Publications by authors named "Stéphane Gasman"

51 Publications

Somatostatin analogue pasireotide (SOM230) inhibits catecholamine secretion in human pheochromocytoma cells.

Cancer Lett 2021 Oct 9. Epub 2021 Oct 9.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000, Strasbourg, France. Electronic address:

Increasingly common, neuroendocrine tumors (NETs) are regarded nowadays as neoplasms potentially causing debilitating symptoms and life-threatening medical conditions. Pheochromocytoma is a NET that develops from chromaffin cells of the adrenal medulla, and is responsible for an excessive secretion of catecholamines. Consequently, patients have an increased risk for clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Somatostatin analogues are among the main anti-secretory medical drugs used in current clinical practice in patient with NETs. However, their impact on pheochromocytoma-associated catecholamine hypersecretion remains incompletely explored. This study investigated the potential efficacy of octreotide and pasireotide (SOM230) on human tumor cells directly cultured from freshly resected pheochromocytomas using an implemented catecholamine secretion measurement by carbon fiber amperometry. SOM230 treatment efficiently inhibited nicotine-induced catecholamine secretion both in bovine chromaffin cells and in human tumor cells whereas octreotide had no effect. Moreover, SOM230 specifically decreased the number of exocytic events by impairing the stimulation-evoked calcium influx as well as the nicotinic receptor-activated inward current in human pheochromocytoma cells. Altogether, our findings indicate that SOM230 acts as an inhibitor of catecholamine secretion through a mechanism involving the nicotinic receptor and might be considered as a potential anti-secretory treatment for patients with pheochromocytoma.
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http://dx.doi.org/10.1016/j.canlet.2021.10.009DOI Listing
October 2021

Leptin brain entry via a tanycytic LepR-EGFR shuttle controls lipid metabolism and pancreas function.

Nat Metab 2021 08 2;3(8):1071-1090. Epub 2021 Aug 2.

Univ. Lille, Inserm, CHU Lille, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Lille Neuroscience & Cognition, UMR-S1172, EGID, DISTALZ, Lille, France.

Metabolic health depends on the brain's ability to control food intake and nutrient use versus storage, processes that require peripheral signals such as the adipocyte-derived hormone, leptin, to cross brain barriers and mobilize regulatory circuits. We have previously shown that hypothalamic tanycytes shuttle leptin into the brain to reach target neurons. Here, using multiple complementary models, we show that tanycytes express functional leptin receptor (LepR), respond to leptin by triggering Ca waves and target protein phosphorylation, and that their transcytotic transport of leptin requires the activation of a LepR-EGFR complex by leptin and EGF sequentially. Selective deletion of LepR in tanycytes blocks leptin entry into the brain, inducing not only increased food intake and lipogenesis but also glucose intolerance through attenuated insulin secretion by pancreatic β-cells, possibly via altered sympathetic nervous tone. Tanycytic LepRb-EGFR-mediated transport of leptin could thus be crucial to the pathophysiology of diabetes in addition to obesity, with therapeutic implications.
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http://dx.doi.org/10.1038/s42255-021-00432-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611554PMC
August 2021

A Lipidomics Approach to Measure Phosphatidic Acid Species in Subcellular Membrane Fractions Obtained from Cultured Cells.

Bio Protoc 2021 Jun 20;11(12):e4066. Epub 2021 Jun 20.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 67000 Strasbourg, France.

Over the last decade, lipids have emerged as possessing an ever-increasing number of key functions, especially in membrane trafficking. For instance, phosphatidic acid (PA) has been proposed to play a critical role in different steps along the secretory pathway or during phagocytosis. To further investigate in detail the precise nature of PA activities, we need to identify the organelles in which PA is synthesized and the PA subspecies involved in these biological functions. Indeed, PA, like all phospholipids, has a large variety based on its fatty acid composition. The recent development of PA sensors has helped us to follow intracellular PA dynamics but has failed to provide information on individual PA species. Here, we describe a method for the subcellular fractionation of RAW264.7 macrophages that allows us to obtain membrane fractions enriched in specific organelles based on their density. Lipids from these membrane fractions are precipitated and subsequently processed by advanced mass spectrometry-based lipidomics analysis to measure the levels of different PA species based on their fatty acyl chain composition. This approach revealed the presence of up to 50 different species of PA in cellular membranes, opening up the possibility that a single class of phospholipid could play multiple functions in any given organelle. This protocol can be adapted or modified and used for the evaluation of other intracellular membrane compartments or cell types of interest.
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http://dx.doi.org/10.21769/BioProtoc.4066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260265PMC
June 2021

Protocol for electron microscopy ultrastructural localization of the fusogenic lipid phosphatidic acid on plasma membrane sheets from chromaffin cells.

STAR Protoc 2021 Jun 12;2(2):100464. Epub 2021 Apr 12.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, 67000 Strasbourg, France.

