Ultrastructural Correlates of Presynaptic Functional Heterogeneity in Hippocampal Synapses.

Although similar in molecular composition, synapses can exhibit strikingly distinct functional transmitter release and plasticity characteristics. To determine whether ultrastructural differences co-define this functional heterogeneity, we combine hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to compare two functionally distinct synapses: hippocampal Schaffer collateral and mossy fiber synapses. We find that mossy fiber synapses, which exhibit a lower release probability and stronger short-term facilitation than Schaffer collateral synapses, harbor lower numbers of docked synaptic vesicles at active zones and a second pool of possibly tethered vesicles in their vicinity. Our data indicate that differences in the ratio of docked versus tethered vesicles at active zones contribute to distinct functional characteristics of synapses.

Linkage between growth retardation and pituitary cell morphology in a dystrophin-deficient pig model of Duchenne muscular dystrophy.

OBJECTIVE: Human patients with Duchenne muscular dystrophy (DMD) commonly exhibit a short stature, but the pathogenesis of this growth retardation is not completely understood. Due to the suspected involvement of the growth hormone/insulin-like growth factor 1 (GH/IGF1) system, controversial therapeutic approaches have been developed, including both GH- administration, as well as GH-inhibition. In the present study, we examined relevant histomorphological and ultrastructural features of adenohypophyseal GH-producing somatotroph cells in a porcine DMD model. METHODS: The numbers and volumes of immunohistochemically labelled somatotroph cells were determined in consecutive semi-thin sections of plastic resin embedded adenohypophyseal tissue samples using unbiased state-of-the-art quantitative stereological analysis methods. RESULTS: DMD pigs displayed a significant growth retardation, accounting for a 55% reduction of body weight, accompanied by a significant 50% reduction of the number of somatotroph cells, as compared to controls. However, the mean volumes of somatotroph cells and the volume of GH-granules per cell were not altered. Western blot analyses of the adenohypophyseal protein samples showed no differences in the relative adenohypophyseal GH-abundance between DMD pigs and controls. CONCLUSION: The findings of this study do not provide evidence for involvement of somatotroph cells in the pathogenesis of growth retardation of DMD pigs. These results are in contrast with previous findings in other dystrophin-deficient animal models, such as the golden retriever model of Duchenne muscular dystrophy, where increased mean somatotroph cell volumes and elevated volumes of intracellular GH-granules were reported and associated with DMD-related growth retardation. Possible reasons for the differences of somatotroph morphology observed in different DMD models are discussed.

Class V chitin synthase and ß(1,3)-glucan synthase co-travel in the same vesicle in Zymoseptoria tritici.

The fungal cell wall consists of proteins and polysaccharides, formed by the co-ordinated activity of enzymes, such as chitin or glucan synthases. These enzymes are delivered via secretory vesicles to the hyphal tip. In the ascomycete Neurospora crassa, chitin synthases and ß(1,3)-glucan synthase are transported in different vesicles, whereas they co-travel along microtubules in the basidiomycete Ustilago maydis. This suggests fundamental differences in wall synthesis between taxa. Here, we visualize the class V chitin synthase ZtChs5 and the ß(1,3)-glucan synthase ZtGcs1 in the ascomycete Zymoseptoria tritici. Live cell imaging demonstrate that both enzymes co-locate to the apical plasma membrane, but are not concentrated in the Spitzenkörper. Delivery involves co-transport along microtubules of the chitin and glucan synthase. Live cell imaging and electron microscopy suggest that both cell wall synthases locate in the same vesicle. Thus, microtubule-dependent co-delivery of cell wall synthases in the same vesicle is found in asco- and basidiomycetes.

In vitro and in vivo effects of insulin-producing cells generated by xeno-antigen free 3D culture with RCP piece.

