Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane

The ADP ribosylation factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore

Exocytosis and Endocytosis in Neuroendocrine Cells: Inseparable Membranes!

International audienceAlthough much has been learned concerning the mechanisms of secretory vesicle formation and fusion at donor and acceptor membrane compartments, relatively little attention has been paid toward understanding how cells maintain a homeostatic membrane balance through vesicular trafficking. In neurons and neuroendocrine cells, release of neurotrans-mitters, neuropeptides, and hormones occurs through calcium-regulated exocytosis at the plasma membrane. To allow recycling of secretory vesicle components and to preserve organelles integrity, cells must initiate and regulate compensatory membrane uptake. This review relates the fate of secretory granule membranes after full fusion exocytosis in neuroendocrine cells. In particular, we focus on the potential role of lipids in preserving and sorting secretory granule membranes after exocytosis and we discuss the potential mechanisms of membrane retrieval

COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells

Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415–5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1α, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region

V-ATPase modulates exocytosis in neuroendocrine cells through the activation of the ARNO-Arf6-PLD pathway and the synthesis of phosphatidic acid

Although there is mounting evidence indicating that lipids serve crucial functions in cells and are implicated in a growing number of human diseases, their precise roles remain largely unknown. This is particularly true in the case of neurosecretion, where fusion with the plasma membrane of specific membrane organelles is essential. Yet, little attention has been given to the role of lipids. Recent groundbreaking research has emphasized the critical role of lipid localization at exocytotic sites and validated the essentiality of fusogenic lipids, such as phospholipase D (PLD)-generated phosphatidic acid (PA), during membrane fusion. Nevertheless, the regulatory mechanisms synchronizing the synthesis of these key lipids and neurosecretion remain poorly understood. The vacuolar ATPase (V-ATPase) has been involved both in vesicle neurotransmitter loading and in vesicle fusion. Thus, it represents an ideal candidate to regulate the fusogenic status of secretory vesicles according to their replenishment state. Indeed, the cytosolic V1 and vesicular membrane-associated V0 subdomains of V-ATPase were shown to dissociate during the stimulation of neurosecretory cells. This allows the subunits of the vesicular V0 to interact with different proteins of the secretory machinery. Here, we show that V0a1 interacts with the Arf nucleotide-binding site opener (ARNO) and promotes the activation of the Arf6 GTPase during the exocytosis in neuroendocrine cells. When the interaction between V0a1 and ARNO was disrupted, it resulted in the inhibition of PLD activation, synthesis of phosphatidic acid during exocytosis, and changes in the timing of fusion events. These findings indicate that the separation of V1 from V0 could function as a signal to initiate the ARNO-Arf6-PLD1 pathway and facilitate the production of phosphatidic acid, which is essential for effective exocytosis in neuroendocrine cells

Phospholipid Scramblase-1-Induced Lipid Reorganization Regulates Compensatory Endocytosis in Neuroendocrine Cells

Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1−/−mice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis

Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A2

Background: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. Methodology/Principal Findings: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaMbinding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. Conclusions/Significance: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems

Implication de l annexine A2 lors de l assemblage des sites d exocytose dans les cellules chromaffines

L exocytose est un processus qui permet aux cellules de sécréter des signaux chimiques. Dans les cellules chromaffines, la libération des catécholamines est régulée par le calcium et requiert nombre d acteurs, parmi lesquels l annexine A2. Cette protéine existe sous forme d un monomère ou d un tétramère avec S100A10, elle est cytosolique et transloque dans l espace subplasmalemmal lors d une stimulation par un sécrétagogue. L annexine A2 liant les phospholipides membranaires et le cytosquelette d actine, acteurs majeurs de la sécrétion, elle pouvait intervenir dans la régulation spatiotemporelle de ce processus. J ai donc cherché quel était le rôle de l annexine A2 au cours de l exocytose, en me focalisant sur son implication dans l organisation des membranes et du cytosquelette d actine. Mes travaux ont montré l intervention de l annexine A2 dans la formation de microdomaines lipidiques nécessaires à l exocytose dans les cellules chromaffines et l importance de l interaction entre S100A10 et VAMP2, protéine de la machinerie d exocytose, dans le recrutement de l annexine A2 au niveau de présites de sécrétion de la membrane plasmique. L annexine A2 agirait sur l actine, en réticulant des microfilaments ancrés à la membrane plasmique, pour former des microdomaines lipidiques nécessaires à l exocytose. Ainsi, l annexine A2 serait une protéine essentielle à la régulation spatiotemporelle de l exocytose dans les cellules chromaffines : en provoquant la formation de microdomaines lipidiques nécessaires à l assemblage et/ou à l activation de la machinerie d exocytose au bon endroit et au bon moment, l annexine A2 permettrait le bon déroulement du processus de sécrétion.Exocytosis is a fundamental biological process of many cellular functions, including the secretion of chemical signals. In neuroendocrine chromaffin cells, catecholamine secretion is calcium-regulated, and requires the concerted action of a number of proteins and lipids. Annexin A2 is one of the proteins implicated in regulated exocytosis. In cells, annexin A2 exists as a monomer or as a tetramer with S100A10, and translocates from the cytosol to the cell periphery when exocytosis is stimulated. As a phospholipid and actin binding protein, annexin A2 probably participates in the regulation of the secretory process. The aim of my thesis was to determine how annexin A2 affects membrane and actin cytoskeleton organization during exocytosis in chromaffin cells. Annexin A2 was found to promote the formation of lipid rafts necessary for exocytosis in chromaffin cells. Its partner S100A10 was shown to interact with VAMP2, a protein of the secretory machinery, and this interaction may serve to target annexin A2 to exocytotic presites in the plasma membrane. Finally, annexin A2 was shown to act on the bundling of actin filaments anchored to the plasma membrane to form lipid rafts. Taken together, these results suggest that annexin A2 is essential for the spatiotemporal regulation of exocytosis in chromaffin cells: annexin A2 triggers the coalescence of lipid rafts required for the recruitment and/or activation of the exocytotic machinery, and ensures that secretion occurs at the right time and place.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Functional Characterization and Potential Applications for Enhanced Green Fluorescent Protein-and Epitope-Fused Human M1 Muscarinic Receptors

International audienceFour recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential gly-cosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild-type hM1 receptor together with confocal micros-copy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N-terminal domain of the fusion proteins. Their functional properties were examined through McN-A-343-evoked Ca 2ϩ release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC 50 values) to mediate the Ca 2ϩ response, although reduced amplitudes (E max values) were obtained for the i3-shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP-fused receptors, their detection, quantitation, and visual-ization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocyto-chemistry. Altogether, this work demonstrates that these EGFP-and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors

Toxines bactériennes : outils pour l’étude des protéines G impliquées dans les mécanismes de l’exocytose dans les cellules neuroendocrines

Dans les cellules neuroendocrines, l’exocytose est un mécanisme complexe impliquant le recrutement des granules de sécrétion, leur arrimage aux sites d’exocytose de la membrane plasmique, puis leur fusion avec la membrane plasmique permettant la libération des produits de sécrétion dans l’espace extracellulaire. L'utilisation de toxines bactériennes au spectre d’action sélectif nous a permis de montrer que ces différentes étapes sont étroitement contrôlées par des protéines G monomériques et trimériques. Ainsi, la mobilisation des granules de sécrétion dans une cellule stimulée par un sécrétagogue nécessite la réorganisation spécifique du cytosquelette d’actine sous la membrane plasmique. Nos résultats suggèrent que cette toile d’actine est en partie contrôlée par une protéine G trimérique de type Go, associée à la membrane des granules de sécrétion, et qui, par l’intermédiaire de la GTPase RhoA couplée à une phosphatidylinositol 4kinase, peut stabiliser les filaments d’actine à la surface granulaire. De plus, les étapes d’arrimage et/ou de fusion des granules à la membrane plasmique requièrent la participation de la GTPase Cdc42 dont la fonction serait d’assurer la mise en place de structures d’actine nécessaires à l’exocytose. L’ensemble de nos travaux souligne l’importance du cytosquelette d’actine et révèle le contrôle séquentiel exercé par les protéines G dans les étapes ultimes de l’exocytose. En outre, ces données illustrent l’intérêt potentiel des toxines bactériennes pour disséquer les voies moléculaires de la sécrétion dans une cellule neuroendocrine