The glycerophospholipid phosphatidic acid (PA) is a key player in regulated exocytosis, but little is known about its localization at the plasma membrane. Here, we provide a protocol for precisely determining the spatial distribution of PA at exocytotic sites by electron microscopy. Using primary bovine chromaffin cells expressing a PA sensor (Spo20p-GFP), we describe the process for cell stimulation and detergent-free preparation of plasma membrane sheets. The protocol can be applied to other cell models and to distinct membrane lipids. For complete details on the use and execution of this protocol, please refer to Tanguy et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2021.100464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065343PMC
June 2021

Phosphatidic acid: Mono- and poly-unsaturated forms regulate distinct stages of neuroendocrine exocytosis.

Adv Biol Regul 2021 01 28;79:100772. Epub 2020 Nov 28.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000 Strasbourg, France. Electronic address:

Lipids have emerged as important actors in an ever-growing number of key functions in cell biology over the last few years. Among them, glycerophospholipids are major constituents of cellular membranes. Because of their amphiphilic nature, phospholipids form lipid bilayers that are particularly useful to isolate cellular content from the extracellular medium, but also to define intracellular compartments. Interestingly, phospholipids come in different flavors based on their fatty acyl chain composition. Indeed, lipidomic analyses have revealed the presence in cellular membranes of up to 50 different species of an individual class of phospholipid, opening the possibility of multiple functions for a single class of phospholipid. In this review we will focus on phosphatidic acid (PA), the simplest phospholipid, that plays both structural and signaling functions. Among the numerous roles that have been attributed to PA, a key regulatory role in secretion has been proposed in different cell models. We review here the evidences that support the idea that mono- and poly-unsaturated PA control distinct steps in hormone secretion from neuroendocrine cells.
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http://dx.doi.org/10.1016/j.jbior.2020.100772DOI Listing
January 2021

Transmission Electron Microscopy and Tomography on Plasma Membrane Sheets to Study Secretory Docking.

Methods Mol Biol 2021 ;2233:301-309

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France.

To study the formation and the architecture of exocytotic site, we generated plasma membrane (PM) sheets on electron microscopy grids to visualize the membrane organization and quantitatively analyze distributions of specific proteins and lipids. This technique allows observing the cytoplasmic face of the plasma membrane by transmission electron microscope. The principle of this approach relies on application of mechanical forces to break open cells. The exposed inner membrane surface can then be visualized with different electron-dense colorations, and specific proteins or lipids can be detected with gold-conjugated probes. Moreover, the membrane sheets are sufficiently resistant to support automated acquisition of multiple-tilt projections, and thus electron tomography allows to obtain three-dimensional (3D) ultrastructural images of secretory granule docked to the plasma membrane.
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http://dx.doi.org/10.1007/978-1-0716-1044-2_20DOI Listing
March 2021

Bovine Chromaffin Cells: Culture and Fluorescence Assay for Secretion.

Methods Mol Biol 2021 ;2233:169-179

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France.

Over the last four decades, chromaffin cells originating from the adrenal medulla have been probably one of the most popular cell models to study neurosecretion at the molecular level. Accordingly, numerous seminal discoveries in the field, including the characterization of role of the cytoskeleton, fusogenic lipids, and soluble N-ethylmaleimide-sensitivefactor attachment protein receptor (SNARE) proteins, have been made using this model. In this chapter, we describe a standard method currently used to isolate and culture bovine chromaffin cells, and we illustrate a catecholamine secretion assay based on the successive transformation of adrenaline into adrenochrome and adrenolutine for fluorescence measurements. We also provide some guidelines for efficient cell recovery and for the use of this assay in the laboratory.
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http://dx.doi.org/10.1007/978-1-0716-1044-2_11DOI Listing
March 2021

Measurements of Compensatory Endocytosis by Antibody Internalization and Quantification of Endocytic Vesicle Distribution in Adrenal Chromaffin Cells.

Methods Mol Biol 2021 ;2233:43-51

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France.

Plasma membrane proteins are amenable to endocytosis assays since they are easily labeled by reagents applied in the extracellular medium. This has been widely exploited to study constitutive endocytosis or ligand-induced receptor endocytosis. Compensatory endocytosis is the mechanism by which components of secretory vesicles are retrieved after vesicle fusion with the plasma membrane in response to cell stimulation and a rise in intracellular calcium. Luminal membrane proteins from secretory vesicles are therefore transiently exposed at the plasma membrane. Here, we described an antibody-based method to monitor compensatory endocytosis in chromaffin cells and present an image-based analysis to quantify endocytic vesicles distribution.
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http://dx.doi.org/10.1007/978-1-0716-1044-2_3DOI Listing
March 2021

Annexin A2 Egress during Calcium-Regulated Exocytosis in Neuroendocrine Cells.

Cells 2020 09 9;9(9). Epub 2020 Sep 9.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000 Strasbourg, France .

Annexin A2 (AnxA2) is a calcium- and lipid-binding protein involved in neuroendocrine secretion where it participates in the formation and/or stabilization of lipid micro-domains required for structural and spatial organization of the exocytotic machinery. We have recently described that phosphorylation of AnxA2 on Tyr is critical for exocytosis. Considering that Tyr phosphorylation is known to promote AnxA2 externalization to the outer face of the plasma membrane in different cell types, we examined whether this phenomenon occurred in neurosecretory chromaffin cells. Using immunolabeling and biochemical approaches, we observed that nicotine stimulation triggered the egress of AnxA2 to the external leaflets of the plasma membrane in the vicinity of exocytotic sites. AnxA2 was found co-localized with tissue plasminogen activator, previously described on the surface of chromaffin cells following secretory granule release. We propose that AnxA2 might be a cell surface tissue plasminogen activator receptor for chromaffin cells, thus playing a role in autocrine or paracrine regulation of exocytosis.
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http://dx.doi.org/10.3390/cells9092059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564067PMC
September 2020

Mono- and Poly-unsaturated Phosphatidic Acid Regulate Distinct Steps of Regulated Exocytosis in Neuroendocrine Cells.

Cell Rep 2020 08;32(7):108026

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, UPR-3212 67000 Strasbourg, France. Electronic address:

Specific forms of fatty acids are well known to have beneficial health effects, but their precise mechanism of action remains elusive. Phosphatidic acid (PA) produced by phospholipase D1 (PLD1) regulates the sequential stages underlying secretory granule exocytosis in neuroendocrine chromaffin cells, as revealed by pharmacological approaches and genetic mouse models. Lipidomic analysis shows that secretory granule and plasma membranes display distinct and specific composition in PA. Secretagogue-evoked stimulation triggers the selective production of several PA species at the plasma membrane near the sites of active exocytosis. Rescue experiments in cells depleted of PLD1 activity reveal that mono-unsaturated PA restores the number of exocytotic events, possibly by contributing to granule docking, whereas poly-unsaturated PA regulates fusion pore stability and expansion. Altogether, this work provides insight into the roles that subspecies of the same phospholipid may play based on their fatty acyl chain composition.
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http://dx.doi.org/10.1016/j.celrep.2020.108026DOI Listing
August 2020

The atypical Rho GTPase RhoU interacts with intersectin-2 to regulate endosomal recycling pathways.

J Cell Sci 2020 08 27;133(16). Epub 2020 Aug 27.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, F-67000 Strasbourg, France

Rho GTPases play a key role in various membrane trafficking processes. RhoU is an atypical small Rho GTPase related to Rac/Cdc42, which possesses unique N- and C-terminal domains that regulate its function and its subcellular localization. RhoU localizes at the plasma membrane, on endosomes and in cell adhesion structures where it governs cell signaling, differentiation and migration. However, despite its endomembrane localization, RhoU function in vesicular trafficking has been unexplored. Here, we identified intersectins (ITSNs) as new binding partners for RhoU and showed that the second PxxP motif at the N terminus of RhoU mediated interactions with the SH3 domains of ITSNs. To evaluate the function of RhoU and ITSNs in vesicular trafficking, we used fluorescent transferrin as a cargo for uptake experiments. We showed that silencing of either RhoU or ITSN2, but not ITSN1, increased transferrin accumulation in early endosomes, resulting from a defect in fast vesicle recycling. Concomitantly, RhoU and ITSN2 colocalized to a subset of Rab4-positive vesicles, suggesting that a RhoU-ITSN2 interaction may occur on fast recycling endosomes to regulate the fate of vesicular cargos.
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http://dx.doi.org/10.1242/jcs.234104DOI Listing
August 2020

Hormones Secretion and Rho GTPases in Neuroendocrine Tumors.

Cancers (Basel) 2020 Jul 10;12(7). Epub 2020 Jul 10.

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique, Université de Strasbourg, F-67000 Strasbourg, France.

Neuroendocrine tumors (NETs) belong to a heterogeneous group of neoplasms arising from hormone secreting cells. These tumors are often associated with a dysfunction of their secretory activity. Neuroendocrine secretion occurs through calcium-regulated exocytosis, a process that is tightly controlled by Rho GTPases family members. In this review, we compiled the numerous mutations and modification of expression levels of Rho GTPases or their regulators (Rho guanine nucleotide-exchange factors and Rho GTPase-activating proteins) that have been identified in NETs. We discussed how they might regulate neuroendocrine secretion.
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http://dx.doi.org/10.3390/cancers12071859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408961PMC
July 2020

Phosphatidic acid metabolism regulates neuroendocrine secretion but is not under the direct control of lipins.

IUBMB Life 2020 04 22;72(4):533-543. Epub 2020 Jan 22.

Centre National de la Recherche Scientifique, Université de Strasbourg, Institut des Neurosciences Cellulaires et Intégratives, Strasbourg, France.

Phosphatidic acid (PA) produced by phospholipase D1 has been shown to contribute to secretory vesicle exocytosis in a large number of cell models. Among various hypotheses, PA may contribute to recruit and/or activate at the exocytotic site a set of proteins from the molecular machinery dedicated to secretion, but also directly influence membrane curvature thereby favoring membrane rearrangements required for membrane fusion. The release of informative molecules by regulated exocytosis is a tightly controlled process. It is thus expected that PA produced to trigger membrane fusion should be rapidly metabolized and converted in a lipid that does not present similar characteristics. PA-phosphatases of the lipin family are possible candidates as they convert PA into diacylglycerol. We show here that lipin 1 and lipin 2 are expressed in neuroendocrine cells where they are cytosolic, but also partially associated with the endoplasmic reticulum. Silencing of lipin 1 or 2 did not affect significantly either basal or evoked secretion from PC12 cells, suggesting that it is unlikely that conversion of PA into a secondary lipid by lipins might represent a regulatory step in exocytosis in neurosecretory cells. However, in agreement with a model in which PA-metabolism could contribute to prevent entering into exocytosis of additional secretory vesicles, ectopic expression of lipin1B-GFP in bovine chromaffin cells reduced the number of exocytotic events as revealed by carbon fiber amperometry recording. Furthermore, individual spike parameters reflecting fusion pore dynamics were also modified by lipin1B-GFP, suggesting that a tight control of PA levels represents an important regulatory step of the number and kinetic of exocytotic events.
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http://dx.doi.org/10.1002/iub.2229DOI Listing
April 2020

αII-spectrin controls calcium-regulated exocytosis in neuroendocrine chromaffin cells through neuronal Wiskott-Aldrich Syndrome protein interaction.

IUBMB Life 2020 04 20;72(4):544-552. Epub 2019 Dec 20.

Centre National de la Recherche Scientifique, Institut des Neurosciences Cellulaires et Intégratives, Université de Strasbourg, Strasbourg, France.

Besides a fundamental structural role at the plasma membrane, spectrin- and actin-based skeletons have been proposed to participate in various processes including vesicular trafficking. Neuroendocrine cells release hormones and neuropeptides through calcium-regulated exocytosis, a process that is coordinated by a fine remodeling of the actin cytoskeleton. We describe here that calcium-regulated exocytosis is impaired in chromaffin and PC12 cells with reduced αII-spectrin expression levels. Using yeast two-hybrid screening, we show that neuronal Wiskott-Aldrich Syndrome protein (N-WASP) is a partner of the αII-spectrin SH3 domain and demonstrate that secretagogue-evoked N-WASP recruitment at cell periphery is blocked in the absence of αII-spectrin. Additionally, experiments performed with ectopically expressed αII-spectrin mutant unable to bind N-WASP indicated that the interaction between SH3 domain and N-WASP is pivotal for neuroendocrine secretion. Our results extend the list of spectrin interactors and strengthen the idea that αII-spectrin is an important scaffold protein that gathers crucial actin-related players of the exocytic machinery.
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http://dx.doi.org/10.1002/iub.2217DOI Listing
April 2020

Phosphorylation cycling of Annexin A2 Tyr23 is critical for calcium-regulated exocytosis in neuroendocrine cells.

Biochim Biophys Acta Mol Cell Res 2019 07 2;1866(7):1207-1217. Epub 2019 Jan 2.

INCI, UPR3212 CNRS, Université de Strasbourg, France; Plateforme Imagerie In Vitro, Neuropôle de Strasbourg, 5 rue Blaise Pascal, F-6708 Strasbourg, France. Electronic address:

Annexin A2 (AnxA2) is a calcium and lipid binding protein involved in neuroendocrine secretion. We have previously demonstrated that AnxA2 participates in the formation and/or stabilization of lipid microdomains required for structural and spatial organization of the exocytotic machinery in chromaffin cells. However, the regulation of AnxA2 is not fully understood. Numerous phosphorylation sites have been identified in the amino-terminal domain of AnxA2. Phosphorylation of Ser25 and Tyr23 are well established and confirmed to be functionally significant. In particular, phosphorylation of Tyr23 by the tyrosine kinase pp60Src reduces the binding of AnxA2 to both actin filaments and the plasma membrane, two major actors of exocytosis, thus, we examined whether AnxA2 was phosphorylated on Tyr23 during exocytosis. Using immunolabelling and a biochemical approach, we found that nicotine stimulation triggered the phosphorylation of Anx A2 on Tyr23. The expression of two AnxA2 mutants carrying phosphorylation deficient (Y23A) or phosphomimetic (Y23E) mutations reduced the number exocytotic sites. Furthermore, expression of AnxA2-Y23A inhibited the formation of lipid microdomains, whereas the expression of AnxA2-Y23E altered actin filaments associated with docked granules. These results suggest that phosphorylation/dephosphorylation switch at Tyr23 in AnxA2 is critical for calcium-regulated exocytosis in neuroendocrine cells. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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http://dx.doi.org/10.1016/j.bbamcr.2018.12.013DOI Listing
July 2019

The caveolae-associated coiled-coil protein, NECC2, regulates insulin signalling in Adipocytes.

J Cell Mol Med 2018 11 30;22(11):5648-5661. Epub 2018 Aug 30.

Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain.

Adipocyte dysfunction in obesity is commonly associated with impaired insulin signalling in adipocytes and insulin resistance. Insulin signalling has been associated with caveolae, which are coated by large complexes of caveolin and cavin proteins, along with proteins with membrane-binding and remodelling properties. Here, we analysed the regulation and function of a component of caveolae involved in growth factor signalling in neuroendocrine cells, neuroendocrine long coiled-coil protein-2 (NECC2), in adipocytes. Studies in 3T3-L1 cells showed that NECC2 expression increased during adipogenesis. Furthermore, NECC2 co-immunoprecipitated with caveolin-1 (CAV1) and exhibited a distribution pattern similar to that of the components of adipocyte caveolae, CAV1, Cavin1, the insulin receptor and cortical actin. Interestingly, NECC2 overexpression enhanced insulin-activated Akt phosphorylation, whereas NECC2 downregulation impaired insulin-induced phosphorylation of Akt and ERK2. Finally, an up-regulation of NECC2 in subcutaneous and omental adipose tissue was found in association with human obesity and insulin resistance. This effect was also observed in 3T3-L1 adipocytes exposed to hyperglycaemia/hyperinsulinemia. Overall, the present study identifies NECC2 as a component of adipocyte caveolae that is regulated in response to obesity and associated metabolic complications, and supports the contribution of this protein as a molecular scaffold modulating insulin signal transduction at these membrane microdomains.
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http://dx.doi.org/10.1111/jcmm.13840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201366PMC
November 2018

18F-FDOPA PET/CT Uptake Parameters Correlate with Catecholamine Secretion in Human Pheochromocytomas.

Neuroendocrinology 2018 27;107(3):228-236. Epub 2018 Jun 27.

Aix-Marseille Université, INSERM, U1251, Marseille Medical Genetics and AP-HM, Department of Endocrinology, Hôpital de la Conception, Centre de Référence des Maladies Rares Hypophysaires HYPO, Marseille,

Background: 18F-FDOPA positron emission tomography/computed tomography (PET/CT) is a sensitive nuclear imaging technology for the diagnosis of pheochromocytomas (PHEO). However, its utility in determining predictive factors for the secretion of catecholamines remains poorly studied.

Methods: Thirty-nine histologically confirmed PHEO were included in this retrospective single-center study. Patients underwent 18F-FDOPA PET/CT before surgery, with an evaluation of several uptake parameters (standardized uptake values [SUVmax and SUVmean] and the metabolic burden [MB] calculated as follows: MB = SUVmean × tumor volume) and measurement of plasma and/or urinary metanephrine (MN), normetanephrine (NM), and chromogranin A. Thirty-five patients were screened for germline mutations in the RET, SDHx, and VHL genes. Once resected, primary cultures of 5 PHEO were used for real-time measurement of catecholamine release by carbon fiber amperometry.

Results: The MB of the PHEO positively correlated with 24-h urinary excretion of NM (r = 0.64, p < 0.0001), MN (r = 0.49, p = 0.002), combined MN and NM (r = 0.75, p < 0.0001), and eventually plasma free levels of NM (r = 0.55, p = 0.006). In the mutated patients (3 SDHD, 2 SDHB, 3 NF1, 1 VHL, and 3 RET), a similar correlation was observed between MB and 24-h urinary combined MN and NM (r = 0.86, p = 0.0012). For the first time, we demonstrate a positive correlation between the PHEO-to-liver SUVmax ratio and the mean number of secretory granule fusion events of the corresponding PHEO cells revealed by amperometric spikes (p = 0.01).

Conclusion: While the 18F-FDOPA PET/CT MB of PHEO strongly correlates with the concentration of MN, amperometric recordings suggest that 18F-FDOPA uptake could be enhanced by overactivity of catecholamine exocytosis.
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http://dx.doi.org/10.1159/000491578DOI Listing
January 2019

Lipid remodelling in neuroendocrine secretion.

Biol Cell 2017 Nov 18;109(11):381-390. Epub 2017 Oct 18.

Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR3212, Université de Strasbourg, Strasbourg, France.

Neuroendocrine cells secrete hormones and polypeptides through a complex membrane trafficking process that involves the transport of specific organelles, called large dense core secretory granules, from the Golgi apparatus to specialised sites at the plasma membrane where these vesicles are successively exocytosed and recaptured by endocytosis through tightly coupled reactions. The minimal machinery required for exocytosis has been defined as SNARE proteins associated with few accessory proteins. On the other side, clathrin and dynamin constitute major components of some of the most important endocytotic pathways. Although many protein contributors of both exocytosis and endocytosis are now identified, their actual interplay is not well resolved. Furthermore, the necessary tight coupling of exocytosis and compensatory endocytosis to maintain membrane homeostasis in neuroendocrine cells is far from being understood. In this review, we focus on the more recently identified role of lipids in these important processes that are above all membrane remodelling events.
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http://dx.doi.org/10.1111/boc.201700030DOI Listing
November 2017

Meeting after meeting: 20 years of discoveries by the members of the Exocytosis-Endocytosis Club.

Biol Cell 2017 Sep 22;109(9):339-353. Epub 2017 Aug 22.

Institut Curie - Centre de Recherche, PSL Research University, Membrane Dynamics and Mechanics of Intracellular Signaling Laboratory, Paris, France.

Twenty years ago, a group of French cell biologists merged two scientific clubs with the aim of bringing together researchers in the fields of Endocytosis and Exocytosis. Founded in 1997, the first annual meeting of the Exocytosis Club was held in 1998. The Endocytosis Club held quarterly meetings from its founding in 1999. The first joint annual meeting of the Exocytosis-Endocytosis Club took place in Paris in April, 2001. What started as a modest gathering of enthusiastic scientists working in the field of cell trafficking has gone from strength to strength, rapidly becoming an unmissable yearly meeting, vividly demonstrating the high quality of science performed in our community and beyond. On the occasion of the 20th meeting of our club, we want to provide historic insight into the fields of exocytosis and endocytosis, and by extension, to subcellular trafficking, highlighting how French scientists have contributed to major advances in these fields. Today, the Exocytosis-Endocytosis Club represents a vibrant and friendly community that will hold its 20th meeting at the Presqu'Ile de Giens, near Toulon in the South of France, on May 11-13, 2017.
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http://dx.doi.org/10.1111/boc.201700026DOI Listing
September 2017

Myosin 1b and F-actin are involved in the control of secretory granule biogenesis.

Sci Rep 2017 07 12;7(1):5172. Epub 2017 Jul 12.

Normandie Univ, UNIROUEN, INSERM, U1239, Laboratoire de Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et d'Innovation Biomédicale de Normandie, 76000, Rouen, France.

Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.
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http://dx.doi.org/10.1038/s41598-017-05617-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507975PMC
July 2017

Inhibition of Cdc42 and Rac1 activities in pheochromocytoma, the adrenal medulla tumor.

Small GTPases 2017 04 29;8(2):122-127. Epub 2016 Jun 29.

a Institut des Neurosciences Cellulaires et Intégratives (INCI) , Strasbourg , France.

Altered Rho GTPase signaling has been linked to many types of cancer. As many small G proteins, Rho GTPases cycle between an active and inactive state thanks to specific regulators that catalyze exchange of GDP into GTP (Rho-GEF) or hydrolysis of GTP into GDP (Rho-GAP). Recent studies have shown that alteration takes place either at the level of Rho proteins themselves (expression levels, point mutations) or at the level of their regulators, mostly RhoGEFs and RhoGAPs. Most reports describe Rho GTPases gain of function that may participate to the tumorigenesis processes. In contrast, we have recently reported that decreased activities of Cdc42 and Rac1 as well as decreased expression of 2 Rho-GEFs, FARP1 and ARHGEF1, correlate with pheochromocytomas, a tumor developing in the medulla of the adrenal gland (Croisé et al., Endocrine Related Cancer, 2016). Here we highlight the major evidence and further study the correlation between Rho GTPases activities and expression levels of ARHGEF1 and FARP1. Finally we also discuss how the decrease of Cdc42 and Rac1 activities may help human pheochromocytomas to develop and comment the possible relationship between FARP1, ARHGEF1 and the 2 Rho GTPases Cdc42 and Rac1 in tumorigenesis.
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http://dx.doi.org/10.1080/21541248.2016.1202634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464121PMC
April 2017

Cdc42 and Rac1 activity is reduced in human pheochromocytoma and correlates with FARP1 and ARHGEF1 expression.

Endocr Relat Cancer 2016 Apr 24;23(4):281-93. Epub 2016 Feb 24.

Institut des Neurosciences Cellulaires et Intégratives (INCI)CNRS UPR 3212, Strasbourg, France

Among small GTPases from the Rho family, Cdc42, RAC, and Rho are well known to mediate a large variety of cellular processes linked with cancer biology through their ability to cycle between an inactive (GDP-bound) and an active (GTP-bound) state. Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GDP for GTP to generate the activated form, whereas the GTPase-activating proteins (GAPs) catalyze GTP hydrolysis, leading to the inactivated form. Modulation of Rho GTPase activity following altered expression of RHO-GEFs and/or RHO-GAPs has already been reported in various human tumors. However, nothing is known about the Rho GTPase activity or the expression of their regulators in human pheochromocytomas, a neuroendocrine tumor (NET) arising from chromaffin cells of the adrenal medulla. In this study, we demonstrate, through an ELISA-based activity assay, that Rac1 and Cdc42 activities decrease in human pheochromocytomas (PCCs) compared with the matched adjacent non-tumor tissue. Furthermore, through quantitative mass spectrometry (MS) approaches, we show that the expression of two RHO-GEF proteins, namely ARHGEF1 and FARP1, is significantly reduced in tumors compared with matched non-tumor tissue, whereas ARHGAP36 expression is increased. Moreover, siRNA-based knockdown of ARHGEF1 and FARP1 in PC12 cells leads to a significant inhibition of Rac1 and Cdc42 activities, respectively. Finally, a principal component analysis (PCA) of our dataset was able to discriminate PCC from non-tumor tissue and indicates a close correlation between Cdc42/Rac1 activity and FARP1/ARHGEF1 expression. Altogether, our findings reveal for the first time the importance of modulation of Rho GTPase activities and expression of their regulators in human PCCs.
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http://dx.doi.org/10.1530/ERC-15-0502DOI Listing
April 2016

Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages.

PLoS One 2016 8;11(1):e0145617. Epub 2016 Jan 8.

Inserm U1016, Institut Cochin, Paris, France.

Phospholipid Scramblase 1 (PLSCR1) was initially characterized as a type II transmembrane protein involved in bilayer movements of phospholipids across the plasma membrane leading to the cell surface exposure of phosphatidylserine, but other cellular functions have been ascribed to this protein in signaling processes and in the nucleus. In the present study, expression and functions of PLSCR1 were explored in specialized phagocytic cells of the monocyte/macrophage lineage. The expression of PLSCR1 was found to be markedly increased in monocyte-derived macrophages compared to undifferentiated primary monocytes. Surprisingly, this 3-fold increase in PLSCR1 expression correlated with an apparent modification in the membrane topology of the protein at the cell surface of differentiated macrophages. While depletion of PLSCR1 in the monocytic THP-1 cell-line with specific shRNA did not inhibit the constitutive cell surface exposure of phosphatidylserine observed in differentiated macrophages, a net increase in the FcR-mediated phagocytic activity was measured in PLSCR1-depleted THP-1 cells and in bone marrow-derived macrophages from PLSCR1 knock-out mice. Reciprocally, phagocytosis was down-regulated in cells overexpressing PLSCR1. Since endogenous PLSCR1 was recruited both in phagocytic cups and in phagosomes, our results reveal a specific role for induced PLSCR1 expression in the modulation of the phagocytic process in differentiated macrophages.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145617PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712888PMC
July 2016

Oligophrenin-1 Connects Exocytotic Fusion to Compensatory Endocytosis in Neuroendocrine Cells.

J Neurosci 2015 Aug;35(31):11045-55

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique, UPR 3212, and Université de Strasbourg, 67084 Strasbourg, France, and

Oligophrenin-1 (OPHN1) is a protein with multiple domains including a Rho family GTPase-activating (Rho-GAP) domain, and a Bin-Amphiphysin-Rvs (BAR) domain. Involved in X-linked intellectual disability, OPHN1 has been reported to control several synaptic functions, including synaptic plasticity, synaptic vesicle trafficking, and endocytosis. In neuroendocrine cells, hormones and neuropeptides stored in large dense core vesicles (secretory granules) are released through calcium-regulated exocytosis, a process that is tightly coupled to compensatory endocytosis, allowing secretory granule recycling. We show here that OPHN1 is expressed and mainly localized at the plasma membrane and in the cytosol in chromaffin cells from adrenal medulla. Using carbon fiber amperometry, we found that exocytosis is impaired at the late stage of membrane fusion in Ophn1 knock-out mice and OPHN1-silenced bovine chromaffin cells. Experiments performed with ectopically expressed OPHN1 mutants indicate that OPHN1 requires its Rho-GAP domain to control fusion pore dynamics. On the other hand, compensatory endocytosis assessed by measuring dopamine-β-hydroxylase (secretory granule membrane) internalization is severely inhibited in Ophn1 knock-out chromaffin cells. This inhibitory effect is mimicked by the expression of a truncated OPHN1 mutant lacking the BAR domain, demonstrating that the BAR domain implicates OPHN1 in granule membrane recapture after exocytosis. These findings reveal for the first time that OPHN1 is a bifunctional protein that is able, through distinct mechanisms, to regulate and most likely link exocytosis to compensatory endocytosis in chromaffin cells.
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http://dx.doi.org/10.1523/JNEUROSCI.4048-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605274PMC
August 2015

The RhoGEF DOCK10 is essential for dendritic spine morphogenesis.

Mol Biol Cell 2015 Jun 7;26(11):2112-27. Epub 2015 Apr 7.

Centre de Recherche en Biochimie Macromoléculaire, CNRS-UMR 5237, Université de Montpellier, 34293 Montpellier, France

By regulating actin cytoskeleton dynamics, Rho GTPases and their activators RhoGEFs are implicated in various aspects of neuronal differentiation, including dendritogenesis and synaptogenesis. Purkinje cells (PCs) of the cerebellum, by developing spectacular dendrites covered with spines, represent an attractive model system in which to decipher the molecular signaling underlying these processes. To identify novel regulators of dendritic spine morphogenesis among members of the poorly characterized DOCK family of RhoGEFs, we performed gene expression profiling of fluorescence-activated cell sorting (FACS)-purified murine PCs at various stages of their postnatal differentiation. We found a strong increase in the expression of the Cdc42-specific GEF DOCK10. Depleting DOCK10 in organotypic cerebellar cultures resulted in dramatic dendritic spine defects in PCs. Accordingly, in mouse hippocampal neurons, depletion of DOCK10 or expression of a DOCK10 GEF-dead mutant led to a strong decrease in spine density and size. Conversely, overexpression of DOCK10 led to increased spine formation. We show that DOCK10 function in spinogenesis is mediated mainly by Cdc42 and its downstream effectors N-WASP and PAK3, although DOCK10 is also able to activate Rac1. Our global approach thus identifies an unprecedented function for DOCK10 as a novel regulator of dendritic spine morphogenesis via a Cdc42-mediated pathway.
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http://dx.doi.org/10.1091/mbc.E14-08-1310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472020PMC
June 2015

Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension.

Mol Biol Cell 2014 Oct 20;25(20):3195-209. Epub 2014 Aug 20.

Université Paris Descartes, Sorbonne Paris Cité, Centre National de la Recherche Scientifique, UMR 8250, 75270 Paris Cedex 06, France

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.
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http://dx.doi.org/10.1091/mbc.E14-07-1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196869PMC
October 2014

Rho GTPases, phosphoinositides, and actin: a tripartite framework for efficient vesicular trafficking.

Small GTPases 2014 10;5:e29469. Epub 2014 Jun 10.

CNRS UPR 3212; Institut des Neurosciences Cellulaires et Intégratives; Université de Strasbourg; Strasbourg, France.

Rho GTPases are well known regulators of the actin cytoskeleton that act by binding and activating actin nucleators. They are therefore involved in many actin-based processes, including cell migration, cell polarity, and membrane trafficking. With the identification of phosphoinositide kinases and phosphatases as potential binding partners or effectors, Rho GTPases also appear to participate in the regulation of phosphoinositide metabolism. Since both actin dynamics and phosphoinositide turnover affect the efficiency and the fidelity of vesicle transport between cell compartments, Rho GTPases have emerged as critical players in membrane trafficking. Rho GTPase activity, actin remodeling, and phosphoinositide metabolism need to be coordinated in both space and time to ensure the progression of vesicles along membrane trafficking pathways. Although most molecular pathways are still unclear, in this review, we will highlight recent advances made in our understanding of how Rho-dependent signaling pathways organize actin dynamics and phosphoinositides and how phosphoinositides potentially provide negative feedback to Rho GTPases during endocytosis, exocytosis and membrane exchange between intracellular compartments.
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http://dx.doi.org/10.4161/sgtp.29469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114633PMC
February 2015

The regulated secretory pathway in neuroendocrine cells.

Front Endocrinol (Lausanne) 2014 8;5:48. Epub 2014 Apr 8.

Centre National de la Recherche Scientifique (CNRS UPR 3212), Institut des Neurosciences Cellulaires et Intégratives (INCI), Université de Strasbourg , Strasbourg , France.

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http://dx.doi.org/10.3389/fendo.2014.00048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986512PMC
April 2014

Exocytosis and endocytosis in neuroendocrine cells: inseparable membranes!

Front Endocrinol (Lausanne) 2013 Oct 2;4:135. Epub 2013 Oct 2.

Institut des Neurosciences Cellulaires et Intégratives (INCI), Centre National de la Recherche Scientifique (CNRS UPR 3212), Université de Strasbourg , Strasbourg , France.

Although 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 neurotransmitters, 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.
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http://dx.doi.org/10.3389/fendo.2013.00135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788349PMC
October 2013

The long coiled-coil protein NECC2 is associated to caveolae and modulates NGF/TrkA signaling in PC12 cells [corrected].

PLoS One 2013 6;8(9):e73668. Epub 2013 Sep 6.

Department of Cell Biology, Physiology and Immunology, Instituto Maimónides de Investigación Biomédica (IMIBIC)/Reina Sofia University Hospital/University of Córdoba, Córdoba, Spain ; CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), Spain.

TrkA-mediated NGF signaling in PC12 cells has been shown to be compartimentalized in specialized microdomains of the plasma membrane, the caveolae, which are organized by scaffold proteins including the member of the caveolin family of proteins, caveolin-1. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of the neuroendocrine long coiled-coil protein 2 (NECC2), a novel long coiled-coil protein selectively expressed in neuroendocrine tissues that contains a predicted caveolin-binding domain and displays structural characteristics of a scaffolding factor. NECC2 distributes in caveolae, wherein it colocalizes with the TrkA receptor, and behaves as a caveolae-associated protein in neuroendocrine PC12 cells. In addition, stimulation of PC12 cells with nerve growth factor (NGF) increased the expression and regulated the distribution of NECC2. Interestingly, knockdown as well as overexpression of NECC2 resulted in a reduction of NGF-induced phosphorylation of the TrkA downstream effector extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) but not of Akt. Altogether, our results identify NECC2 as a novel component of caveolae in PC12 cells and support the contribution of this protein in the maintenance of TrkA-mediated NGF signaling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073668PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765260PMC
July 2014
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