To establish widespread cell therapy for type 1 diabetes mellitus, we aimed to develop an effective protocol for generating insulin-producing cells (IPCs) from adipose-derived stem cells (ADSCs). We established a 3D culture using a human recombinant peptide (RCP) petaloid µ-piece with xeno-antigen free reagents. Briefly, we employed our two-step protocol to differentiate ADSCs in 96-well dishes and cultured cells in xeno-antigen free reagents with 0.1 mg/mL RCP µ-piece for 7 days (step 1), followed by addition of histone deacetylase inhibitor for 14 days (step 2). Generated IPCs were strongly stained with dithizone, anti-insulin antibody at day 21, and microstructures resembling insulin secretory granules were detected by electron microscopy. Glucose stimulation index (maximum value, 4.9) and MAFA mRNA expression were significantly higher in 3D cultured cells compared with conventionally cultured cells (P < 0.01 and P < 0.05, respectively). The hyperglycaemic state of streptozotocin-induced diabetic nude mice converted to normoglycaemic state around 14 days after transplantation of 96 IPCs under kidney capsule or intra-mesentery. Histological evaluation revealed that insulin and C-peptide positive structures existed at day 120. Our established xeno-antigen free and RCP petaloid µ-piece 3D culture method for generating IPCs may be suitable for clinical application, due to the proven effectiveness in vitro and in vivo.

Blood Circulating Exosomes Contain Distinguishable Fractions of Free and Cell-Surface-Associated Vesicles.

BACKGROUND: Considering exosomes as intercellular transporters, inevitably interacting with the plasma membrane and the large available surface of blood cells, we wonder if a fraction of circulating exosomes is associated with the surface of blood cells. OBJECTIVE: The aim of this study was to develop an efficient protocol for isolating exosomes associated with the surface of blood cells and to further investigate the characteristics of this fraction in a healthy state and during the development of breast cancer, as well as its possible implication for use in diagnostic applications. METHODS: Blood samples were collected from Healthy Females (HFs) and breast cancer patients (BCPs). Exosomes extracted from blood plasma and eluted from the surface of blood cells were isolated by ultrafiltration with subsequent ultracentrifugation. RESULTS: Transmission Electron Microscopy (TEM), along with immunogold labeling, demonstrated the presence of exosomes among membrane-wrapped extracellular vesicles (EVs) isolated from both plasma and blood cell eluates. TEM, nanoparticle tracking analysis, and NanoOrange protein quantitation data showed that cell-associated exosomes constituted no less than 2/3 of total blood exosome number. Exosomes, ranging from 50-70 nm in size, prevailed in the blood of breast cancer patients, whereas smaller exosomes (30-50 nm) were mostly observed in the blood of healthy women. Analysis of specific proteins and RNAs in exosomes circulating in blood demonstrated the significant differences in the packing density of the polymers in exosomes of HFs and BCPs. Preliminary data indicated that detection of cancer-specific miRNA (miR-103, miR-191, miR-195) in exosomes associated with the fraction of red blood cells allowed to discriminate HFs and BCPs more precisely compared to cell-free exosomes circulating in plasma. CONCLUSION: Our data provide the basis for using blood cell-associated exosomes for diagnostic applications.

Vesicle-Mediated Dendritic Cell Activation in Acinetobacter baumannii Clinical Isolate, which Contributes to Th2 Response.

Acinetobacter baumannii, as a nonfermentation Gram-negative bacterium, mainly cause nosocomial infections in critically ill patients. With the widespread of multidrug-resistant Acinetobacter baumannii, the urgency of developing effective therapy options has been emphasized nowadays. Outer membrane vesicles derived from bacteria show potential vaccine effects against bacterial infection in recent study. Our present research is aimed at investigating the mechanisms involved in immune protection of mice after outer membrane vesicle immunization. As our data showed, the outer membrane vesicle from an Acinetobacter baumannii clinical strain could activate bone marrow-derived dendritic cells (BMDCs) to promote Th2 activity together with humoral immune responses to Acinetobacter baumannii-induced sepsis, which might enlighten people to have a better understanding of OMVs' role as a vaccine to prevent bacterial infections.

Kinesin-1 Is a New Actor Involved in Platelet Secretion and Thrombus Stability.

OBJECTIVE: Platelet secretion is crucial for many physiological platelet responses. Even though several regulators of the fusion machinery for secretory granule exocytosis have been identified in platelets, the underlying mechanisms are not yet fully characterized. APPROACH AND RESULTS: By studying a mouse model (cKO [conditional knockout]Kif5b) lacking Kif5b (kinesin-1 heavy chain) in its megakaryocytes and platelets, we evidenced unstable hemostasis characterized by an increase of blood loss associated to a marked tendency to rebleed in a tail-clip assay and thrombus instability in an in vivo thrombosis model. This instability was confirmed in vitro in a whole-blood perfusion assay under blood flow conditions. Aggregations induced by thrombin and collagen were also impaired in cKOKif5b platelets. Furthermore, P-selectin exposure, PF4 (platelet factor 4) secretion, and ATP release after thrombin stimulation were impaired in cKOKif5b platelets, highlighting the role of kinesin-1 in α-granule and dense granule secretion. Importantly, exogenous ADP rescued normal thrombin induced-aggregation in cKOKif5b platelets, which indicates that impaired aggregation was because of defective release of ADP and dense granules. Last, we demonstrated that kinesin-1 interacts with the molecular machinery comprising the granule-associated Rab27 (Ras-related protein Rab-27) protein and the Slp4 (synaptotagmin-like protein 4/SYTL4) adaptor protein. CONCLUSIONS: Our results indicate that a kinesin-1-dependent process plays a role for platelet function by acting into the mechanism underlying α-granule and dense granule secretion.

Identification of Piecemeal Degranulation and Vesicular Transport of MBP-1 in Liver-Infiltrating Mouse Eosinophils During Acute Experimental Schistosoma mansoni Infection.

Eosinophils have been long associated with helminthic infections, although their functions in these diseases remain unclear. During schistosomiasis caused by the trematode Schistosoma mansoni, eosinophils are specifically recruited and migrate to sites of granulomatous responses where they degranulate. However, little is known about the mechanisms of eosinophil secretion during this disease. Here, we investigated the degranulation patterns, including the cellular mechanisms of major basic protein-1 (MBP-1) release, from inflammatory eosinophils in a mouse model of S. mansoni infection (acute phase). Fragments of the liver, a major target organ of this disease, were processed for histologic analyses (whole slide imaging), conventional transmission electron microscopy (TEM), and immunonanogold EM using a pre-embedding approach for precise localization of major basic protein 1 (MBP-1), a typical cationic protein stored pre-synthesized in eosinophil secretory (specific) granules. A well-characterized granulomatous inflammatory response with a high number of infiltrating eosinophils surrounding S. mansoni eggs was observed in the livers of infected mice. Moreover, significant elevations in the levels of plasma Th2 cytokines (IL-4, IL-13, and IL-10) and serum enzymes (alanine aminotransferase and aspartate aminotransferase) reflecting altered liver function were detected in response to the infection. TEM quantitative analyses revealed that while 19.1% of eosinophils were intact, most of them showed distinct degranulation processes: cytolysis (13.0%), classical and/or compound exocytosis identified by granule fusions (1.5%), and mainly piecemeal degranulation (PMD) (66.4%), which is mediated by vesicular trafficking. Immunonanogold EM showed a consistent labeling for MBP-1 associated with secretory granules. Most MBP-1-positive granules had PMD features (79.0 ± 4.8%). MBP-1 was also present extracellularly and on vesicles distributed in the cytoplasm and attached to/surrounding the surface of emptying granules. Our data demonstrated that liver-infiltrating mouse eosinophils are able to degranulate through different secretory processes during acute experimental S. mansoni infections with PMD being the predominant mechanism of eosinophil secretion. This means that a selective secretion of MBP-1 is occurring. Moreover, our study demonstrates, for the first time, a vesicular trafficking of MBP-1 within mouse eosinophils elicited by a helminth infection. Vesicle-mediated secretion of MBP-1 may be relevant for the rapid release of small concentrations of MBP-1 under cell activation.

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells.

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.

Germline heterozygous variants in genes associated with familial hemophagocytic lymphohistiocytosis as a cause of increased bleeding.

