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Sample records for cell secretory granules

  1. Characterization of mast cell secretory granules and their cell biology.

    PubMed

    Azouz, Nurit Pereg; Hammel, Ilan; Sagi-Eisenberg, Ronit

    2014-10-01

    Exocytosis and secretion of secretory granule (SG) contained inflammatory mediators is the primary mechanism by which mast cells exert their protective immune responses in host defense, as well as their pathological functions in allergic reactions and anaphylaxis. Despite their central role in mast cell function, the molecular mechanisms underlying the biogenesis and secretion of mast cell SGs remain largely unresolved. Early studies have established the lysosomal nature of the mast cell SGs and implicated SG homotypic fusion as an important step occurring during both their biogenesis and compound secretion. However, the molecular mechanisms that account for key features of this process largely remain to be defined. A novel high-resolution imaging based methodology allowed us to screen Rab GTPases for their phenotypic and functional impact and identify Rab networks that regulate mast cell secretion. This screen has identified Rab5 as a novel regulator of homotypic fusion of the mast cell SGs that thereby regulates their size and cargo composition.

  2. Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules.

    PubMed

    Santodomingo, Jaime; Vay, Laura; Camacho, Marcial; Hernández-Sanmiguel, Esther; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Moreno, Alfredo; Alvarez, Javier

    2008-10-01

    The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50-100 microM Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.

  3. Formation of secretory granules by chromogranins.

    PubMed

    Inomoto, Chie; Osamura, Robert Yoshiyuki

    2009-12-01

    This review article covers the molecular mechanisms of secretory granule formation by chromogranin transfection. Recently, a few investigators have reported that the transfection of chromogranin A and B produces the structures of secretory granules. We used the GFP-chromogranin A transfection method to nonendocrine cells, COS-7 cells, which are not equipped with secretory granules. Despite the absence of endogenous secretory granules in nontransfected COS-7 cells, COS-7 cells transfected with chromogranin A contained granule-like structures in electron micrographs. The granules were composed of an outer limiting membrane with core structures that were interpreted as secretory granules. Human chromogranin A (CgA) labeled with 5-nm gold particles was present in several dense-core granules in our previous electron microscopy study. This review depicts the role of chromogranin A in the formation of secretory granules. It emphasizes the application of recently developed new technologies and the genesis of secretory granules.

  4. Secretory granule biogenesis and neuropeptide sorting to the regulated secretory pathway in neuroendocrine cells.

    PubMed

    Loh, Y Peng; Kim, Taeyoon; Rodriguez, Yazmin M; Cawley, Niamh X

    2004-01-01

    Neuropeptide precursors synthesized at the rough endoplasmic reticulum are transported and sorted at the trans-Golgi network (TGN) to the granules of the regulated secretory pathway (RSP) of neuroendocrine cells. They are then processed into active peptides and stored in large dense-core granules (LDCGs) until secreted upon stimulation. We have studied the regulation of biogenesis of the LDCGs and the mechanism by which neuropeptide precursors, such as pro-opiomelanocortin (POMC), are sorted into these LDCGs of the RSP in neuroendocrine and endocrine cells. We provide evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, plays an important role in the regulation of LDCG biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of secretory granule formation. Exogenously expressed POMC was neither stored nor secreted in a regulated manner in these CgA-deficient PC12 cells. Overexpression of CgA in a CgA- and LDCG-deficient endocrine cell line, 6T3, restored regulated secretion of transfected POMC and the presence of immunoreactive CgA at the tips of the processes of these cells. Unlike CgA, CgB, another granin protein, could not substitute for the role of CgA in regulating LDCG biogenesis. Thus, we conclude that CgA is a key player in the regulation of the biogenesis of LDCGs in neuroendocrine cells. To examine the mechanism of sorting POMC to the LDCGs, we carried out site-directed mutagenesis, transfected the POMC mutants into PC12 cells, and assayed for regulated secretion. Our previous molecular modeling studies predicted a three-dimensional sorting motif in POMC that can bind to a sorting receptor, membrane carboxypeptidase E (CPE). The sorting signal consists of four conserved residues at the N-terminal loop structure of POMC: two acidic residues and two hydrophobic residues. The two acidic residues were predicted to bind to a

  5. Synthesis of Prostaglandins and Eicosanoids by the Mast Cell Secretory Granule

    DTIC Science & Technology

    1988-01-01

    various lipid-derived mediators during exocytosis. MATERIALS AND METHODS The procedure for granule preparation is similar to that which has been described...Press, Inc. Printed in U.S.A. SYNTHESIS OF PROSTACLANDINS AND KICOSANOIDS BY THE M&ST CELL SECRETORY GRANULE Stephen P. Chock and Elsa A. Schmauder-Chock...SCISNTIFIC XeiOT Received September 30, 1988 SR88-32 The identification of a non-bilayer phospholipid storage in the secretory granule and the linking of

  6. Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

    SciTech Connect

    Baconnais, S.; Delavoie, F. |; Zahm, J.M.; Milliot, M.; Castillon, N.; Terryn, C.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E. , E-Mail: edith.puchelle@univ-reims.fr; Balossier, G.

    2005-10-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

  7. Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells.

    PubMed

    Rindler, Michael J; Xu, Chong-Feng; Gumper, Iwona; Cen, Chuan; Sonderegger, Peter; Neubert, Thomas A

    2008-04-01

    Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.

  8. Secretory granules of mast cells accumulate mature and immature MHC class II molecules.

    PubMed

    Vincent-Schneider, H; Théry, C; Mazzeo, D; Tenza, D; Raposo, G; Bonnerot, C

    2001-01-01

    Bone marrow-derived mast cells as well as dendritic cells, macrophages and B lymphocytes express major histocompatibility complex (MHC) class II molecules. In mast cells, the majority of MHC class II molecules reside in intracellular cell type-specific compartments, secretory granules. To understand the molecular basis for the localisation of MHC class II molecules in secretory granules, MHC class II molecules were expressed, together with the invariant chain, in the mast cell line, RBL-2H3. Using electron and confocal microscopy, we observed that in RBL-2H3 cells, mature and immature class II molecules accumulate in secretory granules. Two particular features of class II transport accounted for this intracellular localization: first, a large fraction of newly synthesized MHC class II molecules remained associated with invariant chain fragments. This defect, resulting in a slower rate of MHC class II maturation, was ascribed to a low cathepsin S activity. Second, although a small fraction of class II dimers matured (i.e. became free of invariant chain), allowing their association with antigenic peptides, they were retained in secretory granules. As a consequence of this intracellular localization, cell surface expression of class II molecules was strongly increased by cell activation stimuli which induced the release of the contents of secretory granules. Our results suggest that antigen presentation, and thereby antigen specific T cell stimulation, are regulated in mast cells by stimuli which induce mast cell activation.

  9. Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

    PubMed

    Barg, S; Knowles, M K; Chen, X; Midorikawa, M; Almers, Wolfhard

    2010-11-30

    Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

  10. Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay

    PubMed Central

    Urbé, Sylvie; Page, Lesley J.; Tooze, Sharon A.

    1998-01-01

    The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [35S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process. PMID:9864358

  11. Avian minor salivary glands: an ultrastructural study of the secretory granules in mucous and seromucous cells.

    PubMed

    Olmedo, L A; Samar, M E; Avila, R E; de Crosa, M G; Dettin, L

    2000-01-01

    Ultrastructural descriptions in birds are scarce thus, in this study we have characterized the secretory granules of mucous and seromucous cells from the palatine and lingual salivary glands of birds with different diets. The samples were taken from the tongue and palatine mucosa of chicken (Gallus gallus), quail (Coturnix coturnix), chimango (Milvago chimango) and white heron (Egretta thula). The samples were processed for observation by transmission electron microscopy (TEM) employing 4% Karnovsky solution for fixation. The most noteworthy finding was the heterogeneous ultrastructural appearance of the secretory granules. Differences in substructure were found between the four species, between the palatine and lingual glands in the same species and even within the same acinus and the same cell. At variance with other authors, these differences cannot be attributed to the type of fixative solution used taking into account that all the samples were processed in the same way. Previous histochemical studies have shown the presence of sulfated and non sulfated glycoconjugates in these glands which can be associated to the maturation of the granules. These granules are probably representative of peculiar storage of the secretory products that would give rise to a heterogeneous and complex ultrastructural pattern of granules in the mucosa and seromucosa cells of these avian species.

  12. Three-dimensional tracking of single secretory granules in live PC12 cells.

    PubMed

    Li, Dongdong; Xiong, Jun; Qu, Anlian; Xu, Tao

    2004-09-01

    Deconvolution wide-field fluorescence microscopy and single-particle tracking were used to study the three-dimensional mobility of single secretory granules in live PC12 cells. Acridine orange-labeled granules were found to travel primarily in random and caged diffusion, whereas only a small fraction of granules traveled in directed fashion. High K(+) stimulation increased significantly the percentage of granules traveling in directed fashion. By dividing granules into the near-membrane group (within 1 microm from the plasma membrane) and cytosolic group, we have revealed significant differences between these two groups of granules in their mobility. The mobility of these two groups of granules is also differentially affected by disruption of F-actin, suggesting different mechanisms are involved in the motion of the two groups of granules. Our results demonstrate that combined deconvolution and single-particle tracking may find its application in three-dimensional tracking of long-term motion of granules and elucidating the underlying mechanisms.

  13. Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

    PubMed

    Harthoorn, L F; Diederen, J H; Oudejans, R C; Verstegen, M M; Vullings, H G; Van der Horst, D J

    2000-01-01

    The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

  14. Pasteurella multocida toxin: Targeting mast cell secretory granules during kiss-and-run secretion.

    PubMed

    Danielsen, Elisabeth M; Christiansen, Nina; Danielsen, E Michael

    2016-02-01

    Pasteurella multocida toxin (PMT), a virulence factor of the pathogenic Gram-negative bacterium P. multocida, is a 146 kDa protein belonging to the A-B class of toxins. Once inside a target cell, the A domain deamidates the α-subunit of heterotrimeric G-proteins, thereby activating downstream signaling cascades. However, little is known about how PMT selects and enters its cellular targets. We therefore studied PMT binding and uptake in porcine cultured intestinal mucosal explants to identify susceptible cells in the epithelium and underlying lamina propria. In comparison with Vibrio cholera B-subunit, a well-known enterotoxin taken up by receptor-mediated endocytosis, PMT binding to the epithelial brush border was scarce, and no uptake into enterocytes was detected by 2h, implying that none of the glycolipids in the brush border are a functional receptor for PMT. However, in the lamina propria, PMT distinctly accumulated in the secretory granules of mast cells. This also occurred at 4 °C, ruling out endocytosis, but suggestive of uptake via pores that connect the granules to the cell surface. Mast cell granules are known to secrete their contents by a "kiss-and-run" mechanism, and we propose that PMT may exploit this secretory mechanism to gain entry into this particular cell type.

  15. Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells.

    PubMed Central

    Lang, T; Wacker, I; Wunderlich, I; Rohrbach, A; Giese, G; Soldati, T; Almers, W

    2000-01-01

    In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion. PMID:10827968

  16. Secretory granule biogenesis: rafting to the SNARE.

    PubMed

    Tooze, S A; Martens, G J; Huttner, W B

    2001-03-01

    Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.

  17. BACE2 is stored in secretory granules of mouse and rat pancreatic beta cells.

    PubMed

    Finzi, Giovanna; Franzi, Francesca; Placidi, Claudia; Acquati, Francesco; Palumbo, Elisa; Russo, Antonella; Taramelli, Roberto; Sessa, Fausto; La Rosa, Stefano

    2008-01-01

    BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of beta cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of beta cells suggests that it may play a role in diabetes-associated amyloidogenesis.

  18. Accumulation of Major Histocompatibility Complex Class II Molecules in Mast Cell Secretory Granules and Their Release upon Degranulation

    PubMed Central

    Raposo, Graça; Tenza, Danielle; Mecheri, Salahedine; Peronet, Roger; Bonnerot, Christian; Desaymard, Catherine

    1997-01-01

    To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60–80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles. PMID:9398681

  19. Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation.

    PubMed

    Raposo, G; Tenza, D; Mecheri, S; Peronet, R; Bonnerot, C; Desaymard, C

    1997-12-01

    To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60-80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

  20. Dense-core granules in neuroendocrine cells and neurons release their secretory constituents by piecemeal degranulation (review).

    PubMed

    Crivellato, Enrico; Nico, Beatrice; Bertelli, Eugenio; Nussdorfer, Gastone G; Ribatti, Domenico

    2006-12-01

    The term piecemeal degranulation (PMD) refers to a slow releasing process mediated by vesicular transport of stored secretory granule contents. This form of cell secretion was first proposed for basophils, mast cells and eosinophils, but evidence has begun to accumulate that PMD also occurs in dense-core granules of neuroendocrine cells and neurons. This review summarizes the electron-microscopic evidence that has been gathered in support of this view and also discusses the possible physiological significance of PMD in this class of secretory organelles in comparison with 'full fusion' and 'kiss-and-run' exocytosis.

  1. Toxoplasma secretory granules: one population or more?

    PubMed

    Mercier, Corinne; Cesbron-Delauw, Marie-France

    2015-02-01

    In Toxoplasma gondii, dense granules are known as the storage secretory organelles of the so-called GRA proteins (for dense granule proteins), which are destined to the parasitophorous vacuole (PV) and the PV-derived cyst wall. Recently, newly annotated GRA proteins targeted to the host cell nucleus have enlarged this view. Here we provide an update on the latest developments on the Toxoplasma secreted proteins, which to date have been mainly studied at both the tachyzoite and bradyzoite stages, and we point out that recent discoveries could open the issue of a possible, yet uncharacterized, distinct secretory pathway in Toxoplasma.

  2. Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

    PubMed

    Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-Ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-Ichiro

    2016-10-31

    Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

  3. Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution

    PubMed Central

    Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-ichiro

    2016-01-01

    Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules. PMID:27796315

  4. Identification of a Chromogranin A Domain That Mediates Binding to Secretogranin III and Targeting to Secretory Granules in Pituitary Cells and Pancreatic β-Cells

    PubMed Central

    Hosaka, Masahiro; Watanabe, Tsuyoshi; Sakai, Yuko; Uchiyama, Yasuo; Takeuchi, Toshiyuki

    2002-01-01

    Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca2+-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48–111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214–373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells. PMID:12388744

  5. Spiperone: evidence for uptake into secretory granules.

    PubMed Central

    Dannies, P S; Rudnick, M S; Fishkes, H; Rudnick, G

    1984-01-01

    Spiperone, a dopamine antagonist widely used as a specific ligand for dopamine and serotonin receptors, is actively accumulated into the F4C1 strain of rat pituitary tumor cells. The accumulation of 10 nM [3H]spiperone was linear for 3 min and reached a steady state after 10 min. Spiperone accumulation was reduced 50% by preincubation with 5 microM reserpine, an inhibitor of biogenic amine transport into secretory granules, and was also blocked by monensin and ammonium chloride, both of which increase the pH of intracellular storage organelles. Uptake was not affected by replacing sodium in the buffer with lithium at equimolar concentrations. Spiperone at 1 microM inhibited by over 50% serotonin transport into membrane vesicles isolated from platelet dense granules; this concentration inhibited the Na+-dependent plasma membrane transport system less than 10%. The data indicate spiperone specifically interacts with the secretory granule amine transport system and suggest that this transport system is found in the F4C1 pituitary cell strain as well as in platelets and neurons. The data also suggest that experiments utilizing spiperone to measure dopamine and serotonin receptors be interpreted with caution. PMID:6584920

  6. Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest.

    PubMed

    Diederen, J H; Vullings, H G

    1995-03-01

    The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

  7. Hydrogen sulfide induces hyperpolarization and decreases the exocytosis of secretory granules of rat GH3 pituitary tumor cells.

    PubMed

    Mustafina, Alsu N; Yakovlev, Aleksey V; Gaifullina, Aisylu Sh; Weiger, Thomas M; Hermann, Anton; Sitdikova, Guzel F

    2015-10-02

    The aim of the present study was to evaluate the effects of hydrogen sulfide (H2S) on the membrane potential, action potential discharge and exocytosis of secretory granules in neurosecretory pituitary tumor cells (GH3). The H2S donor - sodium hydrosulfide (NaHS) induced membrane hyperpolarization, followed by truncation of spontaneous electrical activity and decrease of the membrane resistance. The NaHS effect was dose-dependent with an EC50 of 152 μM (equals effective H2S of 16-19 μM). NaHS effects were not altered after inhibition of maxi conductance calcium-activated potassium (BK) channels by tetraethylammonium or paxilline, but were significantly reduced after inhibition or activation of ATP-dependent potassium channels (KATP) by glibenclamide or by diazoxide, respectively. In whole-cell recordings NaHS increased the amplitude of KATP currents, induced by hyperpolarizing pulses and subsequent application of glibenclamide decreased currents to control levels. Using the fluorescent dye FM 1-43 exocytosis of secretory granules was analyzed in basal and stimulated conditions (high K(+) external solution). Prior application of NaHS decreased the fluorescence of the cell membrane in both conditions which links with activation of KATP currents (basal secretion) and activation of KATP currents and BK-currents (stimulated exocytosis). We suggest that H2S induces hyperpolarization of GH3 cells by activation of KATP channels which results in a truncation of spontaneous action potentials and a decrease of hormone release.

  8. Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

    PubMed Central

    Brown, H; Larsson, O; Bränström, R; Yang, S N; Leibiger, B; Leibiger, I; Fried, G; Moede, T; Deeney, J T; Brown, G R; Jacobsson, G; Rhodes, C J; Braun, J E; Scheller, R H; Corkey, B E; Berggren, P O; Meister, B

    1998-01-01

    Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels. PMID:9724640

  9. Mefloquine, an anti-malaria agent, causes reactive oxygen species-dependent cell death in mast cells via a secretory granule-mediated pathway

    PubMed Central

    Paivandy, Aida; Calounova, Gabriela; Zarnegar, Behdad; Öhrvik, Helena; Melo, Fabio R; Pejler, Gunnar

    2014-01-01

    Mast cells are known to have a detrimental impact on a variety of pathological conditions. There is therefore an urgent need of developing strategies that limit their harmful effects. The aim of this study was to accomplish this by developing a means of inducing mast cell apoptosis. The strategy was to identify novel compounds that induce mast cell apoptosis by permeabilization of their secretory lysosomes (granules). As a candidate, we assessed mefloquine, an anti-malarial drug that has been proposed to have lysosome-permeabilizing activity. Mefloquine was added to mast cells and administered in vivo, followed by assessment of the extent and mechanisms of mast cell death. Mefloquine was cytotoxic to murine and human mast cells. Mefloquine induced apoptotic cell death of wild-type mast cells whereas cells lacking the granule compounds serglycin proteoglycan or tryptase were shown to undergo necrotic cell death, the latter finding indicating a role of the mast cell granules in mefloquine-induced cell death. In support of this, mefloquine was shown to cause compromised granule integrity and to induce leakage of granule components into the cytosol. Mefloquine-induced cell death was refractory to caspase inhibitors but was completely abrogated by reactive oxygen species inhibition. These findings identify mefloquine as a novel anti-mast cell agent, which induces mast cell death through a granule-mediated pathway. Mefloquine may thus become useful in therapy aiming at limiting harmful effects of mast cells. PMID:25505612

  10. AP-1A controls secretory granule biogenesis and trafficking of membrane secretory granule proteins.

    PubMed

    Bonnemaison, Mathilde; Bäck, Nils; Lin, Yimo; Bonifacino, Juan S; Mains, Richard; Eipper, Betty

    2014-10-01

    The adaptor protein 1A complex (AP-1A) transports cargo between the trans-Golgi network (TGN) and endosomes. In professional secretory cells, AP-1A also retrieves material from immature secretory granules (SGs). The role of AP-1A in SG biogenesis was explored using AtT-20 corticotrope tumor cells expressing reduced levels of the AP-1A μ1A subunit. A twofold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non-condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue-stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α-amidating monooxygenase-1 (PAM-1), integral membrane enzymes that enter immature SGs. The non-condensing SGs contained POMC products and PAM-1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM-1 into PHM was unaltered, but PHM basal secretion was increased in sh-μ1A PAM-1 cells. Despite lacking a canonical AP-1A binding motif, yeast two-hybrid studies demonstrated an interaction between the PAM-1 cytosolic domain and AP-1A. Coimmunoprecipitation experiments with PAM-1 mutants revealed an influence of the luminal domains of PAM-1 on this interaction. Thus, AP-1A is crucial for normal SG biogenesis, function and composition.

  11. HID-1 is required for homotypic fusion of immature secretory granules during maturation.

    PubMed

    Du, Wen; Zhou, Maoge; Zhao, Wei; Cheng, Dongwan; Wang, Lifen; Lu, Jingze; Song, Eli; Feng, Wei; Xue, Yanhong; Xu, Pingyong; Xu, Tao

    2016-10-18

    Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules.

  12. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells.

    PubMed Central

    Stevens, R L; Fox, C C; Lichtenstein, L M; Austen, K F

    1988-01-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of 35S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although [35S]heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. We here demonstrate that human lung mast cells of 96% purity incorporate [35S] sulfate into separate heparin and chondroitin sulfate proteoglycans in an approximately equal to 2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin [35S]sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin [35S]sulfate E proteoglycans and the [35S]heparin proteoglycans were exocytosed from the [35S]sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of 35S-labeled proteoglycans reside in the secretory granules of these human lung mast cells. PMID:3353378

  13. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

    SciTech Connect

    Stevens, R.L.; Austen, K.F. ); Fox, C.C.; Lichtenstein, L.M. )

    1988-04-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of {sup 35}S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although ({sup 35}S)heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate ({sup 35}S)sulfate into separate heparin and chondroitin sulfate proteoglycans in an {approx}2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin ({sup 35}S)sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin ({sup 35}S)sulfate E proteoglycans and the ({sup 35}S)heparin proteoglycans were exocytosed from the ({sup 35}S)sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of {sup 35}S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

  14. The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

    PubMed Central

    Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

    1995-01-01

    The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626033

  15. Secretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaera.

    PubMed Central

    Pouli, A E; Emmanouilidou, E; Zhao, C; Wasmeier, C; Hutton, J C; Rutter, G A

    1998-01-01

    To image the behaviour in real time of single secretory granules in neuroendocrine cells we have expressed cDNA encoding a fusion construct between the dense-core secretory-granule-membrane glycoprotein, phogrin (phosphatase on the granule of insulinoma cells), and enhanced green fluorescent protein (EGFP). Expressed in INS-1 beta-cells and pheochromocytoma PC12 cells, the chimaera was localized efficiently (up to 95%) to dense-core secretory granules (diameter 200-1000 nm), identified by co-immunolocalization with anti-(pro-)insulin antibodies in INS-1 cells and dopamine beta-hydroxylase in PC12 cells. Using laser-scanning confocal microscopy and digital image analysis, we have used this chimaera to monitor the effects of secretagogues on the dynamics of secretory granules in single living cells. In unstimulated INS-1 beta-cells, granule movement was confined to oscillatory movement (dithering) with period of oscillation 5-10 s and mean displacement <1 microm. Both elevated glucose concentrations (30 mM), and depolarization of the plasma membrane with K+, provoked large (5-10 microm) saltatory excursions of granules across the cell, which were never observed in cells maintained at low glucose concentration. By contrast, long excursions of granules occurred in PC12 cells without stimulation, and occurred predominantly from the cell body towards the cell periphery and neurite extensions. Purinergic-receptor activation with ATP provoked granule movement towards the membrane of PC12 cells, resulting in the transfer of fluorescence to the plasma membrane consistent with fusion of the granule and diffusion of the chimaera in the plasma membrane. These results illustrate the potential use of phogrin-EGFP chimeras in the study of secretory-granule dynamics, the regulation of granule-cytoskeletal interactions and the trafficking of a granule-specific transmembrane protein during the cycle of exocytosis and endocytosis. PMID:9639579

  16. EFTEM cytochemistry and sexual dimorphism of secretory granules in male and female hamster submandibular glands.

    PubMed

    Moriguchi, Keiichi; Utsumi, Michiya; Ohno, Norikazu

    2011-05-01

    After glutaraldehyde fixation followed by osmium tetroxide postfixing, the secretory granules of acinar cells in male hamster submandibular glands (SGs) exhibit a characteristic bipartite substructure, with an electron-lucid rim and a more electron-dense central core. In female hamsters, the reverse is seen, with the larger portion of the granules forming an electron-lucid core and an outer electron-dense crescent rim. In the present study of endogenous peroxidase (PO) activity of male and female hamster SGs, secretory granules in the acinar cells were studied by DAB cytochemical technique. Individual granules showed bipartite substructure with the PO activity in a positive center core and unreacted lucid rim in both the male and the female acinar cells. Through isolation of granular fractions, the male and the female granules exhibited the same bipartite structure. We also examined the relation between the PO activity and counterstained areas in male and female hamster SGs, and the secretory granules of acinar cells by using EFTEM. In the male SG, the secretory granules exhibited the characteristic bipartite substructure to carry out parallel-EELS, nitrogen reflecting the presence of DAB moieties and uranium from counterstaing the presence the central core but not in the rim. On the other hand, the female bipartite secretory granules of the SG, exhibit the nitrogen reflecting the presence in the central core and uranium in the rim.

  17. Kinetics of early TCR signaling regulate the pathway of lytic granule delivery to the secretory domain

    PubMed Central

    Beal, Allison M.; Anikeeva, Nadia; Varma, Rajat; Cameron, Thomas O.; Vasiliver-Shamis, Gaia; Norris, Philip J.; Dustin, Michael L.; Sykulev, Yuri

    2009-01-01

    SUMMARY Cytolytic granule mediated killing of virus-infected cells is an essential function of cytotoxic T lymphocytes. Analysis of lytic granule delivery shows that the granules can take long or short paths to the secretory domain where they are released. Both paths utilize the same intracellular molecular events, which have different spatial and temporal arrangements in each path and are regulated by the kinetics of downstream Ca2+ mediated signaling. Rapid and robust signaling causes swift granule concentration near the MTOC and subsequent delivery by the polarized MTOC directly to the secretory domain - the shortest and fastest path. Indolent signaling leads to late recruitment of granules that move along microtubules to the periphery of the synapse and then move tangentially to fuse at the outer edge of the secretory domain - a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. PMID:19833088

  18. Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells.

    PubMed

    Hong-Geller, E; Cerione, R A

    2000-02-07

    We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP(3) production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP(3) receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCgamma1, the enzyme immediately upstream of IP(3) formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP(3) formation and calcium mobilization upon antigen stimulation.

  19. Changes in the Paneth cell population of human small intestine assessed by image analysis of the secretory granule area.

    PubMed Central

    Elmes, M E; Jones, J G; Stanton, M R

    1983-01-01

    Estimates of the Paneth cell population in human jejunum and ileum were made using measurement of the granule area in micron2 by image analysis in a defined number of crypts. This figure was preferable to granule area per mm as there was a significant difference in crypts per mm between biopsies and surgical samples. In the jejunum no significant difference was found between normal children and adults with and without peptic ulcer. In adults with subtotal or partial villous atrophy the decrease in area was not statistically significant and there was no decrease in area in children with partial villous atrophy and coeliac disease. There was a marked increase in granule area in the jejunum of patients who had had a previous partial gastrectomy which was statistically significant. In the ileum patients with carcinoma of the caecum had higher values than patients with non-inflammatory non-malignant conditions but this was not statistically significant and two patients with Crohn's disease had an increased granule area. Paneth cell populations are affected by alterations in the intestinal luminal environment due to previous surgery or neoplastic or inflammatory disease. Images PMID:6875016

  20. Separation of rat pituitary secretory granules by continuous flow electrophoresis

    NASA Technical Reports Server (NTRS)

    Hayes, Daniel; Exton, Carrie; Salada, Thomas; Shellenberger, Kathy; Waddle, Jenny; Hymer, W. C.

    1990-01-01

    The separation of growth hormone-containing cytoplasmic secretory granules from the rat pituitary gland by continuous flow electrophoresis is described. The results are consistent with the hypothesis that granule subpopulations can be separated due to differences in surface charge; these, in turn, may be related to the oligomeric state of the hormone.

  1. HID-1 is required for homotypic fusion of immature secretory granules during maturation

    PubMed Central

    Du, Wen; Zhou, Maoge; Zhao, Wei; Cheng, Dongwan; Wang, Lifen; Lu, Jingze; Song, Eli; Feng, Wei; Xue, Yanhong; Xu, Pingyong; Xu, Tao

    2016-01-01

    Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules. DOI: http://dx.doi.org/10.7554/eLife.18134.001 PMID:27751232

  2. Secretory Granule Membrane Protein Recycles Through Multivesicular Bodies

    PubMed Central

    Bäck, Nils; Rajagopal, Chitra; Mains, Richard E.; Eipper, Betty A.

    2010-01-01

    The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine α-amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation demonstrated efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr946, Ser949) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100 kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins. PMID:20374556

  3. A hydrophobic patch in a charged alpha-helix is sufficient to target proteins to dense core secretory granules.

    PubMed

    Dikeakos, Jimmy D; Lacombe, Marie-Josée; Mercure, Chantal; Mireuta, Matei; Reudelhuber, Timothy L

    2007-01-12

    Many endocrine and neuroendocrine cells contain specialized secretory organelles called dense core secretory granules. These organelles are the repository of proteins and peptides that are secreted in a regulated manner when the cell receives a physiological stimulus. The targeting of proteins to these secretory granules is crucial for the generation of certain peptide hormones, including insulin and ACTH. Although previous work has demonstrated that proteins destined to a variety of cellular locations, including secretory granules, contain targeting sequences, no single consensus sequence for secretory granule-sorting signals has emerged. We have shown previously that alpha-helical domains in the C-terminal tail of the prohormone convertase PC1/3 play an important role in the ability of this region of the protein to direct secretory granule targeting (Jutras, I. Seidah, N. G., and Reudelhuber, T. L. (2000) J. Biol. Chem. 275, 40337-40343). In this study, we show that a variety of alpha-helical domains are capable of directing a heterologous secretory protein to granules. By testing a series of synthetic alpha-helices, we also demonstrate that the presence of charged (either positive or negative) amino acids spatially segregated from a hydrophobic patch in the alpha-helices of secretory proteins likely plays a critical role in the ability of these structures to direct secretory granule sorting.

  4. New class of cargo protein in Tetrahymena thermophila dense core secretory granules.

    PubMed

    Haddad, Alex; Bowman, Grant R; Turkewitz, Aaron P

    2002-08-01

    Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules.

  5. Lumenal protein within secretory granules affects fusion pore expansion.

    PubMed

    Weiss, Annita Ngatchou; Anantharam, Arun; Bittner, Mary A; Axelrod, Daniel; Holz, Ronald W

    2014-07-01

    It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200 ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ∼2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only

  6. Chromaffin granules in the rat adrenal medulla release their secretory content in a particulate fashion.

    PubMed

    Crivellato, Enrico; Belloni, Anna; Nico, Beatrice; Nussdorfer, Gastone G; Ribatti, Domenico

    2004-03-01

    Exocytosis is considered the main route of granule discharge in chromaffin cells. We recently provided ultrastructural evidence suggesting that piecemeal degranulation (PMD) occurs in mouse adrenal chromaffin cells. In the present study, we processed rat adrenal glands for transmission electron microscopy (TEM), and examined chromaffin cells for changes characteristic of PMD. Both adrenaline (A)- and noradrenaline (NA)-storing cells express ultrastructural features suggestive of a slow and particulate mode of granule discharge. In adrenaline-containing cells, some granules present enlarged dimensions accompanied by eroded or dissolved matrices. Likewise, a number of granules in NA-releasing cells show content reduction with variably expanded granule chambers. Dilated, empty granule containers are recognizable in the cytoplasm of both cell types. Characteristically, altered granules and empty containers are seen intermingled with normal, resting granules. In addition, chromaffin granules often show irregular profiles, with budding or tail-like projections of their limiting membranes. Thirty 150-nm-diameter membrane-bound vesicles with a moderately electron-dense or -lucent internal structure are observable in the cytoplasm of both cell types. These vesicles are seen among the granules and some of them are fused with the perigranule membranes in the process of attachment to or budding from the granules. These data add further support to the concept that PMD may be an alternative secretory pathway in adrenal chromaffin cells.

  7. Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.

    PubMed

    Ohara-Imaizumi, Mica; Nakamichi, Yoko; Tanaka, Toshiaki; Katsuta, Hidenori; Ishida, Hitoshi; Nagamatsu, Shinya

    2002-04-01

    The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic beta-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH 7.4, but not at approximately pH 5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy (5 or 10 s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50 mM KCl and 22 mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin-pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

  8. Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland

    SciTech Connect

    Marino, C.R.; Castle, J.D.; Gorelick, F.S. )

    1990-07-01

    An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with ({sup 32}P)orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.

  9. Two Dimensional Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically-Labeled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Radiolabeled newly synthesized proteins can be analyzed by a number of techniques such as two dimensional gel electrophoresis (2DE). This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine in the presence of basal and stimulatory concentrations of glucose, followed by subcellular fractionation to produce a secretory granule fraction and analysis of the granule protein contents by 2DE. This provides a means of determining whether or not the biosynthetic rates of the entire granule constituents are coordinately regulated.

  10. 2D Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically Labelled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabelling of cells with radioactive amino acids such is a common method for investigating the biosynthetic rates of proteins. In this way, the abundance of newly synthesized proteins can be determined by several proteomic techniques including 2D gel electrophoresis (2DE). This chapter describes a protocol for labelling pancreatic islets with (35)S-methionine in the presence of low and high concentrations of glucose, followed by subcellular fractionation enrichment of secretory granule proteins and analysis of the granule protein contents by 2DE. This demonstrated that the biosynthetic rates of most of the granule proteins are co-ordinately regulated in the presence of stimulatory glucose concentrations.

  11. P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells

    PubMed Central

    1992-01-01

    P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to alpha granules of platelets and Weibel-Palade bodies of endotheial cells. Upon stimulation of these cells, P-selectin is translocated to the plasma membrane where it functions as a receptor for monocytes and neutrophils. To investigate whether the mechanism of targeting of P- selectin to granules is specific for megakaryocytes and endothelial cells and/or dependent on von Willebrand factor, a soluble adhesive protein that is stored in the same granules, we have expressed the cDNA for P-selectin in AtT-20 cells. AtT-20 cells are a mouse pituitary cell line that can store proteins in a regulated fashion. By double-label immunofluorescence, P-selectin was visible as a punctate pattern at the tips of cell processes. This pattern closely resembled the localization of ACTH, the endogenous hormone produced and stored by the AtT-20 cells. Fractionation of the transfected cells resulted in the codistribution of P-selectin and ACTH in cellular compartments of the same density. Immunoelectron microscopy using a polyclonal anti-P- selectin antibody demonstrated immunogold localization in dense granules, morphologically indistinguishable from the ACTH granules. Binding experiments with radiolabeled monoclonal antibody to P-selectin indicated that there was also surface expression of P-selectin on the AtT-20 cells. After stimulation with the secretagogue 8-Bromo-cAMP the surface expression increased twofold, concomitant with the release of ACTH. In contrast, the surface expression of P-selectin transfected into CHO cells, which do not have a regulated pathway of secretion, did not change with 8-Br-cAMP treatment. In conclusion, we provide evidence for the regulated secretion of a transmembrane protein (P-selectin) in a heterologous cell line, which indicates that P-selectin contains an independent sorting signal directing it to storage granules. PMID:1370497

  12. Electron microprobe analysis of human labial gland secretory granules in cystic fibrosis

    SciTech Connect

    Izutsu, K.; Johnson, D.; Schubert, M.; Wang, E.; Ramsey, B.; Tamarin, A.; Truelove, E.; Ensign, W.; Young, M.

    1985-06-01

    X-ray microanalysis of freeze-dried labial gland cryosections revealed that Na concentration was doubled and the Ca/S concentration ratio was decreased in secretory granules of labial glands from patients with cystic fibrosis (CF) when compared with glands from normal subjects. Other results suggested that the decrease in the Ca/S concentration ratio resulted from an increase in S concentration. These findings imply that mucous granules in labial saliva showed a CF-related increase in Na and S content, and such changes would be expected to affect the rheology of the mucus after exocytosis. In contrast with a previous study in human parotid glands, no evidence was found for CF-related changes in cytoplasmic or nuclear Na, K, and Ca concentrations. Significant elemental differences were found between secretory granules and nuclei and cytoplasm of control cells.

  13. Porosome: The Universal Secretory Portal in Cells

    NASA Astrophysics Data System (ADS)

    Jena, Bhanu

    2012-10-01

    , and only 20-45% increase in porosome diameter is demonstrated following the docking and fusion of 0.2-1.2 μm in diameter secretory vesicles, it is concluded that secretory vesicles ``transiently'' dock and fuse, rather than completely merge at the base of the porosome complex to release their contents to the outside. In agreement, it has been demonstrated that ``secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells''; that ``single synaptic vesicles fuse transiently and successively without loss of identity''; and that``zymogen granule (the secretory vesicle in exocrine pancreas) exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity.'' In this presentation, the discovery of the porosome, resulting in a paradigm shift in our understanding of cell secretion will be briefly discussed.

  14. α-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion

    PubMed Central

    Geng, Xuehui; Lou, Haiyan; Wang, Jian; Li, Lehong; Swanson, Alexandra L.; Sun, Ming; Beers-Stolz, Donna; Watkins, Simon; Perez, Ruth G.

    2011-01-01

    α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic β-cells, α-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for α-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for α-synuclein localization to insulin granules in β-cells, immunoprecipitation with Western blot analysis for interaction between α-synuclein and KATP channels, and ELISA assays for the effect of altering α-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between α-synuclein knockout and wild-type β-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-α-synuclein antibodies labeled secretory organelles within β-cells. Anti-α-synuclein antibodies colocalized with KATP channel, anti-insulin, and anti-C-peptide antibodies. α-Synuclein coimmunoprecipitated in complexes with KATP channels. Expression of α-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. α-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the β-cell surface membranes observed in these islets. These findings demonstrate that α-synuclein interacts with KATP channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. α-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases. PMID:20858756

  15. Multiplex Sequential Immunoprecipitation of Insulin Secretory Granule Proteins from Radiolabeled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Specific proteins can then be immunoprecipitated and analyzed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine, followed by multiplex sequential immunoprecipitation of insulin and three other secretory granule accessory proteins. This provided a means of distinguishing those pancreatic islet proteins with different biosynthetic rates in response to the media glucose concentrations.

  16. Signaling from the Secretory Granule to the Nucleus: Uhmk1 and PAM

    PubMed Central

    Francone, Victor P.; Ifrim, Marius F.; Rajagopal, Chitra; Leddy, Christopher J.; Wang, Yanping; Carson, John H.; Mains, Richard E.; Eipper, Betty A.

    2010-01-01

    Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine α-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent. PMID:20573687

  17. Sorting and storage during secretory granule biogenesis: looking backward and looking forward.

    PubMed Central

    Arvan, P; Castle, D

    1998-01-01

    Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained. PMID:9620860

  18. The secretory granule matrix: a fast-acting smart polymer.

    PubMed

    Nanavati, C; Fernandez, J M

    1993-02-12

    The secretory granule matrix is a miniature biopolymer that consists of a charged polymer network that traps peptides and transmitters when it condenses and releases them on exocytotic decondensation. Models of exocytotic fusion have treated this matrix as a short circuit and have neglected its electrical contributions. This matrix responded to negative voltages by swelling, which was accompanied by a large increase in conductance, and to positive voltages by condensing. Thus, the matrix resembled a diode. The swollen matrix exerted large pressures on the order of 12 bar. The responses took place within milliseconds of the application of the electric field. These findings suggest that matrix decondensation, and therefore product release, is controlled by potential gradients.

  19. Effects of estrogen and androgen on the ultrastructure of secretory granules and intercellular junctions in regressed canine prostate.

    PubMed

    Merk, F B; Leav, I; Kwan, P W; Ofner, P

    1980-06-01

    Epithelial cells in the prostate of the castrated or hypophysectomized dog were studied by thin-section and freeze-fracture electron microscopy to determine in vivo responses to estradiol-17 beta 17-cyclopentylpropionate (ECP) and testosterone cyclopentylpropionate (TCP). Particular attention was given to changes in specific organelles and intercellular junctions that might reflect hormone action. The few secretory granules that remain in the regressed epithelium (vestigial granules) serve as markers of prior androgen responsiveness. Pharmacologic doses of ECP caused regressed glandular cells to acquire a novel phenotype. Characteristic features of these estrogen-modified glandular (EMG) cells are newly formed secretory granules and tonofilament bundles that coexist with vestigial granules, thus demonstrating bipotentiality of response. Glandular cell-tight junctions appear unaltered by the endocrine manipulations. Although EMG cells have squamous cell features, tight junctions remain intact. Desmosomes in the canine prostate are dimorphic and are classified 70F and 100F according to the width of the filaments that converge on the dense plaques. In intact dogs, 100F desmosomes are associated with basal-reserve cells, whereas only the 70F variety is found between glandular cells. TCP treatment does not alter this distribution. Following ECP administration, both 70F and 100F desmosomes are present between EMG cells. The coexistence of newly formed secretory granules and tonofilaments of 100F desmosomes in the same EMG cell represents estrogen-induced bidirectional differentiation. Our findings indicate that androgens and estrogens are individually capable of controlling the expression of secretory granules and desmosomes. In intact animals, male and female sex hormones may act in concert to direct epithelial cell differentiation of the prostate.

  20. Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

    PubMed Central

    Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

    2016-01-01

    In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals’ endocrine system. PMID:27775083

  1. Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs.

    PubMed

    Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

    2016-10-24

    In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4(+) cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals' endocrine system.

  2. The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals.

    PubMed

    McGirr, Rebecca; Guizzetti, Leonardo; Dhanvantari, Savita

    2013-05-01

    Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1-6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.

  3. Sending proteins to dense core secretory granules: still a lot to sort out.

    PubMed

    Dikeakos, Jimmy D; Reudelhuber, Timothy L

    2007-04-23

    The intracellular sorting of peptide hormone precursors to the dense core secretory granules (DCSGs) is essential for their bioactivation. Despite the fundamental importance of this cellular process, the nature of the sorting signals for entry of proteins into DCSGs remains a source of vigorous debate. This review highlights recent discoveries that are consistent with a model in which several protein domains, acting in a cell-specific fashion and at different steps in the sorting process, act in concert to regulate the entry of proteins into DCSGs.

  4. A functional cyclic AMP response element plays a crucial role in neuroendocrine cell type-specific expression of the secretory granule protein chromogranin A.

    PubMed Central

    Wu, H; Mahata, S K; Mahata, M; Webster, N J; Parmer, R J; O'Connor, D T

    1995-01-01

    Chromogranin A, a soluble acidic protein, is a ubiquitous component of secretory vesicles throughout the neuroendocrine system. We reported previously the cloning and initial characterization of the mouse chromogranin A gene promoter, which showed that the promoter contains both positive and negative domains and that a proximal promoter spanning nucleotides -147 to +42 bp relative to the transcriptional start site is sufficient for neuroendocrine cell type-specific expression. The current study was undertaken to identify the particular elements within this proximal promoter that control tissue-specific expression. We found that deletion or point mutations in the potential cAMP response element (CRE) site at -68 bp virtually abolished promoter activity specifically in neuroendocrine (PC12 chromaffin or AtT20 corticotrope) cells, with little effect on activity in control (NIH3T3 fibroblast) cells; thus, the CRE box is necessary for neuroendocrine cell type-specific activity of the chromogranin A promoter. Furthermore, the effect of the CRE site is enhanced in the context of intact (wild-type) promoter sequences between -147 and -100 bp. DNase I footprint analysis showed that these regions (including the CRE box) bind nuclear proteins present in both neuroendocrine (AtT20) and control (NIH3T3) cells. In AtT20 cells, electrophoretic mobility shift assays and factor-specific antibody supershifts showed that an oligonucleotide containing the chromogranin A CRE site formed a single, homogeneous protein-DNA complex containing the CRE-binding protein CREB. However, in control NIH3T3 cells we found evidence for an additional immunologically unrelated protein in this complex. A single copy of this oligonucleotide was able to confer neuroendocrine-specific expression to a heterologous (thymidine kinase) promoter, albeit with less fold selectivity than the full proximal chromogranin A promoter. Hence, the CRE site was partially sufficient to explain the neuroendocrine cell type

  5. Secretion from Myeloid Cells: Secretory Lysosomes.

    PubMed

    Griffiths, Gillian M

    2016-08-01

    Many cells of the myeloid lineage use an unusual secretory organelle to deliver their effector mechanisms. In these cells, the lysosomal compartment is often modified not only to fulfill the degradative functions of a lysosome but also as a mechanism for secreting additional proteins that are found in the lysosomes of each specialized cell type. These extra proteins vary from one cell type to another according to the specialized function of the cell. For example, mast cells package histamine; cytotoxic T cells express perforin; azurophilic granules in neutrophils express antimicrobial peptides, and platelets von Willebrand factor. Upon release, these very different proteins can trigger inflammation, cell lysis, microbial death, and clotting, respectively, and hence deliver the very different effector mechanisms of these different myeloid cells.

  6. Amyloid formation of growth hormone in presence of zinc: Relevance to its storage in secretory granules

    PubMed Central

    Jacob, Reeba S.; Das, Subhadeep; Ghosh, Saikat; Anoop, Arunagiri; Jha, Narendra Nath; Khan, Tuhin; Singru, Praful; Kumar, Ashutosh; Maji, Samir K.

    2016-01-01

    Amyloids are cross-β-sheet fibrillar aggregates, associated with various human diseases and native functions such as protein/peptide hormone storage inside secretory granules of neuroendocrine cells. In the current study, using amyloid detecting agents, we show that growth hormone (GH) could be stored as amyloid in the pituitary of rat. Moreover, to demonstrate the formation of GH amyloid in vitro, we studied various conditions (solvents, glycosaminoglycans, salts and metal ions) and found that in presence of zinc metal ions (Zn(II)), GH formed short curvy fibrils. The amyloidogenic nature of these fibrils was examined by Thioflavin T binding, Congo Red binding, transmission electron microscopy and X-ray diffraction. Our biophysical studies also suggest that Zn(II) initiates the early oligomerization of GH that eventually facilitates the fibrillation process. Furthermore, using immunofluorescence study of pituitary tissue, we show that GH in pituitary significantly co-localizes with Zn(II), suggesting the probable role of zinc in GH aggregation within secretory granules. We also found that GH amyloid formed in vitro is capable of releasing monomers. The study will help to understand the possible mechanism of GH storage, its regulation and monomer release from the somatotrophs of anterior pituitary. PMID:27004850

  7. Flow cytometry-assisted purification and proteomic analysis of the corticotropes dense-core secretory granules.

    PubMed

    Gauthier, Daniel J; Sobota, Jacqueline A; Ferraro, Francesco; Mains, Richard E; Lazure, Claude

    2008-09-01

    The field of organellar proteomics has emerged as an attempt to minimize the complexity of the proteomics data obtained from whole cell and tissue extracts while maximizing the resolution on the protein composition of a single subcellular compartment. Standard methods involve lengthy density-based gradient and/or immunoaffinity purification steps followed by extraction, 1-DE or 2-DE, gel staining, in-gel tryptic digestion, and protein identification by MS. In this paper, we present an alternate approach to purify subcellular organelles containing a fluorescent reporter molecule. The gel-free procedure involves fluorescence-assisted sorting of the secretory granules followed by gentle extraction in a buffer compatible with tryptic digestion and MS. Once the subcellular organelle labeled, this procedure can be done in a single day, requires no major modification to any instrumentation and can be readily adapted to the study of other organelles. When applied to corticotrope secretory granules, it led to a much enriched granular fraction from which numerous proteins could be identified through MS.

  8. Morphological and morphometric study of atrial specific granules and other secretory components in dogs experimentally infected with Trypanosoma cruzi.

    PubMed Central

    Caliari, M. V.; Lana, M.; Leite, V. H.; Tafuri, W. L.

    1995-01-01

    Changes in blood volume can induce morphometric and morphological alterations in the secretory complex of the myoendocrine cells due to the stretching of atrial walls. These alterations were studied by electron microscopy, using dogs infected intraperitonially with Trypanosoma cruzi and necropsied during the acute phase of the infection when congestive heart failure was present. Several changes were observed in the myoendocrine cells of the heart: hypertrophy and hyperplasia of rough endoplasmic reticulum and Golgi complex, increase in telenuclear secretory complex, increase in fusion of type B atrial specific granules (ASG), decrease of the total number of ASG, enlargement of the maximum diameter of type A ASG and a relative increase in the number of type B ASG. These alterations suggest a larger secretory activity of the atrial myoendocrine cells with a larger secretion of atrial natriuretic peptide (ANP). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7547444

  9. Platelet secretory behaviour: as diverse as the granules … or not?

    PubMed

    Heijnen, H; van der Sluijs, P

    2015-12-01

    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.

  10. Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells

    PubMed Central

    1992-01-01

    Regulated secretory cells have two pathways that transport secreted proteins from the Golgi complex to the cell surface. To identify carrier vesicles involved in regulated and constitutive secretion, PC12 pheochromocytoma cells were labeled with [35S]sulfate to identify markers for the two secretory pathways, then mechanically permeabilized and incubated in vitro. Small constitutive secretory vesicles, containing mostly sulfated proteoglycans, accumulated during an in vitro incubation with ATP. In the presence of GTP gamma S, the constitutive vesicles became significantly more dense, suggesting that a coated intermediate was stabilized. Larger immature regulated secretory granules, enriched in sulfated secretogranin II, also escaped from the permeabilized cells in vitro. During granule maturation, their density increased and the amount of cofractionating proteoglycans diminished. The data suggest that sorting continues during secretory granule maturation. PMID:1572894

  11. [Chromogranin A: immunocytochemical localization in secretory granules of frog atrial cardiomyocytes].

    PubMed

    Krylova, M I

    2007-01-01

    Chromogranin A (CgA) is a member of the granin family of acidic proteins that present in the secretory granules (SGs) of many endocrine, neuroendocrine and neuronal cells. Atrial natriuretic peptide (ANP)-storing SGs in atrial cardiomyocytes of rat heart also contain CgA. Cardiosuppressive effect of CgA-derived peptides (vasostatins) on in vitro isolated and perfused working frog and rat hearts has been shown under both basal conditions and beta-adrenergic stimulation. More recently it has been revealed that rat heart produces and processes CgA-derived vasostatin-containing peptides. Until now nothing has been known about the presence of CgA in an amphibian heart. We have investigated the subcellular localization of CgA in atrial myocytes of adult frog Rana temporaria heart using ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP has shown that out of three morphologically different types (A, B and D) of specific cytoplasmic granules (SCGs) present in myocytes only two (A and B)--large (120-200 nm in diameter) granules with more and with less electron dense core--exhibit immunoreactivity (IR) to these two antigens. The third type (D) of granules (80-100 nm in diameter) are small membrane bound granules characterized by highly electron dense core surrounded with a thin halo. These granules revealed negative reaction on immunostaining for both CgA and ANP. The presence of CgA- and ANP-IR in the same SCGs in frog atrial myocytes is consistent with the endocrine nature of these granules. Taking into account our and literature data we propose that CgA present in frog atrial cardiomyocite SCGs might be a precursor of vasostatin-containing peptides, as it takes place in rat heart. It is possible that these CgA-derived peptides together with ANP exert their regulatory function through the autocrine and/or paracrine mechanisms and play important cardioprotective role in frog heart under stress condition.

  12. Myosin VI small insert isoform maintains exocytosis by tethering secretory granules to the cortical actin.

    PubMed

    Tomatis, Vanesa M; Papadopulos, Andreas; Malintan, Nancy T; Martin, Sally; Wallis, Tristan; Gormal, Rachel S; Kendrick-Jones, John; Buss, Folma; Meunier, Frédéric A

    2013-02-04

    Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.

  13. Serous cutaneous glands in anurans: Fourier transform analysis of the repeating secretory granule substructure

    NASA Astrophysics Data System (ADS)

    Nosi, D.; Delfino, G.; Quercioli, F.

    2013-03-01

    A combined transmission electron microscopy (TEM) and Fourier transform analysis has been performed on the secretory granules storing active peptides/proteins in serous cutaneous glands of n = 12 anuran species. Previous TEM investigation showed that the granules are provided with remarkable repeating substructures based on discrete subunits, arranged into a consistent framework. Furthermore, TEM findings revealed that this recurrent arrangement is acquired during a prolonged post-Golgian (or maturational) processing that affects the secretory product. Maturation leads to a variety of patterns depending on the degree of subunit clustering. This variety of recurrent patterns has been plotted into a range of frequency spectra. Through this quantitative approach, we found that the varying granule substructure can be reduced to a single mechanism of peptide/protein aggregation.

