²⁺-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs.

Principal Findings

Ca²⁺-driven WPB exocytosis occurred at all temperatures studied (7–37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s⁻¹ (37°C) to 0.80±0.14 s⁻¹ (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca²⁺-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75–80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60–120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself.

Conclusions

We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.     

Actin dynamics during Ca2+-dependent exocytosis of endothelial Weibel-Palade bodies

Weibel-Palade bodies (WPBs) are specialized secretory organelles of endothelial cells that serve important functions in the response to inflammation and vascular injury. WPBs actively respond to different stimuli by regulated exocytosis leading to full or selective release of their contents. Cellular conditions and mechanisms that distinguish between these possibilities are only beginning to emerge. To address this we analyzed dynamic rearrangements of the actin cytoskeleton during histamine-stimulated, Ca²⁺-dependent WPB exocytosis. We show that most WPB fusion events are followed by a rapid release of von-Willebrand factor (VWF), the large WPB cargo, and that this occurs concomitant with a softening of the actin cortex by the recently described Ca²⁺-dependent actin reset (CaAR). However, a considerable fraction of WPB fusion events is characterized by a delayed release of VWF and observed after the CaAR reaction peak. These delayed VWF secretions are accompanied by an assembly of actin rings or coats around the WPB post-fusion structures and are also seen following direct elevation of intracellular Ca²⁺ by plasma membrane wounding. Actin ring/coat assembly at WPB post-fusion structures requires Rho GTPase activity and is significantly reduced upon expression of a dominant-active mutant of the formin INF2 that triggers a permanent CaAR peak-like sequestration of actin to the endoplasmic reticulum. These findings suggest that a rigid actin cortex correlates with a higher proportion of fused WPB which assemble actin rings/coats most likely required for efficient VWF expulsion and/or stabilization of a WPB post-fusion structure.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

Hypoxia Causes Aggregation of Serine Palmitoyltransferase followed by Non-Apoptotic Death of Human Lymphocytes

In the central nervous system chronic hypoxia has been suggested to cause neurodegenerations and protein aggregation, as in Alzheimer’s disease. Here we have shown protein aggregation during acute hypoxia in human primary cells. Clinically relevant acute hypoxia (pO2 = 25 mmHg) was produced by incubation of venous blood in vitro, where 18-hour incubation resulted in raise of pCO2 to 90 mmHg, accumulation of lactate and acidosis (pH 7.06). In hypoxic samples the number of necrotic, but not apoptotic, white blood cells increased to 9.6%. Viable cells displayed hypoxia-related changes such as a drop of mitochondrial membrane potential and changes in the plasma membrane. These changes coincided with the 2.6-fold increase in immunoreactivity of serine palmitoyltransferase subunit 1 (SPT1), which is the enzyme involved in HSN1-type neurodegeneration. SPT1 immunoreactivity was presented as large cytosolic aggregates, which appeared in viable hypoxic cells and remained in dead cells. SPT-positive aggregates were also found in cell nuclei. This data suggests that SPT1 aggregation preceded cell death in hypoxia and represents the first evidence of acute protein aggregation during hypoxia.     

Arginine-specific gingipain A from Porphyromonas gingivalis induces Weibel-Palade body exocytosis and enhanced activation of vascular endothelial cells through protease-activated receptors

Gingipains, cysteine proteases derived from Porphyromonas gingivalis, are important virulence factors in periodontal diseases. We found that arginine-specific gingipain A (RgpA) increased the responsiveness of vascular endothelial cells to P. gingivalis lipopolysaccharides (LPS) and P. gingivalis whole cells to induce enhanced IL-8 production through protease-activated receptors (PARs) and phospholipase C (PLC) gamma. We therefore investigated whether RgpA-induced enhanced cell activation is mediated through exocytosis of Weibel-Palade bodies (WPBs) because they store vasoactive substances. RgpA rapidly activated PAR- and PLCgamma-dependent WPB exocytosis. In addition, angiopoietin (Ang)-2, a substance of WPB, enhanced IL-8 production by P. gingivalis LPS, suggesting that Ang-2 mediates the RgpA-induced enhanced cell responses. Thus, we propose a novel role for RgpA in induction of a proinflammatory event through PAR-mediated WPB exocytosis, which may be an important step for enhanced endothelial responses to P. gingivalis.     

Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140

We have examined the cell activation-dependent redistribution of the intracellular granule membrane protein GMP-140 of human endothelial cells. By dual-label immunofluorescence, the distribution of GMP-140 within cultured human umbilical vein endothelial cells was found to coincide with the distribution of von Willebrand factor (vWF), suggesting that GMP-140 is located in the membranes of vWF-containing storage granules. Stimulation of vWF secretion resulted in an increase in GMP-140 on the cell surface, as detected by increased binding of the monoclonal antibody S12 which recognizes the extracytoplasmic domain of GMP-140. For each agonist tested (histamine, thrombin, phorbol 12-myristate 13-acetate, and the calcium ionophore A23187) a dose-dependent redistribution of GMP-140 to the endothelial surface was observed which closely paralleled the dose-dependent secretion of vWF into the cell supernatant. When cells were maximally stimulated by histamine in the presence of antibody S12, a 4-fold increase in S12 uptake by the cells was observed. This increase occurred rapidly and reached a plateau by 10 min. In contrast, when histamine-stimulated cells were first fixed with paraformaldehyde or chilled to 4 degrees C before addition of antibody S12, only a transient increase in cell surface GMP-140 was detected. Under these conditions of arrested membrane turnover during antibody binding, cell surface GMP-140 was maximal 3 min after histamine stimulation and then declined to control levels by 20 min. These data suggest that stimulated secretion of vWF from endothelial cells entails fusion of vWF-containing storage granules with the plasma membrane. Once inserted into the plasma membrane, GMP-140 is subsequently removed from the endothelial surface, most likely by an endocytic mechanism.     

Reconstituted fusion pore

Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles.     

Regulation of fusion pore closure and compound exocytosis in neuroendocrine PC12 cells by SCAMP1

During exocytosis, neuroendocrine cells can achieve partial release of stored secretory products from dense core vesicles (DCVs) by coupling endocytosis directly at fusion sites and without full discharge. The physiological role of partial secretion is of substantial interest. Much is known about SNARE-mediated initiation of exocytosis and dynamin-mediated completion of endocytosis, but little is known about coupling events. We have used real-time microscopy to examine the role of secretory carrier membrane protein SCAMP1 in exo-endocytic coupling in PC12 cells. While reduced SCAMP1 expression is known to impede dilation of newly opened fusion pores during onset of DCV exocytosis, we now show that SCAMP1 deficiency also inhibits closure of fusion pores after they have opened. Inhibition causes accumulation of fusion figures at the plasma membrane. Closure is recovered by restoring expression and accelerated slightly by overexpression. Interestingly, inhibited pore closure resulting from loss of SCAMP1 appears to increase secondary fusion of DCVs to already-fused DCVs (compound exocytosis). Unexpectedly, reinternalization of expanded DCV membranes following compound exocytosis appears to proceed normally in SCAMP1-deficient cells. SCAMP1's apparent dual role in facilitating dilation and closure of fusion pores implicates its function in exo-endocytic coupling and in the regulation of partial secretion. Secondarily, SCAMP1 may serve to limit the extent of compound exocytosis.