Exocytosis Of Dense-core Granules Research Articles

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction.

Commentary: Granins, Secretory Granule Biogenesis, and Transport

Secretory granules are the hallmark of secretory cells that secrete granins, peptide hormones, and enzymes for various physiological functions in endocrine, exocrine, and neurons. The granin family of proteins are highly acidic and are the major components in dense core secretory granules, including those in chromaffin cells. Granins include chromogranin (Cg) A, B, and secretogranin (Sg) II which are processed to various derived peptides. In this 16th International Symposium of Chromaffin Cell Biology, a number of papers focusing on the mechanisms of biogenesis and transport of secretory granules in chromaffin cells, other endocrine cells and astrocytes were presented. As well, new physiological roles of granin-derived peptides were discussed. Maite Montero-Hadjadje (France) demonstrated that via its terminal domains, CgA induced granule biogenesis and generated a regulated secretory pathway in fibroblasts. These granules recruited cytoskeletal proteins, actin/myosin and microtubule motors to their surface and exhibited Ca sensitive transport to the plasma membrane for release. Peng Loh (USA) showed that a C-terminal CgA-derived peptide, serpinin, upon being released after stimulation of the cells, upregulated the transcription of a protease inhibitor, protease nexin-1 (PN-1) in a cAMP-PKA-sp1 dependent pathway. This in turn stabilized and augmented the level of granule proteins in the Golgi complex, leading to enhanced granule biogenesis. Ricardo Borges (Spain) using chromaffin cells from CgA knockout (KO), CgB KO and Cg double KO mice showed that the absence of granins led to changes in the accumulation and secretion of catecholamines. They concluded that these Cgs are highly efficient in facilitatingmonoamine and calcium accumulation and in the kinetics of exocytosis of dense core granules (for more details, see review by Borges group below). Seung Yoo (Korea) demonstrated the presence of inositol 1,4,5, triphosphate (IP3) sensitive Ca stores and three forms of IP3 receptors/channels in secretory granules in astrocytes, that presumably interacts with the Cgs to modulate Ca channels and Ca release as in chromaffin granules. Several papers were presented demonstrating the role of cytoskeletal network/proteins in granule transport to the plasma membrane for exocytosis. Luis Gutierrez (Spain) presented confocal microscopy and 3D reconstructed images revealing the complex F-actin structure at the cortex of chromaffin cells. He showed that this F-actin structure underwent reorganization during secretion to reposition the vesicles for exocytosis, (for more details, see their review below). Heidi de Wit (The Netherlands) recapped her extensive studies on the morphology of synaptic protein knockout cells at the electron microscopy level, suggesting that syntaxin, SNAP25 and synaptogamin form the minimal docking machinery. She also discussed the role of Munc 18 in reorganizing cortical F-actin for vesicle docking at the plasma membrane, prior to secretion, in chromaffin cells of embryonic mice. (See her review below). Frederic Meunier (Australia) described a novel phosphatidylinisitol (4,5) biphosphate pathway that controls actin-mediated mobilization and translocation of secretory vesicles to the plasma membrane. Small GTPases such as the Rho GTPase family are involved in actin remodeling during exocytosis. Stephane Gasman (France) showed in chromaffin cells and PC12 cells that Rho A, which is associated with secretory Y. P. Loh (*) Section on Cellular Neurobiology, Program on Developmental Neuroscience, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA e-mail: lohp@mail.nih.gov

Submembranous cytoskeleton and neuroplasticity in hypothalamo-neurohypophysial system of rats and mice

Event Abstract Back to Event Submembranous cytoskeleton and neuroplasticity in hypothalamo-neurohypophysial system of rats and mice Roza Benabdesselam1, Ouahiba Benmessaoud2 and Latifa Dorbani-Mamine2* 1 University M. Mammeri, ISB, Algeria 2 University of Sciences and Technologies H. Boumediene, Neurochemistry group, Loboratory of Biology and Physiology of Organisms, Faculty of Biology, Algeria The regulation of water in the body is vital. It occurs due to the sensation of thirst, which itself is caused by intra- and extracellular dehydration. The baroreceptors and peripheral and central voloreceptors detect modifications of blood hyperosmolarity and hypovolemia and communicate with osmosensitive nerve centers that send efferents to magnocellular neurons. These hypothalamic neurons produce and secrete antidiuretic hormone, arginine vasopressin (AVp) and oxytocin (Ox) hormones. Secretion occurs by exocytosis of dense core granules in the neurohypophysial capillary surrounding space and then in the general circulation. The magnocellular neurons are closely associated with glial cells such as pituicytes in neural lobe and tanicytes in median eminence. The molecular processes that generate and regulate secretion are based mainly on the submembranous and cytoplasmic cytoskeletal and membranous domains components. For the first time, we describe the distribution of dystrophins (Dp), utrophins (Up) and their associated proteins (dystrophin-associated proteins, DAPs) distribution in glial, neuronal and vascular endothelial cells of hypothalamo-neurohypophysial system. Dystrophins are the products of the gene altered in Duchenne muscular dystrophy, a debilitating lethal disease. The utrophins are autosomal gene products homologous to dystrophins. These two family proteins have binding capacities even to cytoplasmic and membranous DAPs forming a molecular bridge between the intracellular environment by binding to actin filaments and the extracellular space through their interaction with extracellular matrix components. Previous and present results obtained by immunohistochemistry, Western blotting and in situ hybridization approaches using rats, Dp71 knock out mice and their wild type have identified, in part, the molecular composition of these complexes in rat and mice hypothalamo-neurohypophysial systems compared to that of muscle, used as a reference. Otherwise, the distribution of these complexes is modified under dehydration conditions and suggests different roles as in receiving signals, by implication in the receptor clustering, in granules secretion. All these roles are accompanied with plasticity modifications, either of neurons or of glial cells. Acknowledgment: We thank Professor D. Mornet, University of Montpellier (France) for the donation of antibodies, and Drs A. Rendon and Professor H. Hardin-Pouzet, University Paris (France) Conference: 2nd NEUROMED Workshop, Fez, Morocco, 10 Jun - 12 Jun, 2010. Presentation Type: Oral Presentation Topic: Oral Session 3: The plastic brain: implications for learning and education Citation: Benabdesselam R, Benmessaoud O and Dorbani-Mamine L (2010). Submembranous cytoskeleton and neuroplasticity in hypothalamo-neurohypophysial system of rats and mice. Front. Neurosci. Conference Abstract: 2nd NEUROMED Workshop. doi: 10.3389/conf.fnins.2010.12.00029 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Jun 2010; Published Online: 04 Jun 2010. * Correspondence: Latifa Dorbani-Mamine, University of Sciences and Technologies H. Boumediene, Neurochemistry group, Loboratory of Biology and Physiology of Organisms, Faculty of Biology, Algiers, Algeria, latifa.dorbani@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Roza Benabdesselam Ouahiba Benmessaoud Latifa Dorbani-Mamine Google Roza Benabdesselam Ouahiba Benmessaoud Latifa Dorbani-Mamine Google Scholar Roza Benabdesselam Ouahiba Benmessaoud Latifa Dorbani-Mamine PubMed Roza Benabdesselam Ouahiba Benmessaoud Latifa Dorbani-Mamine Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Synaptotagmin-2 Controls Regulated Exocytosis but Not Other Secretory Responses of Mast Cells

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Role of Munc18 and Rab proteins in the dynamics of dense-core granule exocytosis

Role of Munc18 and Rab proteins in the dynamics of dense-core granule exocytosis

Intersectin-1L nucleotide exchange factor regulates secretory granule exocytosis by activating Cdc42

Rho GTPases are key regulators of the actin cytoskeleton in membrane trafficking events. We previously reported that Cdc42 facilitates exocytosis in neuroendocrine cells by stimulating actin assembly at docking sites for secretory granules. These findings raise the question of the mechanism activating Cdc42 in exocytosis. The neuronal guanine nucleotide exchange factor, intersectin-1L, which specifically activates Cdc42 and is at an interface between membrane trafficking and actin dynamics, appears as an ideal candidate to fulfill this function. Using PC12 and chromaffin cells, we now show the presence of intersectin-1 at exocytotic sites. Moreover, through an RNA interference strategy coupled with expression of various constructs encoding the guanine nucleotide exchange domain, we demonstrate that intersectin-1L is an essential component of the exocytotic machinery. Silencing of intersectin-1 prevents secretagogue-induced activation of Cdc42 revealing intersectin-1L as the factor integrating Cdc42 activation to the exocytotic pathway. Our results extend the current role of intersectin-1L in endocytosis to a function in exocytosis and support the idea that intersectin-1L is an adaptor that coordinates exo-endocytotic membrane trafficking in secretory cells.

Amisyn Regulates Exocytosis and Fusion Pore Stability by Both Syntaxin-dependent and Syntaxin-independent Mechanisms

Amisyn and tomosyn are related by the possession of a C-terminal vesicle-associated membrane protein-like domain that allows them to bind to syntaxin 1 and assemble into SNARE complexes. The formation of inactive complexes may sequester syntaxin and allow tomosyn and amisyn to act as inhibitors of exocytosis. We aimed to use adrenal chromaffin and PC12 cells to probe this possible mode of action of amisyn and tomosyn in dense core granule exocytosis. Although tomosyn is expressed by adrenal chromaffin and PC12 cells, amisyn expression could not be detected allowing examination of the effect of introduction of amisyn expression onto a neuronal-like background. Overexpression of m-tomosyn1 and expression of amisyn both inhibited Ca2+-induced exocytosis in transfected PC12 cells. Surprisingly, this inhibition was not removed when amisyn and tomosyn constructs were used in which key residues required for efficient binding to syntaxin1 were mutated. The effect of amisyn was further characterized using carbon fiber amperometry in chromaffin cells. Expression of amisyn had no effect on the basic characteristics of the amperometric spikes but reduced the number of spikes elicited. This inhibitory action on the extent of exocytosis was also seen with the amisyn mutant deficient in syntaxin1 binding. In addition, expression of amisyn resulted in an increase in the lifetime of the prespike foot, and this effect was abolished by the mutations. These results show that tomosyn and amisyn can negatively regulate exocytosis independently of syntaxin and also that amisyn can regulate the stability of the fusion pore.

Mechanisms of Transport and Exocytosis of Dense-Core Granules Containing Tissue Plasminogen Activator in Developing Hippocampal Neurons

Dense-core granules (DCGs) are organelles found in specialized secretory cells, including neuroendocrine cells and neurons. Neuronal DCGs facilitate many critical processes, including the transport and secretion of proteins involved in learning, and yet their transport and exocytosis are poorly understood. We have used wide-field and total internal reflection fluorescence microscopy, in conjunction with transport theory, to visualize the transport and exocytosis of DCGs containing a tissue plasminogen activator-green fluorescent protein hybrid in cell bodies, neurites, and growth cones of developing hippocampal neurons and to quantify the roles that diffusion, directed motion, and immobility play in these processes. Our results demonstrate that shorter-ranged transport of DCGs near sites of exocytosis in hippocampal neurons and neuroendocrine cells differs markedly. Specifically, the immobile fraction of DCGs within growth cones and near the plasma membrane of hippocampal neurons is small and relatively unaltered by actin disruption, unlike in neuroendocrine cells. Moreover, transport of DCGs in these domains of hippocampal neurons is unusually heterogeneous, being significantly rapid and directed as well as slow and diffusive. Our results also demonstrate that exocytosis is preceded by substantial movement and heterogeneous transport; this movement may facilitate delivery of DCG cargo in hippocampal neurons, given the relatively low abundance of neuronal DCGs. In addition, the extensive mobility of DCGs in hippocampal neurons argues strongly against the hypothesis that cortical actin is a major barrier to membrane-proximal DCGs in these cells. Instead, our results suggest that extended release of DCG cargo from hippocampal neurons arises from heterogeneity in DCG mobility.

Involvement of Rab27b in the regulated secretion of pituitary hormones.

We recently identified a novel set of Rab and its effector, Rab27a/granuphilin, that possibly regulates the exocytosis of insulin-containing, dense core granules in pancreatic beta-cells. In the present study we characterized another isoform, Rab27b, to further investigate the function of the Rab27 subfamily. Among the tissues examined, Rab27b is most abundantly expressed in pituitary tissue, where Rab27a is preferentially expressed. It is also significantly expressed in brain and spleen, where Rab27a is minimally expressed. Rab27a and Rab27b are differentially expressed in pituitary cell types that secrete different peptide hormones. Rab27b associates with secretory granules and granuphilin in the pituitary endocrine cell line AtT20. Furthermore, overexpression of its inactive mutant, Rab27b N133I, significantly inhibits basal and forskolin-induced ACTH secretion in AtT20 cells. These findings indicate that Rab27b is involved in pituitary hormone secretion, which further supports the idea that members of the Rab27 subfamily regulate the exocytosis of dense core granules containing peptide hormones in endocrine cells.

A Direct Inhibitory Role for the Rab3-specific Effector, Noc2, in Ca2+-regulated Exocytosis in Neuroendocrine Cells

Rab proteins comprise a family of GTPases, conserved from yeast to mammals, which are integral components of membrane trafficking pathways. Rab3A is a neural/neuroendocrine-specific member of the Rab family involved in Ca(2+) -regulated exocytosis, where it functions in an inhibitory capacity controlling recruitment of secretory vesicles into a releasable pool at the plasma membrane. The effector by which Rab3A exerts its inhibitory effect is unclear as the Rab3A effectors Rabphilin and RIM have been excluded from for this role. One putative Rab3A effector in dense-core granule exocytosis is the cytosolic zinc finger protein, Noc2. We have established that overexpression of Noc2 in PC12 cells has a direct inhibitory effect upon Ca(2+)-triggered exocytosis in permeabilized cells. We demonstrate specific nucleotide-dependent binding of Noc2 to Rab3A and show that the inhibition of exocytosis is dependent upon this interaction since Rab3A binding-deficient mutants of Noc2 do not inhibit exocytosis. We propose that Noc2 may be a negative effector for Rab3A in regulated exocytosis of dense-core granules from endocrine cells.

Comparison of Cysteine String Protein (Csp) and Mutant α-SNAP Overexpression Reveals a Role for Csp in Late Steps of Membrane Fusion in Dense-Core Granule Exocytosis in Adrenal Chromaffin Cells

Assembly of the SNARE complex and its disassembly caused by the action of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) and NSF is crucial for the maintenance of vesicular traffic, including fusion of regulated exocytotic vesicles. Various other proteins may also have important roles in the processes leading to membrane fusion via interaction with the SNARE proteins, including the secretory vesicle cysteine string protein (Csp). Here we have examined the effect of overexpression of a dominant negative alpha-SNAP mutant or Csp on exocytosis of dense-core granules in single chromaffin cells monitored using amperometry to detect released catecholamine. Exocytosis of trans-Golgi network (TGN)-derived dense-core granules was substantially inhibited by expression of alpha-SNAP(L294A). The amplitude and characteristics of the individual release events were unaffected by expression of alpha-SNAP(L294A), consistent with an essential role for alpha-SNAP in early steps of priming but not in the fusion process. In contrast, Csp overexpression, which also inhibited the extent of exocytosis, also modified the kinetics of the individual release events seen as an increase in the rise time and a broadening of the residual amperometric spikes in Csp-transfected cells. These results suggest that unlike alpha-SNAP, Csp plays a key role in the protein interactions close to the fusion process or fusion pore opening during Ca(2+)-regulated exocytosis.

Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release

To characterize the molecular mechanisms of platelet secretion, we focused on the calcium-induced exocytosis of dense core granules. Platelets contain several known t-SNAREs (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptors) such as syntaxins 2, 4, and 7 and SNAP-23 (synaptosomal associated protein 23). By using an in vitro exocytosis assay, we have been able to assign roles for some of these t-SNAREs in dense core granule release. This calcium-induced secretion relies on the SNARE proteins because it is stimulated by the addition of recombinant alpha-SNAP and inhibited by a dominant negative alpha-SNAP-L294A mutant or by anti-alpha-SNAP and anti-NSF antibodies. SNAP-23 antibodies and an inhibitory C-terminal SNAP-23 peptide both blocked dense core granule release, demonstrating a role for SNAP-23. Unlike other cell types, platelets contain a significant pool of soluble SNAP-23, which does not partition into Triton X-114. Of the anti-syntaxin antibodies tested, only anti-syntaxin 2 antibody inhibited dense core granule release. Immunoprecipitation studies showed that the 2 t-SNAREs syntaxin 2 and SNAP-23 do form a complex in vivo. These data clearly show that SNAPs, NSF, and specific t-SNAREs are used for dense core granule release; these data provide a greater understanding of regulated exocytosis in platelets.

Neuronal Ca2+ Sensor 1, the Mammalian Homologue of Frequenin, Is Expressed in Chromaffin and PC12 Cells and Regulates Neurosecretion from Dense-core Granules

Neuronal Ca2+ sensor 1 (NCS-1) is the mammalian homologue of the Ca2+-binding protein frequenin previously implicated in regulation of neurotransmission in Drosophila (Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Endelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K. W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J. , Canal, I., Barbas, J. A., and Ferrus, A. (1993) Neuron 11, 15-28). NCS-1 has been considered to be expressed only in neurons, but we show that NCS-1 expression can be detected in bovine adrenal chromaffin and PC12 cells, two widely studied model neuroendocrine cells. NCS-1 was present in both cytosolic and membrane fractions including purified chromaffin granules, and in immunofluorescence, its distribution overlapped with peripheral punctate staining seen with the synaptic-like microvesicle marker synaptophysin in PC12 cells. The possible functional role of NCS-1 in exocytosis of dense-core granules was tested using transient transfection in PC12 cells and assay of co-transfected growth hormone (GH) release. Overexpression of NCS-1 increased evoked GH release in intact cells in response to ATP. No effect of overexpression was seen on GH release because of Ca2+ in permeabilized cells suggesting that NCS-1 may have a regulatory but not direct role in neurosecretion.

Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: correlation with its blockade of catecholamine release.

The seven types (A--G) of botulinum neurotoxin (BoNT) are Zn2+ -dependent endoproteases that potently block neurosecretion. Syntaxin is presently thought to be the sole substrate for BoNT/C1, and synaptosomal-associated protein of Mr = 25 000 (SNAP-25) is selectively proteolyzed by types A and E. In this study, the effects of C1 on Ca2+ -regulated exocytosis of dense core granules from adreno-chromaffin cells were examined together with its underlying molecular action. Intact chromaffin cells were exposed to the toxin, and catecholamine release therefrom was then measured in conjunction with the monitoring of syntaxin cleavage by Western blotting. A good correlation was obtained between degradation of syntaxin 1A/B and reduction in Ca2+- or Ba2+-dependent secretion. However, blotting with antibodies against a C-terminal peptide of SNAP-25 revealed the additional disappearance of immunoreactivity, with the same toxin concentration dependency as syntaxin breakdown. Notably, the cleaved SNAP-25 product was similar in size to that produced by BoNT/A; however, contamination of BoNT/C1 by serotypes A or E was eliminated. Therefore, it is concluded that syntaxin 1A/B and SNAP-25 are cleaved in intact cells poisoned with only C1. Notably, C1 treatment of chromaffin cells abolished Ca2+ -evoked secretion following digitonin permeabilization, compared with partial inhibition by BoNT/A, suggesting the importance of syntaxin for catecholamine release. Unexpectedly, C1 failed to proteolyze a soluble recombinant SNAP-25, even though it served as an efficient substrate for BoNT/A. These interesting observations suggest that C1 can only efficiently cleave SNAP-25 in intact cells, possibly due to the existence therein of a unique conformation and/or the participation of accessory factors.