  14. P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

    PubMed

    Kaur, Jasber; Cutler, Daniel F

    2002-03-22

    The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.

  15. Reversible condensation of mast cell secretory products in vitro.

    PubMed Central

    Fernandez, J M; Villalón, M; Verdugo, P

    1991-01-01

    We have investigated the mechanisms responsible for the condensation and decondensation of secretory products that occur in mast cell secretion. We show here that the hydrated matrix of an exocytosed secretory granule can be recondensed to its original volume by exposure to acidic solutions containing histamine at concentrations that mimic those found in vivo. Recondensation by acidic histamine began in the range of 1-10 mM with a dose response curve that was accurately predicted by a Hill type equation with four highly cooperative binding sites and a half maximum concentration of [Hi++] = 3.9 mM. Recondensation by histamine showed a sigmoidal dependency on pH (critical range pH 5.5-6.5) and was fully reversible. These experiments suggest that histamine, possibly by binding to anionic sites in the protein-heparin complex of the granule matrix, triggers a change in the polymeric structures of the granule matrix from an extended coil to a collapsed globular state. This may be a useful model for understanding the condensation of secretory products into dense core granules and their subsequent decondensation upon exocytosis. Images FIGURE 1 FIGURE 4 PMID:1868152

  16. Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies.

    PubMed

    Dias, Felipe F; Amaral, Kátia B; Carmo, Lívia A S; Shamri, Revital; Dvorak, Ann M; Weller, Peter F; Melo, Rossana C N

    2014-06-01

    Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.

  17. Three-Dimensional Tracking of Single Granules in Living PC-12 Cells Employing TIRFM and WFFM.

    PubMed

    Xiong, Jun; Li, Dongdong; Zhu, Dan; Qu, Anlian

    2005-01-01

    A comparative study was carried out on evaluating the performance of total internal reflection fluorescence microscopy (TIRFM) and deconvolution wide-field fluorescence microscopy (WFFM) in tracking single secretory granules. Both techniques have been applied to follow the three-dimensional mobility of single secretory granules in living neuroendocrine PC-12 cells. Both techniques return the similar result that most acridine orange-labeled granules were found to travel in random and caged diffusion, and only a small fraction of granules traveled in directed diffusion. Furthermore, the size and 3-D diffusion coefficient of secretory granules, obtained by these two imaging techniques, yield the same value. Together, our results demonstrate the potential of the combination TIRFM and WFFM in tracking long-termed motion of granules throughout live whole cells.

  18. A Nibbling Mechanism for Clathrin-mediated Retrieval of Secretory Granule Membrane after Exocytosis*

    PubMed Central

    Bittner, Mary A.; Aikman, Rachel L.; Holz, Ronald W.

    2013-01-01

    Clathrin-mediated endocytosis is the major pathway for recycling of granule membrane components after strong stimulation and high exocytotic rates. It resembles “classical” receptor-mediated endocytosis but has a trigger that is unique to secretion, the sudden appearance of the secretory granule membrane in the plasma membrane. The spatial localization, the relationship to individual fusion events, the nature of the cargo, and the timing and nature of the nucleation events are unknown. Furthermore, a size mismatch between chromaffin granules (∼300-nm diameter) and typical clathrin-coated vesicles (∼90 nm) makes it unlikely that clathrin-mediated endocytosis internalizes as a unit the entire fused granule membrane. We have used a combination of total internal reflection fluorescence microscopy of transiently expressed proteins and time-resolved quantitative confocal imaging of endogenous proteins along with a fluid-phase marker to address these issues. We demonstrate that the fused granule membrane remains a distinct entity and serves as a nucleation site for clathrin- and dynamin-mediated endocytosis that internalizes granule membrane components in small increments. PMID:23386611

  19. The econobiology of pancreatic acinar cells granule inventory and the stealthy nano-machine behind it.

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2016-03-01

    The pancreatic gland secretes most of the enzymes and many other macromolecules needed for food digestion in the gastrointestinal tract. These molecules play an important role in digestion, host defense and lubrication. The secretion of pancreatic proteins ensures the availability of the correct mix of proteins when needed. This review describes model systems available for the study of the econobiology of secretory granule content. The secretory pancreatic molecules are stored in large dense-core secretory granules that may undergo either constitutive or evoked secretion, and constitute the granule inventory of the cell. It is proposed that the Golgi complex functions as a distribution center for secretory proteins in pancreatic acinar cells, packing the newly formed secretory molecules into maturing secretory granules, also known functionally as condensing vacuoles. Mathematical modelling brings forward a process underlying granule inventory maintenance at various physiological states of condensation and aggregation by homotypic fusion. These models suggest unique but simple mechanisms accountable for inventory buildup and size, as well as for the distribution of secretory molecules into different secretory pathways in pancreatic acinar cells.

  20. A Dynamic Analysis of Secretory Granules Containing Proteins Involved In Learning

    NASA Astrophysics Data System (ADS)

    Prahl, Louis; Simon, Alex; Jacobs, Conor; Fulwiler, Audrey; Hilken, Lindsay; Scalettar, Bethe; Lochner, Janis

    2010-10-01

    Formation and encoding of long-term memories requires a series of structural changes at synapses, or sites of neuronal communication, in the hippocampus; these changes are mediated by neuromodulatory proteins and serve to strengthen synapses to improve communication. Two prominent neuromodulators, tissue plasminogen activator (tPA) and brain-derived neurotrophic factor (BDNF), are copackaged into secretory granules (SGs) in the body of nerve cells and are transported to distal synapses by motor proteins. At synapses, particularly presynaptic sites, the fate of tPA and BDNF is largely unknown. Motivated by this, and by recent data implicating presynaptic BDNF in early phases of learning, we used fluorescence microscopy to elucidate dynamic properties of presynaptic tPA and BDNF. We find that presynaptic SGs containing tPA and/or BDNF undergo Brownian and anomalous diffusive motion that, in 75% of cases, is so slow that it typically would be classified as immobility. These results suggest that tPA and BDNF are retained at presynaptic sites to facilitate their corelease and role in learning.

  1. Targeted ablation of the chromogranin a (Chga) gene: normal neuroendocrine dense-core secretory granules and increased expression of other granins.

    PubMed

    Hendy, Geoffrey N; Li, Tong; Girard, Martine; Feldstein, Richard C; Mulay, Shree; Desjardins, Roxane; Day, Robert; Karaplis, Andrew C; Tremblay, Michel L; Canaff, Lucie

    2006-08-01

    Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.

  2. Ca2+ dynamics in the secretory vesicles of neurosecretory PC12 and INS1 cells.

    PubMed

    SantoDomingo, Jaime; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Moreno, Alfredo; Alvarez, Javier

    2010-11-01

    We have investigated the dynamics of the free [Ca(2+)] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca(2+)-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca(2+)] ([Ca(2+)](SG)] was around 20-40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca(2+) pumps with thapsigargin largely blocked Ca(2+) uptake by the granules in Ca(2+)-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca(2+) pump inhibitor benzohydroquinone induced a rapid release of Ca(2+) from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca(2+) pumps is essential to maintain the steady-state [Ca(2+)](SG). Both inositol 1,4,5-trisphosphate (InsP(3)) and caffeine produced a rapid Ca(2+) release from the granules, suggesting the presence of InsP(3) and ryanodine receptors in the granules. The response to high-K(+) depolarization was different in both cell types, a decrease in [Ca(2+)](SG) in PC12 cells and an increase in [Ca(2+)](SG) in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca(2+)](SG) in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca(2+) through SERCA-type Ca(2+) pumps and can release it through InsP(3) and ryanodine receptors, supporting the hypothesis that secretory granule Ca(2+) may be released during cell stimulation and contribute to secretion.

  3. Molecular mechanism of myosin Va recruitment to dense core secretory granules.

    PubMed

    Brozzi, Flora; Diraison, Frederique; Lajus, Sophie; Rajatileka, Shavanthi; Philips, Thomas; Regazzi, Romano; Fukuda, Mitsunori; Verkade, Paul; Molnár, Elek; Váradi, Anikó

    2012-01-01

    The brain-spliced isoform of Myosin Va (BR-MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide-producing cells. The molecular composition of the protein complex that recruits BR-MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG-associated proteins granuphilin-a/b (Gran-a/b), BR-MyoVa and Rab27a, a member of the Rab family of GTPases. Gran-a/b-BR-MyoVa interaction is direct, involves regions downstream of the Rab27-binding domain, and the C-terminal part of Gran-a determines exon specificity. MyoVa and Gran-a/b are partially colocalised on SGs and disruption of Gran-a/b-BR-MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient-stimulated hormone secretion in pancreatic beta-cells. These results indicate the existence of at least another binding partner of BR-MyoVa that was identified as rabphilin-3A (Rph-3A). BR-MyoVa-Rph-3A interaction is also direct and enhanced when secretion is activated. The BR-MyoVa-Rph-3A and BR-MyoVa-Gran-a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR-MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.

  4. Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway.

    PubMed

    Braun, Laurence; Travier, Laetitia; Kieffer, Sylvie; Musset, Karine; Garin, Jérôme; Mercier, Corinne; Cesbron-Delauw, Marie-France

    2008-01-01

    Dense granules are Apicomplexa specific secretory organelles. In Toxoplasma gondii, the dense granules proteins, named GRA proteins, are massively secreted into the parasitophorous vacuole (PV) shortly after invasion. Despite the presence of hydrophobic membrane segments, they are stored as both soluble and aggregated forms within the dense granules and are secreted as soluble forms into the vacuolar space where they further stably associate with PV membranes. In this study, we explored the unusual biochemical behavior of GRA proteins during their trafficking. Conventional chromatography indicated that the GRA proteins form high globular weight complexes within the parasite. To confirm these results, DeltaGRA knocked-out parasites were stably complemented with their respective HA-FLAG tagged GRA2 or GRA5. Purification of the tagged proteins by affinity chromatography showed that within the parasite and the PV soluble fraction, both the soluble GRA2-HA-FLAG and GRA5-HA-FLAG associate with several GRA proteins, the major ones being GRA3, GRA6 and GRA7. Following their insertion into the PV membranes, GRA2-HA-FLAG associated with GRA5 and GRA7 while GRA5-HA-FLAG associated with GRA7 only. Taken together, these data suggest that the GRA proteins form oligomeric complexes that may explain their solubility within the dense granules and the vacuolar matrix by sequestering their hydrophobic domains within the interior of the complex. Insertion into the PV membranes correlates with the decrease of the GRA partners number.

  5. PC1/3, PC2 and PC5/6A are targeted to dense core secretory granules by a common mechanism.

    PubMed

    Dikeakos, Jimmy D; Mercure, Chantal; Lacombe, Marie-Josée; Seidah, Nabil G; Reudelhuber, Timothy L

    2007-08-01

    There are seven members of the proprotein convertase (PC) family of secreted serine proteases that cleave their substrates at basic amino acids, thereby activating a variety of hormones, growth factors, and viruses. PC1/3, PC2 and PC5/6A are the only members of the PC family that are targeted to dense core secretory granules, where they carry out the processing of proteins that are secreted from the cell in a regulated manner. Previous studies have identified alpha-helices in the C-termini of the PC1/3 and PC2 proteases that are required for this subcellular targeting. In the current study, we demonstrate that a predicted alpha-helix in the C-terminus of PC5/6A is also critical for the ability of this domain to target a heterologous protein to the regulated secretory pathway of mouse endocrine AtT-20 cells. Analysis of the subcellular distribution of fusion proteins containing the C-terminal domains of PC1/3, PC2 and PC5/6A confirmed that all three domains have the capacity to redirect a constitutively secreted protein to the granule-containing cytoplasmic extensions. Analysis of the predicted structures formed by these three granule-sorting helices shows a correlation between their granule-sorting efficiency and the clustering of hydrophobic amino acids in their granule-targeting helices.

  6. Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

    PubMed Central

    González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

    2013-01-01

    Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

  7. Prolactin and growth hormone aggregates in secretory granules: the need to understand the structure of the aggregate.

    PubMed

    Dannies, Priscilla S

    2012-04-01

    Prolactin and GH form reversible aggregates in the trans-Golgi lumen that become the dense cores of secretory granules. Aggregation is an economical means of sorting, because self-association removes the hormones from other possible pathways. Secretory granules containing different aggregates show different behavior, such as the reduction in stimulated release of granules containing R183H-GH compared with release of those containing wild-type hormone. Aggregates may facilitate localization of membrane proteins necessary for transport and exocytosis of secretory granules, and therefore understanding their properties is important. Three types of self-association have been characterized: dimers of human GH that form with Zn(2+), low-affinity self-association of human prolactin caused by acidic pH and Zn(2+) with macromolecular crowding, and amyloid fibers of prolactin. The best candidate for the form in most granules may be low-affinity self-association because it occurs rapidly at Zn(2+) concentrations that are likely to be in granules and reverses rapidly in neutral pH. Amyloid may form in older granules. Determining differences between aggregates of wild type and those of R183H-GH should help to understand why granules containing the mutant behave differently from those containing wild-type hormone. If reversible aggregation of other hormones, including those that are proteolytically processed, is the crucial act in forming granules, rather than use of a sorting signal, then prohormones should form reversible aggregates in solution in conditions that resemble those of the trans-Golgi lumen, including macromolecular crowding.

  8. Independent transport and sorting of functionally distinct protein families in Tetrahymena thermophila dense core secretory granules.

    PubMed

    Rahaman, Abdur; Miao, Wei; Turkewitz, Aaron P

    2009-10-01

    Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, DeltaGRT1 DeltaGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in DeltaGRT1 DeltaGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from DeltaGRT1 DeltaGRT2 cells appear less adhesive than those from the wild type.

  9. Platelet Granule Exocytosis: A Comparison with Chromaffin Cells

    PubMed Central

    Fitch-Tewfik, Jennifer L.; Flaumenhaft, Robert

    2013-01-01

    The rapid secretion of bioactive amines from chromaffin cells constitutes an important component of the fight or flight response of mammals to stress. Platelets respond to stresses within the vasculature by rapidly secreting cargo at sites of injury, inflammation, or infection. Although chromaffin cells derive from the neural crest and platelets from bone marrow megakaryocytes, both have evolved a heterogeneous assemblage of granule types and a mechanism for efficient release. This article will provide an overview of granule formation and exocytosis in platelets with an emphasis on areas in which the study of chromaffin cells has influenced that of platelets and on similarities between the two secretory systems. Commonalities include the use of transporters to concentrate bioactive amines and other cargos into granules, the role of cytoskeletal remodeling in granule exocytosis, and the use of granules to provide membrane for cytoplasmic projections. The SNAREs and SNARE accessory proteins used by each cell type will also be considered. Finally, we will discuss the newly appreciated role of dynamin family proteins in regulated fusion pore formation. This evaluation of the comparative cell biology of regulated exocytosis in platelets and chromaffin cells demonstrates a convergence of mechanisms between two disparate cell types both tasked with responding rapidly to physiological stimuli. PMID:23805129

  10. The organization of the secretory machinery in chromaffin cells as a major factor in modeling exocytosis

    PubMed Central

    Villanueva, José; Torregrosa-Hetland, Cristina J.; Gil, Amparo; González-Vélez, Virginia; Segura, Javier; Viniegra, Salvador; Gutiérrez, Luis M.

    2010-01-01

    The organization of cytoplasm in excitable cells was a largely ignored factor when mathematical models were developed to understand intracellular calcium and secretory behavior. Here we employed a combination of fluorescent evanescent and transmitted light microscopy to explore the F-actin cytoskeletal organization in the vicinity of secretory sites in cultured bovine chromaffin cells. This technique and confocal fluorescent microscopy show chromaffin granules associated with the borders of cortical cytoskeletal cages forming an intricate tridimensional network. Furthermore, the overexpression of SNAP-25 in these cells also reveals the association of secretory machinery clusters with the borders of these cytoskeletal cages. The importance of these F-actin cage borders is stressed when granules appear to interact and remain associated during exocytosis visualized in acridin orange loaded vesicles. These results will prompt us to propose a model of cytoskeletal cages, where the secretory machinery is associated with its borders. Both the calcium level and the secretory response are enhanced in this geometrical arrangement when compared with a random distribution of the secretory machinery that is not restricted to the borders of the cage. PMID:20885775

  11. Thrombopoietin-induced Dami cells as a model for alpha-granule biogenesis.

    PubMed

    Briquet-Laugier, Véronique; El Golli, Nargès; Nurden, Paquita; Lavenu-Bombled, Cécile; Dubart-Kupperschmitt, Anne; Nurden, Alan; Rosa, Jean-Philippe

    2004-09-01

    Megakaryocytic alpha-granules contain secretory proteins relevant to megakaryocyte and platelet functions. Understanding alpha-granule biogenesis is hampered because human primary megakaryocytes are difficult to manipulate. Existing promegakaryocytic cell lines do not spontaneously exhibit mature alpha-granules. Dami cells, transfected with the megakaryocytic platelet factor 4, fused to GFP (PF4-GFP), were induced in the presence of thrombopoietin (TPO), a megakaryocyte cytokine and PMA. Using confocal microscopy, PF4-GFP colocalized with von Willebrand Factor (vWF) in newly formed storage granules. Immunoelectron microscopy demonstrated alpha-granule-like features, including a dense core or parallel tubules and colocalization of PF4-GFP and vWF. Hence, TPO-treated Dami cells are a suitable model to study alpha-granules and their biogenesis.

  12. The stealthy nano-machine behind mast cell granule size distribution.

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2015-01-01

    The classical model of mast cell secretory granule formation suggests that newly synthesized secretory mediators, transported from the rough endoplasmic reticulum to the Golgi complex, undergo post-transitional modification and are packaged for secretion by condensation within membrane-bound granules of unit size. These unit granules may fuse with other granules to form larger granules that reside in the cytoplasm until secreted. A novel stochastic model for mast cell granule growth and elimination (G&E) as well as inventory management is presented. Resorting to a statistical mechanics approach in which SNAP (Soluble NSF Attachment Protein) REceptor (SNARE) components are viewed as interacting particles, the G&E model provides a simple 'nano-machine' of SNARE self-aggregation that can perform granule growth and secretion. Granule stock is maintained as a buffer to meet uncertainty in demand by the extracellular environment and to serve as source of supply during the lead time to produce granules of adaptive content. Experimental work, mathematical calculations, statistical modeling and a rationale for the emergence of nearly last-in, first out inventory management, are discussed.

  13. BDNF-GFP containing secretory granules are localized in the vicinity of synaptic junctions of cultured cortical neurons.

    PubMed

    Haubensak, W; Narz, F; Heumann, R; Lessmann, V

    1998-06-01

    The protein family of mammalian neurotrophins, comprising nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 and -4/5 (NT-3, NT-4/5), supports the survival and the phenotype of neurons from the central as well as the peripheral nervous system (CNS, PNS). In addition, exogenous application of neurotrophins has recently been found to modulate synaptic transmission in the rodent CNS. However, to provide evidence for a role of neurotophins as endogenous fast acting modulators of synaptic transmission, the synaptic localization and secretion of neurotrophins needs to be shown. We have now constructed a fusion protein consisting of N-terminal BDNF (the most abundant neurotrophin in the rodent hippocampus and neocortex) and C-terminal green fluorescent protein (GFP) to elucidate the cellular localization of BDNF in cortical neurons. Transient expression of BDNF-GFP in COS-7 cells revealed that the cellular localization in the trans-Golgi network (TGN), the processing of precursor proteins and the secretion of mature BDNF-GFP is indistinguishable from the properties of untagged BDNF. Upon transient transfection of primary rat cortical neurons, BDNF-GFP was found in secretory granules of the regulated pathway of secretion, as indicated by colocalization with the secretory granule marker secretogranin II. BDNF-GFP vesicles were found in the neurites of transfected neurons with a pattern reminiscent of the localization of endogenous BDNF in untransfected cortical neurons. BDNF-GFP vesicles were found predominantly in the somatodendritic compartment of the neurons, whereas additional axonal localization was found less frequently. Immunocytochemical staining of synaptic terminals with synapsin I antibodies revealed that the density of BDNF-GFP vesicles is elevated in the vicinity of synaptic junctions, indicating that BDNF is localized appropriately to function as an acute modulator of synaptic transmission. These data suggest that BDNF-GFP will

  14. Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

    PubMed Central

    Gabel, Marion; Delavoie, Franck; Demais, Valérie; Royer, Cathy; Bailly, Yannick; Vitale, Nicolas; Bader, Marie-France

    2015-01-01

    Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites. PMID:26323692

  15. Stimulation of mast cells leads to cholesterol accumulation in macrophages in vitro by a mast cell granule-mediated uptake of low density lipoprotein

    SciTech Connect

    Kokkonen, J.O.; Kovanen, P.T.

    1987-04-01

    The uptake of low density lipoprotein (LDL) by cultured mouse macrophages was markedly promoted by isolated rat mast cell granules present in the culture medium. The granule-mediated uptake of /sup 125/I-LDL enhanced the rate of cholesteryl ester synthesis in the macrophages, the result being accumulation of cholesteryl esters in these cells. Binding of LDL to the granules was essential for the granule-mediated uptake of LDL by macrophages, for the uptake process was prevented by treating the granules with avidin or protamine chloride or by treating LDL with 1,2-cyclohexanedione, all of which inhibit the binding of LDL to the granules. Inhibition of granule phagocytosis by the macrophages with cytochalasin B also abolished the granule-mediated uptake of LDL. Finally, mouse macrophage monolayers and LDL were incubated in the presence of isolated rat serosal mast cells. Stimulation of the mast cells with compound 48/80, a degranulating agent, resulted in dose-dependent release of secretory granules from the mast cells and a parallel increase in /sup 14/C cholesteryl ester synthesis in the macrophages. The results show that, in this in vitro model, the sequence of events leading to accumulation of cholesteryl esters in macrophages involves initial stimulation of mast cells, subsequent release of their secretory granules, binding of LDL to the exocytosed granules, and, finally, phagocytosis of the LDL-containing granules by macrophages.

  16. Myosin 2 Maintains an Open Exocytic Fusion Pore in Secretory Epithelial Cells

    PubMed Central

    Bhat, Purnima

    2009-01-01

    Many studies have implicated F-actin and myosin 2 in the control of regulated secretion. Most recently, evidence suggests a role for the microfilament network in regulating the postfusion events of vesicle dynamics. This is of potential importance as postfusion behavior can influence the loss of vesicle content and may provide a new target for drug therapy. We have investigated the role of myosin 2 in regulating exocytosis in secretory epithelial cells by using novel assays to determine the behavior of the fusion pore in individual granules. We immunolocalize myosin 2A to the apical region of pancreatic acinar cells, suggesting it is this isoform that plays a role in granule exocytosis. We further show myosin 2 phosphorylation increased on cell stimulation, consistent with a regulatory role in secretion. Importantly, in a single-cell, single-granule secretion assay, neither the myosin 2 inhibitor (−)-blebbistatin nor the myosin light chain kinase inhibitor ML-9 had any effect on the numbers of granules stimulated to fuse after cell stimulation. These data indicate that myosin 2, if it has any action on secretion, must be targeting postfusion granule behavior. This interpretation is supported by direct study of fusion pore opening in which we show that (−)-blebbistatin and ML-9 promote fusion pore closure and decrease fusion pore lifetimes. Our work now adds to a growing body of evidence showing that myosin 2 is an essential regulator of postfusion granule behavior. In particular, in the case of the secretory epithelial cells, myosin 2 activity is necessary to maintain fusion pore opening. PMID:19158378

  17. Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

    PubMed

    Yokawa, Satoru; Furuno, Tadahide; Suzuki, Takahiro; Inoh, Yoshikazu; Suzuki, Ryo; Hirashima, Naohide

    2016-09-01

    Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 μm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 μm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity.

  18. In vitro conditions modify immunoassayability of bovine pituitary prolactin and growth hormone: insights into their secretory granule storage forms

    SciTech Connect

    Lorenson, M.Y.

    1985-04-01

    The amount of immunoassayable intracellular bovine (b) PRL and GH varies depending on treatment conditions. The present studies were designed to characterize the mechanisms involved and to compare immunoassayability of both hormones under similar conditions. Pituitary homogenate and secretory granule hormones displayed both time- and temperature-dependent increases when incubated at pH 10.5 with reduced glutathione. Changes in immunoassayability seem to reflect conversion from poorly immunoactive tissue hormone oligomers to monomeric hormone. The data indicate that oligomeric bPRL is stabilized primarily by intermolecular disulfide bonds, although it is also susceptible to urea, SDS, and EDTA; granule thiols may also influence the conversion to monomer. The storage form of bGH appears to be stabilized differently. Maneuvers demonstrated in these studies to influence immunoassayability correlate very well with their previously established effects on hormone release and secretion, strengthening the likelihood that a functional link exists between assayability and secretion.

  19. Transient fusion ensures granule replenishment to enable repeated release after IgE-mediated mast cell degranulation.

    PubMed

    Balseiro-Gomez, Santiago; Flores, Juan A; Acosta, Jorge; Ramirez-Ponce, M Pilar; Ales, Eva

    2016-11-01

    To ensure normal immune function, mast cells employ different pathways to release mediators. Here, we report a thus far unknown capacity of mast cells to recycle and reuse secretory granules after an antigen-evoked degranulation process under physiological conditions; this phenomenon involves the existence of a recycling secretory granule pool that is available for release in a short time scale. Rapid endocytic modes contributed to the recycling of ∼60% of the total secretory granule population, which involved kiss-and-run and cavicapture mechanisms, causing retention of the intragranular matrix. We found the presence of normal-size granules and giant actomyosin- and dynamin-dependent granules, which were characterized by large quantal content. These large structures allowed the recovered mast cells to release a large amount of 5-HT, compensating for the decrease in the number of exocytosed secretory granules. This work uncovers a new physiological role of the exo-endocytosis cycle in the immunological plasticity of mast cells and reveals a new property of their biological secretion.

  20. Hippocampal granule cells opt for early retirement.

    PubMed

    Alme, C B; Buzzetti, R A; Marrone, D F; Leutgeb, J K; Chawla, M K; Schaner, M J; Bohanick, J D; Khoboko, T; Leutgeb, S; Moser, E I; Moser, M-B; McNaughton, B L; Barnes, C A

    2010-10-01

    Increased excitability and plasticity of adult-generated hippocampal granule cells during a critical period suggests that they may "orthogonalize" memories according to time. One version of this "temporal tag" hypothesis suggests that young granule cells are particularly responsive during a specific time period after their genesis, allowing them to play a significant role in sculpting CA3 representations, after which they become much less responsive to any input. An alternative possibility is that the granule cells active during their window of increased plasticity, and excitability become selectively tuned to events that occurred during that time and participate in later reinstatement of those experiences, to the exclusion of other cells. To discriminate between these possibilities, rats were exposed to different environments at different times over many weeks, and cell activation was subsequently assessed during a single session in which all environments were revisited. Dispersing the initial experiences in time did not lead to the increase in total recruitment at reinstatement time predicted by the selective tuning hypothesis. The data indicate that, during a given time frame, only a very small number of granule cells participate in many experiences, with most not participating significantly in any. Based on these and previous data, the small excitable population of granule cells probably correspond to the most recently generated cells. It appears that, rather than contributing to the recollection of long past events, most granule cells, possibly 90-95%, are effectively "retired." If granule cells indeed sculpt CA3 representations (which remains to be shown), then a possible consequence of having a new set of granule cells participate when old memories are reinstated is that new representations of these experiences might be generated in CA3. Whatever the case, the present data may be interpreted to undermine the standard "orthogonalizer" theory of the role of

  1. The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin.

    PubMed

    Borisova, Tatiana; Kasatkina, Ludmila; Ostapchenko, Ludmila

    2011-11-01

    Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na(+)-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated platelets with cholesterol-depleting agent methyl-β-cyclodextrin (MβCD). Confocal imaging with the cholesterol-sensitive fluorescent dye filipin showed a quick reduction of cholesterol level in platelets. Using pH-sensitive fluorescent dye acridine orange, we demonstrated that the acidification of secretory granules of human and rabbit platelets was decreased by ∼15% and 51% after the addition of 5 and 15mM MβCD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex MβCD-cholesterol (1:0.2) led to the additional accumulation of acridine orange in secretory granules indicating an increase in the proton pumping activity of vesicular H(+)-ATPase. MβCD did not evoke release of glutamate from platelets that was measured with glutamate dehydrogenase assay. Flow cytometric analysis did not reveal alterations in platelet size and granularity in the presence of MβCD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused MβCD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of MβCD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets.

  2. Ca-dependent Nonsecretory Vesicle Fusion in a Secretory Cell

    PubMed Central

    Wang, Tzu-Ming; Hilgemann, Donald W.

    2008-01-01

    We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2–4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100–200 μM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P2) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P2, PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII−/− mouse embryonic fibroblasts (MEFs), as well as in PLCδ1, PLC δ1/δ4, and PLCγ1−/− MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (KD ∼71 μM) than expected for these C2 domain–containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP

  3. Studies on the pH gradient and histamine uptake of isolated mast cell granules

    SciTech Connect

    De Young, M.B.; Nemeth, E.F.; Scarpa, A.

    1986-05-01

    A purified preparation of mast cell granules with intact perigranular membranes was obtained using a method involving probe sonication of rat serosal mast cells followed by differential centrifugation and Percoll gradient separation of the granules. Purification was assessed with histamine and mast cell granule protease assays. Granule integrity was demonstrated by light and electron microscopy and quantitated with a ruthenium red binding assay. The low yield of granules (20 ..mu..g protein/4 rats) necessitated the development of two microanalytical techniques to demonstrate the existence of a pH gradient across the membrane: 9-aminoacridine fluorescence studies in a cuvet with 50 ..mu..l capacity and /sup 14/C-methylamine distribution studies on microgram quantities of granule protein. Quantitation of results from isotope studies were confounded by the presence of oil used for separating granules from the aqueous phase. Nonetheless, an extrapolation procedure calibrated by external pH yielded an internal pH value of 5.46 +/- .03 (n = 4), consistent with values observed in granules obtained from other secretory cells. Collapse of the pH gradient by NH/sub 4//sup +/ or nigericin/KCl was demonstrated using either technique. Addition of histamine depressed intragranular pH, suggesting that histamine transport may utilize the ..delta..pH as a driving force.

  4. Ectopic Granule Cells of the Rat Dentate Gyrus

    PubMed Central

    Scharfman, Helen; Goodman, Jeffrey; McCloskey, Daniel

    2007-01-01

    Granule cells of the mammalian dentate gyrus normally form a discrete layer, and virtually all granule cells migrate to this location. Exceptional granule cells that are positioned incorrectly, in ‘ectopic’ locations, are rare. Although the characteristics of such ectopic granule cells appear similar in many respects to granule cells located in the granule cell layer, their rare occurrence has limited a full evaluation of their structure and function. More information about ectopic granule cells has been obtained by studying those that develop after experimental manipulations that increase their number. For example, after severe seizures, the number of ectopic granule cells located in the hilus increases dramatically. These experimentally induced ectopic granule cells may not be equivalent to normal ectopic granule cells necessarily, but the vastly increased numbers have allowed much more information to be obtained. Remarkably, the granule cells that are positioned ectopically develop intrinsic properties and an axonal projection that are similar to granule cells that are located normally, i.e., in the granule cell layer. However, dendritic structure and synaptic structure/function appear to differ. These studies have provided new insight into a rare type of granule cell in the dentate gyrus, and the plastic characteristics of dentate granule cells that appear to depend on the location of the cell body. PMID:17148946

  5. Tracheobronchial epithelium of the sheep: IV. Lectin histochemical characterization of secretory epithelial cells.

    PubMed

    Mariassy, A T; Plopper, C G; St George, J A; Wilson, D W

    1988-09-01

    Conventional histochemical characterization of the mucus secretory apparatus is often difficult to reconcile with the biochemical analysis of respiratory secretions. This study was designed to examine the secretory glycoconjugates in airways using lectins with biochemically defined affinities for main sugar residues of mucus. We used five biotinylated lectins--DBA (Dolichos biflorus) and SBA (Glycine max) for N-acetyl galactosamine (galNAc), BSA I (Bandeiraea simplicifolia) and PNA (Arachis hypogea) for galactose (gal), and UEA I (Ulex europeus)--for detection of fucose (fuc) in HgCl2-fixed, paraffin-embedded, serially sectioned trachea, lobar and segmental bronchi and bronchioles of nine sheep. Lectins selectively localized the carbohydrate residues in luminal secretions, on epithelial cell surfaces, and in secretory cells. In proximal airways, the major carbohydrate residues in luminal secretions, cell surfaces, goblet cells, and glands were fuc and gal-NAc. PNA reacted mainly with apical granules of less than 10% of goblet cells, and gal residues were only detected in some of the mucous cells and on basolateral cell surfaces. Distal airways contained sparse secretion in the lumen, mucous cells contained weakly reactive fuc and gal-NAc, and the epithelial surfaces of Clara cells contained gal. Sugars abundant in the airway secretions were also the major component of cells in glands. We conclude that there is a correlation between specific sugar residues in secretory cells, glycocalyx, and luminal secretions in proximal and distal airways. This suggests that lectins may be used to obtain information about airway secretory cell composition from respiratory secretions.

  6. Mechanisms of granule membrane recapture following exocytosis in intact mast cells.

    PubMed

    Cabeza, Jose M; Acosta, Jorge; Alés, Eva

    2013-07-12

    In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified "large-capacitance flickers" that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent "compound cavicapture," most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.

  7. Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells*

    PubMed Central

    Cabeza, Jose M.; Acosta, Jorge; Alés, Eva

    2013-01-01

    In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified “large-capacitance flickers” that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent “compound cavicapture,” most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event. PMID:23709219

  8. Calelectrin, a calcium-dependent membrane-binding protein associated with secretory granules in Torpedo cholinergic electromotor nerve endings and rat adrenal medulla.

    PubMed

    Walker, J H; Obrocki, J; Südhof, T C

    1983-07-01

    Calelectrin, a calcium-dependent membrane-binding protein of subunit molecular weight 32,000 has been isolated from the electric organ of Torpedo, and shown to occur in cholinergic neurones and in bovine adrenal medulla. In this study a monospecific antiserum against the Torpedo protein has been used to study the localization of calelectrin in the rat adrenal gland. The cortex was not stained, whereas in the medulla the cytoplasm of the chromaffin cells was stained in a particulate manner. An identical staining pattern was obtained with an antiserum against the chromaffin granule enzyme dopamine beta-hydroxylase, although the two antisera did not cross-react with the same antigen. The purified protein aggregates bovine chromaffin granule membranes and cholinergic synaptic vesicles and also self aggregates in a calcium-dependent manner. Negative staining results demonstrate that calcium induces a transformation of the purified protein from circular structures 30-80 nm in diameter into a highly aggregated structure. Calelectrin may have a structural or regulatory role in the intracellular organization of secretory cells.

  9. The effect of androgen and estrogen on secretory epithelial cells and basal cells of the rat ventral prostate after long-term castration.

    PubMed

    Kawamura, H; Kimura, M; Ichihara, I

    1993-12-01

    After long-term castration, rats were injected with cotton seed oil, testosterone- and estradiol-17 beta-cypionate (CS, TC and EC). The height of the epithelial cells of the ventral prostates from the castrated rats increased after TC and EC-injection. The secretory and basal cells formed two layers of epithelium, an inner layer near the lumen with pale nuclei and another layer with dark nuclei. These two layers could result from a reduction of secretory epithelial cells. Castration decreased the ratio of secretory cells to basal cells (S/B). TC-injection increased the ratio of S/B because of the secretory epithelial cell growth. Longer dark cells may be transient cells, appearing during the differentiation of basal cells into secretory epithelial cells. A sheet branching off from the basal lamina was observed. Androgen may stimulate the synthesis of the lamina, but whether it induces the synthesis or turnover of the basal lamina has not been established. EC increased the ventral prostatic weight and secretory epithelial cell height and induced the appearance of crystalline granules. Increase in S/B ratio may result from an increase in the secretory epithelial cells, but not from basal cell multiplication due to squamous metaplasia. The ratio is significantly correlated to the weight of the ventral prostate, but not to the secretory epithelial cell height. Its value could indicate the multiplication of secretory epithelial cells, differentiation of basal cells into epithelial cells, or both. It is probable that basal cells do not change in number, but control the size of the rat ventral prostate in response to the hormone level.

  10. Orai-STIM–mediated Ca2+ release from secretory granules revealed by a targeted Ca2+ and pH probe

    PubMed Central

    Dickson, Eamonn J.; Duman, Joseph G.; Moody, Mark W.; Chen, Liangyi; Hille, Bertil

    2012-01-01

    Secretory granules (SGs) sequester significant calcium. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty of measuring SG calcium directly in living cells. We adapted the Förster resonance energy transfer-based D1-endoplasmic reticulum (ER) probe to develop a unique probe (D1-SG) to measure calcium and pH in secretory granules. It significantly localizes to SGs and reports resting free Ca2+ of 69 ± 15 μM and a pH of 5.8. Application of extracellular ATP to activate P2Y receptors resulted in a slow monotonic decrease in SG Ca2+ temporally correlated with the occurrence of store-operated calcium entry (SOCE). Further investigation revealed a unique receptor-mediated mechanism of calcium release from SGs that involves SG store-operated Orai channels activated by their regulator stromal interaction molecule 1 (STIM1) on the ER. SG Ca2+ release is completely antagonized by a SOCE antagonist, by switching to Ca2+-free medium, and by overexpression of a dominant-negative Orai1(E106A). Overexpression of the CRAC activation domain (CAD) of STIM1 resulted in a decrease of resting SG Ca2+ by ∼75% and completely abolished the ATP-mediated release of Ca2+ from SGs. Overexpression of a dominant-negative CAD construct (CAD-A376K) induced no significant changes in SG Ca2+. Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels and that during SG Ca2+ release, colocalization between SGs and STIM1 increases. We propose Orai channel opening on SG membranes as a potential mode of calcium release from SGs that may serve to raise local cytoplasmic calcium concentrations and aid in refilling intracellular calcium stores of the ER and exocytosis. PMID:23184982

  11. Selective nucleotide-release from dense-core granules in insulin-secreting cells.

    PubMed

    Obermüller, Stefanie; Lindqvist, Anders; Karanauskaite, Jovita; Galvanovskis, Juris; Rorsman, Patrik; Barg, Sebastian

    2005-09-15

    Secretory granules of insulin-secreting cells are used to store and release peptide hormones as well as low-molecular-weight compounds such as nucleotides. Here we have compared the rate of exocytosis with the time courses of nucleotide and peptide release by a combination of capacitance measurements, electrophysiological detection of ATP release and single-granule imaging. We demonstrate that the release of nucleotides and peptides is delayed by approximately 0.1 and approximately 2 seconds with respect to membrane fusion, respectively. We further show that in up to 70% of the cases exocytosis does not result in significant release of the peptide cargo, likely because of a mechanism that leads to premature closure of the fusion pore. Release of nucleotides and protons occurred regardless of whether peptides were secreted or not. These observations suggest that insulin-secreting cells are able to use the same secretory vesicles to release small molecules either alone or together with the peptide hormone.

  12. Beta-agonists and secretory cell number and intracellular glycoproteins in airway epithelium. The effect of isoproterenol and salbutamol.

    PubMed Central

    Jones, R.; Reid, L.

    1979-01-01

    This study describes the effect of systemic administration of the beta-adrenergic agonists isoproterenol and salbutamol on the secretory cell populations in seven regions of rat airway epithelium (three extrapulmonary and four intrapulmonary) and on the size of salivary glands and heart. Isoproterenol (a nonselective beta-adrenergic agonist) significantly increases secretory cell number in all airway regions except the midtrachea; salbutamol (a selective beta 2 agonist) increases secretory cell number only in proximal and peripheral regions. The absolute number of secretory cells is greatest in the most peripheral region after isoproterenol administration and in the most proximal region after salbutamol, although both drugs produce the greatest relative increase at the periphery. In proximal and, particularly, peripheral regions, the increase by isoproterenol (less than 3- and 14-fold, respectively) is greater than by salbutamol (less than 2- and less than 3-fold, respectively). In all airway regions, both drugs modify intracellular glycoprotein in the secretory cell population; within a given region, modification is much the same. In the most proximal region, the population of cells synthesizing only granules of neutral glycoprotein significantly increases while in other regions increase is in cells synthesizing only granules of acid. A significant shift in glycoprotein synthesis occurs whether or not the secretory cell population is increased, which suggests that existing as well as newly appearing cells modify their product. Isoproterenol significantly increases the size of the parotid and submaxillary glands; salbutamol increases the size of the parotid only. Isoproterenol significantly increases the weight of both ventricles of the heart; salbutamol has no such effect. PMID:36762

  13. Two modes of lytic granule fusion during degranulation by natural killer cells.

    PubMed

    Liu, Dongfang; Martina, Jose A; Wu, Xufeng S; Hammer, John A; Long, Eric O

    2011-08-01

    Lytic granules in cytotoxic lymphocytes, which include T cells and natural killer (NK) cells, are secretory lysosomes that release their content upon fusion with the plasma membrane (PM), a process known as degranulation. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Here, we used total internal reflection fluorescence microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand (FasL) in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptor-ligand interactions. Two fusion modes were observed: complete fusion, characterized by loss of granule content and rapid diffusion of FasL at the PM; and incomplete fusion, characterized by transient fusion pore opening and retention of FasL at the fusion site. The pH-sensitive green fluorescence protein (pHluorin) fused to the lumenal domain of FasL was used to visualize fusion pore opening with a time resolution of 30 ms. Upon incomplete fusion, pHluorin emission lasted several seconds in the absence of noticeable diffusion. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the PM, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.

  14. Vaccine adjuvants: Tailor-made mast-cell granules

    NASA Astrophysics Data System (ADS)

    Gunzer, Matthias

    2012-03-01

    Mast cells induce protective immune responses through secretion of stimulatory granules. Microparticles modelled after mast-cell granules are now shown to replicate and enhance the functions of their natural counterparts and to direct the character of the resulting immunity.

  15. Monoclonal Antibodies as Probes for Unique Antigens in Secretory Cells of Mixed Exocrine Organs

    NASA Astrophysics Data System (ADS)

    Basbaum, C. B.; Mann, J. K.; Chow, A. W.; Finkbeiner, W. E.

    1984-07-01

    In the past, it has been difficult to identify the secretory product and control mechanisms associated with individual cell types making up mixed exocrine organs. This report establishes the feasibility of using immunological methods to characterize both the biochemical constituents and regulatory mechanisms associated with secretory cells in the trachea. Monoclonal antibodies directed against components of tracheal mucus were produced by immunizing mice with dialyzed, desiccated secretions harvested from tracheal organ culture. An immunofluorescence assay revealed that of the total 337 hybridomas screened, 100 produced antibodies recognizing goblet cell granules; 64, gland cell granules; and 3, antigen confined to the ciliated apical surface of the epithelium. The tracheal goblet cell antibody described in this report was strongly cross-reactive with intestinal goblet cells, as well as with a subpopulation of submandibular gland cells, but not with cells of Brunner's glands or the ciliated cell apical membrane. The serous cell antibody was not cross-reactive with goblet, Brunner's gland, or submandibular cells, or the ciliated cell apical membrane. The antibody directed against the apical membrane of ciliated cells did not cross-react with gland or goblet cells or the apical membrane of epithelial cells in the duodenum. Monoclonal antibodies, therefore, represent probes by which products unique to specific cells or parts of cells in the trachea can be distinguished. The antibodies, when used in enzyme immunoassays, can be used to quantitatively monitor secretion by individual cell types under a variety of physiological and pathological conditions. They also provide the means for purification and characterization of cell-specific products by immunoaffinity chromatography.

  16. Cell-specific analysis of tracheobronchial secretory cells and secretions

    SciTech Connect

    Finkbeiner, W.E.

    1989-01-01

    In these studies, two methods (cell culture and monoclonal antibody production) that allowed cell-specific analysis of tracheobronchial secretion were used. Bovine tracheal submucosal gland cells were isolated, placed into culture and serially propagated. In culture, the cells maintained features of serous cells. The cells incorporated {sup 35}S into high molecular weight molecules. {beta}-adrenergic agonists stimulated release of radiolabeled molecules and elevations in intracellular cAMP levels, responses that could be blocked by the {beta}-adrenergic antagonist propranolol. Cyclic AMP appeared to be involved in the stimulus-secretion coupling events in serous cells since the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine potentiated the effects of isoproterenol on the secretory response and the elevation of intracellular cAMP levels. Furthermore, cAMP analogues elicited a secretory response in the absence of cAMP. The phosphorylation state of several cytosolic and particulate phosphoproteins was altered by cAMP-activated kinase activity. Monoclonal antibodies were produced against human airway secretions.

  17. Isolation and characterization of secretory granules storing a vasoactive intestinal polypeptide-like peptide in Torpedo cholinergic electromotor neurones.

    PubMed

    Agoston, D V; Dowe, G H; Whittaker, V P

    1989-06-01

    Previous immunocytochemical work showed that the cholinergic electromotor neurones of Torpedo marmorata contain a vasoactive intestinal polypeptide-like immunoreactivity (VIPLI) that is conveyed to the terminals by axonal transport from the cell bodies where it is presumably synthesized. In extension of this work, we have now succeeded in isolating the VIPLI storage granules from both the terminals and the axons of these neurones and characterizing them morphologically and biochemically. They were readily separated from synaptic vesicles but contained several components in common that had previously been regarded as specific for synaptic vesicles. Among these were a heparan sulphate type of proteoglycan, synaptophysin, and a Mg2+-dependent ATPase. The VIPLI concentration in lobe tissue and the amount of tissue available were both insufficient to permit the isolation of granules from the electromotor cell bodies by the same technique but it was possible to establish the presence of such granules by particle-exclusion chromatography, using the stable markers mentioned above. In contrast to the VIPLI-containing granules, axonal synaptic vesicles differed from their terminal counterparts in having a very low acetylcholine content relative to stable vesicle markers: they presumably fill up on reaching the terminal where they are exposed to higher concentrations of cytoplasmic acetylcholine.

  18. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure

    PubMed Central

    LaSarge, Candi L.; Santos, Victor R; Danzer, Steve C.

    2015-01-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offset the reduction in boutons per axon length. These morphological changes predicts a net increase in granule cell >> CA3 innervation. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell >> CA3 communication. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. PMID:25600212

  19. Identification of a membrane-bound, glycol-stimulated phospholipase A sub 2 located in the secretory granules of the adrenal medulla

    SciTech Connect

    Hildebrandt, E.; Albanesi, J.P. )

    1991-01-01

    Chromaffin granule membranes prepared from bovine adrenal medullae showed Ca{sup 2+}-stimulated phospholipase A{sub 2} (PLA{sub 2}) activity when assayed at pH 9.0 with phosphatidylcholine containing an ({sup 14}C)-arachidonyl group in the 2-position. However, the activity occurred in both soluble and particulate subcellular fractions, and did not codistribute with markers for the secretory granule. PLA{sub 2} activity in the granule membrane preparation was stimulated dramatically by addition of glycerol, ethylene glycole, or poly(ethylene glycol). This glycol-stimulated PLA{sub 2} activity codistributed with membrane-bound dopamine {beta}-hydroxylase, a marker for the granule membranes, through the sequence of differential centrifugation steps employed to prepare the granule membrane fraction, as well as on a sucrose density gradient which resolved the granules from mitochondria, lysosomes, and plasma membrane. The glycol-stimulated PLA{sub 2} of the chromaffin granule was membrane-bound, exhibited a pH optimum of 7.8, retained activity in the presence of EDTA, and was inactivated by p-bromophenacyl bromide. When different {sup 14}C-labeled phospholipids were incorporated into diarachidonylphosphatidylcholine liposomes, 1-palmitoyl-2-arachidonylphosphatidylcholine was a better substrate for this enzyme than 1-palmitoyl-2-oleylphosphatidylcholine or 1-acyl-2-arachidonyl-phosphatidylethhanolamine, and distearoylphosphatidylcholine was not hydrolyzed.

  20. Single Granule Cells Excite Golgi Cells and Evoke Feedback Inhibition in the Cochlear Nucleus

    PubMed Central

    Yaeger, Daniel B.

    2015-01-01

    In cerebellum-like circuits, synapses from thousands of granule cells converge onto principal cells. This fact, combined with theoretical considerations, has led to the concept that granule cells encode afferent input as a population and that spiking in individual granule cells is relatively unimportant. However, granule cells also provide excitatory input to Golgi cells, each of which provide inhibition to hundreds of granule cells. We investigated whether spiking in individual granule cells could recruit Golgi cells and thereby trigger widespread inhibition in slices of mouse cochlear nucleus. Using paired whole-cell patch-clamp recordings, trains of action potentials at 100 Hz in single granule cells was sufficient to evoke spikes in Golgi cells in ∼40% of paired granule-to-Golgi cell recordings. High-frequency spiking in single granule cells evoked IPSCs in ∼5% of neighboring granule cells, indicating that bursts of activity in single granule cells can recruit feedback inhibition from Golgi cells. Moreover, IPSPs mediated by single Golgi cell action potentials paused granule cell firing, suggesting that inhibitory events recruited by activity in single granule cells were able to control granule cell firing. These results suggest a previously unappreciated relationship between population coding and bursting in single granule cells by which spiking in a small number of granule cells may have an impact on the activity of a much larger number of granule cells. PMID:25788690

  1. Immunocytochemical detection of beta-defensins and cathelicidins in the secretory granules of the tongue in the lizard Anolis carolinensis.

    PubMed

    Alibardi, Lorenzo

    2015-04-01

    Previous molecular studies indicated that antimicrobial peptides in lizard are expressed in the skin and tongue among other epithelial organs. The present ultrastructural immunogold study aimed to detect the specific location of three broadly expressed antimicrobial peptides in the tongue of the lizard Anolis carolinensis. The immunocytochemical study indicated that beta-defensin-15, the likely main defensin of granulocytes and skin, is poorly expressed in some dense and medium-dense granules of glandular cells of the papillated tongue. Conversely beta-defensin-27 appears highly expressed in numerous pale and cribriform dense granules of glandular cells and is also secreted on the tongue surface. The immunostaining for cathelicidin-1 indicated a variable but however positive immunolabeling in numerous granules in the tongue glands, suggesting that this antimicrobial peptide previously found on the epidermal surface is also present in the tongue secretions and participates to the formation of the anti-microbial oral barrier. The study suggests that among the numerous beta-defensins and cathelicidins identified in the genome of this lizard is present a specific distribution of different peptide subtypes in various body regions, including the tongue, and that these peptides contribute to the formation of local antimicrobial barriers.

  2. Secretory mechanism of fibroin, a silk protein, in the posterior silk gland cells of Bombyx mori.

    PubMed

    Sasaki, S; Nakagaki, I

    1980-01-01

    There are two microtubule-microfilament systems in the posterior silk gland cells of Bombyx mori. One is a radial microtubule system; the other is a circular microtubule-microfilament system. These two systems are presumably concerned with the intracellular transport of secretory granules of fibroin and the secretion of fibroin into the lumen, respectively. Conventional and scanning electron microscopic observations of the two microtubule-microfilament systems in the posterior silk gland cells are reported. Scanning electron micrographs showed that a number of parallel linear cytoplasmic processes ran circularly on the luminal surface of the posterior silk gland cells. These processes were assumed to correspond to the circular microtubule-microfilament systems. The effects of cytochalasin (B or D), a secretion stimulating agent of fibroin, on the intracellular recording of membrane potential from the posterior silk gland cells are also reported. Exposure to cytochalasin resulted in depolarization of the membrane potential of the gland cells. Possible functional roles of the two microtubule-microfilament systems in the secretory mechanism of fibroin are discussed with reference to the effects of antimitotic reagents and cytochalasin on these two systems.

  3. The Small GTPase RalA controls exocytosis of large dense core secretory granules by interacting with ARF6-dependent phospholipase D1.

    PubMed

    Vitale, Nicolas; Mawet, Jacques; Camonis, Jacques; Regazzi, Romano; Bader, Marie-France; Chasserot-Golaz, Sylvette

    2005-08-19

    RalA and RalB constitute a family of highly similar Ras-related GTPases widely distributed in different tissues. Recently, active forms of Ral proteins have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. Since RalA is present on the plasma membrane in neuroendocrine chromaffin and PC12 cells, we investigated the potential role of RalA in calcium-regulated exocytotic secretion. We show here that endogenous RalA is activated during exocytosis. Expression of the constitutively active RalA (G23V) mutant enhances secretagogue-evoked secretion from PC12 cells. Conversely, expression of the constitutively inactive GDP-bound RalA (G26A) or silencing of the RalA gene by RNA interference led to a strong impairment of the exocytotic response. RalA was found to co-localize with phospholipase D1 (PLD1) at the plasma membrane in PC12 cells. We demonstrate that cell stimulation triggers a direct interaction between RalA and ARF6-activated PLD1. Moreover, reduction of endogenous RalA expression level interfered with the activation of PLD1 observed in secretagogue-stimulated cells. Finally, using various RalA mutants selectively impaired in their ability to activate downstream effectors, we show that PLD1 activation is essential for the activation of secretion by GTP-loaded RalA. Together, these results provide evidence that RalA is a positive regulator of calcium-evoked exocytosis of large dense core secretory granules and suggest that stimulation of PLD1 and consequent changes in plasma membrane phospholipid composition is the major function RalA undertakes in calcium-regulated exocytosis.

  4. Secretory and basal cells of the epithelium of the tubular glands in the male Mullerian gland of the caecilian Uraeotyphlus narayani (Amphibia: Gymnophiona).

    PubMed

    George, Jancy M; Smita, Matthew; Kadalmani, Balamuthu; Girija, Ramankutty; Oommen, Oommen V; Akbarsha, Mohammad A

    2004-12-01

    Caecilians are exceptional among the vertebrates in that males retain the Mullerian duct as a functional glandular structure. The Mullerian gland on each side is formed from a large number of tubular glands connecting to a central duct, which either connects to the urogenital duct or opens directly into the cloaca. The Mullerian gland is believed to secrete a substance to be added to the sperm during ejaculation. Thus, the Mullerian gland could function as a male accessory reproductive gland. Recently, we described the male Mullerian gland of Uraeotyphlus narayani using light and transmission electron microscopy (TEM) and histochemistry. The present TEM study reports that the secretory cells of both the tubular and basal portions of the tubular glands of the male Mullerian gland of this caecilian produce secretion granules in the same manner as do other glandular epithelial cells. The secretion granules are released in the form of structured granules into the lumen of the tubular glands, and such granules are traceable to the lumen of the central duct of the Mullerian gland. This is comparable to the situation prevailing in the epididymal epithelium of several reptiles. In the secretory cells of the basal portion of the tubular glands, mitochondria are intimately associated with fabrication of the secretion granules. The structural and functional organization of the epithelium of the basal portion of the tubular glands is complicated by the presence of basal cells. This study suggests the origin of the basal cells from peritubular tissue leukocytes. The study also indicates a role for the basal cells in acquiring secretion granules from the neighboring secretory cells and processing them into lipofuscin material in the context of regression of the Mullerian gland during the period of reproductive quiescence. In these respects the basal cells match those in the epithelial lining of the epididymis of amniotes.

  5. Unremitting Cell Proliferation in the Secretory Phase of Eutopic Endometriosis

    PubMed Central

    Franco-Murillo, Yanira; Miranda-Rodríguez, José Antonio; Rendón-Huerta, Erika; Montaño, Luis F.; Cornejo, Gerardo Velázquez; Gómez, Lucila Poblano; Valdez-Morales, Francisco Javier; Gonzalez-Sanchez, Ignacio

    2014-01-01

    Objective: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. Design: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3β), and phosphorylated glycogen synthase kinase 3 beta (pGSK3β), throughout the menstrual cycle. Results: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3β was identical in all samples and cycle phases, whereas pAkt and pGSK3β, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. Conclusion: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways. PMID:25194152

  6. Quantification of endocrine cells and ultrastructural study of insulin granules in the large intestine of opossum Didelphis aurita (Wied-Neuwied, 1826).

    PubMed

    dos Santos, Daiane Cristina Marques; Cupertino, Marli do Carmo; Fialho, Maria do Carmo Queiroz; Barbosa, Alfredo Jose Afonso; Fonseca, Cláudio Cesar; Sartori, Sirlene Souza Rodrigues; da Matta, Sérgio Luis Pinto

    2014-02-01

    This study aimed to investigate the distribution of argyrophil, argentaffin, and insulin-immunoreactive endocrine cells in the large intestine of opossums (Didelphis aurita) and to describe the ultrastructure of the secretory granules of insulin-immunoreactive endocrine cells. Fragments of the large intestine of 10 male specimens of D. aurita were collected, processed, and subjected to staining, immunohistochemistry, and transmission electron microscopy. The argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells were sparsely distributed in the intestinal glands of the mucous layer, among other cell types of the epithelium in all regions studied. Proportionally, the argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells represented 62.75%, 36.26%, and 0.99% of the total determined endocrine cells of the large intestine, respectively. Quantitatively, there was no difference between the argyrophil and the argentaffin endocrine cells, whereas insulin-immunoreactive endocrine cells were less numerous. The insulin-immunoreactive endocrine cells were elongated or pyramidal, with rounded nuclei of irregularly contoured, and large amounts of secretory granules distributed throughout the cytoplasm. The granules have different sizes and electron densities and are classified as immature and mature, with the mature granules in predominant form in the overall granular population. In general, the granule is shown with an external electron-lucent halo and electron-dense core. The ultrastructure pattern in the granules of the insulin-immunoreactive endocrine cells was similar to that of the B cells of pancreatic islets in rats.

  7. Dense-core granules: a specific hallmark of the neuronal/neurosecretory cell phenotype.

    PubMed

    Malosio, Maria Luisa; Giordano, Tiziana; Laslop, Andrea; Meldolesi, Jacopo

    2004-02-15

    Expression of dense-core granules, a typical exocytic organelle, is widely believed to be controlled by coordinate gene expression mechanisms specific to neurones and neurosecretory cells. Recent studies in PC12 cells, however, have suggested the number of granules/cells depends on the levels of only one of their cargo proteins, chromogranin A, regulating the metabolism of the other proteins, and thus the composition of the organelles, by an on/off switch mechanism. In addition, transfection of chromogranin A was reported to induce appearance of dense-core granules in the non-neurosecretory fibroblasts of the CV-1 line. Here the role of chromogranin A has been reinvestigated using not the heterogeneous PC12 line but several clones isolated therefrom. In these clones, investigated as such or after transfection with chromogranin A antisense sequences, the ratio between chromogranin A and its secretory protein mate, chromogranin B, was not constant but highly and apparently randomly variable. Variability of the chromogranin A/chromogranin B ratio was seen by confocal immunofluorescence also among the cells of single clones and subclones and among the granules of single cells. Moreover, stable and transient transfections of chromogranin A in a PC12 clone characterised by a low number of dense-core granules (one fifth of the reference clone) failed to modify significantly the number of the organelles, despite the several-fold increase of the granin. Finally, in three types of non-neurosecretory cells (CV-1, adenocarcinoma TS/A and a clone of PC12 incompetent for secretion) the transfected chromogranin A accumulated mostly in the Golgi/transGolgi area and was released rapidly from resting cells (constitutive secretion) as revealed by both immunofluorescence during cycloheximide treatment and pulse-chase experiments. Only a minor fraction was sorted to discrete organelles that were not dense-core granules, but primarily lysosomes because they contained no chromogranin B

  8. Corticotropin-releasing factor binding protein enters the regulated secretory pathway in neuroendocrine cells and cortical neurons.

    PubMed

    Blanco, Elías H; Zúñiga, Juan Pablo; Andrés, María Estela; Alvarez, Alejandra R; Gysling, Katia

    2011-08-01

    Corticotropin releasing factor binding protein (CRF-BP) is a 37kDa glycoprotein that binds CRF with high affinity. CRF-BP controls CRF levels within plasma during human pregnancy. It has also been shown that CRF-BP is expressed in various brain nuclei. Main actions that have been proposed for brain CRF-BP are either decreasing available CRF or facilitating CRF ligand-induced activation of CRF-R2 receptors. For both actions, it is necessary the release of CRF-BP from CRF-BP expressing neurons. However, the secretion mode of CRF-BP is currently unknown. We used heterologous expression of CRF-BP-Flag in PC12 cells and in primary culture of rat cortical neurons to study CRF-BP secretion mode. We observed that CRF-BP-Flag immunoreactivity presents the typical cytoplasmatic punctuate pattern that has been described for neuropeptides and proteins that enter the regulated secretory pathway in PC12 cells. Quantitative analysis of double immunofluorescence confocal images showed that CRF-BP-Flag colocalizes with secretogranin II, marker of secretory granules, both in PC12 and in primary-cultured rat neurons. Furthermore, CRF-BP-Flag is released from PC12 cells upon high K(+)-depolarization. Thus, our results show that CRF-BP is efficiently sorted to the regulated secretory pathway in two cellular contexts, suggesting that the extracellular levels of CRF-BP in the central nervous system depends on neuronal activity.

  9. Carboxypeptidase E and Secretogranin III Coordinately Facilitate Efficient Sorting of Proopiomelanocortin to the Regulated Secretory Pathway in AtT20 Cells

    PubMed Central

    Rathod, Trushar; Young, Sigrid; Lou, Hong; Birch, Nigel; Loh, Y. Peng

    2016-01-01

    Proopiomelanocortin (POMC) is a multivalent prohormone that can be processed into at least 7 biologically active peptide hormones. Processing can begin in the trans-Golgi network (TGN) and continues in the secretory granules of the regulated secretory pathway (RSP). Sorting of POMC into these granules is a complex process. Previously, a membrane-associated form of carboxypeptidase E (CPE) was shown to bind to POMC and facilitate its trafficking into these granules. More recently, secretogranin III (SgIII) was also found to affect POMC trafficking. Here, we show by RNA silencing that CPE and SgIII play a synergistic role in the trafficking of POMC to granules of the RSP in AtT20 cells. Reduction of either protein resulted in increased constitutive secretion of POMC and chromogranin A, which was increased even further when both proteins were reduced together, indicative of missorting at the TGN. In SgIII-reduced cells, POMC accumulated in a compartment that cofractionated and colocalized with syntaxin 6, a marker of the TGN, on sucrose density gradients and in immunocytochemistry, respectively, indicating an accumulation of this protein in the presumed sorting compartment. Regulated secretion of ACTH, as a measure of sorting and processing of POMC in mature granules, was reduced in the SgIII down-regulated cells but was increased in the CPE down-regulated cells. These results suggest that multiple sorting systems exist, providing redundancy to ensure the important task of continuous and accurate trafficking of prohormones to the granules of the RSP for the production of peptide hormones. PMID:26646096

  10. Fallopian tube secretory cell expansion: a sensitive biomarker for ovarian serous carcinogenesis.

    PubMed

    Wang, Yiying; Li, Li; Wang, Yue; Tang, Sarah Ngocvi; Zheng, Wenxin

    2015-01-01

    Recent advances suggest that precancerous lesions of pelvic serous carcinoma originate from tubal secretory cells. The purpose of our study was to determine if an increased number of secretory cells varies with age or location in the fallopian tube and to examine its association with serous neoplasia. Three groups (benign control, high-risk, and pelvic serous carcinoma) of age-matched patients were studied. The age data were stratified into 10-year intervals ranging from 20-29 to older than 80. The number of secretory and ciliated cells from both tubal fimbria and ampulla segments was counted by microscopy and immunohistochemical staining methods. The data were analyzed by standard contingency table and Poisson distribution methods after age justification. We found that the absolute number of tubal secretory cells increased significantly with age in all three groups. But a more dramatic increase of secretory cells was observed in high-risk and pelvic serous carcinoma patients. Secretory cell expansion is more prevalent than secretory cell outgrowth in both fimbria and ampulla tubal segments and is significantly associated with serous neoplasia (P < 0.001). Furthermore, age remained a significant risk factor for serous neoplasia after age adjustment. These findings suggest that secretory cell expansion could serve as a potential sensitive biomarker for early serous carcinogenesis within the fallopian tube. The study also supports a relationship between serous neoplasia and increased secretory to ciliated cell ratios, and the relationship between frequency of secretory cell expansion within the fallopian tube and increasing age and-more significantly-presence of high-risk factors or co-existing serous cancers.

  11. Fallopian tube secretory cell expansion: a sensitive biomarker for ovarian serous carcinogenesis.

    PubMed

    Wang, Yiying; Li, Li; Wang, Yue; Tang, Sarah Ngocvi; Zheng, Wenxin

    2016-01-01

    Recent advances suggest that precancerous lesions of pelvic serous carcinoma originate from tubal secretory cells. The purpose of our study was to determine if an increased number of secretory cells vary with age or location in the fallopian tube and to examine its association with serous neoplasia. Three groups (benign control, high-risk, and pelvic serous carcinoma) of age-matched patients were studied. The age data were stratified into 10-year intervals ranging from 20-29 to older than 80. The number of secretory and ciliated cells from both tubal fimbria and ampulla segments was counted by microscopy and immunohistochemical staining methods. The data were analyzed by standard contingency table and Poisson distribution methods after age justification. We found that the absolute number of tubal secretory cells increased significantly with age in all three groups. But a more dramatic increase of secretory cells was observed in high-risk and pelvic serous carcinoma patients. Secretory cell expansion is more prevalent than secretory cell outgrowth in both fimbria and ampulla tubal segments and is significantly associated with serous neoplasia (p < 0.001). Furthermore, age remained a significant risk factor for serous neoplasia after age adjustment. These findings suggest that secretory cell expansion could serve as a potential sensitive biomarker for early serous carcinogenesis within the fallopian tube. The study also supports a relationship between serous neoplasia and increased secretory to ciliated cell ratios, and the relationship between frequency of secretory cell expansion within the fallopian tube and increasing age and-more significantly-presence of high-risk factors or co-existing serous cancers.

  12. Integration of quanta in cerebellar granule cells during sensory processing.

    PubMed

    Chadderton, Paul; Margrie, Troy W; Häusser, Michael

    2004-04-22

    To understand the computations performed by the input layers of cortical structures, it is essential to determine the relationship between sensory-evoked synaptic input and the resulting pattern of output spikes. In the cerebellum, granule cells constitute the input layer, translating mossy fibre signals into parallel fibre input to Purkinje cells. Until now, their small size and dense packing have precluded recordings from individual granule cells in vivo. Here we use whole-cell patch-clamp recordings to show the relationship between mossy fibre synaptic currents evoked by somatosensory stimulation and the resulting granule cell output patterns. Granule cells exhibited a low ongoing firing rate, due in part to dampening of excitability by a tonic inhibitory conductance mediated by GABA(A) (gamma-aminobutyric acid type A) receptors. Sensory stimulation produced bursts of mossy fibre excitatory postsynaptic currents (EPSCs) that summate to trigger bursts of spikes. Notably, these spike bursts were evoked by only a few quantal EPSCs, and yet spontaneous mossy fibre inputs triggered spikes only when inhibition was reduced. Our results reveal that the input layer of the cerebellum balances exquisite sensitivity with a high signal-to-noise ratio. Granule cell bursts are optimally suited to trigger glutamate receptor activation and plasticity at parallel fibre synapses, providing a link between input representation and memory storage in the cerebellum.

  13. Event-driven simulation of cerebellar granule cells.

    PubMed

    Carrillo, Richard R; Ros, Eduardo; Tolu, Silvia; Nieus, Thierry; D'Angelo, Egidio

    2008-01-01

    Around half of the neurons of a human brain are granule cells (approximately 10(11)granule neurons) [Kandel, E.R., Schwartz, J.H., Jessell, T.M., 2000. Principles of Neural Science. McGraw-Hill Professional Publishing, New York]. In order to study in detail the functional role of the intrinsic features of this cell we have developed a pre-compiled behavioural model based on the simplified granule-cell model of Bezzi et al. [Bezzi, M., Nieus, T., Arleo, A., D'Angelo, E., Coenen, O.J.-M.D., 2004. Information transfer at the mossy fiber-granule cell synapse of the cerebellum. 34th Annual Meeting. Society for Neuroscience, San Diego, CA, USA]. We can use an efficient event-driven simulation scheme based on lookup tables (EDLUT) [Ros, E., Carrillo, R.R., Ortigosa, E.M., Barbour, B., Ags, R., 2006. Event-driven simulation scheme for spiking neural networks using lookup tables to characterize neuronal dynamics. Neural Computation 18 (12), 2959-2993]. For this purpose it is necessary to compile into tables the data obtained through a massive numerical calculation of the simplified cell model. This allows network simulations requiring minimal numerical calculation. There are three major features that are considered functionally relevant in the simplified granule cell model: bursting, subthreshold oscillations and resonance. In this work we describe how the cell model is compiled into tables keeping these key properties of the neuron model.

  14. Massively augmented hippocampal dentate granule cell activation accompanies epilepsy development.

    PubMed

    Dengler, Christopher G; Yue, Cuiyong; Takano, Hajime; Coulter, Douglas A

    2017-02-20

    In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulation-induced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses.

  15. Massively augmented hippocampal dentate granule cell activation accompanies epilepsy development

    PubMed Central

    Dengler, Christopher G.; Yue, Cuiyong; Takano, Hajime; Coulter, Douglas A.

    2017-01-01

    In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulation-induced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses. PMID:28218241

  16. Mast Cell Mediators: Their Differential Release and the Secretory Pathways Involved

    PubMed Central

    Moon, Tae Chul; Befus, A. Dean; Kulka, Marianna

    2014-01-01

    Mast cells (MC) are widely distributed throughout the body and are common at mucosal surfaces, a major host–environment interface. MC are functionally and phenotypically heterogeneous depending on the microenvironment in which they mature. Although MC have been classically viewed as effector cells of IgE-mediated allergic diseases, they are also recognized as important in host defense, innate and acquired immunity, homeostatic responses, and immunoregulation. MC activation can induce release of pre-formed mediators such as histamine from their granules, as well as release of de novo synthesized lipid mediators, cytokines, and chemokines that play diverse roles, not only in allergic reactions but also in numerous physiological and pathophysiological responses. Indeed, MC release their mediators in a discriminating and chronological manner, depending upon the stimuli involved and their signaling cascades (e.g., IgE-mediated or Toll-like receptor-mediated). However, the precise mechanisms underlying differential mediator release in response to these stimuli are poorly known. This review summarizes our knowledge of MC mediators and will focus on what is known about the discriminatory release of these mediators dependent upon diverse stimuli, MC phenotypes, and species of origin, as well as on the intracellular synthesis, storage, and secretory processes involved. PMID:25452755

  17. ICA 512, an autoantigen of type I diabetes, is an intrinsic membrane protein of neurosecretory granules.

    PubMed Central

    Solimena, M; Dirkx, R; Hermel, J M; Pleasic-Williams, S; Shapiro, J A; Caron, L; Rabin, D U

    1996-01-01

    Islet cell autoantigen (ICA) 512 is a novel autoantigen of insulin-dependent diabetes mellitus (IDDM) which is homologous to receptor-type protein tyrosine phosphatases (++PTPases). We show that ICA 512 is an intrinsic membrane protein of secretory granules expressed in insulin-producing pancreatic beta-cells as well as in virtually all other peptide-secreting endocrine cells and neurons containing neurosecretory granules. ICA 512 is cleaved at its luminal domain and, following exposure at the cell surface, recycles to the Golgi complex region and is sorted into newly formed secretory granules. By immunoprecipitation, anti-ICA 512 autoantibodies were detected in 15/17 (88%) newly diagnosed IDDM patients, but not in 10/10 healthy subjects. These results suggest that tyrosine phosphorylation participates in some aspect of secretory granule function common to all neuroendocrine cells and that a subset of autoantibodies in IDDM is directed against an integral membrane protein of insulin-containing granules. Images PMID:8641276

  18. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    PubMed

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

  19. Isolating stromal stem cells from periodontal granulation tissues.

    PubMed

    Hung, Tzu-Yuan; Lin, Hsiang-Chun; Chan, Ying-Jen; Yuan, Kuo

    2012-08-01

    Stem cell therapy is a promising area in regenerative medicine. Periodontal granulation tissues are often discarded during conventional surgery. If stromal stem cells can be isolated from these tissues, they can be used for subsequent surgery on the same patient. Fifteen human periodontal granulation tissue samples were obtained from intrabony defects during surgery. Immunohistochemistry (IHC) was carried out on five of the samples to identify STRO-1, a marker of mesenchymal stem cells. Five samples underwent flow cytometry analysis for the same marker. The remaining five samples were characterized by "colony formation unit-fibroblast" (CFU-f) assay and selected for proliferation assay, flow cytometry of stem cell markers, immunocytochemistry (ICC), multipotent differentiation assays, and repairing critical-size defects in mice. The ratio of STRO-1(+) cells detected by IHC was 5.91 ± 1.50%. The analysis of flow cytometry for STRO-1 was 6.70 ± 0.81%. Approximately two thirds of the CFU-f colonies had a strong reaction to STRO-1 in ICC staining. The cells were multipotent both in vitro and in vivo. Mice given bone grafts and stem cells showed significantly better bone healing than those without stem cells. Multipotent stromal stem cells can be isolated from human periodontal granulation tissues. These cells improve new bone formation when transplanted in mouse calvarial defects. Isolating stem cells from relatively accessible sites without extra procedures is clinically advantageous. This study demonstrated that human periodontal granulation tissues contain isolatable multipotent stem cells. The cells may be a good source for autotransplantation in subsequent treatment.

  20. The use of lectins as markers for differentiated secretory cells in planarians.

    PubMed

    Zayas, Ricardo M; Cebrià, Francesc; Guo, Tingxia; Feng, Junjie; Newmark, Phillip A

    2010-11-01

    Freshwater planarians have reemerged as excellent models to investigate mechanisms underlying regeneration. The introduction of molecular tools has facilitated the study of planarians, but cell- and tissue-specific markers are still needed to examine differentiation of most cell types. Here we report the utility of fluorescent lectin-conjugates to label tissues in the planarian Schmidtea mediterranea. We show that 16 lectin-conjugates stain planarian cells or tissues; 13 primarily label the secretory cells, their cytoplasmic projections, and terminal pores. Thus, we examined regeneration of the secretory system using lectin markers and functionally characterized two genes expressed in the secretory cells: marginal adhesive gland-1 (mag-1) and Smed-reticulocalbin1 (Smed-rcn1). RNAi knockdown of these genes caused a dramatic reduction of secretory cell lectin staining, suggesting a role for mag-1 and Smed-rcn1 in secretory cell differentiation. Our results provide new insights into planarian secretory system regeneration and add new markers for labeling several planarian tissues.

  1. Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens

    PubMed Central

    Ogbomo, Henry; Mody, Christopher H.

    2017-01-01

    Natural killer (NK) cells kill or inhibit the growth of a number of fungi including Cryptococcus, Candida, Aspergillus, Rhizopus, and Paracoccidioides. Although many fungi are not dangerous, invasive fungal pathogens, such as Cryptococcus neoformans, cause life-threatening disease in individuals with impaired cell-mediated immunity. While there are similarities to cell-mediated killing of tumor cells, there are also important differences. Similar to tumor killing, NK cells directly kill fungi in a receptor-mediated and cytotoxic granule-dependent manner. Unlike tumor cell killing where multiple NK cell-activating receptors cooperate and signal events that mediate cytotoxicity, only the NKp30 receptor has been described to mediate signaling events that trigger the NK cell to mobilize its cytolytic payload to the site of interaction with C. neoformans and Candida albicans, subsequently leading to granule exocytosis and fungal killing. More recently, the NKp46 receptor was reported to bind Candida glabrata adhesins Epa1, 6, and 7 and directly mediate fungal clearance. A number of unanswered questions remain. For example, is only one NK cell-activating receptor sufficient for signaling leading to fungal killing? Are the signaling pathways activated by fungi similar to those activated by tumor cells during NK cell killing? How do the cytolytic granules traffic to the site of interaction with fungi, and how does this process compare with tumor killing? Recent insights into receptor use, intracellular signaling and cytolytic granule trafficking during NK cell-mediated fungal killing will be compared to tumor killing, and the implications for therapeutic approaches will be discussed. PMID:28123389

  2. Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion.

    PubMed

    Verkhratsky, Alexei; Matteoli, Michela; Parpura, Vladimir; Mothet, Jean-Pierre; Zorec, Robert

    2016-02-01

    Astrocytes are housekeepers of the central nervous system (CNS) and are important for CNS development, homeostasis and defence. They communicate with neurones and other glial cells through the release of signalling molecules. Astrocytes secrete a wide array of classic neurotransmitters, neuromodulators and hormones, as well as metabolic, trophic and plastic factors, all of which contribute to the gliocrine system. The release of neuroactive substances from astrocytes occurs through several distinct pathways that include diffusion through plasmalemmal channels, translocation by multiple transporters and regulated exocytosis. As in other eukaryotic cells, exocytotic secretion from astrocytes involves divergent secretory organelles (synaptic-like microvesicles, dense-core vesicles, lysosomes, exosomes and ectosomes), which differ in size, origin, cargo, membrane composition, dynamics and functions. In this review, we summarize the features and functions of secretory organelles in astrocytes. We focus on the biogenesis and trafficking of secretory organelles and on the regulation of the exocytotic secretory system in the context of healthy and diseased astrocytes.

  3. Estradiol increases cAMP in the oviductal secretory cells through a nongenomic mechanism.

    PubMed

    Oróstica, María L; Lopez, John; Rojas, Israel; Rocco, Jocelyn; Díaz, Patricia; Reuquén, Patricia; Cardenas, Hugo; Parada-Bustamante, Alexis; Orihuela, Pedro A

    2014-09-01

    In the rat oviduct, estradiol (E2) accelerates egg transport by a nongenomic action that requires previous conversion of E2 to methoxyestrogens via catechol-O-methyltranferase (COMT) and activation of estrogen receptor (ER) with subsequent production of cAMP and inositol triphosphate (IP3). However, the role of the different oviductal cellular phenotypes on this E2 nongenomic pathway remains undetermined. The aim of this study was to investigate the effect of E2 on the levels of cAMP and IP3 in primary cultures of secretory and smooth muscle cells from rat oviducts and determine the mechanism by which E2 increases cAMP in the secretory cells. In the secretory cells, E2 increased cAMP but not IP3, while in the smooth muscle cells E2 decreased cAMP and increased IP3. Suppression of protein synthesis by actinomycin D did not prevent the E2-induced cAMP increase, but this was blocked by the ER antagonist ICI 182 780 and the inhibitors of COMT OR 486, G protein-α inhibitory (Gαi) protein pertussis toxin and adenylyl cyclase (AC) SQ 22536. Expression of the mRNA for the enzymes that metabolizes estrogens, Comt, Cyp1a1, and Cyp1b1 was found in the secretory cells, but this was not affected by E2. Finally, confocal immunofluorescence analysis showed that E2 induced colocalization between ESR1 (ERα) and Gαi in extranuclear regions of the secretory cells. We conclude that E2 differentially regulates cAMP and IP3 in the secretory and smooth muscle cells of the rat oviduct. In the secretory cells, E2 increases cAMP via a nongenomic action that requires activation of COMT and ER, coupling between ESR1 and Gαi, and stimulation of AC.

  4. Development of secretory cells and crystal cells in Eichhornia crassipes ramet shoot apex.

    PubMed

    Xu, Guo Xin; Tan, Chao; Wei, Xiao Jing; Gao, Xiao Yan; Zheng, Hui Qiong

    2011-04-01

    The distribution and development of secretory cells and crystal cells in young shoot apexes of water hyacinth were investigated through morphological and cytological analysis. The density of secretory cells and crystal cells were high in parenchyma tissues around the vascular bundles of shoot apexes. Three developmental stages of the secretory cells can be distinguished under transmission electron microscopy. Firstly, a large number of electron-dense vesicles formed in the cytoplasm, then fused with the tonoplast and released into the vacuole in the form of electron-dense droplets. As these droplets fused together, a large mass of dark material completely filled the vacuole. To this end, a secretion storage vacuole (SSV) formed. Secondly, an active secretion stage accompanied with degradation of the large electron-dense masses through an ill-defined autophagic process at periphery and in the limited internal regions of the SSV. Finally, after most storage substances were withdrawn, the materials remaining in the spent SSV consisted of an electron-dense network structure. The distribution and development of crystal cells in shoot apical tissue of water hyacinth were also studied by light and electron microscopy. Crystals initially formed at one site in the vacuole, where tube-like membrane structures formed crystal chambers. The chamber enlarged as the crystal grew in bidirectional manner and formed needle-shaped raphides. Most of these crystals finally occurred as raphide bundles, and the others appeared as block-like rhombohedral crystals in the vacuole. These results suggest that the formation of both secretory cells and crystal cells are involved in the metamorphosis of vacuoles and a role for vacuoles in water hyacinth rapid growth and tolerance.

  5. Newborn granule cells in the ageing dentate gyrus

    PubMed Central

    Morgenstern, Nicolás A; Lombardi, Gabriela; Schinder, Alejandro F

    2008-01-01

    The dentate gyrus of the hippocampus generates neurons throughout life, but adult neurogenesis exhibits a marked age-dependent decline. Although the decrease in the rate of neurogenesis has been extensively documented in the ageing hippocampus, the specific characteristics of dentate granule cells born in such a continuously changing environment have received little attention. We have used retroviral labelling of neural progenitor cells of the adult mouse dentate gyrus to study morphological properties of neurons born at different ages. Dendritic spine density was measured to estimate glutamatergic afferent connectivity. Fully mature neurons born at the age of 2 months display ∼2.3 spines μm−1 and maintain their overall morphology and spine density in 1-year-old mice. Surprisingly, granule cells born in 10-month-old mice, at which time the rate of neurogenesis has decreased by ∼40-fold, reach a density of dendritic spines similar to that of neurons born in young adulthood. Therefore, in spite of the sharp decline in cell proliferation, differentiation and overall neuronal number, the ageing hippocampus presents a suitable environment for new surviving neurons to reach a high level of complexity, comparable to that of all other dentate granule cells. PMID:18565998

  6. Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo

    PubMed Central

    Schnell, Eric; Bensen, AeSoon L.; Washburn, Eric K.; Westbrook, Gary L.

    2012-01-01

    Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons. PMID:23110172

  7. Spatial Representations of Granule Cells and Mossy Cells of the Dentate Gyrus.

    PubMed

    GoodSmith, Douglas; Chen, Xiaojing; Wang, Cheng; Kim, Sang Hoon; Song, Hongjun; Burgalossi, Andrea; Christian, Kimberly M; Knierim, James J

    2017-02-08

    Granule cells in the dentate gyrus of the hippocampus are thought to be essential to memory function by decorrelating overlapping input patterns (pattern separation). A second excitatory cell type in the dentate gyrus, the mossy cell, forms an intricate circuit with granule cells, CA3c pyramidal cells, and local interneurons, but the influence of mossy cells on dentate function is often overlooked. Multiple tetrode recordings, supported by juxtacellular recording techniques, showed that granule cells fired very sparsely, whereas mossy cells in the hilus fired promiscuously in multiple locations and in multiple environments. The activity patterns of these cell types thus represent different environments through distinct computational mechanisms: sparse coding in granule cells and changes in firing field locations in mossy cells.

  8. FIB/SEM cell sectioning for intracellular metal granules characterization

    NASA Astrophysics Data System (ADS)

    Milani, Marziale; Brundu, Claudia; Santisi, Grazia; Savoia, Claudio; Tatti, Francesco

    2009-05-01

    Focused Ion Beams (FIBs) provide a cross-sectioning tool for submicron dissection of cells and subcellular structures. In combination with Scanning Electron Microscope (SEM), FIB provides complementary morphological information, that can be further completed by EDX (Energy Dispersive X-ray Spectroscopy). This study focus onto intracellular microstructures, particularly onto metal granules (typically Zn, Cu and Fe) and on the possibility of sectioning digestive gland cells of the terrestrial isopod P. scaber making the granules available for a compositional analysis with EDX. Qualitative and quantitative analysis of metal granules size, amount and distribution are performed. Information is made available of the cellular storing pattern and, indirectly, metal metabolism. The extension to human level is of utmost interest since some pathologies of relevance are metal related. Apart from the common metal-overload-diseases (hereditary hemochromatosis, Wilson's and Menkes disease) it has been demonstrated that metal in excess can influence carcinogenesis in liver, kidney and breast. Therefore protocols will be established for the observation of mammal cells to improve our knowledge about the intracellular metal amount and distribution both in healthy cells and in those affected by primary or secondary metal overload or depletion.

  9. Secretory activity of mast cell during stress: effect of prolyl-glycyl-proline and Semax.

    PubMed

    Umarova, B A; Kopylova, G N; Smirnova, E A; Guseva, A A; Zhuikova, S E

    2003-10-01

    Stress increased secretory activity of mast cells in the mesentery and subcutaneous fat of rats. Intraperitoneal injection of Semax and prolyl-glycyl-proline in doses of 0.05 and 1 mg/kg, respectively, 1 h before stress abolished this effect. The test preparations did not modulate secretory activity of mast cells in unstressed animals. Semax and prolyl-glycyl-proline in vitro prevented activation of mast cells with synacten and acetylcholine. The stabilizing effect of peptides on mast cells probably determines their antiulcer activity.

  10. Single granule cells reliably discharge targets in the hippocampal CA3 network in vivo.

    PubMed

    Henze, Darrell A; Wittner, Lucia; Buzsáki, György

    2002-08-01

    Processing of neuronal information depends on interactions between the anatomical connectivity and cellular properties of single cells. We examined how these computational building blocks work together in the intact rat hippocampus. Single spikes in dentate granule cells, controlled intracellularly, generally failed to discharge either interneurons or CA3 pyramidal cells. In contrast, trains of spikes effectively discharged both CA3 cell types. Increasing the discharge rate of the granule cell increased the discharge probability of its target neuron and decreased the delay between the onset of a granule cell train and evoked firing in postsynaptic targets. Thus, we conclude that the granule cell to CA3 synapses are 'conditional detonators,' dependent on granule cell firing pattern. In addition, we suggest that information in single granule cells is converted into a temporal delay code in target CA3 pyramidal cells and interneurons. These data demonstrate how a neural circuit of the CNS may process information.

  11. Intestinal Mucus Gel and Secretory Antibody are Barriers to Campylobacter jejuni Adherence to INT 407 Cells

    DTIC Science & Technology

    1987-06-01

    An in vitro mucus assay was developed to study the role of mucus gel and secretory immunoglobulin A (sIgA) in preventing attachment of Campylobacter ... jejuni to INT 407 cells. An overlay of rabbit small intestinal mucus was found to impede the attachment of C. jejuni to a monolayer of INT 407 cells

  12. Cerebellar granule cells acquire a widespread predictive feedback signal during motor learning.

    PubMed

    Giovannucci, Andrea; Badura, Aleksandra; Deverett, Ben; Najafi, Farzaneh; Pereira, Talmo D; Gao, Zhenyu; Ozden, Ilker; Kloth, Alexander D; Pnevmatikakis, Eftychios; Paninski, Liam; De Zeeuw, Chris I; Medina, Javier F; Wang, Samuel S-H

    2017-03-20

    Cerebellar granule cells, which constitute half the brain's neurons, supply Purkinje cells with contextual information necessary for motor learning, but how they encode this information is unknown. Here we show, using two-photon microscopy to track neural activity over multiple days of cerebellum-dependent eyeblink conditioning in mice, that granule cell populations acquire a dense representation of the anticipatory eyelid movement. Initially, granule cells responded to neutral visual and somatosensory stimuli as well as periorbital airpuffs used for training. As learning progressed, two-thirds of monitored granule cells acquired a conditional response whose timing matched or preceded the learned eyelid movements. Granule cell activity covaried trial by trial to form a redundant code. Many granule cells were also active during movements of nearby body structures. Thus, a predictive signal about the upcoming movement is widely available at the input stage of the cerebellar cortex, as required by forward models of cerebellar control.

  13. Control over the morphology and segregation of Zebrafish germ cell granules during embryonic development

    PubMed Central

    Strasser, Markus J; Mackenzie, Natalia C; Dumstrei, Karin; Nakkrasae, La-Iad; Stebler, Jürg; Raz, Erez

    2008-01-01

    Background Zebrafish germ cells contain granular-like structures, organized around the cell nucleus. These structures share common features with polar granules in Drosophila, germinal granules in Xenopus and chromatoid bodies in mice germ cells, such as the localization of the zebrafish Vasa, Piwi and Nanos proteins, among others. Little is known about the structure of these granules as well as their segregation in mitosis during early germ-cell development. Results Using transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7) is required for determination of granule morphology and number. Conclusion Our results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network. PMID:18507824

  14. An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

    PubMed Central

    Bertrand, Florie; Müller, Sabina; Roh, Kyung-Ho; Laurent, Camille; Dupré, Loïc; Valitutti, Salvatore

    2013-01-01

    It is presently assumed that lethal hit delivery by cytotoxic T lymphocytes (CTLs) is mechanistically linked to centrosome polarization toward target cells, leading to dedicated release of lytic granules within a confined secretory domain. Here we provide three lines of evidence showing that this mechanism might not apply as a general paradigm for lethal hit delivery. First, in CTLs stimulated with immobilized peptide–MHC complexes, lytic granules and microtubule organizing center localization into synaptic areas are spatio-temporally dissociated, as detected by total internal reflection fluorescence microscopy. Second, in many CTL/target cell conjugates, lytic granule secretion precedes microtubule polarization and can be detected during the first minute after cell–cell contact. Third, inhibition of microtubule organizing center and centrosome polarization impairs neither lytic granule release at the CTL synapse nor killing efficiency. Our results broaden current views of CTL biology by revealing an extremely rapid step of lytic granule secretion and by showing that microtubule organizing center polarization is dispensable for efficient lethal hit delivery. PMID:23536289

  15. Ultrastructural Characterization of Mammary Analogue Secretory Carcinoma of the Salivary Glands: A Distinct Entity from Acinic Cell Carcinoma?

    PubMed

    Guilmette, Julie; Nielsen, Gunnlaugur P; Faquin, William C; Selig, Martin; Nosé, Vânia; Chi, Anthony W S; Sadow, Peter M

    2017-02-13

    Mammary analogue secretory carcinoma (MASC) of the salivary glands is a recently described neoplasm of the salivary glands with a characteristic morphology complemented by a specific cytogenetic translocation and gene rearrangements. Although immunophenotypic and cytogenetic differences allow for a more reliable distinction, ultrastructural features can also provide important information about the relationship between MASC, classic acinic cell carcinoma (AciCC), and AciCC intercalated duct cell-predominant variant. Following approval from the hospital's institutional review board, 7 cases of MASC, 8 cases of classic AciCC, and 4 cases of AciCC intercalated duct cell-predominant variant were retrieved from the pathology files of Massachusetts General Hospital from 2012 to 2015. Electron microscopy was performed using formalin-fixed, paraffin-embedded tissue. Ultrastructural features of all 19 neoplasms of the salivary glands were recorded. The predominant cell-types observed in MASC are those with intercalated/striated duct cell differentiation. These features include prominent invaginations of the cell surface studded with microvilli, and some intra- and intercellular lumina also with a microvillous surface. Classic AciCC dominant cell-type recapitulates acinar cell differentiation. These cells contain large intracytoplasmic zymogen-like granules. AciCC intercalated duct cell-predominant variant showed both cell populations in various proportions with the intercalated/striated duct cell type usually being the dominant one. MASC presents with distinctive ultrastructural features that allows its proper differentiation from classic AciCC. However, significant ultrastructural features overlaps between both AciCC intercalated duct cells-predominant and classic AciCC and MASC. These findings indicate a very close proximity between these tumors.

  16. LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration

    PubMed Central

    Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

    2015-01-01

    The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

  17. Gray platelet syndrome. Dissociation between abnormal sorting in megakaryocyte alpha-granules and normal sorting in Weibel-Palade bodies of endothelial cells.

    PubMed Central

    Gebrane-Younès, J; Cramer, E M; Orcel, L; Caen, J P

    1993-01-01

    The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which megakaryocytes and platelets are deficient in alpha-granule secretory proteins. Since the Weibel-Palade bodies (WPB) of endothelial cells as well as the alpha-granules contain the von Willebrand Factor (vWF) and P-selectin, we examined by transmission electron microscopy the dermis capillary network of two patients with GPS. Endothelial cells showed the presence of normal WPB with typical internal tubules. Using single and double immunogold labeling for vWF and P-selectin, we detected vWF within WPB, where it was codistributed with the tubules, whereas P-selectin delineated the outline of WPB. Therefore, the fundamental targeting defect in GPS is specific to the megakaryocytic cell line. Images PMID:7504696

  18. Automated insulin granule segmentation from electron photomicrographs of rat pancreatic β-cells

    NASA Astrophysics Data System (ADS)

    McClanahan, Timothy P.; Straub, Susanne G.; Sharp, Geoffrey W. G.; Loew, Murray

    2005-04-01

    Increased blood glucose stimulates pancreatic β-cells and induces an exocytotic release of insulin. The β-cell, which contains ~10^4 insulin-containing granules, releases only a few percent of the granules during a given stimulus such as a meal. The temporal response function to a square wave increase in the concentration of glucose is characteristically biphasic. It is not known whether the granules exhibit random or directed migration patterns as a function of phase. Directed migration would suggest the development of an intracellular gradient directing the path and velocity of insulin granule movement. Our ongoing research investigates this process using manual morphometric analysis of electron micrographs of rat pancreatic β-cells. This is a tedious and time-consuming stereological process. Consequently, we have developed an automated algorithm for accurately segmenting and deriving granule counts, areas, and measuring distance to the plasma membrane. The method is a data-driven image processing approach that implements Mahalanobis classifiers to hierarchically classify pixel candidates and subsequently pixel aggregates as insulin granules. Granule cores and halos are classified independently and fused by intersecting the convex difference of granule halos with core candidates. Once fused, total and individual granule areas and distance metrics to the β-cell plasma membrane are obtained. This algorithm provides a rapid and accurate method for the determination of granule numbers, location, and potential gradients in the pancreatic β-cell under different experimental conditions.

  19. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

    PubMed

    Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

    2014-03-01

    Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression.

  20. Differentiation of apical and basal dendrites in pyramidal cells and granule cells in dissociated hippocampal cultures.

    PubMed

    Wu, You Kure; Fujishima, Kazuto; Kengaku, Mineko

    2015-01-01

    Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.

  1. Probing platelet factor 4 alpha-granule targeting.

    PubMed

    Briquet-Laugier, V; Lavenu-Bombled, C; Schmitt, A; Leboeuf, M; Uzan, G; Dubart-Kupperschmitt, A; Rosa, J-P

    2004-12-01

    The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.

  2. Granuphilin exclusively mediates functional granule docking to the plasma membrane

    PubMed Central

    Mizuno, Kouichi; Fujita, Takuji; Gomi, Hiroshi; Izumi, Tetsuro

    2016-01-01

    In regulated exocytosis, it is generally assumed that vesicles must stably “dock” at the plasma membrane before they are primed to become fusion-competent. However, recent biophysical analyses in living cells that visualize fluorescent secretory granules have revealed that exocytic behaviors are not necessarily uniform: some granules beneath the plasma membrane are resistant to Ca2+ -triggered release, while others are accelerated to fuse without a pause for stable docking. These findings suggest that stable docking is unnecessary, and can even be inhibitory or nonfunctional, for fusion. Consistently, pancreatic β cells deficient in the Rab27 effector, granuphilin, lack insulin granules directly attached to the plasma membrane in electron micrographs but nevertheless exhibit augmented exocytosis. Here we directly compare the exocytic behaviors between granuphilin-positive and -negative insulin granules. Although granuphilin makes granules immobile and fusion-reluctant beneath the plasma membrane, those granuphilin-positive, docked granules release a portion of granuphilin upon fusion, and fuse at a frequency and time course similar to those of granuphilin-negative undocked granules. Furthermore, granuphilin forms a 180-nm cluster at the site of each docked granule, along with granuphilin-interacting Rab27a and Munc18-1 clusters. These findings indicate that granuphilin is an exclusive component of the functional and fusion-inhibitory docking machinery of secretory granules. PMID:27032672

  3. Isolation of chromaffin granules.

    PubMed

    Creutz, Carl E

    2010-09-01

    Adrenal medullary chromaffin granules (dense core secretory vesicles) have been a valuable model system for the study of the proteins and membrane components involved in the process of exocytosis. Because of the abundance of chromaffin granules in a readily available tissue source, bovine adrenal medullae, and their unique sedimentation properties, it is possible to obtain large quantities of highly purified granules and granule membranes in a short period of time. Two protocols are presented here for the isolation of chromaffin granules: a basic protocol based on differential centrifugation in an iso-osmotic medium that yields intact chromaffin granules, and an alternate protocol based on sedimentation through a density step gradient that provides a greater yield of more highly purified chromaffin granules. Since in the latter case the granules cannot be returned to a medium of physiological osmolarity without lysis after purification on the step gradient, the alternate protocol is more useful to obtain the granule membranes or contents for further study.

  4. Functional extracellular eosinophil granules: a bomb caught in a trap.

    PubMed

    Muniz, Valdirene S; Baptista-Dos-Reis, Renata; Neves, Josiane S

    2013-01-01

    Eosinophils store a wide range of preformed proteins, including cationic proteins and cytokines, within their morphologically unique granules. Recently, we have demonstrated that cell-free eosinophil granules are functional, independent, secretory organelles and that clusters of cell-free granules are commonly found at tissue sites associated with various pathologic conditions. Cytolytic release of intact eosinophil granules produces extracellular organelles that are fully capable of ligand-elicited, active, secretory responses and are hence able to act as 'cluster bombs' that amplify the differential secretory properties of eosinophils. Herein, we review recent progress in elucidating the molecular mechanisms involved in the cytolytical release of intact cell-free functional eosinophil granules in a process associated with the liberation of eosinophil DNA traps (nets), a known aspect of the innate response recognized in various immune cells and pathological conditions. We also discuss the importance of clusters of cell-free eosinophil granules trapped in eosinophil DNA nets in disease and speculate on their potential role(s) in immunity as well as compare available data on DNA-releasing neutrophils.

  5. The plant secretory pathway seen through the lens of the cell wall.

    PubMed

    van de Meene, A M L; Doblin, M S; Bacic, Antony

    2017-01-01

    Secretion in plant cells is often studied by looking at well-characterised, evolutionarily conserved membrane proteins associated with particular endomembrane compartments. Studies using live cell microscopy and fluorescent proteins have illuminated the highly dynamic nature of trafficking, and electron microscopy studies have resolved the ultrastructure of many compartments. Biochemical and molecular analyses have further informed about the function of particular proteins and endomembrane compartments. In plants, there are over 40 cell types, each with highly specialised functions, and hence potential variations in cell biological processes and cell wall structure. As the primary function of secretion in plant cells is for the biosynthesis of cell wall polysaccharides and apoplastic transport complexes, it follows that utilising our knowledge of cell wall glycosyltransferases (GTs) and their polysaccharide products will inform us about secretion. Indeed, this knowledge has led to novel insights into the secretory pathway, including previously unseen post-TGN secretory compartments. Conversely, our knowledge of trafficking routes of secretion will inform us about polarised and localised deposition of cell walls and their constituent polysaccharides/glycoproteins. In this review, we look at what is known about cell wall biosynthesis and the secretory pathway and how the different approaches can be used in a complementary manner to study secretion and provide novel insights into these processes.

  6. The secretory synapse: the secrets of a serial killer.

    PubMed

    Bossi, Giovanna; Trambas, Christina; Booth, Sarah; Clark, Richard; Stinchcombe, Jane; Griffiths, Gillian M

    2002-11-01

    Cytotoxic T lymphocytes (CTLs) destroy their targets by a process involving secretion of specialized granules. The interactions between CTLs and target can be very brief; nevertheless, adhesion and signaling proteins segregate into an immunological synapse. Secretion occurs in a specialized secretory domain. Use of live and fixed cell microscopy allows this secretory synapse to be visualized both temporally and spatially. The combined use of confocal and electron microscopy has produced some surprising findings, which suggest that the secretory synapse may be important both in delivering the lethal hit and in facilitating membrane transfer from target to CTL. Studies on the secretory synapse in wild-type and mutant CTLs have been used to identify proteins involved in secretion. Further clues as to the signals required for secretion are emerging from comparisons of inhibitory and activating synapses formed by natural killer cells.

  7. Regulated and constitutive protein targeting can be distinguished by secretory polarity in thyroid epithelial cells

    PubMed Central

    1991-01-01

    We have studied concurrent apical/basolateral and regulated/constitutive secretory targeting in filter-grown thyroid epithelial monolayers in vitro, by following the exocytotic routes of two newly synthesized endogenous secretory proteins, thyroglobulin (Tg) and p500. Tg is a regulated secretory protein as indicated by its acute secretory response to secretagogues. Without stimulation, pulse-labeled Tg exhibits primarily two kinetically distinct routes: less than or equal to 80% is released in an apical secretory phase which is largely complete by 6-10 h, with most of the remaining Tg retained in intracellular storage from which delayed apical discharge is seen. The rapid export observed for most Tg is unlikely to be because of default secretion, since its apical polarity is preserved even during the period (less than or equal to 10 h) when p500 is released basolaterally by a constitutive pathway unresponsive to secretagogues. p500 also exhibits a second, kinetically distinct secretory route: at chase times greater than 10 h, a residual fraction (less than or equal to 8%) of p500 is secreted with an apical preponderance similar to that of Tg. It appears that this fraction of p500 has failed to be excluded from the regulated pathway, which has a predetermined apical polarity. From these data we hypothesize that a targeting hierarchy may exist in thyroid epithelial cells such that initial sorting to the regulated pathway may be a way of insuring apical surface delivery from one of two possible exocytotic routes originating in the immature storage compartment. PMID:1991788

  8. Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT-20 cells.

    PubMed Central

    Gross, D J; Halban, P A; Kahn, C R; Weir, G C; Villa-Komaroff, L

    1989-01-01

    A patient with type II diabetes associated with hyperproinsulinemia has been shown to have a point mutation in one insulin gene allele, resulting in replacement of histidine with aspartic acid at position 10 of the B-chain. To investigate the basis of the proinsulin processing defect, we introduced an identical mutation in the rat insulin II gene and expressed both the normal and the mutant genes in the AtT-20 pituitary corticotroph cell line. Cells expressing the mutant gene showed increased secretion of proinsulin relative to insulin and rapid release of newly synthesized proinsulin. Moreover, the mutant cell lines did not store the prohormone nor did they release it upon stimulation with secretagogues. These data indicate that a significant fraction of the mutant prohormone is released via the constitutive secretory pathway rather than the regulated pathway, thereby bypassing granule-related processing and regulated release. PMID:2657740

  9. BDNF over-expression increases olfactory bulb granule cell dendritic spine density in vivo

    PubMed Central

    McDole, Brittnee; Isgor, Ceylan; Pare, Christopher; Guthrie, Kathleen

    2015-01-01

    Olfactory bulb granule cells are axon-less, inhibitory interneurons that regulate the activity of the excitatory output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses located on granule cell spines. These contacts are established in the distal apical dendritic compartment, while granule cell basal dendrites and more proximal apical segments bear spines that receive glutamatergic inputs from the olfactory cortices. This synaptic connectivity is vital to olfactory circuit function and is remodeled during development, and in response to changes in sensory activity and lifelong granule cell neurogenesis. Manipulations that alter levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in vivo have significant effects on dendritic spine morphology, maintenance and activity-dependent plasticity for a variety of CNS neurons, yet little is known regarding BDNF effects on bulb granule cell spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF produces a marked increase in granule cell spine density that includes an increase in mature spines on their apical dendrites. Morphometric analysis demonstrated that changes in spine density were most notable in the distal and proximal apical domains, indicating that multiple excitatory inputs are potentially modified by BDNF. Our results indicate that increased levels of endogenous BDNF can promote the maturation and/or maintenance of dendritic spines on granule cells, suggesting a role for this factor in modulating granule cell functional connectivity within adult olfactory circuitry. PMID:26211445

  10. Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

    SciTech Connect

    Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

    1985-03-01

    Incubation of (/sup 35/S)heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of /sup 35/S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of (/sup 35/S)heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular (/sup 35/S)heparin proteoglycan after 24 hours and the appearance of free (/sup 35/S)sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading (/sup 35/S)heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule.

  11. Immunohistochemical and ultrastructural study of anterior pituitary cells in the female Afghan pika, Ochotona rufescens rufescens.

    PubMed

    Nakamura, F; Suzuki, Y; Yoshimura, F

    1986-01-01

    An immunohistochemical study of the anterior pituitary gland of the female Afghan pika was carried out to distinguish the ultrastructural features of GH, PRL, ACTH, TSH and LH cells. The histochemically identified GH cells resembled ultrastructurally oval or round GH cells of the rat laden with large, dense secretory granules. PRL cells were divided into three subtypes based on differences in the diameter of their spherical secretory granules. They lacked polymorphic or irregularly shaped secretory granules. ACTH cells resembled ultrastructurally, in some respects, Siperstein's "corticotrophs" of the rat with peripheral arrangement of secretory granules. However, they were not always stellate, but elongate or angular in shape. The dense secretory granules were concentrated in the peripheral area of cytoplasm. TSH cells were non-stellate, but usually oval in shape, containing the smallest spherical secretory granules (100-200 nm in diameter). Almost all LH cells reacted also with FSH antiserum. They were irregular in shape, sometimes in contact with or surrounded the GH cells. They contained an abundance of medium-sized secretory granules (140-260 nm in diameter) which were larger than those in the LH cells of the female rat throughout the estrous cycle. Large secretory granules in the LH cells of the female pika seemed to be related to the endocrine state of persistent estrus.

  12. Effect of oral acetylcysteine on tobacco smoke-induced secretory cell hyperplasia.

    PubMed

    Jeffery, P K; Rogers, D F; Ayers, M M

    1985-01-01

    The present investigation explores whether N-acetylcysteine (NAC) inhibits the secretory cell hyperplasia known to occur experimentally in specific pathogen-free (SPF) bronchitic rats. The animals were divided into 4 groups: no tobacco smoke (TS), no drug, no TS but NAC (1040 mg/kg body weight), TS but no drug, and TS plus NAC. NAC-treated animals showed no ill effects, TS exposed animals showed an initial fall in weight gain which never fully recovered (P less than 0.01): NAC did not protect. TS caused a significant increase (62-421%) in secretory cell number at all airway levels distal to the upper trachea (P less than 0.01) and NAC significantly inhibited it (P less than 0.01-0.05) in all, mostly in secretory cells containing acidic glycoprotein. TS exposure also induced a significant rise in epithelial cell concentration and of ciliated, mucous and especially basal cell number (P less than 0.001). NAC inhibited the mucous cell increase (P less than 0.001) and had 3 effects on the peak of dividing cells: it was (a) delayed until 3 days (b) greatly reduced in size and (c) prolonged at a lower level until its return to control values at 10 days of TS exposure.

  13. Enhanced CREB phosphorylation in immature dentate gyrus granule cells precedes neurotrophin expression and indicates a specific role of CREB in granule cell differentiation

    PubMed Central

    Bender, R. A.; Lauterborn, J. C.; Gall, C. M.; Cariaga, W.; Baram, T. Z.

    2011-01-01

    Differentiation and maturation of dentate gyrus granule cells requires coordinated interactions of numerous processes. These must be regulated by protein factors capable of integrating signals mediated through diverse signalling pathways. Such integrators of inter and intracellular physiological stimuli include the cAMP-response element binding protein (CREB), a leucine-zipper class transcription factor that is activated through phosphorylation. Neuronal activity and neurotrophic factors, known to be involved in granule cell differentiation, are major physiologic regulators of CREB function. To examine whether CREB may play a role in governing coordinated gene transcription during granule cell differentiation, we determined the spatial and temporal profiles of phosphorylated (activated) CREB throughout postnatal development in immature rat hippocampus. We demonstrate that CREB activation is confined to discrete, early stages of granule cell differentiation. In addition, CREB phosphorylation occurs prior to expression of the neurotrophins BDNF and NT-3. These data indicate that in a signal transduction cascade connecting CREB and neurotrophins in the process of granule cell maturation, CREB is located upstream of neurotrophins. Importantly, CREB may be a critical component of the machinery regulating the coordinated transcription of genes contributing to the differentiation of granule cells and their integration into the dentate gyrus network. PMID:11207803

  14. Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X-ray tomography

    PubMed Central

    Chen, Huan-Yuan; Chiang, Dapi Meng-Lin; Lin, Zi-Jing; Hsieh, Chia-Chun; Yin, Gung-Chian; Weng, I.-Chun; Guttermann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Lai, Lee-Jene; Liu, Fu-Tong

    2016-01-01

    Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level. PMID:27748356

  15. The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells.

    PubMed

    Villanueva, José; Viniegra, Salvador; Gimenez-Molina, Yolanda; García-Martinez, Virginia; Expósito-Romero, Giovanna; del Mar Frances, Maria; García-Sancho, Javier; Gutiérrez, Luis M

    2014-12-01

    Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca(2+) microdomains described previously.

  16. Pancreatic endoproteases and pancreatic secretory trypsin inhibitor immunoreactivity in human Paneth cells.

    PubMed Central

    Bohe, M; Borgström, A; Lindström, C; Ohlsson, K

    1986-01-01

    Normal and metaplastic gastrointestinal mucosa obtained at surgical resection were studied by light microscopy, using the unlabelled antibody enzyme method for immunohistochemical staining of lysozyme, pancreatic endoproteases, and pancreatic secretory trypsin inhibitor (PSTI). Paneth cells in the mucosa of normal small intestine, gastric mucosa with intestinal metaplasia, and colonic metaplastic mucosa were found to contain anionic trypsin, cationic trypsin, lysozyme, and PSTI immunoreactivity, but not chymotrypsin and elastase immunoreactivity. Normal gastric and colonic mucosa and some goblet cells in the small intestine showed positive PSTI immunoreactivity but no endoprotease immunoreactivity. The presence of immunoreactive trypsin and immunoreactive PSTI in the Paneth cells, which are of secretory type, probably indicates an important extrapancreatic source of these proteins rather than a storage of endocytosed material. Images PMID:3525612

  17. Sparse activity of identified dentate granule cells during spatial exploration

    PubMed Central

    Diamantaki, Maria; Frey, Markus; Berens, Philipp; Preston-Ferrer, Patricia; Burgalossi, Andrea

    2016-01-01

    In the dentate gyrus – a key component of spatial memory circuits – granule cells (GCs) are known to be morphologically diverse and to display heterogeneous activity profiles during behavior. To resolve structure–function relationships, we juxtacellularly recorded and labeled single GCs in freely moving rats. We found that the vast majority of neurons were silent during exploration. Most active GCs displayed a characteristic spike waveform, fired at low rates and showed spatial activity. Primary dendritic parameters were sufficient for classifying neurons as active or silent with high accuracy. Our data thus support a sparse coding scheme in the dentate gyrus and provide a possible link between structural and functional heterogeneity among the GC population. DOI: http://dx.doi.org/10.7554/eLife.20252.001 PMID:27692065

  18. Ultrastructure and cytochemistry of lipid granules in the many-celled magnetotactic prokaryote, 'Candidatus Magnetoglobus multicellularis'.

    PubMed

    Silva, Karen Tavares; Abreu, Fernanda; Keim, Carolina N; Farina, Marcos; Lins, Ulysses

    2008-12-01

    Conspicuous cytoplasmic granules are reported in a magnetotactic multicellular prokaryote named 'Candidatus Magnetoglobus multicellularis'. Unfortunately, this microorganism, which consists of an assembly of gram-negative bacterial cells, cannot yet be cultivated, limiting the biochemical analysis of the granules and preventing in vitro studies with starvation/excess of nutrients. In this scenario, light and electron microscopy techniques were used to partially address the nature of the granules. Besides magnetosomes, three types of inclusions were observed: small (mean diameter=124 nm) polyhydroxyalkanoate-like (PHA) granules, large (diameters ranging from 0.11 to 2.5 microm) non-PHA lipid granules, and rare phosphorus-rich granules, which probably correspond to polyphosphate bodies. The PHA granules were rounded in projection, non-reactive with OsO(4), and suffered the typical plastic deformation of PHAs after freeze fracturing. The nature of the large granules, consisting of round globular structures (mean diameter=0.76 microm), was classified as non-PHA based on the following data: (a) multilayered structure in freeze-fracture electron microscopy, typical of non-PHA lipids; (b) Nile blue fluorescence imaging detected non-PHA lipids; (c) imidazole buffered osmium tetroxide and ruthenium red cytochemistry stained the globules, which appeared as electron-dense granules instead of electron lucent as PHAs do. Most likely, 'Candidatus Magnetoglobus multicellularis' stores carbon mainly as unusual lipid granules, together with smaller amounts of PHAs.

  19. Delayed release of neurotransmitter from cerebellar granule cells.

    PubMed

    Atluri, P P; Regehr, W G

    1998-10-15

    At fast chemical synapses the rapid release of neurotransmitter that occurs within a few milliseconds of an action potential is followed by a more sustained elevation of release probability, known as delayed release. Here we characterize the role of calcium in delayed release and test the hypothesis that facilitation and delayed release share a common mechanism. Synapses between cerebellar granule cells and their postsynaptic targets, stellate cells and Purkinje cells, were studied in rat brain slices. Presynaptic calcium transients were measured with calcium-sensitive fluorophores, and delayed release was detected with whole-cell recordings. Calcium influx, presynaptic calcium dynamics, and the number of stimulus pulses were altered to assess their effect on delayed release and facilitation. Following single stimuli, delayed release can be separated into two components: one lasting for tens of milliseconds that is steeply calcium-dependent, the other lasting for hundreds of milliseconds that is driven by low levels of calcium with a nearly linear calcium dependence. The amplitude, calcium dependence, and magnitude of delayed release do not correspond to those of facilitation, indicating that these processes are not simple reflections of a shared mechanism. The steep calcium dependence of delayed release, combined with the large calcium transients observed in these presynaptic terminals, suggests that for physiological conditions delayed release provides a way for cells to influence their postsynaptic targets long after their own action potential activity has subsided.

  20. Giant cytoplasmic granules in Langerhans cells of Chediak-Higashi syndrome.

    PubMed

    Carrillo-Farga, J; Gutiérrez-Palomera, G; Ruiz-Maldonado, R; Rondán, A; Antuna, S

    1990-02-01

    Giant membrane-bound cytoplasmic granules were found in the epidermal Langerhans cells of a patient with the Chediak-Higashi syndrome. These cells also contained normal-appearing Birbeck granules. The giant granules had a granular or sometimes globular internal structure; they are believed to derive from fusion of lysosomes or some portion of Birbeck granules. It is unclear whether this morphologic change in Langerhans cell interferes with their antigen-presenting function; it may be, in part, responsible for the frequent infections seen in patients with Chediak-Higashi syndrome that are otherwise more clearly related to the abnormalities in neutrophils and lymphocytes. The Langerhans cell is another cellular type in Chediak-Higashi syndrome in which giant cytoplasmic granules are found.

  1. Differentiated cytoplasmic granule formation in quiescent and non-quiescent cells upon chronological aging

    PubMed Central

    Lee, Hsin-Yi; Cheng, Kuo-Yu; Chao, Jung-Chi; Leu, Jun-Yi

    2016-01-01

    Stationary phase cultures represent a complicated cell population comprising at least two different cell types, quiescent (Q) and non-quiescent (NQ) cells. Q and NQ cells have different lifespans and cell physiologies. However, less is known about the organization of cytosolic protein structures in these two cell types. In this study, we examined Q and NQ cells for the formation of several stationary phase-prevalent granule structures including actin bodies, proteasome storage granules, stress granules, P-bodies, the compartment for unconventional protein secretion (CUPS), and Hsp42-associated stationary phase granules (Hsp42-SPGs). Most of these structures preferentially form in NQ cells, except for Hsp42-SPGs, which are enriched in Q cells. When nutrients are provided, NQ cells enter mitosis less efficiently than Q cells, likely due to the time requirement for reorganizing some granule structures. We observed that heat shock-induced misfolded proteins often colocalize to Hsp42-SPGs, and Q cells clear these protein aggregates more efficiently, suggesting that Hsp42-SPGs may play an important role in the stress resistance of Q cells. Finally, we show that the cell fate of NQ cells is largely irreversible even if they are allowed to reenter mitosis. Our results reveal that the formation of different granule structures may represent the early stage of cell type differentiation in yeast stationary phase cultures. PMID:28357341

  2. Statistical Frailty Modeling for Quantitative Analysis of Exocytotic Events Recorded by Live Cell Imaging: Rapid Release of Insulin-Containing Granules Is Impaired in Human Diabetic β-cells

    PubMed Central

    Cortese, Giuliana; Gandasi, Nikhil R.; Barg, Sebastian; Pedersen, Morten Gram

    2016-01-01

    Hormones and neurotransmitters are released when secretory granules or synaptic vesicles fuse with the cell membrane, a process denoted exocytosis. Modern imaging techniques, in particular total internal reflection fluorescence (TIRF) microscopy, allow the investigator to monitor secretory granules at the plasma membrane before and when they undergo exocytosis. However, rigorous statistical approaches for temporal analysis of such exocytosis data are still lacking. We propose here that statistical methods from time-to-event (also known as survival) analysis are well suited for the problem. These methods are typically used in clinical settings when individuals are followed over time to the occurrence of an event such as death, remission or conception. We model the rate of exocytosis in response to pulses of stimuli in insulin-secreting pancreatic β-cell from healthy and diabetic human donors using piecewise-constant hazard modeling. To study heterogeneity in the granule population we exploit frailty modeling, which describe unobserved differences in the propensity to exocytosis. In particular, we insert a discrete frailty in our statistical model to account for the higher rate of exocytosis in an immediately releasable pool (IRP) of insulin-containing granules. Estimates of parameters are obtained from maximum-likelihood methods. Since granules within the same cell are correlated, i.e., the data are clustered, a modified likelihood function is used for log-likelihood ratio tests in order to perform valid inference. Our approach allows us for example to estimate the size of the IRP in the cells, and we find that the IRP is deficient in diabetic cells. This novel application of time-to-event analysis and frailty modeling should be useful also for the study of other well-defined temporal events at the cellular level. PMID:27907065

  3. Down-regulation of zinc transporter 8 (SLC30A8) in pancreatic beta-cells promotes cell survival

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pancreatic islet contains high levels of zinc in granular vesicles of beta-cells where insulin is matured, crystallized, and stored before secretion. Zinc is an essential co-factor for insulin crystallization forming dense core in secretory granules. In insulin-containing secretory granules, zin...

  4. Down-regulation of zinc transporter 8 (SLC30A8) in pancreatic beta-cells promotes cell survival.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pancreatic islet contains high levels of zinc in granular vesicles of ß-cells where insulin is matured, crystallized, and stored before secretion. Zinc is an essential co-factor for insulin crystallization forming dense cores in secretory granules. In insulin-containing secretory granules, zinc ...

  5. Analysis of Spine Motility of Newborn Granule Cells in Acute Brain Slices.

    PubMed

    Tashiro, Ayumu; Zhao, Chunmei; Suh, Hoonkyo; Gage, Fred H

    2015-10-01

    In this protocol, acute brain slices are prepared from mice in which newborn granule cells have been labeled using retroviral vector technology. Using a live-cell imaging stage and confocal microscopy coupled to imaging software, dendritic spines are analyzed.

  6. Modulation of Sertoli cell secretory function by rat round spermatid protein(s).

    PubMed

    Onoda, M; Djakiew, D

    1990-10-01

    The influence of rat round spermatid protein(s) (RSP) on protein synthesis and secretory function of Sertoli cells was used in the bicameral chamber system. Round spermatids (RS) were purified from 90-day-old rats by centrifugal elutriation. RS were incubated in a supplement-enriched culture medium that lacked exogenous proteins. The RS-conditioned media were dialysed and lyophilized to obtain RSP. Most de novo protein synthesized under basal conditions by Sertoli cells (18-day-old) was secreted into the apical chamber (apical/basal ratio: 3.42). Follicle-stimulating hormone (FSH, 100 ng/ml) stimulated total protein secretion from Sertoli cells by a factor of 1.54. The RSP (100 micrograms/ml) stimulated total protein secretion from Sertoli cells by a factor of 2.33. The enhancement of total Sertoli cell protein secretion by FSH and RSP additively increased by a factor of 2.82. The combined effect of FSH and RSP on total protein secretion from Sertoli cells was dose dependent and saturated at approximately 200 micrograms/ml of RSP. Polarity of total protein secretion from Sertoli cells (apical/basal ratio: 3.42) was stimulated by RSP predominantly in the apical direction (apical/basal ratio: 8.48). The modulation of radiolabeled Sertoli cell secretory proteins (ceruloplasmin, CP; sulfated glycoprotein-2, SGP-2; testins and transferrin, Tf) by cold (non-labeled) RSP was investigated by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The secretion of CP, SGP-2 and Tf was stimulated in a dose-dependent manner by the addition of RSP up to a saturating concentration of between 200 and 300 micrograms/ml, whereas the secretion of Sertoli cell testins did not reach saturation at 300 micrograms/ml RSP. These results indicate that FSH and RSP independently modulate Sertoli cell protein secretion, and that Sertoli cell secretory proteins may differentially respond to RSP stimulation.

  7. Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells.

    PubMed

    Cesaratto, Francesca; López-Requena, Alejandro; Burrone, Oscar R; Petris, Gianluca

    2015-10-20

    Tobacco etch virus protease (TEVp) is a unique endopeptidase with stringent substrate specificity. TEVp has been widely used as a purified protein for in vitro applications, but also as a biological tool directly expressing it in living cells. To adapt the protease to diverse applications, several TEVp mutants with different stability and enzymatic properties have been reported. Herein we describe the development of a novel engineered TEVp mutant designed to be active in the secretory pathway. While wild type TEVp targeted to the secretory pathway of mammalian cells is synthetized as an N-glycosylated and catalytically inactive enzyme, a TEVp mutant with selected mutations at two verified N-glycosylation sites and at an exposed cysteine was highly efficient. This mutant was very active in the endoplasmic reticulum (ER) of living cells and can be used as a biotechnological tool to cleave proteins within the secretory pathway. As an immediate practical application we report the expression of a complete functional monoclonal antibody expressed from a single polypeptide, which was cleaved by our TEVp mutant into the two antibody chains and secreted as an assembled and functional molecule. In addition, we show active TEVp mutants lacking auto-cleavage activity.

  8. Intracellular ion concentrations and cell volume during cholinergic stimulation of eccrine secretory coil cells

    SciTech Connect

    Takemura, T.; Sato, F.; Saga, K.; Suzuki, Y.; Sato, K. )

    1991-02-01

    Methacholine (MCh)-induced changes in intracellular concentrations of Na, K, and Cl (( Na)i, (K)i, and (Cl)i, respectively) and in cellular dry mass (a measure of cell shrinkage) were examined in isolated monkey eccrine sweat secretory coils by electron probe X-ray microanalysis using the peripheral standard method. To further confirm the occurrence of cell shrinkage during MCh stimulation, the change in cell volume of dissociated clear and dark cells were directly determined under a light microscope equipped with differential interference contrast (DIC) optics. X-ray microanalysis revealed a biphasic increase in cellular dry mass in clear cells during continuous MCh stimulation; an initial increase of dry mass to 158% (of control) followed by a plateau at 140%, which correspond to the decrease in cell volume of 37 and 29%, respectively. The latter agrees with the MCh-induced cell shrinkage of 29% in dissociated clear cells. The MCh-induced increase in dry mass in myoepithelial cells was less than half that of clear cells. During the steady state of MCh stimulation, both (K+)i and (Cl)i of clear cells decreased by about 45%, whereas (Na)i increased in such a way to maintain the sum of (Na) i + (K)i constant. There was a small (12-15 mM) increase in (Na)i and a decrease in (K)i in myoepithelial cells during stimulation with MCh. Dissociated dark cells failed to significantly shrink during MCh stimulation. The decrease in (Cl)i in the face of constant (Na)i + (K)i suggests the accumulation of unknown anion(s) inside the clear cell during MCh stimulation.

  9. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus.

    PubMed

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-03-19

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7*nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7*nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2-3 week-old Wistar rats, and 2-9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7*nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7*nicotinic receptor modulator, which were blocked by a specific α7*nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7*nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7*nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain.

  10. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus

    PubMed Central

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-01-01

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7⁎nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7⁎nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2–3 week-old Wistar rats, and 2–9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7⁎nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7⁎nicotinic receptor modulator, which were blocked by a specific α7⁎nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7⁎nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7⁎nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain. PMID:25553616

  11. Internalized Tau sensitizes cells to stress by promoting formation and stability of stress granules

    PubMed Central

    Brunello, Cecilia A.; Yan, Xu; Huttunen, Henri J.

    2016-01-01

    Stress granules are membrane-less RNA- and RNA-binding protein-containing complexes that are transiently assembled in stressful conditions to promote cell survival. Several stress granule-associated RNA-binding proteins have been associated with neurodegenerative diseases. In addition, a close link was recently identified between the stress granule core-nucleating protein TIA-1 and Tau. Tau is a central pathological protein in Alzheimer’s disease and other tauopathies, and misfolded, aggregated Tau is capable of propagating pathology via cell-to-cell transmission. Here we show that following internalization hyperphosphorylated extracellular Tau associates with stress granules in a TIA-1 dependent manner. Cytosolic Tau normally only weakly interacts with TIA-1 but mutations mimicking abnormal phosphorylation promote this interaction. We show that internalized Tau significantly delays normal clearance of stress granules in the recipient cells sensitizing them to secondary stress. These results suggest that secreted Tau species may have properties, likely related to its hyperphosphorylation and oligomerization, which promote pathological association of internalized Tau with stress granules altering their dynamics and reducing cell viability. We suggest that stress granules and TIA-1 play a central role in the cell-to-cell transmission of Tau pathology. PMID:27460788

  12. Sequential Immunoprecipitation of Secretory Vesicle Proteins from Biosynthetically Labelled Cells.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.

  13. Somatic translocation: a novel mechanism of granule cell dendritic dysmorphogenesis and dispersion

    PubMed Central

    Murphy, Brian L.; Danzer, Steve C.

    2011-01-01

    Pronounced neuronal remodeling is a hallmark of temporal lobe epilepsy. Here, we use real-time confocal imaging of tissue from mouse brain to demonstrate that remodeling can involve fully-differentiated granule cells following translocation of the soma into an existing apical dendrite. Somatic translocation converts dendritic branches into primary dendrites and shifts adjacent apical dendrites to the basal pole of the cell. Moreover, somatic translocation contributes to the dispersion of the granule cell body layer in vitro, and when granule cell dispersion is induced in vivo, the dispersed cells exhibit virtually identical derangements of their dendritic structures. Together, these findings identify novel forms of neuronal plasticity which contribute to granule cell dysmorphogenesis in the epileptic brain. PMID:21414917

  14. Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults

    SciTech Connect

    Gianotti, A.J.; Clark, D.T.; Dash, J. )

    1991-04-01

    A source of confusion has existed since the turn of the century about the reddish brown, weakly birefringent 'sphaerocrystals' located in the intestines of strongyle nematodes, Strongylus and Ancylostoma. X-ray diffraction and energy dispersive spectrometric analyses were used for accurate determination of the crystalline order and elemental composition of the granules in the canine hookworm Ancylostoma caninum. The composition of the intestinal pigmented granules was identified unequivocally as zinc sulfide. It seems most probable that the granules serve to detoxify high levels of metallic ions (specifically zinc) present due to the large intake of host blood.

  15. Potassium currents in acutely isolated human hippocampal dentate granule cells.

    PubMed Central

    Beck, H; Clusmann, H; Kral, T; Schramm, J; Heinemann, U; Elger, C E

    1997-01-01

    1. Properties of voltage- and Ca(2+)-dependent K+ currents were investigated in thirty-four dentate granule cells acutely isolated from the resected hippocampus of eleven patients with therapy-refractory temporal lobe epilepsy (TLE). 2. When intracellular Ca2+ was strongly buffered with 11.5 mM EGTA-1 mM Ca2+ in the recording pipette, K+ currents (IK) with a slow activation and biexponential time-dependent decay could be elicited, which showed a threshold for activation around -30 mV. 3. A contribution of Ca(2+)-dependent K+ currents became apparent with intracellular solution containing 1 mM BAPTA-0.1 mM Ca2+. Superfusion of low-Ca2+ extracellular solution blocked 43% of outward currents in this recording configuration. Outward current components could also be blocked by substituting 5 mM Ba2+ for extracellular Ca2+ (78%), or by application of 100 microM Cd2+ (25%). 4. The Ca(2+)-dependent K+ currents could be pharmacologically subdivided into two components. One component was sensitive to 500 microM tetraethylammmonium (TEA; 41%) and 10 nM charybdotoxin (CTX; 47.2%). The blocking effects of 10 nM CTX and 500 microM TEA were not additive, suggesting that both agents block the same conductance. A second, smaller outward current component was blocked by 50 nM apamin (13%). 5. A transient A-type K+ current could be observed in six neurones and showed a fast monoexponential time-dependent inactivation with a steady-state voltage dependence that was distinct from that of IK. The A-type current was blocked by 4-aminopyridine (4-AP) but not by TEA or low-Ca2+ solution. 6. We conclude that outward currents in human hippocampal dentate granule cells can be separated into at least four types by their kinetic and pharmacological properties. These include at least one voltage-dependent current similar to those observed in mammalian hippocampal neurones, and two Ca(2+)-dependent K+ currents that most probably correspond to SK- and BK-type currents. A classical A-type current

  16. Functional differentiation of cholinergic and noradrenergic modulation in a biophysical model of olfactory bulb granule cells

    PubMed Central

    Linster, Christiane

    2015-01-01

    Olfactory bulb granule cells are modulated by both acetylcholine (ACh) and norepinephrine (NE), but the effects of these neuromodulators have not been clearly distinguished. We used detailed biophysical simulations of granule cells, both alone and embedded in a microcircuit with mitral cells, to measure and distinguish the effects of ACh and NE on cellular and microcircuit function. Cholinergic and noradrenergic modulatory effects on granule cells were based on data obtained from slice experiments; specifically, ACh reduced the conductance densities of the potassium M current and the calcium-dependent potassium current, whereas NE nonmonotonically regulated the conductance density of an ohmic potassium current. We report that the effects of ACh and NE on granule cell physiology are distinct and functionally complementary to one another. ACh strongly regulates granule cell firing rates and afterpotentials, whereas NE bidirectionally regulates subthreshold membrane potentials. When combined, NE can regulate the ACh-induced expression of afterdepolarizing potentials and persistent firing. In a microcircuit simulation developed to investigate the effects of granule cell neuromodulation on mitral cell firing properties, ACh increased spike synchronization among mitral cells, whereas NE modulated the signal-to-noise ratio. Coapplication of ACh and NE both functionally improved the signal-to-noise ratio and enhanced spike synchronization among mitral cells. In summary, our computational results support distinct and complementary roles for ACh and NE in modulating olfactory bulb circuitry and suggest that NE may play a role in the regulation of cholinergic function. PMID:26334007

  17. Functional differentiation of cholinergic and noradrenergic modulation in a biophysical model of olfactory bulb granule cells.

    PubMed

    Li, Guoshi; Linster, Christiane; Cleland, Thomas A

    2015-12-01

    Olfactory bulb granule cells are modulated by both acetylcholine (ACh) and norepinephrine (NE), but the effects of these neuromodulators have not been clearly distinguished. We used detailed biophysical simulations of granule cells, both alone and embedded in a microcircuit with mitral cells, to measure and distinguish the effects of ACh and NE on cellular and microcircuit function. Cholinergic and noradrenergic modulatory effects on granule cells were based on data obtained from slice experiments; specifically, ACh reduced the conductance densities of the potassium M current and the calcium-dependent potassium current, whereas NE nonmonotonically regulated the conductance density of an ohmic potassium current. We report that the effects of ACh and NE on granule cell physiology are distinct and functionally complementary to one another. ACh strongly regulates granule cell firing rates and afterpotentials, whereas NE bidirectionally regulates subthreshold membrane potentials. When combined, NE can regulate the ACh-induced expression of afterdepolarizing potentials and persistent firing. In a microcircuit simulation developed to investigate the effects of granule cell neuromodulation on mitral cell firing properties, ACh increased spike synchronization among mitral cells, whereas NE modulated the signal-to-noise ratio. Coapplication of ACh and NE both functionally improved the signal-to-noise ratio and enhanced spike synchronization among mitral cells. In summary, our computational results support distinct and complementary roles for ACh and NE in modulating olfactory bulb circuitry and suggest that NE may play a role in the regulation of cholinergic function.

  18. Granulated peripolar epithelial cells in the renal corpuscle of marine elasmobranch fish.

    PubMed

    Lacy, E R; Reale, E

    1989-07-01

    Granulated epithelial cells at the vascular pole of the renal corpuscle, peripolar cells, have been found in the kidneys of five species of elasmobranchs, the little skate (Raja erinacea), the smooth dogfish shark (Mustelus canis), the Atlantic sharpnose shark (Rhizoprionodon terraenovae), the scalloped hammerhead shark (Sphyrna lewini), and the cow-nosed ray (Rhinoptera bonasus). In a sixth elasmobranch, the spiny dogfish shark (Squalus acanthias), the peripolar cells could not be identified among numerous other granulated epithelial cells. The peripolar cells are located at the transition between the parietal epithelium of Bowman's capsule and the visceral epithelium (podocytes) of the glomerulus, thus forming a cuff-like arrangement surrounding the hilar vessels of the renal corpuscle. These cells may have granules and/or vacuoles. Electron microscopy shows that the granules are membrane-bounded, and contain either a homogeneous material or a paracrystalline structure with a repeating period of about 18 nm. The vacuoles are electron lucent or may contain remnants of a granule. These epithelial cells lie close to the granulated cells of the glomerular afferent arteriole. They correspond to the granular peripolar cells of the mammalian, avian and amphibian kidney. The present study is the first reported occurrence of peripolar cells in a marine organism or in either bony or cartilagenous fish.

  19. The Fas pathway is involved in pancreatic β cell secretory function

    PubMed Central

    Schumann, Desiree M.; Maedler, Kathrin; Franklin, Isobel; Konrad, Daniel; Størling, Joachim; Böni-Schnetzler, Marianne; Gjinovci, Asllan; Kurrer, Michael O.; Gauthier, Benoit R.; Bosco, Domenico; Andres, Axel; Berney, Thierry; Greter, Melanie; Becher, Burkhard; Chervonsky, Alexander V.; Halban, Philippe A.; Mandrup-Poulsen, Thomas; Wollheim, Claes B.; Donath, Marc Y.

    2007-01-01

    Pancreatic β cell mass and function increase in conditions of enhanced insulin demand such as obesity. Failure to adapt leads to diabetes. The molecular mechanisms controlling this adaptive process are unclear. Fas is a death receptor involved in β cell apoptosis or proliferation, depending on the activity of the caspase-8 inhibitor FLIP. Here we show that the Fas pathway also regulates β cell secretory function. We observed impaired glucose tolerance in Fas-deficient mice due to a delayed and decreased insulin secretory pattern. Expression of PDX-1, a β cell-specific transcription factor regulating insulin gene expression and mitochondrial metabolism, was decreased in Fas-deficient β cells. As a consequence, insulin and ATP production were severely reduced and only partly compensated for by increased β cell mass. Up-regulation of FLIP enhanced NF-κB activity via NF-κB-inducing kinase and RelB. This led to increased PDX-1 and insulin production independent of changes in cell turnover. The results support a previously undescribed role for the Fas pathway in regulating insulin production and release. PMID:17299038

  20. Formation of tRNA granules in the nucleus of heat-induced human cells

    SciTech Connect

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

  1. Fine structure of the Caenorhabditis elegans secretory-excretory system.

    PubMed

    Nelson, F K; Albert, P S; Riddle, D L

    1983-02-01

    The secretory-excretory system of C. elegans, reconstructed from serial-section electron micrographs of larvae, is composed of four cells, the nuclei of which are located on the ventral side of the pharynx and adjacent intestine. (1) The pore cell encloses the terminal one-third of the excretory duct which leads to an excretory pore at the ventral midline. (2) The duct cell surrounds the excretory duct with a lamellar membrane from the origin of the duct at the excretory sinus to the pore cell boundary. (3) A large H-shaped excretory cell extends bilateral canals anteriorly and posteriorly nearly the entire length of the worm. The excretory sinus within the cell body joins the lumena of the canals with the origin of the duct. (4) A binucleate, A-shaped gland cell extends bilateral processes anteriorly from cell bodies located just behind the pharynx. These processes are fused at the anterior tip of the cell, where the cell enters the circumpharyngeal nerve ring. The processes are also joined at the anterior edge of the excretory cell body, where the excretory cell and gland are joined to the duct cell at the origin of the duct. Secretory granules may be concentrated in the gland near this secretory-excretory junction. Although the gland cells of all growing developmental stages stain positively with paraldehyde-fuchsin, the gland of the dauer larva stage (a developmentally arrested third-stage larva) does not stain, nor do glands of starved worms of other stages. Dauer larvae uniquely lack secretory granules, and the gland cytoplasm is displaced by a labyrinth of large, transparent spaces. Exit from the dauer stage results in the return of active secretory morphology in fourth-stage larvae.

  2. Prox1 postmitotically defines dentate gyrus cells by specifying granule cell identity over CA3 pyramidal cell fate in the hippocampus.

    PubMed

    Iwano, Tomohiko; Masuda, Aki; Kiyonari, Hiroshi; Enomoto, Hideki; Matsuzaki, Fumio

    2012-08-01

    The brain is composed of diverse types of neurons that fulfill distinct roles in neuronal circuits, as manifested by the hippocampus, where pyramidal neurons and granule cells constitute functionally distinct domains: cornu ammonis (CA) and dentate gyrus (DG), respectively. Little is known about how these two types of neuron differentiate during hippocampal development, although a set of transcription factors that is expressed in progenitor cells is known to be required for the survival of granule cells. Here, we demonstrate in mice that Prox1, a transcription factor constitutively expressed in the granule cell lineage, postmitotically functions to specify DG granule cell identity. Postmitotic elimination of Prox1 caused immature DG neurons to lose the granule cell identity and in turn terminally differentiate into the pyramidal cell type manifesting CA3 neuronal identity. By contrast, Prox1 overexpression caused opposing effects on presumptive hippocampal pyramidal cells. These results indicate that the immature DG cell has the potential to become a granule cell or a pyramidal cell, and Prox1 defines the granule cell identity. This bi-potency is lost in mature DG cells, although Prox1 is still required for correct gene expression in DG granule cells. Thus, our data indicate that Prox1 acts as a postmitotic cell fate determinant for DG granule cells over the CA3 pyramidal cell fate and is crucial for maintenance of the granule cell identity throughout the life.

  3. [Effect of plant hormones on the components of secretory pathway in human normal and tumor cells].

    PubMed

    Vil'danova, M S; Savitskaia, M A; Onishchenko, G E; Smirnova, E A

    2014-01-01

    Plant hormones play a key role in plant growth and differentiation. Many hormones are known as potential antitumor agents, yet others appear to affect the secretory activity and are produced by mammalian cells as pro-inflammatory cytokines. The goal of this research was to study the effect of abscisic and gibberellic acids on the secretory system of human cultured epidermoid carcinoma cells A431 and keratinocytes HaCat. Immunocytochemical and morphometric analysis demonstrated that subtoxic concentration of plant hormones induced the broadening of the ER network and increased the size of Golgi complex. Electron microscopy studies confirmed the hypertrophic changes of the Golgi apparatus, specifically, the swelling of cisternae in the trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartments of dictyosomes after exposure to gibberellic acid. Using of Click-iT technique allowed to detect the elevation of the total protein synthesis only in A431 cells exposed to abscisic acid. Cumulative data suggests that, under these conditions, the hypertrophy of Golgi apparatus may reflect the enhanced secretory activity of cells. In other experiments, the hypertrophy of Golgi is not related to increased protein synthesis and therefore may suggest the stress-related changes of ER and Golgi apparatus. Our results demonstrate that morphologically similar reaction of cellular organelles, such as hypertrophy of Golgi apparatus, is the result of different functional activities, and that molecular mechanisms underlying the changes induced in cells need further investigations.

  4. Automatic detection of large dense-core vesicles in secretory cells and statistical analysis of their intracellular distribution.

    PubMed

    Díaz, Ester; Ayala, Guillermo; Díaz, María Elena; Gong, Liang-Wei; Toomre, Derek

    2010-01-01

    Analyzing the morphological appearance and the spatial distribution of large dense-core vesicles (granules) in the cell cytoplasm is central to the understanding of regulated exocytosis. This paper is concerned with the automatic detection of granules and the statistical analysis of their spatial locations in different cell groups. We model the locations of granules of a given cell as a realization of a finite spatial point process and the point patterns associated with the cell groups as replicated point patterns of different spatial point processes. First, an algorithm to segment the granules using electron microscopy images is proposed. Second, the relative locations of the granules with respect to the plasma membrane are characterized by two functional descriptors: the empirical cumulative distribution function of the distances from the granules to the plasma membrane and the density of granules within a given distance to the plasma membrane. The descriptors of the different cells for each group are compared using bootstrap procedures. Our results show that these descriptors and the testing procedure allow discriminating between control and treated cells. The application of these novel tools to studies of secretion should help in the analysis of diseases associated with dysfunctional secretion, such as diabetes.

  5. Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells

    PubMed Central

    Fu, Zhanyan; Vicini, Stefano

    2009-01-01

    Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2(NLG2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABAA (γ-aminobutyric acid) receptor mediated miniature postsynaptic currents (mISPCs). NLG2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV 7-8). Down-regulation of NLG2 by the isoform specific shRNA-NLG2 resulted in an opposite effect. We and others have shown that the switch of α subunits of GABAA Rs from α2/3 to α1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG2 is due to the recruitment of more α1 containing GABAARs at the synapses, we examined the prolongation of current decay by the zolpidem, which has been shown to preferentially enhance the activity of α1 subunit containing GABA channel. The application of zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG2 over-expressing cells than control cells, suggesting that NLG2 over-expression accelerates synapse maturation by promoting incorporation of the α1 subunit-containing GABAARs at postsynaptic sites in immature cells. In addition, the effect of NLG2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from α1-/- mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG2 could be also due to the impacts of NLG2 on the GABA transient in synaptic cleft, we measured the sensitivity of mIPSCs to the fast-off competitive antagonists TPMPA. We found that TPMPA similarly inhibits mIPSCs in control and NLG2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of cultures. However, we confirm our previous

  6. Reduced inhibition of dentate granule cells in a model of temporal lobe epilepsy.

    PubMed

    Kobayashi, Masayuki; Buckmaster, Paul S

    2003-03-15

    Patients and models of temporal lobe epilepsy have fewer inhibitory interneurons in the dentate gyrus than controls, but it is unclear whether granule cell inhibition is reduced. We report the loss of GABAergic inhibition of granule cells in the temporal dentate gyrus of pilocarpine-induced epileptic rats. In situ hybridization for GAD65 mRNA and immunocytochemistry for parvalbumin and somatostatin confirmed the loss of inhibitory interneurons. In epileptic rats, granule cells had prolonged EPSPs, and they discharged more action potentials than controls. Although the conductances of evoked IPSPs recorded in normal ACSF were not significantly reduced and paired-pulse responses showed enhanced inhibition of granule cells from epileptic rats, more direct measures of granule cell inhibition revealed significant deficiencies. In granule cells from epileptic rats, evoked monosynaptic IPSP conductances were <40% of controls, and the frequency of GABA(A) receptor-mediated spontaneous and miniature IPSCs (mIPSCs) was <50% of controls. Within 3-7 d after pilocarpine-induced status epilepticus, miniature IPSC frequency had decreased, and it remained low, without functional evidence of compensatory synaptogenesis by GABAergic axons in chronically epileptic rats. Both parvalbumin- and somatostatin-immunoreactive interneuron numbers and the frequency of both fast- and slow-rising GABA(A) receptor-mediated mIPSCs were reduced, suggesting that loss of inhibitory synaptic input to granule cells occurred at both proximal/somatic and distal/dendritic sites. Reduced granule cell inhibition in the temporal dentate gyrus preceded the onset of spontaneous recurrent seizures by days to weeks, so it may contribute, but is insufficient, to cause epilepsy.

  7. Cysteamine depletes prolactin (PRL) but does not alter the structure of PRL-containing granules in the anterior pituitary

    SciTech Connect

    Weinstein, L.A.; Landis, D.M.; Sagar, S.M.; Millard, W.J.; Martin, J.B.

    1984-10-01

    Cysteamine causes a profound depletion of PRL in the anterior pituitary and in the systemic circulation, as measured by RIA and bioassay. However, electron microscopic study of PRL-containing cells in rat anterior pituitary does not reveal changes in secretory granule or cytoplasmic structure during the interval of depressed PRL content and of subsequent recovery to normal levels. In contrast to the results obtained by RIA, PRL-like immunoreactivity as detected by immunocyto-chemistry is present and similar to that of control preparations after cysteamine administration. We suggest that cysteamine alters PRL structure in secretory granules, probably by interacting with the disulfide bonds of PRL, thereby altering bioactivity and immunoreactivity. The presence of cysteamine-altered PRL in secretory granules does not seem to trigger degradation of granules by the lysosomal system.

  8. A micromethod for the assay of cellular secretory physiology: Application to rabbit parietal cells

    SciTech Connect

    Adrian, T.E.; Goldenring, J.R.; Oddsdottir, M.; Zdon, M.J.; Zucker, K.A.; Lewis, J.J.; Modlin, I.M. )

    1989-11-01

    A micromethod for investigating secretory physiology in isolated cells was evaluated. The method utilized a specially designed polycarbonate incubation chamber to provide constant oxygenation to cells incubating in a 96-well microtiter plate. Cells were rapidly separated from media by vacuum filtration. Isolated parietal cells were utilized to demonstrate the versatility of the method for assay of intracellular accumulation of ({sup 14}C)-aminopyrine, secretion of intrinsic factor into the medium, and assay of intracellular cAMP. Histamine stimulated the uptake of ({sup 14}C)aminopyrine and intrinsic factor secretion in a sustained and linear fashion. At the end of the 2-h period uptake of aminopyrine and secretion of intrinsic factor were increased 17- and 5-fold, respectively. This response to histamine was accompanied by a rapid and sustained 3-fold rise in intracellular cyclic AMP. In contrast, carbamylcholine caused a transient increase in ({sup 14}C)aminopyrine accumulation and intrinsic factor secretion which was most pronounced during the first 10 min and had almost ceased by 30 min. Carbamylcholine had no effect on intracellular cAMP levels. This new method, which can handle 400 replicates using parietal cells from the fundic mucosa of a single rabbit, is suitable for studying the time course of intracellular events which accompany general secretory processes.

  9. The biology and dynamics of mammalian cortical granules.

    PubMed

    Liu, Min

    2011-11-17

    Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals.

  10. The biology and dynamics of mammalian cortical granules

    PubMed Central

    2011-01-01

    Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals. PMID:22088197

  11. De novo epidermal regeneration using human eccrine sweat gland cells: higher competence of secretory over absorptive cells.

    PubMed

    Pontiggia, Luca; Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Oliveira, Carol; Braziulis, Erik; Klar, Agnieszka S; Meuli-Simmen, Claudia; Meuli, Martin; Reichmann, Ernst

    2014-06-01

    In our previous work, we showed that human sweat gland-derived epithelial cells represent an alternative source of keratinocytes to grow a near normal autologous epidermis. The role of subtypes of sweat gland cells in epidermal regeneration and maintenance remained unclear. In this study, we compare the regenerative potential of both secretory and absorptive sweat gland cell subpopulations. We demonstrate the superiority of secretory over absorptive cells in forming a new epidermis on two levels: first, the proliferative and colony-forming efficiencies in vitro are significantly higher for secretory cells (SCs), and second, SCs show a higher frequency of successful epidermis formation as well as an increase in the thickness of the formed epidermis in the in vitro and in vivo functional analyses using a 3D dermo-epidermal skin model. However, the ability of forming functional skin substitutes is not limited to SCs, which supports the hypothesis that multiple subtypes of sweat gland epithelial cells hold regenerative properties, while the existence and exact localization of a keratinocyte stem cell population in the human eccrine sweat gland remain elusive.

  12. Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

    SciTech Connect

    Choi, Bongkun; Kang, Soon-Suk; Kang, Sang-Wook; Min, Bon-Hong; Lee, Eun-Jin; Song, Da-Hyun; Kim, Sang-Min; Song, Youngsup; Yoon, Seung-Yong; Chang, Eun-Ju

    2014-07-18

    Highlights: • We describe the expression and secretion of clusterin in osteoclasts. • Endogenous clusterin deficiency does not affect osteoclast formation. • Exogenous treatment with secretory clusterin decreases osteoclast differentiation. • Secretory clusterin attenuates osteoclast precursor cell proliferation by inhibiting M-CSF-mediated ERK activation. - Abstract: Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU{sup −/−} mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

  13. Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

    SciTech Connect

    Kierszenbaum, A.L.; Crowell, J.A.; Shabanowitz, R.B.; DePhilip, R.M.; Tres, L.L.

    1986-08-01

    An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates.

  14. Mature eosinophils stimulated to develop in human-cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. II. Vesicular transport of specific granule matrix peroxidase, a mechanism for effecting piecemeal degranulation.

    PubMed Central

    Dvorak, A. M.; Ackerman, S. J.; Furitsu, T.; Estrella, P.; Letourneau, L.; Ishizaka, T.

    1992-01-01

    The mechanism of piecemeal degranulation by human eosinophils was investigated. Mature eosinophils that developed in rhIL-5-containing conditioned media from cultured human cord blood mononuclear cells were prepared for ultrastructural studies using a combined technique to image eosinophil peroxidase by cytochemistry in the same sections on which postembedding immunogold was used to demonstrate Charcot-Leyden crystal protein. Vesicular transport of eosinophil peroxidase from the specific granule matrix compartment to the cell surface was associated with piecemeal degranulation. This process involved budding of eosinophil peroxidase-loaded vesicles and tubules from specific granules. Some eosinophil peroxidase that was released from eosinophils remained bound to the cell surface; some was free among the cultured cells. Macrophages and basophils bound the released eosinophil peroxidase to their plasma membranes, internalized it in endocytotic vesicles, and stored it in their respective phagolysosomes and secretory granules. Charcot-Leyden crystal protein was diffusely present in the nucleus and cytoplasm of IL-5-stimulated mature eosinophils. Extensive amounts were generally present in granule-poor and subplasma membrane areas of the cytoplasm in contrast to eosinophil peroxidase, which was secreted and bound to the external surface of eosinophil plasma membranes. These studies establish vesicular transport as a mechanism for emptying the specific eosinophil granule matrix compartment during IL-5-associated piecemeal degranulation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:1562046

  15. A hypothesis for temporal coding of young and mature granule cells

    PubMed Central

    Rangel, Lara M.; Quinn, Laleh K.; Chiba, Andrea A.; Gage, Fred H.; Aimone, James B.

    2013-01-01

    While it has been hypothesized that adult neurogenesis (NG) plays a role in the encoding of temporal information at long time-scales, the temporal relationship of immature cells to the highly rhythmic network activity of the hippocampus has been largely unexplored. Here, we present a theory for how the activity of immature adult-born granule cells relates to hippocampal oscillations. Our hypothesis is that theta rhythmic (5–10 Hz) excitatory and inhibitory inputs into the hippocampus could differentially affect young and mature granule cells due to differences in intrinsic physiology and synaptic inhibition between the two cell populations. Consequently, immature cell activity may occur at broader ranges of theta phase than the activity of their mature counterparts. We describe how this differential influence on young and mature granule cells could separate the activity of differently aged neurons in a temporal coding regime. Notably, this process could have considerable implications on how the downstream CA3 region interprets the information conveyed by young and mature granule cells. To begin to investigate the phasic behavior of granule cells, we analyzed in vivo recordings of the rat dentate gyrus (DG), observing that the temporal behavior of granule cells with respect to the theta rhythm is different between rats with normal and impaired levels of NG. Specifically, in control animals, granule cells exhibit both strong and weak coupling to the phase of the theta rhythm. In contrast, the distribution of phase relationships in NG-impaired rats is shifted such that they are significantly stronger. These preliminary data support our hypothesis that immature neurons could distinctly affect the temporal dynamics of hippocampal encoding. PMID:23717259

  16. Mucous granule exocytosis and CFTR expression in gallbladder epithelium.

    PubMed

    Kuver, R; Klinkspoor, J H; Osborne, W R; Lee, S P

    2000-02-01

    A mechanistic model of mucous granule exocytosis by columnar epithelial cells must take into account the unique physical-chemical properties of mucin glycoproteins and the resultant mucus gel. In particular, any model must explain the intracellular packaging and the kinetics of release of these large, heavily charged species. We studied mucous granule exocytosis in gallbladder epithelium, a model system for mucus secretion by columnar epithelial cells. Mucous granules released mucus by merocrine exocytosis in mouse gallbladder epithelium when examined by transmission electron microscopy. Spherules of secreted mucus larger than intracellular granules were noted on scanning electron microscopy. Electron probe microanalysis demonstrated increased calcium concentrations within mucous granules. Immunofluorescence microscopic studies revealed intracellular colocalization of mucins and the cystic fibrosis transmembrane conductance regulator (CFTR). Confocal laser immunofluorescence microscopy confirmed colocalization. These observations suggest that calcium in mucous secretory granules provides cationic shielding to keep mucus tightly packed. The data also suggests CFTR chloride channels are present in granule membranes. These observations support a model in which influx of chloride ions into the granule disrupts cationic shielding, leading to rapid swelling, exocytosis and hydration of mucus. Such a model explains the physical-chemical mechanisms involved in mucous granule exocytosis.

  17. Multiple effects of the phenylhydrazone derivative FCCP on the secretory pathway in rat plasma cells.

    PubMed

    Antoine, J C; Jouanne, C

    1986-10-01

    We studied the sensitivity of the last steps of the secretory process of antibody-producing cells to carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and sodium azide (NaN3), agents which lower the cellular ATP content by inhibiting oxidative phosphorylation and mitochondrial electron transport, respectively. Popliteal lymph node cells or purified plasma cells from rats immunized against horseradish peroxidase were incubated with the drugs. The rate of secretion of anti-HRP antibodies was measured by an enzyme-linked immunoadsorbent assay or after biosynthetic labeling with L-[3H]fucose. FCCP as well as NaN3 were shown to rapidly inhibit (in less than 5 min) the secretion of immunoglobulins (Ig) and to partially block the release of fucosylated Ig. This indicates that the drugs inhibit the transport of Ig from the Golgi apparatus (GA) (where fucose is added to Ig) to the plasma membrane. However, the degree of inhibition reached 40 to 50% with NaN3 and 70 to 80% with FCCP, whereas both drugs similarly depleted ATP stores by 45 to 55%. These results are consistent with multiple effects of FCCP on the secretion pathway of Ig. As a tentative explanation, we suggest that FCCP, because of its protonophore properties, not only reduces cellular ATP levels but may also neutralize the Golgi or post-Golgi acidic compartments recently shown to be involved in the transport of plasma membrane and secretory proteins.

  18. The transcription factor Cux1 in cerebellar granule cell development and medulloblastoma pathogenesis.

    PubMed

    Topka, Sabine; Glassmann, Alexander; Weisheit, Gunnar; Schüller, Ulrich; Schilling, Karl

    2014-12-01

    Cux1, also known as Cutl1, CDP or Cut is a homeodomain transcription factor implicated in the regulation of normal and oncogenic development in diverse peripheral tissues and organs. We studied the expression and functional role of Cux1 in cerebellar granule cells and medulloblastoma. Cux1 is robustly expressed in proliferating granule cell precursors and in postmitotic, migrating granule cells. Expression is lost as postmigratory granule cells mature. Moreover, Cux1 is also strongly expressed in a well-established mouse model of medulloblastoma. In contrast, expression of CUX1 in human medulloblastoma tissue samples is lower than in normal fetal cerebellum. In these tumors, CUX1 expression tightly correlates with a set of genes which, when mapped on a global protein-protein interaction dataset, yields a tight network that constitutes a cell cycle control signature and may be related to p53 and the DNA damage response pathway. Antisense-mediated reduction of CUX1 levels in two human medulloblastoma cell lines led to a decrease in proliferation and altered motility. The developmental expression of Cux1 in the cerebellum and its action in cell lines support a role in granule cell and medulloblastoma proliferation. Its expression in human medulloblastoma shifts that perspective, suggesting that CUX1 is part of a network involved in cell cycle control and maintenance of DNA integrity. The constituents of this network may be rational targets to therapeutically approach medulloblastomas.

  19. Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells.

    PubMed

    Wu, Xin-Sheng; Zhang, Zhen; Zhao, Wei-Dong; Wang, Dongsheng; Luo, Fujun; Wu, Ling-Gang

    2014-05-22

    Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), we found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. We therefore suggest that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis.

  20. Activation of protein kinase Ceta triggers cortical granule exocytosis in Xenopus oocytes.

    PubMed

    Gundersen, Cameron B; Kohan, Sirus A; Chen, Qian; Iagnemma, Joseph; Umbach, Joy A

    2002-03-15

    Previous work has shown that phorbol esters or diacylglycerol trigger cortical granule exocytosis in Xenopus oocytes. We sought to identify the isoform(s) of protein kinase C (PKC) that mediate(s) this regulated secretory event. Because this process is initiated by lipid activators of PKC but is independent of calcium ions, we focused on the family of novel (calcium-independent) PKCs. Pharmacological investigations using Gö6976 and Gö6983 tended to exclude PKCdelta, epsilon and mu as secretory triggers. Subcellular fractionation and immunoblot data revealed that these oocytes expressed all five members of the novel PKC family, but it was only PKCeta that colocalized with cortical granules. Finally, expression of wild type or constitutively active forms of PKCdelta and eta strongly supported the conclusion that it is PKCeta that initiates cortical granule exocytosis in these cells. These observations represent an important step in identifying the mechanism of secretory triggering in this system.

  1. Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques

    SciTech Connect

    De Young, M.B.; Nemeth, E.F.; Scarpa, A.

    1987-04-01

    The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and (/sup 14/C)methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca/sup 2 +/ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.

  2. Regulation by L channels of Ca(2+)-evoked secretory responses in ouabain-treated chromaffin cells.

    PubMed

    De Pascual, Ricardo; Colmena, Inés; Ruiz-Pascual, Lucía; Baraibar, Andrés Mateo; Egea, Javier; Gandía, Luis; García, Antonio G

    2016-10-01

    It is known that the sustained depolarisation of adrenal medullary bovine chromaffin cells (BCCs) with high K(+) concentrations produces an initial sharp catecholamine release that subsequently fades off in spite depolarisation persists. Here, we have recreated a sustained depolarisation condition of BCCs by treating them with the Na(+)/K(+) ATPase blocker ouabain; in doing so, we searched experimental conditions that permitted the development of a sustained long-term catecholamine release response that could be relevant during prolonged stress. BCCs were perifused with nominal 0Ca(2+) solution, and secretion responses were elicited by intermittent application of short 2Ca(2+) pulses (Krebs-HEPES containing 2 mM Ca(2+)). These pulses elicited a biphasic secretory pattern with an initial 30-min period with secretory responses of increasing amplitude and a second 30-min period with steady-state, non-inactivating responses. The initial phase was not due to gradual depolarisation neither to gradual increases of the cytosolic calcium transients ([Ca(2+)]c) elicited by 2Ca(2+) pulses in BBCs exposed to ouabain; both parameters increased soon after ouabain addition. Νifedipine blocked these responses, and FPL64176 potentiated them, suggesting that they were triggered by Ca(2+) entry through non-inactivating L-type calcium channels. This was corroborated by nifedipine-evoked blockade of the L-type Ca(2+) channel current and the [Ca(2+)]c transients elicited by 2Ca(2+) pulses. Furthermore, the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) blocker SEA0400 caused a mild inhibition followed by a large rebound increase of the steady-state secretory responses. We conclude that these two phases of secretion are mostly contributed by Ca(2+) entry through L calcium channels, with a minor contribution of Ca(2+) entry through the reverse mode of the NCX.

  3. Disrupted Dentate Granule Cell Chloride Regulation Enhances Synaptic Excitability during Development of Temporal Lobe Epilepsy

    PubMed Central

    Pathak, Hemal R.; Weissinger, Florian; Terunuma, Miho; Carlson, Gregory C.; Hsu, Fu-Chun; Moss, Stephen J.; Coulter, Douglas A.

    2008-01-01

    GABAA receptor-mediated inhibition depends on the maintenance of intracellular Cl− concentration ([Cl−]in ) at low levels. In neurons in the developing CNS, [Cl−]in is elevated, EGABA is depolarizing, and GABA consequently is excitatory. Depolarizing GABAergic synaptic responses may be recapitulated in various neuropathological conditions, including epilepsy. In the present study, rat hippocampal dentate granule cells were recorded using gramicidin perforated patch techniques at varying times (1–60 d) after an epileptogenic injury, pilocarpine-induced status epilepticus (STEP). In normal, non-epileptic animals, these strongly inhibited dentate granule cells act as a gate, regulating hippocampal excitation, controlling seizure initiation and/or propagation. For 2 weeks after STEP, we found that EGABA was positively shifted in granule cells. This shift in EGABA altered synaptic integration, increased granule cell excitability, and resulted in compromised “gate” function of the dentate gyrus. EGABA recovered to control values at longer latencies post-STEP (2–8 weeks), when animals had developed epilepsy. During this period of shifted EGABA, expression of the Cl− extruding K+/Cl− cotransporter, KCC2 was decreased. Application of the KCC2 blocker, furosemide, to control neurons mimicked EGABA shifts evident in granule cells post-STEP. Furthermore, post-STEP and furosemide effects interacted occlusively, both on EGABA in granule cells, and on gatekeeper function of the dentate gyrus. This suggests a shared mechanism, reduced KCC2 function. These findings demonstrate that decreased expression of KCC2 persists for weeks after an epileptogenic injury, reducing inhibitory efficacy and enhancing dentate granule cell excitability. This pathophysiological process may constitute a significant mechanism linking injury to the subsequent development of epilepsy. PMID:18094240

  4. Distinct kainate receptor phenotypes in immature and mature mouse cerebellar granule cells

    PubMed Central

    Smith, T Caitlin; Wang, Lu-Yang; Howe, James R

    1999-01-01

    Although glutamate receptors have been shown to be involved in neuronal maturation, a developmental role for kainate-type receptors has not been described. In addition, the single-channel properties of native kainate receptors have not been studied in situ. We have characterized the electrophysiological properties of native kainate receptors of granule cell neurons at two distinct stages in postnatal development, using whole-cell and outside-out patch-clamp recordings in acute cerebellar slices. Kainate-type currents were detected in both immature and mature granule cells. However, noise analysis showed that the apparent unitary conductance of kainate-type channels is significantly higher in proliferating than post-migratory granule cells. The conductance and rectification behaviour of the channels in immature granule cells indicate that they contain unedited GluR5 and GluR6 subunits and are likely to be calcium permeable. Single-channel kainate-type currents were observed in outside-out patches from proliferating granule cells in the external germinal layer. The kinetic behaviour of kainate receptors in immature cells was complex. Openings to multiple conductance levels were observed, although our analysis indicates that the channels spend most of their open time in a 4 pS state. PMID:10226148

  5. Depletion of primary cilia from mature dentate granule cells impairs hippocampus-dependent contextual memory

    PubMed Central

    Rhee, Soyoung; Kirschen, Gregory W.; Gu, Yan; Ge, Shaoyu

    2016-01-01

    The primary cilium, a sensory organelle, regulates cell proliferation and neuronal development of dentate granule cells in the hippocampus. However, its role in the function of mature dentate granule cells remains unknown. Here we specifically depleted and disrupted ciliary proteins IFT20 and Kif3A (respectively) in mature dentate granule cells and investigated hippocampus-dependent contextual memory and long-term plasticity at mossy fiber synapses. We found that depletion of IFT20 in these cells significantly impaired context-dependent fear-related memory. Furthermore, we tested synaptic plasticity of mossy fiber synapses in area CA3 and found increased long-term potentiation upon depletion of IFT20 or disruption of Kif3A. Our findings suggest a role of primary cilia in the memory function of mature dentate granule cells, which may result from abnormal mossy fiber synaptic plasticity. A direct link between the primary cilia of mature dentate granule cells and behavior will require further investigation using independent approaches to manipulate primary cilia. PMID:27678193

  6. Altered morphology of hippocampal dentate granule cell presynaptic and postsynaptic terminals following conditional deletion of TrkB.

    PubMed

    Danzer, Steve C; Kotloski, Robert J; Walter, Cynthia; Hughes, Maya; McNamara, James O

    2008-01-01

    Dentate granule cells play a critical role in the function of the entorhinal-hippocampal circuitry in health and disease. Dentate granule cells are situated to regulate the flow of information into the hippocampus, a structure required for normal learning and memory. Correspondingly, impaired granule cell function leads to memory deficits, and, interestingly, altered granule cell connectivity may contribute to the hyperexcitability of limbic epilepsy. It is important, therefore, to understand the molecular determinants of synaptic connectivity of these neurons. Brain-derived neurotrophic factor and its receptor TrkB are expressed at high levels in the dentate gyrus (DG) of the hippocampus, and are implicated in regulating neuronal development, neuronal plasticity, learning, and the development of epilepsy. Whether and how TrkB regulates granule cell structure, however, is incompletely understood. To begin to elucidate the role of TrkB in regulating granule cell morphology, here we examine conditional TrkB knockout mice crossed to mice expressing green fluorescent protein in subsets of dentate granule cells. In stratum lucidum, where granule cell mossy fiber axons project, the density of giant mossy fiber boutons was unchanged, suggesting similar output to CA3 pyramidal cell targets. However, filopodial extensions of giant boutons, which contact inhibitory interneurons, were increased in number in TrkB knockout mice relative to wildtype controls, predicting enhanced feedforward inhibition of CA3 pyramidal cells. In knockout animals, dentate granule cells possessed fewer primary dendrites and enlarged dendritic spines, indicative of disrupted excitatory synaptic input to the granule cells. Together, these findings demonstrate that TrkB is required for development and/or maintenance of normal synaptic connectivity of the granule cells, thereby implying an important role for TrkB in the function of the granule cells and hippocampal circuitry.

  7. The novel secretory protein CGREF1 inhibits the activation of AP-1 transcriptional activity and cell proliferation.

    PubMed

    Deng, Weiwei; Wang, Lan; Xiong, Ying; Li, Jing; Wang, Ying; Shi, Taiping; Ma, Dalong

    2015-08-01

    The transcription factor AP-1 plays an important role in inflammation and cell survival. Using a dual-luciferase reporter assay system and a library of 940 candidate human secretory protein cDNA clones, we identified that CGREF1 can inhibit the transcriptional activity of AP-1. We demonstrated that CGREF1 is secreted via the classical secretory pathway through the ER-to-Golgi apparatus. Functional investigations revealed that overexpression of CGREF1 can significantly inhibit the phosphorylation of ERK and p38 MAPK, and suppress the proliferation of HEK293T and HCT116 cells. Conversely, specific siRNAs against CGREF1 can increase the transcriptional activity of AP-1. These results clearly indicated that CGREF1 is a novel secretory protein, and plays an important role in regulation of AP-1 transcriptional activity and cell proliferation.

  8. Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.

    PubMed

    Love, Kerry Routenberg; Politano, Timothy J; Panagiotou, Vasiliki; Jiang, Bo; Stadheim, Terrance A; Love, J Christopher

    2012-01-01

    Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

  9. Mitochondrial calcium in the life and death of exocrine secretory cells.

    PubMed

    Voronina, Svetlana; Tepikin, Alexei

    2012-07-01

    The remarkable recent discoveries of the proteins mediating mitochondrial Ca(2+) transport (reviewed in this issue) provide an exciting opportunity to utilise this new knowledge to improve our fundamental understanding of relationships between Ca(2+) signalling and bioenergetics and, importantly, to improve the understanding of diseases in which Ca(2+) toxicity and mitochondrial malfunction play a crucial role. Ca(2+) is an important activator of exocrine secretion, a regulator of the bioenergetics of exocrine cells and a contributor to exocrine cell damage. Exocrine secretory cells, exocrine tissues and diseases affecting exocrine glands (like Sjögren's syndrome and acute pancreatitis) will, therefore, provide worthy research areas for the application of this new knowledge of the Ca(2+) transport mechanisms in mitochondria.

  10. Intestinal label-retaining cells are secretory precursors expressing Lgr5.

    PubMed

    Buczacki, Simon J A; Zecchini, Heather Ireland; Nicholson, Anna M; Russell, Roslin; Vermeulen, Louis; Kemp, Richard; Winton, Douglas J

    2013-03-07

    The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation.

  11. An LRRTM4-HSPG complex mediates excitatory synapse development on dentate gyrus granule cells.

    PubMed

    Siddiqui, Tabrez J; Tari, Parisa Karimi; Connor, Steven A; Zhang, Peng; Dobie, Frederick A; She, Kevin; Kawabe, Hiroshi; Wang, Yu Tian; Brose, Nils; Craig, Ann Marie

    2013-08-21

    Selective synapse development determines how complex neuronal networks in the brain are formed. Complexes of postsynaptic neuroligins and LRRTMs with presynaptic neurexins contribute widely to excitatory synapse development, and mutations in these gene families increase the risk of developing psychiatric disorders. We find that LRRTM4 has distinct presynaptic binding partners, heparan sulfate proteoglycans (HSPGs). HSPGs are required to mediate the synaptogenic activity of LRRTM4. LRRTM4 shows highly selective expression in the brain. Within the hippocampus, we detected LRRTM4 specifically at excitatory postsynaptic sites on dentate gyrus granule cells. LRRTM4(-/-) dentate gyrus granule cells, but not CA1 pyramidal cells, exhibit reductions in excitatory synapse density and function. Furthermore, LRRTM4(-/-) dentate gyrus granule cells show impaired activity-regulated AMPA receptor trafficking. These results identifying cell-type-specific functions and multiple presynaptic binding partners for different LRRTM family members reveal an unexpected complexity in the design and function of synapse-organizing proteins.

  12. Olfactory experiences dynamically regulate plasticity of dendritic spines in granule cells of Xenopus tadpoles in vivo

    PubMed Central

    Zhang, Li; Huang, Yubin; Hu, Bing

    2016-01-01

    Granule cells, rich in dendrites with densely punctated dendritic spines, are the most abundant inhibitory interneurons in the olfactory bulb. The dendritic spines of granule cells undergo remodeling during the development of the nervous system. The morphological plasticity of the spines’ response to different olfactory experiences in vivo is not fully known. In initial studies, a single granule cell in Xenopus tadpoles was labeled with GFP plasmids via cell electroporation; then, morphologic changes of the granule cell spines were visualized by in vivo confocal time-lapse imaging. With the help of long-term imaging, the total spine density, dynamics, and stability of four types of dendritic spines (mushroom, stubby, thin and filopodia) were obtained. Morphological analysis demonstrated that odor enrichment produced a remarkable increase in the spine density and stability of large mushroom spine. Then, with the help of short-term imaging, we analyzed the morphological transitions among different spines. We found that transitions between small spines (thin and filopodia) were more easily influenced by odor stimulation or olfactory deprivation. These results indicate that different olfactory experiences can regulate the morphological plasticity of different dendritic spines in the granule cell. PMID:27713557

  13. Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase.

    PubMed

    Bembenek, Joshua N; Richie, Christopher T; Squirrell, Jayne M; Campbell, Jay M; Eliceiri, Kevin W; Poteryaev, Dmitry; Spang, Anne; Golden, Andy; White, John G

    2007-11-01

    In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.

  14. Common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

    SciTech Connect

    Cameron, R.S.; Cameron, P.L.; Castle, J.D.

    1986-10-01

    A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues - pancreatic, lacrimal, and submandibular - from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pI and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.

  15. MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

    PubMed

    Constantin, Lena; Wainwright, Brandon J

    2015-12-01

    MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

  16. Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells.

    PubMed

    Ohashi, Ryo; Sakata, Shin-ichi; Naito, Asami; Hirashima, Naohide; Tanaka, Masahiko

    2014-04-01

    Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca(2+) release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single-cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain-derived neurotrophic factor (BDNF) in the culture medium. The ryanodine-induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells.

  17. Culturing of cerebellar granule cells to study neuronal migration: gradient and local perfusion assays.

    PubMed

    Guijarro, Patricia; Jiang, Jian; Yuan, Xiao-bing

    2012-07-01

    Cultures of cerebellar granule cells are a suitable model to analyze the mechanisms governing neuronal migration. In this unit, we describe a protocol to obtain cultures of dissociated granule cells at a low density, where individual cells can be easily observed. In addition, we include a protocol for studying neuronal migration in these cultures, using single, actively migrating cerebellar granule cells. Following this protocol, a factor of interest can be applied either in a gradient concentration by means of a micropipet located near the neuron, or in a homogeneous concentration by locally perfusing a certain region of the neuron. Time-lapse images are taken to analyze changes in the speed and/or directionality of the observed neuron. Overall, the two protocols take more or less a day and a half to perform, and are a useful way to evaluate a certain factor/drug for its chemotactic activity or its capacity to alter migration speed.

  18. Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells.

    PubMed

    Chaiyadet, Sujittra; Smout, Michael; Laha, Thewarach; Sripa, Banchob; Loukas, Alex; Sotillo, Javier

    2016-02-09

    The association between liver fluke infection caused by Opisthorchis viverrini and cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium) has been well established. Multiple mechanisms play a role in the development of CCA, but the excretory/secretory products released by O. viverrini (OvES) represent the major interface between the parasite and its host, and their uptake by biliary epithelial cells has been suggested to be responsible for proliferation of cholangiocytes, the cells that line the biliary epithelium. Despite recent progress in the study of the molecular basis of O. viverrini-host interactions, little is known about the effects that OvES induces upon internalization by host cells. In the present study we incubated non-cancerous human cholangiocytes (H69) and human colon cancer (CaCo-2) cells with OvES and performed a time-course quantitative proteomic analysis on the cells to determine the early changes induced by the parasite. Different KEGG pathways were altered in H69 cells compared to Caco-2 cells: glycolysis/gluconeogenesis and protein processing in the endoplasmic reticulum. In addition, the Reactome pathway analysis showed a predominance of proteins involved in cellular pathways related to apoptosis and apoptotic execution phase in H69 cells after incubation with OvES. The present study provides the first proteomic analysis to address the molecular mechanisms by which OvES products interact with host cells, and Sheds light on the cellular processes involved in O. viverrini-induced CCA.

  19. Acute insulin responses to glucose and arginine as predictors of beta-cell secretory capacity in human islet transplantation.

    PubMed

    Rickels, Michael R; Naji, Ali; Teff, Karen L

    2007-11-27

    Islet transplantation for type 1 diabetes can enable the achievement of near-normal glycemic control without severe hypoglycemic episodes. How much an islet (beta-cell) graft may be contributing to glycemic control can be quantified by stimulatory tests of insulin (or C-peptide) secretion. Glucose-potentiation of arginine-induced insulin secretion provides a measure of functional beta-cell mass, the beta-cell secretory capacity, as either AIR(pot) or AIR(max), but requires conduct of a hyperglycemic clamp. We sought to determine whether acute insulin responses to intravenous glucose (AIR(glu)) or arginine (AIR(arg)) could predict beta-cell secretory capacity in islet recipients. AIR(arg) was a better predictor of both AIR(pot) and AIR(max) (n=10, r2=0.98, P<0.0001 and n=7, r2=0.97, P<0.0001) than was AIR(glu) (n=9, r2=0.78, P=0.002 and n=6, r2=0.76, P=0.02). Also, the measures of beta-cell secretory capacity were highly correlated (n=7, r2=0.98, P<0.0001). These results support the use of AIR(arg) as a surrogate indicator of beta-cell secretory capacity in islet transplantation.

  20. Identification of miRNAs differentially expressed in human epilepsy with or without granule cell pathology.

    PubMed

    Zucchini, Silvia; Marucci, Gianluca; Paradiso, Beatrice; Lanza, Giovanni; Roncon, Paolo; Cifelli, Pierangelo; Ferracin, Manuela; Giulioni, Marco; Michelucci, Roberto; Rubboli, Guido; Simonato, Michele

    2014-01-01

    The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets.

  1. von Willebrand factor multimerization and the polarity of secretory pathways in endothelial cells

    PubMed Central

    Lopes da Silva, Mafalda

    2016-01-01

    The von Willebrand factor (VWF) synthesized and secreted by endothelial cells is central to hemostasis and thrombosis, providing a multifunctional adhesive platform that brings together components needed for these processes. VWF secretion can occur from both apical and basolateral sides of endothelial cells, and from constitutive, basal, and regulated secretory pathways, the latter two via Weibel-Palade bodies (WPB). Although the amount and structure of VWF is crucial to its function, the extent of VWF release, multimerization, and polarity of the 3 secretory pathways have only been addressed separately, and with conflicting results. We set out to clarify these relationships using polarized human umbilical vein endothelial cells (HUVECs) grown on Transwell membranes. We found that regulated secretion of ultra–large (UL)-molecular-weight VWF predominantly occurred apically, consistent with a role in localized platelet capture in the vessel lumen. We found that constitutive secretion of low-molecular-weight (LMW) VWF is targeted basolaterally, toward the subendothelial matrix, using the adaptor protein complex 1 (AP-1), where it may provide the bulk of collagen-bound subendothelial VWF. We also found that basally-secreted VWF is composed of UL-VWF, released continuously from WPBs in the absence of stimuli, and occurs predominantly apically, suggesting this could be the main source of circulating plasma VWF. Together, we provide a unified dataset reporting the amount and multimeric state of VWF secreted from the constitutive, basal, and regulated pathways in polarized HUVECs, and have established a new role for AP-1 in the basolateral constitutive secretion of VWF. PMID:27106123

  2. [Identification of Ca2+ release channels in salivary glands secretory cells of Chironomus plumosus L].

    PubMed

    Man'ko, V V; Bychkova, S V; Klevets', M Iu

    2004-01-01

    The presence of two types of well-characterised Ca2+ release channels, namely IP3-receptors (Ins(1,4,5)P3Rs) and ryanodine-receptors (RyRs), was detected in the salivary glands secretory cells of Chironomus plumosus L. For this aim different blockators and activators of these Ca2+ -transport systems were used. The conditions for permeabilization of these cells by saponine were experimentally chosen for their more intensive action. It was shown that IP3 decreased calcium content in saponine-treated gland tissue by (41.14 +/- 11.75)%. The effect of IP3 was not observed under condition of heparin and eosin Y presence in the incubation medium, but heparin alone did not cause any action on calcium content in saponine-treated gland tissue. The observed effects of IP3 are supposed to be the evidences of Ins (1,4,5)P3Rs presence in the intracellular membrane of this object. It was also shown that calcium content in intact gland tissue increased by (67.12 +/- 22.60)% in presence of heparin (500 mkg/ml) in the incubation medium. This effect of heparin was also observed with presence of verapamil (100 mkM) and eosin Y (5, 20 mkM) in incubation medium. So, this effect is not connected with function of voltage-gated Ca2+ -channels and Ca2+ -pumps. Ryanodine in concentration of 5nM decreased calcium content in saponine-treated gland tissue by (35.18 +/- 3.87)% but it caused the increase of calcium content at high concentration (500 nM) by (40.72 +/- 12.52)%. It improved the presence of RyRs in intracellular membrane of secretory cells of this object. Besides, these channels, perhaps, belong to "non-sensitive" to caffeine, because caffeine did not affect calcium content in the gland tissue neither in presence nor with absence of eosin Y.

  3. Seizure-Induced Motility of Differentiated Dentate Granule Cells Is Prevented by the Central Reelin Fragment

    PubMed Central

    Orcinha, Catarina; Münzner, Gert; Gerlach, Johannes; Kilias, Antje; Follo, Marie; Egert, Ulrich; Haas, Carola A.

    2016-01-01

    Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSCs) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus, as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate toward the hilar region, where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3–6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together, these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons. PMID:27516734

  4. Dependence of structure stability and integrity of aerobic granules on ATP and cell communication.

    PubMed

    Jiang, Bo; Liu, Yu

    2013-06-01

    Aerobic granules are dense and compact microbial aggregates with various bacterial species. Recently, aerobic granulation technology has been extensively explored for treatment of municipal and industrial wastewaters. However, little information is currently available with regard to their structure stability and integrity at levels of energy metabolism and cell communication. In the present study, a typical chemical uncoupler, 3,3',4',5-tetrachlorosalicylanilide with the power to dissipate proton motive force and subsequently inhibit adenosine triphosphate (ATP) generation, was used to investigate possible roles of ATP and cell communication in maintaining the structure stability and integrity of aerobic granules. It was found that inhibited ATP synthesis resulted in the reduced production of autoinducer-2 and N-acylhomoserine lactones essential for cell communication, while lowered extracellular polymeric substance (EPS) production was also observed. As a consequence, aerobic granules appeared to break up. This study showed that ATP-dependent quorum sensing and EPS were essential for sustaining the structure stability and integrity of aerobic granules.

  5. Mast cell differentiation depends on T cells and granule synthesis on fibroblasts.

    PubMed Central

    Davidson, S; Mansour, A; Gallily, R; Smolarski, M; Rofolovitch, M; Ginsburg, H

    1983-01-01

    Mast cell differentiation was generated in the following three experimental situations: (i) infection of mice with Schistosoma Mansoni or with Nippostrongylus brasiliensis and growth of the lymph node cells in the presence of the corresponding helminth antigen; (ii) immunization with horse serum and growth of blood and lymph node cells in the presence of the horse serum; (iii) exposure of T-cell-depleted suspensions of lymph node cells from unimmunized mice to T-cell factor (TCF) released into medium of the young cultures of (i) and (ii). This differentiation was also obtained when lymph node cells from athymic nude mice were exposed to TCF. The cell suspensions were plated on X-irradiated fibroblast monolayers prepared from embryonic mouse skin. Screening of the suspensions before plating on the fibroblasts in culture revealed no young forms of mast cells, and none were present in culture of nude mice lymph node cells maintained without TCF. Primordial appearance of metachromatic granules generally in the golgi zone was first seen in many 'large lymphoid cells' as early as 18 hr after plating. This was followed by increase in the cytoplasm volume, increase in granule number and mitosis, ending at 10-18 days with homogeneous populations of mature mast cells. When the mesenteric lymph node cells from mice infected with the helminths were grown in the absence of fibroblasts but in the presence of the antigen, homogeneous populations of cells with extended cytoplasm, filled with unstained vacuoles developed during days 7-13. These cells did not contain histamine (or at most 0.2 microgram per 10(6) vacuolated cells). When these cells were plated on fibroblast monolayers clear granule formation in all the vacuoles was seen 2 days later. It increased progressively in size and staining intensity, until the vacuoles transformed into typical mast cell granules. By the fourth day the vacuolated cells attained the typical mast cell morphology and the histamine content greatly

  6. [Basic proteins in the granules of mast cells. Demonstration of masked proteins, acidophilic staining of the granules].

    PubMed

    Anikó, K; Lajos, K

    1976-07-01

    Basic proteins of the granules of mast cells in nativ, formalin-, alcohol- and aceton fixed preparations without any preliminary treatment, when stained with acidic dye at the pH 9 cytochemically seem to be masked. After various preliminary treatment (treatment with acid, with cetylpiridinumchlorid, CPC) mast-cell granula stained with acidic-dye at pH 9 appear intensively acidophile. This phenomenon can be explained by the presence of basic proteins in the mast cell granula. Preliminary treatment with CPC inhibits acid radicals of the heparin. This may lead to the disintegration of the linkage between proteins of the heparin, thus amino-group of the basic proteins become reactivated and can be identified by acidic dyes. It can not be excluded as well, that CPC linked to the heparin with free positive radicals reveals acidic-dye-binding capacity. In cases of preliminary treatment with various acids this mechanism does not seem possible to lay on the base of changing of the dye binding capacity.

  7. Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV-2 Microglial Cells.

    PubMed

    Lee, Jinyoung; Kang, Jung-Mi; Kim, Tae Im; Kim, Jong-Hyun; Sohn, Hae-Jin; Na, Byoung-Kuk; Shin, Ho-Joon

    2017-03-01

    Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV-2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV-2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL-1α and TNF-α. NfESP-induced IL-1α and TNF-α expression in BV-2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP-induced IL-1α and TNF-α production in BV-2 cells were effectively downregulated by inhibition of NF-kB and AP-1. These results collectively suggest that NfESP stimulates BV-2 cells to release IL-1α and TNF-α via NF-kB- and AP-1-dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection.

  8. Cytocompatibility of porous biphasic calcium phosphate granules with human mesenchymal cells by a multiparametric assay.

    PubMed

    Mitri, Fabio; Alves, Gutemberg; Fernandes, Gustavo; König, Bruno; Rossi, Alexandre J R; Granjeiro, Jose

    2012-06-01

    This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/beta-tricalcium phosphate (β-TCP) (60:40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100°C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/β-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications.

  9. Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells

    PubMed Central

    Huang, Cheng-Chiu; Sugino, Ken; Shima, Yasuyuki; Guo, Caiying; Bai, Suxia; Mensh, Brett D; Nelson, Sacha B; Hantman, Adam W

    2013-01-01

    Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control. DOI: http://dx.doi.org/10.7554/eLife.00400.001 PMID:23467508

  10. Excretory-secretory products (ESP) from Fasciola hepatica induce tolerogenic properties in myeloid dendritic cells.

    PubMed

    Falcón, Cristian; Carranza, Franco; Martínez, Fernando F; Knubel, Carolina P; Masih, Diana T; Motrán, Claudia C; Cervi, Laura

    2010-09-15

    Fasciola hepatica is a helminth trematode that migrates through the host tissues until reaching bile ducts where it becomes an adult. During its migration the parasite releases different excretory-secretory products (ESP), which are in contact with the immune system. In this study, we focused on the effect of ESP on the maturation and function of murine bone marrow derived-dendritic cells (DC). We found that the treatment of DC with ESP failed to induce a classical maturation of these cells, since ESP alone did not activate DC to produce any cytokines, although they impaired the ability of DC to be activated by TLR ligands and also their capacity to stimulate an allospecific response. In addition, using an in vitro ovalbumin peptide-restricted priming assay, ESP-treated DC exhibited a capacity to drive Th2 and regulatory T cell (Treg) polarization of CD4(+) cells from DO11.10 transgenic mice. This was characterized by increased IL-4, IL-5, IL-10 and TGF-beta production and the expansion of CD4(+)CD25(+)Foxp3(+) cells. Our results support the hypothesis that ESP from F. hepatica modulate the maturation and function of DC as part of a generalized immunosuppressive mechanism that involves a bias towards a Th2 response and Treg development.

  11. Corticosterone activity during early weaning reprograms molecular markers in rat gastric secretory cells

    PubMed Central

    Zulian, Juliana Guimarães; Hosoya, Larissa Yukari Massarenti; Figueiredo, Priscila Moreira; Ogias, Daniela; Osaki, Luciana Harumi; Gama, Patricia

    2017-01-01

    Gastric epithelial cells differentiate throughout the third postnatal week in rats, and become completely functional by weaning time. When suckling is interrupted by early weaning (EW), cell proliferation and differentiation change in the gastric mucosa, and regulatory mechanisms might involve corticosterone activity. Here we used EW and RU486 (glucocorticoid receptor antagonist) to investigate the roles of corticosterone on differentiation of mucous neck (MNC) and zymogenic cells (ZC) in rats, and to evaluate whether effects persisted in young adults. MNC give rise to ZC, and mucin 6, Mist1, pepsinogen a5 and pepsinogen C are produced to characterize these cells. We found that in pups, EW augmented the expression of mucins, Mist1 and pepsinogen C at mRNA and protein levels, and it changed the number of MNC and ZC. Corticosterone regulated pepsinogen C expression, and MNC and ZC distributions. Further, the changes on MNC population and pepsinogen C were maintained until early- adult life. Therefore, by using EW as a model for altered corticosterone activity in rats, we demonstrated that the differentiation of secretory epithelial cells is sensitive to the type of nutrient in the lumen. Moreover, this environmental perception activates corticosterone to change maturation and reprogram cellular functions in adulthood. PMID:28361902

  12. Xbp1-based engineering of secretory capacity enhances the productivity of Chinese hamster ovary cells.

    PubMed

    Tigges, Marcel; Fussenegger, Martin

    2006-05-01

    A variety of successful transcription and translation engineering strategies implemented during the past decade have driven the specific productivity of mammalian cells to an apparent limit. Restricted post-translation competence has since been considered the major bottleneck preventing mammalian cells from fully exploiting their physiologic production capacity in a biopharmaceutical manufacturing scenario. Through ectopic expression of the human transcription factor Xbp1 (X-box-binding-protein 1), evolved to manage plasma cell differentiation and coordinate the unfolded protein response, we have specifically expanded the endoplasmic reticulum and the Golgi of transgenic Chinese hamster ovary (CHO-K1)-derived cell lines with a resulting increase in overall production capacity. Xbp-1-based engineering of secretory bottlenecks was compatible with a variety of different promoter–product gene configurations suggesting that Xbp-1 induces generic production increases in CHO-K1 cell derivatives. Secretion engineering, illustrated here by Xbp1-based reprogramming of the post-translational processing machinery, provides a first insight into mastering a major system bottleneck which impacts biopharmaceutical manufacturing of secreted protein therapeutics.

  13. Mouse granulated metrial gland cells require contact with stromal cells to maintain viability

    PubMed Central

    STEWART, I. J.

    2000-01-01

    Granulated metrial gland (GMG) cells differentiate in the uterine wall in pregnancy in mice but the mechanisms which control their differentiation and maintenance are unknown. In vivo, GMG cells share an intimate association with fibroblast-like stromal cells. The importance of this association has been assessed by examining the effects of withdrawal of stromal cell contact on GMG cell maintenance in vitro. When single cell suspensions of cells were prepared from mouse metrial glands there was a steady decline in numbers with days of culture but usually some remained at 7 d of culture. The ability of metrial gland cells to kill Wehi 164 target cells in 51Cr-release cytotoxicity assays was retained by cells cultured for at least 3 d. When explants of metrial gland were maintained in culture to allow GMG cell migration onto the culture flask, the attached GMG cells were lost by 1 d later. Overall, these results suggest that a juxtacrine regulatory mechanism maintains GMG cells. The rapid loss of unsupported GMG cells in culture has major implications in the design of assays to examine GMG cell function. PMID:11117633

  14. Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA

    PubMed Central

    Nakanishi, Katsuhiro; Morikane, Shota; Ichikawa, Shiori; Kurohane, Kohta; Niwa, Yasuo; Akimoto, Yoshihiro; Matsubara, Sachie; Kawakami, Hayato; Kobayashi, Hirokazu; Imai, Yasuyuki

    2017-01-01

    Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection. PMID:28368034

  15. Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA.

    PubMed

    Nakanishi, Katsuhiro; Morikane, Shota; Ichikawa, Shiori; Kurohane, Kohta; Niwa, Yasuo; Akimoto, Yoshihiro; Matsubara, Sachie; Kawakami, Hayato; Kobayashi, Hirokazu; Imai, Yasuyuki

    2017-04-03

    Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection.

  16. Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon

    PubMed Central

    Sasaki, Nobuo; Sachs, Norman; Wiebrands, Kay; Ellenbroek, Saskia I. J.; Fumagalli, Arianna; Lyubimova, Anna; Begthel, Harry; van den Born, Maaike; van Es, Johan H.; Karthaus, Wouter R.; Li, Vivian S. W.; López-Iglesias, Carmen; Peters, Peter J.; van Rheenen, Jacco; van Oudenaarden, Alexander; Clevers, Hans

    2016-01-01

    Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5+) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5+ stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5+ stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4+ DCS cells promote organoid formation of single Lgr5+ colon stem cells. DCS cells can be massively produced from Lgr5+ colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4+ DCS cells serve as Paneth cell equivalents in the colon crypt niche. PMID:27573849

  17. Linking local circuit inhibition to olfactory behavior: a critical role for granule cells in olfactory discrimination.

    PubMed

    Strowbridge, Ben W

    2010-02-11

    In this issue of Neuron, Abraham et al. report a direct connection between inhibitory function and olfactory behavior. Using molecular methods to alter glutamate receptor subunit composition in olfactory bulb granule cells, the authors found a selective modulation in the time required for difficult, but not simple, olfactory discrimination tasks.

  18. Morphological changes among hippocampal dentate granule cells exposed to early kindling-epileptogenesis

    PubMed Central

    Singh, Shatrunjai P.; He, Xiaoping; McNamara, James O.; Danzer, Steve C.

    2013-01-01

    Temporal lobe epilepsy is associated with changes in the morphology of hippocampal dentate granule cells. These changes are evident in numerous models that are associated with substantial neuron loss and spontaneous recurrent seizures. By contrast, previous studies have shown that in the kindling model, it is possible to administer a limited number of stimulations sufficient to produce a lifelong enhanced sensitivity to stimulus evoked seizures without associated spontaneous seizures and minimal neuronal loss. Here we examined whether stimulation of the amygdala sufficient to evoke five convulsive seizures (class IV or greater on Racine’s scale) produce morphological changes similar to those observed in models of epilepsy associated with substantial cell loss. The morphology of GFP-expressing granule cells from Thy-1 GFP mice was examined either one day or one month after the last evoked seizure. Interestingly, significant reductions in dendritic spine density were evident one day after the last seizure, the magnitude of which had diminished by one month. Further, there was an increase in the thickness of the granule cell layer one day after the last evoked seizure, which was absent a month later. We also observed an increase in the area of the proximal axon, which again returned to control levels a month later. No differences in the number of basal dendrites were detected at either time point. These findings demonstrate that the early stages of kindling epileptogenesis produce transient changes in the granule cell body layer thickness, molecular layer spine density and axon proximal area, but do not produce striking rearrangements of granule cell structure. PMID:23893783

  19. Free and complexed‐secretory immunoglobulin A triggers distinct intestinal epithelial cell responses

    PubMed Central

    Safavie, F.; Fasano, A.; Sztein, M. B.

    2016-01-01

    Summary Secretory immunoglobulin A (SIgA) antibodies play an important role in protecting the mucosal surfaces against pathogens and maintaining homeostasis with the commensal microbiota. Because a substantial portion of the gut microbiota is coated with SIgA, we hypothesized that microbiota–SIgA complexes are important for the maintenance of gut homeostasis. Here we investigated the relationship between microbiota–SIgA complexes and inflammatory epithelial cell responses. We used a multi‐cellular three‐dimensional (3D) organotypical model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes/monocytes, endothelial cells and fibroblasts. We also used human SIgA from human colostrum, and a prominent bacterial member of the first colonizers, Escherichia coli, as a surrogate commensal. We found that free and microbiota‐complexed SIgA triggered different epithelial responses. While free SIgA up‐regulated mucus production, expression of polymeric immunoglobulin receptor (pIgR) and secretion of interleukin‐8 and tumoir necrosis factor‐α, microbiota‐complexed SIgA mitigated these responses. These results suggest that free and complexed SIgA have different functions as immunoregulatory agents in the gut and that an imbalance between the two may affect gut homeostasis. PMID:27084834

  20. Recruitment of an inhibitory hippocampal network after bursting in a single granule cell.

    PubMed

    Mori, Masahiro; Gähwiler, Beat H; Gerber, Urs

    2007-05-01

    The hippocampal CA3 area, an associational network implicated in memory function, receives monosynaptic excitatory as well as disynaptic inhibitory input through the mossy-fiber axons of the dentate granule cells. Synapses made by mossy fibers exhibit low release probability, resulting in high failure rates at resting discharge frequencies of 0.1 Hz. In recordings from functionally connected pairs of neurons, burst firing of a granule cell increased the probability of glutamate release onto both CA3 pyramidal cells and inhibitory interneurons, such that subsequent low-frequency stimulation evoked biphasic excitatory/inhibitory responses in a CA3 pyramidal cell, an effect lasting for minutes. Analysis of the unitary connections in the circuit revealed that granule cell bursting caused powerful activation of an inhibitory network, thereby transiently suppressing excitatory input to CA3 pyramidal cells. This phenomenon reflects the high incidence of spike-to-spike transmission at granule cell to interneuron synapses, the numerically much greater targeting by mossy fibers of inhibitory interneurons versus principal cells, and the extensively divergent output of interneurons targeting CA3 pyramidal cells. Thus, mossy-fiber input to CA3 pyramidal cells appears to function in three distinct modes: a resting mode, in which synaptic transmission is ineffectual because of high failure rates; a bursting mode, in which excitation predominates; and a postbursting mode, in which inhibitory input to the CA3 pyramidal cells is greatly enhanced. A mechanism allowing the transient recruitment of inhibitory input may be important for controlling network activity in the highly interconnected CA3 pyramidal cell region.

  1. Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism.

    PubMed

    Wang, Rong; Yu, Zhen; Sunchu, Bharath; Shoaf, James; Dang, Ivana; Zhao, Stephanie; Caples, Kelsey; Bradley, Lynda; Beaver, Laura M; Ho, Emily; Löhr, Christiane V; Perez, Viviana I

    2017-03-31

    Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-β-galactosidase (β-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β-gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.

  2. Hedgehog antagonist REN(KCTD11) regulates proliferation and apoptosis of developing granule cell progenitors.

    PubMed

    Argenti, Beatrice; Gallo, Rita; Di Marcotullio, Lucia; Ferretti, Elisabetta; Napolitano, Maddalena; Canterini, Sonia; De Smaele, Enrico; Greco, Azzura; Fiorenza, Maria Teresa; Maroder, Marella; Screpanti, Isabella; Alesse, Edoardo; Gulino, Alberto

    2005-09-07

    During the early development of the cerebellum, a burst of granule cell progenitor (GCP) proliferation occurs in the outer external granule layer (EGL), which is sustained mainly by Purkinje cell-derived Sonic Hedgehog (Shh). Shh response is interrupted once GCPs move into the inner EGL, where granule progenitors withdraw proliferation and start differentiating and migrating toward the internal granule layer (IGL). Failure to interrupt Shh signals results in uncoordinated proliferation and differentiation of GCPs and eventually leads to malignancy (i.e., medulloblastoma). The Shh inhibitory mechanisms that are responsible for GCP growth arrest and differentiation remain unclear. Here we report that REN, a putative tumor suppressor frequently deleted in human medulloblastoma, is expressed to a higher extent in nonproliferating inner EGL and IGL granule cells than in highly proliferating outer EGL cells. Accordingly, upregulated REN expression occurs along GCP differentiation in vitro, and, in turn, REN overexpression promotes growth arrest and increases the proportion of p27/Kip1+ GCPs. REN also impairs both Gli2-dependent gene transcription and Shh-enhanced expression of the target Gli1 mRNA, thus antagonizing the Shh-induced effects on the proliferation and differentiation of cultured GCPs. Conversely, REN functional knock-down impairs Hedgehog antagonism and differentiation and sustains the proliferation of GCPs. Finally, REN enhances caspase-3 activation and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling apoptotic GCP numbers; therefore, the pattern of REN expression, its activity, and its antagonism on the Hedgehog pathway suggest that this gene may represent a restraint of Shh signaling at the outer to inner EGL GCP transitions. Medulloblastoma-associated REN loss of function might withdraw such a limiting signal for immature cell expansion, thus favoring tumorigenesis.

  3. Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis.

    PubMed

    Donelan, Matthew J; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A; Molkentin, Jeffery D; Brady, Scott T; Rhodes, Christopher J

    2002-07-05

    The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.

  4. Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

    NASA Technical Reports Server (NTRS)

    Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.; Rhodes, Christopher J.

    2002-01-01

    The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.

  5. P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.

    PubMed

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R

    2011-04-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca(2+)-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca(2+) concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca(2+) and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.

  6. P2X7 Receptors Trigger ATP Exocytosis and Modify Secretory Vesicle Dynamics in Neuroblastoma Cells*

    PubMed Central

    Gutiérrez-Martín, Yolanda; Bustillo, Diego; Gómez-Villafuertes, Rosa; Sánchez-Nogueiro, Jesús; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutiérrez, Luis Miguel; Miras-Portugal, María Teresa; Artalejo, Antonio R.

    2011-01-01

    Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca2+ concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca2+ and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation. PMID:21292765

  7. Kinetic Evaluation of Cell Membrane Hydrolysis during Apoptosis by Human Isoforms of Secretory Phospholipase A2*

    PubMed Central

    Olson, Erin D.; Nelson, Jennifer; Griffith, Katalyn; Nguyen, Thaothanh; Streeter, Michael; Wilson-Ashworth, Heather A.; Gelb, Michael H.; Judd, Allan M.; Bell, John D.

    2010-01-01

    Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform. PMID:20139082

  8. Expression of S100 beta in sensory and secretory cells of the vertebrate inner ear

    NASA Technical Reports Server (NTRS)

    Fermin, C. D.; Martin, D. S.

    1995-01-01

    We evaluated anti-S100 beta expression in the chick (Gallus domesticus) inner ear and determined that: 1) the monomer anti-S100 beta is expressed differentially in the vestibular and auditory perikarya; 2) expression of S100 beta in the afferent nerve terminals is time-related to synapse and myelin formation; 3) the expression of the dimer anti-S100 alpha alpha beta beta and monomer anti-S100 beta overlaps in most inner ear cell types. Most S100 alpha alpha beta beta positive cells express S100 beta, but S100 beta positive cells do not always express S100 alpha alpha beta beta. 4) the expression of S100 beta is diffused over the perikaryal cytoplasm and nuclei of the acoustic ganglia but is concentrated over the nuclei of the vestibular perikarya. 6) S100 beta is expressed in secretory cells, and it is co-localized with GABA in sensory cells. 7) Color thresholding objective quantitation indicates that the amount of S100 beta was higher (mean 22, SD +/- 4) at E19 than at E9 (mean 34, SD +/- 3) in afferent axons. 8) Moreover, S100 beta was unchanged between E11-E19 in the perikaryal cytoplasm, but did change over the nuclei. At E9, 74%, and at E21, 5% of vestibular perikarya were positive. The data suggest that S100 beta may be physically associated with neuronal and ionic controlling cells of the vertebrate inner ear, where it could provide a dual ionic and neurotrophic modulatory function.

  9. Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus.

    PubMed

    Wittner, L; Maglóczky, Z; Borhegyi, Z; Halász, P; Tóth, S; Eross, L; Szabó, Z; Freund, T F

    2001-01-01

    Temporal lobe epilepsy is known to be associated with hyperactivity that is likely to be generated or amplified in the hippocampal formation. The majority of granule cells, the principal cells of the dentate gyrus, are found to be resistant to damage in epilepsy, and may serve as generators of seizures if their inhibition is impaired. Therefore, the parvalbumin-containing subset of interneurons, known to provide the most powerful inhibitory input to granule cell somata and axon initial segments, were examined in human control and epileptic dentate gyrus. A strong reduction in the number of parvalbumin-containing cells was found in the epileptic samples especially in the hilar region, although in some patches of the granule cell layer parvalbumin-positive terminals that form vertical clusters characteristic of axo-axonic cells were more numerous than in controls. Analysis of the postsynaptic target elements of parvalbumin-positive axon terminals showed that they form symmetric synapses with somata, dendrites, axon initial segments and spines as in the control, but the ratio of axon initial segment synapses was increased in the epileptic tissue (control: 15.9%, epileptic: 31.3%). Furthermore, the synaptic coverage of granule cell axon initial segments increased more than three times (control: 0.52, epileptic: 2.10 microm synaptic length/100 microm axon initial segment membrane) in the epileptic samples, whereas the amount of somatic symmetric synapses did not change significantly. Although the number of parvalbumin-positive interneurons is decreased, the perisomatic inhibitory input of dentate granule cells is preserved in temporal lobe epilepsy. Basket and axo-axonic cell terminals - whether positive or negative for parvalbumin - are present, moreover, the axon collaterals targeting axon initial segments sprout in the epileptic dentate gyrus. We suggest that perisomatic inhibitory interneurons survive in epilepsy, but their somadendritic compartment and partly the

  10. YAP Induces High-Grade Serous Carcinoma in Fallopian Tube Secretory Epithelial Cells

    PubMed Central

    Hua, Guohua; Lv, Xiangmin; He, Chunbo; Remmenga, Steven W.; Rodabough, Kerry J.; Dong, Jixin; Yang, Liguo; Lele, Subodh M.; Yang, Peixin; Zhou, Jin; Karst, Alison; Drapkin, Ronny I.; Davis, John S.; Wang, Cheng

    2015-01-01

    Accumulating evidence indicates that ovarian high-grade serous carcinoma (HGSC) originates from Fallopian tube secretory epithelial cells (FTSECs). However, the molecular mechanisms underlying the initiation and progression of HGSC derived from FTSECs remains unclear. In the present study, we found that the Hippo/YAP signaling pathway plays a critical role in the initiation and progression of Fallopian tube and ovarian HGSC. Importantly, YAP was overexpressed in inflammatory and cancerous Fallopian tube tissues. Further, overexpression of wild-type YAP, or constitutively active YAP in immortalized FTSECs, induced cell proliferation, migration, colony formation, and tumorigenesis. Moreover, the Hippo/YAP and the fibroblast growth factor (FGF) signaling pathways formed an autocrine/paracrine positive feedback loop to drive the progression of the FTSECs-derived HGSC. Evidence in this study strongly suggests that combined therapy with inhibitors of YAP (such as verteporfin) and FGFRs (such as BGJ398) can provide a novel therapeutic strategy to treat Fallopian tube and ovarian HGSC. PMID:26364602

  11. Active dentate granule cells encode experience to promote the addition of adult-born hippocampal neurons.

    PubMed

    Kirschen, Gregory W; Shen, Jia; Tian, Mu; Schroeder, Bryce; Wang, Jia; Man, Guoming; Wu, Song; Ge, Shaoyu

    2017-04-03

    The continuous addition of new dentate granule cells, exquisitely regulated by brain activity, renders the hippocampus plastic. However, how neural circuits encode experiences to impact the addition of adult-born neurons remains unknown. Here, we used endoscopic Ca(2+) imaging to track the real-time activity of individual dentate granule cells in freely-behaving mice. For the first time, we found that active dentate granule cells responded to a novel experience by preferentially increasing their Ca(2+) event frequency. This elevated activity, which we found to be associated with object exploration, returned to baseline by one hour in the same environment, but could be dishabituated via introduction to a novel environment. To seamlessly transition between environments, we next established a freely-controllable virtual reality system for unrestrained mice. We again observed increased firing of active neurons in a virtual enriched environment. Interestingly, multiple novel virtual experiences accumulatively increased the number of newborn neurons when compared to a single experience. Finally, optogenetic silencing of existing dentate granule cells during novel environmental exploration perturbed experience-induced neuronal addition. Together, our study shows that the adult brain conveys novel, enriched experiences to increase the addition of adult-born hippocampal neurons by increasing the firing of active dentate granule cells.SIGNIFICANCE STATEMENTAdult brains are constantly reshaping themselves from synapses to circuits as we encounter novel experiences from moment to moment. Importantly, this reshaping includes the addition of newborn hippocampal neurons. However, it remains largely unknown how our circuits encode experience-induced brain activity to govern the addition of new hippocampal neurons. By coupling in vivo Ca(2+) imaging of dentate granule neurons with a novel unrestrained virtual reality system for rodents, we discovered that a new experience rapidly

  12. Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis.

    PubMed

    Yagami, Tatsurou; Ueda, Keiichi; Asakura, Kenji; Hata, Satoshi; Kuroda, Takayuki; Sakaeda, Toshiyuki; Takasu, Nobuo; Tanaka, Kazushige; Gemba, Takefumi; Hori, Yozo

    2002-01-01

    Expression of group IIA secretory phospholipase A2 (sPLA2-IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA2-IIA is associated with neurodegeneration. However, how sPLA2-IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA2-IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA2 activity was increased in the CTX. An sPLA2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA2-IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD2 generation. Indoxam prevented neurons from sPLA2-IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA2-IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA2-IIA-induced PGD2 generation and neuronal cell death. In conclusion, sPLA2-IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD2. Furthermore, sPLA2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA2-IIA inhibitors for stroke.

  13. Pseudomonas aeruginosa and tumor necrosis factor-alpha attenuate Clara cell secretory protein promoter function.

    PubMed

    Harrod, Kevin S; Jaramillo, Richard J

    2002-02-01

    The Clara cell secretory protein (CCSP, also CC-10/uterglobin) is a 16-kD homodimeric protein abundantly expressed in the airways of mammals. Although the molecular function is unknown, gene-targeting studies indicate CCSP as a regulator of lung inflammation following acute respiratory infection or injury. CCSP is decreased in the lungs of mice following acute Pseudomonas aeruginosa (P.a.) infection. In the present study, the role of decreased promoter function in the regulation of CCSP by P.a. was assessed using an in vitro co-culture system and in vivo studies of transgenic mice. CCSP promoter activity in lung epithelial cells was markedly decreased by P.a. or tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner. Regulation of CCSP promoter function by either P.a. or TNF-alpha was localized to the proximal 166 bp flanking region of the CCSP promoter activity. Decreased regulation of the CCSP promoter by P.a. or TNF-alpha was specific to CCSP, as human surfactant protein D (SP-D) promoter activity was unaffected or increased by P.a. or TNF-alpha, respectively. A neutralizing antibody against human TNF-alpha was able to reverse both the TNF-alpha- mediated as well as P.a.-mediated decrease in CCSP promoter function in lung epithelial cells. TNF-alpha secretion by lung epithelial cells coincided with the decrease in CCSP promoter function following P.a. administration. Using a transgenic mouse model, P.a. administration to the lung markedly attenuated CCSP promoter-conferred gene expression in vivo. The attenuation of CCSP promoter activity in lung epithelial cells by P.a. involves, in part, autocrine/paracrine secretion of TNF-alpha, which in turn regulates CCSP transcription through cis-active elements in the proximal promoter region.

  14. Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium

    PubMed Central

    Gerbe, François; van Es, Johan H.; Makrini, Leila; Brulin, Bénédicte; Mellitzer, Georg; Robine, Sylvie; Romagnolo, Béatrice; Shroyer, Noah F.; Bourgaux, Jean-François; Pignodel, Christine; Clevers, Hans

    2011-01-01

    The unique morphology of tuft cells was first revealed by electron microscopy analyses in several endoderm-derived epithelia. Here, we explore the relationship of these cells with the other cell types of the intestinal epithelium and describe the first marker signature allowing their unambiguous identification. We demonstrate that although mature tuft cells express DCLK1, a putative marker of quiescent stem cells, they are post-mitotic, short lived, derive from Lgr5-expressing epithelial stem cells, and are found in mouse and human tumors. We show that whereas the ATOH1/MATH1 transcription factor is essential for their differentiation, Neurog3, SOX9, GFI1, and SPDEF are dispensable, which distinguishes these cells from enteroendocrine, Paneth, and goblet cells, and raises from three to four the number of secretory cell types in the intestinal epithelium. Moreover, we show that tuft cells are the main source of endogenous intestinal opioids and are the only epithelial cells that express cyclooxygenase enzymes, suggesting important roles for these cells in the intestinal epithelium physiopathology. PMID:21383077

  15. Mechanisms and benefits of granule cell latency coding in the mouse olfactory bulb

    PubMed Central

    Giridhar, Sonya; Urban, Nathaniel N.

    2012-01-01

    Inhibitory circuits are critical for shaping odor representations in the olfactory bulb. There, individual granule cells can respond to brief stimulation with extremely long (up to 1000 ms), input-specific latencies that are highly reliable. However, the mechanism and function of this long timescale activity remain unknown. We sought to elucidate the mechanism responsible for long-latency activity, and to understand the impact of widely distributed interneuron latencies on olfactory coding. We used a combination of electrophysiological, optical, and pharmacological techniques to show that long-latency inhibition is driven by late onset synaptic excitation to granule cells. This late excitation originates from tufted cells, which have intrinsic properties that favor longer latency spiking than mitral cells. Using computational modeling, we show that widely distributed interneuron latency increases the discriminability of similar stimuli. Thus, long-latency inhibition in the olfactory bulb requires a combination of circuit- and cellular-level mechanisms that function to improve stimulus representations. PMID:22754503

  16. Recurrent mossy fibers preferentially innervate parvalbumin-immunoreactive interneurons in the granule cell layer of the rat dentate gyrus.

    PubMed

    Blasco-Ibáñez, J M; Martínez-Guijarro, F J; Freund, T F

    2000-09-28

    Detection of vesicular zinc and immunohistochemistry against markers for different interneuron subsets were combined to study the postsynaptic target selection of zinc-containing recurrent mossy fiber collaterals in the dentate gyrus. Mossy fiber collaterals in the granule cell layer selectively innervated parvalbumin-containing cells, with numerous contacts per cell, whereas the granule cells were avoided. Under the electron microscope, those boutons made asymmetrical contacts on dendrites and somata. These findings suggest that, in addition to the hilar perforant path-associated (HIPP) interneurons, the basket and chandelier cells also receive a powerful feed-back drive from the granule cells, and thereby are able to control population synchrony in the dentate gyrus. On the other hand, the amount of monosynaptic excitatory feed-back among granule cells is shown to be negligible.

  17. A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes.

    PubMed

    Moreno, Alfredo; SantoDomingo, Jaime; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Alvarez, Javier

    2010-12-01

    Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2-EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange, neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH(3) cells. We find that all the acidic dyes labelled the same population of vesicles. However, that population was largely different from the one labelled by the targeted proteins, with very little colocalization among them, in all the cell types studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles are not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide (GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting that they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle dynamics is studied using these techniques.

  18. On the origins of the universal dynamics of endogenous granules in mammalian cells.

    PubMed

    Vanapalli, Siva A; Li, Yixuan; Mugele, Frieder; Duits, Michel H G

    2009-12-01

    Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

  19. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

    PubMed

    Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

    2013-04-01

    Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

  20. Identification of glucagon-producing cells (A cells) in dog gastric mucosa

    PubMed Central

    1976-01-01

    An immunocytochemical technique using specific antiglucagon serum reveals the presence of glucagon-containing cells situated exclusively in the oxyntic glandular mucosa of the dog stomach. Electron microscope examination of the mucosa demonstrated endocrine cells containing secretory granules with a round dense core surrounded by a clear halo, indistinguishable from secretory granules of pancreatic A cells. Like the alpha granules of pancreatic A cells, the granules of these gastric endocrine cells exhibited a peripheral distribution of silver grains after Grimelius silver staining. Moreover, the granules of these cells were found to be specifically labeled with reaction product, using the peroxidase immunocytochemical technique at the ultrastructural level. Accordingly, these cells were named gastric A cells. These data suggest that the gastric oxyntic mucosa contains cells indistinguishable cytologically, cytochemically, and immunocytochemically from pancreatic A cells. It is believed that gastric A cells are responsible for the secretion of the gastric glucagon. PMID:770482

  1. Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells

    PubMed Central

    Madroñal, Noelia; Delgado-García, José M.; Fernández-Guizán, Azahara; Chatterjee, Jayanta; Köhn, Maja; Mattucci, Camilla; Jain, Apar; Tsetsenis, Theodoros; Illarionova, Anna; Grinevich, Valery; Gross, Cornelius T.; Gruart, Agnès

    2016-01-01

    The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories. PMID:26988806

  2. Cerebellar cortical degeneration with selective granule cell loss in Bavarian mountain dogs.

    PubMed

    Flegel, T; Matiasek, K; Henke, D; Grevel, V

    2007-08-01

    Three Bavarian mountain dogs aged between 18 and 20 months, not related to each other, were presented with chronic signs of cerebellar dysfunction. On sagittal T2-weighted magnetic resonance imaging brain images, the tentative diagnosis of cerebellar hypoplasia was established based on an enlarged cerebrospinal fluid space around the cerebellum and an increased cerebrospinal fluid signal between the folia. Post-mortem examination was performed in one dog and did show an overall reduction of cerebellar size. On histopathologic examination, a selective loss of cerebellar granule cells with sparing of Purkinje cells was evident. Therefore, the Bavarian mountain dog is a breed where cerebellar cortical degeneration caused by the rather exceptional selective granule cell loss can be seen as cause of chronic, slowly progressive cerebellar dysfunction starting at an age of several months.

  3. Targeting vaccinia virus-expressed secretory beta subunit of human chorionic gonadotropin to the cell surface induces antibodies.

    PubMed Central

    Srinivasan, J; Singh, O; Chakrabarti, S; Talwar, G P

    1995-01-01

    We carried out experiments designed to study the effect of a protein's localization on its immunogenicity. A novel cell-surface protein was generated from a small, glycosylated secretory protein. The DNA sequence encoding the entire precursor of the human chorionic gonadotropin beta (beta hCG) subunit was fused in the correct reading frame to the DNA sequence encoding the transmembrane and cytoplasmic domains of vesicular stomatitis virus glycoprotein. This chimeric gene was introduced into the vaccinia virus genome to generate a recombinant virus. The recombinant virus, when used to infect animal cells, expressed a 135-amino-acid beta hCG subunit anchored in cellular membranes by the 48 carboxy-terminal amino acids of vesicular stomatitis virus glycoprotein. The immunogenicity of this recombinant virus with respect to its ability to generate anti-hCG antibodies was compared with that of a second recombinant vaccinia virus expressing the native secretory form of beta hCG. All animals immunized with the vaccinia virus expressing beta hCG on the cell surface elicited high titers of anti-hCG antibodies. Even after a single immunization with the recombinant vaccinia virus, the anti-hCG antibody titers persisted for a long period of time (more than 6 months). None of the animals immunized with vaccinia virus expressing the native secretory form of beta hCG showed any hCG-specific antibody response. PMID:7591154

  4. Granule cargo release from bone marrow-derived cells sustains cardiac hypertrophy.

    PubMed

    Yang, Fanmuyi; Dong, Anping; Ahamed, Jasimuddin; Sunkara, Manjula; Smyth, Susan S

    2014-11-15

    Bone marrow-derived inflammatory cells, including platelets, may contribute to the progression of pressure overload-induced left ventricular hypertrophy (LVH). However, the underlying mechanisms for this are still unclear. One potential mechanism is through release of granule cargo. Unc13-d(Jinx) (Jinx) mice, which lack Munc13-4, a limiting factor in vesicular priming and fusion, have granule secretion defects in a variety of hematopoietic cells, including platelets. In the current study, we investigated the role of granule secretion in the development of LVH and cardiac remodeling using chimeric mice specifically lacking Munc13-4 in marrow-derived cells. Pressure overload was elicited by transverse aortic constriction (TAC). Chimeric mice were created by bone marrow transplantation. Echocardiography, histology staining, immunohistochemistry, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and mass spectrometry were used to study LVH progression and inflammatory responses. Wild-type (WT) mice that were transplanted with WT bone marrow (WT→WT) and WT mice that received Jinx bone marrow (Jinx→WT) developed LVH and a classic fetal reprogramming response early (7 days) after TAC. However, at late times (5 wk), mice lacking Munc13-4 in bone marrow-derived cells (Jinx→WT) failed to sustain the cardiac hypertrophy observed in WT chimeric mice. No difference in cardiac fibrosis was observed at early or late time points. Reinjection of WT platelets or platelet releasate partially restored cardiac hypertrophy in Jinx chimeric mice. These results suggest that sustained LVH in the setting of pressure overload depends on one or more factors secreted from bone marrow-derived cells, possibly from platelets. Inhibiting granule cargo release may represent a novel target for preventing sustained LVH.

  5. Elemental levels in mast cell granules differ in sections from normal and diabetic rats: an X-ray microanalysis study

    SciTech Connect

    Kendall, M.D.

    1988-03-01

    Mast cells around the thymus of rats stain red with alcian blue and safranin indicating that the mast cells are probably of the peritoneal (connective tissue) type. After the onset of streptozotocin induced diabetes some cells contain both red and blue granules and blue staining cells may appear. X-ray microanalysis of frozen freeze-dried sections from diabetic male CSE Wistar rats showed electron dense granules to have similar amounts of S to normal rat mast cell granules but reduced levels of Na, Mg, P, Cl and K. Two cells also had electron lucent granules with very high levels of Na, Cl, K and Ca and reduced concentrations of S. The differences in elemental composition suggest that the mast cells from diabetic rats are not immature, but are related to the condition of induced diabetes, and that granules of very different composition can occur within a single cell. X-ray microanalysis has given an insight into mast cell granule elemental content which was not possible by conventional biochemical methods.

  6. Secretory phospholipases A2 induce neurite outgrowth in PC12 cells.

    PubMed Central

    Nakashima, Satoru; Ikeno, Yutaka; Yokoyama, Tatsuya; Kuwana, Masakazu; Bolchi, Angelo; Ottonello, Simone; Kitamoto, Katsuhiko; Arioka, Manabu

    2003-01-01

    sPLA(2)s (secretory phospholipases A(2)) belong to a broad and structurally diverse family of enzymes that hydrolyse the sn -2 ester bond of glycerophospholipids. We previously showed that a secreted fungal 15 kDa protein, named p15, as well as its orthologue from Streptomyces coelicolor (named Scp15) induce neurite outgrowth in PC12 cells at nanomolar concentrations. We report here that both p15 and Scp15 are members of a newly identified group of fungal/bacterial sPLA(2)s. The phospholipid-hydrolysing activity of p15 is absolutely required for neurite outgrowth induction. Mutants with a reduced PLA(2) activity exhibited a comparable reduction in neurite-inducing activity, and the ability to induce neurites closely matched the capacity of various p15 forms to promote fatty acid release from live PC12 cells. A structurally divergent member of the sPLA(2) family, bee venom sPLA(2), also induced neurites in a phospholipase activity-dependent manner, and the same effect was elicited by mouse group V and X sPLA(2)s, but not by group IB and IIA sPLA(2)s. Lysophosphatidylcholine, but not other lysophospholipids, nor arachidonic acid, elicited neurite outgrowth in an L-type Ca(2+) channel activity-dependent manner. In addition, p15-induced neuritogenesis was unaffected by various inhibitors that block arachidonic acid conversion into bioactive eicosanoids. Altogether, these results delineate a novel, Ca(2+)- and lysophosphatidylcholine-dependent neurotrophin-like role of sPLA(2)s in the nervous system. PMID:12967323

  7. Structural Plasticity of Dentate Granule Cell Mossy Fibers During the Development of Limbic Epilepsy

    PubMed Central

    Danzer, Steve C.; He, Xiaoping; Loepke, Andreas W.; McNamara, James O.

    2009-01-01

    Altered granule cell≫CA3 pyramidal cell synaptic connectivity may contribute to the development of limbic epilepsy. To explore this possibility, granule cell giant mossy fiber bouton plasticity was examined in the kindling and pilocarpine models of epilepsy using green fluorescent protein-expressing transgenic mice. These studies revealed significant increases in the frequency of giant boutons with satellite boutons 2 days and 1 month after pilocarpine status epilepticus, and increases in giant bouton area at 1 month. Similar increases in giant bouton area were observed shortly after kindling. Finally, both models exhibited plasticity of mossy fiber giant bouton filopodia, which contact GABAergic interneurons mediating feedforward inhibition of CA3 pyramids. In the kindling model, however, all changes were fleeting, having resolved by 1 month after the last evoked seizure. Together, these findings demonstrate striking structural plasticity of granule cell mossy fiber synaptic terminal structure in two distinct models of adult limbic epileptogenesis. We suggest that these plasticities modify local connectivities between individual mossy fiber terminals and their targets, inhibitory interneurons, and CA3 pyramidal cells potentially altering the balance of excitation and inhibition during the development of epilepsy. PMID:19294647

  8. Induction of stress granule-like structures in vesicular stomatitis virus-infected cells.

    PubMed

    Dinh, Phat X; Beura, Lalit K; Das, Phani B; Panda, Debasis; Das, Anshuman; Pattnaik, Asit K

    2013-01-01

    Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.

  9. Neuroendocrine secretory protein 7B2: structure, expression and functions.

    PubMed Central

    Mbikay, M; Seidah, N G; Chrétien, M

    2001-01-01

    7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2-7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin ('ACTH') hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2 has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation. PMID:11439082

  10. Activation of basophil and mast cell histamine release by eosinophil granule major basic protein

    PubMed Central

    1983-01-01

    Major basic protein (MBP) is a primary constituent of eosinophil granules. In this report, we demonstrate that MBP from human eosinophil granules initiates a nonlytic histamine release from human leukocytes. A direct effect of MBP on basophils was confirmed using purified human basophils. The kinetics of release were similar to those reported for poly-L-arginine, although MBP was less potent than poly-L-arginine of similar molecular weight. Reduction and alkylation of MBP diminished both the potency and efficacy of the molecule. Native MBP also stimulated histamine secretion from purified rat peritoneal mast cells in a manner characteristic of other polycations. These results emphasize the bidirectional nature of the basophil/mast cell-eosinophil regulatory axis. PMID:6854212

  11. The secretory pathway calcium ATPase PMR-1/SPCA1 has essential roles in cell migration during Caenorhabditis elegans embryonic development.

    PubMed

    Praitis, Vida; Simske, Jeffrey; Kniss, Sarah; Mandt, Rebecca; Imlay, Leah; Feddersen, Charlotte; Miller, Michael B; Mushi, Juliet; Liszewski, Walter; Weinstein, Rachel; Chakravorty, Adityarup; Ha, Dae-Gon; Schacht Farrell, Angela; Sullivan-Wilson, Alexander; Stock, Tyson

    2013-05-01

    Maintaining levels of calcium in the cytosol is important for many cellular events, including cell migration, where localized regions of high calcium are required to regulate cytoskeletal dynamics, contractility, and adhesion. Studies show inositol-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), which release calcium into the cytosol, are important regulators of cell migration. Similarly, proteins that return calcium to secretory stores are likely to be important for cell migration. The secretory protein calcium ATPase (SPCA) is a Golgi-localized protein that transports calcium from the cytosol into secretory stores. SPCA has established roles in protein processing, metal homeostasis, and inositol-trisphosphate signaling. Defects in the human SPCA1/ATP2C1 gene cause Hailey-Hailey disease (MIM# 169600), a genodermatosis characterized by cutaneous blisters and fissures as well as keratinocyte cell adhesion defects. We have determined that PMR-1, the Caenorhabditis elegans ortholog of SPCA1, plays an essential role in embryogenesis. Pmr-1 strains isolated from genetic screens show terminal phenotypes, such as ventral and anterior enclosure failures, body morphogenesis defects, and an unattached pharynx, which are caused by earlier defects during gastrulation. In Pmr-1 embryos, migration rates are significantly reduced for cells moving along the embryo surface, such as ventral neuroblasts, C-derived, and anterior-most blastomeres. Gene interaction experiments show changing the activity of itr-1/IP3R and unc-68/RyR modulates levels of embryonic lethality in Pmr-1 strains, indicating pmr-1 acts with these calcium channels to regulate cell migration. This analysis reveals novel genes involved in C. elegans cell migration, as well as a new role in cell migration for the highly conserved SPCA gene family.

  12. The Secretory Pathway Calcium ATPase PMR-1/SPCA1 Has Essential Roles in Cell Migration during Caenorhabditis elegans Embryonic Development

    PubMed Central

    Praitis, Vida; Simske, Jeffrey; Kniss, Sarah; Mandt, Rebecca; Imlay, Leah; Feddersen, Charlotte; Miller, Michael B.; Mushi, Juliet; Liszewski, Walter; Weinstein, Rachel; Chakravorty, Adityarup; Ha, Dae-Gon; Schacht Farrell, Angela; Sullivan-Wilson, Alexander; Stock, Tyson

    2013-01-01

    Maintaining levels of calcium in the cytosol is important for many cellular events, including cell migration, where localized regions of high calcium are required to regulate cytoskeletal dynamics, contractility, and adhesion. Studies show inositol-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), which release calcium into the cytosol, are important regulators of cell migration. Similarly, proteins that return calcium to secretory stores are likely to be important for cell migration. The secretory protein calcium ATPase (SPCA) is a Golgi-localized protein that transports calcium from the cytosol into secretory stores. SPCA has established roles in protein processing, metal homeostasis, and inositol-trisphosphate signaling. Defects in the human SPCA1/ATP2C1 gene cause Hailey-Hailey disease (MIM# 169600), a genodermatosis characterized by cutaneous blisters and fissures as well as keratinocyte cell adhesion defects. We have determined that PMR-1, the Caenorhabditis elegans ortholog of SPCA1, plays an essential role in embryogenesis. Pmr-1 strains isolated from genetic screens show terminal phenotypes, such as ventral and anterior enclosure failures, body morphogenesis defects, and an unattached pharynx, which are caused by earlier defects during gastrulation. In Pmr-1 embryos, migration rates are significantly reduced for cells moving along the embryo surface, such as ventral neuroblasts, C-derived, and anterior-most blastomeres. Gene interaction experiments show changing the activity of itr-1/IP3R and unc-68/RyR modulates levels of embryonic lethality in Pmr-1 strains, indicating pmr-1 acts with these calcium channels to regulate cell migration. This analysis reveals novel genes involved in C. elegans cell migration, as well as a new role in cell migration for the highly conserved SPCA gene family. PMID:23696750

  13. Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production.

    PubMed

    Chaiyadet, Sujittra; Smout, Michael; Johnson, Michael; Whitchurch, Cynthia; Turnbull, Lynne; Kaewkes, Sasithorn; Sotillo, Javier; Loukas, Alex; Sripa, Banchob

    2015-10-01

    Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south

  14. Dentate granule cell modulation in freely moving rats: vigilance state effects.

    PubMed

    Bronzino, J D; Blaise, J H; Mokler, D J; Morgane, P J

    1999-04-12

    Dentate granule cell population responses to paired-pulse stimulation applied to the perforant pathway across a range of interpulse intervals (IPIs) were examined during different vigilance states-quiet waking (QW), slow-wave sleep (SWS), and rapid-eye movement (REM) sleep-in freely moving rats at 15, 30 and 90 days of age. Using these evoked field potentials, the paired-pulse index (PPI), a measure of the type and degree of modulation of dentate granule cell excitability, was computed and shown to be altered as a function of age. Animals, 15 days old, showed significantly lower levels of early inhibition (20-40 ms IPIs), i.e., greater PPI values, during all three vigilance states when compared to both the 30- and 90-day old animals. Adult, i.e, 90-day old animals, on the other hand, showed significantly greater levels of late inhibition (300-1000 ms IPIs), i.e., lower PPI values, than the younger animals (15- and 30-day old) during QW and SWS. These results indicate that as the dentate field of the hippocampal formation matures there are significant alterations in the modulation of dentate granule cell activity.

  15. A Critical Role for Toxoplasma gondii Vacuolar Protein Sorting VPS9 in Secretory Organelle Biogenesis and Host Infection

    PubMed Central

    Sakura, Takaya; Sindikubwabo, Fabien; Oesterlin, Lena K.; Bousquet, Hugo; Slomianny, Christian; Hakimi, Mohamed-Ali; Langsley, Gordon; Tomavo, Stanislas

    2016-01-01

    Accurate sorting of proteins to the three types of parasite-specific secretory organelles namely rhoptry, microneme and dense granule in Toxoplasma gondii is crucial for successful host cell invasion by this obligate intracellular parasite. Despite its tiny body architecture and limited trafficking machinery, T. gondii relies heavily on transport of vesicles containing proteins, lipids and important virulence-like factors that are delivered to these secretory organelles. However, our understanding on how trafficking of vesicles operates in the parasite is still limited. Here, we show that the T. gondii vacuolar protein sorting 9 (TgVps9), has guanine nucleotide exchange factor (GEF) activity towards Rab5a and is crucial for sorting of proteins destined to secretory organelles. Our results illuminate features of TgVps9 protein as a key trafficking facilitator that regulates protein maturation, secretory organelle formation and secretion, thereby ensuring a primary role in host infection by T. gondii. PMID:27966671

  16. Epithelial Cell Transforming 2 and Aurora Kinase B Modulate Formation of Stress Granule-Containing Transcripts from Diverse Cellular Pathways in Astrocytoma Cells.

    PubMed

    Weeks, Adrienne; Agnihotri, Sameer; Lymer, Jennifer; Chalil, Alan; Diaz, Roberto; Isik, Semra; Smith, Christian; Rutka, James T

    2016-06-01

    Stress granules are small RNA-protein granules that modify the translational landscape during cellular stress to promote survival. The RhoGTPase RhoA is implicated in the formation of RNA stress granules. Our data demonstrate that the cytokinetic proteins epithelial cell transforming 2 and Aurora kinase B (AurkB) are localized to stress granules in human astrocytoma cells. AurkB and its downstream target histone-3 are phosphorylated during arsenite-induced stress. Chemical (AZD1152-HQPA) and siRNA inhibition of AurkB results in fewer and smaller stress granules when analyzed using high-throughput fluorescent-based cellomics assays. RNA immunoprecipitation with the known stress granule aggregates TIAR and G3BP1 was performed on astrocytoma cells, and subsequent analysis revealed that astrocytoma stress granules harbor unique mRNAs for various cellular pathways, including cellular migration, metabolism, translation, and transcriptional regulation. Human astrocytoma cell stress granules contain mRNAs that are known to be involved in glioma signaling and the mammalian target of rapamycin pathway. These data provide evidence that RNA stress granules are a novel form of epigenetic regulation in astrocytoma cells, which may be targetable by chemical inhibitors and enhance astrocytoma susceptibility to conventional therapy, such as radiation and chemotherapy.

  17. A 24,500 Da protein derived from rat germ cells is associated with sertoli cell secretory function.

    PubMed

    Onoda, M; Djakiew, D

    1993-12-15

    A function and identify of a 24,500 Da protein derived from round spermatids of the rat testis was investigated with a specific polyclonal antiserum raised against RSP-24.5. The proteins released from cultured round spermatids significantly stimulated the secretion of de novo synthesized protein from cultured immature rat Sertoli cells 2.4-fold above control levels. Immunoprecipitation of RSP-24.5 from round spermatid protein further enhanced the stimulation of Sertoli cell protein secretion up to 3.1-fold above control levels, indicating that RSP-24.5 plays a role in the down regulation of Sertoli cell secretion. The antiserum recognized the 24,500 Da protein in Western blots of round spermatid protein, pachytene spermatoctye protein, Sertoli cell lysate and peritubular myoid cell lysate. A 40 amino acid sequence of a cyanogen bromide cleaved internal fragment of RSP-24.5 showed 80.5% homology to a phosphatidylethanolamine binding protein. These results suggest that phosphatidylethanolamine binding protein participates in the negative regulation of Sertoli cell secretory function during spermatogenesis.

  18. Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation.

    PubMed

    Vester-Christensen, Malene Bech; Hachem, Maher Abou; Naested, Henrik; Svensson, Birte

    2010-01-01

    Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on beta-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98kDa was estimated by SDS-PAGE in excellent agreement with the theoretical value of 97419Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K(m,app)=0.16+/-0.02 mg/mL and k(cat,app)=79+/-10s(-1) by fitting the uncompetitive substrate inhibition Michaelis-Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k(cat,app)/K(m,app)=488+/-23mL/(mgs) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed alpha-, beta-, and gamma-cyclodextrin binding to LD with K(d) of 27.2, 0.70, and 34.7 microM, respectively.

  19. Studies of dentate granule cell modulation: paired-pulse responses in freely moving rats at three ages.

    PubMed

    Bronzino, J D; Blaise, J H; Austin-LaFrance, R J; Morgane, P J

    1996-10-23

    Dentate granule cell population responses to paired-pulse stimulations applied to the perforant pathway across a range of interpulse intervals (IPI) were examined in freely moving rats at 15, 30, and 90 days of age. The profile of the paired-pulse index (PPI), a measure of the type and degree of modulation of dentate granule cell excitability, was shown to change significantly as a function of age.

  20. High-frequency stimulation induces gradual immediate early gene expression in maturing adult-generated hippocampal granule cells.

    PubMed

    Jungenitz, Tassilo; Radic, Tijana; Jedlicka, Peter; Schwarzacher, Stephan W

    2014-07-01

    Increasing evidence shows that adult neurogenesis of hippocampal granule cells is advantageous for learning and memory. We examined at which stage of structural maturation and age new granule cells can be activated by strong synaptic stimulation. High-frequency stimulation of the perforant pathway in urethane-anesthetized rats elicited expression of the immediate early genes c-fos, Arc, zif268 and pCREB133 in almost 100% of mature, calbindin-positive granule cells. In contrast, it failed to induce immediate early gene expression in immature doublecortin-positive granule cells. Furthermore, doublecortin-positive neurons did not react with c-fos or Arc expression to mild theta-burst stimulation or novel environment exposure. Endogenous expression of pCREB133 was increasingly present in young cells with more elaborated dendrites, revealing a close correlation to structural maturation. Labeling with bromodeoxyuridine revealed cell age dependence of stimulation-induced c-fos, Arc and zif268 expression, with only a few cells reacting at 21 days, but with up to 75% of cells activated at 35-77 days of cell age. Our results indicate an increasing synaptic integration of maturing granule cells, starting at 21 days of cell age, but suggest a lack of ability to respond to activation with synaptic potentiation on the transcriptional level as long as immature cells express doublecortin.

  1. Effects of prenatal protein malnutrition on kindling-induced alterations in dentate granule cell excitability. II. Paired-pulse measures.

    PubMed

    Bronzino, J D; Austin-LaFrance, R J; Morgane, P J; Galler, J R

    1991-05-01

    The effects of prenatal protein malnutrition on kindling-induced changes in inhibitory modulation of dentate granule cell activity were examined by analysis of extracellular field potentials recorded from the granule cell layer of the dentate gyrus in response to paired-pulse stimulation of the perforant pathway in freely-moving rats. Since we have shown that kindling results in enhanced synaptic transmission at the level of the perforant path/granule cell synapse (see preceding paper), we sought to determine if the kindling process might induce changes in inhibitory modulation of granule cell excitability which could be involved in the slower acquisition of the kindled state we have previously reported in malnourished animals. Beginning at 120-150 days of age, the response of dentate granule cells to paired-pulse stimulation of the perforant path was examined at interpulse intervals (IPIs) ranging from 20-1000 ms. A paired-pulse index (PPI) was constructed based on the mean percent change in population spike amplitudes of the two responses resulting from application of the pulse pair. PPI measures obtained during the kindling process were compared with individual prekindling measures to determine the mean percent change in excitatory/inhibitory modulation of granule cell activity. Significant inhibition of the second population response was apparent at all IPIs tested for both diet groups following the first kindled afterdischarge.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Study of Mast Cells and Granules from Primo Nodes Using Scanning Ionic Conductance Microscopy.

    PubMed

    Yoo, Yeong-Yung; Jung, Goo-Eun; Kwon, Hee-Min; Bae, Kyoung-Hee; Cho, Sang-Joon; Soh, Kwang-Sup

    2015-12-01

    Acupuncture points have a notable characteristic in that they have a higher density of mast cells (MCs) compared with nonacupoints in the skin, which is consistent with the augmentation of the immune function by acupuncture treatment. The primo vascular system, which was proposed as the anatomical structure of the acupuncture points and meridians, also has a high density of MCs. We isolated the primo nodes from the surfaces of internal abdominal organs, and the harvested primo nodes were stained with toluidine blue. The MCs were easily recognized by their stained color and their characteristic granules. The MCs were classified into four stages according to the degranulation of histamine granules in the MCs. Using conventional optical microscopes details of the degranulation state of MCs in each stage were not observable. However, we were able to investigate the distribution of the granules on the surfaces of the MCs in each stage, and to demonstrate the height profiles and three-dimensional structures of the MCs without disturbance of the cell membrane by using the scanning ion conductance microscopy.

  3. Use of granzyme B-based fluorescent protein reporters to monitor granzyme distribution and granule integrity in live cells.

    PubMed

    Bird, Catherina H; Rizzitelli, Alexandra; Harper, Ian; Prescott, Mark; Bird, Phillip I

    2010-08-01

    Reporter proteins comprising granzyme B (GrB) fused to eGFP, ecliptic pHluorin or mCherry, were generated and used to study granule (lysosome) distribution and properties in COS-1 cells and natural killer cells. The reporters resembled native GrB in biosynthesis and localization, and accumulated in granules. In live cells both the eGFP and pHluorin reporters were dark in lysosomes, but fluoresced when the granule integrity or pH was perturbed by Leu-Leu methyl ester, hydrogen peroxide, naphthazarin, or sphingosine treatment. By contrast, fluorescence of the mCherry reporter was not pH-dependent. The quenching of eGFP within granules indicates that this commonly-used fluorescent protein is not appropriate as a vital intra-lysosomal marker.

  4. Synthetic mast-cell granules as adjuvants to promote and polarize immunity in lymph nodes

    NASA Astrophysics Data System (ADS)

    St. John, Ashley L.; Chan, Cheryl Y.; Staats, Herman F.; Leong, Kam W.; Abraham, Soman N.

    2012-03-01

    Granules of mast cells (MCs) enhance adaptive immunity when, on activation, they are released as stable particles. Here we show that submicrometre particles modelled after MC granules augment immunity when used as adjuvants in vaccines. The synthetic particles, which consist of a carbohydrate backbone with encapsulated inflammatory mediators such as tumour necrosis factor, replicate attributes of MCs in vivo including the targeting of draining lymph nodes and the timed release of the encapsulated mediators. When used as an adjuvant during vaccination of mice with haemagglutinin from the influenza virus, the particles enhanced adaptive immune responses and increased survival of mice on lethal challenge. Furthermore, differential loading of the particles with the cytokine IL-12 directed the character of the response towards Th1 lymphocytes. The synthetic MC adjuvants replicate and enhance the functions of MCs during vaccination, and can be extended to polarize the resulting immunity.

  5. Direct Imaging of RAB27B-Enriched Secretory Vesicle Biogenesis in Lacrimal Acinar Cells Reveals Origins on a Nascent Vesicle Budding Site

    PubMed Central

    Chiang, Lilian; Karvar, Serhan; Hamm-Alvarez, Sarah F.

    2012-01-01

    This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the “nascent vesicle site,” from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules. PMID:22363735

  6. Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces cerevisiae.

    PubMed

    Petrovic, Uros; Sribar, Jernej; Matis, Maja; Anderluh, Gregor; Peter-Katalinić, Jasna; Krizaj, Igor; Gubensek, Franc

    2005-10-15

    Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to g and e isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms. Thus yeast Saccharomyces cerevisiae can be used as a model eukaryotic cell, which lacks endogenous phospholipases A2, to assess the in vivo relevance of this interaction. Atx was expressed in yeast cells and shown to be biologically active inside the cells. It inhibited G2 cell-cycle arrest in yeast, which is regulated by 14-3-3 proteins. Interference with the cell cycle indicates a possible mechanism by which sPLA2s are able to cause the opposing effects, proliferation and apoptosis, in mammalian cells.

  7. Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor.

    PubMed

    Zanin, Juan Pablo; Abercrombie, Elizabeth; Friedman, Wilma J

    2016-07-19

    Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75(NTR)) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75(NTR) in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75(NTR), GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits.

  8. Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro; Nakamichi, Yoko; Nishiwaki, Chiyono; Kawakami, Hayato; Nagamatsu, Shinya

    2009-12-04

    To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic {beta} cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.

  9. Granule cell hyperexcitability in the early post-traumatic rat dentate gyrus: the 'irritable mossy cell' hypothesis.

    PubMed

    Santhakumar, V; Bender, R; Frotscher, M; Ross, S T; Hollrigel, G S; Toth, Z; Soltesz, I

    2000-04-01

    1. Cytochemical and in vitro whole-cell patch clamp techniques were used to investigate granule cell hyperexcitability in the dentate gyrus 1 week after fluid percussion head trauma. 2. The percentage decrease in the number of hilar interneurones labelled with either GAD67 or parvalbumin mRNA probes following trauma was not different from the decrease in the total population of hilar cells, indicating no preferential survival of interneurones with respect to the non-GABAergic hilar cells, i.e. the mossy cells. 3. Dentate granule cells following trauma showed enhanced action potential discharges, and longer-lasting depolarizations, in response to perforant path stimulation, in the presence of the GABAA receptor antagonist bicuculline. 4. There was no post-traumatic alteration in the perforant path-evoked monosynaptic excitatory postsynaptic currents (EPSCs), or in the intrinsic properties of granule cells. However, after trauma, the monosynaptic EPSC was followed by late, polysynaptic EPSCs, which were not present in controls. 5. The late EPSCs in granule cells from fluid percussion-injured rats were not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were eliminated by both the non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the AMPA receptor antagonist GYKI 53655. 6. In addition, the late EPSCs were not present in low (0.5 mM) extracellular calcium, and they were also eliminated by the removal of the dentate hilus from the slice. 7. Mossy hilar cells in the traumatic dentate gyrus responded with significantly enhanced, prolonged trains of action potential discharges to perforant path stimulation. 8. These data indicate that surviving mossy cells play a crucial role in the hyperexcitable responses of the post-traumatic dentate gyrus.

  10. Differential dendritic Ca2+ signalling in young and mature hippocampal granule cells

    PubMed Central

    Stocca, Gabriella; Schmidt-Hieber, Christoph; Bischofberger, Josef

    2008-01-01

    Neuronal activity is critically important for development and plasticity of dendrites, axons and synaptic connections. Although Ca2+ is an important signal molecule for these processes, not much is known about the regulation of the dendritic Ca2+ concentration in developing neurons. Here we used confocal Ca2+ imaging to investigate dendritic Ca2+ signalling in young and mature hippocampal granule cells, identified by the expression of the immature neuronal marker polysialated neural cell adhesion molecule (PSA-NCAM). Using the Ca2+-sensitive fluorescent dye OGB-5N, we found that both young and mature granule cells showed large action-potential evoked dendritic Ca2+ transients with similar amplitude of ∼200 nm, indicating active backpropagation of action potentials. However, the decay of the dendritic Ca2+ concentration back to baseline values was substantially different with a decay time constant of 550 ms in young versus 130 ms in mature cells, leading to a more efficient temporal summation of Ca2+ signals during theta-frequency stimulation in the young neurons. Comparison of the peak Ca2+ concentration and the decay measured with different Ca2+ indicators (OGB-5N, OGB-1) in the two populations of neurons revealed that the young cells had an ∼3 times smaller endogenous Ca2+-binding ratio (∼75 versus∼220) and an ∼10 times slower Ca2+ extrusion rate (∼170 s−1versus∼1800 s−1). These data suggest that the large dendritic Ca2+ signals due to low buffer capacity and slow extrusion rates in young granule cells may contribute to the activity-dependent growth and plasticity of dendrites and new synaptic connections. This will finally support differentiation and integration of young neurons into the hippocampal network. PMID:18591186

  11. Impact of simulated microgravity on the secretory and adhesive activity of cultured human vascular endothelial cells.

    NASA Astrophysics Data System (ADS)

    Rudimov, Evgeny; Buravkova, Ludmila; Pogodina, Margarita; Andrianova, Irina

    The layer of vascular endothelial cells (ECs) is a dynamic,disseminated organ that perform the function of an interface between the blood and vascular wall. The endothelial monolayer is able to quickly respond to changes in the microenvironment due to its synthesis of vasoactive substances, chemokines, adhesion molecules expression, etc. ECs are highly sensitive to gravitational changes and capable of short-term and long-term responses (Sangha et al., 2001; Buravkova et al., 2005; Infanger et al., 2006, 2007. However, the question remains how to reflect the impact of microgravity on endothelium under the inflammatory process. Therefore, the aim of this study was to investigate secretory and adhesive activity of human umbilical vein endothelial cells (HUVECs) during simulated microgravity and TNF-a activation. HUVECs were isolated according to Gimbrone et al. (1978) in modification A. Antonov (1981) and used for experiments at 2-4 passages. HUVECs were activated by low level of TNF-a (2 ng/ml). Microgravity was generated by Random Positioning Machine (RPM, Dutch Space, Leiden) placed into the thermostat at 37°C. After 24 hours of clinorotation we measured adhesion molecules expression on the cell surface (ICAM-1, VCAM-1, PECAM-1, E-selectin, CD144, endoglin (CD105)) and cell viability using a flow cytometry. To evaluate the level of target gene expression was used the real time RT-PCR. IL-6 and IL-8 concentration was measured in the conditioned medium of HUVECs by using the ELISA test. We found that simulated microgravity within 24 hours caused a decrease of ICAM-1, CD144, and E-selectin expression, at the same time not affect the cell viability, endoglin and PECAM-1 expression on the surface HUVEC. Furthermore, there were no changes of the level of IL-6 and IL-8 gene expression and their products in the culture medium. TNF-activated HUVECs showed an increase in gene expression of interleukins and molecules involved in the adhesion process, which also was confirmed

  12. Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein

    SciTech Connect

    Plopper, C.G. . E-mail: cgplopper@ucdavis.edu; Mango, G.W.; Hatch, G.E.; Wong, V.J.; Toskala, E.; Reynolds, S.D.; Tarkington, B.K.; Stripp, B.R.

    2006-05-15

    Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O{sub 3}). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.

  13. Excitability changes within transverse lamellae of dentate granule cells and their longitudinal spread following orthodromic or antidromic activation.

    PubMed

    Lømo, Terje

    2009-07-01

    The functional organization of the perforant path input to the dentate gyrus of the exposed hippocampus was studied in adult rabbits anesthetized with urethane and chloralose. Electrical stimulation of perforant path fibers caused excitation of granule cells along narrow, nearly transverse strips (lamellae) of tissue. Stimulation of granule cell axons (mossy fibers) in CA3 caused antidromic activation of granule cells along similar strips. Paired-pulse stimulation revealed marked changes in granule cell excitability both within a lamella (on-line) and for several mm off-line along the septo-temporal axis of the dentate gyrus. After the first pulse, granule cells were inhibited for up to about 100 ms and then facilitated for up to hundreds of ms. Feedback activity along mossy fiber collaterals exciting local inhibitory and excitatory neurons appeared to dominate in producing on- and off-line inhibition and facilitation. Neurons mediating these effects could be inhibitory basket cells and other inhibitory interneurons targeting granule cells on- and off-line. In addition, excitatory mossy cells with far reaching, longitudinally running axons could affect off-line granule cells by exciting them directly or inhibit them indirectly by exciting local inhibitory interneurons. A scheme for dentate gyrus function is proposed whereby information to the dentate gyrus becomes split into interacting transverse strips of neuronal assemblies along which temporal processing occurs. A matrix of neuronal assemblies thus arises within which fragments of events and experiences is stored through the plasticity of synapses within and between the assemblies. Similar fragments may then be recognized at later times allowing memories of the whole to be created by pattern completion at subsequent computational stages in the hippocampus.

  14. Calcium dynamics in catecholamine-containing secretory vesicles.

    PubMed

    Moreno, Alfredo; Lobatón, Carmen D; Santodomingo, Jaime; Vay, Laura; Hernández-SanMiguel, Esther; Rizzuto, Rosario; Montero, Mayte; Alvarez, Javier

    2005-06-01

    We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.

  15. Rab27b regulates number and secretion of platelet dense granules.

    PubMed

    Tolmachova, Tanya; Abrink, Magnus; Futter, Clare E; Authi, Kalwant S; Seabra, Miguel C

    2007-04-03

    The Rab27 GTPase subfamily consists of two closely related homologs, Rab27a and Rab27b. Rab27a has been shown previously to regulate organelle movement and regulated exocytosis in a wide variety of secretory cells. However, the role of the more restrictedly expressed Rab27b remains unclear. Here we describe the creation of Rab27b knockout (KO) strain that was subsequently crossed with the naturally occurring Rab27a KO line, ashen, to produce double KO (Rab27a(ash/ash) Rab27b(-/-)) mice. Rab27b KO (and double KO) exhibit significant hemorrhagic disease in contrast to ashen mice. In vitro assays demonstrated impaired aggregation with collagen and U46619 and reduced secretion of dense granules in both Rab27b and double KO strains. Additionally, we detected a 50% reduction in the number of dense granules per platelet and diminished platelet serotonin content, possibly due to a dense granule packaging defect into proplatelets during megakaryocyte maturation. The presence of Rab27a partially compensated for the secretory defect but not the reduced granule number. The morphology and function of platelet alpha-granules were unaffected. Our data suggest that Rab27b is a key regulator of dense granule secretion in platelets and thus a candidate gene for delta-storage pool deficiency in humans.

  16. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

    PubMed

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S; Greenstein, David; Navarro, Rosa E

    2016-04-07

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures.

  17. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans

    PubMed Central

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S.; Greenstein, David; Navarro, Rosa E.

    2016-01-01

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline—the immortal cell lineage required for sexual reproduction—protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. PMID:26865701

  18. Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

    NASA Astrophysics Data System (ADS)

    Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

    2014-02-01

    In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

  19. A secretory cell type develops alongside multiciliated cells, ionocytes and goblet cells, and provides a protective, anti-infective function in the frog embryonic mucociliary epidermis

    PubMed Central

    Dubaissi, Eamon; Rousseau, Karine; Lea, Robert; Soto, Ximena; Nardeosingh, Siddarth; Schweickert, Axel; Amaya, Enrique; Thornton, David J.; Papalopulu, Nancy

    2014-01-01

    The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences. PMID:24598166

  20. Staufen1 impairs stress granule formation in skeletal muscle cells from myotonic dystrophy type 1 patients

    PubMed Central

    Ravel-Chapuis, Aymeric; Klein Gunnewiek, Amanda; Bélanger, Guy; Crawford Parks, Tara E.; Côté, Jocelyn; Jasmin, Bernard J.

    2016-01-01

    Myotonic dystrophy (DM1) is caused by an expansion of CUG repeats (CUGexp) in the DMPK mRNA 3′UTR. CUGexp-containing mRNAs become toxic to cells by misregulating RNA-binding proteins. Here we investigated the consequence of this RNA toxicity on the cellular stress response. We report that cell stress efficiently triggers formation of stress granules (SGs) in proliferating, quiescent, and differentiated muscle cells, as shown by the appearance of distinct cytoplasmic TIA-1– and DDX3-containing foci. We show that Staufen1 is also dynamically recruited into these granules. Moreover, we discovered that DM1 myoblasts fail to properly form SGs in response to arsenite. This blockage was not observed in DM1 fibroblasts, demonstrating a cell type–specific defect. DM1 myoblasts display increased expression and sequestration of toxic CUGexp mRNAs compared with fibroblasts. Of importance, down-regulation of Staufen1 in DM1 myoblasts rescues SG formation. Together our data show that Staufen1 participates in the inhibition of SG formation in DM1 myoblasts. These results reveal that DM1 muscle cells fail to properly respond to stress, thereby likely contributing to the complex pathogenesis of DM1. PMID:27030674

  1. Serine proteinases of mast cell and leukocyte granules. A league of their own.

    PubMed

    Caughey, G H

    1994-12-01

    Serine proteinases are hydrolases that use serine's side chain hydroxyl group to attack and cleave internal peptide bonds in peptides and proteins. They reside in all mammalian tissues, including the lung and airway. As a group, they vary tremendously in form and target specificity and have a vast repertoire of functions, many of which are critical for life. A subset of these proteinases is expressed primarily in the cytosolic granules of leukocytes from bone marrow, including mast cells. Examples are elastase-related proteinases and cathepsin G of monocytes and neutrophils, the many "granzymes" of cytotoxic T lymphocytes and natural killer (NK) cells, and the tryptases and chymases of mast cells. The pace of discovery and characterization of these granule-associated serine proteinases, fueled by technical advances in molecular biology, has accelerated rapidly in the past few years. Progress has been made in assigning possible functions to individual proteinases. However, the burgeoning numbers of these enzymes; their cell, tissue and species-dependent differences in expression; and their variety of action in vitro (despite, in many cases, shared modes of activation and recent divergence in protein evolution) have vexed and challenged those of us who are anxious to establish their roles in mammalian biology. Certainly, much remains to be discovered and clarified. The purpose of this overview is to capture the state of the art in this field, stressing the similarities as well as the differences among individual granule-associated proteinases and focusing particularly on those enzymes likely to be important in the human lung and airways.

  2. Secretory protein decondensation as a distinct, Ca2+-mediated event during the final steps of exocytosis in Paramecium cells

    PubMed Central

    1981-01-01

    The contents of secretory vesicles ("trichocysts") were isolated in the condensed state from Paramecium cells. It is well known that the majority portion of trichocysts perform a rapid decondensation process during exocytosis, which is visible in the light microscope. We have analyzed this condensed leads to decondensed transition in vitro and determined some relevant parameters. In the condensed state, free phosphate (and possibly magnesium) ions screen local surplus charges. This is supported by x-ray spectra recorded from individual trichocysts (prepared by physical methods) in a scanning transmission electron microscope. Calcium, as well as other ions that eliminate phosphate by precipitation, produces decondensation in vitro. Under in vivo conditions, Ca2+ enters the vesicle lumen from the outside medium, once an exocytic opening has been formed. Consequently, within the intact cell, membrane fusion and protein decondensation take place with optimal timing. Ca2+ might then trigger decondensation in the same way by precipitating phosphate ions (as it does in vitro) and, indeed, such precipitates (again yielding Ca and P signals in x-ray spectra) can be recognized in situ under trigger conditions. As decondensation is a unidirectional, rapid process in Paramecium cells, it would contribute to drive the discharge of the secretory contents to the outside. Further implications on the energetics of exocytosis are discussed. PMID:7204486

  3. The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms.

    PubMed

    Karsten, V; Qi, H; Beckers, C J; Reddy, A; Dubremetz, J F; Webster, P; Joiner, K A

    1998-06-15

    All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli beta-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.

  4. Glucose Toxic Effects on Granulation Tissue Productive Cells: The Diabetics' Impaired Healing

    PubMed Central

    Berlanga-Acosta, Jorge; Schultz, Gregory S.; López-Mola, Ernesto; Guillen-Nieto, Gerardo; García-Siverio, Marianela; Herrera-Martínez, Luis

    2013-01-01

    Type 2 diabetes mellitus is a metabolic noncommunicable disease with an expanding pandemic magnitude. Diabetes predisposes to lower extremities ulceration and impairs the healing process leading to wound chronification. Diabetes also dismantles innate immunity favoring wound infection. Amputation is therefore acknowledged as one of the disease's complications. Hyperglycemia is the proximal detonator of systemic and local toxic effectors including proinflammation, acute-phase proteins elevation, and spillover of reactive oxygen and nitrogen species. Insulin axis deficiency weakens wounds' anabolism and predisposes to inflammation. The systemic accumulation of advanced glycation end-products irreversibly impairs the entire physiology from cells-to-organs. These factors in concert hamper fibroblasts and endothelial cells proliferation, migration, homing, secretion, and organization of a productive granulation tissue. Diabetic wound bed may turn chronically inflammed, procatabolic, and an additional source of circulating pro-inflammatory cytokines, establishing a self-perpetuating loop. Diabetic fibroblasts and endothelial cells may bear mitochondrial damages becoming prone to apoptosis, which impairs granulation tissue cellularity and perfusion. Endothelial progenitor cells recruitment and tubulogenesis are also impaired. Failure of wound reepithelialization remains a clinical challenge while it appears to be biologically multifactorial. Ulcer prevention by primary care surveillance, education, and attention programs is of outmost importance to reduce worldwide amputation figures. PMID:23484099

  5. Increased expression of BDNF and proliferation of dentate granule cells after bacterial meningitis.

    PubMed

    Tauber, Simone C; Stadelmann, Christine; Spreer, Annette; Brück, Wolfgang; Nau, Roland; Gerber, Joachim

    2005-09-01

    Proliferation and differentiation of neural progenitor cells is increased after bacterial meningitis. To identify endogenous factors involved in neurogenesis, expression of brain-derived neurotrophic factor (BDNF), TrkB, nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF) was investigated. C57BL/6 mice were infected by intracerebral injection of Streptococcus pneumoniae. Mice were killed 30 hours later or treated with ceftriaxone and killed 4 days after infection. Hippocampal BDNF mRNA levels were increased 2.4-fold 4 days after infection (p = 0.026). Similarly, BDNF protein levels in the hippocampal formation were higher in infected mice than in control animals (p = 0.0003). This was accompanied by an elevated proliferation of dentate granule cells (p = 0.0002). BDNF protein was located predominantly in the hippocampal CA3/4 area and the hilus of the dentate gyrus. The density of dentate granule cells expressing the BDNF receptor TrkB as well as mRNA levels of TrkB in the hippocampal formation were increased 4 days after infection (p = 0.027 and 0.0048, respectively). Conversely, NGF mRNA levels at 30 hours after infection were reduced by approximately 50% (p = 0.004). No significant changes in GDNF expression were observed. In conclusion, increased synthesis of BDNF and TrkB suggests a contribution of this neurotrophic factor to neurogenesis after bacterial meningitis.

  6. Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

    SciTech Connect

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.; Paulsen, Ragnhild E.

    2010-09-15

    Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

  7. Organization of spinocerebellar projection map in three types of agranular cerebellum: Purkinje cells vs. granule cells as organizer element

    SciTech Connect

    Arsenio Nunes, M.L.; Sotelo, C.; Wehrle, R.

    1988-07-01

    The organization of the spinocerebellar projection was analysed by the anterograde axonal WGA-HRP (horseradish peroxidase-wheat germ agglutinin conjugate) tracing method in three different types of agranular cerebellar cortex either induced experimentally by X-irradiation or occurring spontaneously in weaver (wv/wv) and staggerer (sg/sg) mutant mice. The results of this study show that in the X-irradiated rat and weaver mouse, in both of which the granule cells are directly affected and die early in development, the spinal axons reproduce, with few differences, the normal spinocerebellar pattern. Conversely, in staggerer mouse, in which the Purkinje cells are intrinsically affected and granule neurons do not seem to be primarily perturbed by the staggerer gene action, the spinocerebellar organization is severely modified. These findings appear somewhat paradoxical because if granule cells, the synaptic targets of mossy spinocerebellar fibers, were necessary for the organization of spinocerebellar projection, the staggerer cerebellum would exhibit a much more normal projectional map than the weaver and the X-irradiated cerebella. It is, therefore, obvious that granule cells, and even specific synaptogenesis, are not essential for the establishment of the normal spinocerebellar topography. On the other hand, the fact that the Purkinje cells are primarily affected in the unique agranular cortex in which the spinocerebellar organization is severely modified suggests that these neurons could be the main element in the organization of the spinocerebellar projection map. This hypothesis is discussed in correlation with already-reported findings on the zonation of the cerebellar cortex by biochemically different clusters of Purkinje cells.

  8. Altered patterning of dentate granule cell mossy fiber inputs onto CA3 pyramidal cells in limbic epilepsy

    PubMed Central

    McAuliffe, John J.; Bronson, Stefanie L.; Hester, Michael S.; Murphy, Brian L.; Dahlquist-Topalá, Renée; Richards, David A.; Danzer, Steve C.

    2009-01-01

    Impaired gating by hippocampal dentate granule cells may promote the development of limbic epilepsy by facilitating seizure spread through the hippocampal trisynaptic circuit. The second synapse in this circuit, the dentate granule cell≫CA3 pyramidal cell connection, may be of particular importance because pathological changes occurring within the dentate likely exert their principal effect on downstream CA3 pyramids. Here, we utilized GFP-expressing mice and immunolabeling for the zinc transporter ZnT-3 to reveal the pre- and postsynaptic components of granule cell≫CA3 pyramidal cell synapses following pilocarpine-epileptogenesis. Confocal analyses of these terminals revealed that while granule cell presynaptic giant boutons increased in size and complexity one month after status epilepticus, individual thorns making up the postsynaptic thorny excrescences of the CA3 pyramidal cells were reduced in number. This reduction, however, was transient, and three months after status, thorn density recovered. This recovery was accompanied by a significant change in the distribution of thorns along pyramidal cells dendrites. While thorns in control animals tended to be tightly clustered, thorns in epileptic animals were more evenly distributed. Computational modeling of thorn distributions predicted an increase in the number of boutons required to cover equivalent numbers of thorns in epileptic vs. control mice. Confirming this prediction, ZnT-3 labeling of presynaptic giant boutons apposed to GFP-expressing thorns revealed a near doubling in bouton density, while the number of individual thorns per bouton was reduced by half. Together, these data provide clear evidence of novel plastic changes occurring within the epileptic hippocampus. PMID:20014385

  9. Distinct fusion properties of synaptotagmin-1 and synaptotagmin-7 bearing dense core granules.

    PubMed

    Rao, Tejeshwar C; Passmore, Daniel R; Peleman, Andrew R; Das, Madhurima; Chapman, Edwin R; Anantharam, Arun

    2014-08-15

    Adrenal chromaffin cells release hormones and neuropeptides that are essential for physiological homeostasis. During this process, secretory granules fuse with the plasma membrane and deliver their cargo to the extracellular space. It was once believed that fusion was the final regulated step in exocytosis, resulting in uniform and total release of granule cargo. Recent evidence argues for nonuniform outcomes after fusion, in which cargo is released with variable kinetics and selectivity. The goal of this study was to identify factors that contribute to the different outcomes, with a focus on the Ca(2+)-sensing synaptotagmin (Syt) proteins. Two Syt isoforms are expressed in chromaffin cells: Syt-1 and Syt-7. We find that overexpressed and endogenous Syt isoforms are usually sorted to separate secretory granules and are differentially activated by depolarizing stimuli. In addition, overexpressed Syt-1 and Syt-7 impose distinct effects on fusion pore expansion and granule cargo release. Syt-7 pores usually fail to expand (or reseal), slowing the dispersal of lumenal cargo proteins and granule membrane proteins. On the other hand, Syt-1 diffuses from fusion sites and promotes the release of lumenal cargo proteins. These findings suggest one way in which chromaffin cells may regulate cargo release is via differential activation of synaptotagmin isoforms.

  10. Feedback inhibition of thymic secretory activity in mice treated by the thymic extract TP-1 (thymostimulin).

    PubMed Central

    Shoham, J; Ben-David, E; Sandbank, U

    1982-01-01

    The ultrastructural changes occurring in the medullary epithelium of the thymus of young mice, as a result of repeated injections of thymic extract, TP-1 (thymostimulin) was investigated. After daily injection of TP-1 for 3 weeks, no changes in thymus architecture could be observed by light microscopy. However, by electron microscopy, specific changes were noticed in the epithelial cells. The secretory granules became dilated and engorged; diameter of granules in normal control thymus was approximately 200-250 nm, but reached 1000 nm in treated mice. Degenerative changes appeared in some of these granules, including myelin bodies, distorted configuration and fat droplets. Signs of involution of whole cells and presence of cellular debri within macrophages were observed. Acid phosphatase staining disclosed many lysosomes containing ingested granules. No such findings were observed in control untreated mice, or in mice treated by a heart extract similarly prepared to TP-1. All these findings can be taken as ultrastructural evidence for feedback inhibition of thymic secretory activity, in analogy to the changes occurring other feedback inhibited, peptide hormone secreting glands. The data indicate that (i) the thymus respond to feedback inhibitory stimuli, as other endocrine glands do; (ii)TP-1, the thymic extract under study, contains a physiologically significant thymic hormone, which, when introduced in high doses can exert specific feedback inhibition. This can be taken as an additional, new criterion for the definition of thymic hormones. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7056566

  11. High-pressure potato starch granule gelatinization: synchrotron radiation micro-SAXS/WAXS using a diamond anvil cell.

    PubMed

    Gebhardt, R; Hanfland, M; Mezouar, M; Riekel, C

    2007-07-01

    Potato starch granules have been examined by synchrotron radiation small- and wide-angle scattering in a diamond anvil cell (DAC) up to 750 MPa. Use of a 1 microm synchrotron radiation beam allowed the mapping of individual granules at several pressure levels. The data collected at 183 MPa show an increase in the a axis and lamellar period from the edge to the center of the granule, probably due to a gradient in water content of the crystalline and amorphous lamellae. The average granules radius increases up to the onset of gelatinization at about 500 MPa, but the a axis and the lamellar periodicity remain constant or even show a decrease, suggesting an initial hydration of amorphous growth rings. The onset of gelatinization is accompanied by (i) an increase in the average a axis and lamellar periodicity, (ii) the appearance of an equatorial SAXS streak, and (iii) additional short-range order peaks.

  12. The paired-pulse index: a measure of hippocampal dentate granule cell modulation.

    PubMed

    Bronzino, J D; Blaise, J H; Morgane, P J

    1997-01-01

    This study was undertaken to assess whether the paired-pulse index (PPI) is an effective measure of the modulation of dentate granule cell excitability during normal development. Paired-pulse stimulations of the perforant path were, therefore, used to construct a PPI for 15-, 30-, and 90-day old, freely moving male rats. Significant age-dependent differences in the PPI were obtained. Fifteen-day old rats showed significantly less inhibition at short interpulse intervals [interpulse interval (IPI): 20 to 30 msec), a lack of facilitation at intermediate IPIs (50 to 150 msec), and significantly less inhibition at longer IPIs (300 to 1,000 msec) than adults.

  13. Enhanced acoustic startle responding in rats with radiation-induced hippocampal granule cell hypoplasia

    SciTech Connect

    Mickley, G.A.; Ferguson, J.L.

    1989-01-01

    Irradiation of the neonatal rat hippocampus reduces the proliferation of granule cells in the dentate gyrus and results in locomotor hyperactivity, behavioral preservation, and deficits on some learned tasks. In order to address the role of changes in stimulus salience and behavioral inhibition in animals with this type of brain damage, irradiated and normal rats were compared in their startle reactions to an acoustic stimulus. Irradiated rats startled with a consistently higher amplitude than control and were more likely to exhibit startle responses. These animals with hippocampal damage also failed to habituate to the startle stimulus and, under certain circumstances, showed potentiated startle responses after many tone presentations.

  14. MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability

    PubMed Central

    Toriseva, Mervi; Laato, Matti; Carpén, Olli; Ruohonen, Suvi T.; Savontaus, Eriika; Inada, Masaki; Krane, Stephen M.; Kähäri, Veli-Matti

    2012-01-01

    Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13−/−) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13−/− mice. Granulation tissue in Mmp13−/− mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13−/− mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13−/− mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13−/− granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13−/− mice compared to WT mice. Mmp13−/− mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis. PMID:22880047

  15. Prox1 is required for granule cell maturation and intermediate progenitor maintenance during brain neurogenesis.

    PubMed

    Lavado, Alfonso; Lagutin, Oleg V; Chow, Lionel M L; Baker, Suzanne J; Oliver, Guillermo

    2010-08-17

    The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the ectopic expression of Prox1 induces premature differentiation of neural stem cells.

  16. Mutant torsinA interferes with protein processing through the secretory pathway in DYT1 dystonia cells

    PubMed Central

    Hewett, Jeffrey W.; Tannous, Bakhos; Niland, Brian P.; Nery, Flavia C.; Zeng, Juan; Li, Yuqing; Breakefield, Xandra O.

    2007-01-01

    TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope responsible for early onset torsion dystonia (DYT1). Most cases of this dominantly inherited movement disorder are caused by deletion of a glutamic acid in the carboxyl terminal region of torsinA. We used a sensitive reporter, Gaussia luciferase (Gluc) to evaluate the role of torsinA in processing proteins through the ER. In primary fibroblasts from controls and DYT1 patients most Gluc activity (95%) was released into the media and processed through the secretory pathway, as confirmed by inhibition with brefeldinA and nocodazole. Fusion of Gluc to a fluorescent protein revealed coalignment and fractionation with ER proteins and association of Gluc with torsinA. Notably, fibroblasts from DYT1 patients were found to secrete markedly less Gluc activity as compared with control fibroblasts. This decrease in processing of Gluc in DYT1 cells appear to arise, at least in part, from a loss of torsinA activity, because mouse embryonic fibroblasts lacking torsinA also had reduced secretion as compared with control cells. These studies demonstrate the exquisite sensitivity of this reporter system for quantitation of processing through the secretory pathway and support a role for torsinA as an ER chaperone protein. PMID:17428918

  17. Coupling of Insulin Secretion and Display of a Granule-resident Zinc Transporter ZnT8 on the Surface of Pancreatic Beta Cells.

    PubMed

    Huang, Qiong; Merriman, Chengfeng; Zhang, Hao; Fu, Dax

    2017-03-10

    The islet-specific zinc transporter ZnT8 mediates zinc enrichment in the insulin secretory granules of the pancreatic beta cell. This granular zinc transporter is also a major self-antigen found in type 1 diabetes patients. It is not clear whether ZnT8 can be displayed on the cell surface and how insulin secretion may regulate the level of ZnT8 exposure to extracellular immune surveillance. Here we report specific antibody binding to the extracellular surface of rat insulinoma INS-1E cells that stably expressed a tagged human zinc transporter ZnT8. Flow cytometry analysis after fluorescent antibody labeling revealed strong correlations among the levels of ZnT8 expression, its display on the cell surface, and glucose-stimulated insulin secretion (GSIS). Glucose stimulation increased the surface display of endogenous ZnT8 from a basal level to 32.5% of the housekeeping Na(+)/K(+) ATPase on the cell surface, thereby providing direct evidence for a GSIS-dependent surface exposure of the ZnT8 self-antigen. Moreover, the variation in tagged-ZnT8 expression and surface labeling enabled sorting of heterogeneous beta cells to subpopulations that exhibited marked differences in GSIS with parallel changes in endogenous ZnT8 expression. The abundant surface display of endogenous ZnT8 and its coupling to GSIS demonstrated the potential of ZnT8 as a surface biomarker for tracking and isolating functional beta cells in mixed cell populations.

  18. Excretory/secretory products of Fasciola hepatica but not recombinant phosphoglycerate kinase induce death of human hepatocyte cells.

    PubMed

    Bąska, Piotr; Norbury, Luke J; Wiśniewski, Marcin; Januszkiewicz, Kamil; Wędrychowicz, Halina

    2013-06-01

    The liver fluke Fasciola hepatica infects a wide range of hosts, and has a considerable impact on the agriculture industry, mainly through infections of sheep and cattle. Further, human infection is now considered of public health importance and is hyperendemic in some regions. The fluke infection causes considerable damage to the hosts' liver. However, the mechanisms of liver destruction have not yet been completely elucidated. In the present report we incubated a human liver cell line in the presence of either F. hepatica excretory/secretory material (FhES) or recombinant phosphoglycerate kinase (FhPGK). Dosedependent cytotoxicity in the presence of FhES was observed, indicating that FhES is capable of killing human hepatocytes, supporting a role for FhES in damaging host liver cells during infection; while treatment with a recombinant intracellular protein - FhPGK, had no impact on cell survival.

  19. Adaptor protein-3: A key player in RBL-2H3 mast cell mediator release

    PubMed Central

    da Silva, Elaine Zayas Marcelino; Freitas-Filho, Edismauro Garcia; de Souza-Júnior, Devandir Antonio; daSilva, Luis Lamberti Pinto; Jamur, Maria Celia

    2017-01-01

    Mast cell (MC) secretory granules are Lysosome-Related Organelles (LROs) whose biogenesis is associated with the post-Golgi secretory and endocytic pathways in which the sorting of proteins destined for a specific organelle relies on the recognition of sorting signals by adaptor proteins that direct their incorporation into transport vesicles. The adaptor protein 3 (AP-3) complex mediates protein trafficking between the trans-Golgi network (TGN) and late endosomes, lysosomes, and LROs. AP-3 has a recognized role in LROs biogenesis and regulated secretion in several cell types, including many immune cells such as neutrophils, natural killer cells, and cytotoxic T lymphocytes. However, the relevance of AP-3 for these processes in MCs has not been previously investigated. AP-3 was found to be expressed and distributed in a punctate fashion in rat peritoneal mast cells ex vivo. The rat MC line RBL-2H3 was used as a model system to investigate the role of AP-3 in mast cell secretory granule biogenesis and mediator release. By immunofluorescence and immunoelectron microscopy, AP-3 was localized both to the TGN and early endosomes indicating that AP-3 dependent sorting of proteins to MC secretory granules originates in these organelles. ShRNA mediated depletion of the AP-3 δ subunit was shown to destabilize the AP-3 complex in RBL-2H3 MCs. AP-3 knockdown significantly affected MC regulated secretion of β-hexosaminidase without affecting total cellular enzyme levels. Morphometric evaluation of MC secretory granules by electron microscopy revealed that the area of MC secretory granules in AP-3 knockdown MCs was significantly increased, indicating that AP-3 is involved in MC secretory granule biogenesis. Furthermore, AP-3 knockdown had a selective impact on the secretion of newly formed and newly synthesized mediators. These results show for the first time that AP-3 plays a critical role in secretory granule biogenesis and mediator release in MCs. PMID:28273137

  20. Mammary Analogue Secretory Carcinoma.

    PubMed

    Stevens, Todd M; Parekh, Vishwas

    2016-09-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor that shares the same histologic appearance and ETV6 gene (12p13) rearrangement as secretory carcinoma of the breast. Prior to its recognition, MASC cases were commonly labeled acinic cell carcinoma and adenocarcinoma, not otherwise specified. Despite distinctive histologic features, MASC may be difficult to distinguish from other salivary gland tumors, in particular zymogen-poor acinic cell carcinoma and low-grade salivary duct carcinoma. Although characteristic morphologic and immunohistochemical features form the basis of a diagnosis of MASC, the presence of an ETV6-NTRK3 gene fusion is confirmatory. Given its recent recognition the true prognostic import of MASC is not yet clearly defined.

  1. Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy

    PubMed Central

    Zhang, Wei; Huguenard, John R.; Buckmaster, Paul S.

    2012-01-01

    One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked excitatory postsynaptic currents (EPSCs) in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared to controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy. PMID:22279204

  2. Ontogeny of calbindin immunoreactivity in the human hippocampal formation with a special emphasis on granule cells of the dentate gyrus.

    PubMed

    Abrahám, Hajnalka; Veszprémi, Béla; Kravják, András; Kovács, Krisztina; Gömöri, Eva; Seress, László

    2009-04-01

    Calbindin (CB) is a calcium-binding protein that is present in principal cells as well as in interneurons of the hippocampal formation of various species including humans. Studies with transgenic mice revealed that CB is essential for long-term potentiation and synaptic plasticity which are the cellular basis of learning and memory. In a previous study we have shown that CB expression in granule cells of the dentate gyrus correlates with the functional maturation of the hippocampal formation in the rat. In the present study we examined the ontogeny of CB using immunohistochemistry in the human hippocampal formation paying special attention to the granule cells of the dentate gyrus. As early as the 14(th) week of gestation (GW), CB was being expressed by pyramidal cells of CA1-3 regions in the deepest cell rows of the pyramidal layer towards the ventricular zone. Later, CB sequentially appears in more superficial cell rows. After midgestation, CB disappears from CA3 pyramidal neurons. Expression of CB by granule cells starts at the 22(nd)-23(rd) GW, first by the most superficial neurons of the ectal end of the dorsal blade. At the 24(th) GW, CB is expressed by granule cells of the crest and medial portion of the ventral blade whereas later the entire ventral blade revealed CB immunoreactivity. At term, and in the first few postnatal months, CB-immunoreaction is detected in granule cells of both blades except for those neurons in the deepest cell rows at the hilar border. At around 2-3 years of age, all granule cells of the entire cell layer are CB-immunoreactive. Axons of granule cells, the mossy fibers, start to express CB around the 30(th) GW in stratum lucidum of CA3a. With further development, CB is expressed in CA3b and c, as well as in the hilus. An adult-like pattern of CB-immunoreactivity could be observed at 11 years of age. Our results indicate that (i) CB is expressed by hippocampal pyramidal cells a few weeks before midgestation; (ii) similarly to

  3. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    ERIC Educational Resources Information Center

    Hood-DeGrenier, Jennifer K.

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

  4. Targeted deletion of AKAP7 in dentate granule cells impairs spatial discrimination

    PubMed Central

    Weisenhaus, Michael; Sanford, Christina A; Slack, Margaret C; Chin, Jenesa; Nachmanson, Daniela; McKennon, Alex; Castillo, Pablo E; McKnight, G Stanley

    2016-01-01

    Protein Kinase A (PKA) mediates synaptic plasticity and is widely implicated in learning and memory. The hippocampal dentate gyrus (DG) is thought to be responsible for processing and encoding distinct contextual associations in response to highly similar inputs. The mossy fiber (MF) axons of the dentate granule cells convey strong excitatory drive to CA3 pyramidal neurons and express presynaptic, PKA-dependent forms of plasticity. Here, we demonstrate an essential role for the PKA anchoring protein, AKAP7, in mouse MF axons and terminals. Genetic ablation of AKAP7 specifically from dentate granule cells results in disruption of MF-CA3 LTP directly initiated by cAMP, and the AKAP7 mutant mice are selectively deficient in pattern separation behaviors. Our results suggest that the AKAP7/PKA complex in the MF projections plays an essential role in synaptic plasticity and contextual memory formation. DOI: http://dx.doi.org/10.7554/eLife.20695.001 PMID:27911261

  5. Interleukin-1 mediates long-term hippocampal dentate granule cell loss following postnatal viral infection.

    PubMed

    Orr, Anna G; Sharma, Anup; Binder, Nikolaus B; Miller, Andrew H; Pearce, Bradley D

    2010-05-01

    Viral infections of the developing CNS can cause long-term neuropathological sequela through undefined mechanisms. Proinflammatory cytokines such as IL-1beta have gained attention in mediating neurodegeneration in corticohippocampal structures due to a variety of insults in adults, though there is less information on the developing brain. Little is known concerning the spatial-temporal pattern of IL-1beta induction in the developing hippocampus following live virus infection, and there are few studies addressing the long-term consequences of this cytokine induction. We report that infection of rats with lymphocytic choriomeningitis virus on postnatal day 4 induces IL-1beta protein in select regions of the hippocampus on 6, 15, 21, and 45 days after infection. This infection resulted in a 71% reduction of dentate granule cell neurons by the time the rats reached mid-adulthood. We further investigated the causative role of IL-1 in this dentate granule cell loss by blocking IL-1 activity using an IL-1ra-expressing adenoviral vector administered at the time of infection. Blockade of IL-1 abrogated the infection-associated neuron loss in this vivo model. Considering that IL-1 can be triggered by multiple perinatal insults, our findings suggest that early therapy with anti-inflammatory agents that block IL-1 may be effective for reducing adulthood neuropathology.

  6. Antimicrobial peptide gramicidin S is accumulated in granules of producer cells for storage of bacterial phosphagens

    PubMed Central

    Berditsch, Marina; Trapp, Mareike; Afonin, Sergii; Weber, Christian; Misiewicz, Julia; Turkson, Joana; Ulrich, Anne S.

    2017-01-01

    Many antimicrobial peptides are synthesized non-ribosomally in bacteria, but little is known about their subcellular route of biosynthesis, their mode of intracellular accumulation, or their role in the physiology of the producer cells. Here, we present a comprehensive view on the biosynthesis of gramicidin S (GS) in Aneurinibacillus migulanus, having observed a peripheral membrane localization of its synthetases. The peptide gets accumulated in nano-globules, which mature by fusion into larger granules and end up within vacuolar structures. These granules serve as energy storage devices, as they contain GS molecules that are non-covalently attached to alkyl phosphates and protect them from dephosphorylation and premature release of energy. This finding of a fundamentally new type of high-energy phosphate storage mechanism can explain the curious role of GS biosynthesis in the physiology of the bacterial producer cells. The unknown role of the GrsT protein, which is part of the non-ribosomal GS synthetase operon, can thus be assumed to be responsible for the biosynthesis of alkyl phosphates. GS binding to alkyl phosphates may suggest its general affinity to phosphagens such as ATP and GTP, which can represent the important intracellular targets in pathogenic bacteria. PMID:28295017

  7. In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

    SciTech Connect

    Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B. . E-mail: jbarnett@hsc.wvu.edu

    2007-06-01

    The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

  8. The immunomodulatory properties of periodontal ligament stem cells isolated from inflamed periodontal granulation.

    PubMed

    Li, Chenghua; Wang, Xinwen; Tan, Jun; Wang, Tao; Wang, Qintao

    2014-01-01

    Periodontitis is currently the main cause of tooth loss and as yet there is no appropriate method for establishing a functional and predictable periodontal regeneration. Tissue engineering involving seed cells provides a new prospect for periodontal regeneration. While periodontal ligament stem cells (PDLSCs) are a good choice for seed cells, it is not always possible to obtain the patients' own PDLSCs. We and others have found a type of stromal cells from inflamed periodontal granulation. These cells displayed similar differentiation properties to PDLSCs. Inflammation has a profound influence on the immunomodulatory properties of mesenchymal stem cells, which may affect therapeutic outcome. In this study, we assessed the immunomodulatory characteristics of these inflamed human (ih)PDLSCs. Along with the similarity in cell surface marker expressions, they also displayed immunomodulatory properties comparable to those in healthy human (hh)PDLSCs. Both hhPDLSCs and ihPDLSCs can suppress the proliferation and secretion of IFN-γ in peripheral blood mononuclear cells by indirect soluble mediators and direct cell-cell contact. Albeit with some quantitative variances, the gene expressions of inducible nitric oxide synthases, indoleamine 2,3 dioxygenase, cyclooxygenase-2, TNF-α-induced protein 6 and IL-10 in ihPDLSCs displayed similar patterns as those in hhPDLSCs. Taken together, our results suggest that ihPDLSCs can provide a promising alternative to hhPDLSCs in terms of evident similarities in immunomodulatory properties as well as their easier accessibility and availability.

  9. The chemokine growth-related gene product β protects rat cerebellar granule cells from apoptotic cell death through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors

    PubMed Central

    Limatola, Cristina; Ciotti, Maria Teresa; Mercanti, Delio; Vacca, Fabrizio; Ragozzino, Davide; Giovannelli, Aldo; Santoni, Angela; Eusebi, Fabrizio; Miledi, Ricardo

    2000-01-01

    Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K+. We have found that the growth-related gene product β (GROβ) partially prevents the K+-depletion-induced cell death, and that the neuroprotective action of GROβ on granule cells is mediated through the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GROβ-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-d-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 μM) treatment. Moreover, GROβ-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GROβ is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors. PMID:10811878

  10. The secretory clear cell of the eccrine sweat gland as the probable source of excess sweat production in hyperhidrosis.

    PubMed

    Bovell, Douglas L; MacDonald, Alison; Meyer, Barbara A; Corbett, Alistair D; MacLaren, William M; Holmes, Susan L; Harker, Mark

    2011-12-01

    Primary hyperhidrosis is characterized by excessive sweating in palmar, plantar and axillary body regions. Gland hypertrophy and the existence of a third type of sweat gland, the apoeccrine gland, with high fluid transporting capabilities have been suggested as possible causes. This study investigated whether sweat glands were hypertrophied in axillary hyperhidrotic patients and if mechanisms associated with fluid transport were found in all types of axillary sweat glands. The occurrence of apoeccrine sweat glands was also investigated. Axillary skin biopsies from control and hyperhidrosis patients were examined using immunohistochemistry, image analysis and immunofluorescence microscopy. Results showed that glands were not hypertrophied and that only the clear cells in the eccrine glands expressed proteins associated with fluid transport. There was no evidence of the presence of apoeccrine glands in the tissues investigated. Preliminary findings suggest the eccrine gland secretory clear cell as the main source of fluid transport in hyperhidrosis.

  11. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

    PubMed

    Brown, Alice C N; Oddos, Stephane; Dobbie, Ian M; Alakoskela, Juha-Matti; Parton, Richard M; Eissmann, Philipp; Neil, Mark A A; Dunsby, Christopher; French, Paul M W; Davis, Ilan; Davis, Daniel M

    2011-09-01

    Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

  12. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

    SciTech Connect

    Coppé, Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith

    2008-10-24

    Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

  13. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

    PubMed Central

    Coppé, Jean-Philippe; Sun, Yu; Muñoz, Denise P; Goldstein, Joshua; Nelson, Peter S; Desprez, Pierre-Yves; Campisi, Judith

    2008-01-01

    Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment. PMID:19053174

  14. A Human-Like Senescence-Associated Secretory Phenotype Is Conserved in Mouse Cells Dependent on Physiological Oxygen

    PubMed Central

    Coppé, Jean-Philippe; Krtolica, Ana; Beauséjour, Christian M.; Parrinello, Simona; Hodgson, J. Graeme; Chin, Koei; Desprez, Pierre-Yves; Campisi, Judith

    2010-01-01

    Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP), which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that “senescent” mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen), do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3%) oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-α. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP. PMID:20169192

  15. Adverse influence of coumestrol on secretory function of bovine luteal cells in the first trimester of pregnancy.

    PubMed

    Młynarczuk, J; Wróbel, M H; Kotwica, J

    2013-07-01

    Coumestrol is one of a few biologically active substances present in leguminous plants, which are widely used as fodder for ruminants. Depending on the doses, coumestrol acts on the reproductive processes as an estrogen-like factor or antiestrogen to evoke a decrease in ovulation frequency, elongation of estrous cycle duration. The aim of the current investigations was to study the influence of coumestrol on secretory function of luteal cells obtained from first trimester of pregnant cows. Luteal cells (2.5 × 10(5) /mL) from 3rd to 5th, 6th to 8th, and 9th to 12th week of pregnancy were preincubated for 24 h and incubated with coumestrol (1 × 10(-6) M) for successive 48 h and the medium concentrations of progesterone (P4), oxytocin (OT), prostaglandin (PG) E2 and F2α were determined. Moreover, the expression of mRNA for neurophysin-I/oxytocin (NP-I/OT; precursor of OT) and peptidyl-glycine-α-amidating mono-oxygenase (PGA, an enzyme responsible for post-translational OT synthesis) was determined after 8 h of treatment. Coumestrol did not affect P4 secretion but increased the secretion of OT from the cells collected at all stages of gestation studied. Hence, the ratio of P4 to OT was markedly decreased. Simultaneously, coumestrol increased the expression of NP-I/OT mRNA during 9th to 12th weeks of pregnancy, and mRNA for PGA during 3rd to 5th and 9th to 12th weeks of gestation. Furthermore, coumestrol decreased PGE2 secretion from luteal cells in all studied stages of pregnancy, while it affected PGF2α metabolite (PGFM) concentration only from week 3 to 5 of pregnancy. Obtained results suggest that coumestrol impairs secretory function of the corpus luteum (CL) and this way it can affect the maintenance of pregnancy in the cow.

  16. Free radicals enzymatically triggered by Clonorchis sinensis excretory-secretory products cause NF-κB-mediated inflammation in human cholangiocarcinoma cells.

    PubMed

    Nam, Joo-Hyun; Moon, Ju Hyun; Kim, In Ki; Lee, Myoung-Ro; Hong, Sung-Jong; Ahn, Joong Ho; Chung, Jong Woo; Pak, Jhang Ho

    2012-01-01

    Chronic clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis worms and their excretory-secretory products, is associated with hepatobiliary damage, inflammation, periductal fibrosis and even development of cholangiocarcinoma. Our previous report revealed that intracellular reactive oxygen species were generated in C. sinensis excretory-secretory product-treated human cholangiocarcinoma cells; however, their endogenous sources and pathophysiological roles in host cells were not determined. In the present study, we found that treatment of human cholangiocarcinoma cells with excretory-secretory products triggered increases in free radicals via a time-dependent activation of NADPH oxidase, xanthine oxidase and inducible nitric oxide synthase. This increase in free radicals substantially promoted the degradation of cytosolic IκB-α, nuclear translocation of nuclear factor-κB subunits (RelA and p50), and increased κB consensus DNA-binding activity. Excretory-secretory product-induced nuclear factor-κB activation was markedly attenuated by preincubation with specific inhibitors of each free radical-producing enzyme or the antioxidant, N-acetylcysteine. Moreover, excretory-secretory products induced an increase in the mRNA and protein expression of the proinflammatory cytokines, IL-1β and IL-6, in an nuclear factor-κB-dependent manner, indicating that enzymatic production of free radicals in ESP-treated cells participates in nuclear factor-κB-mediated inflammation. These findings provide new insights into the pathophysiological role of C. sinensis excretory-secretory products in host chronic inflammatory processes, which are initial events in hepatobiliary diseases.

  17. Developmental changes in membrane properties and postsynaptic currents of granule cells in rat dentate gyrus.

    PubMed

    Liu, Y B; Lio, P A; Pasternak, J F; Trommer, B L

    1996-08-01

    1. Whole cell patch-clamp recordings were used to study dentate gyrus granule cells in hippocampal slices from juvenile rats (postnatal days 8-32). Membrane properties were measured with the use of current-clamp recordings and were correlated with the morphology of a subgroup of neurons filled with biocytin. The components of the postsynaptic currents (PSCs) induced by medial perforant path stimulation were characterized with the use of specific receptor antagonists in voltage-clamp recordings. 2. Granule cells located in the middle third of the superior blade of stratum granulosum from the rostral third of hippocampus were divided into three groups according to their input resistance (IR). Neurons with low IR (206 +/- 182 M omega, mean +/- SD) had hyperpolarized resting membrane potentials (-82 +/- 7 mV) and high-amplitude action potentials (108 +/- 23 mV). Neurons were high IR (1,259 +/- 204 M omega) had more depolarized resting membrane potentials (-54 +/- 6 mV) and lower-amplitude action potentials (71 +/- 10 mV). Neurons with intermediate IR (619 +/- 166 M omega) also had intermediate resting membrane potentials (-63 +/- 7 mV) and action potential amplitudes (86 +/- 14 mV). Low-IR neurons became increasingly prevalent with advancing postnatal age, but neurons from each group could be found throughout the entire period under study. 3. Morphological studies of low-IR neurons revealed an extensive dendritic arborization that traversed the entire molecular layer and was characteristic of mature granule cells. High-IR cells had smaller somata and short, simple dendritic arborization that incompletely penetrated the molecular layer and were classified as immature. Intermediate-IR cells had morphological features of intermediate maturity. 4. The initial phase of the PSC evoked at -80 mV was a fast inward current that was comparable with respect to latency to peak, latency to onset, and 10-90% rise time in neurons of all maturities held at -80 mV. This current was 6

  18. The role of calcium and cyclic nucleotide signaling in cerebellar granule cell migration under normal and pathological conditions.

    PubMed

    Komuro, Yutaro; Galas, Ludovic; Lebon, Alexis; Raoult, Emilie; Fahrion, Jennifer K; Tilot, Amanda; Kumada, Tasturo; Ohno, Nobuhiko; Vaudry, David; Komuro, Hitoshi

    2015-04-01

    In the developing brain, immature neurons migrate from their sites of origin to their final destination, where they reside for the rest of their lives. This active movement of immature neurons is essential for the formation of normal neuronal cytoarchitecture and proper differentiation. Deficits in migration result in the abnormal development of the brain, leading to a variety of neurological disorders. A myriad of extracellular guidance molecules and intracellular effector molecules is involved in controlling the migration of immature neurons in a cell type, cortical layer and birth-date-specific manner. To date, little is known about how extracellular guidance molecules transfer their information to the intracellular effector molecules, which regulate the migration of immature neurons. In this article, to fill the gap between extracellular guidance molecules and intracellular effector molecules, using the migration of cerebellar granule cells as a model system of neuronal cell migration, we explore the role of second messenger signaling (specifically Ca(2+) and cyclic nucleotide signaling) in the regulation of neuronal cell migration. We will, first, describe the cortical layer-specific changes in granule cell migration. Second, we will discuss the roles of Ca(2+) and cyclic nucleotide signaling in controlling granule cell migration. Third, we will present recent studies showing the roles of Ca(2+) and cyclic nucleotide signaling in the deficits in granule cell migration in mouse models of fetal alcohol spectrum disorders and fetal Minamata disease.

  19. Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

    PubMed

    Chang, Hsin-Fang; Bzeih, Hawraa; Schirra, Claudia; Chitirala, Praneeth; Halimani, Mahantappa; Cordat, Emmanuelle; Krause, Elmar; Rettig, Jens; Pattu, Varsha

    2016-09-15

    CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

  20. Revisiting the single cell protein application of Cupriavidus necator H16 and recovering bioplastic granules simultaneously.

    PubMed

    Kunasundari, Balakrishnan; Murugaiyah, Vikneswaran; Kaur, Gurjeet; Maurer, Frans H J; Sudesh, Kumar

    2013-01-01

    Cupriavidus necator H16 (formerly known as Hydrogenomonas eutropha) was famous as a potential single cell protein (SCP) in the 1970s. The drawback however was the undesirably efficient accumulation of non-nutritive polyhydroxybutyrate (PHB) storage compound in the cytoplasm of this bacterium. Eventually, competition from soy-based protein resulted in SCP not receiving much attention. Nevertheless, C. necator H16 remained in the limelight as a producer of PHB, which is a material that resembles commodity plastics such as polypropylene. PHB is a 100% biobased and biodegradable polyester. Although tremendous achievements have been attained in the past 3 decades in the efficient production of PHB, this bioplastic is still costly. One of the main problems has been the recovery of PHB from the cell cytoplasm. In this study, we showed for the first time that kilogram quantities of PHB can be easily recovered in the laboratory without the use of any solvents and chemicals, just by using the cells as SCP. In addition, the present study also demonstrated the safety and tolerability of animal model used, Sprague Dawley given lyophilized cells of C. necator H16. The test animals readily produced fecal pellets that were whitish in color, as would be expected of PHB granules. The pellets were determined to contain about 82-97 wt% PHB and possessed molecular mass of around 930 kg/mol. The PHB granules recovered biologically possessed similar molecular mass compared to chloroform extracted PHB [950 kg/mol]. This method now allows the production and purification of substantial quantities of PHB for various experimental trials. The method reported here is easy, does not require expensive instrumentation, scalable and does not involve extensive use of solvents and strong chemicals.

  1. Dissecting the molecular mechanisms that impair stress granule formation in aging cells.

    PubMed

    Moujaber, Ossama; Mahboubi, Hicham; Kodiha, Mohamed; Bouttier, Manuella; Bednarz, Klaudia; Bakshi, Ragini; White, John; Larose, Louise; Colmegna, Inés; Stochaj, Ursula

    2017-03-01

    Aging affects numerous aspects of cell biology, but the senescence-associated changes in the stress response are only beginning to emerge. To obtain mechanistic insights into these events, we examined the formation of canonical and non-canonical stress granules (SGs) in the cytoplasm. SG generation is a key event after exposure to physiological or environmental stressors. It requires the SG-nucleating proteins G3BP1 and TIA-1/TIAR and stress-related signaling events. To analyze SG formation, we used two independent models of somatic cell aging. In both model systems, cellular senescence impaired the assembly of two SG classes: (i) it compromised the formation of canonical SGs, and (ii) skewed the production of non-canonical SGs. We dissected the mechanisms underlying these senescence-dependent changes in granule biogenesis and identified several specific targets that were modulated by aging. Thus, we demonstrate a depletion of G3BP1 and TIA-1/TIAR in senescent cells and show that the loss of G3BP1 contributed to impaired SG formation. We further reveal that aging reduced Sp1 levels; this transcription factor regulated G3BP1 and TIA-1/TIAR abundance. The assembly of canonical SGs relies on the phosphorylation of translation initiation factor eIF2α. We show that senescence can cause eIF2α hyperphosphorylation. CReP is a subunit of protein phosphatase 1 and critical to reverse the stress-dependent phosphorylation of eIF2α. We demonstrate that the loss of CReP correlated with the aging-related hyperphosphorylation of eIF2α. Together, we have identified significant changes in the stress response of aging cells and provide mechanistic insights. Based on our work, we propose that the decline in SG formation can provide a new biomarker to evaluate cellular aging.

  2. Demonstration of Birbeck (Langerhans cells) granules in the normal chicken epidermis

    PubMed Central

    PÉREZ-TORRES, ARMANDO; USTARROZ-CANO, MARTHA

    2001-01-01

    Mammalian Langerhans cells (LC) are epidermal dendritic cells which originate in bone marrow and migrate toward the T cell area of lymph nodes, where they act as professional antigen-presenting cells. A variety of cell surface markers, such as the ectoenzyme adenosine triphosphatase (ATPase), Ia and CD1a antigens, have been used extensively to identify LC. Ultrastructural identification of this cell type in the mammalian epidermis is made by the demonstration of a typical and unique cytoplasmic organelle, the Birbeck granule (BG). Although we had earlier demonstrated the coexpression of ATPase and Ia antigens on epidermal dendritic cells of the chicken epidermis, the presence of the BG has not previously been documented. The aim of the present study was to investigate whether chicken epidermal LC-like cells possess an organelle similar to the BG, and thus to complete their identification. Our findings are the first demonstration of characteristic rod-shaped, racket-shaped and disc-shaped intracytoplasmic organelles, morphologically similar to the mammalian BG, in avian LC. PMID:11693310

  3. Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

    PubMed Central

    Titorenko, V I; Ogrydziak, D M; Rachubinski, R A

    1997-01-01

    We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum. PMID:9271399

  4. Regional development of Langerhans cells and formation of Birbeck granules in human embryonic and fetal skin.

    PubMed

    Fujita, M; Furukawa, F; Horiguchi, Y; Ueda, M; Kashihara-Sawami, M; Imamura, S

    1991-07-01

    The regional development of Langerhans cells (LC) and the formation of Birbeck granules (BG) were examined in human embryonic and fetal skin. Samples were obtained from multiple anatomic sites and stained with anti-CD36, anti-CD1a, and anti-HLA-DR antibody as well as Lag antibody specifically reactive to BG and some vacuoles of human LC. In the first trimester, CD36+ dendritic epidermal cells were identified before the appearance of CD1a+ cells and Lag+ cells. Some of the former co-expressed HLA-DR antigens but not CD1a antigens. In the second trimester, regional variations in LC development were observed. Epidermal LC of palms and soles reached a peak in number in the first trimester but were rarely detected after 18 weeks estimated gestation age (EGA), whereas, in other regions, their number increased with age. In the second trimester, CD1a+ cells and Lag+ cells were also identified in the epidermis, although Lag+ cells appeared later than CD1a+ cells. The Lag+ cells until 17 weeks EGA showed a variety of staining intensities and immunoelectron microscopy revealed that they contained various amounts of Lag-reactive BG. Flow cytometric analysis showed that relative amounts of Lag antigens in LC increased during the second trimester and that fetal LC of 18 weeks EGA expressed the same amounts of HLA-DR, CD1a, and Lag antigens as did adult human LC. In the dermis, in the second trimester, numerous CD36+ cells and HLA-DR+ cells were found, whereas CD1a+ cells and Lag+ cells were rarely detected. Taken together, it is suggested that HLA-DR+ dendritic cells acquire CD1a+ antigens first and then form BG after migration to the epidermis and that fetal LC are phenotypically mature in the second trimester.

  5. Stress Granules Modulate SYK to Cause Microglial Cell Dysfunction in Alzheimer's Disease

    PubMed Central

    Ghosh, Soumitra; Geahlen, Robert L.

    2015-01-01

    Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (Aβ) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or Aβ(1–42) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or Aβ fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to Aβ, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells. PMID:26870803

  6. Expression of the AMPA Receptor Subunits GluR1 and GluR2 is Associated with Granule Cell Maturation in the Dentate Gyrus

    PubMed Central

    Hagihara, Hideo; Ohira, Koji; Toyama, Keiko; Miyakawa, Tsuyoshi

    2011-01-01

    The dentate gyrus produces new granule neurons throughout adulthood in mammals from rodents to humans. During granule cell maturation, defined markers are expressed in a highly regulated sequential process, which is necessary for directed neuronal differentiation. In the present study, we show that α-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) receptor subunits GluR1 and GluR2 are expressed in differentiated granule cells, but not in stem cells, in neonatal, and adult dentate gyrus. Using markers for neural progenitors, immature and mature granule cells, we found that GluR1 and GluR2 were expressed mainly in mature cells and in some immature cells. A time-course analysis of 5-bromo-2′-deoxyuridine staining revealed that granule cells express GluR1 around 3 weeks after being generated. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, a putative animal model of schizophrenia and bipolar disorder in which dentate gyrus granule cells fail to mature normally, GluR1 and GluR2 immunoreactivities were substantially downregulated in the dentate gyrus granule cells. In the granule cells of mutant mice, the expression of both presynaptic and postsynaptic markers was decreased, suggesting that GluR1 and GluR2 are also associated with synaptic maturation. Moreover, GluR1 and GluR2 were also expressed in mature granule cells of the neonatal dentate gyrus. Taken together, these findings indicate that GluR1 and GluR2 expression closely correlates with the neuronal maturation state, and that GluR1 and GluR2 are useful markers for mature granule cells in the dentate gyrus. PMID:21927594

  7. Isolation of a sesquiterpene synthase expressing in specialized epithelial cells surrounding the secretory cavities in rough lemon (Citrus jambhiri).

    PubMed

    Uji, Yuya; Ozawa, Rika; Shishido, Hodaka; Taniguchi, Shiduku; Takabayashi, Junji; Akimitsu, Kazuya; Gomi, Kenji

    2015-05-15

    Volatile terpenoids such as monoterpenes and sesquiterpenes play multiple roles in plant responses and are synthesized by terpene synthases (TPSs). We have previously isolated a partial TPS gene, RlemTPS4, that responds to microbial attack in rough lemon. In this study, we isolated a full length RlemTPS4 cDNA from rough lemon. RlemTPS4 localized in the cytosol. The recombinant RlemTPS4 protein was obtained using a prokaryotic expression system and GC-MS analysis of the terpenes produced by the RlemTPS4 enzymatic reaction determined that RlemTPS4 produces some sesquiterpenes such as δ-elemene. The RlemTPS4 gene was specifically expressed in specialized epithelial cells surrounding the oil secretory cavities in rough lemon leaf tissue.

  8. Granulator Selection

    SciTech Connect

    Gould, T H; Armantrout, G

    1999-08-02

    Following our detailed review of the granulation reports and additional conversations with process and development personnel, we have reached a consensus position regarding granulator selection. At this time, we recommend going forward with implementation of the tumbling granulator approach (GEMCO) based on our assessment of the tested granulation techniques using the established criteria. The basis for this selection is summarized in the following sections, followed by our recommendations for proceeding with implementation of the tumbling granulation approach. All five granulation technologies produced granulated products that can be made into acceptable sintered pucks. A possible exception is the product from the fluidized bed granulator. This material has been more difficult to press into uniform pucks without subsequent cracking of the puck during the sintering cycle for the pucks in this series of tests. This problem may be an artifact of the conditions of the particular granulation demonstration run involved, but earlier results have also been mixed. All granulators made acceptable granulated feed from the standpoint of transfer and press feeding, though the roller compactor and fluidized bed products were dustier than the rest. There was also differentiation among the granulators in the operational areas of (1) potential for process upset, (2) plant implementation and operational complexity, and (3) maintenance concerns. These considerations will be discussed further in the next section. Note that concerns also exist regarding the extension of the granulation processes to powders containing actinides. Only the method that involves tumbling and moisture addition has been tested with uranium, and in that instance, significant differences were found in the granulation behavior of the powders.

  9. Increased number of cerebellar granule cells and astrocytes in the internal granule layer in sheep following prenatal intra-amniotic injection of lipopolysaccharide.

    PubMed

    Strackx, Eveline; Gantert, Markus; Moers, Veronique; van Kooten, Imke A J; Rieke, Rebecca; Hürter, Hanna; Lemmens, Marijke A M; Steinbusch, Harry W M; Zimmermann, L J I; Vles, Johannes S H; Garnier, Yves; Gavilanes, Antonio W D; Kramer, Boris W

    2012-03-01

    Chorioamnionitis is an important problem in perinatology today, leading to brain injury and neurological handicaps. However, there are almost no data available regarding chorioamnionitis and a specific damage of the cerebellum. Therefore, this study aimed at determining if chorioamnionitis causes cerebellar morphological alterations. Chorioamnionitis was induced in sheep by the intra-amniotic injection of lipopolysaccharide (LPS) at a gestational age (GA) of 110 days. At a GA of 140 days, we assessed the mean total and layer-specific volume and the mean total granule cell (GCs) and Purkinje cell (PC) number in the cerebelli of LPS-exposed and control animals using high-precision design-based stereology. Astrogliosis was assessed in the gray and white matter (WM) using a glial fibrillary acidic protein staining combined with gray value image analysis. The present study showed an unchanged volume of the total cerebellum as well as the molecular layer, outer and inner granular cell layers (OGL and IGL, respectively), and WM. Interestingly, compared with controls, the LPS-exposed brains showed a statistically significant increase (+20.4%) in the mean total number of GCs, whereas the number of PCs did not show any difference between the two groups. In addition, LPS-exposed animals showed signs of astrogliosis specifically affecting the IGL. Intra-amniotic injection of LPS causes morphological changes in the cerebellum of fetal sheep still detectable at full-term birth. In this study, changes were restricted to the inner granule layer. These cerebellar changes might correspond to some of the motor or non-motor deficits seen in neonates from compromised pregnancies.

  10. Stress Granules contribute to α-globin homeostasis in differentiating erythroid cells

    PubMed Central

    Ghisolfi, Laura; Dutt, Shilpee; McConkey, Marie E.; Ebert, Benjamin L.; Anderson, Paul

    2012-01-01

    Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin protein and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2α and inhibiting the translation of globin protein when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2α-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells. PMID:22452989

  11. Effect of heart failure on catecholamine granule morphology and storage in chromaffin cells

    PubMed Central

    Mahata, Sushil K; Zheng, Hong; Mahata, Sumana; Liu, Xuefei

    2016-01-01

    One of the key mechanisms involved in sympathoexcitation in chronic heart failure (HF) is the activation of the adrenal glands. Impact of the elevated catecholamines on the hemodynamic parameters has been previously demonstrated. However, studies linking the structural effects of such overactivation with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not been previously reported. In this study, HF was induced in male Sprague-Dawley rats by ligation of the left coronary artery. Five weeks after surgery, cardiac function was assessed by ventricular hemodynamics. HF rats showed increased adrenal weight and adrenal catecholamine levels (norepinephrine, epinephrine and dopamine) compared with sham-operated rats. Rats with HF demonstrated increased small synaptic and dense core vesicle in splanchnic–adrenal synapses indicating trans-synaptic activation of catecholamine biosynthetic enzymes, increased endoplasmic reticulum and Golgi lumen width to meet the demand of increased catecholamine synthesis and release, and more mitochondria with dilated cristae and glycogen to accommodate for the increased energy demand for the increased biogenesis and exocytosis of catecholamines from the adrenal medulla. These findings suggest that increased trans-synaptic activation of the chromaffin cells within the adrenal medulla may lead to increased catecholamines in the circulation which in turn contributes to the enhanced neurohumoral drive, providing a unique mechanistic insight for enhanced catecholamine levels in plasma commonly observed in chronic HF condition. PMID:27402067

  12. Effect of heart failure on catecholamine granule morphology and storage in chromaffin cells.

    PubMed

    Mahata, Sushil K; Zheng, Hong; Mahata, Sumana; Liu, Xuefei; Patel, Kaushik P

    2016-09-01

    One of the key mechanisms involved in sympathoexcitation in chronic heart failure (HF) is the activation of the adrenal glands. Impact of the elevated catecholamines on the hemodynamic parameters has been previously demonstrated. However, studies linking the structural effects of such overactivation with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not been previously reported. In this study, HF was induced in male Sprague-Dawley rats by ligation of the left coronary artery. Five weeks after surgery, cardiac function was assessed by ventricular hemodynamics. HF rats showed increased adrenal weight and adrenal catecholamine levels (norepinephrine, epinephrine and dopamine) compared with sham-operated rats. Rats with HF demonstrated increased small synaptic and dense core vesicle in splanchnic-adrenal synapses indicating trans-synaptic activation of catecholamine biosynthetic enzymes, increased endoplasmic reticulum and Golgi lumen width to meet the demand of increased catecholamine synthesis and release, and more mitochondria with dilated cristae and glycogen to accommodate for the increased energy demand for the increased biogenesis and exocytosis of catecholamines from the adrenal medulla. These findings suggest that increased trans-synaptic activation of the chromaffin cells within the adrenal medulla may lead to increased catecholamines in the circulation which in turn contributes to the enhanced neurohumoral drive, providing a unique mechanistic insight for enhanced catecholamine levels in plasma commonly observed in chronic HF condition.

  13. Dendrites of dentate gyrus granule cells contribute to pattern separation by controlling sparsity

    PubMed Central

    Chavlis, Spyridon; Petrantonakis, Panagiotis C.

    2016-01-01

    ABSTRACT The hippocampus plays a key role in pattern separation, the process of transforming similar incoming information to highly dissimilar, nonverlapping representations. Sparse firing granule cells (GCs) in the dentate gyrus (DG) have been proposed to undertake this computation, but little is known about which of their properties influence pattern separation. Dendritic atrophy has been reported in diseases associated with pattern separation deficits, suggesting a possible role for dendrites in this phenomenon. To investigate whether and how the dendrites of GCs contribute to pattern separation, we build a simplified, biologically relevant, computational model of the DG. Our model suggests that the presence of GC dendrites is associated with high pattern separation efficiency while their atrophy leads to increased excitability and performance impairments. These impairments can be rescued by restoring GC sparsity to control levels through various manipulations. We predict that dendrites contribute to pattern separation as a mechanism for controlling sparsity. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc. PMID:27784124

  14. FHF-independent conduction of action potentials along the leak-resistant cerebellar granule cell axon

    PubMed Central

    Dover, Katarzyna; Marra, Christopher; Solinas, Sergio; Popovic, Marko; Subramaniyam, Sathyaa; Zecevic, Dejan; D'Angelo, Egidio; Goldfarb, Mitchell

    2016-01-01

    Neurons in vertebrate central nervous systems initiate and conduct sodium action potentials in distinct subcellular compartments that differ architecturally and electrically. Here, we report several unanticipated passive and active properties of the cerebellar granule cell's unmyelinated axon. Whereas spike initiation at the axon initial segment relies on sodium channel (Nav)-associated fibroblast growth factor homologous factor (FHF) proteins to delay Nav inactivation, distal axonal Navs show little FHF association or FHF requirement for high-frequency transmission, velocity and waveforms of conducting action potentials. In addition, leak conductance density along the distal axon is estimated as <1% that of somatodendritic membrane. The faster inactivation rate of FHF-free Navs together with very low axonal leak conductance serves to minimize ionic fluxes and energetic demand during repetitive spike conduction and at rest. The absence of FHFs from Navs at nodes of Ranvier in the central nervous system suggests a similar mechanism of current flux minimization along myelinated axons. PMID:27666389

  15. Protective Effect of Edaravone in Primary Cerebellar Granule Neurons against Iodoacetic Acid-Induced Cell Injury

    PubMed Central

    Zhou, Xinhua; Zhu, Longjun; Wang, Liang; Guo, Baojian; Zhang, Gaoxiao; Sun, Yewei; Zhang, Zaijun; Lee, Simon Ming-Yuen; Yu, Pei; Wang, Yuqiang

    2015-01-01

    Edaravone (EDA) is clinically used for treatment of acute ischemic stroke in Japan and China due to its potent free radical-scavenging effect. However, it has yet to be determined whether EDA can attenuate iodoacetic acid- (IAA-) induced neuronal death in vitro. In the present study, we investigated the effect of EDA on damage of IAA-induced primary cerebellar granule neurons (CGNs) and its possible underlying mechanisms. We found that EDA attenuated IAA-induced cell injury in CGNs. Moreover, EDA significantly reduced intracellular reactive oxidative stress production, loss of mitochondrial membrane potential, and caspase 3 activity induced by IAA. Taken together, EDA protected CGNs against IAA-induced neuronal damage, which may be attributed to its antiapoptotic and antioxidative activities. PMID:26557222

  16. Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites?

    PubMed

    Mercier, Corinne; Adjogble, Koku D Z; Däubener, Walter; Delauw, Marie-France-Cesbron

    2005-07-01

    Together with micronemes and rhoptries, dense granules are specialised secretory organelles of Apicomplexa parasites. Among Apicomplexa, Plasmodium represents a model of parasites propagated by way of an insect vector, whereas Toxoplasma is a model of food borne protozoa forming cysts. Through comparison of both models, this review summarises data accumulated over recent years on alternative strategies chosen by these parasites to develop within a parasitophorous vacuole and explores the role of dense granules in this process. One of the characteristics of the Plasmodium erythrocyte stages is to export numerous parasite proteins into both the host cell cytoplasm and/or plasma membrane via the vacuole used as a step trafficking compartment. Whether this feature can be correlated to few storage granules and a restricted number of dense granule proteins, is not yet clear. By contrast, the Toxoplasma developing vacuole is decorated by abundantly expressed dense granule proteins and is characterised by a network of membranous nanotubes. Although the exact function of most of these proteins remains currently unknown, recent data suggest that some of these dense granule proteins could be involved in building the intravacuolar membranous network. Conserved expression of the Toxoplasma dense granule proteins throughout most of the parasite stages suggests that they could also be key elements of the cyst formation.

  17. The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in α-Granule Secretion†

    PubMed Central

    Woronowicz, Kamil; Dilks, James R.; Rozenvayn, Nataliya; Dowal, Louisa; Blair, Price S.; Peters, Christian G.; Woronowicz, Lucyna; Flaumenhaft, Robert

    2010-01-01

    Following platelet activation, platelets undergo a dramatic shape change mediated by the actin cytoskeleton and accompanied by secretion of granule contents. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism for this putative regulation is not known. We found that disruption of the actin cytoskeleton by latrunculin A inhibited α-granule secretion induced by several different platelet agonists without significantly affecting activation-induced platelet aggregation. In a cell-free secretory system, platelet cytosol was required for α-granule secretion. Inhibition of actin polymerization prevented α-granule secretion in this system and purified platelet actin could substitute for platelet cytosol to support α–granule secretion. To determine whether SNAREs physically associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with cytoskeletons isolated from either resting or activated platelets. When syntaxin-4 and SNAP-23 were tested for actin binding in a purified protein system, only syntaxin-4 associated directly with polymerized platelet actin. These data show that the platelet cytoskeleton interacts with select SNAREs and that actin polymerization facilitates α-granule release. PMID:20429610

  18. Object/Context-Specific Memory Deficits Associated with Loss of Hippocampal Granule Cells after Adrenalectomy in Rats

    ERIC Educational Resources Information Center

    Spanswick, Simon C.; Sutherland, Robert J.

    2010-01-01

    Chronic adrenalectomy (ADX) causes a gradual and selective loss of granule cells in the dentate gyrus (DG) of the rat. Here, we administered replacement corticosterone to rats beginning 10 wk after ADX. We then tested them in three discrimination tasks based on object novelty, location, or object/context association. Only during testing of the…

  19. Lack of MHC class I surface expression on neoplastic cells and poor activation of the secretory pathway of cytotoxic cells in oral squamous cell carcinomas

    PubMed Central

    Cruz, I; Meijer, C J L M; Walboomers, J M M; Snijders, P J F; Waal, I Van der

    1999-01-01

    Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the secretory pathway of perforin/granzymes to kill their target cells. In contrast to NK cells, CTL responses are MHC class I restricted. In this study we analysed the relative activation of CTL and NK cells in relation with MHC class I expression on oral squamous cell carcinomas (OSCCs). MHC class I expression was investigated in 47 OSCCs by immunohistochemistry using HCA2, HC10 and β2-m antibodies. The presence of CTLs, NK cells, and its activation, was investigated in 21 of these OSCCs using respectively, CD8, CD57 and GrB7 antibodies. The Q-Prodit measuring system was used for quantification of cytotoxic cells. All OSCCs showed weak or absent staining of β2-m on the cell surface. The absence of β2-m was significantly associated with absent expression of MHC class I heavy chain as detected by HC10 antibody (P = 0.004). In tumour infiltrates CTLs always outnumbered NK cells, as reflected by the ratio CD57/CD8 being always inferior to one (mean: 0.19; SD: 0.15). The proportion of activated cytotoxic cells as detected by granzyme B expression was generally low (mean: 8.6%; SD 8.9). A clear correlation between MHC class I expression and the relative proportion of NK cells/CTLs was not found. This study shows that the majority of OSCCs show weak or absent expression of MHC class I molecules on the cell surface, possibly due to alterations in the normal β2-m pathway. The low proportion of granzyme B-positive CTLs/NK cells indicates that the secretory pathway of cytotoxicity is poor in these patients. The lack of correlation between MHC class I expression and CTL/NK cell activation as detected by granzyme B expression suggests that, next to poor antigen presentation, also local factors seem to determine the final outcome of the cytotoxic immune response. © 1999 Cancer Research Campaign PMID:10555762

  20. Palmitoylethanolamide protects dentate gyrus granule cells via peroxisome proliferator-activated receptor-α.

    PubMed

    Koch, Marco; Kreutz, Susanne; Böttger, Charlotte; Benz, Alexander; Maronde, Erik; Ghadban, Chalid; Korf, Horst-Werner; Dehghani, Faramarz

    2011-02-01

    Endocannabinoids like 2-arachidonoylglycerol strongly modulate the complex machinery of secondary neuronal damage and are shown to improve neuronal survival after excitotoxic lesion. Palmitoylethanolamide (PEA), the naturally occurring fatty acid amide of ethanolamine and palmitic acid, is an endogenous lipid known to mimic several effects of endocannabinoids even without binding to cannabinoid receptors. Here we show that PEA (0.001-1 μM) and the synthetic peroxisome proliferator-activated receptor (PPAR)-alpha agonist 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (Wy-14,643; 0.1-1 μM) reduced the number of microglial cells and protected dentate gyrus granule cells in excitotoxically lesioned organotypic hippocampal slice cultures (OHSCs). Treatment with the PPAR-alpha antagonist N-((2S)-2-(((1Z)-1-Methyl-3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-enyl)amino)-3-(4-(2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy)phenyl)propyl)propanamide (GW6471; 0.05-5 μM) blocked PEA-mediated neuroprotection and reduction of microglial cell numbers whereas the PPAR-gamma antagonist 2-chloro-5-nitro-N-phenyl-benzamide (GW9662; 0.01-1 μM) showed no effects. Immunocytochemistry and Western blot analyses revealed a strong PPAR-alpha immunoreaction in BV-2 microglial cells and in HT22 hippocampal cells. Intensity and location of PPAR-alpha immunoreaction remained constant during stimulation with PEA (0.01 μM; 1-36 h). In conclusion our data provide evidence that (1) PEA counteracted excitotoxically induced secondary neuronal damage of dentate gyrus granule cells, (2) PPAR-alpha but not PPAR-gamma is the endogenous binding site for PEA-mediated neuroprotection, and (3) PEA may activate PPAR-alpha in microglial cells and hippocampal neurons to exert its neuroprotective effects. In addition to classical endocannabinoids, PEA-mediated PPAR-alpha activation represents a possible target for therapeutic interventions to mitigate symptoms of secondary neuronal damage.

  1. Clonal analysis reveals granule cell behaviors and compartmentalization that determine the folded morphology of the cerebellum.

    PubMed

    Legué, Emilie; Riedel, Elyn; Joyner, Alexandra L

    2015-05-01

    The mammalian cerebellum consists of folds of different sizes and shapes that house distinct neural circuits. A crucial factor underlying foliation is the generation of granule cells (gcs), the most numerous neuron type in the brain. We used clonal analysis to uncover global as well as folium size-specific cellular behaviors that underlie cerebellar morphogenesis. Unlike most neural precursors, gc precursors divide symmetrically, accounting for their massive expansion. We found that oriented cell divisions underlie an overall anteroposteriorly polarized growth of the cerebellum and gc clone geometry. Clone geometry is further refined by mediolateral oriented migration and passive dispersion of differentiating gcs. Most strikingly, the base of each fissure acts as a boundary for gc precursor dispersion, which we propose allows each folium to be regulated as a developmental unit. Indeed, the geometry and size of clones in long and short folia are distinct. Moreover, in engrailed 1/2 mutants with shorter folia, clone cell number and geometry are most similar to clones in short folia of wild-type mice. Thus, the cerebellum has a modular mode of development that allows the plane of cell division and number of divisions to be differentially regulated to ensure that the appropriate number of cells are partitioned into each folium.

  2. PERK Activation Promotes Medulloblastoma Tumorigenesis by Attenuating Premalignant Granule Cell Precursor Apoptosis.

    PubMed

    Ho, Yeung; Li, Xiting; Jamison, Stephanie; Harding, Heather P; McKinnon, Peter J; Ron, David; Lin, Wensheng

    2016-07-01

    Evidence suggests that activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress negatively or positively influences cell transformation by regulating apoptosis. Patched1 heterozygous deficient (Ptch1(+/-)) mice reproduce human Gorlin's syndrome and are regarded as the best animal model to study tumorigenesis of the sonic hedgehog subgroup of medulloblastomas. It is believed that medulloblastomas in Ptch1(+/-) mice results from the transformation of granule cell precursors (GCPs) in the developing cerebellum. Here, we determined the role of PERK signaling on medulloblastoma tumorigenesis by assessing its effects on premalignant GCPs and tumor cells. We found that PERK signaling was activated in both premalignant GCPs in young Ptch1(+/-) mice and medulloblastoma cells in adult mice. We demonstrated that PERK haploinsufficiency reduced the incidence of medulloblastomas in Ptch1(+/-) mice. Interestingly, PERK haploinsufficiency enhanced apoptosis of premalignant GCPs in young Ptch1(+/-) mice but had no significant effect on medulloblastoma cells in adult mice. Moreover, we showed that the PERK pathway was activated in medulloblastomas in humans. These results suggest that PERK signaling promotes medulloblastoma tumorigenesis by attenuating apoptosis of premalignant GCPs during the course of malignant transformation.

  3. Ultrastructure of the surface structures and secretory glands of the rosette attachment organ of Gyrocotyle urna (Cestoda:Gyrocotylidea).

    PubMed

    Poddubnaya, Larisa G; Scholz, Tomás; Kuchta, Roman; Levron, Céline; Gibson, David I

    2008-09-01

    The surface structures and gland cells of the posterior rosette organ of Gyrocotyle urna Grube et Wagener, 1852, a member of the group presumed to be the most basal of the tapeworms (Cestoda: Gyrocotylidea), was studied by scanning electron and transmission electron microscopy. Surface structures on the outer (oriented away from the intestinal wall) and inner (in contact with the intestinal wall) rosette surfaces differ from each other and represent a transitional form between microvilli and microtriches typical of tapeworms (Eucestoda). The inner surface of the rosette possesses numerous glands. On the basis of the size and electron-density of their secretory granules, three types of unicellular gland cells can be distinguished. The least common type (Type I) is characterized by the production of small, round, electron-dense granules of about 0.3 microm in diameter, whereas another type of secretion (Type II) is formed from homogenous, moderately electron-dense, spheroidal granules of about 0.7 microm in diameter. The most common type of glands (Type III) is recognized by a secretion comprising large, elongate, electron-dense granules of about 1 microm long and 0.5 microm broad. The secretory granules of the three types of the glands are liberated by an eccrine mechanism and the gland ducts open via small pores on the inner rosette surface. The complex of secretory glands of the posterior rosette of G. urna is similar to those in the anterior attachment glands of monogeneans (as opposed to the types of glands present in other helminth groups). However, the tegumental surface structures of Gyrocotyle are supporting evidence for the relationship between the Gyrocotylidea and Eucestoda.

  4. Two dipolar α-helices within hormone-encoding regions of proglucagon are sorting signals to the regulated secretory pathway.

    PubMed

    Guizzetti, Leonardo; McGirr, Rebecca; Dhanvantari, Savita

    2014-05-23

    Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229-240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting "signature."

  5. Cytoskeletal Dependence of Insulin Granule Movement Dynamics in INS-1 Beta-Cells in Response to Glucose

    PubMed Central

    Heaslip, Aoife T.; Nelson, Shane R.; Lombardo, Andrew T.; Beck Previs, Samantha; Armstrong, Jessica; Warshaw, David M.

    2014-01-01

    For pancreatic β-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 β-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited. PMID:25310693

  6. FYCO1 and autophagy control the integrity of the haploid male germ cell-specific RNP granules

    PubMed Central

    Da Ros, Matteo; Lehtiniemi, Tiina; Olotu, Opeyemi; Fischer, Daniel; Zhang, Fu-Ping; Vihinen, Helena; Jokitalo, Eija; Sironen, Anu; Toppari, Jorma; Kotaja, Noora

    2017-01-01

    ABSTRACT Ribonucleoprotein (RNP) granules play a major role in compartmentalizing cytoplasmic RNA regulation. Haploid round spermatids that have exceptionally diverse transcriptomes are characterized by a unique germ cell-specific RNP granule, the chromatoid body (CB). The CB shares many characteristics with somatic RNP granules but also has germline-specific features. The CB appears to be a central structure in PIWI-interacting RNA (piRNA)-targeted RNA regulation. Here, we identified a novel CB component, FYCO1, which is involved in the intracellular transport of autophagic vesicles in somatic cells. We demonstrated that the CB is associated with autophagic activity. Induction of autophagy leads to the recruitment of lysosomal vesicles onto the CB in a FYCO1-dependent manner as demonstrated by the analysis of a germ cell-specific Fyco1 conditional knockout mouse model. Furthermore, in the absence of FYCO1, the integrity of the CB was affected and the CB was fragmented. Our results suggest that RNP granule homeostasis is regulated by FYCO1-mediated autophagy. PMID:27929729

  7. Translation suppression promotes stress granule formation and cell survival in response to cold shock

    PubMed Central

    Hofmann, Sarah; Cherkasova, Valeria; Bankhead, Peter; Bukau, Bernd; Stoecklin, Georg

    2012-01-01

    Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock–induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia. PMID:22875991

  8. Granule cell hyperexcitability in the early post-traumatic rat dentate gyrus: the ‘irritable mossy cell’ hypothesis

    PubMed Central

    Santhakumar, Vijayalakshmi; Bender, Roland; Frotscher, Michael; Ross, Stephen T; Hollrigel, Greg S; Toth, Zsolt; Soltesz, Ivan

    2000-01-01

    Cytochemical and in vitro whole-cell patch clamp techniques were used to investigate granule cell hyperexcitability in the dentate gyrus 1 week after fluid percussion head trauma. The percentage decrease in the number of hilar interneurones labelled with either GAD67 or parvalbumin mRNA probes following trauma was not different from the decrease in the total population of hilar cells, indicating no preferential survival of interneurones with respect to the non-GABAergic hilar cells, i.e. the mossy cells. Dentate granule cells following trauma showed enhanced action potential discharges, and longer-lasting depolarizations, in response to perforant path stimulation, in the presence of the GABAA receptor antagonist bicuculline. There was no post-traumatic alteration in the perforant path-evoked monosynaptic excitatory postsynaptic currents (EPSCs), or in the intrinsic properties of granule cells. However, after trauma, the monosynaptic EPSC was followed by late, polysynaptic EPSCs, which were not present in controls. The late EPSCs in granule cells from fluid percussion-injured rats were not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were eliminated by both the non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the AMPA receptor antagonist GYKI 53655. In addition, the late EPSCs were not present in low (0·5 mM) extracellular calcium, and they were also eliminated by the removal of the dentate hilus from the slice. Mossy hilar cells in the traumatic dentate gyrus responded with significantly enhanced, prolonged trains of action potential discharges to perforant path stimulation. These data indicate that surviving mossy cells play a crucial role in the hyperexcitable responses of the post-traumatic dentate gyrus. PMID:10747187

  9. Novel CD200 homologues iSEC1 and iSEC2 are gastrointestinal secretory cell-specific ligands of inhibitory receptor CD200R

    PubMed Central

    Kojima, Toshiyuki; Tsuchiya, Kiichiro; Ikemizu, Shinji; Yoshikawa, Soichiro; Yamanishi, Yoshinori; Watanabe, Mamoru; Karasuyama, Hajime

    2016-01-01

    CD200R is an inhibitory receptor expressed on myeloid cells and some lymphoid cells, and plays important roles in negatively regulating immune responses. CD200 is the only known ligand of CD200R and broadly distributed in a variety of cell types. Here we identified novel CD200 homologues, designated iSEC1 and iSEC2, that are expressed exclusively by secretory cell lineages in the gastrointestinal epithelium while authentic CD200 is expressed by none of epithelial cells including secretory cells. Both iSEC1 and iSEC2 could bind to CD200R but not other members of the CD200R family. Notably, CD200R expression was confined to intraepithelial lymphocytes (IELs) among cells in the gastrointestinal epithelium. Binding of iSEC1 to CD200R on IELs resulted in the suppression of cytokine production and cytolytic activity by activated IELs. Thus, iSEC1 is a previously unappreciated CD200R ligand with restricted expression in gastrointestinal secretory cells and may negatively regulate mucosal immune responses. PMID:27819346

  10. Potential implications of a monosynaptic pathway from mossy cells to adult-born granule cells of the dentate gyrus

    PubMed Central

    Scharfman, Helen E.; Bernstein, Hannah L.

    2015-01-01

    The dentate gyrus (DG) is important to many aspects of hippocampal function, but there are many aspects of the DG that are incompletely understood. One example is the role of mossy cells (MCs), a major DG cell type that is glutamatergic and innervates the primary output cells of the DG, the granule cells (GCs). MCs innervate the GCs as well as local circuit neurons that make GABAergic synapses on GCs, so the net effect of MCs on GCs – and therefore the output of the DG – is unclear. Here we first review fundamental information about MCs and the current hypotheses for their role in the normal DG and in diseases that involve the DG. Then we review previously published data which suggest that MCs are a source of input to a subset of GCs that are born in adulthood (adult-born GCs). In addition, we discuss the evidence that adult-born GCs may support the normal inhibitory ‘gate’ functions of the DG, where the GCs are a filter or gate for information from the entorhinal cortical input to area CA3. The implications are then discussed in the context of seizures and temporal lobe epilepsy (TLE). In TLE, it has been suggested that the DG inhibitory gate is weak or broken and MC loss leads to insufficient activation of inhibitory neurons, causing hyperexcitability. That idea was called the “dormant basket cell hypothesis.” Recent data suggest that loss of normal adult-born GCs may also cause disinhibition, and seizure susceptibility. Therefore, we propose a reconsideration of the dormant basket cell hypothesis with an intervening adult-born GC between the MC and basket cell and call this hypothesis the “dormant immature granule cell hypothesis.” PMID:26347618

  11. Spontaneous proliferative lesions of the adrenal medulla in aging Long-Evans rats. Comparison to PC12 cells, small granule-containing cells, and human adrenal medullary hyperplasia.

    PubMed

    Tischler, A S; DeLellis, R A; Perlman, R L; Allen, J M; Costopoulos, D; Lee, Y C; Nunnemacher, G; Wolfe, H J; Bloom, S R

    1985-10-01

    Aging rats of the Long-Evans strain spontaneously develop diffuse and nodular hyperplasia of the adrenal medulla in association with other abnormalities commonly encountered in human multiple endocrine neoplasia syndromes. The cells which comprise the adrenal nodules resemble those in the parent tumor of the rat PC12 pheochromocytoma cell line in that they show varying degrees of spontaneous or nerve growth factor-induced neurite outgrowth in culture and they contain little or no epinephrine. In addition, cells from at least some of the nodules contain immunoreactive neurotensin and neuropeptide-Y, which are also found in PC12 cells. There are a number of striking resemblances between the cells in adrenal nodules and the small granule-containing cells in the normal rodent adrenal. The findings suggest that spontaneous rat adrenal medullary nodules and PC12 cells might be derived from small granule-containing cells, or that cells within the nodules might regain properties of immature chromaffin cells and acquire characteristics of small granule-containing cells and of PC12 cells in the course of neoplastic progression. They further suggest a possible relationship between proliferative capacity and neurotransmitter phenotype in the adult rat adrenal medulla. By virtue of their sparse epinephrine content and their small granules, the cells in adrenal medullary nodules of Long-Evans rats differ from those in adrenal medullary nodules of humans with multiple endocrine neoplasia syndromes.

  12. Degranulation of mast cells and inhibition of the response to secretory agents by phototoxic compounds and ultraviolet radiation

    SciTech Connect

    Gendimenico, G.J.; Kochevar, I.E.

    1984-11-01

    The symptoms of cutaneous phototoxicity from coal tar compounds and the nonsteroidal anti-inflammatory drug benoxaprofen are characterized by wheal and flare formation which is mediated by histamine released from dermal mast cells. Rat serosal mast cells were used as an in vitro model system to study the direct effect of phototoxic compounds on mast cell degranulation. The coal tar compounds studied included acridine and pyrene. Combined exposure of cells to acridine and UVA (320 to 400 nm) radiation caused mast cells to degranulate, as assayed by the release of (/sup 3/H)serotonin. Maximum (/sup 3/H)serotonin release (70 to 80%) was obtained with 50 microM acridine and 300 kJ/m2 UVA. Pyrene (25 microM), when photoexcited with UVB (280 to 360 nm) radiation, caused about 80% release of (/sup 3/H)serotonin. No degranulation occurred with 20 microM benoxaprofen and UVB doses up to 7.2 kJ/m2. Trypan blue staining correlated well with degranulation caused by acridine plus UVA; however, with pyrene plus UVB there was greater (/sup 3/H)serotonin release than dye uptake. Excitation of photosensitizers with doses of UV radiation that did not cause trypan blue staining suppressed degranulation of mast cells in response to chemical stimulation. Acridine, pyrene, and benoxaprofen in the presence of UV radiation inhibited the mast cells from responding to compound 48/80 or the calcium ionophore, chlortetracycline. Two other phototoxic compounds, chlorpromazine and deoxytetracycline, also abolished degranulation by compound 48/80. These findings indicate that phototoxic compounds: (1) cause degranulation in the presence of high doses of UV radiation; and (2) suppress degranulation of mast cells in response to secretory stimuli at doses of UV radiation that do not cause release of mediator.

  13. Recovery of amorphous polyhydroxybutyrate granules from Cupriavidus necator cells grown on used cooking oil.

    PubMed

    Martino, Lucrezia; Cruz, Madalena V; Scoma, Alberto; Freitas, Filomena; Bertin, Lorenzo; Scandola, Mariastella; Reis, Maria A M

    2014-11-01

    Used cooking oil (UCO) was employed as the sole carbon source for the production of polyhydroxybutyrate (PHB) by cultivation in batch mode of Cupriavidus necator DSM 428. The produced biomass was used for extraction of the PHB granules with a solvent-free approach using sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and the enzyme Alcalase in an aqueous medium. The recovered PHB granules showed a degree of purity higher than 90% and no crystallization (i.e., granules were recovered in their 'native' amorphous state) as demonstrated by wide angle X-ray diffraction (WAXS). Granules were characterized according to their thermal properties and stability by differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Results show that UCO can be used as a renewable resource to produce amorphous PHB granules with excellent properties in a biocompatible manner.

  14. How do secretory products cross the plant cell wall to be released? A new hypothesis involving cyclic mechanical actions of the protoplast

    PubMed Central

    Paiva, Elder Antônio Sousa

    2016-01-01

    Background In plants, the products of secretory activity leave the protoplast and cross the plasma membrane by means of transporters, fusion with membranous vesicles or, less commonly, as result of disintegration of the cell. These mechanisms do not address an intriguing question: How do secretory products cross the cell wall? Furthermore, how do these substances reach the external surface of the plant body? Such diverse substances as oils, polysaccharides or nectar are forced to cross the cell wall and, in fact, do so. How are chemical materials that are repelled by the cell wall or that are sufficiently viscous to not cross passively released from plant cells? Scope and Conclusions I propose a cell-cycle model developed based on observations of different secreting systems, some unpublished results and an extensive literature review, aiming to understand the processes involved in both the secretory process and the release of secretion products. In the absence of facilitated diffusion, a mechanical action of the protoplast is necessary to ensure that some substances can cross the cell wall. The mechanical action of the protoplast, in the form of successive cycles of contraction and expansion, causes the material accumulated in the periplasmic space to cross the cell wall and the cuticle. This action is particularly relevant for the release of lipids, resins and highly viscous hydrophilic secretions. The proposed cell-cycle model and the statements regarding exudate release will also apply to secretory glands not elaborated upon here. Continuous secretion of several days, as observed in extrafloral nectaries, salt glands and some mucilage-producing glands, is only possible because the process is cyclical. PMID:26929201

  15. JAM-A promotes wound healing by enhancing both homing and secretory activities of mesenchymal stem cells.

    PubMed

    Wu, Minjuan; Ji, Shizhao; Xiao, Shichu; Kong, Zhengdong; Fang, He; Zhang, Yunqing; Ji, Kaihong; Zheng, Yongjun; Liu, Houqi; Xia, Zhaofan

    2015-10-01

    The homing ability and secretory function of mesenchymal stem cells (MSCs) are key factors that influence cell involvement in wound repair. These factors are controlled by multilayer regulatory circuitry, including adhesion molecules, core transcription factors (TFs) and certain other regulators. However, the role of adhesion molecules in this regulatory circuitry and their underlying mechanism remain undefined. In the present paper, we demonstrate that an adhesion molecule, junction adhesion molecule A (JAM-A), may function as a key promoter molecule to regulate skin wound healing by MSCs. In in vivo experiments, we show that JAM-A up-regulation promoted both MSC homing to full-thickness skin wounds and wound healing-related cytokine secretion by MSCs. In vitro experiments also showed that JAM-A promoted MSC proliferation and migration by activating T-cell lymphoma invasion and metastasis 1 (Tiam1). We suggest that JAM-A up-regulation can increase the proliferation, cytokine secretion and wound-homing ability of MSCs, thus accelerating the repair rate of full-thickness skin defects. These results may provide insights into a novel and potentially effective approach to improve the efficacy of MSC treatment.

  16. Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells

    PubMed Central

    Bahk, Young Yil; Pak, Jhang Ho

    2016-01-01

    Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases. PMID:27853127

  17. Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells.

    PubMed

    Bahk, Young Yil; Pak, Jhang Ho

    2016-10-01

    Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.

  18. Differential inhibition onto developing and mature granule cells generates high-frequency filters with variable gain

    PubMed Central

    Pardi, María Belén; Ogando, Mora Belén; Schinder, Alejandro F; Marin-Burgin, Antonia

    2015-01-01

    Adult hippocampal neurogenesis provides the dentate gyrus with heterogeneous populations of granule cells (GC) originated at different times. The contribution of these cells to information encoding is under current investigation. Here, we show that incoming spike trains activate different populations of GC determined by the stimulation frequency and GC age. Immature GC respond to a wider range of stimulus frequencies, whereas mature GC are less responsive at high frequencies. This difference is dictated by feedforward inhibition, which restricts mature GC activation. Yet, the stronger inhibition of mature GC results in a higher temporal fidelity compared to that of immature GC. Thus, hippocampal inputs activate two populations of neurons with variable frequency filters: immature cells, with wide‐range responses, that are reliable transmitters of the incoming frequency, and mature neurons, with narrow frequency response, that are precise at informing the beginning of the stimulus, but with a sparse activity. DOI: http://dx.doi.org/10.7554/eLife.08764.001 PMID:26163657

  19. Cell type-dependent trafficking of neuropeptide Y-containing dense core granules in CNS neurons.

    PubMed

    Ramamoorthy, Prabhu; Wang, Qian; Whim, Matthew D

    2011-10-12

    Neuropeptide transmitters are synthesized throughout the CNS and play important modulatory roles. After synthesis in the neuronal cell body, it is generally assumed that peptides are transported to nonspecialized sites of release. However, apart from a few cases, this scenario has not been thoroughly examined. Using wild-type and NPY(GFP) transgenic mice, we have studied the subcellular distribution of neuropeptide Y (NPY), a prototypical and broadly expressed neuropeptide. NPY puncta were found in the dendrites and axons of hippocampal GABAergic interneurons in situ. In contrast in hypothalamic GABAergic interneurons, NPY was restricted to the axon. Surprisingly this differential trafficking was preserved when the neurons were maintained in vitro. When hippocampal and hypothalamic neurons were transfected with NPY-Venus, the distribution of the fluorescent puncta replicated the cell type-specific distribution of endogenous neuropeptide Y. The NPY puncta in the axons of hippocampal and hypothalamic neurons colocalized with the sites of classical transmitter release (identified by staining for synapsin and the vesicular GABAergic transporter, VGAT). In hippocampal neurons, most of the postsynaptic NPY puncta were clustered opposite synapsin-containing varicosities. When neurons were stained for a second neuropeptide, agouti-related protein, immunoreactivity was found in the axon and dendrites of hippocampal neurons but only in the axons of hypothalamic neurons, thus mimicking the polarized distribution of NPY. These results indicate that the trafficking of neuropeptide-containing dense core granules is markedly cell type specific and is not determined entirely by the characteristics of the particular peptide per se.

  20. Brefeldin A sensitive mechanisms contribute to endocytotic membrane retrieval and vesicle recycling in cerebellar granule cells.

    PubMed

    Rampérez, Alberto; Sánchez-Prieto, José; Torres, Magdalena

    2017-03-11

    The recycling of synaptic vesicle (SV) proteins and transmitter release both occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the AP-1/AP-3 complex. As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7 days in vitro. We used Brefeldin A (BFA) to inhibit AP-1/AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5 minutes after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone (AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. This article is protected by copyright. All rights reserved.

  1. Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells

    PubMed Central

    Redhai, Siamak; Hellberg, Josephine E. E. U.; Wainwright, Mark; Perera, Sumeth W.; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C. I.

    2016-01-01

    Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion. PMID:27727275

  2. Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells.

    PubMed

    Redhai, Siamak; Hellberg, Josephine E E U; Wainwright, Mark; Perera, Sumeth W; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C I; Wilson, Clive

    2016-10-01

    Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.

  3. Incoming synapses and size of small granule-containing cells in a rat sympathetic ganglion after post-ganglionic axotomy.

    PubMed Central

    Case, C P; Matthews, M R

    1986-01-01

    A quantitative ultrastructural study has been made of the reaction of the incoming synapses of small granule-containing cells after axotomy of the major post-ganglionic branches of the superior cervical ganglion of the young adult rat. These cells are intrinsic and interneurone-like in this ganglion, receiving a preganglionic input and giving outgoing synapses to principal post-ganglionic neurones. Unlike their outgoing synapses, which are lost after post-ganglionic axotomy (Case & Matthews, 1986), the incoming synapses of the small granule-containing cells in axotomized ganglia increased in incidence post-operatively. The increase first became clearly evident 5-7 days post-operatively and was greater, being both more sustained and progressive, after bilateral than after unilateral axotomy. After bilateral axotomy the incidence of incoming synapses rose to more than four times that of normal ganglia and was still elevated at 128 days post-operatively, but was within normal limits at 390 days. After a unilateral lesion, increases of similar extent and time course to those in the axotomized ganglia were seen in the incoming synapses of small granule-containing cells in the uninjured contralateral ganglia. The incoming synapses of the small granule-containing cells are multifocal, i.e. show several points or active foci of synaptic specialization. The increase in synapses expressed itself both through an increased incidence of these synaptic active foci per nerve terminal and through an increase in the number of presynaptic nerve terminal profiles associated with the cells. Control observations indicated that the increase in synapses was not due to surgical stress, nor was it attributable solely to post-operative ageing. The nerve terminals which were presynaptic to the small granule-containing cells post-operatively were all of preganglionic origin: no incoming synapses or presynaptic nerve terminals remained at 2 days after a preganglionic denervation of axotomized

  4. Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells

    PubMed Central

    Sakai, H; Jingushi, S; Shuto, T; Urabe, K; Ikenoue, T; Okazaki, K; Kukita, T; Kukita, A; Iwamoto, Y

    2002-01-01

    Background: It has previously been shown that many osteoclast precursors are included in the granulation tissue within the pseudocapsule obtained at revision arthroplasty from hips with osteolysis. In vitro culture of only cells isolated from the granulation tissue has been previously shown to generate many mature osteoclasts. Objective: To investigate the presence or otherwise of supporting cells, similar to stromal cells, which differentiate osteoclasts within the granulation tissue. Methods: Cells isolated from the granulation tissue were cultured alone, and after four weeks fibroblast-like cells (granulation fibroblasts) remained. Rat non-adherent bone marrow cells (NA-BMCs) were co-cultured with the granulation fibroblasts with or without 1α,25(OH)2D3 (10-8 M) or heat treated ROS 17/2.8 cell conditioned medium (ht ROSCM), or both. Multinucleated cells (MNCs), which formed, were assessed by biochemical and functional characterisation of osteoclasts. Receptor activator of NFκB ligand (RANKL) was investigated by immunohistochemistry. Results: Co-culture of NA-BMCs and granulation fibroblasts caused the formation of tartrate resistant acid phosphatase (TRAP) positive MNCs, which had the calcitonin receptor (CTR), the Kat-1 antigen, which is specific to the surface of rat osteoclasts, and the ability to form pits in the presence of both 1α,25(OH)2D3 and ht ROSCM or in the presence of just ht ROSCM. RANKL was detected in fibroblast-like cells in the granulation tissue. Conclusion: These data suggest that granulation fibroblasts support osteoclast differentiation, as do osteoblasts/stromal cells, and may play a part in aseptic loosening. PMID:11796394

  5. Mitochondrial oxidative stress mediates high-phosphate-induced secretory defects and apoptosis in insulin-secreting cells.

    PubMed

    Nguyen, Tuyet Thi; Quan, Xianglan; Hwang, Kyu-Hee; Xu, Shanhua; Das, Ranjan; Choi, Seong-Kyung; Wiederkehr, Andreas; Wollheim, Claes B; Cha, Seung-Kuy; Park, Kyu-Sang

    2015-06-01

    Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin-releasing cells, Pi transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Pi concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pi on secretory function and apoptosis in INS-1E clonal β-cells and rat pancreatic islets. Elevated extracellular Pi (1~5 mM) increased the mitochondrial membrane potential (ΔΨm), superoxide generation, caspase activation, and cell death. Depolarization of the ΔΨm abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented ΔΨm hyperpolarization, superoxide generation, and cytotoxicity caused by Pi. High Pi also promoted the opening of the mitochondrial permeability transition (PT) pore, leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pi diminished ATP synthesis, cytosolic Ca(2+) oscillations, and insulin content and secretion in INS-1E cells as well as in dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high-Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Pi causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content and defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states.

  6. Kruppel-Like Factor 4 Regulates Granule Cell Pax6 Expression and Cell Proliferation in Early Cerebellar Development

    PubMed Central

    Zhang, Peter; Ha, Thomas; Larouche, Matt; Swanson, Douglas; Goldowitz, Dan

    2015-01-01

    Kruppel-like factor 4 (Klf4) is a transcription factor that regulates many important cellular processes in stem cell biology, cancer, and development. We used histological and molecular methods to study the expression of Klf4 in embryonic development of the normal and Klf4 knockout cerebellum. We find that Klf4 is expressed strongly in early granule cell progenitor development but tails-off considerably by the end of embryonic development. Klf4 is also co-expressed with Pax6 in these cells. In the Klf4-null mouse, which is perinatal lethal, Klf4 positively regulates Pax6 expression and regulates the proliferation of neuronal progenitors in the rhombic lip, external granular layer and the neuroepithelium. This paper is the first to describe a role for Klf4 in the cerebellum and provides insight into this gene’s function in neuronal development. PMID:26226504

  7. Simulated Responses of Cerebellar Purkinje Cells are Independent of the Dendritic Location of Granule Cell Synaptic Inputs

    NASA Astrophysics Data System (ADS)

    de Schutter, Erik; Bower, James M.

    1994-05-01

    Cerebellar Purkinje cell responses to granule cell synaptic inputs were examined with a computer model including active dendritic conductances. Dendritic P-type Ca2+ channels amplified postsynaptic responses when the model was firing at a physiological rate. Small synchronous excitatory inputs applied distally on the large dendritic tree resulted in somatic responses of similar size to those generated by more proximal inputs. In contrast, in a passive model the somatic postsynaptic potentials to distal inputs were 76% smaller. The model predicts that the somatic firing response of Purkinje cells is relatively insensitive to the exact dendritic location of synaptic inputs. We describe a mechanism of Ca2+-mediated synaptic amplification, based on the subspiking threshold recruitment of P-type Ca2+ channels in the dendritic branches surrounding the input site.

  8. Urethral Reconstruction Using Mesothelial Cell-Seeded Autogenous Granulation Tissue Tube: An Experimental Study in Male Rabbits

    PubMed Central

    Jiang, Shiwei; Xu, Zhonghua; Zhao, Yuanyuan; Yan, Lei; Zhou, Zunlin

    2017-01-01

    Objective. This study was to evaluate the utility of the compound graft for tubularized urethroplasty by seeding mesothelial cells onto autogenous granulation tissue. Methods. Silastic tubes were implanted subcutaneously in 18 male rabbits, of which nine underwent omentum biopsies simultaneously for in vitro expansion of mesothelial cells. The granulation tissue covering the tubes was harvested 2 weeks after operation. Mesothelial cells were seeded onto and cocultured with the tissue for 7 days. A pendulous urethral segment of 1.5 cm was totally excised. Urethroplasty was performed with mesothelial cell-seeded tissue tubes in an end-to-end fashion in nine rabbits and with unseeded grafts in others as controls. Serial urethrograms were performed at 1, 2, and 6 months postoperatively. Meanwhile, the neourethra was harvested and analyzed grossly and histologically. Results. Urethrograms showed cell-seeded grafts maintained wide at each time point, while strictures formation was found in unseeded grafts. Histologically, layers of urothelium surrounded by increasingly organized smooth muscles were observed in seeded grafts. In contrast, myofibroblasts accumulation and extensive scarring occurred in unseeded grafts. Conclusions. Mesothelial cell-seeded granulation tissue tube can be successfully used for tubularized urethroplasty in male rabbits. PMID:28337443

  9. Gastrin upregulates the prosurvival factor secretory clusterin in adenocarcinoma cells and in oxyntic mucosa of hypergastrinemic rats.

    PubMed

    Fjeldbo, Christina Sæten; Bakke, Ingunn; Erlandsen, Sten Even; Holmseth, Jannicke; Lægreid, Astrid; Sandvik, Arne K; Thommesen, Liv; Bruland, Torunn

    2012-01-01

    We show that the gastric hormone gastrin induces the expression of the prosurvival secretory clusterin (sCLU) in rat adenocarcinoma cells. Clusterin mRNA was still upregulated in the presence of the protein synthesis inhibitor cycloheximide, although at a lower level. This indicates that gastrin induces clusterin transcription independently of de novo protein synthesis but requires de novo protein synthesis of signal transduction pathway components to achieve maximal expression level. Luciferase reporter assay indicates that the AP-1 transcription factor complex is involved in gastrin-mediated activation of the clusterin promoter. Gastrin-induced clusterin expression and subsequent secretion is dependent on sustained treatment, because removal of gastrin after 1-2 h abolished the response. Neutralization of secreted clusterin by a specific antibody abolished the antiapoptotic effect of gastrin on serum starvation-induced apoptosis, suggesting that extracellular clusterin is involved in gastrin-mediated inhibition of apoptosis. The clusterin response to gastrin was validated in vivo in hypergastrinemic rats, showing increased clusterin expression in the oxyntic mucosa, as well as higher levels of clusterin in plasma. In normal rat oxyntic mucosa, clusterin protein was strongly expressed in chromogranin A-immunoreactive neuroendocrine cells, of which the main cell type was the histidine decarboxylase-immunoreactive enterochromaffin-like (ECL) cell. The association of clusterin with neuroendocrine differentiation was further confirmed in human gastric ECL carcinoids. Interestingly, in hypergastrinemic rats, clusterin-immunoreactive cells formed distinct groups of diverse cells at the base of many glands. Our results suggest that clusterin may contribute to gastrin's growth-promoting effect on the oxyntic mucosa.

  10. Effect of oral N-acetylcysteine (NAC) on volume and albumin content of respiratory tract fluid but not on epithelial secretory cell number in "smoking" rats.

    PubMed

    Robinson, N; Brattsand, R; Dahlbäck, M

    1990-03-01

    This study was designed to look at the effect of N-acetylcysteine (NAC) on epithelial secretory cells and the respiratory tract fluid volume and albumin content from the lower airways of "bronchitic" rats. Rats were exposed either to tobacco smoke (TS), TS and NAC, or NAC alone. TS caused a significant increase in epithelial secretory cell number which was not reduced by concomitant NAC administration; NAC alone had no effect on cell numbers. TS increased respiratory tract fluid volume and albumin content by a small but non-significant amount, whereas TS and NAC increased the volume and albumin content by a greater and significant amount; NAC alone was also shown to significantly increase both fluid volume and albumin content.

  11. Cancer metastasis-suppressing peptide metastin upregulates excitatory synaptic transmission in hippocampal dentate granule cells.

    PubMed

    Arai, Amy C; Xia, Yan-Fang; Suzuki, Erika; Kessler, Markus; Civelli, Olivier; Nothacker, Hans-Peter

    2005-11-01

    Metastin is an antimetastatic peptide encoded by the KiSS-1 gene in cancer cells. Recent studies found that metastin is a ligand for the orphan G-protein-coupled receptor GPR54, which is highly expressed in specific brain regions such as the hypothalamus and parts of the hippocampus. This study shows that activation of GPR54 by submicromolar concentrations of metastin reversibly enhances excitatory synaptic transmission in hippocampal dentate granule cells in a mitogen-activated protein (MAP) kinase-dependent manner. Synaptic enhancement by metastin was suppressed by intracellular application of the G-protein inhibitor GDP-beta-S and the calcium chelator BAPTA. Analysis of miniature excitatory postsynaptic currents (mEPSCs) revealed an increase in the mean amplitude but no change in event frequency. This indicates that GPR54 and the mechanism responsible for the increase in EPSCs are postsynaptic. Metastin-induced synaptic potentiation was abolished by 50 microM PD98059 and 20 microM U0126, two inhibitors of the MAP kinases ERK1 and ERK2. The effect was also blocked by inhibitors of calcium/calmodulin-dependent kinases and tyrosine kinases. RT-PCR experiments showed that both KiSS-1 and GPR54 are expressed in the hippocampal dentate gyrus. Metastin is thus a novel endogenous factor that modulates synaptic excitability in the dentate gyrus through mechanisms involving MAP kinases, which in turn may be controlled upstream by calcium-activated kinases and tyrosine kinases.

  12. Semaphorin 5A inhibits synaptogenesis in early postnatal- and adult-born hippocampal dentate granule cells

    PubMed Central

    Duan, Yuntao; Wang, Shih-Hsiu; Song, Juan; Mironova, Yevgeniya; Ming, Guo-li; Kolodkin, Alex L; Giger, Roman J

    2014-01-01

    Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A−/− GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A−/− mutants, PlexinA2−/− mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A−/− mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders. DOI: http://dx.doi.org/10.7554/eLife.04390.001 PMID:25313870

  13. BDNF over-expression increases olfactory bulb granule cell dendritic spine density in vivo.

    PubMed

    McDole, B; Isgor, C; Pare, C; Guthrie, K

    2015-09-24

    Olfactory bulb granule cells (GCs) are axon-less, inhibitory interneurons that regulate the activity of the excitatory output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses located on GC spines. These contacts are established in the distal apical dendritic compartment, while GC basal dendrites and more proximal apical segments bear spines that receive glutamatergic inputs from the olfactory cortices. This synaptic connectivity is vital to olfactory circuit function and is remodeled during development, and in response to changes in sensory activity and lifelong GC neurogenesis. Manipulations that alter levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in vivo have significant effects on dendritic spine morphology, maintenance and activity-dependent plasticity for a variety of CNS neurons, yet little is known regarding BDNF effects on bulb GC spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF in transgenic mice produces a marked increase in GC spine density that includes an increase in mature spines on their apical dendrites. Morphometric analysis demonstrated that changes in spine density were most notable in the distal and proximal apical domains, indicating that multiple excitatory inputs are potentially modified by BDNF. Our results indicate that increased levels of endogenous BDNF can promote the maturation and/or maintenance of dendritic spines on GCs, suggesting a role for this factor in modulating GC functional connectivity within adult olfactory circuitry.

  14. Electrophysiological characterization of granule cells in the dentate gyrus immediately after birth

    PubMed Central

    Pedroni, Andrea; Minh, Do Duc; Mallamaci, Antonello; Cherubini, Enrico

    2014-01-01

    Granule cells (GCs) in the dentate gyrus are generated mainly postnatally. Between embryonic day 10 and 14, neural precursors migrate from the primary dentate matrix to the dentate gyrus where they differentiate into neurons. Neurogenesis reaches a peak at the end of the first postnatal week and it is completed at the end of the first postnatal month. This process continues at a reduced rate throughout life. Interestingly, immediately after birth, GCs exhibit a clear GABAergic phenotype. Only later they integrate the classical glutamatergic trisynaptic hippocampal circuit. Here, whole cell patch clamp recordings, in current clamp mode, were performed from immature GCs, intracellularly loaded with biocytin (in hippocampal slices from P0 to P3 old rats) in order to compare their morphological characteristics with their electrophysiological properties. The vast majority of GCs were very immature with small somata, few dendritic branches terminating with small varicosities and growth cones. In spite of their immaturity their axons reached often the cornu ammonis 3 area. Immature GCs generated, upon membrane depolarization, either rudimentary sodium spikes or more clear overshooting action potentials that fired repetitively. They exhibited also low threshold calcium spikes. In addition, most spiking neurons showed spontaneous synchronized network activity, reminiscent of giant depolarizing potentials (GDPs) generated in the hippocampus by the synergistic action of glutamate and GABA, both depolarizing and excitatory. This early synchronized activity, absent during adult neurogenesis, may play a crucial role in the refinement of local neuronal circuits within the developing dentate gyrus. PMID:24592213

  15. Forward transport of proteins in the plasma membrane of migrating cerebellar granule cells.

    PubMed

    Wang, Dong; She, Liang; Sui, Ya-nan; Yuan, Xiao-bing; Wen, Yunqing; Poo, Mu-ming

    2012-12-18

    Directional flow of membrane components has been detected at the leading front of fibroblasts and the growth cone of neuronal processes, but whether there exists global directional flow of plasma membrane components over the entire migrating neuron remains largely unknown. By analyzing the trajectories of antibody-coated single quantum dots (QDs) bound to two membrane proteins, overexpressed myc-tagged synaptic vesicle-associated membrane protein VAMP2 and endogenous neurotrophin receptor TrkB, we found that these two proteins exhibited net forward transport, which is superimposed upon Brownian motion, in both leading and trailing processes of migrating cerebellar granule cells in culture. Furthermore, no net directional transport of membrane proteins was observed in nonmigrating cells with either growing or stalling leading processes. Analysis of the correlation of motion direction between two QDs on the same process in migrating neurons also showed a higher frequency of correlated forward than rearward movements. Such correlated QD movements were markedly reduced in the presence of myosin II inhibitor blebbistatin,suggesting the involvement of myosin II-dependent active transport processes. Thus, a net forward transport of plasma membrane proteins exists in the leading and trailing processes of migrating neurons, in line with the translocation of the soma.

  16. Single Neuron Optimization as a Basis for Accurate Biophysical Modeling: The Case of Cerebellar Granule Cells.

    PubMed

    Masoli, Stefano; Rizza, Martina F; Sgritta, Martina; Van Geit, Werner; Schürmann, Felix; D'Angelo, Egidio

    2017-01-01

    In realistic neuronal modeling, once the ionic channel complement has been defined, the maximum ionic conductance (Gi-max) values need to be tuned in order to match the firing pattern revealed by electrophysiological recordings. Recently, selection/mutation genetic algorithms have been proposed to efficiently and automatically tune these parameters. Nonetheless, since similar firing patterns can be achieved through different combinations of Gi-max values, it is not clear how well these algorithms approximate the corresponding properties of real cells. Here we have evaluated the issue by exploiting a unique opportunity offered by the cerebellar granule cell (GrC), which is electrotonically compact and has therefore allowed the direct experimental measurement of ionic currents. Previous models were constructed using empirical tuning of Gi-max values to match the original data set. Here, by using repetitive discharge patterns as a template, the optimization procedure yielded models that closely approximated the experimental Gi-max values. These models, in addition to repetitive firing, captured additional features, including inward rectification, near-threshold oscillations, and resonance, which were not used as features. Thus, parameter optimization using genetic algorithms provided an efficient modeling strategy for reconstructing the biophysical properties of neurons and for the subsequent reconstruction of large-scale neuronal network models.

  17. Single Neuron Optimization as a Basis for Accurate Biophysical Modeling: The Case of Cerebellar Granule Cells

    PubMed Central

    Masoli, Stefano; Rizza, Martina F.; Sgritta, Martina; Van Geit, Werner; Schürmann, Felix; D'Angelo, Egidio

    2017-01-01

    In realistic neuronal modeling, once the ionic channel complement has been defined, the maximum ionic conductance (Gi-max) values need to be tuned in order to match the firing pattern revealed by electrophysiological recordings. Recently, selection/mutation genetic algorithms have been proposed to efficiently and automatically tune these parameters. Nonetheless, since similar firing patterns can be achieved through different combinations of Gi-max values, it is not clear how well these algorithms approximate the corresponding properties of real cells. Here we have evaluated the issue by exploiting a unique opportunity offered by the cerebellar granule cell (GrC), which is electrotonically compact and has therefore allowed the direct experimental measurement of ionic currents. Previous models were constructed using empirical tuning of Gi-max values to match the original data set. Here, by using repetitive discharge patterns as a template, the optimization procedure yielded models that closely approximated the experimental Gi-max values. These models, in addition to repetitive firing, captured additional features, including inward rectification, near-threshold oscillations, and resonance, which were not used as features. Thus, parameter optimization using genetic algorithms provided an efficient modeling strategy for reconstructing the biophysical properties of neurons and for the subsequent reconstruction of large-scale neuronal network models. PMID:28360841

  18. Neurotoxicity of amphetamine derivatives is mediated by caspase pathway activation in rat cerebellar granule cells.

    PubMed

    Jiménez, Andrés; Jordà, Elvira G; Verdaguer, Ester; Pubill, David; Sureda, Francesc X; Canudas, Anna M; Escubedo, Elena; Camarasa, Jordi; Camins, Antoni; Pallàs, Mercè

    2004-04-15

    The neurotoxic action of the abuse drugs methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) on cerebellar granule neurones (CGNs) culture was examined. Treatment for 48 h with METH or MDMA (1-5 mM) induced a higher decrease in viability than 24 h treatment. z.VAD.fmk (100 microM) but not MK-801 nor NBQX recovered control viability values. In both cases, cell death was characterised as apoptotic rather than necrotic by morphology cell observation. Apoptosis measured by flow cytometry indicated an increase in the hypodiploid population after 48 h treatment with METH and MDMA. Apoptosis was reverted by the presence of z.VAD.fmk (100 microM) but not by 10 microM MK-801 or NBQX. Similar results were obtained by analysing nuclear chromatine condensation. These results ruled out excitotoxic participation in amphetamine derivative-induced neurotoxicity in CGNs. Participation of radical oxygen species (ROS) was evaluated using alpha-tocopherol (1-15 microM) and cytometric studies. The co-treatment with 4 mM METH or MDMA for 48 h partially reverted neurotoxic action and apoptotic features, indicating ROS implication in CGNs death by amphetamine derivatives. Alteration of mitochondrial function induced cytochrome C (Cyt C) release after 48-h treatment with METH and MDMA (4 mM). There was also indication of caspase-3-like activation, measured by immunoanalysis and biochemically. Finally, neurodegenerative action caused by amphetamine derivatives may be prevented by using caspase inhibitors.

  19. Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography.

    PubMed

    Xue, Yanhong; Zhao, Wei; Du, Wen; Zhang, Xiang; Ji, Gang; Ying, Wang; Xu, Tao

    2012-07-01

    Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic is