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Mast cell secretory granules: armed for battle.  


Mast cells are important effector cells of the immune system and recent studies show that they have immunomodulatory roles in diverse processes in both health and disease. Mast cells are distinguished by their high content of electron-dense secretory granules, which are filled with large amounts of preformed and pre-activated immunomodulatory compounds. When appropriately activated, mast cells undergo degranulation, a process by which these preformed granule compounds are rapidly released into the surroundings. In many cases, the effects that mast cells have on an immune response are closely associated with the biological actions of the granule compounds that they release, as exemplified by the recent studies showing that mast cell granule proteases account for many of the protective and detrimental effects of mast cells in various inflammatory settings. In this Review, we discuss the current knowledge of mast cell secretory granules. PMID:24903914

Wernersson, Sara; Pejler, Gunnar



Biogenesis of secretory granules in the trans-Golgi network of neuroendocrine and endocrine cells  

Microsoft Academic Search

Secretory granule formation requires selection of soluble and membrane proteins into nascent secretory granules, and exclusion of proteins not required for the function of secretory granules. Both selection and exclusion presumably can occur in the compartment where assembly of the secretory granule begins, the trans most cisternae of the Golgi complex. Current research focused on the initial stages of secretory

Sharon A Tooze



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

SciTech Connect

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.

Baconnais, S.; Delavoie, F. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France)]|[INSERM UMRS 514, IFR 53, CHU Maison Blanche, 45, rue Cognac-Jay, 51092 Reims Cedex (France); Zahm, J.M.; Milliot, M.; Castillon, N. [INSERM UMRS 514, IFR 53, CHU Maison Blanche, 45, rue Cognac-Jay, 51092 Reims Cedex (France); Terryn, C. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France); Banchet, V. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France); Michel, J. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France); Danos, O. [Genethon, CNRS UMR 8115, 1bis rue de l'Internationale, Evry (France); Merten, M. [INSERM EMI 0014, Faculte de Medecine, 9, Avenue de la Foret de Haye, BP 184, 54505 Vandoeuvre Les Nancy cedex, (France); Chinet, T. [Laboratoire de Biologie et Pharmacologie des Epitheliums Respiratoires, UFR Paris Ile de France Ouest, Boulogne-Billancourt (France); Zierold, K. [Max Planck Institute of Molekular Physiology, Laboratory for Analytical Microscopy, Otto-Hahn-Strasse 11, D-44227 Dortmund (Germany); Bonnet, N. [INSERM UMRS 514, IFR 53, CHU Maison Blanche, 45, rue Cognac-Jay, 51092 Reims Cedex (France); Puchelle, E. [INSERM UMRS 514, IFR 53, CHU Maison Blanche, 45, rue Cognac-Jay, 51092 Reims Cedex (France)], E-Mail:; Balossier, G. [INSERM ERM 203, Laboratoire de Microscopie Electronique Analytique, IFR53, Universite de Reims Champagne-Ardenne, 21 rue Clement Ader, 51685 Reims Cedex 2 (France)



Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy.  

PubMed Central

We have observed secretory granules beneath the plasma membrane of chromaffin cells. Using evanescent-field excitation by epiillumination, we have illuminated a thin layer of cytosol where cells adhere to glass coverslips. Up to 600 frames could be recorded at diffraction-limited resolution without appreciable photodynamic damage. We localized single granules with an uncertainty of approximately 30 nm and tracked their motion in three dimensions. Granules in resting cells wander randomly as if imprisoned in a cage that leaves approximately 70 nm space around a granule. The "cage" itself moves only slowly (D = 2 x 10(-12) cm2/s). Rarely do granules arrive at or depart from the plasma membrane of resting cells. Stimulation increases lateral motion only slightly. After the plasma membrane has been depleted of granules by exocytosis, fresh granules can be seen to approach it at an angle. The method will be useful for exploring the molecular steps preceding exocytosis at the level of single granules.

Steyer, J A; Almers, W



Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage  

PubMed Central

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins—including amylase, proline-rich proteins, and parotid secretory protein (PSP)—to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.

Gorr, S.-U.; Venkatesh, S.G.; Darling, D.S.



Rab3D Is Critical for Secretory Granule Maturation in PC12 Cells  

PubMed Central

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.

Kogel, Tanja; Rudolf, Rudiger; Hodneland, Erlend; Copier, John; Regazzi, Romano; Tooze, Sharon A.; Gerdes, Hans-Hermann



Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells  

PubMed Central

Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuro-peptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP–phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH4Cl. We conclude that most granules reseal in <10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.

Taraska, Justin W.; Perrais, David; Ohara-Imaizumi, Mica; Nagamatsu, Shinya; Almers, Wolfhard



Serous cells in the parotid glands of two species of tamarins: polarized secretory granules.  


The parotid glands of two species of tamarins were examined by electron microscopy. Endpiece cells are typical in appearance, with an extensive rough endoplasmic reticulum, prominent Golgi apparatuses, and numerous serous granules. In the saddleback tamarin, the secretory granules contain a dense spherule pressed against the inner aspect of the limiting membrane, leading to a surface bulge. During the course of merocrine secretion (a form of exocytosis), such morphologically polarized granules approach the luminal plasma membranes with the bulge in the vanguard. It is these protuberances that fuse with the plasmalemma. In contrast, although serous granules in the cotton top tamarin contain a spherule, they lack surface bulges and their docking on luminal membranes seems to be a random event with respect to their surface morphology. Moreover, certain other types of cells in a taxonomically wide spectrum of species have granules with a less obvious structural polarity, as well as cells whose granules lack morphological polarity but have a functional polarity that comes into play during exocytosis of such secretory granules. PMID:18780306

Tandler, Bernard



Simultaneous Electrical and Optical Measurements Show that Membrane Fusion Precedes Secretory Granule Swelling during Exocytosis of Beige Mouse Mast Cells  

Microsoft Academic Search

Mast cells show dramatic morphological changes when undergoing exocytosis. We have investigated whether the first of those morphological changes, swelling of the secretory granule, precedes--and therefore possibly initiates--secretion or whether it occurs after fusion of the granule and plasma membranes. We used cell membrane capacitance to detect the moment when granule and plasma membrane become continuous. We measured large capacitance

Joshua Zimmerberg; Michael Curran; Fredric S. Cohen; Malcolm Brodwick



Presence of dynamin--syntaxin complexes associated with secretory granules in adrenal chromaffin cells.  


Dynamin proteins have been implicated in many aspects of endocytosis, including clathrin-mediated endocytosis, internalization of caveolae, synaptic vesicle recycling, and, more recently, vesicular trafficking to and from the Golgi complex. To provide further insight into the function(s) of dynamin in neuroendocrine cells, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation, immunoreplica analysis, and confocal immunofluorescence. We found that dynamin, presumably the dynamin-2 isoform, is associated specifically with the membrane of purified secretory chromaffin granules. Oligomerization state analysis by sucrose density velocity gradients indicated that the granule-associated dynamin is in a monomeric form. Immunoprecipitation experiments coupled to double-labeling immunofluorescence cytochemistry revealed that the granular dynamin is associated with a syntaxin component that is not involved in the granule-bound SNARE complex. The possibility that dynamin participates in the coupling of the exocytotic and endocytotic reaction through the building of a granular membrane subset of proteins is discussed. PMID:10987831

Galas, M C; Chasserot-Golaz, S; Dirrig-Grosch, S; Bader, M F



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

PubMed Central

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.

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



Protein Mobility within Secretory Granules.  


We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (?64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10(-10) cm(2)/s (?1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit. PMID:24988337

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



The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer  

PubMed Central

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br- cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.



Expression of regulated secretory proteins is sufficient to generate granule-like structures in constitutively secreting cells.  


The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein alpha(1)-protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30-70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas alpha(1)-protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation. PMID:14996840

Beuret, Nicole; Stettler, Hansruedi; Renold, Anja; Rutishauser, Jonas; Spiess, Martin



Secretory Granule to the Nucleus  

PubMed Central

Intrinsically unstructured domains occur in one-third of all proteins and are characterized by conformational flexibility, protease sensitivity, and the occurrence of multiple phosphorylation. They provide large interfaces for diverse protein-protein interactions. Peptidylglycine ?-amidating monooxygenase (PAM), an enzyme essential for neuropeptide biosynthesis, is a secretory granule membrane protein. As one of the few proteins spanning the granule membrane, PAM is a candidate to relay information about the status of the granule pool and conditions in the granule lumen. Here, we show that the PAM cytosolic domain is unstructured. Mass spectroscopy and two-dimensional gel electrophoresis demonstrated phosphorylation at 10–12 sites in the cytosolic domain. Stimulation of exocytosis resulted in coupled phosphorylation and dephosphorylation of specific sites and in the endoproteolytic release of a soluble, proteasome-sensitive cytosolic domain fragment. Analysis of granule-rich tissues, such as pituitary and heart, showed that a similar fragment was generated endogenously and translocated to the nucleus. This multiply phosphorylated unstructured domain may act as a signaling molecule that relays information from secretory granules to both cytosol and nucleus.

Rajagopal, Chitra; Stone, Kathryn L.; Francone, Victor P.; Mains, Richard E.; Eipper, Betty A.



Quercetin-induced expression of rat mast cell protease II and accumulation of secretory granules in rat basophilic leukemia cells.  


Rat basophilic leukemia (RBL) cells are considered to be similar to bone-marrow derived mast cells and to mucosal mast cells (MMC), the latter of which may be involved in inflammatory bowel diseases. RBL cells are not able to accumulate histamine and secretory granules under regular growing conditions. Here we show that the flavonoid quercetin, which inhibits mast cell secretion of histamine, also inhibited RBL cell proliferation and constitutive histamine release while it induced synthesis of rat mast cell protease (RMCP) II and triggered processes leading to accumulation of secretory granules. Cell viability was also retained in the presence of quercetin, whereas untreated cells did not survive past 6 days of growth. Quercetin did not affect the expression of mRNA for alpha-subunit of immunoglobulin E (IgE) receptor, but led to increased expression of mRNA for, and synthesis of RMCP II, which is a marker protein for MMC. Many of these granules showed metachromasia with toluidine blue after 3 days of growth, stained red with alcian blue counterstained with safranin after 8 days of growth, and contained electron dense material. Our results suggest that RBL cells have the capacity to progress to a more mature state and may lend themselves to further analysis of a growth regulator(s) with action similar to that of quercetin. PMID:7506028

Trnovsky, J; Letourneau, R; Haggag, E; Boucher, W; Theoharides, T C



Ca(2+)-regulated secretory granule exocytosis in pancreatic and parotid acinar cells.  


Protein secretion from acinar cells of the pancreas and parotid glands is controlled by G-protein coupled receptor activation and generation of the cellular messengers Ca(2+), diacylglycerol and cAMP. Secretory granule (SG) exocytosis shares some common characteristics with nerve, neuroendocrine and endocrine cells which are regulated mainly by elevated cell Ca(2+). However, in addition to diverse signaling pathways, acinar cells have large ?1?m diameter SGs (?30 fold larger diameter than synaptic vesicles), respond to stimulation at slower rates (seconds versus milliseconds), demonstrate significant constitutive secretion, and in isolated acini, undergo sequential compound SG-SG exocytosis at the apical membrane. Exocytosis proceeds as an initial rapid phase that peaks and declines over 3min followed by a prolonged phase that decays to near basal levels over 20-30min. Studies indicate the early phase is triggered by Ca(2+) and involves the SG proteins VAMP2 (vesicle associated membrane protein2), Ca(2+)-sensing protein synatotagmin 1 (syt1) and the accessory protein complexin 2. The molecular details for regulation of VAMP8-mediated SG exocytosis and the prolonged phase of secretion are still emerging. Here we review the known regulatory molecules that impact the sequential exocytic process of SG tethering, docking, priming and fusion in acinar cells. PMID:24742357

Messenger, Scott W; Falkowski, Michelle A; Groblewski, Guy E



Evidence of de novo Membrane Generation in the Mechanism of Mast Cell Secretory Granule Activation.  

National Technical Information Service (NTIS)

Overwhelming evidence has suggested that granule matrix swelling and the enlargement of the perigranular membrane are early morphological indicators of mast cell granula activation (1-3). Since excess membrane in the form of folds or inclusions has not be...

S. P. Chock E. A. Schmauder-Chock



Elemental composition of secretory granules in pancreatic islets of Langerhans.  

PubMed Central

We have characterized, by electron probe microanalysis, rapidly frozen cultured rat islets at the level of individual secretory granules. Elemental analysis of thin, dried cryosections showed that beta granules could be distinguished by high Zn, Ca, and S, whereas non-beta (mainly alpha) granules contained elevated P and Mg. Although a single granule type predominated in a particular cell, some rebel granules were found in A cells that had the compositional fingerprint of B cell granules. Zn, which was found in millimolar concentrations in B cell granules, was considered a marker for the insulin storage complex. The data indicate that non-B islet cells in the adult pancreas may produce insulin-containing organelles and that, when glucagon and insulin are coexpressed, these hormones are packaged in separate granules. Images FIGURE 2

Foster, M C; Leapman, R D; Li, M X; Atwater, I



Inositol 1,4,5-trisphosphate receptor subtype 3 in pancreatic islet cell secretory granules revisited.  

PubMed Central

It has been reported that the inositol 1,4,5-trisphosphate receptor subtype 3 is expressed in islet cells and is localized to both insulin and somatostatin granules [Blondel, O., Moody, M. M., Depaoli, A. M., Sharp, A. H., Ross, C. A., Swift, H. & Bell, G. I. (1994) Proc. Natl. Acad. Sci. USA 91, 7777-7781]. This subcellular localization was based on electron microscope immunocytochemistry using antibodies (affinity-purified polyclonal antiserum AB3) directed to a 15-residue peptide of rat inositol trisphosphate receptor subtype 3. We now show that these antibodies cross-react with rat, but not human, insulin. Accordingly, the anti-inositol trisphosphate receptor subtype 3 (AB3) antibodies label electron dense cores of mature (insulin-rich) granules of rat pancreatic beta cells, and rat granule labeling was blocked by preabsorption of the AB3 antibodies with rat insulin. The immunostaining of immature, Golgi-associated proinsulin-rich granules with AB3 antibodies was very weak, indicating that cross-reactivity is limited to the hormone and not its precursor. Also, the AB3 antibodies labeled pure rat insulin crystals grown in vitro but failed to stain crystals grown from pure human insulin. By immunoprecipitation, the antibodies similarly displayed a higher affinity for rat than for human insulin. We could not confirm the labeling of somatostatin granules using AB3 antibodies. Images Fig. 1

Ravazzola, M; Halban, P A; Orci, L



Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells  

PubMed Central

Background Cytotoxic cells of the immune system have evolved a lysosomal compartment to store and mobilize effector molecules. In T lymphocytes and NK cells, the death factor FasL is one of the characteristic marker proteins of these so-called secretory lysosomes, which combine properties of conventional lysosomes and exocytotic vesicles. Although these vesicles are crucial for immune effector function, their protein content in T cells has so far not been investigated in detail. Results In the present study, intact membranous vesicles were enriched from homogenates of polyclonally activated T cells and initially characterized by Western blotting and electron microscopic inspection. The vesicular fraction that contained the marker proteins of secretory lysosomes was subsequently analyzed by 2D electrophoresis and mass spectrometry. The proteome analysis and data evaluation revealed that 70% of the 397 annotated proteins had been associated with different lysosome-related organelles in previous proteome studies. Conclusion We provide the first comprehensive proteome map of T cell-derived secretory lysosomes with only minor contaminations by cytosolic, nuclear or other proteins. This information will be useful to more precisely address the activation-dependent maturation and the specific distribution of effector organelles and proteins in individual T or NK cell populations in future studies.



Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells  

SciTech Connect

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.

Fujita-Yoshigaki, Junko [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan)]. E-mail:; Katsumata, Osamu [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan); Matsuki, Miwako [Department of Pathology, Tokyo Dental College, Chiba 261-8502 (Japan); Yoshigaki, Tomoyoshi [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan); Furuyama, Shunsuke [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan); Sugiya, Hiroshi [Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587 (Japan)



Heterologous processing of rat prosomatostatin to somatostatin-14 by PC2: requirement for secretory cell but not the secretion granule.  

PubMed Central

The role of PC2 in prosomatostatin (PSS) processing was investigated in GH3/GH4C1 pituitary cells. These cells are sparsely granulated, express different amounts of PC2 and no PC1. We described heterologous processing of rat PSS (rPSS) co-expressed with PC2 in stably transfected cells, correlate PC2 protein levels under different conditions of transfection with efficiency of PSS processing to somatostatin-14 (SS-14), determine the effect of modulating cell granularity on enzyme expression and PSS processing, and compare the relative potency of PC2 with that of PC1, PSS and cleavage products were monitored by HPLC and radioimmunoassay of SS-like immunoreactivity (SSLI). Radioimmunoassay analysis of N-terminal PC2-like immunoreactivity (PC2 LI) in GH4C1:rPSS, GH4C1:rPSS + PC2 and GH3:rPSS transfectants showed a gradient of PC2 protein of 1:2.6:3.4 in cell extracts and 1:4.7:9 in secretion media from these cells respectively. The concentration of PC2 protein correlated with SS-14 conversion efficiency was 36 +/- 3% in GH4C1:rPSS cells, 56 +/- 7% in GH4C1:rPSS-PC2 cells and 100% in GH3:rPSS cells. Treatment of GH4C1:rPSS + PC2 cells with epidermal growth factor, insulin, and beta-estradiol to induce granules, significantly increased basal and forskolin-stimulated co-release of SS LI and PC2 LI, but had no influence on SS-14 processing efficiency. Hormone treatment led to a small increase in the ratio of mature PC2 (68 kDa) to proPC2 (75 kDa) forms. PC1 stably transfected in GH4C1 cells produced significantly greater SS-14 conversion (62% in cells, 66% in media) compared with PC2 transfectants (53% in cells, 47% in media) These results provide the first proof that PC2 can effect dibasic processing of mammalian PSS, and, along with PC1, qualifies as an authentic SS-14 convertase. The activity of PC2 requires the milieu of the secretory cell but not the secretory granule. Images Figure 1 Figure 2 Figure 3 Figure 6

Galanopoulou, A S; Seidah, N G; Patel, Y C



Differential expression of secretory granule proteases in mouse mast cells exposed to interleukin 3 and c-kit ligand  

PubMed Central

It is now established that the subclasses of mast cells (MC) that reside in mucosal and serosal environments can be distinguished from one another in terms of their expression of specific secretory granule- localized proteases and proteoglycans. Further, the hematopoietic- and connective tissue-derived cytokines that regulate expression of the genes that encode these constituents of the granule can now be identified using recently developed gene-specific probes and recombinant cytokines. When bone marrow-derived MC (BMMC) were developed with recombinant interleukin 3 (rIL-3) and maintained with this cytokine in the absence or presence of recombinant c-kit ligand (rKL), they remained safranin-, produced almost no 35S-labeled heparin proteoglycans, and contained greater levels of mouse MC protease (MMCP) -5 mRNA and mast cell carboxypeptidase A (MC-CPA) mRNA than MMCP-6 mRNA. They did not contain MMCP-4 or -2 mRNA, genes expressed late in the differentiation of progenitor cells into serosal and mucosal MCs, respectively. In contrast, BMMC developed with rKL alone or by sequential culture in medium containing rIL-3 followed by rKL expressed high levels of MMCP-4 and -6 mRNA, as well as the transcripts that encode MMCP-5 and MC-CPA. Although rKL-developed BMMC were safranin+ and produced substantial amounts of 35S-labeled heparin proteoglycans, they contained only minimal amounts of histamine and MC-CPA enzymatic activity relative to serosal MC. These are the first studies to characterize the transcriptional granule phenotype of a population of BMMC derived using any recombinant cytokine, to demonstrate a dissociation between histochemical staining and granule maturation, and to demonstrate antagonistic regulation of late expressed protease genes by a cytokine.



The localization of phospholipase A2 in the secretory granule.  

PubMed Central

A heat-resistant phospholipase A2 has been detected in the secretory granules of the mast cell [Chock, Rhee, Tang and Schmauder-Chock (1991) Eur. J. Biochem. 195, 707-713]. By using ultrastructural immunocytochemical techniques, we have now localized this enzyme to the matrix of the secretory granule. Like the cyclo-oxygenase [Schmauder-Chock and Chock (1989) J. Histochem. Cytochem. 37, 1319-1328], this enzyme also adheres tightly to the ribbon-like granule matrix components. The results from Western-blot analysis suggest that it has a molecular mass of about 14 kDa. The localization of the phospholipase A2, the presence of a phospholipid store with millimolar concentrations of calcium and the localization of the enzymes of the arachidonic acid cascade make the secretory granule a natural site for lipid-mediator synthesis. The packaging of phospholipase A2, together with its substrate and the components of the arachidonic acid cascade, in the secretory granule represents a physical arrangement by which the initiation of the cascade and the release of mediators can be directly linked to the stimulation of cell-surface receptors. Images Figure 1 Figure 2 Figure 3

Chock, S P; Schmauder-Chock, E A; Cordella-Miele, E; Miele, L; Mukherjee, A B



Secretory lysosomes  

Microsoft Academic Search

Regulated secretion of stored secretory products is important in many cell types. In contrast to professional secretory cells, which store their secretory products in specialized secretory granules, some secretory cells store their secretory proteins in a dual-function organelle, called a secretory lysosome. Functionally, secretory lysosomes are unusual in that they serve both as a degradative and as a secretory compartment.

Emma J. Blott; Gillian M. Griffiths



A synthetic mimic of the secretory granule for drug delivery.  


Secretory cells contain submicroscopic granules composed of a polyanionic polymer network that is collapsed owing to the presence of hydronium ions and weak base cations. The network is encapsulated within a lipid membrane, and functions as a vehicle for the osmotically inert storage of a variety of granule-bound endogenous mediator species, such as histamine, serotonin and proteases. These species are excreted from the granule and thence from the cell in response to external biochemical signals. Hydrogels that swell and shrink in response to external stimuli might serve as synthetic analogues of secretory granules. Here we describe the systematic engineering of multi-component, environmentally responsive hydrogel microspheres, coated with a lipid bilayer to mimic more closely the natural secretory granule. These microspheres exhibit pH- and ion-dependent volume phase transitions and ion-sensitive exchange of bound cations when the encapsulating lipid membrane is porated. We stimulated poration electrically in individual microgel particles immobilized and manipulated with a micropipette. This system could find use for the triggered release of encapsulated drugs in the body. PMID:9697768

Kiser, P F; Wilson, G; Needham, D



Rapid ATP-dependent priming of secretory granules precedes Ca(2+)-induced exocytosis in mouse pancreatic B-cells.  

PubMed Central

1. The glucose and ATP dependence of exocytosis were investigated in single mouse pancreatic B-cells by monitoring changes in cell capacitance evoked by voltage-clamp depolarizations, infusion of high [Ca2+]i buffers or photorelease of caged Ca2+ or ATP. 2. In intact B-cells, using the perforated patch whole-cell technique, glucose (5 mM) increased exocytotic responses evoked by membrane depolarization 5-fold over that observed in the absence of the sugar. Increasing the glucose concentration to 20 mM produced a further doubling of exocytosis. The stimulatory action of glucose was attributable to glucose metabolism and abolished by mannoheptulose, an inhibitor of glucose phosphorylation. 3. Exocytosis triggered by infusion of high [Ca2+]i and ATP was reduced by 80% when ATP was replaced by its non-hydrolysable analogue adenosine 5'-[beta, gamma-methylene]triphosphate (AMP-PCP) in standard whole-cell experiments. Exocytosis elicited by GTP gamma S was similarly affected by replacement of ATP with the stable analogue. 4. Photoreleasing ATP in the presence of 170 nM [Ca2+]i, following the complete wash-out of endogenous ATP produced a prompt (latency, < 400 ms) and biphasic stimulation of exocytosis. 5. Elevation of [Ca2+]i to exocytotic levels by photorelease from Ca(2+)-nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg-ATP. The response consisted of an initial rapid (completed in < 200 ms) phase followed by a slower (lasting > or = 10 s) sustained component. Replacement of ATP with AMP-PCP abolished the late component but did not affect the initial phase. The latency between elevation of [Ca2+]i and exocytosis was determined as < 45 ms. Inclusion of N-ethylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exerted effects similar to those obtained by substituting AMP-PCP for ATP. 6. We conclude that the B-cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP-independent fashion. We propose that this pool of granules is preferentially released during first phase glucose-stimulated insulin secretion. The short latency between the application of ATP and the onset of exocytosis finally suggests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B-cell. Images Figure 1 Figure 4 Figure 8

Eliasson, L; Renstrom, E; Ding, W G; Proks, P; Rorsman, P



The AP1 adaptor complex binds to immature secretory granules from PC12 cells, and is regulated by ADP-ribosylation factor  

Microsoft Academic Search

Immature secretory granules (ISGs) in endo- crine and neuroendocrine cells have been shown by morphological techniques to be partially clathrin coated (Orci, L., M. Ravazzola, M. Amherdt, D. Lon- vard, A. Perrelet. 1985a. Proc. Natl. Acad. Sci. USA. 82: 5385-5389; Tooze, J., and S.A. Tooze. 1986. J. Cell Biol. 103:839-850). The function, and composition, of this clathrin coat has remained

A. S. Dittie; Nasser Hajibagheri; Sharon A. Tooze



Rab5 is a novel regulator of mast cell secretory granules: impact on size, cargo, and exocytosis.  


Secretion of inflammatory mediators prestored in mast cells secretory granules (SGs) enhances immune responses such as in allergy and host defense. However, the mechanisms underlying the biogenesis of the SGs remain largely unresolved. By combining high-resolution live cell imaging and quantitative morphometric analyses, we show that the small GTPase Rab5 controls the SG size and cargo composition by a VAMP8-dependent fusion mechanism. Knockdown of the endogenous Rab5, or expression of constitutively negative mutants, significantly reduces the size of SGs and increases their number. Conversely, expression of constitutively active Rab5 mutants induces few, but giant, SGs. Both the small and giant SGs maintain their exocytosis competence. Finally, we show that Rab5-mediated fusion between Golgi-derived SGs and early endosomes precedes the maturation of the SGs, as reflected by the recruitment of Rab27B, and allows the incorporation of cargo, such as CD63, that traffics through endosomes. Collectively, our results assign Rab5 a key role in mediating mast cell SG fusion during biogenesis, thereby controlling the amount and composition of the SGs content and maintaining the communication between new and pre-existing SGs. PMID:24696234

Azouz, Nurit P; Zur, Neta; Efergan, Adi; Ohbayashi, Norihiko; Fukuda, Mitsunori; Amihai, Dina; Hammel, Ilan; Rothenberg, Marc E; Sagi-Eisenberg, Ronit



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

Microsoft Academic Search

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the rans-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 horseradish peroxidase and by the endogenous marker

J. H. B. Diederen; H. G. B. Vullings



Statistical analysis of the quantal basis of secretory granule formation.  


The size distribution of vesicles exocytosed from secretory cells displays quantal nature, vesicle volume is periodic multi-modal, suggesting that these heterogeneous vesicles are aggregate sums of a variable number of homogeneous basic granules. Whether heterogeneity is a lumping-together artifact of the measurement or an inherent intra-cell feature of the vesicles is an unresolved question. Recent empirical evidence will be provided for the quantal nature of intra-cell vesicle volume, supporting the controversial paradigm of homotypic fusion: basic cytoplasmic granules fuse with each other to create heterogeneously sized vesicles. An EM-algorithm-based method is presented for the conversion of multi-modal to quantal data that provides as by-product estimates of means and variances of basic granule packaging. PMID:24185612

K?epelová-Dror, Marika; Hammel, Ilan; Meilijson, Isaac



Kalirin/Trio Rho Guanine Nucleotide Exchange Factors Regulate a Novel Step in Secretory Granule Maturation  

PubMed Central

The molecular mechanisms involved in the maturation of secretory granules, organelles that store hormones and neuropeptides, are poorly understood. As granule content proteins are processed, the composition of granule membranes changes, yielding constitutive-like secretion of immature content proteins and producing secretagogue-responsive mature granules. Constitutive-like secretion was not previously recognized as a process subject to regulation. We show that Kalirin and Trio, homologous Rho guanine nucleotide exchange factors (GEFs), which interact with a secretory granule resident protein, modulate cargo secretion from immature granules. Some of the Kalirin and Trio isoforms expressed in neuroendocrine cells colocalize with immature granules. Overexpression of their N-terminal GEF domain (GEF1) enhances secretion from immature granules, depleting cells of secretory cargo in the absence of secretagogue. This response requires GEF1 activity and is mimicked by Kalirin/Trio substrates Rac1 and RhoG. Accordingly, selective pharmacological inhibition of endogenous GEF1 activity decreases secretagogue-independent release of hormone precursors, accumulating product peptide in mature secretory granules. Kalirin/Trio modulation of cargo secretion from immature granules provides secretory cells with an extra layer of control over the sets of peptides released. Control of this step enhances the range of physiological responses that can be elicited, whereas lack of control could have pathological consequences.

Ferraro, Francesco; Ma, Xin-Ming; Sobota, Jacqueline A.; Eipper, Betty A.



Lipopolysaccharide-binding protein: localization in secretory granules of Paneth cells in the mouse small intestine  

Microsoft Academic Search

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host’s response to endotoxin and\\u000a mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including\\u000a the enterocyte-like cell line Caco-2. To study in closer detail the synthesis and storage of LBP in the intestinal mucosal\\u000a epithelium, we performed

Gert H. Hansen; Karina Rasmussen; Lise-Lotte Niels-Christiansen; E. Michael Danielsen



Not All Secretory Granules Are Created Equal: Partitioning of Soluble Content Proteins  

PubMed Central

Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. We fused the soluble secretory protein peptidylglycine ?-hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Importantly, we found that segregation of content proteins is not an artifact of overexpression nor peculiar to GFP-tagged proteins. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Our data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple “self-aggregation default” theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules.

Sobota, Jacqueline A.; Ferraro, Francesco; Back, Nils; Eipper, Betty A.



Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore.  

PubMed Central

For fusion to occur the repulsive forces between two interacting phospholipid bilayers must be reduced. In model systems, this can be achieved by increasing the surface tension of at least one of the membranes. However, there has so far been no evidence that the secretory granule membrane is under tension. We have been studying exocytosis by using the patch-clamp technique to measure the surface area of the plasma membrane of degranulating mast cells. When a secretory granule fuses with the plasma membrane there is a step increase in the cell surface area. Some fusion events are reversible, in which case we have found that the backstep is larger than the initial step, indicating that there is a net decrease in the area of the plasma membrane. The decrease has the following properties: (i) the magnitude is strongly dependent on the lifetime of the fusion event and can be extensive, representing as much as 40% of the initial granule surface area; (ii) the rate of decrease is independent of granule size; and (iii) the decrease is not dependent on swelling of the secretory granule matrix. We conclude that the granule membrane is under tension and that this tension causes a net transfer of membrane from the plasma membrane to the secretory granule, while they are connected by the fusion pore. The high membrane tension in the secretory granule may be the critical stress necessary for bringing about exocytotic fusion.

Monck, J R; Alvarez de Toledo, G; Fernandez, J M



Separation of rat pituitary secretory granules by continuous flow electrophoresis  

NASA Technical Reports Server (NTRS)

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.

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



Rab3D, a small GTPase, is localized on mast cell secretory granules and translocates to the plasma membrane upon exocytosis.  


Although mast cell secretion has been intensively studied because of its pivotal role in allergic reactions and its advantages as a physiologic model, the molecular composition of the secretory machine is virtually unknown. In view of the guanine-nucleotide dependency of mast cell exocytosis and the participation of Rab3 proteins in synaptic vesicle release, we hypothesized that a Rab3 isoform regulates mast cell secretion. Fragments of Rab3A, 3B, and 3D were cloned from RBL-2H3 mast cells by reverse transcription- polymerase chain reaction (RT-PCR). Northern blot analysis revealed Rab3D transcripts to be relatively abundant, Rab3B substantially less so, and Rab3A and 3C undetectable. By ribonuclease (RNase) protection assay, Rab3D transcripts were at least 10-fold more abundant than those of other isoforms, and by immunoblot analysis, Rab3D protein was at least 60-fold more abundant than that of Rab3B. Rab3D was more abundant in RBL cells than in brain, but the total mass of Rab3 proteins in RBL cells was 10-fold less than in brain. Rab3D only partly colocalized with secretory granules in RBL cells, but fully colocalized in mature peritoneal mast cells. There was a descending concentration gradient of Rab3D from peripheral to central granules, and no cytoplasmic pool was detectable in resting mast cells. Following exocytotic degranulation, Rab3D translocated to the plasma membrane and remained there for at least 15 min. These studies suggest that Rab3D is a component of the regulated exocytotic machine of mast cells, and identify differences between mast cells and neurons in Rab3 expression and trafficking. PMID:9870920

Tuvim, M J; Adachi, R; Chocano, J F; Moore, R H; Lampert, R M; Zera, E; Romero, E; Knoll, B J; Dickey, B F



Association with Nitric Oxide Synthase on Insulin Secretory Granules Regulates Glucokinase Protein Levels  

PubMed Central

Glucokinase (GCK) association with insulin-secretory granules is controlled by interaction with nitric oxide synthase (NOS) and is reversed by GCK S-nitrosylation. Nonetheless, the function of GCK sequestration on secretory granules is unknown. Here we report that the S-nitrosylation blocking V367M mutation prevents GCK accumulation on secretory granules by inhibiting association with NOS. Expression of this mutant is reduced compared with a second S-nitrosylation blocking GCK mutant (C371S) that accumulates to secretory granules and is expressed at levels greater than wild type. Even so, the rate of degradation for wild type and mutant GCK proteins were not significantly different from one another, and neither mutation disrupted the ability of GCK to be ubiquitinated. Furthermore, gene silencing of NOS reduced endogenous GCK content but did not affect ?-actin content. Treatment of GCK(C371S) expressing cells with short interfering RNA specific for NOS also blocked accumulation of this protein to secretory granules and reduced expression levels to that of GCK(V367M). Conversely, cotransfection of catalytically inactive NOS increased GCK-mCherry levels. Expression of GCK(C371S) in ?TC3 cells enhanced glucose metabolism compared with untransfected cells and cells expressing wild type GCK, even though this mutant has slightly reduced enzymatic activity in vitro. Finally, molecular dynamics simulations revealed that V367M induces conformational changes in GCK that are similar to S-nitrosylated GCK, thereby suggesting a mechanism for V367M-inhibition of NOS association. Our findings suggest that sequestration of GCK on secretory granules regulates cellular GCK protein content, and thus cellular GCK activity, by acting as a storage pool for GCK proteins.

Markwardt, Michele L.; Nkobena, Andongfac; Ding, Shi-Ying



Involvement of AQP6 in the Mercury-sensitive osmotic lysis of rat parotid secretory granules.  


In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73-80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²?, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²? (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5-2.0 ?M Hg²?, concentrations that activate AQP6. The Hg lysis was completely blocked by ?-mercaptoethanol which disrupts Hg²?-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO(3) (-) > Br? > I? > Cl? and was facilitated by acidic pH. The anion selectivity for NO(3) (-) and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²?-sensitive anion channel in rat parotid secretory granules. PMID:23183829

Matsuki-Fukushima, Miwako; Fujita-Yoshigaki, Junko; Murakami, Masataka; Katsumata-Kato, Osamu; Yokoyama, Megumi; Sugiya, Hiroshi



Differential dynamics of Rab3A and Rab27A on secretory granules.  


We have assessed the dynamics of the association of Rab3A and Rab27A with secretory granules at various stages of their life in PC12 cells. Endogenous Rab3A colocalised with the secretory granule marker secretogranin II (SGII) and expressed EGFP-Rab3A and ECFP-Rab27A colocalised with one another. The extent of colocalisation between EGFP-Rab3A or EGFP-Rab27 and SGII increased after longer times post transfection suggesting that these Rab proteins are preferentially recruited to newly synthesised granules. Following the release of immature secretory granules from the trans-Golgi network, Rab3A and Rab27A became associated with the immature granules after a lag period of around 20 minutes. Rab dynamics on granules were analysed in fluorescence recovery after photobleaching (FRAP) experiments. The recovery profile of EGFP-Rab27A was comparable to that of ppANF-EGFP, whereas the recovery profile of EGFP-Rab3A was significantly faster, indicating that Rab3A but not Rab27A might be rapidly exchanged between granules and cytosol. Inhibition of heat-shock protein 90 with 10 muM geldanamycin did not affect the exchange process or regulated exocytosis. Rab dynamics during stimulation with 300 muM ATP were analysed in live cells. Loss of granular ppANF-EGFP fluorescence was seen at the cell periphery after stimulation but only limited changes in EGFP-Rab3A and EGFP-Rab27A fluorescence was observed, indicating that the Rab proteins do not immediately dissociate or disperse on stimulation. The data suggest potentially distinct roles for Rab3A and Rab27A and we suggest that the finding that young secretory granules have a higher capacity for binding Rab3A and Rab27A is functionally important for preferential exocytosis from these granules. PMID:17311845

Handley, Mark T W; Haynes, Lee P; Burgoyne, Robert D



Fibroblast Growth Factor Receptor Like-1 (FGFRL1) Interacts with SHP-1 Phosphatase at Insulin Secretory Granules and Induces Beta-cell ERK1/2 Protein Activation*  

PubMed Central

FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and ?TC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-?His) or entire intracellular sequence (RL1-?C) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor's intracellular SH2-binding motif.

Silva, Pamuditha N.; Altamentova, Svetlana M.; Kilkenny, Dawn M.; Rocheleau, Jonathan V.



Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors.  


Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes. PMID:23650620

Rørvig, Sara; Østergaard, Ole; Heegaard, Niels H H; Borregaard, Niels



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

SciTech Connect

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.

Marino, C.R.; Castle, J.D.; Gorelick, F.S. (Yale Univ. School of Medicine, New Haven, CT (USA))



Characterization of cytoplasmic secretory granules (PSG), in prostatic epithelium and their transformation-induced loss in dysplasia and adenocarcinoma  

Microsoft Academic Search

Cytoplasmic clarity is a histological feature of normal prostatic secretory cells, but in this study, tissue fixation in strong (>2.5%) glutaraldehyde dramatically altered cytological staining. Secretory cytoplasm appeared red and granular on routine stains because of myriad intensely staining eosinophilic granules (PSG). Immunostaining for prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) showed their exclusive localization to the PSG. Electron

Ronald J Cohen; John E McNeal; Stephen G Edgar; Terry Robertson; Hugh J. S Dawkins



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

SciTech Connect

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.

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



Role of Adaptor Proteins in Secretory Granule Biogenesis and Maturation  

PubMed Central

In the regulated secretory pathway, secretory granules (SGs) store peptide hormones that are released on demand. SGs are formed at the trans-Golgi network and must undergo a maturation process to become responsive to secretagogues. The production of mature SGs requires concentrating newly synthesized soluble content proteins in granules whose membranes contain the appropriate integral membrane proteins. The mechanisms underlying the sorting of soluble and integral membrane proteins destined for SGs from other proteins are not yet well understood. For soluble proteins, luminal pH and divalent metals can affect aggregation and interaction with surrounding membranes. The trafficking of granule membrane proteins can be controlled by both luminal and cytosolic factors. Cytosolic adaptor proteins (APs), which recognize the cytosolic domains of proteins that span the SG membrane, have been shown to play essential roles in the assembly of functional SGs. Adaptor protein 1A (AP-1A) is known to interact with specific motifs in its cargo proteins and with the clathrin heavy chain, contributing to the formation of a clathrin coat. AP-1A is present in patches on immature SG membranes, where it removes cargo and facilitates SG maturation. AP-1A recruitment to membranes can be modulated by Phosphofurin Acidic Cluster Sorting protein 1 (PACS-1), a cytosolic protein which interacts with both AP-1A and cargo that has been phosphorylated by casein kinase II. A cargo/PACS-1/AP-1A complex is necessary to drive the appropriate transport of several cargo proteins within the regulated secretory pathway. The Golgi-localized, ?-ear containing, ADP-ribosylation factor binding (GGA) family of APs serve a similar role. We review the functions of AP-1A, PACS-1, and GGAs in facilitating the retrieval of proteins from immature SGs and review examples of cargo proteins whose trafficking within the regulated secretory pathway is governed by APs.

Bonnemaison, Mathilde L.; Eipper, Betty A.; Mains, Richard E.



Mapping Dynamic Protein Interactions to Insulin Secretory Granule Behavior with TIRF-FRET  

PubMed Central

Biological processes are governed by extensive networks of dynamic molecular interactions. Yet, establishing a spatial and temporal map of these interactions and their direct relationship to specific cell functions has remained a challenge. Here, we implement sensitized emission Förster resonance energy transfer (FRET) stoichiometry under total internal reflection fluorescence (TIRF) microscopy. We demonstrate through quantitative analysis and modeling that evanescent fields must be precisely matched between FRET excitation wavelengths to isolate dynamic interactions between bimolecular FRET pairs that are not entirely membrane-delimited. We then use TIRF-FRET to monitor the behavior of individual insulin-containing secretory granules at the plasma membrane of living cells, while simultaneously tracking the dynamic interaction between the GTPase Rab27A and its effector Slp4A, on those same granules. Notably, insulin granules that underwent exocytosis demonstrated a specific increase in Rab27A-GTP/Slp4A FRET in the 5 s before membrane fusion, which coincided temporally with an increase in granule displacement and mobility. These results demonstrate an initial spatiotemporal mapping of a dynamic protein-protein interaction on individual secretory granules that is linked to a specific granule behavior in living cells.

Lam, Alice D.; Ismail, Sahar; Wu, Ray; Yizhar, Ofer; Passmore, Daniel R.; Ernst, Stephen A.; Stuenkel, Edward L.



Degradation of human anaphylatoxin C3a by rat peritoneal mast cells: a role for the secretory granule enzyme chymase and heparin proteoglycan  

SciTech Connect

Purified human C3a was iodinated (/sup 125/I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of /sup 125/I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of /sup 125/I-C3a and the rate of dissociation from the cell were temperature dependent. At 0/sup 0/C, the binding of /sup 125/I-C3a was increased and the rate of dissociation reduced, as compared to 37/sup 0/C. Once /sup 125/I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of /sup 125/I precipitable by TCA and the appearance of /sup 125/I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of /sup 125/I-C3a. Treatment of RMC bearing /sup 125/I-C3a with Bis (sulfosuccinimidyl) suberate (BS/sup 3/) covalently crosslinked the /sup 125/I-Ca to chymase, the predominant enzyme found in the secretory granules. Indirect immunofluorescence of RMC using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. The results indicate that /sup 125/I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan. In addition, this proteolysis of /sup 125/I-C3a by chymase must be blocked in order to detect plasma membrane C3a binding components on RMC.

Gervasoni, J.E. Jr.



Redistribution of a Rab3-1ike GTP-binding Protein from Secretory Granules to the Golgi Complex in Pancreatic Acinar Cells during Regulated Exocytosis  

Microsoft Academic Search

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensa- tion. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ul- timately reaches the TGN. In this study, we analyzed the fate of a low Mr GTP-binding

Bhanu P. Jena; Francine D. Gumkowski; Elisa M. Konieczko; Gabriele Fischer; Reinhard Jahn; James D. Jamieson


Cholesterol biosynthesis pathway intermediates and inhibitors regulate glucose-stimulated insulin secretion and secretory granule formation in pancreatic beta-cells.  


Cholesterol is reportedly abundant in the endocrine secretory granule (SG) membrane. In this study, we examined the involvement of cholesterol biosynthesis intermediates and inhibitors in insulin secretion and SG formation mechanisms. There are two routes for the supply of cholesterol to the cells: one via de novo biosynthesis and the other via low-density lipoprotein receptor-mediated endocytosis. We found that insulin secretion and content are diminished by ?-hydroxy-?-methylglutaryl-coenzyme A inhibitor lovastatin but not by lipoprotein depletion from the culture medium in MIN6 ?-cells. Cholesterol biosynthesis intermediates mevalonate, squalene, and geranylgeranyl pyrophosphate enhanced glucose-stimulated insulin secretion, and the former two increased insulin content. The glucose-stimulated insulin secretion-enhancing effect of geranylgeranyl pyrophosphate was also confirmed in perifusion with rat islets. Morphologically, mevalonate and squalene increased the population of SGs without affecting their size. In contrast, lovastatin increased the SG size with reduction of insulin-accumulating dense cores, leading to a decrease in insulin content. Furthermore, insulin was secreted in a constitutive manner, indicating disruption of regulated insulin secretion. Because secretogranin III, a cholesterol-binding SG-residential granin-family protein, coincides with SG localization based on the cholesterol composition, secretogranin III may be associated with insulin-accumulating mechanisms. Although the SG membrane exhibits a high cholesterol composition, we could not find detergent-resistant membrane regions using a lipid raft-residential protein flotillin and a fluorescent cholesterol-Si-pyrene probe as markers on a sucrose-density gradient fractionation. We suggest that the high cholesterol composition of SG membrane with 40-50 mol% is crucial for insulin secretion and SG formation functions. PMID:20685866

Tsuchiya, Miho; Hosaka, Masahiro; Moriguchi, Tomohisa; Zhang, Shaojuan; Suda, Masayuki; Yokota-Hashimoto, Hiromi; Shinozuka, Kazuo; Takeuchi, Toshiyuki



Myosin Vc is a molecular motor that functions in secretory granule trafficking.  


Class V myosins are actin-based motor proteins that have critical functions in organelle trafficking. Of the three class V myosins expressed in mammals, relatively little is known about Myo5c except that it is abundant in exocrine tissues. Here we use MCF-7 cells to identify the organelles that Myo5c associates with, image the dynamics of Myo5c in living cells, and test the functions of Myo5c. Endogenous Myo5c localizes to two distinct compartments: small puncta and slender tubules. Myo5c often exhibits a highly polarized distribution toward the leading edge in migrating cells and is clearly distinct from the Myo5a or Myo5b compartments. Imaging with GFP-Myo5c reveals that Myo5c puncta move slowly (approximately 30 nm/s) and microtubule independently, whereas tubules move rapidly (approximately 440 nm/s) and microtubule dependently. Myo5c puncta colocalize with secretory granule markers such as chromogranin A and Rab27b, whereas Myo5c tubules are labeled by Rab8a. TIRF imaging indicates that the granules can be triggered to undergo secretion. To test if Myo5c functions in granule trafficking, we used the Myo5c tail as a dominant negative and found that it dramatically perturbs the distribution of granule markers. These results provide the first live-cell imaging of Myo5c and indicate that Myo5c functions in secretory granule trafficking. PMID:19741097

Jacobs, Damon T; Weigert, Roberto; Grode, Kyle D; Donaldson, Julie G; Cheney, Richard E



Myosin Vc Is a Molecular Motor That Functions in Secretory Granule Trafficking  

PubMed Central

Class V myosins are actin-based motor proteins that have critical functions in organelle trafficking. Of the three class V myosins expressed in mammals, relatively little is known about Myo5c except that it is abundant in exocrine tissues. Here we use MCF-7 cells to identify the organelles that Myo5c associates with, image the dynamics of Myo5c in living cells, and test the functions of Myo5c. Endogenous Myo5c localizes to two distinct compartments: small puncta and slender tubules. Myo5c often exhibits a highly polarized distribution toward the leading edge in migrating cells and is clearly distinct from the Myo5a or Myo5b compartments. Imaging with GFP-Myo5c reveals that Myo5c puncta move slowly (?30 nm/s) and microtubule independently, whereas tubules move rapidly (?440 nm/s) and microtubule dependently. Myo5c puncta colocalize with secretory granule markers such as chromogranin A and Rab27b, whereas Myo5c tubules are labeled by Rab8a. TIRF imaging indicates that the granules can be triggered to undergo secretion. To test if Myo5c functions in granule trafficking, we used the Myo5c tail as a dominant negative and found that it dramatically perturbs the distribution of granule markers. These results provide the first live-cell imaging of Myo5c and indicate that Myo5c functions in secretory granule trafficking.

Jacobs, Damon T.; Weigert, Roberto; Grode, Kyle D.; Donaldson, Julie G.



Glucose Transporter (GLUT-4) Is Targeted to Secretory Granules in Rat Atrial Cardiomyocytes  

PubMed Central

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the transGolgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulindependent movement to the cell surface. To examine the relationship between these GLUT-4–containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.

Slot, Jan W.; Garruti, Gabriella; Martin, Sally; Oorschot, Viola; Posthuma, George; Kraegen, Edward W.; Laybutt, Ross; Thibault, Gaetan; James, David E.



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


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. PMID:12456006

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



Kinetics of release of serotonin from isolated secretory granules. I. Amperometric detection of serotonin from electroporated granules.  

PubMed Central

We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance approximately 100 microm) and positioned adjacent (<1 microm) to a carbon fiber microelectrode. A short (approximately 30 micros) high-intensity voltage pulse (electric field of approximately 5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of current and maximum flux, t(max)) with approximately 25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) approximately 21r2) and reflects the time required for membrane rupture. The average t(1/2)spike of the amperometric spikes was found to be approximately 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis. Images FIGURE 1 FIGURE 4 FIGURE 5 FIGURE 6

Marszalek, P E; Farrell, B; Verdugo, P; Fernandez, J M



Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation  

PubMed Central

The ultrastructural changes in electropermeabilized bovine platelets that accompany the Ca2(+)-induced secretion of serotonin were investigated in ultra-thin sections of chemically fixed cells. Such preparations permitted us to study both the localization of and the structures associated with serotonin-containing dense granules. Localization of dense granules within cells was examined by measuring the shortest distances between the granular membranes and the plasma membrane. About 40% of total granules were located close to the plasma membrane at an average distance of 10.8 +/- 1.6 nm. 71% of the total number of granules were localized at a similar average distance of 12.5 +/- 2.7 nm in intact platelets. The percentage of granules apposed to the plasma membrane corresponded closely to the percentage of total serotonin that was maximally secreted after stimulation of the permeabilized (38 +/- 4.9%) and the intact platelets (72 +/- 3.6%). Furthermore, the percentage of granules anchored to the membrane, but not of those in other regions of permeabilized cells, decreased markedly when cells were stimulated for 30 s by extracellularly added Ca2+. The decrease in the numbers of granules in the vicinity of the plasma membrane corresponded to approximately 22% of the total number of dense granules that were used for measurements of the distances between the two membranes and corresponded roughly to the overall decrease (15%) in the average number of the granules per cell. Most dense granules were found to be associated with meshwork structures of microfilaments. Upon secretory stimulation, nonfilamentous, amorphous structures found between the plasma membrane and the apposed granules formed a bridge-like structure that connected both membranes without any obvious accompanying changes in the microfilament structures. These results suggest that the dense granules that are susceptible to secretory stimulation are anchored to the plasma membrane before stimulation, and that the formation of the bridge-like structure may participate in the Ca2(+)-regulated exocytosis.



Exocrine granule specific packaging signals are present in the polypeptide moiety of the pancreatic granule membrane protein GP2 and in amylase: implications for protein targeting to secretory granules.  

PubMed Central

The mechanisms for segregation of secretory and membrane proteins incorporated into storage granules from those transported constitutively have been thought to be conserved in diverse cell types, including exocrine and endocrine cells. However, GP2, the major protein of pancreatic zymogen granule membranes, in its native glycosyl phosphatidylinositol (GPI)-linked form, is incorporated into secretory granules when expressed in exocrine pancreatic AR42J cells, but not in the endocrine cells such as pituitary AtT20. To determine whether the protein moiety of GP2 contains the cell-type specific information for packaging into granules, a secretory form of GP2 (GP2-GPI-), with the GPI attachment site deleted, was generated and introduced into AR42J and AtT20 cells. Like native GP2, GP2-GPI- localized to the zymogen-like granules of AR42J cells and underwent regulated secretion. In AtT20 cells expressing GP2-GPI-, however, the protein was secreted by the constitutive pathway. Thus, a granule packaging signal is present in the luminal portion of GP2 that is functional only in the exocrine cells. However, this cell-type dependent sorting process is not limited to GP2 or membrane proteins. Amylase, a major content protein of pancreatic acinar and serous salivary gland granules, was also secreted exclusively by the constitutive pathway when expressed in AtT20 cells. The cell-type specific targeting of GP2 to granules correlated with its behavior in an in vitro aggregation assay where it co-aggregated more effectively with content proteins from pancreatic zymogen granules than with those from pituitary granules.(ABSTRACT TRUNCATED AT 250 WORDS) Images

Colomer, V; Lal, K; Hoops, T C; Rindler, M J



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


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. PMID:23418362

McGirr, Rebecca; Guizzetti, Leonardo; Dhanvantari, Savita



Granule lattice protein 1 (Grl1p), an acidic, calcium-binding protein in Tetrahymena thermophila dense-core secretory granules, influences granule size, shape, content organization, and release but not protein sorting or condensation  

PubMed Central

The electron-dense cores of regulated secretory granules in the ciliate Tetrahymena thermophila are crystal lattices composed of multiple proteins. Granule synthesis involves a series of steps beginning with protein sorting, followed by the condensation and precise geometric assembly of the granule cargo. These steps may to various degrees be determined by the cargo proteins themselves. A prominent group of granule proteins, in ciliates as well as in vertebrate neuronal and endocrine cells, are acidic, heat-stable, and bind calcium. We focused on a protein with these characteristics named granule lattice protein 1 (Grl1p), which represents 16% of total granule contents, and we have now cloned the corresponding gene. Mutants in which the macronuclear copies of GRL1 have been disrupted continue to synthesize dense-core granules but are nonetheless defective in regulated protein secretion. To understand the nature of this defect, we characterized mutant and wild-type granules. In the absence of Grl1p, the sorting of the remaining granule proteins appears normal, and they condense to form a well-defined core. However, the condensed cores do not demonstrate a visible crystalline lattice, and are notably different from wild type in size and shape. The cellular secretion defect arises from failure of the aberrant granule cores to undergo rapid expansion and extrusion after exocytic fusion of the granule and plasma membranes. The results suggest that sorting, condensation, and precise granule assembly are distinct in their requirements for Grl1p.



RNA Granules in Germ Cells  

PubMed Central

“Germ granules” are cytoplasmic, nonmembrane-bound organelles unique to germline. Germ granules share components with the P bodies and stress granules of somatic cells, but also contain proteins and RNAs uniquely required for germ cell development. In this review, we focus on recent advances in our understanding of germ granule assembly, dynamics, and function. One hypothesis is that germ granules operate as hubs for the posttranscriptional control of gene expression, a function at the core of the germ cell differentiation program.

Voronina, Ekaterina; Seydoux, Geraldine; Sassone-Corsi, Paolo; Nagamori, Ippei



Rab3D Is Not Required for Exocrine Exocytosis but for Maintenance of Normally Sized Secretory Granules  

PubMed Central

Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.

Riedel, Dietmar; Antonin, Wolfram; Fernandez-Chacon, Rafael; Alvarez de Toledo, Guillermo; Jo, Tobias; Geppert, Martin; Valentijn, Jack A.; Valentijn, Karin; Jamieson, James D.; Sudhof, Thomas C.; Jahn, Reinhard




PubMed Central

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, one-dimensional or two-dimensional gel electrophoresis, gel staining, in-gel tryptic digestion and protein identification by mass spectrometry. 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 mass-spectrometry. 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 mass spectrometry.

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



The Arf family G protein Arl1 is required for secretory granule biogenesis in Drosophila.  


The small G protein Arf like 1 (Arl1) is found at the Golgi complex, and its GTP-bound form recruits several effectors to the Golgi including GRIP-domain-containing coiled-coil proteins, and the Arf1 exchange factors Big1 and Big2. To investigate the role of Arl1, we have characterised a loss-of-function mutant of the Drosophila Arl1 orthologue. The gene is essential, and examination of clones of cells lacking Arl1 shows that it is required for recruitment of three of the four GRIP domain golgins to the Golgi, with Drosophila GCC185 being less dependent on Arl1. At a functional level, Arl1 is essential for formation of secretory granules in the larval salivary gland. When Arl1 is missing, Golgi are still present but there is a dispersal of adaptor protein 1 (AP-1), a clathrin adaptor that requires Arf1 for its membrane recruitment and which is known to be required for secretory granule biogenesis. Arl1 does not appear to be required for AP-1 recruitment in all tissues, suggesting that it is crucially required to enhance Arf1 activation at the trans-Golgi in particular tissues. PMID:24610947

Torres, Isabel L; Rosa-Ferreira, Cláudia; Munro, Sean



Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena  

PubMed Central

Secretory granules, such as neuronal dense core vesicles, are specialized for storing cargo at high concentration and releasing it via regulated exocytosis in response to extracellular stimuli. Here, we used expression profiling to identify new components of the machinery for sorting proteins into mucocysts, secretory granule-like vesicles in the ciliate Tetrahymena thermophila. We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis. In particular, the delivery of nonaggregated, but not aggregated, cargo proteins requires classical receptors of the sortilin/VPS10 family, which indicates that dual mechanisms are involved in sorting to this secretory compartment. In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation. Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

Briguglio, Joseph S.; Kumar, Santosh



Evidence for tubulin-binding sites on cellular membranes: plasma membranes, mitochondrial membranes, and secretory granule membranes  

Microsoft Academic Search

We describe the interaction of pure brain tubulin with purified membranes specialized in different cell functions, i.e., plasma membranes and mitochondrial membranes from liver and secretory granule membranes from adrenal medulla. We studied the tubulin- binding activity of cellular membranes using a radiolabeled ligand-receptor assay and an antibody retention assay. The tubulin-membrane interaction was time- and temperature- dependent, reversible, specific,




Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.  


The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a ?-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells. PMID:24029466

Fujita-Yoshigaki, Junko; Matsuki-Fukushima, Miwako; Yokoyama, Megumi; Katsumata-Kato, Osamu



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


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. PMID:23382463

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



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

PubMed Central

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 Ca2+-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.

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



A functioning artificial secretory cell  

NASA Astrophysics Data System (ADS)

We present an amperometric study of content release from individual vesicles in an artificial secretory cell designed with the minimal components required to carry out exocytosis. Here, the membranes of the cell and vesicles are substituted for protein-free giant and large unilamellar vesicles respectively. In replacement of the SNARE-complex, the cell model was equipped with an analog composed of complimentary DNA constructs. The DNA constructs hybridize in a zipper-like fashion to bring about docking of the artificial secretory vesicles and following the addition of Ca2+ artificial exocytosis was completed. Exocytotic events recorded from the artificial cell closely approximate exocytosis in live cells. The results together with simulations of vesicular release demonstrate that the molecular flux in this model is attenuated and we suggest that this is the result of restricted diffusion through a semi-stable fusion pore or a partitioning of the signalling molecule out of the fused vesicle membrane.

Simonsson, Lisa; Kurczy, Michael E.; Trouillon, Raphaël; Hook, Fredrik; Cans, Ann-Sofie



Microscopic nodes and ducts inside lymphatics and on the surface of internal organs are rich in granulocytes and secretory granules.  


The blood and lymphatic systems are the two well-established circulatory systems. The existence of a third circulatory system representing acupuncture meridians was claimed in the 1960s. The very existence and function of the system, however, remained uncertain. We have found that microscopic nodes and ducts inside lymphatics, as well as on the surface of internal organs of the rat. The nodes and ducts are covered by a layer of EMP-3-positive spindle-shaped epithelium with, below, a layer of vWF-positive but CD31-negative endothelium. The nodes contain a variety of immune cells, usually enriched with mast cells, eosinophils, neutrophils and histiocytes, as well as chromaffin cells, other granule-containing cells. Secretory granules originating from the mast cells in the nodes appear to pass along ductules, two or more of which make up a duct. Our results reveal a potential circulatory system whose anatomical structure and cellular content differ from the blood and lymph systems, and which may be involved in the transport of secretory granules. PMID:22884518

Kwon, Byoung S; Ha, Chang M; Yu, Sungsook; Lee, Byung-Cheon; Ro, Jae Y; Hwang, Sunhee



The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation  

PubMed Central

After cell fate specification, differentiating cells must amplify the specific subcellular features required for their specialized function. How cells regulate such subcellular scaling is a fundamental unanswered question. Here, we show that the E3 ubiquitin ligase Mindbomb 1 (MIB1) is required for the apical secretory apparatus established by gastric zymogenic cells as they differentiate from their progenitors. When Mib1 was deleted, death-associated protein kinase–1 (DAPK1) was rerouted to the cell base, microtubule-associated protein 1B (MAP1B) was dephosphorylated, and the apical vesicles that normally support mature secretory granules were dispersed. Consequently, secretory granules did not mature. The transcription factor MIST1 bound the first intron of Mib1 and regulated its expression. We further showed that loss of MIB1 and dismantling of the apical secretory apparatus was the earliest quantifiable aberration in zymogenic cells undergoing transition to a precancerous metaplastic state in mouse and human stomach. Our results reveal a mechanistic pathway by which cells can scale up a specific, specialized subcellular compartment to alter function during differentiation and scale it down during disease.

Capoccia, Benjamin J.; Jin, Ramon U.; Kong, Young-Yun; Peek, Richard M.; Fassan, Matteo; Rugge, Massimo; Mills, Jason C.



Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia.  


The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors. PMID:16611634

Gottig, Natalia; Elías, Eliana V; Quiroga, Rodrigo; Nores, María J; Solari, Alberto J; Touz, María C; Luján, Hugo D



The role of secretory granules in radiation-induced dysfunction of rat salivary glands  

SciTech Connect

To investigate the possible role of secretory granules in radiation-induced salivary gland dysfunction, rats were pretreated with isoproterenol (5 mg/kg intraperitoneally) to degranulate salivary gland acini. At maximal depletion, salivary glands were locally irradiated with a single dose of 15 Gy of X rays. Parotid and submandibular/sublingual saliva samples were collected before and 1-10 days after irradiation. The lag phase, flow rate, concentrations of potassium and sodium, and amylase secretion were determined. Sham-treated, isoproterenol-treated and irradiated animals provided reference data. In the parotid gland, but not in the submandibular gland, protection against radiation-induced changes in flow rate and composition of saliva occurred after pretreatment with isoproterenol. Combining morphological data from a previous study with data from the current study, it is suggested that improvement of parotid gland function is attributed predominantly to a proliferative stimulus on acinar cells by isoproterenol and not to its degranulation effect. After pretreatment with isoproterenol, an earlier expression of radiation-induced acinar cell damage leading to death was observed, followed by a faster tissue recovery. Thus the proliferative stimulus on acinar cells may accelerate the unmasking of latent lethal damage, resulting in the earlier replacement of dead cells by new, functionally intact cells. 33 refs., 2 figs.

Peter, B.; Van Waarde, M.A.W.H.; Konings, A.W.T. [Univ. of Groningen (Netherlands); Vissink, A. [Univ. of Groningen (Netherlands)]|[Univ. Hospital, Groningen (Netherlands); `s-Gravenmade, E.J. [Univ. Hospital, Groningen (Netherlands)



Reversible condensation of mast cell secretory products in vitro.  

PubMed Central

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

Fernandez, J M; Villalon, M; Verdugo, P



A Role for Rab7 in the Movement of Secretory Granules in Cytotoxic T Lymphocytes  

PubMed Central

Cytotoxic T lymphocytes (CTL) are potent killers of virally infected and tumorigenic cells. Upon recognition of target cells, CTL undergo polarized secretion of secretory lysosomes at the immunological synapse (IS) that forms between CTL and target. However, the molecular machinery involved in the polarization of secretory lysosomes is still largely uncharacterized. In this paper, we investigated the role of Rab7 in the polarization of secretory lysosomes. We show that silencing of Rab7 by RNA interference reduces the ability of CTL to kill targets. GTP-bound Rab7 and Rab interacting lysosomal protein, RILP, interact and both localize to secretory lysosomes in CTL. Over-expression of RILP recruits dynein to the membranes of secretory lysosomes and triggers their movement toward the centrosome. Together, these results suggest that Rab7 may play a role in secretory lysosome movement toward the centrosome by interacting with RILP to recruit the minus-end motor, dynein.

Daniele, Tiziana; Hackmann, Yvonne; Ritter, Alex T.; Wenham, Matt; Booth, Sarah; Bossi, Giovanna; Schintler, Michael; Auer-Grumbach, Michaela; Griffiths, Gillian M.



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

Microsoft Academic Search

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

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



beta2-Syntrophin Is a Cdk5 Substrate That Restrains the Motility of Insulin Secretory Granules  

Microsoft Academic Search

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic ?-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of ?2-syntrophin anchors granules to actin filaments and that the phosphorylation\\/dephosphorylation of ?2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-?2-syntrophin through

Sandra Schubert; Klaus-Peter Knoch; Joke Ouwendijk; Shabaz Mohammed; Yury Bodrov; Melanie Jäger; Anke Altkrüger; Carolin Wegbrod; Marvin E. Adams; Yong Kim; Stanley C. Froehner; Ole N. Jensen; Yannis Kalaidzidis; Michele Solimena; Kathrin Maedler



The Novel Insulinotropic Mechanism of Pimobendan: Direct Enhancement of the Exocytotic Process of Insulin Secretory Granules by Increased Ca2+ Sensitivity in  Cells  

Microsoft Academic Search

Pimobendan is a new class of inotropic drug that augments Ca21 sensitivity and inhibits phosphodiesterase (PDE) activity in cardio- myocytes. To examine the insulinotropic effect of pimobendan in pan- creatic b-cells, which have an intracellular signaling mechanism sim- ilar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose- induced insulin release in




Presence of proteinase 3 in secretory vesicles: evidence of a novel, highly mobilizable intracellular pool distinct from azurophil granules.  


Proteinase 3 (PR3), which is also called myeloblastin, the target autoantigen for antineutrophil cytoplasmic antibodies (ANCA) in Wegener's granulomatosis, is a serine proteinase stored in azurophil granules of human neutrophils. We have previously shown that, in contrast to elastase or myeloperoxidase, PR3 is also expressed at the plasma membrane of a subset of unactivated neutrophils and that a high proportion of neutrophils expressing membrane PR3 is a risk factor for vasculitis. The present study demonstrates that the association of PR3 with the plasma membrane is not an ionic interaction and seems to be covalent. Fractionation of neutrophils shows that, besides the azurophil granules, PR3 could be detected both in specific granules and in the plasma membrane-enriched fraction containing secretory vesicles, whereas elastase and myeloperoxidase were exclusively located in azurophil granules. Electron microscopy confirms that PR3 is present along with CR1 in secretory vesicles as well as in some specific granules. In neutrophils stimulated with an increasing dose of FMLP, membrane PR3 expression increased with the degranulation of secretory vesicles, followed by specific granules, and culminated after azurophil granules mobilization. The presence of a readily plasma membrane-mobilizable pool of PR3 contained in the secretory vesicles might play a relevant role in the pathophysiological mechanisms of ANCA-associated vasculitis. PMID:10498622

Witko-Sarsat, V; Cramer, E M; Hieblot, C; Guichard, J; Nusbaum, P; Lopez, S; Lesavre, P; Halbwachs-Mecarelli, L



Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins in Drosophila  

PubMed Central

Type II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues.

Burgess, Jason; Del Bel, Lauren M.; Ma, Cheng-I J.; Barylko, Barbara; Polevoy, Gordon; Rollins, Janet; Albanesi, Joseph P.; Kramer, Helmut; Brill, Julie A.



Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site.  


Docking of secretory vesicles at the plasma membrane is a poorly understood prerequisite for exocytosis. Current models propose raft-like clusters containing syntaxin as docking receptor, but direct evidence for this is lacking. Here we provide quantitative measurements of several exocytosis proteins (syntaxin, SNAP25, munc18, munc13 and rab3) at the insulin granule release site and show that docking coincides with rapid de novo formation of syntaxin1/munc18 clusters at the nascent docking site. Formation of such clusters prevents undocking and is not observed during failed docking attempts. Overexpression of syntaxins' N-terminal Habc-domain competitively interferes with both cluster formation and successful docking. SNAP25 and munc13 are recruited to the docking site more than a minute later, consistent with munc13's reported role in granule priming rather than docking. We conclude that secretory vesicles dock by inducing syntaxin1/munc18 clustering in the target membrane, and find no evidence for preformed docking receptors. PMID:24835618

Gandasi, Nikhil R; Barg, Sebastian



?2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules.  


The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic ?-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of ?2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of ?2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-?2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that ?2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and ?2-syntrophin phosphomutants we found that phosphorylation of ?2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis. PMID:20886068

Schubert, Sandra; Knoch, Klaus-Peter; Ouwendijk, Joke; Mohammed, Shabaz; Bodrov, Yury; Jäger, Melanie; Altkrüger, Anke; Wegbrod, Carolin; Adams, Marvin E; Kim, Yong; Froehner, Stanley C; Jensen, Ole N; Kalaidzidis, Yannis; Solimena, Michele



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

PubMed Central

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.

Gonzalez, Claudia; Espinosa, Marisol; Sanchez, Maria Trinidad; Droguett, Karla; Rios, Mariana; Fonseca, Ximena; Villalon, Manuel



Purinergic Modulation of Granule Cells  

Microsoft Academic Search

Extracellular purines exert their action in the nervous system through purinergic neurotransmission and neuromodulatory processes.\\u000a Among brain areas, efforts have been made to investigate the purinergic modulation of the cerebellar cortex. In addition,\\u000a the use of granule cells in culture as a neuronal in vitro model provided important information about the implications of\\u000a purines in mechanisms such as cell survival

Raphaël Courjaret; María Teresa Miras-Portugal; Joachim W. Deitmer


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

PubMed Central

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.

Villanueva, Jose; Torregrosa-Hetland, Cristina J.; Gil, Amparo; Gonzalez-Velez, Virginia; Segura, Javier; Viniegra, Salvador; Gutierrez, Luis M.



A novel framework for segmentation of secretory granules in electron micrographs.  


It is still a standard practice for biologists to manually analyze transmission electron microscopy images. This is not only time consuming but also not reproducible and prone to induce subjective bias. For large-scale studies of insulin granules inside beta cells of the islet of Langerhans, an automated method for analysis is essential. Due to the complex structure of the images, standard microscopy segmentation techniques cannot be applied. We present a new approach to segment and measure transmission electron microscopy images of insulin granule cores and membranes from beta cells of rat islets of Langerhans. The algorithm is separated into two broad components, core segmentation and membrane segmentation. Core segmentation proceeds through three steps: pre-segmentation using a novel level-set active contour, morphological cleaning and a refining segmentation on each granule using a novel dual level-set active contour. Membrane segmentation is achieved in four steps: morphological cleaning, membrane sampling and scaling, vector field convolution for gap filling and membrane verification using a novel convergence filter. We show results from our algorithm alongside popular microscopy segmentation methods; the advantages of our method are demonstrated. Our algorithm is validated by comparing automated results to a manually defined ground truth. When the number of granules detected is compared to the number of granules in the ground truth a precision of 91% and recall of 87% is observed. The average granule areas differ by 13.35% and 6.08% for core and membranes respectively, when compared to the average areas of the ground truth. These results compare favorably to previously published data. PMID:24444668

Nam, David; Mantell, Judith; Bull, David; Verkade, Paul; Achim, Alin



Granule cells in the CA3 area  

PubMed Central

A fundamental property of neuronal networks in Ammon’s horn is that each area is comprised of a single glutamatergic cell population and various types of GABAergic neurons. Here we describe an exception to this rule, in the form of granule cells that reside within the CA3 area and function as glutamatergic non-principal cells with distinct properties. CA3 granule cells in normal, healthy rats, similarly to dentate gyrus granule cells, co-expressed calbindin and the homeobox protein Prox1. However, CA3 granule cells were located outside of the dentate gyrus, often hundreds of microns from the hilar border, in the lucidum and radiatum layers. CA3 granule cells were present in numbers that were comparable to the rarer GABAergic neuronal subtypes, and their somato-dendritic morphology, intrinsic properties and perforant path inputs were similar to those of dentate gyrus granule cells. CA3 granule cell axons displayed giant mossy fiber terminals with filopodial extensions, demonstrating that not all mossy fibers originate from the dentate gyrus. Somatic paired recordings revealed that CA3 granule cells innervated CA3 pyramidal and GABAergic cells similarly to conventional mossy fiber synapses. However, CA3 granule cells were distinct in the specific organization of their GABAergic inputs. They received GABAergic synapses from cholecystokinin expressing mossy fiber-associated cells that did not innervate the dentate granule cell layer, and these synapses demonstrated unusually strong activity-dependent endocannabinoid-mediated inhibition of GABA release. These results indicate that granule cells in the CA3 constitute a glutamatergic, non-principal neuronal subtype that is integrated into the CA3 synaptic network.

Szabadics, Janos; Varga, Csaba; Brunner, Janos; Chen, Kang; Soltesz, Ivan



Temperature-Sensitive Steps in the Transport of Secretory Proteins through the Golgi Complex in Exocrine Pancreatic Cells  

Microsoft Academic Search

The effect of temperature on secretory protein transport was studied by cell fractionation of rat pancreatic lobules, pulse-labeled in vitro with [35S]methionine and chased for 60 min at 16, 20, or 37 degrees C. Chase at 37 degrees C allowed secretory proteins to reach a zymogen granule fraction, whereas chase at 16 or 20 degrees C led to their extensive

Jaakko Saraste; George E. Palade; Marilyn Gist Farquhar



Regulation of Insulin Granule Turnover in Pancreatic ?-Cells by Cleaved ICA512*S?  

PubMed Central

Insulin maintains homeostasis of glucose by promoting its uptake into cells from the blood. Hyperglycemia triggers secretion of insulin from pancreatic ?-cells. This process is mediated by secretory granule exocytosis. However, how ?-cells keep granule stores relatively constant is still unknown. ICA512 is an intrinsic granule membrane protein, whose cytosolic domain binds ?2-syntrophin, an F-actin-associated protein, and is cleaved upon granule exocytosis. The resulting cleaved cytosolic fragment, ICA512-CCF, reaches the nucleus and up-regulates the transcription of granule genes, including insulin and ICA512. Here, we show that ICA512-CCF also dimerizes with intact ICA512 on granules, thereby displacing it from ?2-syntrophin. This leads to increased granule mobility and insulin release. Based on these findings, we propose a model whereby the generation of ICA512-CCF first amplifies insulin secretion. The ensuing reduction of granule stores would then increase the probability of newly generated ICA512-CCF to reach the nucleus and enhance granule biogenesis, thus allowing ?-cells to constantly adjust production of granules to their storage size and consumption. Pharmacological modulation of these feedback loops may alleviate deficient insulin release in diabetes.

Trajkovski, Mirko; Mziaut, Hassan; Schubert, Sandra; Kalaidzidis, Yannis; Altkruger, Anke; Solimena, Michele



MyRIP interaction with MyoVa on secretory granules is controlled by the cAMP-PKA pathway  

PubMed Central

Myosin- and Rab-interacting protein (MyRIP), which belongs to the protein kinase A (PKA)–anchoring family, is implicated in hormone secretion. However, its mechanism of action is not fully elucidated. Here we investigate the role of MyRIP in myosin Va (MyoVa)-dependent secretory granule (SG) transport and secretion in pancreatic beta cells. These cells solely express the brain isoform of MyoVa (BR-MyoVa), which is a key motor protein in SG transport. In vitro pull-down, coimmunoprecipitation, and colocalization studies revealed that MyRIP does not interact with BR-MyoVa in glucose-stimulated pancreatic beta cells, suggesting that, contrary to previous notions, MyRIP does not link this motor protein to SGs. Glucose-stimulated insulin secretion is augmented by incretin hormones, which increase cAMP levels and leads to MyRIP phosphorylation, its interaction with BR-MyoVa, and phosphorylation of the BR-MyoVa receptor rabphilin-3A (Rph-3A). Rph-3A phosphorylation on Ser-234 was inhibited by small interfering RNA knockdown of MyRIP, which also reduced cAMP-mediated hormone secretion. Demonstrating the importance of this phosphorylation, nonphosphorylatable and phosphomimic Rph-3A mutants significantly altered hormone release when PKA was activated. These data suggest that MyRIP only forms a functional protein complex with BR-MyoVa on SGs when cAMP is elevated and under this condition facilitates phosphorylation of SG-associated proteins, which in turn can enhance secretion.

Brozzi, Flora; Lajus, Sophie; Diraison, Frederique; Rajatileka, Shavanthi; Hayward, Katy; Regazzi, Romano; Molnar, Elek; Varadi, Aniko



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

PubMed Central

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.

Bhat, Purnima



Localization of human intestinal defensin 5 in Paneth cell granules.  

PubMed Central

Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells but recently also found to be produced by epithelial cells. We biosynthesized recombinant human intestinal defensin 5 (rHD-5) using the baculovirus-insect cell expression system. Since insect cells process defensin incompletely and secrete the precursor proHD-5, we substituted a methionine for an alanine at a likely processing site to allow selective chemical cleavage with cyanogen bromide, and rHD-5 was used to elicit polyclonal antibodies. By the immunoperoxidase-staining technique, the antibodies selectively stained Paneth cells of the normal adult small intestine. Immunogold electron microscopy further localized HD-5 to the Paneth cell secretory granules. Since some defensins exert activity cytotoxic to mammalian cells, we assayed the effect of rHD-5 on the human intestinal cell lines Caco2 and Int407. proHD-5 did not exert cytotoxic activity, and rHD-5 showed only minimal activity against Int407 and was inert against Caco2. Since Paneth cells release their granules adjacent to the mitotic cells of the intestinal crypts, HD could protect this cell population against invasion and parasitization by microbes.

Porter, E M; Liu, L; Oren, A; Anton, P A; Ganz, T



Secretory meningioma: immunohistochemical findings and evaluation of mast cell infiltration.  


Secretory meningiomas constitute a relatively rare subtype of meningiomas, accounting for only 1.1% at our institution, with a 6:1 predominance of female patients. This study aimed to obtain more information about the immunohistochemical characteristics of this histological entity, and to analyse the effects of histological factors such as the presence of mast cells on the radiological evidence of surrounding tumour oedema that frequently occurred in this subtype of meningioma. Fourteen cases of secretory meningioma were examined. Relevant clinical information was obtained from the patient files. Peritumoural oedema was determined either by CT or MRI scans and graded as small, moderate and severe. In order to perform the quantitative evaluation of mast cells in secretory meningiomas in a comparison with other meningiomas, 14 non-secretory meningiomas were randomly selected and used as a control group. The immunohistochemical staining of carcinoembryonic antigen was positive within the secretory droplets and the cells surrounding them in all cases. Ki 67 (MIB 1) proliferative index mean values were 2.4%, indicating low expression in all secretory meningiomas. Moreover, from our statistical analysis, there is no clear-cut pattern of various types of cytokeratins emerging in secretory meningiomas. The secretory meningiomas were characterized by a significantly increased number of mast cells as compared with non-secretory meningiomas of different grades. As the present clinical findings and laboratory results could not confirm a correlation between mast cell density and radiological evidence of oedema, further studies of mediators are warranted. PMID:16010579

Tirakotai, Wuttipong; Mennel, Hans-Dieter; Celik, Ilhan; Hellwig, Dieter; Bertalanffy, Helmut; Riegel, Thomas



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


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-?-cyclodextrin (M?CD). 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 M?CD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex M?CD-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. M?CD 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 M?CD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused M?CD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of M?CD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets. PMID:21787821

Borisova, Tatiana; Kasatkina, Ludmila; Ostapchenko, Ludmila



Ectopic granule cells of the rat dentate gyrus.  


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

Scharfman, Helen; Goodman, Jeffrey; McCloskey, Daniel



Kiss-and-run exocytosis and fusion pores of secretory vesicles in human beta-cells.  


Exocytosis of secretory vesicles results in the release of insulin from pancreatic beta-cells, although little is known about this process in humans. We examined the exocytosis of single secretory vesicles and their associated fusion pores in human beta-cells by cell-attached capacitance and conductance measurement. Unitary capacitance steps were observed, consistent with the exocytosis of single secretory vesicles. These were often coincident with increases in patch conductance representing the presence of a stable fusion pore. In some events, the fusion pore closed, mediating kiss-and-run, which contributed 20% of the exocytotic events. The cAMP-raising agent forskolin (5 microM) doubled the relative contribution of kiss-and-run. This effect was confirmed visually in MIN6 cells expressing a fluorescent granule probe. Thus, we demonstrate the unitary capacitance steps and fusion pores during single vesicle exocytosis in human beta-cells. Furthermore, these secretory vesicles can undergo rapid recycling by kiss-and-run, and this process is up-regulated by cAMP. PMID:18795319

Hanna, Salma T; Pigeau, Gary M; Galvanovskis, Juris; Clark, Anne; Rorsman, Patrik; MacDonald, Patrick E



Hilar mossy cell circuitry controlling dentate granule cell excitability  

PubMed Central

Glutamatergic hilar mossy cells of the dentate gyrus can either excite or inhibit distant granule cells, depending on whether their direct excitatory projections to granule cells or their projections to local inhibitory interneurons dominate. However, it remains controversial whether the net effect of mossy cell loss is granule cell excitation or inhibition. Clarifying this controversy has particular relevance to temporal lobe epilepsy, which is marked by dentate granule cell hyperexcitability and extensive loss of dentate hilar mossy cells. Two diametrically opposed hypotheses have been advanced to explain this granule cell hyperexcitability—the “dormant basket cell” and the “irritable mossy cell” hypotheses. The “dormant basket cell” hypothesis proposes that mossy cells normally exert a net inhibitory effect on granule cells and therefore their loss causes dentate granule cell hyperexcitability. The “irritable mossy cell” hypothesis takes the opposite view that mossy cells normally excite granule cells and that the surviving mossy cells in epilepsy increase their activity, causing granule cell excitation. The inability to eliminate mossy cells selectively has made it difficult to test these two opposing hypotheses. To this end, we developed a transgenic toxin-mediated, mossy cell-ablation mouse line. Using these mutants, we demonstrated that the extensive elimination of hilar mossy cells causes granule cell hyperexcitability, although the mossy cell loss observed appeared insufficient to cause clinical epilepsy. In this review, we focus on this topic and also suggest that different interneuron populations may mediate mossy cell-induced translamellar lateral inhibition and intralamellar recurrent inhibition. These unique local circuits in the dentate hilar region may be centrally involved in the functional organization of the dentate gyrus.

Jinde, Seiichiro; Zsiros, Veronika; Nakazawa, Kazu



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


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 Ca(2+) of 69 ± 15 ?M and a pH of 5.8. Application of extracellular ATP to activate P2Y receptors resulted in a slow monotonic decrease in SG Ca(2+) 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 Ca(2+) release is completely antagonized by a SOCE antagonist, by switching to Ca(2+)-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 Ca(2+) by ?75% and completely abolished the ATP-mediated release of Ca(2+) from SGs. Overexpression of a dominant-negative CAD construct(CAD-A376K) induced no significant changes in SG Ca(2+). Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels and that during SG Ca(2+) 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

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



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

PubMed Central

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.

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



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


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. PMID:8297046

Kawamura, H; Kimura, M; Ichihara, I



Vaccine adjuvants: Tailor-made mast-cell granules  

NASA Astrophysics Data System (ADS)

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.

Gunzer, Matthias



Corrosion of Intracellular Granules and Cell Death,  

National Technical Information Service (NTIS)

The snail, Helix aspersa, has large numbers of calcium cells in its hepatopancreas which contain membrane-bound intracellular granules of Ca Mg P2 O7. These inorganic deposits are the sites of accumulation of a wide variety of cations and act as a detoxif...

M. G. Taylor K. Simkiss G. N. Greaves J. Harries



Cerebellar granule cell Cre recombinase expression.  


The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells. PMID:12820171

Aller, M I; Jones, A; Merlo, D; Paterlini, M; Meyer, A H; Amtmann, U; Brickley, S; Jolin, H E; McKenzie, A N J; Monyer, H; Farrant, M; Wisden, W



Human MCTC type of mast cell granule: the uncommon occurrence of discrete scrolls associated with focal absence of chymase.  


Two types of mast cells were previously defined based on neutral protease composition and ultrastructurally distinguished by granule morphology. The MCT cell contains tryptase with little, if any, chymase and was noted to have varying numbers of irregularly-shaped granules with discrete scrolls or particulate or beaded material. The MCTC cell contains both tryptase and chymase and was noted to have more regularly-shaped electron-dense granules with characteristic grating or lattice substructures. This study reports the use of electron microscopy and immunogold staining with antibodies against tryptase and chymase to demonstrate in mature unstimulated MCTC cells in situ, the focal occurrence of discrete or complete scrolls in peripheral regions of certain granules where chymase is deficient. these scrolls often appeared to be protruding from the granule. Granules containing discrete scrolls were observed in 10 of 340 mature MCTC cells, accounting for less than 1% of MCTC granules. Other granules in such cells as well as other regions of the granule under consideration, showed strong staining for both tryptase and chymase. These results strengthen the association of morphology with protease composition in human mast cell secretory granules, but weaken the use of morphology alone to identify the MCTC and MCT types of human mast cells. Whether the uncommon occurrence of focal absence of chymase in MCTC cells arises by chance or as a result of factors relating to mast cell development, interconversion, activation, or regranulation will require further clarification. In conclusion, the appearance of grating or lattice structures in mast cells indicates the presence of chymase and tryptase, characteristic of the MCTC phenotype, whereas multiple discrete scrolls in irregularly shaped granules suggests the MCT phenotype. PMID:2232709

Craig, S S; Schwartz, L B



Primary cilia associated with striated rootlets in granulated and folliculo-stellate cells of the avian adenohypophysis  

Microsoft Academic Search

During observation of the ultrastructure of adenohypophyses of normal and experimentally-manipulated quails, primary cilia were found in secretory cells as well as in non-granulated, folliculo-stellate cells of both cephalic and caudal lobes of the gland. These solitary cilia shared morphological characteristics with those observed in other cell types and species, i.e. they arose from a basal body, which basically had

F. Harrisson



The process of granule exocytosis in non-stimulated atrial granular cells of the snail, Achatina achatina: an ultrastructural, histochemical and immunocytochemical study.  


Abundant secretory granular cells (GCs) in the Giant African land snail atrium harbor a range of bioactive substances and undergo rapid total degranulation in response to stimulation of the cardiac nerve or stressful influences. Here we have analyzed exocytotic events in the non-stimulated GCs. It was shown that the GCs contain three major distinct types of granules that differ histochemically, immunocytochemically and ultrastructurally, each performing specific functions. The type I granules characteristically filled with electron-lucent homogeneous materials exhibit intense immunoreactivity for bioactive proteins and therefore are considered to be storage granules. Histochemistry using vital staining with Acridine Orange and Gomori acid phosphatase technique has revealed lysosomal-related nature of the electron-dense type II granules. Digestion remnants appearing as fine filamentous materials fill the type III granules. Only the type III granules fuse together and with the plasma membrane form degranulation channels and surface pores, through which the debris is removed from the cell. The finding of granules exhibiting intermediate ultrastructural, histochemical and immunocytochemical features suggests that the major granule types represent most stable states along a granule empting continuum. Thus, under physiological conditions, the GCs continuously produce secretory proteins and so maintain readiness for stress-response, but use protein degradation machinery to prevent massive release of these bioactive substances into hemolymph. PMID:23706530

Bystrova, Olga A; Shabelnikov, Sergej V; Martynova, Marina G



Oxytocin stimulates secretory processes in lactating rabbit mammary epithelial cells  

PubMed Central

Oxytocin plays a major role in lactation mainly by its action on milk ejection via the contraction of myoepithelial cells. The effect of oxytocin on milk production and the presence of oxytocin receptors on different epithelial cells suggest that this hormone may play a role in mammary epithelial cells. To determine precisely the various roles of oxytocin, we studied localization of oxytocin receptors in lactating rabbit and rat mammary tissue and the influence of oxytocin on secretory processes in lactating rabbit mammary epithelial cells. Immunolocalization of oxytocin receptors on mammary epithelial cells by immunofluorescence and in mammary tissue by immunogold in addition to in situ hybridization showed that lactating rat and rabbit mammary epithelial cells expressed oxytocin receptors. Moreover, oxytocin bound specifically to epithelial cells. To determine whether oxytocin had an effect on lactating rabbit mammary epithelial cells, isolated mammary fragments were incubated in the presence or absence of 10?6 i.u. ml?1 of oxytocin. After 1 min of incubation with oxytocin, the morphology of epithelial cells and the localization of caseins and proteins associated with the secretory traffic suggested a striking acceleration of the transport leading to exocytosis, whereas the contraction of myoepithelial cells was only detectable after 7 min. Addition of 10?8 g ml?1 of atosiban before the addition of oxytocin prevented the oxytocin effect on secretory processes and on myoepithelial cell contraction. Addition of 10?6 i.u. ml?1 of vasopressin to the incubation medium did not mimic the stimulating effect of oxytocin on secretory traffic. These results show that lactating rabbit and rat mammary epithelial cells express oxytocin receptors and that oxytocin binds to these receptors. They strongly suggest that oxytocin has a dual effect on lactating mammary tissue: an acceleration of the intracellular transfer of caseins in mammary epithelial cells followed by the contraction of myoepithelial cells.

Lollivier, Vanessa; Marnet, Pierre-Guy; Delpal, Serge; Rainteau, Dominique; Achard, Caroline; Rabot, Aline; Ollivier-Bousquet, Michele



Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells  

Microsoft Academic Search

Several types of cells store proteins in secretory vesicles from which they are released by an appropriate stimulus. It might be expected that the secretory vesicles in different cell types use similar molecular machinery. Here we describe a transmembrane glycoprotein (Mr ~100,000) that is present in secretory vesicles in all neurons and endocrine cells studied, in species from elasmobranch fish




Modulating zymogen granule formation in pancreatic AR42J cells.  


Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin™ 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed. PMID:22683857

Rinn, Cornelia; Aroso, Miguel; Prüssing, Judith; Islinger, Markus; Schrader, Michael



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


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. PMID:24359801

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



Gastric neuroendocrine cells and secretory products.  

PubMed Central

The ECL cell is the most common cell type in the oxyntic mucosa of the stomach. It is producing a number of peptides and amines where histamine and chromogranin A seems to be the most important and abundant products. Recent data indicate a direct correlation between ECL-cell mass and circulating chromogranin A levels. Chromogranin A and its splice products might serve as growth promoting agents in ECL-cell hyperplasia or gastric carcinoids.

Oberg, K.



Isolation and characterization of multiple forms of bovine placental lactogen from secretory granules of the fetal cotyledon.  


Previous work has shown that, unlike other species, placental lactogen (PL) in the bovine (bPL) has a mol wt of approximately 32,000 and exists in several different forms with different isoelectric points. This study was carried out to develop a more rapid purification scheme, whereby the yield of bPL obtained was increased while at the same time the possibility of artifacts from a prolonged purification protocol was decreased. A procedure was developed in which a fraction enriched in bPL-containing granules was obtained after gentle disruption of the binucleate cells of the fetal cotyledon. The fetal portion of the placentomes from midpregnant cows was minced with scissors and vigorously stirred in order to remove and disrupt binucleate cells within the fetal villi. The supernatant from this step was fractionated by differential centrifugation followed by a four-step discontinuous Percoll gradient of 1.03-1.08 g/ml. A granule-enriched fraction was isolated from the 1.04 g/ml zone from which membrane-enclosed protein was released by freezing and thawing. Membranes and insoluble proteins were sedimented by high speed centrifugation to yield an extract which contained approximately 20% of the hormone initially in the tissue. Two subsequent chromatographic steps, gel filtration on Sephadex G-75 and high performance reversed phase chromatography with a C-4 column, resulted in a preparation of greater than 98% homogeneity. Two-dimensional gel electrophoresis of purified bPL revealed at least nine protein spots in the 31,000-33,000 mol wt range with isoelectric points ranging from 4.85-6.3. All forms exhibited parallel dilution curves in a RIA for bPL. It would appear, therefore, that multiple forms of bPL exist and that they are not artifacts of the prolonged purification protocol previously used. PMID:3732169

Byatt, J C; Shimomura, K; Duello, T M; Bremel, R D



How helminths use excretory secretory fractions to modulate dendritic cells  

PubMed Central

It is well known that helminth parasites have immunomodulatory effects on their hosts. They characteristically cause a skew toward TH2 immunity, stimulate Treg cells while simultaneously inhibiting TH1 and TH17 responses. Additionally, they induce eosinophilia and extensive IgE release. The exact mechanism of how the worms achieve this effect have yet to be fully elucidated; however, parasite-derived secretions and their interaction with antigen presenting cells have been centrally implicated. Herein, we will review the effects of helminth excretory-secretory fractions on dendritic cells and discuss how this interaction is crucial in shaping the host response.

White, Rhiannon R.; Artavanis-Tsakonas, Katerina



In vitro antioxidant activity of the prostatic secretory granules in rabbit semen after exposure to organic peroxides  

Microsoft Academic Search

BACKGROUND: The prostate gland of rabbits produces numerous granules, which are specifically implicated in the inhibition of sperm capacitation during the first hours after mating. These granules are rich in vitamin E, but their role in the antioxidant protection of rabbit sperm has not been studied. AIM OF STUDY: The objectives of this study were to investigate whether the prostatic

Evangelia Mourvaki; Raffaella Cardinali; Alessandro Dal Bosco; Cesare Castellini



Hilar mossy cells share developmental influences with dentate granule neurons.  


Mossy cells are the major class of excitatory neurons in the dentate hilus. Although mossy cells are involved in a range of physiological and pathological conditions, very little is known about their ontogeny. To gain insight into this issue, we first determined the developmental stage at which mossy cells can be reliably identified with the molecular markers calretinin and GluR2/3 and found that hilar mossy cells were first identifiable around the end of the 1st postnatal week. Birthdating studies combined with staining for these markers revealed that the appearance of mossy cells coincided with the first wave of dentate granule cell production during mid-gestation. Since mossy cells are born as the first granule cells are produced and it is believed that mossy cells originate from the neuroepithelium adjacent to the dentate progenitor zone, we examined to what extent the development of mossy cells is controlled by the same molecular pathways as that of granule cells. To do this, we analyzed the production of mossy cells in Lef1 and NeuroD mutant animals, in which granule cell production is disrupted during precursor proliferation or neuronal differentiation, respectively. The production of mossy cells was almost entirely lost in both mutants. Collectively, these data suggests that hilar mossy cells, unlike CA subfield pyramidal cells, are influenced by many of the same developmental cues as dentate granule cells. PMID:17960053

Li, Guangnan; Berger, Omri; Han, Szu-Min; Paredes, Mercedes; Wu, Ni-Chi; Pleasure, Samuel J




EPA Science Inventory

Proliferative lesions comprised of eosinophilic granule cells (EGCs) extended throughout the gastrointestinal tract of several mature, spawning coho salmon Oncorhynchus kisutch (Walbaum). istological examination of the tumour showed extensive proliferation and infiltration of EGC...


Human NK cell lytic granules and regulation of their exocytosis  

PubMed Central

Natural killer (NK) cells form a subset of lymphocytes that play a key role in immuno-surveillance and host defense against cancer and viral infections. They recognize stressed cells through a variety of germline-encoded activating cell surface receptors and utilize their cytotoxic ability to eliminate abnormal cells. Killing of target cells is a complex, multi-stage process that concludes in the directed secretion of lytic granules, containing perforin and granzymes, at the immunological synapse. Upon delivery to a target cell, perforin mediates generation of pores in membranes of target cells, allowing granzymes to access target cell cytoplasm and induce apoptosis. Therefore, lytic granules of NK cells are indispensable for normal NK cell cytolytic function. Indeed, defects in lytic granule secretion lead or are related to serious and often fatal diseases, such as familial hemophagocytic lymphohistiocytosis (FHL) type 2–5 or Griscelli syndrome type 2. A number of reports highlight the role of several proteins involved in lytic granule release and NK cell-mediated killing of tumor cells. This review focuses on lytic granules of human NK cells and the advancements in understanding the mechanisms controlling their exocytosis.

Krzewski, Konrad; Coligan, John E.



Strategies to investigate gene expression and function in granule cells  

Microsoft Academic Search

Studying gene expression in granule cells is a major route to understanding the factors required for many key cellular processes\\u000a such as specification, proliferation, migration, differentiation, apoptosis, tumour formation and neurodegeneration. A greater\\u000a understanding of these processes will not only provide insight into cerebellum development, but also diseases of the cerebellum.\\u000a Granule cells can be readily grown in culture and

Rebecca M. Savill; Paul J. Scotting; Beth Coyle



Expression of prostasome-like granules by the prostate cancer cell lines PC3, Du145 and LnCaP grown in monolayer.  


Prostasomes are a granular type of secretory product in the human prostate gland cells. It is not known, whether in vitro grown cells derived from human prostate cancers also express prostate secretory components containing granules with properties similar to the prostasomes. Therefore, we carried out the present investigation and found that cytospins of in vitro grown PC3, DU145 and LNCaP cells generally expressed a granular secretion. DU145 demonstrated the highest ratio of cells with granules (about 90%), while cytospins of PC3 and LNCaP contained less stained cells (50-70%). Purified granules from PC3 cells were immunoreactive with a monoclonal antibody (mAb78) originally raised against human seminal prostasomes. The PC3 granules also shared the property with human seminal prostasomes having an elevated UV260/UV280 absorbance ratio. On the other hand we found a low aminopeptidase activity in PC3 granules contrary to that of human prostasomes. Prostasomes may form a heterogeneous group with different properties due to the source from which they are isolated and perhaps it is justified to recognize them as different members of a prostasome family. PMID:10680953

Nilsson, B O; Lennartsson, L; Carlsson, L; Nilsson, S; Ronquist, G



Inhibition of Cerebellar Granule Cell Turning by Alcohol  

PubMed Central

Ectopic neurons are often found in the brains of fetal alcohol spectrum disorders (FASD) and fetal alcohol syndrome (FAS) patients, suggesting that alcohol exposure impairs neuronal cell migration. Although it has been reported that alcohol decreases the speed of neuronal cell migration, little is known about whether alcohol also affects the turning of neurons. Here we show that ethanol exposure inhibits the turning of cerebellar granule cells in vivo and in vitro. First, in vivo studies using P10 mice demonstrated that a single i.p. injection of ethanol not only reduces the number of turning granule cells but also alters the mode of turning at the EGL-ML border of the cerebellum. Second, in vitro analysis using microexplant cultures of P0-P3 mouse cerebella revealed that ethanol directly reduces the frequency of spontaneous granule cell turning in a dose-dependent manner. Third, the action of ethanol on the frequency of granule cell turning was significantly ameliorated by stimulating Ca2+ and cGMP signaling or by inhibiting cAMP signaling. Taken together, these results indicate that ethanol affects the frequency and mode of cerebellar granule cell turning through alteration of the Ca2+ and cyclic nucleotide signaling pathways, suggesting that the abnormal allocation of neurons found in the brains of FASD and FSA patients results, at least in part, from impaired turning of immature neurons by alcohol.

Kumada, Tatsuro; Komuro, Yutaro; Li, Ying; Hu, Taofang; Wang, Zhe; Littner, Yoav; Komuro, Hitoshi



Cholesterol accumulation increases insulin granule size and impairs membrane trafficking  

PubMed Central

The formation of mature secretory granules is essential for proper storage and regulated release of hormones and neuropeptides. In pancreatic ?-cells, cholesterol accumulation causes defects in insulin secretion and may participate in the pathogenesis of type 2 diabetes. Using a novel cholesterol analog, we show for the first time that insulin granules are the major sites of intracellular cholesterol accumulation in live ?-cells. This is distinct from other, non-secretory cell types, in which cholesterol is concentrated in the recycling endosomes and the trans-Golgi network. Excess cholesterol was delivered specifically to insulin granules, which caused granule enlargement and retention of syntaxin 6 and VAMP4 in granule membranes, with concurrent depletion of these proteins from the trans-Golgi network. Clathrin also accumulated in the granules of cholesterol-overloaded cells, consistent with a possible defect in the last stage of granule maturation, during which clathrin-coated vesicles bud from the immature granules. Excess cholesterol also reduced the docking and fusion of insulin granules at the plasma membrane. Together, the data support a model in which cholesterol accumulation in insulin secretory granules impairs the ability of these vesicles to respond to stimuli, and thus reduces insulin secretion.

Bogan, Jonathan S.; Xu, Yingke; Hao, Mingming



Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells  

SciTech Connect

Permeabilized cells treated with the adenosine triphosphate analog, (/sup 35/S)adenosine-5'-0-3(3-thiotriphosphate) ((..gamma..-/sup 35/S)ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated /sup 35/S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of /sup 35/S from the cells over a 20 min period. Treatment of permeabilized cells with ATP..gamma..S inhibited secretion and /sup 35/S incorporation into the cells. Pretreatment with ATP..gamma..S resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate /sup 35/S from (..gamma..-/sup 35/S)ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells. 15 references, 1 figure, 3 tables.

Brooks, J.C.; Brooks, M.



The Chloride Cell: Definitive Identification as the Salt-Secretory Cell in Teleosts  

Microsoft Academic Search

Chloride-secreting isolated opercular membranes from the seawater-adapted teleost Sarotherodon mossambicus contain the several cell types also seen in the branchial epithelium. The vibrating probe technique has been used to localize conductance and chloride current specifically to the so-called chloride cells, thereby establishing these cells definitively as the extrarenal salt-secretory cells.

J. Kevin Foskett; Carl Scheffey



Sparse incomplete representations: a potential role of olfactory granule cells  

PubMed Central

Mitral/tufted cells of the olfactory bulb receive odorant information from receptor neurons and transmit this information to the cortex. Studies in awake behaving animals have found that sustained responses of mitral cells to odorants are rare, suggesting sparse combinatorial representation of the odorants. Careful alignment of mitral cell firing with the phase of the respiration cycle revealed brief transient activity in the larger population of mitral cells, which respond to odorants during a small fraction of the respiration cycle. Responses of these cells are therefore temporally sparse. Here, we propose a mathematical model for the olfactory bulb network that can reproduce both combinatorially and temporally sparse mitral cell codes. We argue that sparse codes emerge as a result of the balance between mitral cells' excitatory inputs and inhibition provided by the granule cells. Our model suggests functional significance for the dendrodendritic synapses mediating interactions between mitral and granule cells.

Koulakov, Alexei A.; Rinberg, Dmitry



Effect of methacholine on ionic permeability of basal membrane of the eccrine secretory cell  

Microsoft Academic Search

In an attempt to clarify the possible change in ionic permeability of the basal membrane of secretory cells during methacholine (MCH) stimulation, the effect of ionic substitution on membrane potential (PD) was studied in the isolated monkey palm eccrine secretory tubule. The mean PD (Vcb) was -73 mV. Stimulation with local iontophoresis of MCH caused a biphasic change in Vcb,

Kenzo Sato



Nordihydroguaiaretic acid blocks secretory and endocytic pathways in human dendritic cells  

Microsoft Academic Search

Nordihydroguaiaretic acid (NDGA), an antioxidant and inhibitor of the lipoxygenase arm of the arachidonic acid metabolism, was recently demonstrated to inhibit transport of secretory pro- teins to the Golgi complex. We have investigated the effects of NDGA on the secretory and endocytic activity of cultured human blood dendritic cells (DC). Treatment with NDGA strongly diminished cytokine secretion by DC. Moreover,

Reinhold Ramoner; Claudia Rieser; Georg Bartsch; Martin Thurnher



A 20-nm Step toward the Cell Membrane Preceding Exocytosis May Correspond to Docking of Tethered Granules  

PubMed Central

In endocrine cells, plasma membrane (PM)-bound secretory granules must undergo a number of maturation stages (i.e., priming) to become fusion-competent. Despite identification of several molecules involved in binding granules to the PM and priming them, the exact nature of events occurring at the PM still largely remains a mystery. In stimulated BON cells, we used evanescent wave microscopy to study trajectories of granules shortly before their exocytoses, which provided a physical description of vesicle-PM interactions at an unprecedented level of detail, and directly lead to an original mechanistic model. In these cells, tethered (T), nonfusogenic, vesicles are prevented from converting to fusogenic, docked (D) ones in resting conditions. Upon elevation of calcium, T-vesicles perform a 21-nm step toward the PM to become D, and fuse ?3 s thereafter. Our ability to directly visualize different modes of PM-attachment paves the way for clarifying the exact role of various molecules implicated in attachment and priming of granules in future studies.

Karatekin, Erdem; Tran, Viet Samuel; Huet, Sebastien; Fanget, Isabelle; Cribier, Sophie; Henry, Jean-Pierre



Syncollin inhibits regulated corticotropin secretion from AtT-20 cells through a reduction in the secretory vesicle population.  

PubMed Central

Syncollin is a 13 kDa protein that is highly expressed in the exocrine pancreas. Syncollin normally exists as a doughnut-shaped homo-oligomer (quite probably a hexamer) in close association with the luminal surface of the zymogen granule membrane. In the present study, we examine the effect of expression of syncollin in AtT-20 neuroendocrine cells, which do not normally express this protein. Efficient expression was achieved by infection of the cells with adenoviral constructs encoding either untagged or GFP (green fluorescent protein)-tagged syncollin. Both forms of the protein were sorted into corticotropin (ACTH)-positive secretory vesicles present mainly at the tips of cell processes. Neither form affected basal corticotropin secretion or the constitutive secretion of exogenously expressed secreted alkaline phosphatase. In contrast, regulated secretion of corticotropin was inhibited (by 49%) by untagged but not by GFP-tagged syncollin. In parallel, untagged syncollin caused a 46% reduction in the number of secretory vesicles present at the tips of the cell processes. Syncollin-GFP was without effect. We could also show that native syncollin purified from rat pancreas was capable of permeabilizing erythrocytes. We suggest that syncollin may induce uncontrolled permeabilization of corticotropin-containing vesicles and subsequently destabilize them. Both forms of syncollin were tightly membrane-associated and appeared to exist as homooligomers. Hence, the lack of effect of syncollin-GFP on regulated exocytosis suggests that the GFP tag interferes in a subtler manner with the properties of the assembled protein.

Wasle, Barbara; Hays, Lori B; Rhodes, Christopher J; Edwardson, J Michael



Identification of the polyhydroxybutyrate granules in mammalian cultured cells.  


Poly-3-hydroxybutyrate (PHB) is a biological polyester present in bacteria and eukaryotic cells. Long-chain (or storage) sPHB (up to 100,000 residues) is typically present in PHB-accumulating bacteria and localized in specialized granules known as carbonosomes. In these organisms, sPHB plays a major role as carbon and energy storage. On the other hand, short-chain (or complexed) cPHB (10-100 residues) is present in eukaryotic organisms, including mammals as well as in many bacteria. Previous studies indicated that cPHB is localized in various subcellular compartments of the eukaryotic organisms. Here, we used fluorescent microscopy to directly investigate the localization of PHB in mammalian cells. PHB was visualized in cultured U87 cells using fluorescent probe BODIPY 493/503. Specificity of PHB staining was confirmed by markedly decreased fluorescence of samples treated with PHB-specific depolymerase (PhaZ7). We found that PHB is associated with granules, and that these PHB-enriched granules do not co-localized with mitochondria, lysosomes, or endoplasmic reticulum. These results suggest that, in mammalian cells, PHB can accumulate in the cytoplasm in granules similar to 'energy storage' carbonosomes found in PHB-accumulating bacteria. PMID:23161637

Elustondo, Pia; Zakharian, Eleonora; Pavlov, Evgeny



Mucin Granule Intraluminal Organization  

PubMed Central

Mucus secretions have played a central role in the evolution of multicellular organisms, enabling adaptation to widely differing environments. In vertebrates, mucus covers and protects the epithelial cells in the respiratory, gastrointestinal, urogenital, visual, and auditory systems, amphibian's epidermis, and the gills in fishes. Deregulation of mucus production and/or composition has important consequences for human health. For example, mucus obstruction of small airways is observed in chronic airway diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis. The major protein component in the mucus is a family of large, disulfide-bonded glycoproteins known as gel-forming mucins. These proteins are accumulated in large, regulated secretory granules (the mucin granules) that occupy most of the apical cytoplasm of specialized cells known as mucous/goblet cells. Since mucin oligomers have contour dimensions larger than the mucin granule average diameter, the question arises how these highly hydrophilic macromolecules are organized within these organelles. I review here the intraluminal organization of the mucin granule in view of our knowledge on the structure, biosynthesis, and biophysical properties of gel-forming mucins, and novel imaging studies in living mucous/goblet cells. The emerging concept is that the mucin granule lumen comprises a partially condensed matrix meshwork embedded in a fluid phase where proteins slowly diffuse.

Perez-Vilar, Juan



Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of reserpine treated guinea pigs  

Microsoft Academic Search

The individual granules of enterochromaffin cells of normal and reserpine treated guinea pigs have been studied by staining slides of the duodenum first by an argentaffin method and subsequently by an argyrophile method. Some argentaffin cells can be shown to contain not only argentaffin granules, but also granules that are purely argyrophile. The relative number of such argentaffin cells is

Inderbir Singh



Mapping Organelle Motion Reveals a Vesicular Conveyor Belt Spatially Replenishing Secretory Vesicles in Stimulated Chromaffin Cells  

PubMed Central

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.

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



Segmental identity and cerebellar granule cell induction in rhombomere 1  

PubMed Central

Background Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment? Results We show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2. Conclusions Different overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip precursor pool. By contrast, a subset of coextensive nuclei is refractory to ectopic Hoxa2 and is presumably induced solely by isthmic organiser activity. Thus, graded (isthmic) and segmental mechanisms may operate exclusively of one another in the specification of different neuronal populations within rhombomere 1. The early designation of an Otx2-negative, Hoxa2-negative region, prior to the appearance of the isthmic organiser, is a key initial step in the specification of the cerebellum.

Eddison, Mark; Toole, Leah; Bell, Esther; Wingate, Richard JT



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


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. PMID:20865784

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



Selective capture of prostatic basal cells and secretory epithelial cells for proteomic and genomic analysis.  


Basal cells play an undefined role in signaling the growth and differentiation of normal secretory epithelial cells in the human prostate. Because basal cells disappear during malignant transformation, we hypothesize that loss of basal cell function may have a permissive role in progression of prostate intraepithelial neoplasia into invasive carcinoma. We describe an immuno-laser capture microdissection approach to selectively capture basal cells. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we identified several protein candidates selectively expressed in microdissected basal cells. We also demonstrate that the RNA derived form this technique is an excellent source for gene-array studies. Thus, we provide evidence that proteomic and microgenomic techniques can be successfully applied to investigate the expression profiles of basal and secretory cells after immuno-capture. PMID:15283892

Diaz, Jose I; Cazares, Lisa H; Corica, Alberto; John Semmes, O



Newborn granule cells in the ageing dentate gyrus  

PubMed Central

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 ?m?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.

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



Volume Density, Distribution, and Ultrastructure of Secretory and Basolateral Membranes and Mitochondria Predict Parietal Cell Secretory (Dys)function  

PubMed Central

Acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking. Histologically, consensus defines acid secretion as the ratio of the volume density (Vd) of canalicular and apical membranes (CAMs) to tubulovesicular (TV) membranes, a value which varies widely under normal conditions. Examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion. Vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion. We confirmed that CAM to TV ratio fairly represented secretory activity in untreated and secretion-inhibited wild-type (WT) mice and in NHE2?/? mice as well, though the response was significantly attenuated in the latter. However, high CAM to TV ratios wrongly posed as active acid secretion in AE2?/?, GHKA??/?, and NHE4?/? mice. Achlorhydric genotypes also had a significantly higher Vd of basolateral membrane than WT mice, and reduced Vd of mitochondria and canaliculi. The Vd of mitochondria, and ratio of the Vd of basolateral membranes/Vd of mitochondria were preferred predictors of the level of acid secretion. Alterations in acid secretion, then, cause significant changes not only in the Vd of secretory membranes but also in mitochondria and basolateral membranes.

Miller, Marian L.; Andringa, Anastasia; Zavros, Yana; Bradford, Emily M.; Shull, Gary E.



FIB/SEM cell sectioning for intracellular metal granules characterization  

NASA Astrophysics Data System (ADS)

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.

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



Human CD8+ T cells store RANTES in a unique secretory compartment and release it rapidly after TcR stimulation.  


The chemokine RANTES is secreted rapidly after activation of human CD8+ T cells, with a cycloheximide-resistant burst during the first hour. This pattern was observed in purified memory and effector phenotype CD8+ cells from blood as well as in blasts. In contrast, secretion of other chemokines and interferon-gamma by these cells was sensitive to cycloheximide and detectable only after a lag. Immunofluorescence microscopy of CD8+ memory and effector cells and blasts showed RANTES present in intracellular vesicles that do not significantly colocalize with cytotoxic granule markers or other markers of defined cytoplasmic compartments. Immunoelectron microscopy confirmed that RANTES is stored in small vesicles distinct from the lysosomal secretory granules. RANTES+ vesicles polarize rapidly in response to TcR engagement and are more rapidly depleted from the cytoplasm. These results show that CD8+ T cells have two distinct TcR-regulated secretory compartments characterized by different mobilization kinetics, effector molecules, and biological function. PMID:14975243

Catalfamo, Marta; Karpova, Tatiana; McNally, James; Costes, Sylvain V; Lockett, Stephen J; Bos, Erik; Peters, Peter J; Henkart, Pierre A



Electrophysiological evidence that dentate hilar mossy cells are excitatory and innervate both granule cells and interneurons.  


1. The hypothesis that dentate hilar "mossy" cells are excitatory was tested by simultaneous intracellular recording in rat hippocampal slices. Mossy cells were recorded simultaneously with their potential targets, granule cells and interneurons. The gamma-amino-butyric acid-A (GABAA) receptor antagonist bicuculline was used in most experiments to block the normally strong inhibitory inputs to granule cells that could mask excitatory effects of mossy cells. Some cells were recorded with electrodes containing the marker Neurobiotin so that their identity could be confirmed morphologically. 2. A mossy cell action potential was immediately followed by a brief depolarization in a granule cell in 20 of 1,316 pairs (1.5%) that were recorded in the presence of bicuculline. The mean amplitude of depolarizations was 1.99 +/- 0.24 (SE) mV when the postsynaptic membrane potential was -55 to -65 mV. Depolarizations could trigger an action potential if the granule cell was depolarized from its resting potential so that its membrane potential was -50 to -60 mV. These data suggest that mossy cells excite granule cells monosynaptically. 3. Monosynaptic excitation of an interneuron by a mossy cell was recorded in 4 of 47 (8.5%) simultaneously recorded mossy cells and interneurons, also in the presence of bicuculline. The mean interneuron depolarization was 1.64 +/- 0.29 mV when the interneuron membrane potential was approximately -60 mV. When an interneuron was at its resting potential (-52 to -63 mV), action potentials were often triggered by the depolarizations. 4. Without bicuculline present, mossy cells had no apparent monosynaptic effects on granule cells, as has been previously reported. However, effects that appeared to be polysynaptic were observed in 5 of 92 pairs (5.4%). Specifically, a small, brief hyperpolarization occurred in granule cells 2.5-7.3 ms after the peak of a mossy cell action potential. Given the results indicating that mossy cells excite interneurons, and the long latency to onset of the hyperpolarization, one possible explanation for the hyperpolarization is that mossy cells excited interneurons that inhibited granule cells. 5. The results suggest that mossy cells are excitatory neurons. In addition, mossy cells appear to innervate both granule cells and interneurons that are located within several hundred micrometers of the mossy cell soma. The only detectable effect on granule cells in this area under normal conditions appears to be disynaptic and inhibitory. However, when GABAA-receptor-mediated inhibition is blocked, monosynaptic excitation of granule cells by mossy cells can be detected. PMID:7472322

Scharfman, H E



Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland  

PubMed Central

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b?/? and Rab27ash/ash/Rab27b?/? mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.

Chiang, Lilian; Ngo, Julie; Schechter, Joel E.; Karvar, Serhan; Tolmachova, Tanya; Seabra, Miguel C.; Hume, Alistair N.



Polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha cells: a computer simulation  

Microsoft Academic Search

Computer simulation of polyhydroxyalkanoate (PHA) granule formation in vivo could help to design strategies to optimize the fermentation process and achieve higher yields of PHA. It could also suggest biotechnological approaches to control the granule size and molecular weight of the polymer. A computer program simulating the formation of PHA granules inside a Ralstonia eutropha cell was developed, based on

L. Jurasek; R. H. Marchessault



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


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. PMID:20461420

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



Histamine synthesis is required for granule maturation in murine mast cells.  


Mast cells are the major sources of histamine, which is released in response to immunological stimulations. The synthesis of histamine is catalyzed by histidine decarboxylase (HDC). Previous studies have shown that Hdc(-/-) mast cells exhibit aberrant granule morphology with severely decreased granule content. Here, we investigated whether the histamine synthesized in mast cells regulates the granule maturation of murine mast cells. Several genes, including those encoding granule proteases and enzymes involved in heparin biosynthesis, were downregulated in Hdc(-/-) peritoneal mast cells. Impaired granule maturation was also found in Hdc(-/-) BM-derived cultured mast cells when they were cocultured with fibroblasts in the presence of c-kit ligand. Exogenous application of histamine and several H4 receptor agonists restored the granule maturation of Hdc(-/-) cultured mast cells. However, the maturation of granules was largely normal in Hrh4(-/-) peritoneal mast cells. Depletion of cellular histamine with tetrabenazine, an inhibitor of vesicular monoamine transporter-2, did not affect granule maturation. In vivo experiments with mast cell deficient Kit(W) /Kit(W-v) mice indicated that the expression of the Hdc gene in mast cells is required for granule maturation. These results suggest that histamine promotes granule maturation in mast cells and acts as an proinflammatory mediator. PMID:24002822

Nakazawa, Shunsuke; Sakanaka, Mariko; Furuta, Kazuyuki; Natsuhara, Mayuko; Takano, Hirotsugu; Tsuchiya, Soken; Okuno, Yasushi; Ohtsu, Hiroshi; Nishibori, Masahiro; Thurmond, Robin L; Hirasawa, Noriyasu; Nakayama, Kazuhisa; Ichikawa, Atsushi; Sugimoto, Yukihiko; Tanaka, Satoshi



Biochemical analysis of secretory proteins synthesized by normal rat pancreas and by pancreatic acinar tumor cells  

PubMed Central

We have examined the secretogogue responsiveness and the pattern of secretory proteins produced by a transplantable rat pancreatic acinar cell tumor. Dispersed tumor cells were found to discharge secretory proteins in vitro when incubated with hormones that act on four different classes of receptors: carbamylcholine, caerulein, secretin- vasoactive intestinal peptide, and bombesin. With all hormones tested, maximal discharge from tumor cells was only about one-half that of control pancreatic lobules, but occurred at the same dose optima except for secretin, whose dose optimum was 10-fold higher. Biochemical analysis of secretory proteins discharged by the tumor cells was carried out by crossed immunoelectrophoresis and by two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis. To establish a baseline for comparison, secretory proteins from normal rat pancreas were identified according to enzymatic activity and correlated with migration position on two-dimensional gels. Our results indicate that a group of basic polypeptides including proelastase, basic trypsinogen, basic chymotrypsinogen, and ribonuclease, two out of three forms of procarboxypeptidase B, and the major lipase species were greatly reduced or absent in tumor cell secretion. In contrast, the amount of acidic chymotrypsinogen was notably increased compared with normal acinar cells. Although the acinar tumor cells are highly differentiated cytologically and express functional receptors for several classes of pancreatic secretagogues, they show quantitative and qualitative differences when compared with normal pancreas with regard to their production of secretory proteins.



Mucin granule-associated proteins in human bronchial epithelial cells: the airway goblet cell "granulome"  

PubMed Central

Background Excess mucus in the airways leads to obstruction in diseases such as chronic bronchitis, asthma, and cystic fibrosis. Mucins, the highly glycosolated protein components of mucus, are stored in membrane-bound granules housed in the cytoplasm of airway epithelial "goblet" cells until they are secreted into the airway lumen via an exocytotic process. Precise mechanism(s) of mucin secretion, including the specific proteins involved in the process, have yet to be elucidated. Previously, we have shown that the Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein regulates mucin secretion by orchestrating translocation of mucin granules from the cytosol to the plasma membrane, where the granules dock, fuse and release their contents into the airway lumen. Associated with MARCKS in this process are chaperone (Heat Shock Protein 70 [HSP70], Cysteine string protein [CSP]) and cytoskeletal (actin, myosin) proteins. However, additional granule-associated proteins that may be involved in secretion have not yet been elucidated. Methods Here, we isolated mucin granules and granule membranes from primary cultures of well differentiated human bronchial epithelial cells utilizing a novel technique of immuno-isolation, based on the presence of the calcium activated chloride channel hCLCA1 (the human ortholog of murine Gob-5) on the granule membranes, and verified via Western blotting and co-immunoprecipitation that MARCKS, HSP70, CSP and hCLCA1 were present on the granule membranes and associated with each other. We then subjected the isolated granules/membranes to liquid chromatography mass spectrometry (LC-MS/MS) to identify other granule associated proteins. Results A number of additional cytoskeletal (e.g. Myosin Vc) and regulatory proteins (e.g. Protein phosphatase 4) associated with the granules and could play a role in secretion were discovered. This is the first description of the airway goblet cell "granulome."



Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment.  


Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment. PMID:10820264

Paumet, F; Le Mao, J; Martin, S; Galli, T; David, B; Blank, U; Roa, M



Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells  

PubMed Central

After exocytosis, chromaffin granules release essentially all their catecholamines in small fractions of a second, but it is unknown how fast they release stored peptides and proteins. Here we compare the exocytic release of fluorescently labelled neuropeptide Y (NPY) and tissue plasminogen activator from single granules. Exocytosis was tracked by measuring the membrane capacitance, and single granules in live cells were imaged by evanescent field microscopy. Neuropeptide Y left most granules in small fractions of a second, while tissue plasminogen activator remained in open granules for minutes. Taking advantage of the dependence on pH of the fluorescence of green fluorescent protein, we used rhythmic external acidification to determine whether and when granules re-sealed. One-third of them re-sealed within 100 s and retained significant levels of tissue plasminogen activator. Re-sealing accounts for only a fraction of the endocytosis monitored in capacitance measurements. When external [Ca2+] was raised, even neuropeptide Y remained in open granules until they re-sealed. It is concluded that a significant fraction of chromaffin granules re-seal after exocytosis, and retain those proteins that leave granules slowly. We suggest that granules vary the stoichiometry of release by varying both granule re-sealing and the association of proteins with the granule matrix.

Perrais, David; Kleppe, Ingo C; Taraska, Justin W; Almers, Wolfhard



Loss of Sonic Hedgehog Leads to Alterations in Intestinal Secretory Cell Maturation and Autophagy  

PubMed Central

Background Intestinal epithelial cells express the Sonic and Indian hedgehog ligands. Despite the strong interest in gut hedgehog signaling in GI diseases, no studies have specifically addressed the singular role of intestinal epithelial cell Sonic hedgehog signaling. The aim of this study was to investigate the specific role of Sonic hedgehog in adult ileal epithelial homeostasis. Methodology/Principal Findings A Sonic hedgehog intestinal epithelial conditional knockout mouse model was generated. Assessment of ileal histological abnormalities, crypt epithelial cell proliferation, epithelial cell fate, junctional proteins, signaling pathways, as well as ultrastructural analysis of intracellular organelles were performed in control and mutant mice. Mice lacking intestinal epithelial Sonic Hedgehog displayed decreased ileal crypt/villus length, decreased crypt proliferation as well as a decrease in the number of ileal mucin-secreting goblet cells and antimicrobial peptide-secreting Paneth cells during adult life. These secretory cells also exhibited disruption of their secretory products in mutant mice. Ultrastructural microscopy analysis revealed a dilated ER lumen in secretory cells. This phenotype was also associated with a decrease in autophagy. Conclusions/Significance Altogether, these findings indicate that the loss of Sonic hedgehog can lead to ileal secretory cell modifications indicative of endoplasmic reticulum stress, accompanied by a significant reduction in autophagy.

Gagne-Sansfacon, Jessica; Allaire, Joannie M.; Jones, Christine; Boudreau, Francois; Perreault, Nathalie



THE CYTOLOGY OF THE NORMAL PARATHYROID GLANDS OF MAN AND VIRGINIA DEER: A Light and Electron Microscopic Study with Morphologic Evidence of Secretory Activity  

Microsoft Academic Search

The normal parathyroids of six humans and a Virginia deer were studied by light and elec- tron microscopy. The parenchyma of the deer parathyroid is composed of uniform chief cells, which contained 100 to 400 m# electron-opaque, membrane-limited granules, pre- sumed to be secretory granules, in addition to the usual cytoplasmic organellcs. Desmosomes are present between adjacent cells, and rare




Mechanisms of Ethanol-induced Death of Cerebellar Granule Cells  

PubMed Central

Maternal ethanol exposure during pregnancy may cause fetal alcohol spectrum disorders (FASD). FASD is the leading cause of mental retardation. The most deleterious effect of fetal alcohol exposure is inducing neuroapoptosis in the developing brain. Ethanol-induced loss of neurons in the central nervous system (CNS) underlies many of the behavioral deficits observed in FASD. The cerebellum is one of the brain areas that is most susceptible to ethanol during development. Ethanol exposure causes a loss of both cerebellar Purkinje cells and granule cells. This review focuses on the toxic effect of ethanol on cerebellar granule cells (CGC) and the underlying mechanisms. Both in vitro and in vivo studies indicate that ethanol induces apoptotic death of CGC. The vulnerability of CGC to ethanol-induced death diminishes over time as neurons mature. Several mechanisms for ethanol-induced apoptosis of CGC have been suggested. These include inhibition of NMDA receptors, interference with signaling by neurotrophic factors, induction of oxidative stress, modulation of retinoid acid signaling, disturbance of potassium channel currents, thiamine deficiency, and disruption of translational regulation. Cultures of CGC provide an excellent system to investigate cellular/molecular mechanisms of ethanol-induced neurodegeneration and to evaluate interventional strategies. This review will also discuss the approaches leading to neuroprotection against ethanol-induced neuroapoptosis.

Luo, Jia



Cellular barcodes increase the efficiency of profiling single-cell secretory responses by microengraving  

PubMed Central

We present a method that uses fluorescent cellular barcodes to increase the number of unique samples that can be analyzed simultaneously by microengraving—a nanowell array-based technique for quantifying the secretory responses of thousands of single cells in parallel. By using n different fluorescent dyes to generate 2n unique cellular barcodes, we achieved a 2n-fold reduction in the number of arrays and quantity of reagents required per sample. The utility of this approach was demonstrated in three applications of interest in clinical and experimental immunology. Using barcoded human peripheral blood mononuclear cells and T cells, we constructed dose-response curves, profiled the secretory behavior of cells treated with mechanistically distinct stimuli, and tracked the secretory behaviors of different lineages of CD4+ T helper cells. In addition to increasing the number of samples analyzed by generating secretory profiles of single cells from multiple populations in a time- and reagent-efficient manner, we expect that cellular barcoding in combination with microengraving will facilitate unique experimental opportunities for quantitatively analyzing interactions among heterogeneous cells isolated in small groups (~2–5 cells).

Yamanaka, Yvonne J.; Szeto, Gregory L.; Gierahn, Todd M.; Forcier, Talitha L.; Benedict, Kelly F.; Brefo, Mavis S.N.; Lauffenburger, Douglas A.; Irvine, Darrell J.; Love, J. Christopher



Endogenous Secretory Receptor for Advanced Glycation End Products in Non-Small Cell Lung Carcinoma  

Microsoft Academic Search

Rationale: The receptor for advanced glycation end products is a multiligand receptor that plays an important role in regulating the invasiveness and metastatic potential of cancer cells. A recently discovered novel splice variant, the endogenous secretory receptor for advanced glycation end products, mediates the receptor for advanced glycation end-product-associated cell responses by func- tioning as a decoy receptor. Objectives: To

Seiichi Kobayashi; Hiroshi Kubo; Takashi Suzuki; Kota Ishizawa; Mitsuhiro Yamada; Mei He; Yasuhiko Yamamoto; Hiroshi Yamamoto; Hironobu Sasano; Hidetada Sasaki; Satoshi Suzuki


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


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. PMID:24373221

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



Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis.  


Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation. PMID:10502067

Höcker, M; John, M; Anagnostopoulos, J; Buhr, H J; Solimena, M; Gasnier, B; Henry, J P; Wang, T C; Wiedenmann, B



Perforant path activation of ectopic granule cells that are born after pilocarpine-induced seizures.  


Granule cells in the dentate gyrus are born throughout life, and various stimuli can affect their development in the adult brain. Following seizures, for instance, neurogenesis increases greatly, and some new cells migrate to abnormal (ectopic) locations, such as the hilus. Previous electrophysiological studies of this population have shown that they have intrinsic properties that are similar to normal granule cells, but differ in other characteristics, consistent with abnormal integration into host circuitry. To characterize the response of ectopic hilar granule cells to perforant path stimulation, intracellular recordings were made in hippocampal slices from rats that had pilocarpine-induced status epilepticus and subsequent spontaneous recurrent seizures. Comparisons were made with granule cells located in the granule cell layer of both pilocarpine- and saline-treated animals. In addition, a few ectopic hilar granule cells were sampled from saline-treated rats. Remarkably, hilar granule cells displayed robust responses, even when their dendrites were not present within the molecular layer, where perforant path axons normally terminate. The evoked responses of hilar granule cells were similar in several ways to those of normally positioned granule cells, but there were some differences. For example, there was an unusually long latency to onset of responses evoked in many hilar granule cells, especially those without molecular layer dendrites. Presumably this is due to polysynaptic activation by the perforant path. These results indicate that synaptic reorganization after seizures can lead to robust activation of newly born hilar granule cells by the perforant path, even when their dendrites are not in the terminal field of the perforant path. Additionally, the fact that these cells can be found in normal tissue and develop similar synaptic responses, suggests that seizures, while not necessary for their formation, strongly promote their generation and the development of associated circuits, potentially contributing to a lowered seizure threshold. PMID:14580952

Scharfman, H E; Sollas, A E; Berger, R E; Goodman, J H; Pierce, J P



Calcineurin dependent lytic granule exocytosis in NK-92 Natural Killer cells  

PubMed Central

Cytotoxic T cells (CTLs) and natural killer cells (NKs) both kill virus-infected cells and tumor cells by releasing the cytoxic contents of their lytic granules. We recently demonstrated a role for calcineurin in lytic granule exocytosis in TALL-104 human leukemic CTLs[1]. However, whether calcineurin plays a similar role in NK lytic granule release is not known. We tested whether calcineurin is involved in lytic granule exocytosis in human leukemic NK-92 cells using immunosuppressive drugs that block calcineurin function and by overexpressing a constitutively active calcineurin fusion protein. Our results indicate that calcineurin does play a role in lytic granule exocytosis in NK-92 cells, and suggest that, as was the case in TALL-104 cells, there are likely to be multiple calcium-dependent steps.

Pores-Fernando, Arun T.; Gaur, Surabhi; Doyon, Michelle Y.; Zweifach, Adam



Lactadherin Inhibits Secretory Phospholipase A2 Activity on Pre-Apoptotic Leukemia Cells  

PubMed Central

Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2’s do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50–60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.

Nyegaard, Steffen; Novakovic, Valerie A.; Rasmussen, Jan T.; Gilbert, Gary E.



Electrophysiological evidence of monosynaptic excitatory transmission between granule cells after seizure-induced mossy fiber sprouting.  


Mossy fiber sprouting is a form of synaptic reorganization in the dentate gyrus that occurs in human temporal lobe epilepsy and animal models of epilepsy. The axons of dentate gyrus granule cells, called mossy fibers, develop collaterals that grow into an abnormal location, the inner third of the dentate gyrus molecular layer. Electron microscopy has shown that sprouted fibers from synapses on both spines and dendritic shafts in the inner molecular layer, which are likely to represent the dendrites of granule cells and inhibitory neurons. One of the controversies about this phenomenon is whether mossy fiber sprouting contributes to seizures by forming novel recurrent excitatory circuits among granule cells. To date, there is a great deal of indirect evidence that suggests this is the case, but there are also counterarguments. The purpose of this study was to determine whether functional monosynaptic connections exist between granule cells after mossy fiber sprouting. Using simultaneous recordings from granule cells, we obtained direct evidence that granule cells in epileptic rats have monosynaptic excitatory connections with other granule cells. Such connections were not obtained when age-matched, saline control rats were examined. The results suggest that indeed mossy fiber sprouting provides a substrate for monosynaptic recurrent excitation among granule cells in the dentate gyrus. Interestingly, the characteristics of the excitatory connections that were found indicate that the pathway is only weakly excitatory. These characteristics may contribute to the empirical observation that the sprouted dentate gyrus does not normally generate epileptiform discharges. PMID:14534276

Scharfman, Helen E; Sollas, Anne L; Berger, Russell E; Goodman, Jeffrey H



Peripolar cells form the majority of granulated cells in the kidneys of antelopes and goats.  


Investigation of renal cortical tissue in 5 adult hartebeests (Alcelaphus buselaphus cokii), 3 impalas (Aepyceros melampus), 1 defassa waterbuck (Kobus defassa) and 5 goats (Capra hircus) revealed large granulated peripolar cells at the junction between the parietal and the visceral epithelial layers of the renal corpuscles. All four animal species under study contained 1 or more peripolar cells for the majority of renal corpuscles sectioned through the vascular pole. In the hartebeests, up to 3 parietal cells and the first podocyte were granulated. Peripolar cells contained intracytoplasmic electron-dense membrane-bounded granules-200-2,800 nm in diameter in the hartebeests, 200-1,740 nm in the impalas, 150-950 nm in the waterbuck and 200-2,140 nm in the goats. Epithelioid granulated juxtaglomerular cells around afferent and efferent arterioles were rarely seen. When observed, they contained smaller granules than those of the peripolar cells. This distribution suggests that peripolar cells play a role in the regulation of body electrolytes and water, probably acting in concert with the renin-angiotensin-aldosterone system. PMID:2750470

Mbassa, G K



Delta1 Expression, Cell Cycle Exit, and Commitment to a Specific Secretory Fate Coincide within a Few Hours in the Mouse Intestinal Stem Cell System  

PubMed Central

The stem cells of the small intestine are multipotent: they give rise, via transit-amplifying cell divisions, to large numbers of columnar absorptive cells mixed with much smaller numbers of three different classes of secretory cells - mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, and bactericide-secreting Paneth cells. Notch signaling is known to control commitment to a secretory fate, but why are the secretory cells such a small fraction of the population, and how does the diversity of secretory cell types arise? Using the mouse as our model organism, we find that secretory cells, and only secretory cells, pass through a phase of strong expression of the Notch ligand Delta1 (Dll1). Onset of this Dll1 expression coincides with a block to further cell division and is followed in much less than a cell cycle time by expression of Neurog3 – a marker of enteroendocrine fate – or Gfi1 – a marker of goblet or Paneth cell fate. By conditional knock-out of Dll1, we confirm that Delta-Notch signaling controls secretory commitment through lateral inhibition. We infer that cells stop dividing as they become committed to a secretory fate, while their neighbors continue dividing, explaining the final excess of absorptive over secretory cells. Our data rule out schemes in which cells first become committed to be secretory, and then diversify through subsequent cell divisions. A simple mathematical model shows how, instead, Notch signaling may simultaneously govern the commitment to be secretory and the choice between alternative modes of secretory differentiation.

Hodgetts, Christine; Jeffery, Rosemary; Nye, Emma; Spencer-Dene, Bradley; Winton, Douglas J.; Lewis, Julian



Programmed cell death: A mechanism for the lysigenous formation of secretory cavities in leaves of Dictamnus dasycarpus.  


The formation of secretory cavities in Rutaceae has been the subject of great interest. In this study, cytological events that are involved in the lysigenous formation of the secretory cavities in the leaves of Dictamnus dasycarpus are characterized by an interesting pattern of programmed cell death (PCD). During the developmental process, clusters of cells from a single protoepidermal cell embark on different trajectories and undergo different cell death fates: the cell walls of the secretory cells have characteristics of thinning or complete breakdown, while the sheath cells present a predominantly thick-walled feature. A DAPI assay shows deformed nuclei that are further confirmed to be TUNEL-positive. Gel electrophoresis indicates that DNA cleavage is random and does not result in ladder-like DNA fragmentation. Ultrastructurally, several remarkable features of PCD have been determined, such as misshapen nuclei with condensed chromatin and a significantly diffused membrane, degenerated mitochondria and plastids with disturbed membrane systems, multivesicular bodies, plastolysomes, vacuole disruption and lysis of the center secretory cell. Cytological evidence and Nile red stains exhibit abundant essential oils accumulated in degenerated outer secretory cells after the dissolution of the center secretory cell. In addition, explanations of taxonomic importance and the relationship between PCD and oil droplet accumulation in the secretory cavities are also discussed. PMID:25017170

Zhou, Ya-Fu; Mao, Shao-Li; Li, Si-Feng; Ni, Xi-Lu; Li, Bin; Liu, Wen-Zhe



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

PubMed Central

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.

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



Lack of DNA degradation in target cells lysed by granules derived from cytolytic T lymphocytes.  


It has been shown previously that fragmentation of target cell DNA is an early event in lysis mediated by cytolytic T lymphocytes (CTL). In this study, we have investigated whether CTL-derived granules that exhibit lytic activity also induce DNA fragmentation in murine target cells. Cytolytic granules isolated from three different alloreactive CTL clones were tested for the induction of DNA fragmentation in P815 and EL4 target cells, by using a Triton X-100-facilitated, radiolabeled DNA release assay. In contrast to the CTL clones from which they were derived, the cytolytic granules did not induce DNA fragmentation. Agarose gel electrophoretic analysis of DNA confirmed the lack of discrete DNA fragments in target cells lysed by CTL-derived granules. Possible explanations for the difference in the ability of CTL and CTL-derived granules to trigger DNA fragmentation are discussed. PMID:3260910

Gromkowski, S H; Brown, T C; Masson, D; Tschopp, J



Secretory activity and cell cycle alteration of alveolar type II cells in the early and late phase after irradiation  

Microsoft Academic Search

Purpose: Type II cells and the surfactant system have been proposed to play a central role in pathogenesis of radiation pneumonitis. We analyzed the secretory function and proliferation parameters of alveolar type II cells in the early (until 24 h) and late phase (1–5 weeks) after irradiation (RT) in vitro and in vivo.Methods and Materials: Type II cells were isolated

Jochen Willner; Dirk Vordermark; Michael Schmidt; Andreamaria Gassel; Michael Flentje; Hubert Wirtz



Dentate Granule Cell Neurogenesis Is Increased by Seizures and Contributes to Aberrant Network Reorganization in the Adult Rat Hippocampus  

Microsoft Academic Search

The dentate granule cell layer of the rodent hippocampal formation has the distinctive property of ongoing neurogen- esis that continues throughout adult life. In both human temporal lobe epilepsy and rodent models of limbic epilepsy, this same neuronal population undergoes extensive remod- eling, including reorganization of mossy fibers, dispersion of the granule cell layer, and the appearance of granule cells

Jack M. Parent; Timothy W. Yu; Rebecca T. Leibowitz; Daniel H. Geschwind; Robert S. Sloviter; Daniel H. Lowenstein


The antioxidant EPC-K1 attenuates NO-induced mitochondrial dysfunction, lipid peroxidation and apoptosis in cerebellar granule cells  

Microsoft Academic Search

In this study we investigated the effects of nitric oxide (NO) on cultured cerebellar granule cells. Exposure to NO donors, S-nitrosoglutathione (GSNO; 250 ?M) or sodium nitroprusside (SNP; 500 ?M), triggered apoptosis in immature cultures of cerebellar granule cells, which was characterized by chromatin condensation, nuclei fragmentation, and DNA laddering. Exposure of cerebellar granule cells to NO donors led to

Taotao Wei; Chang Chen; Jingwu Hou; Baolu Zhao; Wenjuan Xin; Akitane Mori



Primary non-secretory plasma cell leukemia with atypical morphology — a case report  

Microsoft Academic Search

Only one case of primary non-secretory plasma cell leukemia with atypical morphology has been reported thus far. Here we report\\u000a another such case of plasma cell leukemia diagnosed on fl ow cytometry, as morphological heterogeneity and lack of monoclonal\\u000a immunoglobulins in both serum and urine, made it difficult to come to a conclusive diagnosis based purely on morphology.

T. Dadu; A. Rangan; A. Handoo; M. Bhargava



Effect of taxol on secretory cells: functional, morphological, and electrophysiological correlates  

PubMed Central

The effect of 0.5-1.0 microM taxol, a potent promoter of microtubule polymerization in vitro, was studied on the secretory activity of chromaffin cells of the adrenal medulla. Taxol was found to have a dual effect: the long-term effect (after a 1-h incubation) of taxol was to induce almost complete inhibition of catecholamine release, whereas after a short incubation (10 min) a massive, nicotine-independent release of catecholamine was produced. From results obtained using the patch-clamp technique to study the Ca++-dependent K+ channels (Ic channels), it was possible to conclude that taxol probably provokes an augmentation of free [Ca++]i in the cytoplasm, values increasing from 10(-8) M at rest to several 10(-7) M. The increased spontaneous release of stored neurohormones and the increased frequency of opening of Ic channels occur simultaneously and could both originate from a rise of [Ca++]i upon taxol addition. Immunofluorescence and ultrastructural studies showed that 13-h taxol treatment of chromaffin cells led to a different distribution of secretory organelles, and also to microtubule reorganization. In treated cells, microtubules were found to form bundles beneath the cell membrane and, at the ultrastructural level, to be packed along the cell axis. It is concluded that in addition to its action on microtubules, the antitumor drug taxol has side effects on the cell secretory activity, one of them being to modify free [Ca++]i.



Peripolar Cells Form the Majority of Granulated Cells in the Kidneys of Antelopes and Goats  

Microsoft Academic Search

Investigation of renal cortical tissue in 5 adult hartebeests (Alcelaphus buselaphus cokii), 3 impalas (Aepyceros melampus), 1 defassa waterbuck (Kobus defassa) and 5 goats (Capra hircus) revealed large granulated peripolar cells at the junction between the parietal and the visceral epithelial layers of the renal corpuscles. All four animal species under study contained 1 or more peripolar cells for the

Gabriel K. Mbassa



C-Phycocyanin protects cerebellar granule cells from low potassium\\/serum deprivation-induced apoptosis  

Microsoft Academic Search

We tested the potential cytoprotective role of C-phycocyanin in rat cerebellar granule cell cultures. Cell death was induced by potassium and serum (K\\/S) withdrawal. Cell viability was studied using the neutral red assay and laser scanning cytometry with propidium iodide as fluorochrome. C-phycocyanin (1-3 mg\\/ml) showed a neuroprotective effect against 24 h of K\\/S deprivation in cerebellar granule cells. After

Víctor Rimbau; Antoni Camins; David Pubill; Francesc X. Sureda; Cheyla Romay; Ricardo González; Andrés Jiménez; Elena Escubedo; Jordi Camarasa; Mercè Pallàs



Odontogenic Differentiation of Human Dental Pulp Stem Cells Stimulated by the Calcium Phosphate Porous Granules  

PubMed Central

Effects of three-dimensional (3D) calcium phosphate (CaP) porous granules on the growth and odontogenic differentiation of human dental pulp stem cells (hDPSCs) were examined for dental tissue engineering. hDPSCs isolated from adult human dental pulps were cultured for 3-4 passages, and populated on porous granules. Cell growth on the culture dish showed an ongoing increase for up to 21 days, whereas the growth on the 3D granules decreased after 14 days. This reduction in proliferative potential on the 3D granules was more conspicuous under the osteogenic medium conditions, indicating that the 3D granules may induce the odontogenic differentiation of hDPSCs. Differentiation behavior on the 3D granules was confirmed by the increased alkaline phosphatase activity, up-regulation of odontoblast-specific genes, including dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) by quantitative polymerase chain reaction, and greater level of dentin sialoprotein synthesis by western blot. Moreover, the cellular mineralization, as assessed by Alizarin red S and calcium quantification, was significantly higher in the 3D CaP granules than in the culture dish. Taken all, the 3D CaP porous granules should be useful for dental tissue engineering in combination with hDPSCs by providing favorable 3D substrate conditions for cell growth and odontogenic development.

Nam, Sunyoung; Won, Jong-Eun; Kim, Cheol-Hwan; Kim, Hae-Won



Paneth cell granule depletion in the human small intestine under infective and nutritional stress.  


Paneth cells are important contributors to the intestinal antimicrobial barrier through synthesis and release of antimicrobial peptides and proteins. Animal studies indicate that Paneth cell numbers, location and granule morphology are altered by infection and zinc status. We examined human tissue to determine whether Paneth cell numbers, distribution or granule morphology are altered in infective, inflammatory and nutritional disorders. Archival sections from infective disorders (giardiasis, cryptosporidiosis, HIV, helminth infection) were compared with active inflammatory conditions (coeliac, Crohn's and graft-versus-host diseases) and histologically normal tissues. A subset of tissues was studied by electron microscopy and TUNEL staining for apoptosis. Human defensin-5 (HD5) peptide and mRNA was analysed by immunohistochemistry, in situ hybridization and quantitative reverse transcription polymerase chain reaction. Sections from a tropical population cohort study were then analysed to determine the relationship of granule depletion to infection, nutritional status and plasma zinc concentration. In HIV-related cryptosporidiosis, but not other disorders, Paneth cells were reduced in number and markedly depleted of granules. Paneth cell granule depletion was associated with reduced HD5 immunoreactivity, but this was not due to apoptosis and there was no reduction in mRNA transcripts. In the tropical population studied, depletion of granules was associated with reduced body mass index, reduced plasma zinc levels and HIV infection. Paneth cell granules in human small intestine may be depleted in response to infective and nutritional stress. We postulate that this is one mechanism through which zinc status influences host susceptibility to intestinal infection. PMID:14738460

Kelly, P; Feakins, R; Domizio, P; Murphy, J; Bevins, C; Wilson, J; McPhail, G; Poulsom, R; Dhaliwal, W



Paneth cell granule depletion in the human small intestine under infective and nutritional stress  

PubMed Central

Paneth cells are important contributors to the intestinal antimicrobial barrier through synthesis and release of antimicrobial peptides and proteins. Animal studies indicate that Paneth cell numbers, location and granule morphology are altered by infection and zinc status. We examined human tissue to determine whether Paneth cell numbers, distribution or granule morphology are altered in infective, inflammatory and nutritional disorders. Archival sections from infective disorders (giardiasis, cryptosporidiosis, HIV, helminth infection) were compared with active inflammatory conditions (coeliac, Crohn's and graft-versus-host diseases) and histologically normal tissues. A subset of tissues was studied by electron microscopy and TUNEL staining for apoptosis. Human defensin-5 (HD5) peptide and mRNA was analysed by immunohistochemistry, in situ hybridization and quantitative reverse transcription polymerase chain reaction. Sections from a tropical population cohort study were then analysed to determine the relationship of granule depletion to infection, nutritional status and plasma zinc concentration. In HIV-related cryptosporidiosis, but not other disorders, Paneth cells were reduced in number and markedly depleted of granules. Paneth cell granule depletion was associated with reduced HD5 immunoreactivity, but this was not due to apoptosis and there was no reduction in mRNA transcripts. In the tropical population studied, depletion of granules was associated with reduced body mass index, reduced plasma zinc levels and HIV infection. Paneth cell granules in human small intestine may be depleted in response to infective and nutritional stress. We postulate that this is one mechanism through which zinc status influences host susceptibility to intestinal infection.




Gfi1 functions downstream of Math1 to control intestinal secretory cell subtype allocation and differentiation  

PubMed Central

Gfi1 is a transcriptional repressor implicated in lymphomagenesis, neutropenia, and hematopoietic development, as well as ear and lung development. Here, we demonstrate that Gfi1 functions downstream of Math1 in intestinal secretory lineage differentiation. Gfi1-/- mice lack Paneth cells, have fewer goblet cells, and supernumerary enteroendocrine cells. Gfi1-/- mice show gene expression changes consistent with this altered cell allocation. These data suggest that Gfi1 functions to select goblet/Paneth versus enteroendocrine progenitors. We propose a model of intestinal cell fate choice in which ?-catenin and Cdx function upstream of Math1, and lineage-specific genes such as Ngn3 act downstream of Gfi1.

Shroyer, Noah F.; Wallis, Deeann; Venken, Koen J.T.; Bellen, Hugo J.; Zoghbi, Huda Y.



Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults  

SciTech Connect

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.

Gianotti, A.J.; Clark, D.T.; Dash, J. (Portland State Univ., OR (USA))



Non-cell autonomous or secretory tumor suppression.  


Many malignancies result from deletions or loss-of-function mutations in one or more tumor suppressor genes, the products of which curb unrestrained growth or induce cell death in those with dysregulated proliferative capacities. Most tumor suppressors act in a cell autonomous manner, and only very few proteins are shown to exert a non-cell autonomous tumor suppressor function on other cells. Examples of these include members of the secreted frizzled-related protein (SFRP) family and the secreted protein acidic and rich in cysteine (SPARC)-related proteins. Very recent findings have, however, considerably expanded our appreciation of non-cell autonomous tumor suppressor functions. Broadly, this may occur in two ways. Intracellular tumor suppressor proteins within cells could in principle inhibit aberrant growth of neighboring cells by conditioning an antitumor microenvironment through secreted factors. This is demonstrated by an apparent non-cell autonomous tumor suppressing property of p53. On the other hand, a tumor suppressor produced by a cell may be secreted extracellularly, and taken up by another cell with its activity intact. Intriguingly, this has been recently shown to occur for the phosphatase and tensin homolog (PTEN) by both conventional and unconventional modes of secretion. These recent findings would aid the development of therapeutic strategies that seek to reinstate tumor suppression activity in therapeutically recalcitrant tumor cells, which have lost it in the first place. J. Cell. Physiol. 229: 1346-1352, 2014. © 2014 Wiley Periodicals, Inc. PMID:24752809

Chua, Christelle En Lin; Chan, Shu Ning; Tang, Bor Luen



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


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. PMID:2519933

Lacy, E R; Reale, E



Phorbol Myristate Acetate Stimulates Formation of Diphosphoinositide in Rat Mast Cell Granules  

Microsoft Academic Search

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [?32P]ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol\\/chloroform\\/HCl and were separated by thin-layer chromatography on oxalic

Motohiro Kurosawa; Yoshimichi Okayama; Setsuo Kobayashi



NMDA-receptor dependent synaptic activation of TRPC channels in olfactory bulb granule cells  

PubMed Central

TRPC channels are widely expressed throughout the nervous system including the olfactory bulb where their function is largely unknown. Here we describe their contribution to central synaptic processing at the reciprocal mitral and tufted cell - granule cell microcircuit, the most abundant synapse of the mammalian olfactory bulb. Suprathreshold activation of the synapse causes sodium action potentials in mouse granule cells and a subsequent long-lasting depolarization (LLD) linked to a global dendritic postsynaptic calcium signal recorded with two-photon laser scanning microscopy. These signals are not observed after action potentials evoked by current injection in the same cells. The LLD persists in the presence of group I metabotropic glutamate receptor antagonists but is entirely absent from granule cells deficient for the NMDA receptor subunit NR1. Moreover, both depolarization and Ca2+ rise are sensitive to the blockade of NMDA receptors. The LLD and the accompanying Ca2+ rise are also absent in granule cells from mice deficient for both TRPC channel subtypes 1 and 4, whereas the deletion of either TRPC1 or TRPC4 results in only a partial reduction of the LLD. Recordings from mitral cells in the absence of both subunits reveal a reduction of asynchronous neurotransmitter release from the granule cells during recurrent inhibition. We conclude that TRPC1 and TRPC4 can be activated downstream of NMDA receptor activation and contribute to slow synaptic transmission in the olfactory bulb, including the calcium dynamics required for asynchronous release from the granule cell spine.

Stroh, Olga; Freichel, Marc; Kretz, Oliver; Birnbaumer, Lutz; Hartmann, Jana; Egger, Veronica



Cell type specific expression of secretory TFF peptides: colocalization with mucins and synthesis in the brain.  


The "TFF domain" is an ancient cysteine-rich shuffled module forming the basic unit for the family of secretory TFF peptides (formerly P-domain peptides and trefoil factors). It is also an integral component of mosaic proteins associated with mucous surfaces. Three mammalian TFF peptides are known (i.e., TFF1-TFF3); however, in Xenopus laevis the pattern is more complex (xP1, xP4.1, xP4.2, and xP2). TFF peptides are typical secretory products of a variety of mucin-producing epithelial cells (e.g., the conjunctiva, the salivary glands, the gastrointestinal tract, the respiratory tract, and the uterus). Each TFF peptide shows an unique expression pattern and different mucin-producing cells are characterized by their specific TFF peptide/secretory mucin combinations. TFF peptides have a pivotal role in maintaining the surface integrity of mucous epithelia in vivo. They are typical constituents of mucus gels, they modulate rapid mucosal repair ("restitution") by their motogenic and their cell scattering activity, they have antiapoptotic effects, and they probably modulate inflammatory processes. Pathological expression of TFF peptides occurs as a result of chronic inflammatory diseases or certain tumors. TFF peptides are also found in the central nervous system, at least in mammals. In particular, TFF3 is synthesized from oxytocinergic neurons of the hypothalamus and is released from the posterior pituitary into the bloodstream. PMID:11837892

Hoffmann, Werner; Jagla, Wolfgang



Localization of rat endothelin-converting enzyme to vascular endothelial cells and some secretory cells.  

PubMed Central

Endothelin is a potent vasoconstrictive peptide that is produced by vascular endothelial cells; it is formed from its precursor, big endothelin, by endothelin-converting enzyme (ECE). In this work, ECE was studied using specific monoclonal antibodies. In immunoblotting, ECE was estimated to be a 300 kDa protein on SDS/PAGE under non-reducing conditions, and 130 kDa under reducing conditions. Cross-linking experiments revealed that ECE is composed of two disulphide-linked subunits. Localization of ECE was studied at the cellular and subcellular levels in various rat tissues and cells. High-level expression of ECE was observed in membrane fractions of simian virus 40-transformed rat endothelial cells by immunoblotting, but the immunoreactive band was absent form aortic smooth muscle cells and cytosolic fractions of endothelial cells. In immunohistochemical analysis, ECE was found to be localized in the endothelial cells of the aorta, lung, kidney, liver and heart. Confocal immunofluorescent microscopy showed that most of the ECE in endothelial cells and cells transfected with ECE cDNA was clustered along the plasma membrane. Intact COS or CHO cells transfected with ECE cDNA rapidly and efficiently cleaved big endothelin-1 added to the culture medium. Thus endothelial cells express ECE on the plasma membrane and the active site of the enzyme faces outside the cells, i.e. it is an ectoenzyme. Other than endothelial cells, ECE was also present in some secretory cells. The enzyme was abundant in the adrenal gland, and localized in chromaffin cells. ECE was also highly condensed in pancreatic islet beta cells. It is concluded that ECE and endothelin may be involved in the regulated secretion of hormones. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8

Takahashi, M; Fukuda, K; Shimada, K; Barnes, K; Turner, A J; Ikeda, M; Koike, H; Yamamoto, Y; Tanzawa, K



Ultrastructural characteristics, secretory and phagocytotic activity of Paneth cells.  


Paneth cells in the depth of Lieberkühn's crypts were studied ultrastructurally in three human jejunal biopsy material. Fine structural elements were observed indicative of phagocytosis. It is also assumed that the cells had regulatory function of normal intestinal flora. The importance of ultrastructural examinations of Lieberkühn's crypts under pathological conditions is emphasized. PMID:506817

Répássy, G; Lapis, K



Distinct determinants of sparse activation during granule cell maturation.  


Adult neurogenesis continually produces a small population of immature granule cells (GCs) within the dentate gyrus. The physiological properties of immature GCs distinguish them from the more numerous mature GCs and potentially enables distinct network functions. To test how the changing properties of developing GCs affect spiking behavior, we examined synaptic responses of mature and immature GCs in hippocampal slices from adult mice. Whereas synaptic inhibition restricted GC spiking at most stages of maturation, the relative influence of inhibition, excitatory synaptic drive, and intrinsic excitability shifted over the course of maturation. Mature GCs received profuse afferent innervation such that spiking was suppressed primarily by inhibition, whereas immature GC spiking was also limited by the strength of excitatory drive. Although the input resistance was a reliable indicator of maturation, it did not determine spiking probability at immature stages. Our results confirm the existence of a transient period during GC maturation when perforant path stimulation can generate a high probability of spiking, but also reveal that immature GC excitability is tempered by functional synaptic inhibition and reduced excitatory innervation, likely maintaining the sparse population activity observed in vivo. PMID:24305810

Dieni, Cristina V; Nietz, Angela K; Panichi, Roberto; Wadiche, Jacques I; Overstreet-Wadiche, Linda



The biology and dynamics of mammalian cortical granules  

PubMed Central

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.



Calcineurin-dependent lytic granule exocytosis in NK-92 natural killer cells.  


Cytotoxic T cells (CTLs) and natural killer cells (NKs) both kill virus-infected cells and tumor cells by releasing the cytoxic contents of their lytic granules. We recently demonstrated a role for calcineurin in lytic granule exocytosis in TALL-104 human leukemic CTLs [M.J. Grybko, J.P. Bartnik, G.A. Wurth, A.T. Pores-Fernando, A. Zweifach, Calcineurin activation is only one calcium-dependent step in cytotoxic T lymphocyte granule exocytosis, J. Biol. Chem. 282 (2007) 18009-18017]. However, whether calcineurin plays a similar role in NK lytic granule release is not known. We tested whether calcineurin is involved in lytic granule exocytosis in human leukemic NK-92 cells using immunosuppressive drugs that block calcineurin function and by overexpressing a constitutively active calcineurin fusion protein. Our results indicate that calcineurin does play a role in lytic granule exocytosis in NK-92 cells, and suggest that, as was the case in TALL-104 cells, there are likely to be multiple calcium-dependent steps. PMID:18762287

Pores-Fernando, Arun T; Gaur, Surabhi; Doyon, Michelle Y; Zweifach, Adam



The secretory leukocyte protease inhibitor (SLPI) and the secondary granule protein lactoferrin are synthesized in myelocytes, colocalize in subcellular fractions of neutrophils, and are coreleased by activated neutrophils  

Microsoft Academic Search

The secretory leukocyte protease inhibi- tor (SLPI) re-establishes homeostasis at sites of infec- tion by virtue of its ability to exert antimicrobial activity, to suppress LPS-induced cellular immune responses, and to reduce tissue damage through in- hibition of serine proteases released by polymorpho- nuclear neutrophil granulocytes (PMNs). Microarray analysis of bone marrow (BM) populations highly en- riched in promyelocytes, myelocytes\\/metamyelo-

Lars C. Jacobsen; O. E. Sorensen; Jack B. Cowland; Niels Borregaard; Kim Theilgaard-Monch



Neuregulin Induces GABAA Receptor Subunit Expression and Neurite Outgrowth in Cerebellar Granule Cells  

Microsoft Academic Search

Neuregulin (NRG), a growth and differentiation factor that sig- nals via erbB receptor tyrosine kinases, has been shown to have biological effects in both the CNS and the peripheral nervous system. We report here that erbB4 is expressed in mature cerebellar granule cells, where it appears to be concen- trated at the granule cell postsynaptic terminals. We also show that

Heather I. Rieff; Lori T. Raetzman; Douglas W. Sapp; Hermes H. Yeh; Ruth E. Siegel; Gabriel Corfas



Mast Cells and Oral Inflammation  

Microsoft Academic Search

Mast cells are mobile granule-containing secretory cells that are distributed preferentially about the microvascular endothelium in oral mucosa and dental pulp. The enzyme profile of mast cells in oral tissues resembles that of skin, with most mast cells expressing the serine proteases tryptase and chymase. Mast cells in oral tissues contain the pro-inflammatory cytokine tumour necrosis factor-? in their granules,

Laurence J. Walsh



Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells  

PubMed Central

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 (?-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 ? subunits of GABAA Rs from ?2/3 to ?1 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 ?1 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 ?1 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 ?1 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 ?1-/- 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 finding of a greater inhibition of mIPSCs in young (DIV8) than more mature (DIV14) cultures. Together, our results suggest that NLG2 does not alter uniquantal GABA release, and the fast decay of mIPSC induced by NLG2 is due to the differential expression of postsynaptic GABAA receptor subtypes. Taken all together, we propose that NLG-2 plays important functional role in inhibitory synapse development and maturation.

Fu, Zhanyan; Vicini, Stefano



Cell death and immunityThe ABCs of granule-mediated cytotoxicity: new weapons in the arsenal  

Microsoft Academic Search

Granule exocytosis is the main pathway for the immune elimination of virus-infected cells and tumour cells by cytotoxic T lymphocytes and natural killer cells. After target-cell recognition, release of the cytotoxic granule contents into the immunological synapse formed between the killer cell and its target induces apoptosis. The granules contain two membrane-perturbing proteins, perforin and granulysin, and a family of

Judy Lieberman



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

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

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



Kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+.  

PubMed Central

The kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+ were studied by capacitance (Cm) measurements with millisecond time resolution. After elevation of the internal Ca2+ concentration ([Ca2+]i), Cm rises rapidly with one or more exponentials. The time constant of the fastest component decreases for higher [Ca2+]i (range 3-600 microM) over three orders of magnitude before it saturates at approximately 1 ms. The corresponding maximal rates of secretion can be as fast as 100,000 fF/s or 40,000 vesicles/s. There is a Ca(2+)-dependent delay before Cm rises, which may reflect the kinetics of multiple Ca2+ ions binding to the secretory apparatus. The initial rise in Cm is described by models containing a sequence of two to four single Ca(2+)-binding steps followed by a rate-limiting exocytosis step. The predicted Ca2+ dissociation constant (Kd) of a single Ca(2+)-binding site is between 7 and 21 microM. At [Ca2+]i > 30 microM clear indications of a fast endocytotic process complicate the analysis of the secretory response.

Heinemann, C; Chow, R H; Neher, E; Zucker, R S



Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion  

PubMed Central

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet ? cells in part by tethering insulin secretory granules to Ca2+ channels to assist excitosome assembly. Here, we assessed ? cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet ? cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in ? cells are more sensitive than predocked granules to Sec5 regulation.

Xie, Li; Zhu, Dan; Kang, Youhou; Liang, Tao; He, Yu; Gaisano, Herbert Y.



Spatial partitioning of secretory cargo from Golgi resident proteins in live cells  

PubMed Central

Background To maintain organelle integrity, resident proteins must segregate from itinerant cargo during secretory transport. However, Golgi resident enzymes must have intimate access to secretory cargo in order to carry out glycosylation reactions. The amount of cargo and associated membrane may be significant compared to the amount of Golgi membrane and resident protein, but upon Golgi exit, cargo and resident are efficiently sorted. How this occurs in live cells is not known. Results We observed partitioning of the fluorescent Golgi resident T2-CFP and fluorescent cargo proteins VSVG3-YFP or VSVG3-SP-YFP upon Golgi exit after a synchronous pulse of cargo was released from the ER. Golgi elements remained stable in overall size, shape and relative position as cargo emptied. Cargo segregated from resident rapidly by blebbing into micron-sized domains that contained little or no detectable resident protein and that appeared to be continuous with the parent Golgi element. Post-Golgi transport carriers (TCs) exited repeatedly from these domains. Alternatively, entire cargo domains exited Golgi elements, forming large TCs that fused directly with the plasma membrane. However, domain formation did not appear to be an absolute prerequisite for TC exit, since TCs also exited directly from Golgi elements in the absence of large domains. Quantitative cargo-specific photobleaching experiments revealed transfer of cargo between Golgi regions, but no discrete intra-Golgi TCs were observed. Conclusions Our results establish domain formation via rapid lateral partitioning as a general cellular strategy for segregating different transmembrane proteins along the secretory pathway and provide a framework for consideration of molecular mechanisms of secretory transport.

White, Jamie; Keller, Patrick; Stelzer, Ernst HK



Morphometry of Hilar Ectopic Granule Cells in the Rat  

PubMed Central

Granule cell (GC) neurogenesis in the dentate gyrus (DG) does not always proceed normally. After severe seizures (e.g., status epilepticus [SE]) and some other conditions, newborn GCs appear in the hilus. Hilar ectopic GCs (EGCs) can potentially provide insight into the effects of abnormal location and seizures on GC development. Additionally, hilar EGCs that develop after SE may contribute to epileptogenesis and cognitive impairments that follow SE. Thus, it is critical to understand how EGCs differ from normal GCs. Relatively little morphometric information is available on EGCs, especially those restricted to the hilus. This study quantitatively analyzed the structural morphology of hilar EGCs from adult male rats several months after pilocarpineinduced SE, when they are considered to have chronic epilepsy. Hilar EGCs were physiologically identified in slices, intracellularly labeled, processed for light microscopic reconstruction, and compared to GC layer GCs, from both the same post-SE tissue and the NeuroMorpho database (normal GCs). Consistently, hilar EGC and GC layer GCs had similar dendritic lengths and field sizes, and identifiable apical dendrites. However, hilar EGC dendrites were topologically more complex, with more branch points and tortuous dendritic paths. Three-dimensional analysis revealed that, remarkably, hilar EGC dendrites often extended along the longitudinal DG axis, suggesting increased capacity for septotemporal integration. Axonal reconstruction demonstrated that hilar EGCs contributed to mossy fiber sprouting. This combination of preserved and aberrant morphological features, potentially supporting convergent afferent input to EGCs and broad, divergent efferent output, could help explain why the hilar EGC population could impair DG function.

Pierce, Joseph P.; McCloskey, Daniel P.; Scharfman, Helen E.



Morphometry of hilar ectopic granule cells in the rat.  


Granule cell (GC) neurogenesis in the dentate gyrus (DG) does not always proceed normally. After severe seizures (e.g., status epilepticus [SE]) and some other conditions, newborn GCs appear in the hilus. Hilar ectopic GCs (EGCs) can potentially provide insight into the effects of abnormal location and seizures on GC development. Additionally, hilar EGCs that develop after SE may contribute to epileptogenesis and cognitive impairments that follow SE. Thus, it is critical to understand how EGCs differ from normal GCs. Relatively little morphometric information is available on EGCs, especially those restricted to the hilus. This study quantitatively analyzed the structural morphology of hilar EGCs from adult male rats several months after pilocarpine-induced SE, when they are considered to have chronic epilepsy. Hilar EGCs were physiologically identified in slices, intracellularly labeled, processed for light microscopic reconstruction, and compared to GC layer GCs, from both the same post-SE tissue and the NeuroMorpho database (normal GCs). Consistently, hilar EGC and GC layer GCs had similar dendritic lengths and field sizes, and identifiable apical dendrites. However, hilar EGC dendrites were topologically more complex, with more branch points and tortuous dendritic paths. Three-dimensional analysis revealed that, remarkably, hilar EGC dendrites often extended along the longitudinal DG axis, suggesting increased capacity for septotemporal integration. Axonal reconstruction demonstrated that hilar EGCs contributed to mossy fiber sprouting. This combination of preserved and aberrant morphological features, potentially supporting convergent afferent input to EGCs and broad, divergent efferent output, could help explain why the hilar EGC population could impair DG function. PMID:21344409

Pierce, Joseph P; McCloskey, Daniel P; Scharfman, Helen E



Competition from newborn granule cells does not drive axonal retraction of silenced old granule cells in the adult hippocampus.  


In the developing nervous system synaptic refinement, typified by the neuromuscular junction where supernumerary connections are eliminated by axon retraction leaving the postsynaptic target innervated by a single dominant input, critically regulates neuronal circuit formation. Whether such competition-based pruning continues in established circuits of mature animals remains unknown. This question is particularly relevant in the context of adult neurogenesis where newborn cells must integrate into preexisting circuits, and thus, potentially compete with functionally mature synapses to gain access to their postsynaptic targets. The hippocampus plays an important role in memory formation/retrieval and the dentate gyrus (DG) subfield exhibits continued neurogenesis into adulthood. Therefore, this region contains both mature granule cells (old GCs) and immature recently born GCs that are generated throughout adult life (young GCs), providing a neurogenic niche model to examine the role of competition in synaptic refinement. Recent work from an independent group in developing animals indicated that embryonically/early postnatal generated GCs placed at a competitive disadvantage by selective expression of tetanus toxin (TeTX) to prevent synaptic release rapidly retracted their axons, and that this retraction was driven by competition from newborn GCs lacking TeTX. In contrast, following 3-6 months of selective TeTX expression in old GCs of adult mice we did not observe any evidence of axon retraction. Indeed ultrastructural analyses indicated that the terminals of silenced GCs even maintained synaptic contact with their postsynaptic targets. Furthermore, we did not detect any significant differences in the electrophysiological properties between old GCs in control and TeTX conditions. Thus, our data demonstrate a remarkable stability in the face of a relatively prolonged period of altered synaptic competition between two populations of neurons within the adult brain. PMID:23162435

Lopez, Carla M; Pelkey, Kenneth A; Chittajallu, Ramesh; Nakashiba, Toshiaki; Tóth, Katalin; Tonegawa, Susumu; McBain, Chris J



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


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. PMID:20150664

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



Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells.  


von Willebrand factor (vWf) is secreted from endothelial cells by one of two pathways-a constitutive pathway and a regulated pathway originating from the Weibel-Palade bodies. The molecular form of vWf from each of these pathways differs, with the most biologically potent molecules being released from Weibel-Palade bodies (Loesberg, C., M. D. Gonsalves, J. Zandbergen, C. Willems, W. G. Van Aken, H. V. Stel, J. A. Van Mourik, and P. G. DeGroot. 1983. Biochim. Biophys. Acta. 763:160-168; Sporn, L. A., V. J. Marder, and D. D. Wagner. 1987. Cell. 46:185-190). We investigated the polarity of the two secretory pathways using human umbilical vein endothelial cells cultured on polycarbonate membrane filters which allowed sampling of media from both the apical and basolateral compartments. After metabolic labeling of cells, vWf (constitutively secreted during a 10-min period or released during a 10-min treatment with a secretagogue) was purified from the apical and basolateral chambers and subjected to gel analysis. Approximately equal amounts of vWf were constitutively secreted into both chambers, and therefore this secretory pathway appeared to be nonpolarized. On the contrary, an average of 90% of vWf released from Weibel-Palade bodies after treatment with the calcium ionophore A23187 or PMA appeared in the basolateral chamber, indicating that the regulated pathway of secretion is highly polarized. Thrombin, a secretagogue which promotes disruption of the endothelial monolayer, led to release of vWf from cells with no apparent polarity. The presence of microtubule-depolymerizing agents nocodazol and colchicine inhibited the polarized release of vWf. Ammonium chloride treatment did not disrupt the polarity of the regulated secretory pathway, indicating that maintenance of low pH in intracellular compartments was not required for the polarized delivery of preformed Weibel-Palade bodies to the plasma membrane. PMID:2494192

Sporn, L A; Marder, V J; Wagner, D D



Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells  

PubMed Central

von Willebrand factor (vWf) is secreted from endothelial cells by one of two pathways-a constitutive pathway and a regulated pathway originating from the Weibel-Palade bodies. The molecular form of vWf from each of these pathways differs, with the most biologically potent molecules being released from Weibel-Palade bodies (Loesberg, C., M. D. Gonsalves, J. Zandbergen, C. Willems, W. G. Van Aken, H. V. Stel, J. A. Van Mourik, and P. G. DeGroot. 1983. Biochim. Biophys. Acta. 763:160- 168; Sporn, L. A., V. J. Marder, and D. D. Wagner. 1987. Cell. 46:185- 190). We investigated the polarity of the two secretory pathways using human umbilical vein endothelial cells cultured on polycarbonate membrane filters which allowed sampling of media from both the apical and basolateral compartments. After metabolic labeling of cells, vWf (constitutively secreted during a 10-min period or released during a 10- min treatment with a secretagogue) was purified from the apical and basolateral chambers and subjected to gel analysis. Approximately equal amounts of vWf were constitutively secreted into both chambers, and therefore this secretory pathway appeared to be nonpolarized. On the contrary, an average of 90% of vWf released from Weibel-Palade bodies after treatment with the calcium ionophore A23187 or PMA appeared in the basolateral chamber, indicating that the regulated pathway of secretion is highly polarized. Thrombin, a secretagogue which promotes disruption of the endothelial monolayer, led to release of vWf from cells with no apparent polarity. The presence of microtubule- depolymerizing agents nocodazol and colchicine inhibited the polarized release of vWf. Ammonium chloride treatment did not disrupt the polarity of the regulated secretory pathway, indicating that maintenance of low pH in intracellular compartments was not required for the polarized delivery of preformed Weibel-Palade bodies to the plasma membrane.



Susceptibility of S49 lymphoma cell membranes to hydrolysis by secretory phospholipase A 2 during early phase of apoptosis  

Microsoft Academic Search

During cell death, plasma membranes of cells become vulnerable to attack by extracellular secretory phospholipase A2. The purpose of this study was to identify the timing of this phenomenon relative to other events that occur during the process of cell death. Death was induced in S49 murine lymphoma cells by treatment with dexamethasone, dibutyryl cAMP, ionomycin, thapsigargin, or heat shock

Kelli H. Nielson; Cari A. Olsen; Darin V. Allred; Kim L. O’Neill; Gregory F. Burton; John D. Bell



Adult treatment with methamphetamine transiently decreases dentate granule cell proliferation in the gerbil hippocampus  

Microsoft Academic Search

Summary.   The objective of the present study was to examine whether acute treatment with the recreational drug methamphetamine influences\\u000a adult granule cell proliferation in the dentate gyrus of the hippocampus. For that purpose, at the age of postnatal day 90\\u000a adult male gerbils (Meriones unguiculatus) received a single dose of either methamphetamine (25 mg\\/kg; i.p.) or saline. Proliferation\\u000a of granule

G. Teuchert-Noodt; R. R. Dawirs; K. Hildebrandt



Conditions required for polysynaptic excitation of dentate granule cells by area CA3 pyramidal cells in rat hippocampal slices.  


Under control conditions, stimulation of area CA3 pyramidal cells in slices can produce inhibitory postsynaptic potentials in granule cells by a polysynaptic pathway that is likely to involve hilar neurons [Muller W. and Misgeld U. (1990) J. Neurophysiol. 64, 46-56; Muller W. and Misgeld U. (1991) J. Neurophysiol. 65, 141-147; Scharfman H. E. (1993) Neurosci. Lett. 156, 61-66; Scharfman H. F. (1994) Neurosci. Lett. 168, 29-33]. When slices are disinhibited, excitatory postsynaptic potentials occur after the same stimulus [Sharfman H. E. (1994) J. Neurosci. 14, 6041-6057]. The excitatory postsynaptic potentials are likely to be mediated by pyramidal cells that innervate hilar mossy cells, which in turn innervate granule cells. [Scharfman H. F. (1994) J. Neurosci 14, 6041-6057]. These pathways are potentially important, because they could provide positive or negative feedback from area CA3 to the dentate gyrus. However, it is not clear when the CA3-mossy cell-granule cell excitatory pathway operates, because to date it has only been described in detail when GABA(A) receptors are blocked throughout the entire slice [Scharfman H. E. (1994) J. Neurosci 14, 6041-6057]. Furthermore, the monosynaptic excitatory synaptic connections between these cells have only been observed in the presence of bicuculline [Scharfman H. F. (1994) J. Neurophysiol. 72, 2167-2180; Scharfman H. E. (1995) J. Neurophysiol. 74, 179-194]. Yet in vivo data suggest that a CA3-mossy cell-granule cell excitatory pathway may be active under some physiological conditions, because granule cells discharge in association with sharp wave population bursts of CA3 [Ylinen A., et al. (1995) Hippocampus 5, 78-90]. To address whether the CA3-mossy cell-granule cell pathway occurs without global disinhibition of the slice, and where in the network disinhibition may be required, the effects of area CA3 stimulation on granule cells was examined after focal application of the GABAA receptor antagonist bicuculline to restricted areas of hippocampal slices. A micropipette containing 1 mM bicuculline was placed transiently either (i) in the area CA3 cell layer, (ii) the granule cell layer, (iii) the hilus, or (iv) more than one site in succession. If a small segment of the CA3 pyramidal cell layer or the hilus was disinhibited, or bicuculline was applied to both regions, area CA3 stimulation still evoked inhibitory postsynaptic potentials in granule cells. In fact, inhibitory postsynaptic potentials were enhanced under these conditions, probably because excitation of inhibitory cells was increased. When bicuculline was applied just to the area near an impaled granule cell, all inhibitory postsynaptic potentials evoked in that cell were blocked, but no underlying excitatory postsynaptic potential was uncovered. If bicuculline was applied focally to either area CA3 or the hilus and the impaled granule cell, CA3 stimulation subsequently evoked excitatory postsynaptic potentials in that granule cell, presumably because excitatory neurons innervating granule cells were disinhibited while the effects of inhibitory cells on granule cells were blocked. Excitatory postsynaptic potentials were produced without bicuculline application in three of seven cells, simply by stimulating the fimbria repetitively. Thus, if bicuculline is applied to different sites in the slice, different effects occur on the inhibitory postsynaptic potentials of granule cells that are evoked by a fimbria stimulus. If bicuculline is applied to both the granule cell soma and either area CA3 or the hilus, inhibitory postsynaptic potentials are reduced, and reveal that excitatory postsynaptic potentials can be produced by the same stimulus. (ABSTRACT TRUNCATED) PMID:9157312

Scharfman, H E



Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.  

PubMed Central

Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells.

Sanchez, J; Buendia, A J; Salinas, J; Bernabe, A; Rodolakis, A; Stewart, I J



Inflammatory microenvironment changes the secretory profile of mesenchymal stem cells to recruit mesenchymal stem cells.  


Background/Aims: Human bone-marrow mesenchymal stem cells (hBMSCs) are widely transplanted into inflammatory microenvironment to accelerate tissue regeneration. Transplanted hBMSCs recruit host hBMSCs through a poorly understood mechanism. This study was aimed to determine whether and how inflammatory microenvironment influenced the host-hBMSCs-recruiting capability of transplanted hBMSCs. Methods: Pro-inflammatory factors, including IL-1?, IL-6 and TNF-?, were utilized to mimic inflammatory microenvironment. hBMSCs were cultured and conditioned media (CM) were collected. The effects of inflammatory microenvironment on the host-hBMSCs-recruiting capability of cultured hBMSCs were revealed by transwell migration assays. Employing semi-quantitative and quantitative cytokine antibody assays, we examined the secretory profile of cultured hBMSCs. Results: CM from cultured hBMSCs exerted excellent host-hBMSCs-recruiting capability, which was significantly promoted by exposure to inflammatory microenvironment. Within inflammatory microenvironment, hBMSCs secreted more chemokines related to cell migration. Finally, 21 cytokines were verified as potential factors accounting for the enhanced host-hBMSCs-recruiting capability of cultured hBMSCs exposed to inflammatory microenvironment. Conclusion: These results strongly suggested that in clinic, inflammatory microenvironment might promote the host-hBMSCs-recruiting capacity of transplanted hBMSCs by increasing chemokines secretion. Modulation of such characteristics of hBMSCs might provide novel therapeutic ideas in the context of hBMSCs. © 2014 S. Karger AG, Basel. PMID:24713626

Xing, Junchao; Hou, Tianyong; Jin, Huiyong; Luo, Fei; Change, Zhengqi; Li, Zhiqiang; Xie, Zhao; Xu, Jianzhong



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

SciTech Connect

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.

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



In vitro three-dimensional modeling of fallopian tube secretory epithelial cells  

PubMed Central

Background Fallopian tube secretory epithelial cells (FTSECs) have been implicated as a cell-of-origin for high-grade serous epithelial ovarian cancer. However, there are relatively few in vitro models of this tissue type available for use in studies of FTSEC biology and malignant transformation. In vitro three-dimensional (3D) cell culture models aim to recreate the architecture and geometry of tissues in vivo and restore the complex network of cell-cell/cell-matrix interactions that occur throughout the surface of the cell membrane. Results We have established and characterized 3D spheroid culture models of primary FTSECs. FTSEC spheroids contain central cores of hyaline matrix surrounded by mono- or multi-layer epithelial sheets. We found that 3D culturing alters the molecular characteristics of FTSECs compared to 2D cultures of the same cells. Gene expression profiling identified more than a thousand differentially expressed genes between 3D and 2D cultures of the same FTSEC lines. Pathways significantly under-represented in 3D FTSEC cultures were associated with cell cycle progression and DNA replication. This was also reflected in the reduced proliferative indices observed in 3D spheroids stained for the proliferation marker MIB1. Comparisons with gene expression profiles of fresh fallopian tube tissues revealed that 2D FTSEC cultures clustered with follicular phase tubal epithelium, whereas 3D FTSEC cultures clustered with luteal phase samples. Conclusions This 3D model of fallopian tube secretory epithelial cells will advance our ability to study the underlying biology and etiology of fallopian tube tissues and the pathogenesis of high-grade serous epithelial ovarian cancer.



Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells  

Microsoft Academic Search

The relationship between changes in mitochondrial membrane potential (Dcm) and the failure of cytoplasmic Ca 21 homeostasis, delayed Ca 21deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetrameth- ylrhodamine methyl ester (TMRM 1) or rhodamine-123, we de- vised and validated a mathematical simulation with well

Manus W. Ward; A. Cristina Rego; Bruno G. Frenguelli; David G. Nicholls



A role for ADP-ribosylation factor 1, but not COP I, in secretory vesicle biogenesis from the trans-Golgi network  

Microsoft Academic Search

A synthetic N-myristoylated peptide corresponding to the amino-terminal domain of ADP-ribosylation factor 1 (ARF1) markedly increases, in a cell-free system using post-nuclear supernatant from PC12 cells, the biogenesis of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network (TGN). The related N-myristoylated ARF4 peptide only weakly stimulates, and the non-myristoylated ARF1 and ARF4 peptides inhibit, the biogenesis of

Francis A. Barr; Wieland B. Huttner



Cosecretion of secretory protein-I and parathormone by dispersed bovine parathyroid cells  

SciTech Connect

A RIA with a minimal sensitivity of 0.5 ng protein was developed for the measurement of bovine secretory protein-I (SP-I). With this assay and a previously established RIA for parathormone, the secretion and cell content of SP-I and parathormone were determined in dispersed bovine parathyroid cells. SP-I and parathormone were secreted in linear fashion over a 2-h period. The net secretion of both of these proteins diminished progressively as the concentration of calcium was raised from 0.25 mM to 3.0 mM. The molar ratio for the secreted proteins and those remaining in the cell varied from experiment to experiment but on average was 0.70 +/- 0.07 for the secreted proteins and 0.47 +/- 0.03 for the cellular proteins. Possible explanations for the difference in the ratio of SP-I to parathormone between cellular and secreted proteins include 1) a preferential secretion of SP-I; 2) a preferential intracellular degradation of SP-I; 3) a preferential postsecretory degradation of parathormone, or 4) differential affinities of potential fragments of either or both proteins for their antisera. These results suggest that SP-I and parathormone bear close but not identical metabolic and secretory fates.

Cohn, D.V. (Veterans Administration Medical Center, Kansas City, MO); Morrissey, J.J.; Shofstall, R.E.; Chu, L.L.H.



Modeling F-actin cortex influence on the secretory properties of neuroendocrine cells.  


Chromaffin cells are considered as one of the most valuable models to study regulated exocytosis. In these cells, like in other neuroendocrine systems, an intricate cortical cytoskeleton acts as a retentive network impeding vesicle access to plasma membrane. Therefore, during stimulation this structure suffers a transient reorganization allowing active transport of vesicles toward secretory sites. Interestingly, a combination of confocal microscopy studies and mathematical modeling is showing us new aspects of the influence of cortical cytoskeleton in shaping the secretory properties of excitable cells. In this new vision the F-actin-myosin II cortical cytoskeleton is organized forming polygonal cages with the molecular machinery of exocytosis composed by SNARE proteins and voltage-dependent calcium channels associating with its border. In this way the cytoskeleton not only holds together the essential elements acting during secretion, but we proposed that could also act as a structural factor opposing to the free diffusion of the calcium signal and therefore sustains high levels of the intracellular signal triggering exocytosis. PMID:21966558

Gutiérrez, Luis M; Gil, Amparo



Modeling F-actin cortex influence on the secretory properties of neuroendocrine cells  

PubMed Central

Chromaffin cells are considered as one of the most valuable models to study regulated exocytosis. In these cells, like in other neuroendocrine systems, an intricate cortical cytoskeleton acts as a retentive network impeding vesicle access to plasma membrane. Therefore, during stimulation this structure suffers a transient reorganization allowing active transport of vesicles toward secretory sites. Interestingly, a combination of confocal microscopy studies and mathematical modeling is showing us new aspects of the influence of cortical cytoskeleton in shaping the secretory properties of excitable cells. In this new vision the F-actin-myosin II cortical cytoskeleton is organized forming polygonal cages with the molecular machinery of exocytosis composed by SNARE proteins and voltage-dependent calcium channels associating with its border. In this way the cytoskeleton not only holds together the essential elements acting during secretion, but we proposed that could also act as a structural factor opposing to the free diffusion of the calcium signal and therefore sustains high levels of the intracellular signal triggering exocytosis.

Gil, Amparo



Senescence-associated secretory phenotype favors the emergence of cancer stem-like cells  

PubMed Central

The molecular mechanisms underlying cancer resistance remain elusive. One possible explanation is that cancer stem cells (CSCs) elude drug treatment, emerge and reproduce a tumor. Using multiple myeloma as a paradigm, we showed that cancer stem-like cells (CSLCs) appear after genotoxic stress because of their intrinsic properties. However, these properties do not drive the emergence of the CSLCs. Following genotoxic stress, remaining DNA damages lead to a senescence-associated secretory phenotype (SASP). Senescent cells, which are the non-CSLCs, secrete chemokines contributing to the emergence, maintenance and migration of CSLCs. Downregulation of checkpoint protein 2, a key player of SASP, significantly reduced the emergence of CSLCs. Our results unravel a novel molecular mechanism by which SASP might promote malignancy, underlining the dual role of senescence in tumorigenesis. This mechanism, based on mutual cooperation among tumor cells, illustrates how cancer may relapse; its targeting could represent new therapeutic opportunities.

Cahu, J; Bustany, S; Sola, B



Paneth cells of African giant rats (Cricetomys gambianus).  


The ultrastructure of Paneth cells of African giant rats (Cricetomys gambianus), which were captured on the savanna in western Africa, was studied. The Paneth cells of Cricetomys were clustered at the bottom of crypts of the small intestine, but not of the colon. In the normal state, Paneth cells had a few secretory granules showing high electron density. Small clear vesicles which are a characteristic in laboratory albino rats were not conspicuous. Vacuolated Paneth cells and secreted materials from the Paneth cells were frequently found. This suggests that the secretion of Paneth cells of Cricetomys is active in the natural state. No phagocytotic figures were observed. After atropine sulfate treatment, secretory granules increased in size and number, whereas the electron density decreased, similar to that of goblet cell granules. However, the granules were not stained by alcian blue or by PAS. Inhibition of secretory stimuli by atropine can alter the intracellular processing of secretory substances in Paneth cells. PMID:7879593

Satoh, Y; Ono, K; Moutairou, K



Dentate Gyrus Granule Cell Firing Patterns Can Induce Mossy Fiber Long-Term Potentiation In Vitro  

PubMed Central

Hippocampal granule cells transmit information about behaviourally-relevant stimuli to CA3 pyramidal cells via mossy fiber synapses. These synapses express a form of long-term potentiation (mfLTP) that is non-Hebbian and does not require NMDA receptors. mfLTP is thought to be induced and expressed presynaptically, hence, the major determinant of whether mfLTP occurs is activity in the granule cells. However, it remains unclear whether mfLTP can be induced by activity patterns that granule cells exhibit in vivo, and — if so — what context generates these patterns. To address these issues, we examined granule cell activity from in vivo recordings from rats during performance of a delayed nonmatch-to-sample (DNMS) task and found that granule cells exhibit a wide range of spike patterns. In vitro slice experiments in mice demonstrated that some, but not all, of these patterns of activity could induce mfLTP. By further defining the activity thresholds for mfLTP in hippocampal slices, we found that mfLTP can only be induced by spike patterns that fire in high frequency bursts with a low average firing frequency. Using this information, we then screened for supra-threshold bursts of activity during the DNMS task. In a subset of cells, supra-threshold bursts occurred preferentially during the sampling phase of the task. If supra-threshold bursting took place later, during the delay phase, task performance was disrupted. We conclude that mfLTP can be induced by granule cell spike patterns during a memory task, and that the timing of mfLTP induction can predict task performance.

Mistry, Rajen; Dennis, Siobhan; Frerking, Matthew; Mellor, Jack R.



A combined immuno-informatics and structure-based modeling approach for prediction of T cell epitopes of secretory proteins of Mycobacterium tuberculosis  

Microsoft Academic Search

The role of secretory proteins of Mycobacterium tuberculosis in pathogenesis and stimulation of specific host responses is well documented. They are also shown to activate different cell types, which subsequently present mycobacterial antigens to T cells. Therefore identification of T cell epitopes from this set of proteins may serve to define candidate antigens with vaccine potential. Fifty-two secretory proteins of

J. Vani; M. S. Shaila; N. R. Chandra; R. Nayak



Serpinin: a novel chromogranin A-derived, secreted peptide up-regulates protease nexin-1 expression and granule biogenesis in endocrine cells.  


Previously we demonstrated that chromogranin A (CgA) promoted secretory granule biogenesis in endocrine cells by stabilizing and preventing granule protein degradation in the Golgi, through up-regulation of expression of the protease inhibitor, protease nexin-1 (PN-1). However, the mechanism by which CgA signals the increase of PN-1 expression is unknown. Here we identified a 2.9-kDa CgA-C-terminus peptide, which we named serpinin, in conditioned media from AtT-20 cells, a corticotroph cell line, which up-regulated PN-1 mRNA expression. Serpinin was secreted from AtT-20 cells upon high potassium stimulation and increased PN-1 mRNA transcription in these cells, in an actinomycin D-inhibitable manner. CgA itself and other CgA-derived peptides, when added to AtT-20 cell media, had no effect on PN-1 expression. Treatment of AtT-20 cells with 10 nm serpinin elevated cAMP levels and PN-1 mRNA expression, and this effect was inhibited by a protein kinase A inhibitor, 6-22 amide. Serpinin and a cAMP analog, 8-bromo-cAMP, promoted the translocation of the transcription factor Sp1 into the nucleus, which is known to drive PN-1 expression. Additionally, an Sp1 inhibitor, mithramycin A inhibited the serpinin-induced PN-1 mRNA up-regulation. Furthermore, a luciferase reporter assay demonstrated serpinin-induced up-regulation of PN-1 promoter activity in an Sp1-dependent manner. When added to CgB-transfected 6T3 cells, a mutant AtT20 cell line, serpinin induced granule biogenesis as evidenced by the presence of CgB puncta accumulation in the processes and tips. Our findings taken together show that serpinin, a novel CgA-derived peptide, is secreted upon stimulation of corticotrophs and plays an important autocrine role in up-regulating PN-1-dependent granule biogenesis via a cAMP-protein kinase A-Sp1 pathway to replenish released granules. PMID:21436258

Koshimizu, Hisatsugu; Cawley, Niamh X; Kim, Taeyoon; Yergey, Alfred L; Loh, Y Peng



Inhibition of Fas-associated apoptosis in granulation tissue cells accompanies attenuation of postinfarction left ventricular remodeling by olmesartan  

Microsoft Academic Search

remarkable changes during the course of healing: necrotic tissue is infiltrated by inflammatory cells during the acute stage of MI, granulation tissue forms during the subacute stage, and scar tissue forms during the chronic stage (29, 33, 35). Most of the cell components that infiltrate and proliferate within the infarct, including inflammatory and granulation tissue cells, disappear via apoptosis during

Hiromitsu Kanamori; Genzou Takemura; Yiwen Li; Hideshi Okada; Rumi Maruyama; Takuma Aoyama; Shusaku Miyata; Masayasu Esaki; Atsushi Ogino; Munehiro Nakagawa; Hiroaki Ushikoshi; Masanori Kawasaki; Shinya Minatoguchi; Hisayoshi Fujiwara



Cellular geography of IP3 receptors, STIM and Orai: a lesson from secretory epithelial cells.  


Pancreatic acinar cells exhibit a remarkable polarization of Ca2+ release and Ca2+ influx mechanisms. In the present brief review, we discuss the localization of channels responsible for Ca2+ release [mainly IP3 (inositol 1,4,5-trisphosphate) receptors] and proteins responsible for SOCE (store-operated Ca2+ entry). We also place these Ca2+-transporting mechanisms on the map of cellular organelles in pancreatic acinar cells, and discuss the physiological implications of the cellular geography of Ca2+ signalling. Finally, we highlight some unresolved questions stemming from recent observations of co-localization and co-immunoprecipitation of IP3 receptors with Orai channels in the apical (secretory) region of pancreatic acinar cells. PMID:22260674

Dingsdale, Hayley; Voronina, Svetlana; Haynes, Lee; Tepikin, Alexei; Lur, Gyorgy



Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity  

PubMed Central

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.

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



Bone Marrow Cells Expressing Clara Cell Secretory Protein Increase Epithelial Repair After Ablation of Pulmonary Clara Cells  

PubMed Central

We have previously reported a subpopulation of bone marrow cells (BMC) that express Clara cell secretory protein (CCSP), generally felt to be specific to lung Clara cells. Ablation of lung Clara cells has been reported using a transgenic mouse that expresses thymidine kinase under control of the CCSP promoter. Treatment with ganciclovir results in permanent elimination of CCSP+ cells, failure of airway regeneration, and death. To determine if transtracheal delivery of wild-type bone marrow CCSP+ cells is beneficial after ablation of lung CCSP+ cells, transgenic mice were treated with ganciclovir followed by transtracheal administration of CCSP+ or CCSP? BMC. Compared with mice administered CCSP? cells, mice treated with CCSP+ cells had more donor cells lining the airway epithelium, where they expressed epithelial markers including CCSP. Although donor CCSP+ cells did not substantially repopulate the airway, their administration resulted in increased host ciliated cells, better preservation of airway epithelium, reduction of inflammatory cells, and an increase in animal survival time. Administration of CCSP+ BMC is beneficial after permanent ablation of lung Clara cells by increasing bronchial epithelial repair. Therefore, CCSP+ BMC could be important for treatment of lung diseases where airways re-epithelialization is compromised.

Bustos, Martha L; Mura, Marco; Marcus, Paula; Hwang, David; Ludkovski, Olga; Wong, Amy P; Waddell, Thomas K



Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells  

Microsoft Academic Search

Mitochondrial depolarisation has been reported to enhance the generation of superoxide anion (O??2) in a number of cell preparations while an inhibition has been observed with isolated mitochondria. Cerebellar granule cells equilibrated with >1 ?M hydroethidine (dihydroethidium) which is oxidised to the fluorescent ethidium cation by O??2 showed a large increase in fluorescence on protonophore addition. However, controls showed the

Samantha L. Budd; Roger F. Castilho; David G. Nicholls



Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation  

PubMed Central

We used a morphometric and autoradiographic approach to analyze changes in specific cytoplasmic granules and cytoplasmic lipid bodies associated with human lung mast cell degranulation. Mast cells were dissociated from lung tissue by enzymatic digestion and were then enriched to purities of up to 99% by countercurrent centrifugation elutriation and recovery from columns containing specific antigen bound to Sepharose 6 MB. Degranulation was induced by goat anti-IgE. At various intervals after stimulation, parallel aliquots of cells were recovered for determination of histamine release or were fixed for transmission electron microscopy. We found that lipid bodies, electron- dense structures that lack unit membranes, were present in both control and stimulated mast cells. Autoradiographic analysis showed that lipid bodies represented the major repository of 3H-label derived from [3H]arachidonic acid taken up from the external milieu. By contrast, the specific cytoplasmic granules contained no detectable 3H-label. In addition, lipid bodies occurred in intimate association with degranulation channels during mast cell activation, but the total volume of lipid bodies did not change during the 20 min after stimulation with anti-IgE. This result stands in striking contrast to the behavior of specific cytoplasmic granules, the great majority of which (77% according to aggregate volume) exhibited ultrastructural alterations during the first 20 min of mast cell activation. These observations establish that mast cell cytoplasmic granules and cytoplasmic lipid bodies are distinct organelles that differ in ultrastructure, biochemistry, and behavior during mast cell activation.



Responses of cells of the rat fascia dentata to prolonged stimulation of the perforant path: sensitivity of hilar cells and changes in granule cell excitability.  


Recent studies have shown that prolonged stimulation of afferents of the rat fascia dentata in vivo leads to the development of chronic epileptiform activity of the dentate granule cell region, and degeneration of certain cell types in the adjacent hilus. To investigate the development of dentate hyperexcitability and the selective vulnerability of hilar cells, the hippocampal slice preparation offers an in vitro model in which cellular mechanisms can be examined. We have recorded intracellularly from granule cells and hilar cells in tissue slices from rat before, during, and following sustained stimulation of the major afferent input to the dentate gyrus, the perforant path. Results from intracellular studies in slices were consistent with in vivo studies. Hilar cells were far more sensitive to short-term or prolonged perforant path stimulation than granule cells. At a time when the granule cell population response was not affected by prolonged stimulation, simultaneous recordings from hilar cells and some granule layer interneurons showed that these cells were already depolarized, had very low input resistance, and showed other electrophysiological changes indicative of deterioration. In contrast, granule cells generally hyperpolarized during stimulation and their input resistance increased; no signs of injury were evident in granule cells. Some stimulus-induced changes in the physiological characteristics of granule cells, such as decreased spike frequency adaptation and reduced inhibitory postsynaptic potentials, may contribute to the development of dentate hyperexcitability. PMID:2381513

Scharfman, H E; Schwartzkroin, P A




PubMed Central

In the previous paper we presented findings which indicated that enzyme heterogeneity exists among PMN leukocyte granules. From histochemical staining of bone marrow smears, we obtained evidence that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appeared to be restricted to one of the two types. Clear results were obtained with alkaline phosphatase, but those with a number of other enzymes were suggestive rather than conclusive. Since the approach used previously was indirect, it was of interest to localize the enzymes directly in the granules. Toward this end, we carried out cytochemical procedures for five enzymes on normal rabbit bone marrow cells which had been fixed and incubated in suspension. The localization of reaction product in the granules was determined by electron microscopy. In accordance with the results obtained on smears, azurophil granules were found to contain peroxidase and three lysosomal enzymes: acid phosphatase, arylsulfatase, and 5'-nucleotidase; specific granules were found to contain alkaline phosphate. Specific granules also contained small amounts of phosphatasic activity at acid pH. Another finding was that enzyme activity could not be demonstrated in mature granules with metal salt methods (all except peroxidase); reaction product was seen only in immature granules. The findings confirm and extend those obtained previously, indicating that azurophil granules correspond to lysosomes whereas specific granules represent a different secretory product.

Bainton, Dorothy Ford; Farquhar, Marilyn G.



[The ultrastructural patterns of germinal and yolk granules in oocytes and embryonic cells of the holothurian Apostichopus japonicus].  


The yolk germinal granules in oocytes and embryonic cells of Apostichopus japonicus were studied by transmission electron microscopy. Analysis of the features of synthesis and utilization of yolk granules made it possible to reveal ultrastructural criteria to distinguish between granules of the forming and utilized yolk, and germinal granules. Based on these findings, the authors suppose that identification of germ plasm elements in oocytes and embryonic cells of A. japonicus is quite possible with ultrastructural analysis only, and does not require utilizing molecular markers. PMID:16841492

Reunov, A A; Aleksandrova, Ia N



[The ultrastructural patterns of germinal and yolk granules in oocytes and embryonic cells of the holothurian Apostichopus japonicus].  


The yolk germinal granules in oocytes and embryonic cells of Apostichopus japonicus were studied by transmission electron microscopy. Analysis of the features of synthesis and utilization of yolk granules made it possible to reveal ultrastructural criteria to distinguish between granules of the forming and utilized yolk, and germinal granules. Based on these findings, the authors suppose that identification of germ plasm elements in oocytes and embryonic cells of A. japonicus is quite possible with ultrastructural analysis only, and does not require utilizing molecular markers. PMID:16568835

Reunov, A A; Aleksandrova, Ia N



Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells  

PubMed Central

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:

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



Actin cytoskeleton reorganization correlates with polarization of secretory vesicle and cell morphology in the degranulation of mast cell subtypes in human colon tissues.  


Mast cells play a central role in the intestinal immune response. To investigate the relationship between degranulation, cell polarization and the reorganization of actin cytoskeleton of mast cells, we used fluorescence or gold labeling methods to identify different mast cell subtypes in human colon. The reorganization of filamentous actin was visualized and then the polarization of secretory vesicles, as well as cell surfaces, was analyzed by fluorescence microscopy and electron microscopy. Our results first showed a diversity of filamentous actin assembly or disassembly within the contacting cell membrane of different mast cell subtypes. The polarization and degranulation of secretory vesicles was not only accompanied with the assembly and disassembly of filamentous actin at the cell periphery, but also with changes of cell surface polarization. Our study provides an insight into the local membranous structures and suggested correlations of cytoskeleton arrangement with the polarization of secretory vesicles and cell surface configuration during mast cell degranulation. PMID:24161690

Lin, Jue-Long; Chen, Chun-Gui; Shen, Zhi-Zhong; Piao, Zhong-Xian; Li, Wei-Qiu; Liu, Liu; Xu, Li-Yan; Li, En-Min



Barhl1 Regulates Migration and Survival of Cerebellar Granule Cells by Controlling Expression of the Neurotrophin3 Gene  

Microsoft Academic Search

The neurons generated at the germinal rhombic lip undergo long distance migration along divergent pathways to settle in widely dispersed locations within the hindbrain, giving rise to cerebellar granule cells and precerebellar nuclei. Neurotrophin-3 (NT-3) signaling has been shown to be required for proper migration and survival of cerebellar granule cells. The molecular bases that govern NT-3 expression within the

Shengguo Li; Feng Qiu; Anlong Xu; Sandy M. Price; Mengqing Xiang



The plant secretory pathway: an essential factory for building the plant cell wall.  


For building and maintaining the complex structure of the surrounding wall throughout their life, plant cells rely on the endomembrane system, which functions as the main provider and transporter of cell wall constituents. Efforts to understand the mechanisms of synthesis and transport of cell wall materials have been generating valuable information for diverse practical applications. Nonetheless, the identity of the endomembrane components necessary for the transport of cell wall enzymes and polysaccharides is not well known. Evidence indicates that plant cells can accomplish secretion of cell wall constituents through multiple pathways during development or under stress conditions and, that compared with other eukaryotes, they rely on a highly diversified toolkit of proteins for membrane traffic. This suggests that production of the cell wall in plants consists of intricate and highly regulated pathways. In this review, we summarize important discoveries that have allowed the activities of the plant secretory pathway to be linked to the production and deposition of cell wall-synthesizing enzymes and polysaccharides. PMID:24401957

Kim, Sang-Jin; Brandizzi, Federica



Synaptic connections of dentate granule cells and hilar neurons: results of paired intracellular recordings and intracellular horseradish peroxidase injections.  


Simultaneous intracellular recordings were made in the dentate gyrus of rat hippocampal slices, from pairs of the following cell types: granule cells, interneurons located in the granule cell layer, hilar interneurons, and spiny hilar "mossy cells". Granule cells were found to have strong excitatory effects on mossy cells and interneurons. Interneurons inhibited granule cells as well as other interneurons. No synaptic connections from mossy cells onto other cell types were found, within the confines of the slice, using intracellular recording methods. However, at the ultrastructural level, axon terminals of horseradish peroxidase-filled mossy cells were found making synaptic contacts in the hilus on dendrites of interneurons. These studies provide the first step towards determining the functional interactions of the various cell types in the fascia dentata. PMID:2247219

Scharfman, H E; Kunkel, D D; Schwartzkroin, P A



Quantitative analysis of granule cell axons and climbing fiber afferents in the turtle cerebellar cortex  

PubMed Central

The turtle cerebellar cortex is a single flat sheet of gray matter that greatly facilitates quantitative analysis of biotylinated dextran amine labeled granule cell and olivocerebellar axons and Nissl-stained granule and Purkinje neurons. On average, ascending granule cell axons are relatively thicker than their parallel fiber branches (mean±SD: 0.84±0.17 vs 0.64±0.12 µm, respectively). Numerous en passant swellings, the site of presynaptic contact, were present on both ascending and parallel fiber granule cell axons. The swellings on ascending axons (1.82±0.34 µm, n=52) were slightly larger than on parallel fibers (1.43±0.24 µm, n=430). In addition, per unit length (100 µm) there were more swellings on ascending axons (11.2±4.2) than on parallel fibers (9.7±4.2). Each parallel fiber branch from an ascending axon is approximately 1.5 mm long. Olivocerebellar climbing fiber axons followed the highly tortuous dendrites of Purkinje cells in the inner most 15–20% of the molecular layer. Climbing fibers displayed relatively fewer en passant swellings. The spatial perimeter of climbing fiber arbors (area) increased 72% from anteriorly (1797 µm²) to posteriorly (3090 µm²) and 104% from medially (1690 µm²) to laterally (3450 µm²). Differences in the size and spacing of en passant swellings on granule cell axons suggest that ascending axons may have a functionally more significant impact on the excitability of a limited number of radially overlying Purkinje cells than the single contacts by parallel fiber with multiple orthogonally aligned Purkinje cell dendrites. The spatially restricted distribution of climbing fibers to the inner most molecular layer, the paucity of en passant swellings, and different terminal arbor areas are enigmatic. Nevertheless, these finding provide important anatomical information for future optical imaging and electrophysiological experiments.

Tolbert, D. L.; Conoyer, B.; Ariel, M.



RAB26 and RAB3D Are Direct Transcriptional Targets of MIST1 That Regulate Exocrine Granule Maturation? †  

PubMed Central

Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. MIST1, an evolutionarily conserved transcription factor, is required for the formation of large, specialized secretory vesicles in gastric zymogenic (chief) cells (ZCs) as they differentiate from their mucous neck cell progenitors. Here, we show that MIST1 binds to highly conserved CATATG E-boxes to directly activate transcription of 6 genes, including those encoding the small GTPases RAB26 and RAB3D. We next show that RAB26 and RAB3D expression is significantly downregulated in Mist1?/? ZCs, suggesting that MIST1 establishes large secretory granules by inducing RAB transcription. To test this hypothesis, we transfected human gastric cancer cell lines stably expressing MIST1 with red fluorescent protein (RFP)-tagged pepsinogen C, a key secretory product of ZCs. Those cells upregulate expression of RAB26 and RAB3D to form large secretory granules, whereas control, non-MIST1-expressing cells do not. Moreover, granule formation in MIST1-expressing cells requires RAB activity because treatment with a RAB prenylation inhibitor and transfection of dominant negative RAB26 abrogate granule formation. Together, our data establish the molecular process by which a transcription factor can directly induce fundamental cellular architecture changes by increasing transcription of specific cellular effectors that act to organize a unique subcellular compartment.

Tian, Xiaolin; Jin, Ramon U.; Bredemeyer, Andrew J.; Oates, Edward J.; Blazewska, Katarzyna M.; McKenna, Charles E.; Mills, Jason C.



Heterogeneous integration of adult-generated granule cells into the epileptic brain  

PubMed Central

The functional impact of adult-generated granule cells in the epileptic brain is unclear, with data supporting both protective and maladaptive roles. These conflicting findings could be explained if new granule cells integrate heterogeneously, with some cells taking neutral or adaptive roles, while others contribute to recurrent circuitry supporting seizures. Here, we tested this hypothesis by completing detailed morphological characterizations of age- and experience-defined cohorts of adult-generated granule cells from transgenic mice. The majority of newborn cells exposed to an epileptogenic insult exhibited reductions in dendritic spine number, suggesting reduced excitatory input to these cells. A significant subset, however, exhibited higher spine numbers. These latter cells tended to have enlarged cell bodies, long basal dendrites or both. Moreover, cells with basal dendrites received significantly more recurrent mossy fiber input through their apical dendrites, indicating that these cells are robustly integrated into the pathological circuitry of the epileptic brain. These data imply that newborn cells play complex – and potentially conflicting – roles in epilepsy.

Murphy, Brian L.; Pun, Raymund Y.K.; Yin, Hulian; Faulkner, Christian R.; Loepke, Andreas W.; Danzer, Steve C.



Early Neural Grafts Transiently Reduce the Behavioral Effects of Radiation-Induced Fascia Dentata Granule Cell Hypoplasia. (Reannouncement with New Availability Information).  

National Technical Information Service (NTIS)

It is possible to produce selective hypoplasia of the hippocampal granule cells by X-irradiating the partially shielded rat brain. Specificity of damage is assured by conducting the radiation exposure when fascia dentata granule cells are mitotic, but adj...

G. A. Mickley J. L. Ferguson M. A. Mulvihill T. J. Nemeth



Isoprenoid biosynthesis. Metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis.  


Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides. PMID:11553758

Lange, B M; Ketchum, R E; Croteau, R B



Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells  

Microsoft Academic Search

Previous studies have suggested a role for glucocorticoids in the differentiation of the acinar pancreas. We have now used the rat tumor cell line AR42J, derived from the acinar pancreas, to directly study this effect of glucocorticoids in vitro. The steroid hormones dexamethasone, corticosterone, aldosterone, and progesterone, but not estrogen, increased both the amylase content and the number of secretory




Exocytosis of neutrophil granule subsets and activation of prolyl isomerase 1 are required for respiratory burst priming.  


This study tested the hypothesis that priming the neutrophil respiratory burst requires both granule exocytosis and activation of the prolyl isomerase Pin1. Fusion proteins containing the TAT cell permeability sequence and either the SNARE domain of syntaxin-4 or the N-terminal SNARE domain of SNAP-23 were used to examine the role of granule subsets in TNF-mediated respiratory burst priming using human neutrophils. Concentration-inhibition curves for exocytosis of individual granule subsets and for priming of fMLF-stimulated superoxide release and phagocytosis-stimulated H2O2 production were generated. Maximal inhibition of priming ranged from 72 to 88%. Linear regression lines for inhibition of priming versus inhibition of exocytosis did not differ from the line of identity for secretory vesicles and gelatinase granules, while the slopes or the y-intercepts were different from the line of identity for specific and azurophilic granules. Inhibition of Pin1 reduced priming by 56%, while exocytosis of secretory vesicles and specific granules was not affected. These findings indicate that exocytosis of secretory vesicles and gelatinase granules and activation of Pin1 are independent events required for TNF-mediated priming of neutrophil respiratory burst. PMID:23363774

McLeish, Kenneth R; Uriarte, Silvia M; Tandon, Shweta; Creed, Timothy M; Le, Junyi; Ward, Richard A



Exocytosis of Neutrophil Granule Subsets and Activation of Prolyl Isomerase 1 are required for Respiratory Burst Priming  

PubMed Central

This study tested the hypothesis that priming the neutrophil respiratory burst requires both granule exocytosis and activation of the prolyl isomerase, Pin1. Fusion proteins containing the TAT cell permeability sequence and either the SNARE domain of syntaxin-4 or the N-terminal SNARE domain of SNAP-23 were used to examine the role of granule subsets in TNF-mediated respiratory burst priming using human neutrophils. Concentration-inhibition curves for exocytosis of individual granule subsets and for priming of fMLF-stimulated superoxide release and phagocytosis-stimulated H2O2 production were generated. Maximal inhibition of priming ranged from 72% to 88%. Linear regression lines for inhibition of priming versus inhibition of exocytosis did not differ from the line of identity for secretory vesicles and gelatinase granules, while the slopes or the y-intercepts were different from the line of identity for specific and azurophilic granules. Inhibition of Pin1 reduced priming by 56%, while exocytosis of secretory vesicles and specific granules was not affected. These findings indicate that exocytosis of secretory vesicles and gelatinase granules and activation of Pin1 are independent events required for TNF-mediated priming of neutrophil respiratory burst.

McLeish, Kenneth R.; Uriarte, Silvia M.; Tandon, Shweta; Creed, Timothy M.; Le, Junyi; Ward, Richard A.



Imaging activation of adult-generated granule cells in spatial memory  

Microsoft Academic Search

New neurons are continuously generated in the subgranular zone of the hippocampus throughout adulthood, and there is increasing interest as to whether these new neurons become functionally integrated into memory circuits. This protocol describes the immunohistochemical procedures to visualize the recruitment of new neurons into circuits supporting spatial memory in intact mice. To label adult-generated granule cells, mice are injected

Nohjin Kee; Cátia M Teixeira; Afra H Wang; Paul W Frankland



Structural, mass and elemental analyses of storage granules in methanogenic archaeal cells  

PubMed Central

Summary Storage granules are an important component of metabolism in many organisms spanning the bacterial, eukaryal and archaeal domains, but systematic analysis of their organization inside cells is lacking. In this study, we identify and characterize granulelike inclusion bodies in a methanogenic archaeon, Methanospirillum hungatei, an anaerobic microorganism that plays an important role in nutrient recycling in the ecosystem. Using cryo electron microscopy, we show that granules in mature M. hungatei are amorphous in structure with a uniform size. Energy dispersive X-ray spectroscopy analysis establishes that each granule is a polyphosphate body (PPB) that consists of high concentrations of phosphorous and oxygen, and increased levels of iron and magnesium. By scanning transmission electron tomography, we further estimate that the mass density within a PPB is a little less than metal titanium at room temperature and is about four times higher than that of the surrounding cytoplasm. Finally, three-dimensional cryo electron tomography reveals that PPBs are positioned off-centre in their radial locations relative to the cylindrical axis of the cell, and almost uniformly placed near cell ends. This positioning ability points to a genetic program that spatially and temporally directs the accumulation of polyphosphate into a storage granule, perhaps for energy-consuming activities, such as cell maintenance, division or motility.

Toso, Daniel B.; Henstra, Anne M.; Gunsalus, Robert P.; Zhou, Z. Hong



Colchicine induced intraneuronal free zinc accumulation and dentate granule cell degeneration.  


Colchicine has been discovered to inhibit many inflammatory processes such as gout, familial Mediterranean fever, pericarditis and Behcet disease. Other than these beneficial anti-inflammatory effects, colchicine blocks microtubule-assisted axonal transport, which results in the selective loss of dentate granule cells of the hippocampus. The mechanism of the colchicine-induced dentate granule cell death and depletion of mossy fiber terminals still remains unclear. In the present study, we hypothesized that colchicine-induced dentate granule cell death may be caused by accumulation of labile intracellular zinc. 10 ?g kg(-1) of colchicine was injected into the adult rat hippocampus and then brain sections were evaluated at 1 day or 1 week later. Neuronal cell death was evaluated by H&E staining or Fluoro-Jade B. Zinc accumulation and vesicular zinc were detected by N-(6-methoxy-8-quinolyl)-para-toluene sulfonamide (TSQ) staining. To test whether an extracellular zinc chelator can prevent this process, CaEDTA was injected into the hippocampus over a 5 min period with colchicine. To test whether other microtubule toxins also produce similar effects as colchicine, vincristine was injected into the hippocampus. The present study found that colchicine injection induced intracellular zinc accumulation in the dentate granule cells and depleted vesicular zinc from mossy fiber terminals. Injection of a zinc chelator, CaEDTA, did not block the zinc accumulation and neuronal death. Vincristine also produced intracellular zinc accumulation and neuronal death. These results suggest that colchicine-induced dentate granule cell death is caused by blocking axonal zinc flow and accumulation of intracellular labile zinc. PMID:24874779

Choi, Bo Young; Lee, Bo Eun; Kim, Jin Hee; Kim, Hyun Jung; Sohn, Min; Song, Hong Ki; Chung, Tae Nyoung; Suh, Sang Won



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)

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.

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.



Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.  

PubMed Central

Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway. Two- to three-fold overexpression of CgB markedly reduced the constitutive secretion of the 23-kDa fragment, but not beta-lipotropin and tripled the amount of adrenocorticotropin generated and stored in secretory granules. Our results indicate the existence of neuroendocrine-specific helper proteins which promote the sorting from the trans-Golgi network to secretory granules of certain processing intermediates derived from peptide hormone and neuropeptide precursors and demonstrate that CgB functions as such. Images Fig. 2 Fig. 4 Fig. 5

Natori, S; Huttner, W B



Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.  


Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway. Two- to three-fold overexpression of CgB markedly reduced the constitutive secretion of the 23-kDa fragment, but not beta-lipotropin and tripled the amount of adrenocorticotropin generated and stored in secretory granules. Our results indicate the existence of neuroendocrine-specific helper proteins which promote the sorting from the trans-Golgi network to secretory granules of certain processing intermediates derived from peptide hormone and neuropeptide precursors and demonstrate that CgB functions as such. PMID:8633084

Natori, S; Huttner, W B



Dendritic caliber and the 3/2 power relationship of dentate granule cells.  


A quantitative examination of granule cell dendritic caliber and knowledge of dendritic lengths allows assessment of the distribution of dendritic membrane and the 3/2 power relationship at branch points. This paper presents caliber data of Golgi-impregnated rat dentate gyrus. We used camera lucida drawings of the dendritic trees of 15 dorsal leaf and 15 ventral leaf granule cells to quantify mean dendritic caliber, dendritic taper, the 3/2 power relationship of parent and sibling dendritic diameters at branch points, and surface area. First-order dendrites vary substantially in diameter. However, the mean caliber of all other dendrites is uniform across the proximal two-thirds of the molecular layer for the dorsal and ventral leaves. The average diameter here is 1 micron. More distally, only mean ventral leaf dendritic caliber declines. Granule cell dendritic taper is due primarily to caliber decreases at branch points and not to a gradual decline in diameter across the length of a dendritic segment. Comparing the parent segment diameter raised to the 3/2 power with the sum of the 3/2 powers of the two sibling segment diameters reveals, for the dendritic tree located within the distal two-thirds of the molecular layer, the desired 3/2 power relationship for the dorsal and ventral leaves. More proximally, where first-, second-, and third-order dendrites branch sequentially across a 60-100-micron extent, a 3/2 power relationship is not obtained. For the average dorsal leaf granule cell, dendritic surface area (without spines) is 11,984 micron2. The ratio of dendritic to somatic surface area is 28:1. Discussion of these data includes their implications for electrotonic modeling of the dentate granule cell. PMID:6470225

Desmond, N L; Levy, W B



Increased neurogenesis and the ectopic granule cells after intrahippocampal BDNF infusion in adult rats.  


There is evidence that BDNF influences the birth of granule cells in the dentate gyrus, which is one of the few areas of the brain that demonstrates neurogenesis throughout life. However, studies to date have not examined this issue directly. To do so, we compared the effects of BDNF, phosphate-buffered saline (PBS), or bovine serum albumin (BSA) on neurogenesis after infusion into the hippocampus of the normal adult rat, using osmotic pumps that were implanted unilaterally in the dorsal hilus. BDNF, PBS, and BSA were infused for 2 weeks. The mitotic marker bromodeoxyuridine (BrdU) was administered twice daily during the 2-week infusion period. At least 1 month after infusion ended, brains were processed immunocytochemically using antibodies to BrdU, a neuronal nuclear protein (NeuN), or calbindin D28K (CaBP), which labels mature granule cells. Stereology was used to quantify BrdU-labeled cells in the dorsal hippocampus that were double-labeled with NeuN or CaBP. There was a statistically significant increase in BrdU(+)/NeuN(+) double-labeled cells in the granule cell layer after BDNF infusion relative to controls. The values for BrdU(+)/NeuN(+) cells were similar to BrdU(+)/CaBP(+) cells, indicating that most new neurons were likely to be granule cells. In addition, BrdU(+)/NeuN(+)-labeled cells developed in the hilar region after BDNF infusion, which have previously only been identified after severe continuous seizures (status epilepticus) and associated pathological changes. Remarkably, neurogenesis was also increased contralaterally, but BDNF did not appear to spread to the opposite hemisphere. Thus, infusion of BDNF to a local area can have widespread effects on hippocampal neurogenesis. The results demonstrate that BDNF administration to the dentate gyrus leads to increased neurogenesis of granule cells. They also show that ectopic granule cells develop after BDNF infusion, which suggests that ectopic migration is not necessarily confined to pathological conditions. These results are discussed in light of the evidence that BDNF increases neuronal activity in hippocampus. Thus, the mechanisms underlying neurogenesis following BDNF infusion could be due to altered activity as well as direct effects of BDNF itself, and this is relevant to studies of other growth factors because many of them have effects on neuronal excitability that are often not considered. PMID:15755552

Scharfman, Helen; Goodman, Jeffrey; Macleod, Adam; Phani, Sudar; Antonelli, Cara; Croll, Susan



Maturation of postnatally generated olfactory bulb granule cells depends on functional ?-protocadherin expression  

PubMed Central

?-protocadherins (?-pcdhs) are transmembrane receptor proteins ubiquitously expressed in the postnatal and adult mouse brain. ?-pcdhs are required for normal neuronal development as shown for spinal cord interneurons, retinal ganglion cells and cortical neurons. To test the role of ?-pcdhs during development of subventricular zone progenitor cells and their subsequent differentiation into olfactory granule cells we generated a conditional ?-pcdhlox/lox allele (?-pcdhlox/lox) allowing for functional ?-pcdh inactivation upon lentivirus-mediated Cre-recombinase expression selectively in subventricular zone progenitor cells. While ?-pcdh loss did not alter the proliferation of subventricular zone progenitors, ?-pcdh ko progenitors that reached the main olfactory bulb showed a significant reduction in dendritic arborization and failed to develop dendritic spines. Our results suggest that olfactory bulb granule cell maturation necessitates functional ?-pcdh expression.

Ledderose, Julia; Dieter, Sandra; Schwarz, Martin K.



Effect of reconstituted basement membrane on growth and secretory function in pancreatic acinar AR42J cells  

Microsoft Academic Search

Summary  This study investigated the influence of extracellular matrix on growth and secretory function associated with cholecystokinin\\u000a (CCK) receptors in pancreatic acinar AR42J cells, using reconstituted basement membrane derived from Engelbreth-Holm-Swarm\\u000a (EHS) tumor. The cells were cultured with basement membranes of two different thicknesses, 1 mg\\/ml protein equivalent (thick\\u000a RBM) and 50 ?g\\/ml protein equivalent (thin RBM). In cells cultured with

Yutaka Shintani; Tadao Bamba; Hisayuki Inoue; Shiro Hosoda



Ca(2+) signaling and fluid secretion by secretory cells of the airway epithelium.  


Cytoplasmic Ca(2+) is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca(2+) in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca(2+). Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca(2+) as a second messenger. Changes in intracellular Ca(2+) concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca(2+)-activated K(+) channels and Cl(-) channels. We also review evidence of interactions of Ca(2+) signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca(2+) signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways. PMID:24703093

Lee, Robert J; Foskett, J Kevin



Immunohistological comparison of granulated cell proteins in induced immediate urticarial dermographism and delayed pressure urticaria lesions.  


Urticarial dermographism and delayed pressure urticaria are two forms of physical urticaria which are well defined clinically and histologically. Previous studies have shown eosinophil granule protein deposition in urticarial reactions, including chronic urticaria, solar urticaria and delayed pressure urticaria. To evaluate and compare the involvement of granulated inflammatory cells in urticarial dermographism and delayed pressure urticaria, we studied sequential biopsies of induced lesions of urticarial dermographism and delayed pressure urticaria by indirect immunofluorescence, to detect eosinophil granule major basic protein (MBP) and neutrophil granule elastase. Biopsies from dermographic lesions at time 0, 5 min, 15 min, 2 h and 24 h, showed few infiltrating eosinophils, with minimal extracellular MBP deposition, and a few infiltrating neutrophils, with minimal neutrophil elastase deposition, throughout the evolution of the lesions. Sequential biopsies of delayed pressure urticaria at time 0, 20 min, 6, 12 and 24 h, showed eosinophil infiltration with extensive MBP deposition beginning at 20 min, and neutrophil infiltration with variable elastase deposition beginning at 20 min. Control tissue specimens from normal volunteers showed neutrophil infiltration and slight degranulation, but no eosinophil infiltration or degranulation. Comparison of urticarial dermographism with delayed pressure urticaria showed marked differences in the patterns of infiltration. Delayed pressure urticaria, with eosinophil and neutrophil degranulation, was strikingly similar to the IgE-mediated late phase reaction. In contrast, eosinophil and neutrophil involvement in urticarial dermographism was minimal. Considering the extent of eosinophil granule protein deposition and the biological activities of the eosinophil granule proteins, the findings in delayed pressure urticaria point to an important pathophysiological role of eosinophils in the disease. PMID:8547035

McEvoy, M T; Peterson, E A; Kobza-Black, A; English, J S; Dover, J S; Murphy, G M; Bhogal, B; Greaves, M W; Winkelmann, R K; Leiferman, K M



Regulators of Cerebellar Granule Cell Development Act Through Specific Signaling Pathways  

NSDL National Science Digital Library

The proper development of the central nervous system depends upon a finely tuned balance between cell proliferation and programmed cell death (PCD). Although PCD was initially believed to depend solely on the inability of certain neurons to obtain access to a limited supply of trophic factors, it has become apparent that the local production of death signals is also critical. In this Viewpoint, we discuss several pathways implicated in the survival of cerebellar granule cellsâ both pathways that protect from apoptosis and pathways that promote apoptosisâÂÂand describe how these disparate pathways converge on the final common mediators of PCD. Information on other important pathways implicated in granule cell survival may be found in the Connections Maps.

David Vaudry (European Institute for Peptide Research ;Laboratoryof Cellular and Molecular Neuroendocrinology); Anthony Falluel-Morel (European Institute for Peptide Research ;Laboratoryof Cellular and Molecular Neuroendocrinology); Sébastien Leuillet (European Institute for Peptide Research ;Laboratoryof Cellular and Molecular Neuroendocrinology); Hubert Vaudry (European Institute for Peptide Research ;Laboratoryof Cellular and Molecular Neuroendocrinology); Bruno Gonzalez (European Institute for Peptide Research ;Laboratoryof Cellular and Molecular Neuroendocrinology)



Interleukin10 Prevents Glutamate-Mediated Cerebellar Granule Cell Death by Blocking Caspase3Like Activity  

Microsoft Academic Search

Interleukin-10 (IL-10) has been shown to reduce neuronal de- generation after CNS injury. However, the molecular mecha- nisms underlying the neuroprotective properties of this cytokine are still under investigation. Glutamate exacerbates secondary injury caused by trauma. Thus, we examined whether IL-10 prevents glutamate-mediated cell death. We used rat cerebellar granule cells in culture because these neurons undergo apo- ptosis upon

Alessia Bachis; Anna M. Colangelo; Stefano Vicini; Pylord P. Doe; Maria A. De Bernardi; Gary Brooker; Italo Mocchetti



Dynamic changes of [Ca 2+ ] i in cerebellar granule cells exposed to pulsed electric fields  

Microsoft Academic Search

Intracellular free Ca2+ concentration ([Ca2+]i) in embryonic chick cerebellar granule cells loaded with fluo-3\\/AM and exposed to a single pulsed electric field was investigated\\u000a using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system. The results\\u000a showed that [Ca2+]i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed

Ya Chen; Yan Wang; Tong Sun; Jinzhu Zhang; Xianghong Jing; Ruiwu Li



Glutamate-induced protein phosphorylation in cerebellar granule cells: Role of protein kinase C  

Microsoft Academic Search

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells.32P-labelled cells have been stimulated with 100 µM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic

Maria Luisa Eboli; Delio Mercanti; Maria Teresa Ciotti; Angelo Aquino; Loriana Castellani



Topiramate attenuates voltage-gated sodium currents in rat cerebellar granule cells  

Microsoft Academic Search

Whole-cell, voltage-clamp recordings were made from rat cerebellar granule cells in culture under experimental conditions designed to study voltage-gated Na+ currents that were elicited by depolarizing commands from a holding potential of ?60 mV up to +20 mV. These tetrodotoxin-sensitive inward currents were reduced in a dose-related manner by bath application of the structurally novel, anticonvulsant drug topiramate (10–1000 ?M;

Cristina Zona; Maria Teresa Ciotti; Massimo Avoli



Constitutive and basal secretion from the endocrine cell line, AtT-20  

PubMed Central

A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.



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


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. PMID:19899443

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



Stichopin-containing nerves and secretory cells specific to connective tissues of the sea cucumber.  


Stichopin, a 17-amino acid peptide isolated from a sea cucumber, affects the stiffness change of the body-wall catch connective tissues and the contraction of the body-wall muscles. The localization of stichopin in sea cucumbers was studied by indirect immunohistochemistry using antiserum against stichopin. Double staining was performed with both stichopin antiserum and 1E11, the monoclonal antibody specific to echinoderm nerves. A stichopin-like immunoreactivity (stichopin-LI) was exclusively found in the connective tissues of various organs. Many fibres and cells with processes were stained by both the anti-stichopin antibody and 1E11. They were found in the body-wall dermis and the connective tissue layer of the cloacae and were suggested to be connective tissue-specific nerves. Oval cells with stichopin-LI (OCS) without processes were found in the body-wall dermis, the connective tissue sheath of the longitudinal body-wall muscles, the connective tissue layer of the tube feet and tentacles, and the connective tissue in the radial nerves separating the ectoneural part from the hyponeural part. Electron microscopic observations of the OCSs in the radial nerves showed that they were secretory cells. The OCSs were located either near the well-defined neural structures or near the water-filled cavities, such as the epineural sinus and the canals of the tube feet. The location near the water-filled cavities might suggest that stichopin was secreted into these cavities to function as a hormone. PMID:17623636

Tamori, Masaki; Saha, Apurba Kumar; Matsuno, Akira; Noskor, Sukumar Chandra; Koizumi, Osamu; Kobayakawa, Yoshitaka; Nakajima, Yoko; Motokawa, Tatsuo



Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.  

PubMed Central

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Cetin, Y; Kulaksiz, H; Redecker, P; Bargsten, G; Adermann, K; Grube, D; Forssmann, W G



Morphology and monoterpene biosynthetic capabilities of secretory cell clusters isolated from glandular trichomes of peppermint ( Mentha piperita L.)  

Microsoft Academic Search

Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the

David McCaskill; Jonathan Gershenzon; Rodney Croteau



Secretory phospholipase A 2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells  

Microsoft Academic Search

Nonpancreatic secretory phospholipase A2 (sPLA2) displays proinflammatory properties; however, its physiological substrate is not identified. Although inactive toward intact cells, sPLA2 hydrolyzed phospholipids in membrane microvesicles shed from Ca2+-loaded erythrocytes as well as from platelets and from whole blood cells challenged with inflammatory stimuli. sPLA2 was stimulated upon degradation of sphingomyelin (SPH) and produced lysophosphatidic acid (LPA), which induced platelet

Olivier Fourcade; Marie-Françoise Simon; Cécile Viodé; Nathalie Rugani; François Leballe; Ashraf Ragab; Bernard Fournié; Louis Sarda; Hugues Chap



Electron immunocytochemical localization of pepsinogen I (PgI) in chief cells, mucous-neck cells and transitional mucous-neck\\/chief cells, of the human fundic mucosa  

Microsoft Academic Search

Specific PgI antibodies devoid of PgII cross reactivity have been applied to aldehyde-osmium fixed human, fundic-type, gastric mucosa investigated with the protein A-immunogold technique. PgI immunoreactivity has been detected in the homogeneous secretory granules of glandular chief cells, in bipartite granules of mucous-neck cells, in the granules of cells showing intermediate patterns and topography in between chief and mucous-neck cells

M. Cornaggia; C. Capella; C. Riva; G. Finzi; E. Solcia



Hippocampal granule cells are necessary for normal spatial learning but not for spatially-selective pyramidal cell discharge  

Microsoft Academic Search

The effects of massive destruction of granule cells of the fascia dentata on the spatial and temporal firing characteristics of pyramidal cells in the CA1 and CA3 subfields of the hippocampus were examined in freely moving rats. Microinjections of the neurotoxin colchicine were made at a number of levels along the septo-temporal axis of the dentate gyri of both hemispheres,

B. L. McNaughton; C. A. Barnes; J. Meltzer; R. J. Sutherland



Stereological Estimation of Granule Cell Number and Purkinje Cell Volume in the Cerebellum of Noise-Exposed Young Rat  

PubMed Central

In spite of the existing reports on behavioural and biochemical changes related to the cerebellum due to noise stress, not much is known about the effect of noise stress on the neuronal changes in the cerebellum. The present study aims at investigating the effects from one week noise exposure on granule cell number and Purkinje cell volume within the neonate rat cerebellum. 15-day-old male Wistar rats were randomly divided into noise exposed (NE) and control groups (n=8 in each group). NE rats exposed to loud noise (100 dB/30 min/3 times per day) during the third postnatal week. One cerebellar half was selected at random for estimating the volume of the cerebellar layers and neuronal quantifications and the other was used for estimating individual somal volume of Purkinje cells. Cavalieri’s principle, physical disector and nucleator methods were employed respectively for unbiased estimation of the volumes of the cerebellar layers, the numerical density of neurons and the individual volume of Purkinje cells. Results of this study show that noise stress significantly decreases the volume of granule layer together with decreased numerical density and total number of granule cells in the cerebellum. Furthermore, a decrease in somal volume of Purkinje cells was found in NE rats. These results, for the first time, demonstrate an effect of noise stress on the granule cell number and individual volume of Purkinje cells in rat cerebellum.

Hosseini-Sharifabad, Mohammad; Sabahi, Abdoreza



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

PubMed Central

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.

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



GPR56-regulated granule cell adhesion is essential for rostral cerebellar development.  


Mutations in GPR56, an orphan G-protein-coupled receptor (GPCR), cause bilateral frontoparietal polymicrogyria (BFPP), a disorder characterized by mental retardation, seizures, motor developmental delay, and ataxia. BFPP patients have structural abnormalities of the cerebral cortex, cerebellum, and pons. To shed light on the function of GPR56 and the anatomical and behavioral defects underlying BFPP, we analyzed the cerebellum of mice lacking this GPCR. Gpr56(-/-) mice display a severe malformation of the rostral cerebellum that develops perinatally. Defects involve fusion of adjacent lobules, disrupted layering of neurons and glia, and fragmentation of the pial basement membrane. At the age of defect onset, GPR56 expression is restricted specifically to developing granule cells in the rostral cerebellum, suggesting that GPR56 regulates properties of these cells. Indeed, granule cells from the rostral region of perinatal Gpr56(-/-) cerebella show loss of adhesion to extracellular matrix molecules of the pial basement membrane. Interference RNA-mediated knockdown of GPR56 recapitulates the loss of adhesion seen in knock-outs, and reexpression of GPR56 rescues the adhesion defect in knock-out granule cells. Loss of GPR56 does not affect cell proliferation, migration, or neurite outgrowth. These studies establish a novel role for GPR56 in the adhesion of developing neurons to basal lamina molecules and suggest that this adhesion is critical for maintenance of the pia and proper cerebellar morphogenesis. PMID:19515912

Koirala, Samir; Jin, Zhaohui; Piao, Xianhua; Corfas, Gabriel



Loss of Granuphilin and Loss of Syntaxin-1A Cause Differential Effects on Insulin Granule Docking and Fusion*  

PubMed Central

The Rab27 effector granuphilin/Slp4 is essential for the stable attachment (docking) of secretory granules to the plasma membrane, and it also inhibits subsequent fusion. Granuphilin is thought to mediate these processes through interactions with Rab27 on the granule membrane and with syntaxin-1a on the plasma membrane and its binding partner Munc18-1. Consistent with this hypothesis, both syntaxin-1a- and Munc18-1-deficient secretory cells, as well as granuphilin null cells, have been observed to have a deficit of docked granules. However, to date there has been no direct comparative analysis of the docking defects in those mutant cells. In this study, we morphometrically compared granule-docking states between granuphilin null and syntaxin-1a null pancreatic ? cells derived from mice having the same genetic background. We found that loss of syntaxin-1a does not cause a significant granule-docking defect, in contrast to granuphilin deficiency. Furthermore, we newly generated granuphilin/syntaxin-1a double knock-out mice, characterized their phenotypes, and found that the double mutant mice represent a phenocopy of granuphilin null mice and do not represent phenotypes of syntaxin-1a null mice, including their granule-docking behavior. Because granuphilin binds to syntaxin-2 and syntaxin-3 as well as syntaxin-1a, it likely mediates granule docking through interactions with those multiple syntaxins on the plasma membrane.

Wang, Hao; Ishizaki, Ray; Kobayashi, Eri; Fujiwara, Tomonori; Akagawa, Kimio; Izumi, Tetsuro



Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans  

PubMed Central

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane–enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.

Ueki, Shigeharu; Melo, Rossana C. N.; Ghiran, Ionita; Spencer, Lisa A.; Dvorak, Ann M.; Weller, Peter F.



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

PubMed Central

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.

Gutierrez-Martin, Yolanda; Bustillo, Diego; Gomez-Villafuertes, Rosa; Sanchez-Nogueiro, Jesus; Torregrosa-Hetland, Cristina; Binz, Thomas; Gutierrez, Luis Miguel; Miras-Portugal, Maria Teresa; Artalejo, Antonio R.



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

NASA Technical Reports Server (NTRS)

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.

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



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


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. PMID:21292765

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



Cellular and molecular mechanisms of cerebellar granule cell migration  

Microsoft Academic Search

The real-time observation of cell movement in brain slice preparations reveals that in the developing brain, postmitotic neurons\\u000a alter their shape concomitantly with changes in the mode, direction, tempo, and rate of migration as they traverse different\\u000a cortical layers. Although it has been hypothesized that orchestrated activities of multiple external cues and cell-cell contact\\u000a are essential for controlling the cortical-layer-specific

Elina Yacubova; Hitoshi Komuro



Systemic mastocytosis accompanied by a non-secretory plasma cell dyscrasia and nephrotic syndrome-level proteinuria in a 76-year-old patient.  


We report here the interesting case of a 76-year-old man with severe proteinuria who was diagnosed with systemic mastocytosis accompanied by a clonal non-mast-cell lineage haematological disorder (a non-secretory plasma cell dyscrasia). This is a unique report of systemic mastocytosis with a non-secretory plasma cell dyscrasia and nephrotic syndrome. The pathophysiological relevance between these entities along with the probability of occult amyloidosis is discussed. PMID:24081151

Papadopoulou, Vasiliki; Ioannou, Savvas; Levidou, Georgia; Variami, Eleni; Kouzis, Panagiotis; Siakantaris, Marina



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

Microsoft Academic Search

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 ¹²⁵I-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

J. O. Kokkonen; P. T. Kovanen



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

SciTech Connect

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.

Kendall, M.D.



Visualization of Regulated Exocytosis with a Granule-Membrane Probe Using Total Internal Reflection MicroscopyV?  

PubMed Central

Secretory granules labeled with Vamp-green fluorescent protein (GFP) showed distinct signatures upon exocytosis when viewed by total internal reflection fluorescence microscopy. In ?90% of fusion events, we observed a large increase in fluorescence intensity coupled with a transition from a small punctate appearance to a larger, spreading cloud with free diffusion of the Vamp-GFP into the plasma membrane. Quantitation suggests that these events reflect the progression of an initially fused and spherical granule flattening into the plane of the plasma membrane as the Vamp-GFP simultaneously diffuses through the fusion junction. Approximately 10% of the events showed a transition from puncta to ring-like structures coupled with little or no spreading. The ring-like images correspond quantitatively to granules fusing and retaining concavity (recess of ?200 nm). A majority of fusion events involved granules that were present in the evanescent field for at least 12 s. However, ?20% of the events involved granules that were present in the evanescent field for no more than 0.3 s, indicating that the interaction of the granule with the plasma membrane that leads to exocytosis can occur within that time. In addition, ?10% of the exocytotic sites were much more likely to occur within a granule diameter of a previous event than can be accounted for by chance, suggestive of sequential (piggy-back) exocytosis that has been observed in other cells. Overall granule behavior before and during fusion is strikingly similar to exocytosis previously described in the constitutive secretory pathway.

Allersma, Miriam W.; Wang, Li; Axelrod, Daniel; Holz, Ronald W.



Spine morphogenesis in newborn granule cells is differentially regulated in the outer and middle molecular layers.  


New neurons are continuously added to the hippocampus of adult mammals. Their survival and integration into the circuitry are highly dependent on experience. Here we show that mushroom spine formation in newborn granule cells was modulated by experience and that dendritic segments in different areas of the molecular layer were differentially regulated. Specifically, spines of new neurons in the outer molecular layer of the dentate gyrus were more readily influenced by nonspatial features in the living environment. Those in the middle molecular layer were more likely to be influenced by the size of the living environment. Therefore, the activity of cortical inputs into newborn granule cells may be reflected in the formation of mushroom spines in different dendritic segments in the molecular layer. J. Comp. Neurol. 522:2756-2766, 2014. © 2014 Wiley Periodicals, Inc. PMID:24610721

Zhao, Chunmei; Jou, Jessica; Wolff, Lisa J; Sun, Huaiyu; Gage, Fred H



Secretory expression of Lentinula edodes intracellular laccase by yeast high-cell-density system: sub-milligram production of difficult-to-express secretory protein.  


While a number of heterologous expression systems have been reported for extracellular laccases, there are few for the intracellular counterparts. The Lentinula edodes intracellular laccase Lcc4 is an industrially potential enzyme with its unique substrate specificity. The heterologous production of the intracellular laccase, however, had been difficult because of its expression-dependent toxicity. We previously demonstrated that recombinant yeast cells synthesized and, interestingly, secreted Lcc4 only when they were suspended to an inducing medium in a high cell-density (J. Biosci. Bioeng., 113, 154-159, 2012). The high cell-density system was versatile and applicable to other difficult-to-express secretory proteins. Nevertheless, the system's great dependence on aeration, which was a practical obstacle to scale-up production of the enzyme and some other proteins, left the secretion pathway and enzymatic properties of the Lcc4 uncharacterized. In this report, we demonstrate a successful production of Lcc4 by applying a jar-fermentor to the high cell-density system. The elevated yield (0.6 mg L(-1)) due to the sufficient aeration allowed us to prepare and purify the enzyme to homogeneity. The enzyme had been secreted as a hyper-glycosylated protein, resulting in smear band-formations in SDS-PAGE. The amino acid sequencing analysis suggested that the N-terminal 17 residues had been recognized as a secretion signal. The recombinant enzyme showed similar enzymatic properties to the naturally occurring Lcc4. The characteristics of the scale-upped expression system, which includes helpful information for the potential users, have also been described. PMID:24411669

Kurose, Takeshi; Saito, Yuta; Kimata, Koichi; Nakagawa, Yuko; Yano, Akira; Ito, Keisuke; Kawarasaki, Yasuaki



Interactions of total bone marrow cells with increasing quantities of macroporous calcium phosphate ceramic granules.  


The biological properties of synthetic calcium phosphate bioceramics have made them the third choice of material for bone reconstructive surgery, after autologous bone and allografts. Nevertheless, bioceramics lack the osteogenic properties that would allow them to repair large bone defects. One strategy in bone tissue engineering consists of associating a synthetic scaffold with osteogenic cells. Mesenchymal stem cells (MSC) are usually isolated from bone marrow cultured for several weeks and seeded on to a small quantity of bioceramic. We have studied the association of total bone marrow cells, harvested from femurs of rats, with increasing amounts of calcium phosphate ceramic granules (50-250 mg). A cell viability test indicated that a little quantity of bioceramics granules (50 mg) was less detrimental for culturing 1 million nucleated cells from the whole bone marrow population. Cell morphology, viability, adhesion and differentiation were studied after different culture periods. Among the heterogeneous population of bone marrow cells, only a limited amount of cells attached and differentiated on the bioceramics. To explain the influence of the amount of synthetic scaffold on cell viability, media calcium concentrations were measured. Low cell viability could be explained by calcium phosphate precipitation leading to a decrease in calcium concentrations observed with relatively large amounts of scaffold. This study showed that the chemical stability of the ceramic plays a critical role in the viability of bone marrow cells. PMID:17554601

Le Nihouannen, Damien; Duval, Laure; Lecomte, Antoine; Julien, Marion; Guicheux, Jérôme; Daculsi, Guy; Layrolle, Pierre



Methyl iodide toxicity in rat cerebellar granule cells in vitro: the role of glutathione  

Microsoft Academic Search

The monohalomethane methyl iodide (MeI) is toxic to a number of organ systems including the central nervous system. Clinical symptoms of neurotoxicity suggest that the cerebellum is the target within the brain, and we have now modelled the toxicity of MeI in cultured rat cerebellar granule cells. Cytotoxicity is maximal 24 h after a 5 min exposure to MeI, and

Mark P Chamberlain; Nicholas C Sturgess; Edward A Lock; Celia J Reed



Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces cerevisiae  

Microsoft Academic Search

Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to ? and ? 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

Uroš Petrovi?; Jernej Šribar; Maja Matis; Gregor Anderluh; Jasna Peter-Katalini?; Igor Križaj; Franc Gubenšek



Resistance of cholangiocarcinoma cells to parthenolide-induced apoptosis by the excretory–secretory products of Clonorchis sinensis  

Microsoft Academic Search

Infection by Clonorchis sinensis, the Chinese or oriental liver fluke, is a significant risk factor for the development of cholangiocarcinoma, a human epithelial\\u000a carcinoma of the intrahepatic bile duct. Parthenolide is a sesquiterpene lactone that has strong anticancer properties and\\u000a is also known to induce apoptosis in cholangiocarcinoma cells. Many investigators have reported that excretory–secretory (ES)\\u000a products of C. sinensis

Young Ju Kim; Min-Ho Choi; Sung-Tae Hong; Young Mee Bae



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

PubMed Central

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.

Mbikay, M; Seidah, N G; Chretien, M



Secretory phospholipase A2-IIa upregulates HER/HER2-elicited signaling in lung cancer cells.  


Lung cancer is the leading cause of cancer death worldwide. There is an urgent need for early diagnostic tools and novel therapies in order to increase lung cancer survival. Secretory phospholipase A2 group IIa (sPLA2-IIa) is involved in inflammation, tumorigenesis and metastasis. We were the first to uncover that cancer cells secrete sPLA2?IIa. sPLA2?IIa is overexpressed in almost all specimens of human lung cancers examined and is significantly elevated in the plasma of lung cancer patients. High levels of plasma sPLA2-IIa are significantly associated with advanced stage and decreased overall cancer survival. In this study, we further showed that elevated HER/HER2?PI3K-Akt-NF-?B signaling contributes to sPLA2-IIa overexpression in lung cancer cells. sPLA2-IIa in turn phosphorylates and activates HER2 and HER3 in a time- and dose?dependent manner in lung cancer cells. The structure and sequence?based docking analysis revealed that sPLA2-IIa ? hairpin shares structural similarity with the corresponding EGF hairpin. sPLA2-IIa forms an extensive interface with EGFR and brings the two lobes of EGFR into an active conformation. sPLA2-IIa also enhances the NF-?B promoter activity. Anti-sPLA2-IIa antibody, but not the small molecule sPLA2-IIa inhibitor LY315920, significantly inhibits sPLA2?IIa-induced activation of NF-?B promoter. Our findings support the notion that sPLA2-IIa functions as a ligand for the EGFR family of receptors leading to an elevated HER/HER2-elicited signaling. Plasma sPLA2-IIa can potentially serve as lung cancer biomarker and sPLA2?IIa is a potential therapeutic target against lung cancer. PMID:24913497

Dong, Zhongyun; Meller, Jaroslaw; Succop, Paul; Wang, Jiang; Wikenheiser-Brokamp, Kathryn; Starnes, Sandra; Lu, Shan



Mossy cell axon synaptic contacts on ectopic granule cells that are born following pilocarpine-induced seizures.  


Granule cell neurogenesis increases following seizures, and some newly born granule cells develop at abnormal locations within the hilus. These ectopic granule cells (EGCs) demonstrate regular bursts of action potentials that are synchronized with CA3 pyramidal cell burst discharges and the bursts of hilar neurons, including mossy cells. Such findings suggest that mossy cells may participate in circuits that activate EGCs. Electron microscopic immunolabeling was therefore used to determine if mossy cell axon terminals form synapses with hilar EGC dendrites, using animals that underwent pilocarpine-induced status epilepticus. Pilocarpine was administered to adult male rats, and those which developed status epilepticus were perfused 5-7 months later, after the period of EGC genesis. Hippocampal sections were processed for dual electron microscopic immunolabeling (using calcitonin gene-related peptide (CGRP) as a marker for mossy cells and calbindin (CaBP) as a marker for EGCs). Light microscopic analysis revealed large CGRP-immunoreactive cells in the hilus, with the appearance and distribution of mossy cells. Electron microscopic analysis revealed numerous CaBP-immunoreactive dendrites in the hilus, some of which were innervated by CGRP-immunoreactive terminals. The results suggest that mossy cells participate in the excitatory circuits which activate EGCs, providing further insight into the network rearrangements that accompany seizure-induced neurogenesis in this animal model of epilepsy. PMID:17611032

Pierce, Joseph P; Punsoni, Michael; McCloskey, Daniel P; Scharfman, Helen E



Enhancement of the Dense-Core Vesicle Secretory Cycle by Glucocorticoid Differentiation of PC12 Cells: Characteristics of Rapid Exocytosis and Endocytosis  

Microsoft Academic Search

The secretory cycle of dense-core vesicles (DCVs) in physio- logically stimulated patch-clamped PC12 cells was analyzed using both amperometry and capacitance measurements. Un- treated cells had low or undetectable Ca currents and sparse secretory responses to short depolarizations. Dexamethasone (5 mM) treatment for 5-7 d tripled Ca current magnitude and dramatically increased quantal secretion in response to depo- larization with

Abdeladim Elhamdani; Mary E. Brown; Cristina R. Artalejo; H. Clive Palfrey



High-frequency stimulation induces gradual immediate early gene expression in maturing adult-generated hippocampal granule cells.  


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. PMID:23425888

Jungenitz, Tassilo; Radic, Tijana; Jedlicka, Peter; Schwarzacher, Stephan W



Drosophila XBP1 Expression Reporter Marks Cells under Endoplasmic Reticulum Stress and with High Protein Secretory Load  

PubMed Central

Expression of genes in the endoplasmic reticulum (ER) beyond its protein folding capacity activates signaling pathways that are collectively referred to as the Unfolded Protein Response (UPR). A major branch of the UPR pathway is mediated by IRE1, an ER-tethered endonuclease. Upon ER stress-induced activation, IRE1 splices the mRNA of XBP1, thereby generating an active isoform of this transcription factor. During normal Drosophila development, tissues with high protein secretory load show signs of IRE1/XBP1 activity indicative of inherent ER stress associated with those cell types. Here, we report that the XBP1 promoter activity itself is enhanced in secretory tissues of Drosophila, and it can be induced by excessive ER stress. Specifically, we developed a Drosophila XBP1 transcription reporter by placing dsRed under the control of the XBP1 intergenic sequence. DsRed expression in these xbp1p>dsRed transgenic flies showed patterns similar to that of xbp1 transcript distribution. In healthy developing flies, the reporter expression was highest in salivary glands and the intestine. In the adult, the male reproductive organs showed high levels of dsRed. These tissues are known to have high protein secretory load. Consistently, the xbp1p>dsRed reporter was induced by excessive ER stress caused by mutant Rhodopsin-1 overexpression. These results suggest that secretory cells suffer from inherent ER stress, and the xbp1p>dsRed flies provide a useful tool in studying the function and homeostasis of those cells.

Ryoo, Hyung Don; Li, Josepher; Kang, Min-Ji



Combined Inhibition of p97 and the Proteasome Causes Lethal Disruption of the Secretory Apparatus in Multiple Myeloma Cells  

PubMed Central

Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

Auner, Holger W.; Moody, Anne Marie; Ward, Theresa H.; Kraus, Marianne; Milan, Enrico; May, Philippa; Chaidos, Aristeidis; Driessen, Christoph; Cenci, Simone; Dazzi, Francesco; Rahemtulla, Amin; Apperley, Jane F.; Karadimitris, Anastasios; Dillon, Niall



Bmi1 overexpression in the cerebellar granule cell lineage of mice affects cell proliferation and survival without initiating medulloblastoma formation  

PubMed Central

SUMMARY BMI1 is a potent inducer of neural stem cell self-renewal and neural progenitor cell proliferation during development and in adult tissue homeostasis. It is overexpressed in numerous human cancers – including medulloblastomas, in which its functional role is unclear. We generated transgenic mouse lines with targeted overexpression of Bmi1 in the cerebellar granule cell lineage, a cell type that has been shown to act as a cell of origin for medulloblastomas. Overexpression of Bmi1 in granule cell progenitors (GCPs) led to a decrease in cerebellar size due to decreased GCP proliferation and repression of the expression of cyclin genes, whereas Bmi1 overexpression in postmitotic granule cells improved cell survival in response to stress by altering the expression of genes in the mitochondrial cell death pathway and of Myc and Lef-1. Although no medulloblastomas developed in ageing cohorts of transgenic mice, crosses with Trp53?/? mice resulted in a low incidence of medulloblastoma formation. Furthermore, analysis of a large collection of primary human medulloblastomas revealed that tumours with a BMI1high TP53low molecular profile are significantly enriched in Group 4 human medulloblastomas. Our data suggest that different levels and timing of Bmi1 overexpression yield distinct cellular outcomes within the same cellular lineage. Importantly, Bmi1 overexpression at the GCP stage does not induce tumour formation, suggesting that BMI1 overexpression in GCP-derived human medulloblastomas probably occurs during later stages of oncogenesis and might serve to enhance tumour cell survival.

Behesti, Hourinaz; Bhagat, Heeta; Dubuc, Adrian M.; Taylor, Michael D.; Marino, Silvia



Granule cells born in the adult rat hippocampus can regulate the expression of GABAergic markers.  


The granule cells (GCs) of the dentate gyrus transiently express markers of the GABAergic phenotype early during development. However, GCs are generated throughout life, posing the question of whether the newborn neurons in the adult rodent recapitulate the development of the neurotransmitter phenotype of GCs generated during embryonic and early postnatal development. In this work we asked whether newborn GCs transiently express a GABAergic phenotype during their development in the adult rat. Using retroviral infection, we labeled dividing cells in the dorsal hippocampus with GFP, identified them as granule cells, and determined their expression of GABAergic markers at different developmental stages. We found that GFP-positive cells express Prox-1 and calbindin, identifying them as GCs. GABA or GAD(67) was expressed in 13% of GFP-positive cells at 7 dpi, in 16% at 10 dpi and in 20% at 15 dpi. At 30 dpi, however, no GFP-positive cell somata containing GABAergic markers were detected, but their mossy fiber boutons did contain GAD(67). Interestingly, developing GCs detected with doublecortin and PSA-NCAM in non-injected adult rats, did not express GABAergic markers, suggesting that retroviral injection/infection stimulates their transient expression. However, in non-injected rats, a number of mossy fiber boutons of newborn granule cells detected with PSA-NCAM did express GAD(67). Our findings reveal that developing GCs born in the adult are able to transiently up-regulate the expression of GABAergic markers to be detected in their soma in response to insults, while they constitutively express GAD(67) in their mossy fibers. PMID:22750325

Lara, Erika; Beltrán, Jesús Q; Segovia, José; Gutiérrez, Rafael



Decreased ?-cell insulin secretory function in aged rats due to impaired Ca(2+) handling.  


Ageing is associated with an increased impairment in glucose homeostasis and an increased incidence of type 2 diabetes. In this study, we evaluated ?-cell function and its implications for glucose homeostasis in 24-month-old female Wistar rats. Aged rats showed lower plasma glucose levels in the fed and fasting states compared with control rats. In addition, insulinaemia in the fed state was reduced in the older rats. Insulin receptor ? (IR?) expression was lower in the livers of the aged animals, whereas IR? and Akt(1/2/3) protein expressions were higher in the muscles. These effects may contribute to the normal glucose tolerance observed in older rodents. Isolated islets from aged rats secreted less insulin in response to 8.3 and 16.7 mm glucose. Accordingly, this group presented a lower [Ca(2+)](i) in the presence of glucose and a depolarizing stimulus (30 mm K(+)). In addition, islets from aged rats showed reduced insulin secretion in response to 100 ?m carbachol (CCh), 10 nm phorbol 12-myristate 13-acetate and 10 ?m forskolin. The expressions of protein kinase C, protein kinase A and exocytotic proteins, such as syntaxin 1 and synaptosomal-associated protein 25 kDa (SNAP-25), were similar in islets from aged and control rats. In conclusion, our evidence suggests that the increased incidence of type 2 diabetes with age may be due to a progressive decline in ?-cell secretory capacity due to disruption of Ca(2+) handling. Furthermore, the expression of proteins of the insulin transduction cascade showed an adaptive profile, with a compensatory increase in IR? and Akt(1/2/3) in gastrocnemius muscles, which may maintain normal glucose homeostasis in 24-month-old rats. PMID:22542614

Ribeiro, Rosane Aparecida; Batista, Thiago Martins; Coelho, Fernanda Monteiro; Boschero, Antonio Carlos; Lopes, Guiomar Silva; Carneiro, Everardo Magalhães



Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy  

SciTech Connect

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.

Ohara-Imaizumi, Mica; Aoyagi, Kyota [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan); Akimoto, Yoshihiro [Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan); Nakamichi, Yoko; Nishiwaki, Chiyono [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan); Kawakami, Hayato [Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Anatomy, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan); Nagamatsu, Shinya, E-mail: [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)] [Department of Biochemistry, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611 (Japan)



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


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

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



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

PubMed Central

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.

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



NF90 exerts antiviral activity through regulation of PKR phosphorylation and stress granules in infected cells.  


NF90 was shown to exhibit broad antiviral activity against several viruses, but detailed mechanisms remain unclear. In this study, we examined the molecular basis for the inhibitory effect of NF90 on virus replication mediated through protein kinase (PKR)-associated translational regulation. We first verified the interaction between NF90 and PKR in mammalian cells and showed that NF90 interacts with PKR through its C-terminal and that the interaction is independent of NF90 RNA-binding properties. We further showed that knockdown of NF90 resulted in significantly lower levels of PKR phosphorylation in response to dsRNA induction and influenza virus infection. We also showed that high concentrations of NF90 exhibit negative regulatory effects on PKR phosphorylation, presumably through competition for dsRNA via the C-terminal RNA-binding domain. PKR activation is essential for the formation of stress granules in response to dsRNA induction. Our results showed that NF90 is a component of stress granules. In NF90-knockdown cells, dsRNA treatment induced significantly lower levels of stress granules than in control cells. Further evidence for an NF90-PKR antiviral pathway was obtained using an NS1 mutated influenza A virus specifically attenuated in its ability to inhibit PKR activation. This mutant virus replicated indistinguishably from wild-type virus in NF90-knockdown cells, but not in scrambled control cells or Vero cells, indicating that NF90's antiviral function occurs through interaction with PKR. Taken together, these results reveal a yet-to-be defined host antiviral mechanism in which NF90 upregulation of PKR phosphorylation restricts virus infection. PMID:24623135

Wen, Xi; Huang, Xiaofeng; Mok, Bobo Wing-Yee; Chen, Yixin; Zheng, Min; Lau, Siu-Ying; Wang, Pui; Song, Wenjun; Jin, Dong-Yan; Yuen, Kwok-Yung; Chen, Honglin



The transmembrane domain of the prohormone convertase PC3: A key motif for targeting to the regulated secretory pathway  

PubMed Central

The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617–638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC31–638 (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC31–616 (PC3-616) and PC3-?TM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-?TM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

Lou, Hong; Smith, Angela M.; Coates, Leigh C.; Cawley, Niamh X.; Peng Loh, Y.; Birch, Nigel P.



A study of granulated metrial gland cell differentiation in pregnant, macrophage-deficient, osteopetrotic ( op\\/op ) mice  

Microsoft Academic Search

A population of uterine natural killer (NK) cells, commonly called granulated metrial gland (GMG) cells, differentiates in the mouse uterus during normal pregnancy. Little is known regarding the process of differentiation of GMG cells or of other NK cell subsets. It has been suggested that macrophage precursors, under the combined influences of the cytokine growth factors colony stimulating factor-1 (CSF-1)

Y. Kiso; J. W. Pollard; B. A. Croy



Neurotrophins Protect Cultured Cerebellar Granule Neurons against the Early Phase of Cell Death by a Two Component Mechanism  

Microsoft Academic Search

Cerebellar granule neurons cultured with serum develop a ma- ture neuronal phenotype, including stimulus-coupled release of glutamate, and depend on elevated potassium for survival. We find that cells cultured with serum undergo two phases of cell death. By 6 d in vitro, 30 -50% of the cells present are dead; after this time the remaining cells die. Elevated potassium prevents

Michael J. Courtney; Karl E. O. Åkerman; Eleanor T. Coffey



Anion permeation in an apical membrane chloride channel of a secretory epithelial cell  

PubMed Central

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.



Heterogeneity of glycinergic and gabaergic interneurons in the granule cell layer of mouse cerebellum.  


Interneurons of the cerebellum granule cell layer (GCL) form distinct populations. Golgi cells extend dendrites in the molecular layer (ML) and innervate granule cells. In contrast, Lugaro cells have dendrites confined to the GCL but innervate interneurons in the ML, and globular cells have both their dendrites and axons in the ML. The latter cells were described recently and remain poorly characterized. Although several neurochemical markers have been associated selectively with GCL interneurons, it is unclear how they relate to their morphological classification and neurochemical phenotype (glycinergic and/or gamma-aminobutyric acid [GABA]ergic). Here, we performed a detailed characterization of GCL interneurons in mice expressing enhanced green fluorescent protein (GFP) in glycinergic and GABAergic neurons, respectively. By using immunofluorescence for metabotropic glutamate receptor 2 (mGluR2) and neurogranin as markers, we demonstrate the existence of five non-overlapping subsets of Golgi cells: about 65% are glycinergic/GABAergic and co-express both markers. Two small subsets (5-10%) also contain both neurotransmitters but express only mGluR2; they are distinguished by cell body size and location in the GCL. The fourth subset (15%) is GABAergic only and expresses neurogranin. The fifth subset (5%) is glycinergic only and lacks both markers. Thus, the heterogeneity of Golgi cells suggests that they belong to specific functional circuits and are differentially regulated by mGluRs and Ca(2+)-calmodulin-dependent signaling pathways. In contrast to Golgi cells, Lugaro and globular cells are glycinergic/GABAergic and lack mGluR2 and neurogranin. They each represent at least 15% of GCL interneurons and extensively innervate stellate and basket cells, but not Purkinje cells, emphasizing their contribution to inhibitory control of ML interneurons. PMID:17099896

Simat, Marija; Parpan, Franziska; Fritschy, Jean-Marc



Release of ATP during host cell killing by enteropathogenic E. coli and its role as a secretory mediator.  


Enteropathogenic Escherichia coli (EPEC) causes severe, watery diarrhea in children. We investigated ATP release during EPEC-mediated killing of human cell lines and whether released adenine nucleotides function as secretory mediators. EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7, and T84 (colon cells) as measured using a luciferase kit. Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5'-ectonucleotidase was included and was further enhanced if an ATP-regenerating system was added. In the presence of the inhibitor/regenerator, ATP concentrations in the supernatant medium reached 1.5-2 microM 4 h after infection with wild-type EPEC strains. In the absence of the inhibitor/regenerator system, extracellular ATP was rapidly broken down to ADP, AMP, and adenosine. Conditioned medium from EPEC-infected cells triggered a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber (rabbit distal colon and T84 cell monolayers), whereas conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria did not. The short-circuit current response to EPEC-conditioned medium was completely reversed by adenosine receptor blockers, such as 8-(p-sulfophenyl)-theophylline and MRS1754. EPEC killing of host cells releases ATP, which is broken down to adenosine, which in turn stimulates secretion via apical adenosine A2b receptors. These findings provide new insight into how EPEC causes watery diarrhea. PMID:12065294

Crane, John K; Olson, Ruth A; Jones, Heather M; Duffey, Michael E



Altered patterning of dentate granule cell mossy fiber inputs onto CA3 pyramidal cells in limbic epilepsy  

PubMed Central

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.

McAuliffe, John J.; Bronson, Stefanie L.; Hester, Michael S.; Murphy, Brian L.; Dahlquist-Topala, Renee; Richards, David A.; Danzer, Steve C.



EPSPs of dentate gyrus granule cells during epileptiform bursts of dentate hilar "mossy" cells and area CA3 pyramidal cells in disinhibited rat hippocampal slices.  


When hippocampal slices are exposed to GABAA antagonists, area CA3 pyramidal cells and dentate hilar "mossy" cells discharge in synchronized epileptiform bursts (Müller and Misgeld, 1991; Scharfman, 1994b). Dentate interneurons are excited simultaneously, but the degree of discharge varies (Scharfman, 1994b). This study primarily examined the activity of dentate granule cells simultaneous to the epileptiform bursts of pyramidal cells and mossy cells. EPSPs followed by large GABAB receptor-mediated IPSPs were generated in granule cells during all epileptiform bursts of pyramidal cells and mossy cells, regardless of whether they were evoked or spontaneous. By simultaneous recording it was determined that granule cell EPSPs began several milliseconds after the start of pyramidal cell bursts (n = 48 simultaneous recordings) and immediately after the first action potential of a mossy cell burst (n = 77). Interneurons were similar to granule cells in the timing of their depolarizations relative to the onset of pyramidal cell (n = 24; Scharfman, 1994b) and mossy cell (n = 9) bursts. All excitatory activity was blocked by bath application of the glutamatergic AMPA/kainate receptor antagonist CNQX (5 microM, n = 5), but not the NMDA receptor antagonist D-APV (25-50 microM, n = 9). Granule cell EPSPs were decreased after focal application of CNQX to the molecular layer at a site close to the impaled granule cell (n = 5), whereas D-APV had no effect (n = 3). EPSPs also decreased after focal application of CNQX to the hilus, in two of four slices tested. The extracellularly recorded EPSP of granule cells was maximal in the inner molecular layer (n = 33), the site of the mossy cell axonal plexus. Severing the junction of the dentate gyrus and area CA3 blocked all spontaneous and evoked activity of dentate neurons without affecting burst discharges in area CA3a and CA3b (n = 6). None of the excitatory activity of any cell type was affected by cholinergic antagonists (atropine and mecamylamine, 25 microM each, n = 5; pirenzipine and dihydro-beta-erythroidine, 25 microM each, n = 5). The results suggest that there is a glutamatergic, AMPA/kainate receptor-mediated, excitatory pathway from area CA3 to the dentate gyrus in disinhibited slices. The pharmacological results, analyses of latency, as well as the known axonal projections of the sampled cells, suggest that the excitatory pathway begins within area CA3 and leads to granule cells via mossy cells. The data also suggest that dentate interneurons are excited by mossy cells, and possibly by pyramidal cells as well.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7931561

Scharfman, H E



Generation and Characterization of an Nse-CreERT2 Transgenic Line Suitable for Inducible Gene Manipulation in Cerebellar Granule Cells  

PubMed Central

We created an Nse-CreERT2 mouse line expressing the tamoxifen-inducible CreERT2 recombinase under the control of the neuron-specific enolase (Nse) promoter. By using Cre reporter lines we could show that this Nse-CreERT2 line has recombination activity in the granule cells of all cerebellar lobules as well as in postmitotic granule cell precursors in the external granular layer of the developing cerebellum. A few hippocampal dentate gyrus granule cells showed Cre-mediated recombination as well. Cre activity could be induced in both the developing and adult mouse brain. The established mouse line constitutes a valuable tool to study the function of genes expressed by cerebellar granule cells in the developing and adult brain. In combination with reporter lines it is a useful model to analyze the development and maintenance of the cerebellar architecture including granule cell distribution, migration, and the extension of granule cell fibers in vivo.

Pohlkamp, Theresa; Steller, Laura; May, Petra; Gunther, Thomas; Schule, Roland; Frotscher, Michael



Expression of embryonic stem cell markers and osteogenic differentiation potential in cells derived from periodontal granulation tissue.  


The aim of this study was to identify if cells obtained from periodontal granulation tissue possess embryonic stem cell properties and osteogenic capacities in vitro. Periodontal granulation tissue was removed from one furcation and one infrabony defect (FGTC/IGTC-furcation/infrabony defect derived granulation tissue cells) of six patients. The extracted tissues were treated with collagenase/dispase solution, cultured and passaged twice, while a fraction of them was bacteriologically analyzed. Upon reaching confluence, total RNA was extracted, followed by cDNA synthesis and real-time PCR analysis. Gene expression levels of collagen type I, alkaline phosphatase (ALP), and the embryonic stem cell markers Nanog, Oct-4, Rex-1 and Sox-2 were measured, calibrated against the housekeeping gene GAPDH. Further, osteogenic differentiation was induced. Mineralized matrix formation was confirmed by von Kossa staining, and ALP activity was measured colorimetrically. The total bacterial load amounted to 9.4 ± 14.6 × 10(6) counts/mg of tissue for IGTC, and 11.1 ± 6.1 × 10(6) ?counts/of tissue for FGTC. Among the embryonic stem cell markers (FGTC/IGTC), Nanog was most highly expressed (3.48 ± 1.2/5.85 ± 5.7), followed by Oct-4 (1.79 ± 0.69/2.85 ± 2.5), Sox-2 (0.66 ± 0.3/1.26 ± 1.4) and Rex-1 (0.06 ± 0.0/0.04 ± 0.0). The osteogenic differentiation process was positive in both FGTC and IGTC, judged by increased von Kossa staining, and elevated ALP activity and gene expression. This study provides evidence that infected periodontal granulation tissue harbors cells expressing embryonic stem cell markers, and exhibiting osteogenic capacities when in culture in vitro. PMID:24123724

Ronay, Valerie; Belibasakis, Georgios N; Attin, Thomas; Schmidlin, Patrick R; Bostanci, Nagihan



Rapid purification of cationic granule proteases: application to human granzymes.  


This report describes a simple scheme for the simultaneous purification of cationic human granzymes A, B, and 3 from human interleukin 2 (IL-2)-activated lymphocytes. The process, which requires approximately 8 h, includes: (1) cell cavitation, (2) two centrifugation steps, (3) four granule solubilization steps, and (4) cation-exchange chromatography. Granule solubilization consists of three extractions with a hypotonic buffer (25 mM NaCl) that contained Triton X-100 followed by a final extraction in hypertonic detergent-free buffer (390 mM NaCl). We recovered approximately 35% of the trypsin-like (tryptase) activities mediated by granzymes A and 3, respectively, and approximately 25% of the asp-ase activity of granzyme B. The granzymes were identified after elution from the Mono S column by Western blot with a polyclonal antibody that reacts with a conserved amino acid sequence (9-16) of lymphoid/myeloid serine proteases. By amino-terminal sequencing, eluted granzyme A and B were indeed homogeneous. Granzyme 3, although highly enriched, appears to be contaminated with an uncharacterized granzyme. Although we have developed this scheme to rapidly isolate the granzymes, the procedure should assist the purification of secretory granule-associated cationic proteins that reside in neutrophils and mast cells as well as other cells that possess secretory function. PMID:8251751

Hanna, W L; Zhang, X; Turbov, J; Winkler, U; Hudig, D; Froelich, C J



Glucose Toxic Effects on Granulation Tissue Productive Cells: The Diabetics' Impaired Healing  

PubMed Central

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.

Berlanga-Acosta, Jorge; Schultz, Gregory S.; Lopez-Mola, Ernesto; Guillen-Nieto, Gerardo; Garcia-Siverio, Marianela; Herrera-Martinez, Luis



Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons  

SciTech Connect

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.

Aziz, Gulzeb [Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo (Norway); Akselsen, Oyvind W.; Hansen, Trond V. [Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo (Norway); Paulsen, Ragnhild E., E-mail: r.e.paulsen@farmasi.uio.n [Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo (Norway)



Entorhinal Denervation Induces Homeostatic Synaptic Scaling of Excitatory Postsynapses of Dentate Granule Cells in Mouse Organotypic Slice Cultures  

PubMed Central

Denervation-induced changes in excitatory synaptic strength were studied following entorhinal deafferentation of hippocampal granule cells in mature (?3 weeks old) mouse organotypic entorhino-hippocampal slice cultures. Whole-cell patch-clamp recordings revealed an increase in excitatory synaptic strength in response to denervation during the first week after denervation. By the end of the second week synaptic strength had returned to baseline. Because these adaptations occurred in response to the loss of excitatory afferents, they appeared to be in line with a homeostatic adjustment of excitatory synaptic strength. To test whether denervation-induced changes in synaptic strength exploit similar mechanisms as homeostatic synaptic scaling following pharmacological activity blockade, we treated denervated cultures at 2 days post lesion for 2 days with tetrodotoxin. In these cultures, the effects of denervation and activity blockade were not additive, suggesting that similar mechanisms are involved. Finally, we investigated whether entorhinal denervation, which removes afferents from the distal dendrites of granule cells while leaving the associational afferents to the proximal dendrites of granule cells intact, results in a global or a local up-scaling of granule cell synapses. By using computational modeling and local electrical stimulations in Strontium (Sr2+)-containing bath solution, we found evidence for a lamina-specific increase in excitatory synaptic strength in the denervated outer molecular layer at 3–4 days post lesion. Taken together, our data show that entorhinal denervation results in homeostatic functional changes of excitatory postsynapses of denervated dentate granule cells in vitro.

Vlachos, Andreas; Becker, Denise; Jedlicka, Peter; Winkels, Raphael; Roeper, Jochen; Deller, Thomas



Neurite outgrowth of murine cerebellar granule cells can be enhanced by aniracetam with or without ?-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)  

Microsoft Academic Search

To assess the neurotrophic effects of a nootropic drug, aniracetam, we studied neurite extension of mouse cerebellar granule cells in culture with low or with high K+ under different combinations of drugs and then immunohistochemically stained the cells with an antibody against L1, a neural cell adhesion molecule on cerebellar granule cells. Quantitative analyses using parameters of the total neurite

Shinji Fushiki; Kumi Matsumoto; Akihiro Nagata



Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway  

PubMed Central

Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative ?-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane.

Courel, Maite; Vasquez, Michael S.; Hook, Vivian Y.; Mahata, Sushil K.; Taupenot, Laurent



Iron oxide nanoparticles suppress the production of IL-1beta via the secretory lysosomal pathway in murine microglial cells  

PubMed Central

Background Superparamagnetic iron oxide nanoparticles (IONPs) have been used as magnetic resonance imaging contrast agents for various research and diagnostic purposes, such as the detection of neuroinflammation and blood-brain-barrier integrity. As the central resident macrophage-like cells, microglia are responsible for managing foreign agents invading the CNS. The present study investigated the direct effect of IONPs on the production of pro-inflammatory cytokines by murine microglia stimulated with lipopolysaccharide (LPS). Methods Primary murine microglial cells were pretreated with IONPs (1–50 ?g Fe/mL) for 30 min and then stimulated with LPS (100 ng/mL) for 24 h. Confocal microscopy is used to visualize the intracellular IONP distribution and secretory lysosomes after staining with LysoTracker and Rab27a, respectively. The production of interleukin (IL)-1? and tumor necrosis factor (TNF)-? was quantified by ELISA. The activity of IL-1? converting enzyme (ICE) and TNF-? converting enzyme (TACE) was measured by fluorescent microplate assay using specific substrates. The lysosomal number, alkalinity, permeability and cathepsin B activity were determined by flow cytometry with ectodermal dysplasia-1, lysosensor and acridine orange staining, and using cathepsin B specific substrate, respectively. Results Confocal imaging revealed that IONPs were markedly engulfed by microglia. Exposure to IONPs attenuated the production of IL-1?, but not TNF-?. Concordantly, the activity of ICE, but not the TACE, was suppressed in IONP-treated cells. Mechanistic studies showed that IONPs accumulated in lysosomes and the number of lysosomes was increased in IONP-treated cells. In addition, exposure to IONPs increased lysosomal permeability and alkalinity, but decreased the activity of cathepsin B, a secretory lysosomal enzyme involved in the activation of ICE. Conclusions Our results demonstrated a contrasting effect of IONPs on the production of IL-1? and TNF-? by LPS-stimulated microglia, in which the attenuation of IL-1? by IONPs was mediated by inhibiting the secretory lysosomal pathway of cytokine processing.



Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells.  

PubMed Central

Three biologically active monoclonal antibodies (mAbs) specific for the monovalent, high-affinity membrane receptor for IgE (Fc epsilon R) were employed in analysing the secretory response of mast cells of the RBL-2H3 line to crosslinking of their Fc epsilon R. All three mAbs (designated F4, H10 and J17) compete with each other and with IgE for binding to the Fc epsilon R. Their stoichiometry of binding is 1 Fab:1 Fc epsilon R, hence, the intact mAbs can aggregate the Fc epsilon Rs to dimers only. Since all three mAbs induce secretion, we conclude that Fc epsilon R dimers constitute a sufficient 'signal element' for secretion of mediators for RBL-2H3 cells. The secretory dose-response of the cells to these three mAbs are, however, markedly different: F4 caused rather high secretion, reaching almost 80% of the cells' content, while J17 and H10 induced release of only 30-40% mediators content. Both the intrinsic affinities and equilibrium constants for the receptor dimerization were derived from analysis of binding data of the Fab fragments and intact mAbs. These parameters were used to compute the extent of Fc epsilon R dimerization caused by each of the antibodies. However, the different secretory responses to the three mAbs could not be rationalized simply in terms of the extent of Fc epsilon R dimerization which they produce. This suggests that it is not only the number of crosslinked Fc epsilon Rs which determines the magnitude of secretion-causing signal, but rather other constraints imposed by each individual mAb are also important.(ABSTRACT TRUNCATED AT 250 WORDS)

Ortega, E; Schweitzer-Stenner, R; Pecht, I



c-Myc and E1A induced cellular sensitivity to activated NK cells involves cytotoxic granules as death effectors  

Microsoft Academic Search

The contact of natural killer (NK) cells with foreign cells and with certain virus-infected or tumor cells triggers the cytolytic machinery of NK cells. This triggering leads to exocytosis of the cytotoxic NK cell granules. The oncoproteins c-Myc and E1A render cells vulnerable to NK cell mediated cytolysis yet the mechanisms of sensitization are not well understood. In a model

Juha Klefstrom; Panu E Kovanen; Kristina Somersalo; Anne-Odile Hueber; Trevor Littlewood; Gerard I Evan; Arnold H Greenberg; Eero Saksela; Tuomo Timonen; Kari Alitalo



The plasma membrane Q-SNARE syntaxin 2 enters the zymogen granule membrane during exocytosis in the pancreatic acinar cell.  


During exocytosis in the pancreatic acinar cell, zymogen granules fuse directly with the apical plasma membrane and also with granules that have themselves fused with the plasma membrane. Together, these primary and secondary fusion events constitute the process of compound exocytosis. It has been suggested that the sequential nature of primary and secondary fusion is a consequence of the requirement for plasma membrane soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors, such as syntaxin 2, to enter the membrane of the primary fused granule. We have tested this possibility by determining the location of syntaxin 2 in unstimulated and stimulated pancreatic acini. Syntaxin 2 was imaged by confocal immunofluorescence microscopy. Fused granules were detected both through their filling with the aqueous dye lysine-fixable Texas Red-dextran and through the decoration of their cytoplasmic surfaces with filamentous actin. In unstimulated cells, syntaxin 2 was exclusively present on the apical plasma membrane. In contrast, after stimulation, syntaxin 2 had moved into the membranes of fused granules, as judged by its location around dye-filled structures of 1-mum diameter that were coated with filamentous actin. At long times of stimulation (5 min), the majority (85%) of dye-filled granules were also positive for syntaxin 2. In contrast, at shorter times (1 min), more dye-filled granules (29%) were syntaxin 2-negative. We conclude that syntaxin 2 enters the membrane of a fused zymogen granule after the opening of the fusion pore, and we suggest that this movement might permit the onset of secondary fusion. PMID:15536072

Pickett, James A; Thorn, Peter; Edwardson, J Michael



MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability  

PubMed Central

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.

Toriseva, Mervi; Laato, Matti; Carpen, Olli; Ruohonen, Suvi T.; Savontaus, Eriika; Inada, Masaki; Krane, Stephen M.; Kahari, Veli-Matti



Secretagogue-dependent phosphorylation of phogrin, an insulin granule membrane protein tyrosine phosphatase homologue.  

PubMed Central

Phogrin, a 60/64 kDa integral membrane protein localized to dense-core secretory granules of neuroendocrine cells, was found to be reversibly phosphorylated in intact pancreatic beta-cells. Phosphorylation occurred in response to a variety of secretory stimuli, including glucose and depolarizing concentrations of K(+). In MIN6 cells, the glucose dose-response and time course of phogrin phosphorylation paralleled that of insulin secretion. Like secretion, glucose- or K(+)-stimulated phosphorylation required the presence of Ca(2+). The calmodulin antagonist W-7 and the Ca(2+)/calmodulin-dependent kinase II inhibitor KN-93 dose-dependently inhibited both phosphorylation and secretion, while the 'inactive' analogue KN-92 was effective only at significantly higher concentrations. Phosphorylation of phogrin was also stimulated in cells exposed to forskolin, an effect presumably mediated by protein kinase A (cAMP-dependent protein kinase). Under these conditions, phogrin phosphorylation could be dissociated from the secretory response. In MIN6 cells, as in pancreatic islets, cAMP potentiates rather than initiates insulin release. Thus our observations are consistent with a role for phogrin phosphorylation in the signal-transduction pathway at a site proximal to the exocytic event itself, possibly regulating secretory-granule mobilization and recruitment to the exocytic site.

Wasmeier, C; Hutton, J C



Secretagogue-dependent phosphorylation of phogrin, an insulin granule membrane protein tyrosine phosphatase homologue.  


Phogrin, a 60/64 kDa integral membrane protein localized to dense-core secretory granules of neuroendocrine cells, was found to be reversibly phosphorylated in intact pancreatic beta-cells. Phosphorylation occurred in response to a variety of secretory stimuli, including glucose and depolarizing concentrations of K(+). In MIN6 cells, the glucose dose-response and time course of phogrin phosphorylation paralleled that of insulin secretion. Like secretion, glucose- or K(+)-stimulated phosphorylation required the presence of Ca(2+). The calmodulin antagonist W-7 and the Ca(2+)/calmodulin-dependent kinase II inhibitor KN-93 dose-dependently inhibited both phosphorylation and secretion, while the 'inactive' analogue KN-92 was effective only at significantly higher concentrations. Phosphorylation of phogrin was also stimulated in cells exposed to forskolin, an effect presumably mediated by protein kinase A (cAMP-dependent protein kinase). Under these conditions, phogrin phosphorylation could be dissociated from the secretory response. In MIN6 cells, as in pancreatic islets, cAMP potentiates rather than initiates insulin release. Thus our observations are consistent with a role for phogrin phosphorylation in the signal-transduction pathway at a site proximal to the exocytic event itself, possibly regulating secretory-granule mobilization and recruitment to the exocytic site. PMID:10417318

Wasmeier, C; Hutton, J C



Platelet ?-granules: Basic biology and clinical correlates  

PubMed Central

Summary ?–Granules are essential to normal platelet activity. These unusual secretory granules derive their cargo from both regulated secretory and endocytotic pathways in megakaryocytes. Rare, inheritable defects of ?–granule formation in mice and man have enabled identification of proteins that mediate cargo trafficking and ?–granule formation. In platelets, ?–granules fuse with the plasma membrane upon activation, releasing their cargo and increasing platelet surface area. The mechanisms that control ?–granule membrane fusion have begun to be elucidated at the molecular level. SNAREs and SNARE accessory proteins that control ?–granule secretion have been identified. Proteomic studies demonstrate that hundreds of bioactive proteins are released from ?–granules. This breadth of proteins implies a versatile functionality. While initially known primarily for their participation in thrombosis and hemostasis, the role of ?–granules in inflammation, atherosclerosis, antimicrobial host defense, wound healing, angiogenesis, and malignancy has become increasingly appreciated as the function of platelets in the pathophysiology of these processes has been defined. This review will consider the formation, release, and physiologic roles of ?–granules with special emphasis on work performed over the last decade.

Blair, Price; Flaumenhaft, Robert



EPC-K1 attenuates peroxynitrite-induced apoptosis in cerebellar granule cells.  


Apoptosis induced by peroxynitrite in cultured cerebellar granule cells was confirmed morphologically by chromatin condensation and biochemically by DNA laddering. A 30 min exposure to peroxynitrite (10 microM) initiated oxidative stress, which caused the formation of thiobarbituric acid-reactive substances (TBARS) and the alteration of cell membrane fluidity. Peroxynitrite treatment also caused ATP decrease and thus activated the apoptotic program. Pre-treating cells with antioxidant EPC-K1 (L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H -1- benzopyran-6-yl-hydrogen phosphate] potasium salt), a new water-soluble derivative of vitamin C and vitamin E, attenuated oxidative injury and prevents cells from apoptosis. The results suggest that EPC-K1 might be used as a potential therapeutic agent for diseases associated with NO/ONOO(-)-mediated neuronal injury. PMID:9784843

Wei, T; Chen, C; Zhao, B; Xin, W; Mori, A



Pycnogenol and vitamin E inhibit ethanol-induced apoptosis in rat cerebellar granule cells.  


Pycnogenol (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), scavenges free radicals and promotes cellular health. The protective capacity of PYC against ethanol toxicity of neurons has not previously been explored. The present study demonstrates that in postnatal day 9 (P9) rat cerebellar granule cells the antioxidants vitamin E (VE) and PYC (1) dose dependently block cell death following 400, 800, and 1600 mg/dL ethanol exposure (2) inhibit the ethanol-induced activation of caspase-3 in the same model system; and (3) reduce neuronal membrane disruption as assayed by phosphatidylserine translocation to the cell surface. These results suggest that both PYC and VE have the potential to act as therapeutic agents, antagonizing the induction of neuronal cell death by ethanol exposure. PMID:15146544

Siler-Marsiglio, Kendra I; Shaw, Gerry; Heaton, Marieta B



Function suggests nano-structure: electrophysiology supports that granule membranes play dice  

PubMed Central

Cellular communication depends on membrane fusion mechanisms. SNARE proteins play a fundamental role in all intracellular fusion reactions associated with the life cycle of secretory vesicles, such as vesicle–vesicle and vesicle plasma membrane fusion at the porosome base in the cell plasma membrane. We present growth and elimination (G&E), a birth and death model for the investigation of granule growth, its evoked and spontaneous secretion and their information content. Using a statistical mechanics approach in which SNARE components are viewed as interacting particles, the G&E model provides a simple ‘nano-machine’ of SNARE self-aggregation behind granule growth and secretion. Results from experimental work, mathematical calculations and statistical modelling suggest that for vesicle growth a minimal aggregation of three SNAREs is required, while for the evoked secretion one SNARE is enough. Furthermore, the required number of SNARE aggregates (which varies between cell types and is nearly proportional to the square root of the mean granule diameter) affects and is statistically identifiable from the size distributions of spontaneous and evoked secreted granules. The new statistical mechanics approach to granule fusion is bound to have a significant changing effect on the investigation of the pathophysiology of secretory mechanisms and methodologies for the investigation of secretion.

Hammel, Ilan; Meilijson, Isaac



Lactoferrin and free secretory component of human milk inhibit the adhesion of enteropathogenic Escherichia coli to HeLa cells  

PubMed Central

Background Diarrhoea caused by Escherichia coli is an important cause of infant morbidity and mortality in developing countries. Enteropathogenic Escherichia coli (EPEC) is considered one of the major causes of diarrhoea in children living in developing countries. The ability of diarrhoeagenic strains of E. coli to adhere to and colonize the intestine is the first step towards developing the disease. EPEC strains adhere to enterocytes and HeLa cells in a characteristic pattern known as localized adherence. Many epidemiological studies of diarrhoea have shown that breast-feeding protects infants from intestinal infections. Both immunoglobulin and non-immunoglobulin elements of human milk are thought to contribute to the protection from diarrhoeal agents. Results The effects of human milk and its protein components on the localized adherence of EPEC were investigated. Non-immunoglobulin components of human milk responsible for the inhibition of EPEC adhesion to HeLa cells were isolated by chromatographic fractionation of human whey proteins. Besides secretory immunoglobulin A, which has been previously reported to affect the adhesion of EPEC, free secretory component (fSC) and lactoferrin (Lf) were isolated. Even in concentrations lower than those usually found in whole milk, fSC and Lf were able to inhibit the adhesion of EPEC. ?-lactalbumin was also isolated, but showed no activity on EPEC adhesion. Conclusions This study demonstrated that the immunoglobulin fraction, the free secretory component and lactoferrin of human milk inhibit EPEC adhesion to HeLa cells. These results indicate that fSC and Lf may be important non-specific defence factors against EPEC infections.

de Araujo, Andrea Nascimento; Giugliano, Loreny Gimenes



Secretory carrier membrane proteins  

Microsoft Academic Search

Secretory carrier membrane proteins (SCAMPs) are a family of integral membrane proteins that play roles in mediating exocytosis\\u000a in animal cells. However, relatively little is known about the subcellular localization, trafficking, and function of SCAMPs\\u000a in plants. Several recent studies in plant cells indicate that plant SCAMPs share many similarities with their mammalian homologs\\u000a although there are differences. In this

Angus Ho Yin Law; Cheung-Ming Chow; Liwen Jiang


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.  


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

Dubaissi, Eamon; Rousseau, Karine; Lea, Robert; Soto, Ximena; Nardeosingh, Siddarth; Schweickert, Axel; Amaya, Enrique; Thornton, David J; Papalopulu, Nancy



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

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.

Dubaissi, Eamon; Rousseau, Karine; Lea, Robert; Soto, Ximena; Nardeosingh, Siddarth; Schweickert, Axel; Amaya, Enrique; Thornton, David J.; Papalopulu, Nancy



Discovery and progress in our understanding of the regulated secretory pathway in neuroendocrine cells  

Microsoft Academic Search

In this review we start with a historical perspective beginning with the early morphological work done almost 50 years ago.\\u000a The importance of these pioneering studies is underscored by our brief summary of the key questions addressed by subsequent\\u000a research into the mechanism of secretion. We then highlight important advances in our understanding of the formation and maturation\\u000a of neuroendocrine secretory

Joëlle Morvan; Sharon A. Tooze



Differential gene expression profiling in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory-secretory products  

Microsoft Academic Search

Clonorchiasis is associated with bile duct malignancy and the subsequent development of cholangiocarcinoma. Although this\\u000a is likely caused by adult Clonorchis sinensis and its excretory–secretory products (ESP), the precise molecular mechanisms remain obscure. To evaluate the effect of C. sinensis infection on differential gene expression in host hepatocytes, we examined the kinetics of changes in gene expression in\\u000a the human

Jhang Ho Pak; Dong-Wook Kim; Ju Hyun Moon; Joo-Hyun Nam; Jong-Hyeok Kim; Jung Won Ju; Tong-Soo Kim; Sang-Beom Seo



Methods to evaluate Zinc transport into and out of the secretory and endosomal-lysosomal compartments in DT40 cells.  


Zinc plays crucial roles in diverse biological processes. Recently, in addition to zinc mobilization into and out of the cell, zinc mobilization into and out of intracellular organelles, including the secretory and endosomal-lysosomal compartments, has received growing interest. In vertebrate cells, the Zrt/Irt-like proteins (ZIPs) and Zn transporters (ZnTs) are the two major families of zinc transport proteins involved in zinc mobilization across cellular membranes. Importantly, nearly half of them are localized to subcellular compartments. Thus, to elucidate the numerous zinc-related cellular events, understanding those ZIP and ZnT functions is critical. This chapter describes advanced methods used in our laboratory to examine zinc mobilization by them. Specifically, genetic and molecular approaches using chicken DT40 cells deficient in multiple ZIPs and ZnTs are described. Moreover, procedures to evaluate zinc-related phenotypes caused by the impairment of zinc mobilization into and out of the secretory and endosomal-lysosomal compartments are also described. These methods should be useful in characterizing the roles of zinc in diverse cellular events including endosomal signaling. PMID:24359949

Kambe, Taiho



In vitro atrazine-exposure inhibits human natural killer cell lytic granule release  

SciTech Connect

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.

Rowe, Alexander M. [Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506 (United States); Brundage, Kathleen M. [Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506 (United States); Center for Immunopathology and Microbial Pathogenesis, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506 (United States); Barnett, John B. [Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506 (United States) and Center for Immunopathology and Microbial Pathogenesis, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506 (United States)]. E-mail:



A protein induced by NGF in PC12 cells is stored in secretory vesicles and released through the regulated pathway.  

PubMed Central

We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a-beta-galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters. Images

Possenti, R; Eldridge, J D; Paterson, B M; Grasso, A; Levi, A



Core structure, internal osmotic pressure and irreversible structural changes of chromaffin granules during osmometer behaviour.  


In the adrenal medullary cells, catecholamines are stored in and secreted from specialized secretory vesicles, the chromaffin granules. In order to gain some understanding of both functions of chromaffin granules, it is important to characterize their biophysical organization. Using isolated bovine chromaffin granules we have investigated the osmometer behaviour of chromaffin granules by 31P-NMR and fluorescence spectroscopy, by turbidity measurements and by electron-microscopic determination of chromaffin granule size distributions. On the basis of the osmometer model we have formulated equations predicting the behaviour of the native catecholamine fluorescence quenching and of the size of chromaffin granules a a function of osmolarity and have shown experimentally that the granules' behaviour conforms to these. It was possible to estimate the osmotic activity of the chromaffin granule core solution and the mean absolute water space in chromaffin granules from the determination of the size distributions as a function of osmotic pressure. With NMR spectroscopy a selective line-broadening of the alpha-resonances was observed with increasing osmolarities, while the gamma-phosphorus resonances remained virtually unchanged. Possibly there is an increase in core viscosity with osmolarity which affects only the alpha- and beta-phosphorus groups. While suspending chromaffin granules from lower to higher osmolarities causes no lysis, moving them back to their original osmolarity at which they were previously stable lyses them, thereby releasing a maximum of 70% of their releasable protein. This 'hyperosmolar' lysis is independent of preincubation times in the higher osmolarities and of the absolute dilution applied but depends on dilution beyond the 405 to 322 mosM sucrose range. Under the experiment conditions no uptake of sucrose from the medium into the granules could be measured, thereby suggesting that hyperosmolar lysis is a phenomenon not due to solute penetration. Since with NMR and fluorescence spectroscopy no chemical changes in the core composition can be observed, we conclude that hyperosmolar lysis may be caused by irreversible membrane relaxation upon osmotic shrinking. PMID:7055554

Südhof, T C



Detection of chromogranin in neuroendocrine cells with a monoclonal antibody.  

PubMed Central

A monoclonal antibody ( LK2H10 ) produced against a human pheochromocytoma reacted immunohistochemically with 126 normal and neoplastic endocrine tissues with secretory granules which were formalin-fixed and paraffin-embedded. Antibody LK2H10 did not react with 46 other endocrine tissues or tumors without secretory granules nor with 113 normal and neoplastic nonendocrine cells and tumors. Tumors with abundant secretory granules showed intense and diffuse staining, and tumors with few granules, such as Merkel cell carcinomas, neuroblastomas, and small cell carcinomas of lung, showed focal staining. Antibody LK2H10 did not react with melanomas, nevi, posterior pituitary, peripheral nerve tissues, or neurons. The target structure of LK2H10 was identified as human chromogranin, of which the major fraction was chromogranin A (mol wt 68,000 daltons). Preabsorption with purified chromogranin A blocked immunoperoxidase staining by LK2H10 in normal adrenal medulla, in the anterior pituitary, and in a pheochromocytoma. Ultrastructural immunohistochemistry with LK2H10 showed that chromogranin was present in cytoplasmic secretory granules. These results indicate that chromogranin is widely distributed in the secretory granules of most polypeptide-producing endocrine tissues, and it is readily detected with the use of monoclonal antibody LK2H10 . The detection of this marker can be very helpful as a diagnostic aid for neuroendocrine cells and tumors. Images Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 1 Figure 2 Figure 3 Figure 10 Figure 11

Wilson, B. S.; Lloyd, R. V.



Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study.  


The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125?? ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 ?M (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture. PMID:24688815

Toczylowska, Beata; Zieminska, Elzbieta; Goch, Grazyna; Milej, Daniel; Gerega, Anna; Liebert, Adam



Neuropeptidomic Components Generated by Proteomic Functions in Secretory Vesicles for Cell–Cell Communication  

Microsoft Academic Search

Diverse neuropeptides participate in cell–cell communication to coordinate neuronal and endocrine regulation of physiological\\u000a processes in health and disease. Neuropeptides are short peptides ranging in length from ~3 to 40 amino acid residues that\\u000a are involved in biological functions of pain, stress, obesity, hypertension, mental disorders, cancer, and numerous health\\u000a conditions. The unique neuropeptide sequences define their specific biological actions.

Vivian Hook; Steven Bark; Nitin Gupta; Mark Lortie; Weiya D. Lu; Nuno Bandeira; Lydiane Funkelstein; Jill Wegrzyn; Daniel T. O’Connor; Pavel Pevzner



Revisiting the Single Cell Protein Application of Cupriavidus necator H16 and Recovering Bioplastic Granules Simultaneously  

PubMed Central

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.

Kunasundari, Balakrishnan; Murugaiyah, Vikneswaran; Kaur, Gurjeet; Maurer, Frans H. J.; Sudesh, Kumar



Altered Dendritic Integration in Hippocampal Granule Cells of Spatial Learning-Impaired Aged Rats  

PubMed Central

Glutamatergic transmission at central synapses undergoes activity-dependent and developmental changes. In the hippocampal dentate gyrus, the non-N-methyl d-aspartate (NMDA) receptor component of field excitatory postsynaptic potentials (fEPSPs) increases with age in Fischer-344 rats. This effect may not depend on the animal's activity or experience but could be part of the developmental process. Age-dependent differences in synaptic transmission at the perforant path-granule cell synapse may be caused by changes in non-NMDA and NMDA receptor-mediated currents. To test this hypothesis, we compared whole cell excitatory postsynaptic currents (EPSCs) in dentate granule cells evoked by perforant path stimulation in young (3–4 mo) and aged (22–27 mo) Fischer-344 rats using a Cs+-based intracellular solution. Aged animals as a group showed spatial learning and memory deficits in the Morris water maze. Using whole cell recordings, slope conductances of both non-NMDA and NMDA EPSCs at holding potentials ?10 to +50 mV were significantly reduced in aged animals and the non-NMDA/NMDA ratio in aged animals was found to be significantly smaller than in young animals. In contrast, we detected no differences in basic electrophysiological parameters, or absolute amplitudes of non-NMDA and NMDA EPSCs. Extracellular Cs+ increased the fEPSP in young slices to a greater degree than was found in the aged slices, while it increased population spikes to a greater degree in the aged rats. Our results not only provide evidence for reduced glutamatergic synaptic responses in Fischer-344 rats but also point to differential changes in Cs+-sensitive dendritic conductances, such as Ih or inwardly rectifying potassium currents, during aging.

Krause, Michael; Yang, Zhiyong; Rao, Geeta; Houston, Frank P.; Barnes, C. A.



Distinct Temporal Expression of 5-HT1A and 5-HT 2A Receptors on Cerebellar Granule Cells in Mice.  


Serotonin plays an important role of controlling the physiology of the cerebellum. However, serotonin receptor expression has not been fully studied in the developing cerebellum. We have recently shown that cerebellar granule cells transiently express 5-HT3 receptors. In the present study, we investigate expression of 5-HT1 and 5-HT2 receptors in the mouse cerebellum both during postnatal development and in juvenile mice. Here, we show for the first time that 5-HT1A and 5-HT2A receptors are present on cerebellar granule cells with a distinct temporal expression pattern: 5-HT1A receptors are expressed only during the first 2 weeks, while 5-HT2A receptor expression persists until at least 8 weeks after birth. Because of its prolonged expression pattern, we investigated the electrophysiological properties of the 5-HT2A receptor. 5-HT2A receptors expressed by cerebellar granule cells promote stability by reducing variability of the synaptic response, and they modulate the paired-pulse ratio of the parallel fibre-Purkinje cell synapse. Furthermore, pharmacological block of 5-HT2A receptors enhances short-term synaptic plasticity at the parallel fibre-Purkinje cell synapse. We thus show a novel role for serotonin in controlling function of the cerebellum via 5-HT2A receptors expressed by cerebellar granule cells. PMID:24788088

Oostland, Marlies; Buijink, M Renate; Teunisse, Guus M; von Oerthel, Lars; Smidt, Marten P; van Hooft, Johannes A



Dentate hilar cells with dendrites in the molecular layer have lower thresholds for synaptic activation by perforant path than granule cells.  


Neurons in the dentate hilus or area CA3c of rat hippocampal slices were recorded intracellularly with electrodes containing the fluorescent dye Lucifer yellow. Stimulation of perforant path fibers in the molecular layer of the fascia dentata strongly excited most hilar neurons, with a much lower threshold for action potential generation than granule cells and area CA3c pyramidal cells that were recorded in the same area of the slice. Examination of dye-filled hilar neurons with a confocal microscope showed that hilar cells with a low threshold were morphologically heterogeneous: some were spiny "mossy" cells, and others were aspiny interneurons. However, all hilar cells with low thresholds possessed dendrites that penetrated the granule cell layer and passed into the molecular layer, often reaching the outer molecular layer. The few hilar cells that had a threshold similar to, or greater than, granule cells did not possess visible dendrites in the molecular layer. The results suggest that the circuitry of the dentate region allows for (1) excitation of both granule cells and hilar cells by perforant path stimuli, and (2) strong excitation of most hilar cells when most granule cells are subthreshold for action potential generation. Given that hilar neurons project to many different sites in the ipsilateral and contralateral fascia dentata (Blackstad, 1956; Zimmer, 1971; Swanson et al., 1978; Laurberg and Sørensen, 1981), it is quite likely that hilar neurons are involved in the processing of information passing from entorhinal cortex to hippocampus. PMID:2045880

Scharfman, H E



The bHLH transcription factor Ascl1a is essential for the specification of the intestinal secretory cells and mediates Notch signaling in the zebrafish intestine.  


Notch signaling has a fundamental role in stem cell maintenance and in cell fate choice in the intestine of different species. Canonically, Notch signaling represses the expression of transcription factors of the achaete-scute like (ASCL) or atonal related protein (ARP) families. Identifying the ARP/ASCL genes expressed in the gastrointestinal tract is essential to build the regulatory cascade controlling the differentiation of gastrointestinal progenitors into the different intestinal cell types. The expression of the ARP/ASCL factors was analyzed in zebrafish to identify, among all the ARP/ASCL factors found in the zebrafish genome, those expressed in the gastrointestinal tract. ascl1a was found to be the earliest factor detected in the intestine. Loss-of-function analyses using the pia/ascl1a mutant, revealed that ascl1a is crucial for the differentiation of all secretory cells. Furthermore, we identify a battery of transcription factors expressed during secretory cell differentiation and downstream of ascl1a. Finally, we show that the repression of secretory cell fate by Notch signaling is mediated by the inhibition of ascl1a expression. In conclusion, this work identifies Ascl1a as a key regulator of the secretory cell lineage in the zebrafish intestine, playing the same role as Atoh1 in the mouse intestine. This highlights the diversity in the ARP/ASCL family members acting as cell fate determinants downstream from Notch signaling. PMID:23352790

Flasse, Lydie C; Stern, David G; Pirson, Justine L; Manfroid, Isabelle; Peers, Bernard; Voz, Marianne L



Stereological methods reveal the robust size and stability of ectopic hilar granule cells after pilocarpine-induced status epilepticus in the adult rat.  


Following status epilepticus in the rat, dentate granule cell neurogenesis increases greatly, and many of the new neurons appear to develop ectopically, in the hilar region of the hippocampal formation. It has been suggested that the ectopic hilar granule cells could contribute to the spontaneous seizures that ultimately develop after status epilepticus. However, the population has never been quantified, so it is unclear whether it is substantial enough to have a strong influence on epileptogenesis. To quantify this population, the total number of ectopic hilar granule cells was estimated using unbiased stereology at different times after pilocarpine-induced status epilepticus. The number of hilar neurons immunoreactive for Prox-1, a granule-cell-specific marker, was estimated using the optical fractionator method. The results indicate that the size of the hilar ectopic granule cell population after status epilepticus is substantial, and stable over time. Interestingly, the size of the population appears to be correlated with the frequency of behavioral seizures, because animals with more ectopic granule cells in the hilus have more frequent behavioral seizures. The hilar ectopic granule cell population does not appear to vary systematically across the septotemporal axis, although it is associated with an increase in volume of the hilus. The results provide new insight into the potential role of ectopic hilar granule cells in the pilocarpine model of temporal lobe epilepsy. PMID:17042797

McCloskey, Daniel P; Hintz, Tana M; Pierce, Joseph P; Scharfman, Helen E



Observer-independent quantification of insulin granule exocytosis and pre-exocytotic mobility by TIRF microscopy.  


Total internal reflection fluorescence microscopy of fluorescently labeled secretory granules permits monitoring of exocytosis and the preceding granule behavior in one experiment. While observer-dependent evaluation may be sufficient to quantify exocytosis, most of the other information contained in the video files cannot be accessed this way. The present program performs observer-independent detection of exocytosis and tracking of the entire submembrane population of insulin granules. A precondition is the exact localization of the peak of the granule fluorescence. Tracking is based on the peak base radius, peak intensity, and the precrossing itineraries. Robustness of the tracking was shown by simulated tracks of original granule patterns. Mobility in the X-Y dimension is described by the caging diameter which in contrast to the widely used mean square displacement has an inherent time resolution. Observer-independent detection of exocytosis in MIN6 cells labeled with insulin-EGFP is based on the maximal decrease in fluorescence intensity and position of the centroid of the dissipating cloud of released material. Combining the quantification of KCl-induced insulin exocytosis with the analysis of prefusion mobility showed that during the last 3 s pre-exocytotic granules had a smaller caging diameter than control granules and that it increased significantly immediately before fusion. PMID:24230985

Matz, Magnus; Schumacher, Kirstin; Hatlapatka, Kathrin; Lorenz, Dirk; Baumann, Knut; Rustenbeck, Ingo



Loss of ascl1a prevents secretory cell differentiation within the zebrafish intestinal epithelium resulting in a loss of distal intestinal motility  

PubMed Central

The vertebrate intestinal epithelium is renewed continuously from stem cells at the base of the crypt in mammals or base of the fold in fish over the life of the organism. As stem cells divide, newly formed epithelial cells make an initial choice between a secretory or enterocyte fate. This choice has previously been demonstrated to involve Notch signaling as well as Atonal and Her transcription factors in both embryogenesis and adults. Here, we demonstrate that in contrast to the atoh1 in mammals, ascl1a is responsible for formation of secretory cells in zebrafish. ascl1a?/? embryos lack all intestinal epithelial secretory cells and instead differentiate into enterocytes. ascl1a?/? embryos also fail to induce intestinal epithelial expression of deltaD suggesting that ascl1a plays a role in initiation of Notch signaling. Inhibition of Notch signaling increases the number of ascl1a and deltaD expressing intestinal epithelial cells as well as the number of developing secretory cells during two specific time periods: between 30 and 34 hpf and again between 64 and 74 hpf. Loss of enteroendocrine products results in loss of anterograde motility in ascl1a?/? embryos. 5HT produced by enterochromaffin cells is critical in motility and secretion within the intestine. We find that addition of exogenous 5HT to ascl1a?/? embryos at near physiological levels (measured by differential pulse voltammetry) induce anterograde motility at similar levels to wild type velocity, distance, and frequency. Removal or doubling the concentration of 5HT in WT embryos does not significantly affect anterograde motility, suggesting that the loss of additional enteroendocrine products in ascl1a?/? embryos also contributes to intestinal motility. Thus, zebrafish intestinal epithelial cells appear to have a common secretory progenitor from which all subtypes form. Loss of enteroendocrine cells reveals the critical need for enteroendocrine products in maintenance of normal intestinal motility.

Roach, Gillian; Wallace, Rachel Heath; Cameron, Amy; Ozel, Rifat Emrah; Hongay, Cintia F.; Baral, Reshica; Andreescu, Silvana; Wallace, Kenneth N.



Dipeptidyl peptidase-like protein 6 is required for normal electrophysiological properties of cerebellar granule cells.  


In cerebellar granule (CG) cells and many other neurons, A-type potassium currents play an important role in regulating neuronal excitability, firing patterns, and activity-dependent plasticity. Protein biochemistry has identified dipeptidyl peptidase-like protein 6 (DPP6) as an auxiliary subunit of Kv4-based A-type channels and thus a potentially important regulator of neuronal excitability. In this study, we used an RNA interference (RNAi) strategy to examine the role DPP6 plays in forming and shaping the electrophysiological properties of CG cells. DPP6 RNAi delivered by lentiviral vectors effectively disrupts DPP6 protein expression in CG cells. In response to the loss of DPP6, I(SA) peak conductance amplitude is reduced by >85% in parallel with a dramatic reduction in the level of I(SA) channel protein complex found in CG cells. The I(SA) channels remaining in CG cells after suppression of DPP6 show alterations in gating similar to Kv4 channels expressed in heterologous systems without DPP6. In addition to these effects on A-type current, we find that loss of DPP6 has additional effects on input resistance and Na(+) channel conductance that combine with the effects on I(SA) to produce a global change in excitability. Overall, DPP6 expression seems to be critical for the expression of a high-frequency electrophysiological phenotype in CG cells by increasing leak conductance, A-type current levels and kinetics, and Na(+) current amplitude. PMID:20573902

Nadin, Brian M; Pfaffinger, Paul J



NR2B-dependent plasticity of adult born granule cells is necessary for context discrimination  

PubMed Central

Adult generated granule cells (GCs) in the dentate gyrus (DG) exhibit a period of heightened plasticity 4–6 weeks post-mitosis. However, the functional contribution of this critical window of plasticity to hippocampal neurogenesis and behavior remains unknown. Here, we show that deletion of NR2B-containing NMDA receptors from adult born GCs impairs a neurogenesis-dependent form of LTP in the DG, reduces dendritic complexity of adult born GCs, but does not impact their survival. Mice in which the NR2B-containing NMDA receptor was deleted from adult born GCs did not differ from controls in baseline anxiety-like behavior or discrimination of very different contexts, but were impaired in discrimination of highly similar contexts. These results indicate that NR2B-dependent plasticity of adult born GCs is necessary for fine contextual discrimination and is consistent with their proposed role in pattern separation.

Kheirbek, Mazen A.; Tannenholz, Lindsay; Hen, Rene



Excessive activation of mTOR in postnatally generated granule cells is sufficient to cause epilepsy.  


The dentate gyrus is hypothesized to function as a "gate," limiting the flow of excitation through the hippocampus. During epileptogenesis, adult-generated granule cells (DGCs) form aberrant neuronal connections with neighboring DGCs, disrupting the dentate gate. Hyperactivation of the mTOR signaling pathway is implicated in driving this aberrant circuit formation. While the presence of abnormal DGCs in epilepsy has been known for decades, direct evidence linking abnormal DGCs to seizures has been lacking. Here, we isolate the effects of abnormal DGCs using a transgenic mouse model to selectively delete PTEN from postnatally generated DGCs. PTEN deletion led to hyperactivation of the mTOR pathway, producing abnormal DGCs morphologically similar to those in epilepsy. Strikingly, animals in which PTEN was deleted from ? 9% of the DGC population developed spontaneous seizures in about 4 weeks, confirming that abnormal DGCs, which are present in both animals and humans with epilepsy, are capable of causing the disease. PMID:22998871

Pun, Raymund Y K; Rolle, Isaiah J; Lasarge, Candi L; Hosford, Bethany E; Rosen, Jules M; Uhl, Juli D; Schmeltzer, Sarah N; Faulkner, Christian; Bronson, Stefanie L; Murphy, Brian L; Richards, David A; Holland, Katherine D; Danzer, Steve C



Kinetic Analysis of Secretory Protein Traffic and Characterization of Golgi to Plasma Membrane Transport Intermediates in Living Cells  

PubMed Central

Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG–GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG–GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG–GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG–GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments. The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG– GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG–GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG–GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.

Hirschberg, Koret; Miller, Chad M.; Ellenberg, Jan; Presley, John F.; Siggia, Eric D.; Phair, Robert D.; Lippincott-Schwartz, Jennifer



Optofluidic platform for real-time monitoring of live cell secretory activities using Fano resonance in gold nanoslits.  


An optofluidic platform for real-time monitoring of live cell secretory activities is constructed via Fano resonance in a gold nanoslit array. Large-area and highly sensitive gold nanoslits with a period of 500 nm are fabricated on polycarbonate films using the thermal-annealed template-stripping method. The coupling between gap plasmon resonance in the slits and surface plasmon polariton Bloch waves forms a sharp Fano resonance with intensity sensitivity greater than 11 000% per refractive index unit. The nanoslit array is integrated with a cell-trapping microfluidic device to monitor dynamic secretion of matrix metalloproteinase 9 (MMP-9) from human acute monocytic leukemia cells in situ. Upon continuous lipopolysaccharide (LPS) stimulation, MMP-9 secretion is detected within 2 h due to ultrahigh surface sensitivity and close proximity of the sensor to the target cells. In addition to the advantage of detecting early cell responses, the sensor also allows interrogation of cell secretion dynamics. Furthermore, the average secretion per cell measured using our system well matches previous reports while it requires orders of magnitude less cells. The optofluidic platform may find applications in fundamental studies of cell functions and diagnostics based on secretion signals. PMID:23606668

Wu, Shu-Han; Lee, Kuang-Li; Chiou, Arthur; Cheng, Xuanhong; Wei, Pei-Kuen



Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor  

SciTech Connect

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.

Coppé, Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith



Proteomic Analysis of Hippocampal Dentate Granule Cells in Frontotemporal Lobar Degeneration: Application of Laser Capture Technology  

PubMed Central

Frontotemporal lobar degeneration (FTLD) is the most common cause of dementia with pre-senile onset, accounting for as many as 20% of cases. A common subset of FTLD cases is characterized by the presence of ubiquitinated inclusions in vulnerable neurons (FTLD-U). While the pathophysiological mechanisms underlying neurodegeneration in FTLD-U have not yet been elucidated, the presence of inclusions in this disease indicates enhanced aggregation of one or several proteins. Moreover, these inclusions suggest altered expression, processing, or degradation of proteins during FTLD-U pathogenesis. Thus, one approach to understanding disease mechanisms is to delineate the molecular changes in protein composition in FTLD-U brain. Using a combined approach consisting of laser capture microdissection (LCM) and high-resolution liquid chromatography-tandem mass spectrometry (LC–MS/MS), we identified 1252 proteins in hippocampal dentate granule cells excised from three post-mortem FTLD-U and three unaffected control cases processed in parallel. Additionally, we employed a labeling-free quantification technique to compare the abundance of the identified proteins between FTLD-U and control cases. Quantification revealed 54 proteins with selective enrichment in FTLD-U, including TAR–DNA binding protein 43 (TDP-43), a recently identified component of ubiquitinated inclusions. Moreover, 19 proteins were selectively decreased in FTLD-U. Subsequent immunohistochemical analysis of TDP-43 and three additional protein candidates suggests that our proteomic profiling of FTLD-U dentate granule cells reveals both inclusion-associated proteins and non-aggregated disease-specific proteins. Application of LCM is a valuable tool in the molecular analysis of complex tissues, and its application in the proteomic characterization of neurodegenerative disorders such as FTLD-U may be used to identify proteins altered in disease.

Gozal, Yair M.; Dammer, Eric B.; Duong, Duc M.; Cheng, Dongmei; Gearing, Marla; Rees, Howard D.; Peng, Junmin; Lah, James J.; Levey, Allan I.



Dual mechanisms diminishing tonic GABAA inhibition of dentate gyrus granule cells in Noda epileptic rats.  


The Noda epileptic rat (NER), a Wistar colony mutant, spontaneously has tonic-clonic convulsions with paroxysmal discharges. In the present study, we measured phasic and tonic ?-aminobutyric acid A (GABAA) current (I tonic) in NER hippocampal dentate gyrus granule cells and compared the results with those of normal parent strain Wistar rats (WIS). I tonic, revealed by a bicuculline-induced outward shift in holding current, was significantly smaller in NER than in WIS (P < 0.01). The frequency of inhibitory postsynaptic currents (IPSCs) was also significantly lower in NER than in WIS (P < 0.05), without significant differences in the IPSC amplitude or decay time between WIS and NER. I tonic attenuation in NER was further confirmed in the presence of GABA transporter blockers, NO-711 and nipecotic acid, with no difference in neuronal GABA transporter expression between WIS and NER. I tonic responses to extrasynaptic GABAA receptor agonists (THIP and DS-2) were significantly reduced in NER compared with WIS (P < 0.05). Allopregnanolone caused less I tonic increase in NER than in WIS, while it prolonged the IPSC decay time to a similar rate in the two groups. Expression of the GABAA receptor ?-subunit was decreased in the dentate gyrus of NER relative to that of WIS. Taken together, our results showed that a combination of attenuated presynaptic GABA release and extrasynaptic GABAA receptor expression reduced I tonic amplitude and its sensitivity to neurosteroids, which likely diminishes the gating function of dentate gyrus granule cells and renders NER more susceptible to seizure propagation. PMID:23576696

Pandit, Sudip; Jeong, Ji Ae; Jo, Ji Yoon; Cho, Hyun Sill; Kim, Dong Woon; Kim, Jae Moon; Ryu, Pan Dong; Lee, So Yeong; Kim, Hyun Woo; Jeon, Byeong Hwa; Park, Jin Bong



Object/Context-Specific Memory Deficits Associated with Loss of Hippocampal Granule Cells after Adrenalectomy in Rats  

ERIC Educational Resources Information Center

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…

Spanswick, Simon C.; Sutherland, Robert J.



Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B  

PubMed Central

To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granul