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by biallelic variants in genes regulating granule secretion in cytotoxic lymphocytes. In FHL3-5, the affected genes UNC13D, STX11 and STXBP2 have further been shown to regulate the secretion of platelet granules, giving rise to compromised platelet function. Therefore, we aimed to investigate platelet degranulation in patients heterozygous for variants in UNC13D, STX11 and STXBP2. During the work-up of patients referred to the Coagulation Unit, Skåne University Hospital, Malmö, Sweden and the Department of Hematology, Rigshospitalet, Copenhagen, Denmark due to bleeding tendencies, 12 patients harboring heterozygous variants in UNC13D, STX11 or STXBP2 were identified using targeted whole exome sequencing. Transmission electron microscopy (TEM) was used to assess the secretion of platelet dense granules following thrombin stimulation. Platelet degranulation, activation and aggregation were further assessed by flow cytometry (FC) and light transmission aggregometry (LTA) with lumi-aggregometry. In total, eight out of twelve (67%) patients showed impaired degranulation by at least one of the assays (TEM, FC and LTA). In the 12 patients, eight different heterozygous variants were identified. One variant was strongly associated with impaired degranulation, while four of the variants were associated with impaired granule secretion to a slightly lesser extent. One additional variant was found in six out of the twelve patients, and was associated with varying degrees of degranulation impairment. Accordingly, six out of the eight (75%) identified variants were associated with impaired platelet degranulation. Our results suggest that heterozygous variants in UNC13D, STX11 and STXBP2 are sufficient to cause platelet secretion defects resulting in increased bleeding.

Adrenergic regulation of cytoplasmic structures related to secretory processes in pig pinealocytes-an ultrastructural, quantitative study.

Two structures, considered as secretory in nature, are present in the pinealocytes in of the domestic pig show the presence of two structures, which are considered as secretory in nature - the dense core vesicles (DCV) and the membrane bounded (dense) bodies (MBB). The latter are extremely numerous in pig pinealocytes (they occupy 6-20% of the cytoplasm), and the number of MBB changes under different physiological and experimental conditions. Norepinephrine is the main neurotransmitter that regulates the secretion of pineal melatonin. The present study was carried out to 1) clarify whether the DCV and their source - the Golgi apparatus (GA) - as well as the MBB are controlled by norepinephrine, 2) determine the effect of adrenergic stimulation on these structures, and 3) identify the receptors involved in the regulation of these structures. The studies were performed using a static organ culture of pig pineal explants. The explants were incubated in a control medium between 08:00 and 20:00 and in a medium with 10µM norepinephrine or alpha- or beta-adrenoceptor agonists between 20:00 and 08:00 on five consecutive days. The tissues were subsequently prepared for ultrastructural analysis. The results distinctly showed that the DCV, GA and MBB in pig pinealocytes are under adrenergic control. The stimulation of the beta-adrenoceptors resulted in an increase in the numerical density of the DCV and a decrease in the relative volume of the GA in the perikarya, while the incubation with agonists of the alpha1-adrenoceptors was ineffective. The relative volume of the MBB in the perikarya significantly decreased after treatment with both beta-agonists and alpha1-agonists, which suggested the involvement of two types of adrenoceptors in the regulation of these structures.

Abnormal Rab11-Rab8-vesicles cluster in enterocytes of patients with microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo-tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed "secretory granules." However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome-edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra-/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo-tubular organelles as Rab11-Rab8-positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium-hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.

Demethylation of induced pluripotent stem cells from type 1 diabetic patients enhances differentiation into functional pancreatic ß cells.

Type 1 diabetes (T1D) can be managed by transplanting either the whole pancreas or isolated pancreatic islets. However, cadaveric pancreas is scarcely available for clinical use, limiting this approach. As such, there is a great need to identify alternative sources of clinically usable pancreatic tissues. Here, we used induced pluripotent stem (iPS) cells derived from patients with T1D to generate glucose-responsive, insulin-producing cells (IPCs) via 3D culture. Initially, T1D iPS cells were resistant to differentiation, but transient demethylation treatment significantly enhanced IPC yield. The cells responded to high-glucose stimulation by secreting insulin in vitro The shape, size, and number of their granules, as observed by transmission electron microscopy, were identical to those found in cadaveric ß cells. When the IPCs were transplanted into immunodeficient mice that had developed streptozotocin-induced diabetes, they promoted a dramatic decrease in hyperglycemia, causing the mice to become normoglycemic within 28 days. None of the mice died or developed teratomas. Because the cells are derived from "self," immunosuppression is not required, providing a much safer and reliable treatment option for T1D patients. Moreover, these cells can be used for drug screening, thereby accelerating drug discovery. In conclusion, our approach eliminates the need for cadaveric pancreatic tissue.

BAG3 is involved in neuronal differentiation and migration.

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation.

Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGN-independent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles (GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1 follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGN-PM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1 for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent.

The effect of hepatocyte growth factor on secretory functions in human eosinophils.

Hepatocyte growth factor (HGF), originally identified as a potent mitogen for mature hepatocytes, is now recognized as a humoral mediator in inflammatory and immune responses. Previous studies indicated that HGF negatively regulated allergic airway inflammation. In view of eosinophils playing a role in the pathogenesis of asthma, especially in airway remodeling as a rich source of pro-fibrogenic mediators, the effects of HGF on the different types of eosinophil secretory functions were examined in this study. We found that HGF significantly inhibited IL-5-induced secretion of TGF-ß and VEGF from human eosinophils. The inhibitory effect is not associated with TGF-ß transcription; rather, it is associated with ultrastructural granule emptying and loss of intracellular TGF-ß contents, indicating HGF inhibits the process of piecemeal degranulation. The effect of HGF on extracellular trap cell death (ETosis) that mediates cytolytic degranulation was also investigated; however, immobilized IgG- or phorbol myristate acetate-induced ETosis was only minimally attenuated by HGF. These results reveal the effect of HGF on the distinct pathways of eosinophil secretory functions and also provide novel insights into the role of HGF in the pathogenesis of allergic inflammation.

Extension of Helix 12 in Munc18-1 Induces Vesicle Priming.

UNLABELLED: Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (Δ324-339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil-coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or Δ324-339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca(2+) dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming. SIGNIFICANCE STATEMENT: The essential postdocking role of Munc18-1 in vesicular exocytosis has remained elusive, but recent data led to the hypothesis that the extension of helix 12 in Munc18 within domain 3a leads to synaptobrevin-2/VAMP2 interaction and SNARE complex formation. Using both lack-of-function and gain-of-function mutants, we here report that the conformation of helix 12 predicts vesicle priming and secretory amplitude in living chromaffin cells. The effects of mutants on secretion could not be explained by differences in syntaxin-1 chaperoning/localization or vesicle docking, and the fusion kinetics and calcium dependence were unchanged, indicating that the effect of helix 12 extension is specific for the vesicle-priming step. We conclude that a conformational change within helix 12 is responsible for the essential postdocking role of Munc18-1 in neurosecretion.

Platelet secretory behaviour: as diverse as the granules or not?

Platelets play a central role in the arrest of bleeding after damage to a blood vessel and in the development of thrombosis. Platelets rapidly respond after interaction with sub-endothelial components and release cargo from their storage granules. The three principal granule types of platelets are α-granules, dense granules and lysosomes. Timed release of granule contents and regulated expression of critical receptors are essential for maintenance of the platelet thrombus, yet also have important functions beyond hemostasis (i.e. inflammatory reactions and immune responses). α-granules store adhesive molecules such as von Willebrand factor and fibrinogen, growth factors and inflammatory and angiogenic mediators, which play crucial roles in inflammatory responses and tumor genesis. The α-granules comprise a group of subcellular compartments with a unique composition and ultrastructure. Recent studies have suggested that differential secretory kinetics of α-granule subtypes is responsible for a thematic release of adhesive and inflammatory mediators. In addition, new results indicate that activation-dependent synthesis and release of cytokines also contribute to the inflammatory role of platelets. We will discuss the various methods that platelets use to regulate secretory processes and how these relate to potential differential secretion patterns, thereby promoting adhesiveness and/or inflammatory functions. We will focus on the heterogenic granule population, open canalicular system (OCS) plasticity, the role of contractile and mechanobiological forces, and the fusogenic machinery.

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils.

BACKGROUND: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. METHODS: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. RESULTS: STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. CONCLUSIONS: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos.

Impact of the serum- and glucocorticoid-inducible kinase 1 on platelet dense granule biogenesis and secretion.

BACKGROUND: Platelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, biogenesis of platelet granules is poorly understood. OBJECTIVES: Serum- and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far. METHODS AND RESULTS: Electron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1(-/-) ). In sgk1(-/-) platelets serotonin content was significantly reduced and activation-dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3 -induced vascular injury was significantly diminished in sgk1(-/-) mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1(-/-) platelets without affecting Rab27b mRNA stability. In MEG-01 cells transfection with constitutively active (S422) (D) SGK1 but not with inactive (K127) (N) SGK1 significantly enhanced Rab27b mRNA levels. Sgk1(-/-) megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1(+/+) megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1(-/-) platelets independent of Rab27b. CONCLUSIONS: The present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes.