Publications by authors named "Stéphane Ory"

31 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

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

Heteromerization of Endogenous Mu and Delta Opioid Receptors Induces Ligand-Selective Co-Targeting to Lysosomes.

Molecules 2020 Sep 30;25(19). Epub 2020 Sep 30.

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

Increasing evidence indicates that native mu and delta opioid receptors can associate to form heteromers in discrete brain neuronal circuits. However, little is known about their signaling and trafficking. Using double-fluorescent knock-in mice, we investigated the impact of neuronal co-expression on the internalization profile of mu and delta opioid receptors in primary hippocampal cultures. We established ligand selective mu-delta co-internalization upon activation by 1-[[4-(acetylamino)phenyl]methyl]-4-(2-phenylethyl)-4-piperidinecarboxylic acid, ethyl ester (CYM51010), [d-Ala2, NMe-Phe4, Gly-ol5]enkephalin (DAMGO), and deltorphin II, but not (+)-4-[(α)-α-((2,5)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-,-diethylbenzamide (SNC80), morphine, or methadone. Co-internalization was driven by the delta opioid receptor, required an active conformation of both receptors, and led to sorting to the lysosomal compartment. Altogether, our data indicate that mu-delta co-expression, likely through heteromerization, alters the intracellular fate of the mu opioid receptor, which provides a way to fine-tune mu opioid receptor signaling. It also represents an interesting emerging concept for the development of novel therapeutic drugs and strategies.
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http://dx.doi.org/10.3390/molecules25194493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583997PMC
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

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

RCAN1 Knockdown Reverts Defects in the Number of Calcium-Induced Exocytotic Events in a Cellular Model of Down Syndrome.

Front Cell Neurosci 2018 6;12:189. Epub 2018 Jul 6.

Centro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile.

In humans, Down Syndrome (DS) is a condition caused by partial or full trisomy of chromosome 21. Genes present in the DS critical region can result in excess gene dosage, which at least partially can account for DS phenotype. Although regulator of calcineurin 1 (RCAN1) belongs to this region and its ectopic overexpression in neurons impairs transmitter release, synaptic plasticity, learning and memory, the relative contribution of RCAN1 in a context of DS has yet to be clarified. In the present work, we utilized an model of DS, the CTb neuronal cell line derived from the brain cortex of a trisomy 16 (Ts16) fetal mouse, which reportedly exhibits acetylcholine release impairments compared to CNh cells (a neuronal cell line established from a normal littermate). We analyzed single exocytotic events by using total internal reflection fluorescence microscopy (TIRFM) and the vesicular acetylcholine transporter fused to the pH-sensitive green fluorescent protein (VAChT-pHluorin) as a reporter. Our analyses showed that, compared with control CNh cells, the trisomic CTb cells overexpress RCAN1, and they display a reduced number of Ca-induced exocytotic events. Remarkably, RCAN1 knockdown increases the extent of exocytosis at levels comparable to those of CNh cells. These results support a critical contribution of RCAN1 to the exocytosis process in the trisomic condition.
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http://dx.doi.org/10.3389/fncel.2018.00189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6043644PMC
July 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

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

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

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

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

Intersectin: The Crossroad between Vesicle Exocytosis and Endocytosis.

Front Endocrinol (Lausanne) 2013 27;4:109. Epub 2013 Aug 27.

State Key Laboratory of Molecular and Cellular Biology, Institute of Molecular Biology and Genetics , Kyiv , Ukraine ; Department of Functional Genomics, Institute of Molecular Biology and Genetics , Kyiv , Ukraine ; Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique-Unité Propre de Recherche 3212, Université de Strasbourg , Strasbourg , France.

Intersectins (ITSNs) are a family of highly conserved proteins with orthologs from nematodes to mammals. In vertebrates, ITSNs are encoded by two genes (itsn1 and itsn2), which act as scaffolds that were initially discovered as proteins involved in endocytosis. Further investigation demonstrated that ITSN1 is also implicated in several other processes including regulated exocytosis, thereby suggesting a role for ITSN1 in the coupling between exocytosis and endocytosis in excitatory cells. Despite a high degree of conservation amongst orthologs, ITSN function is not so well preserved as they have acquired new properties during evolution. In this review, we will discuss the role of ITSN1 and its orthologs in exo- and endocytosis, in particular in neurons and neuroendocrine cells.
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http://dx.doi.org/10.3389/fendo.2013.00109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753573PMC
August 2013

Dynamin-2 regulates fusion pore expansion and quantal release through a mechanism that involves actin dynamics in neuroendocrine chromaffin cells.

PLoS One 2013 5;8(8):e70638. Epub 2013 Aug 5.

Centro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso, Gran Bretaña, Playa Ancha, Valparaíso, Chile.

Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin's ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070638PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734226PMC
March 2014

Phospholipid scramblase-1-induced lipid reorganization regulates compensatory endocytosis in neuroendocrine cells.

J Neurosci 2013 Feb;33(8):3545-56

Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique-Unité Propre de Recherche 3212 and Université de Strasbourg, 67084 Strasbourg, France.

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.
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http://dx.doi.org/10.1523/JNEUROSCI.3654-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677529PMC
February 2013

Chromogranin A induces the biogenesis of granules with calcium- and actin-dependent dynamics and exocytosis in constitutively secreting cells.

Endocrinology 2012 Sep 31;153(9):4444-56. Epub 2012 Jul 31.

Institut National de la Santé et de la Recherche Médicale (Inserm) U982, University of Rouen, Mont-Saint-Aignan 76821, France.

Chromogranins are a family of acidic glycoproteins that play an active role in hormone and neuropeptide secretion through their crucial role in secretory granule biogenesis in neuroendocrine cells. However, the molecular mechanisms underlying their granulogenic activity are still not fully understood. Because we previously demonstrated that the expression of the major component of secretory granules, chromogranin A (CgA), is able to induce the formation of secretory granules in nonendocrine COS-7 cells, we decided to use this model to dissect the mechanisms triggered by CgA leading to the biogenesis and trafficking of such granules. Using quantitative live cell imaging, we first show that CgA-induced organelles exhibit a Ca(2+)-dependent trafficking, in contrast to native vesicle stomatitis virus G protein-containing constitutive vesicles. To identify the proteins that confer such properties to the newly formed granules, we developed CgA-stably-expressing COS-7 cells, purified their CgA-containing granules by subcellular fractionation, and analyzed the granule proteome by liquid chromatography-tandem mass spectrometry. This analysis revealed the association of several cytosolic proteins to the granule membrane, including GTPases, cytoskeleton-based molecular motors, and other proteins with actin- and/or Ca(2+)-binding properties. Furthermore, disruption of cytoskeleton affects not only the distribution and the transport but also the Ca(2+)-evoked exocytosis of the CgA-containing granules, indicating that these granules interact with microtubules and cortical actin for the regulated release of their content. These data demonstrate for the first time that the neuroendocrine factor CgA induces the recruitment of cytoskeleton-, GTP-, and Ca(2+)-binding proteins in constitutively secreting COS-7 cells to generate vesicles endowed with typical dynamics and exocytotic properties of neuroendocrine secretory granules.
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http://dx.doi.org/10.1210/en.2012-1436DOI Listing
September 2012

How important are Rho GTPases in neurosecretion?

J Neurochem 2011 May 1;117(4):623-31. Epub 2011 Apr 1.

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

Rho GTPases are small GTP binding proteins belonging to the Ras superfamily which act as molecular switches that regulate many cellular function including cell morphology, cell to cell interaction, cell migration and adhesion. In neuronal cells, Rho GTPases have been proposed to regulate neuronal development and synaptic plasticity. However, the role of Rho GTPases in neurosecretion is poorly documented. In this review, we discuss data that highlight the importance of Rho GTPases and their regulators into the control of neurotransmitter and hormone release in neurons and neuroendocrine cells, respectively.
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http://dx.doi.org/10.1111/j.1471-4159.2011.07241.xDOI Listing
May 2011

Rho GTPases and exocytosis: what are the molecular links?

Semin Cell Dev Biol 2011 Feb 8;22(1):27-32. Epub 2010 Dec 8.

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

Delivery of proteins or lipids to the plasma membrane or into the extracellular space occurs through exocytosis, a process that requires tethering, docking, priming and fusion of vesicles, as well as F-actin rearrangements in response to specific extracellular cues. GTPases of the Rho family have been implicated as important regulators of exocytosis, but how Rho proteins control this process is an open question. In this review, we focus on molecular connections that drive Rho-dependent exocytosis in polarized and regulated exocytosis. Specifically, we present data showing that Rho proteins interaction with the exocyst complex and IQGAP mediates polarized exocytosis, whereas interaction with actin-binding proteins like N-WASP mediates regulated exocytosis.
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http://dx.doi.org/10.1016/j.semcdb.2010.12.002DOI Listing
February 2011

Selective recapture of secretory granule components after full collapse exocytosis in neuroendocrine chromaffin cells.

Traffic 2011 Jan 29;12(1):72-88. Epub 2010 Oct 29.

Institut des Neurosciences Cellulaires et Intégratives (UPR 3212), Centre National de la Recherche Scientifique & Université de Strasbourg, 5 rue Blaise Pascal, F-67084 Strasbourg, France.

In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-and-run, cavicapture and full-collapse fusion. During kiss-and-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain. Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-β-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane.
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http://dx.doi.org/10.1111/j.1600-0854.2010.01125.xDOI Listing
January 2011

Calcium-regulated exocytosis in neuroendocrine cells: intersectin-1L stimulates actin polymerization and exocytosis by activating Cdc42.

Ann N Y Acad Sci 2009 Jan;1152:209-14

Département Neurotransmission et Sécrétion Neuroendocrine, Institut des Neurosciences Cellulaires et Intégratives, Centre National de Recherche Scientifique et Université Louis Pasteur, Strasbourg, France.

Actin cytoskeleton remodeling is a critical step of regulated exocytosis in many secretory cell types, including neuroendocrine cells. While the classical model considers the cortical actin network as a physical barrier preventing the uncontrolled recruitment of secretory granules to the plasma membrane docking sites, recent evidence supports the idea that actin polymerization also plays a more active role in the late stages of exocytosis. However, the molecular machinery underlying this positive function of actin in the course of exocytosis remains largely unknown. Here, we propose that the neuronal guanine nucleotide exchange factor, intersectin-1L, activates the GTPase Cdc42, which in turn provides de novo actin filaments that are important for calcium-regulated exocytosis in PC12 cells.
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http://dx.doi.org/10.1111/j.1749-6632.2008.03998.xDOI Listing
January 2009

Rho GTPases in osteoclasts: orchestrators of podosome arrangement.

Eur J Cell Biol 2008 Sep 23;87(8-9):469-77. Epub 2008 Apr 23.

Centre de Recherche de Biochimie Macromoléculaire, UM2, CNRS, Montpellier, France.

Cells from the myeloid lineage, namely macrophages, dendritic cells and osteoclasts, develop podosomes instead of stress fibers and focal adhesions to adhere and migrate. Podosomes share many components with focal adhesions but differ in their molecular organization, with a dense core of polymerized actin surrounded by scaffolding proteins, kinases and integrins. Podosomes are found either isolated both in macrophages and dendritic cells or arranged into superstructures in osteoclasts. When osteoclasts resorb bone, they form an F-actin rich sealing zone, which is a dense array of connected podosomes that firmly anchors osteoclasts to bone. It delineates a compartment in which protons and proteases are secreted to dissolve and degrade the mineralized matrix. Since Rho GTPases have been shown to control F-actin stress fibers and focal adhesions in mesenchymal cells, the question of whether they could also control podosome formation and arrangement in cells from the myeloid lineage, and particularly in osteoclasts, rapidly emerged. This article considers recent advances made in our understanding of podosome arrangements in osteoclasts and how Rho GTPases may control it.
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http://dx.doi.org/10.1016/j.ejcb.2008.03.002DOI Listing
September 2008

Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration.

Biol Cell 2007 Dec;99(12):701-16

CRBM CNRS UMR5237, 1919 route de Mende, 34293 Montpellier cedex 5, France.

Background Information: Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/Wrch-1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N- and C-terminal domains. The N-terminal extension promotes binding to SH3 (Src homology 3)-domain-containing adaptors, whereas the C-terminal extension mediates membrane targeting through palmitoylation of its non-conventional CAAX box. RhoU/Wrch-1 possesses transforming activity, which is negatively regulated by its N-terminal extension and depends on palmitoylation.

Results: In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c-Src-expressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N-terminal extension and the palmitoylation site were dispensable, whereas the C-terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked-down.

Conclusions: Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C-terminal domains are critical for these functions.
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http://dx.doi.org/10.1042/BC20070058DOI Listing
December 2007

Expression profile of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis: identification of essential genes in osteoclasts.

J Bone Miner Res 2006 Sep;21(9):1387-98

Centre de Recherches en Biochimie Macromoléculaire, CNRS FRE 2593, Montpellier, France.

Unlabelled: RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology.

Introduction: During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis.

Materials And Methods: The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest.

Results: Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts.

Conclusions: We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.
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http://dx.doi.org/10.1359/jbmr.060613DOI Listing
September 2006

A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts.

J Cell Sci 2005 Jul;118(Pt 13):2901-11

Laboratoire de Biologie Moléculaire et Cellulaire, UMR 5665 CNRS/ENS, INRA 913, Ecole Normale Supérieure de Lyon, 46, allée d'Italie, 69364 Lyon Cedex 7, France.

Osteoclast maturation is accompanied by changes in podosome patterning, resulting in the formation of a peripheral belt, which requires an intact microtubule network. Here, we report that by inhibiting Rho, the podosome belt is maintained at the cell periphery despite depolymerisation of microtubules by nocodazole. Rho inhibition was correlated to the increase in microtubule stabilisation and microtubule acetylation. By microinjecting activated Rho or its activated effector mDia2 in osteoclasts, we found that the podosome belt was disrupted and the level of microtubule acetylation dramatically decreased. We further characterised the molecular mechanism responsible for microtubule deacetylation by co-immunoprecipitation experiments. We found that not only was mDia2 coprecipitating with the recently identified microtubule deacetylase HDAC6 but that it also activated the microtubule deacetylase activity of HDAC6 in an in vitro deacetylase assay. Finally, we found that during osteoclastogenesis, there is a correlation between the increase in microtubule acetylation and the podosome belt stabilisation and that if Rho is inhibited in the early stages of osteoclast differentiation, it accelerates both microtubule acetylation and podosome belt stabilisation. Altogether, our data reveal a pathway in which Rho interferes with the osteoclast maturation process by controlling the level of microtubule acetylation and actin organisation through mDIA2 and HDAC6.
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http://dx.doi.org/10.1242/jcs.02425DOI Listing
July 2005

Signal transduction: implications for Ras-dependent ERK signaling.

Curr Biol 2004 Apr;14(7):R277-8

Laboratory of Protein Dynamics and Signaling, NCI-Frederick, Frederick, Maryland 21702, USA.

Ras interacts with numerous downstream effectors to transmit a diverse array of cellular signals. A new study shows that a protein known as Impedes Mitogenic signal Propagation, IMP, is an E3 ubiquitin ligase that binds Ras and modulates MAP kinase signaling by regulating the scaffolding activity of KSR.
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http://dx.doi.org/10.1016/j.cub.2004.03.023DOI Listing
April 2004

Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites.

Curr Biol 2003 Aug;13(16):1356-64

Regulation of Cell Growth Laboratory, NCI-Frederick, Frederick, MD 21702, USA.

Background: Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated.

Results: Using mass spectrometry to analyze the KSR1-scaffolding complex, we identify the serine/threonine protein phosphatase PP2A as a KSR1-associated protein and show that PP2A is a critical regulator of KSR1 activity. We find that the enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, we find that PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation.

Conclusions: These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites.
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http://dx.doi.org/10.1016/s0960-9822(03)00535-9DOI Listing
August 2003

Microtubule dynamics differentially regulates Rho and Rac activity and triggers Rho-independent stress fiber formation in macrophage polykaryons.

Eur J Cell Biol 2002 Jun;81(6):351-62

Laboratoire de Biologie Moléculaire et Cellulaire, Ecole Normale Supérieure de Lyon UMR5665, CNRS, ENS Equipe INRA, France.

Multinucleated giant cells (MNGC) derived from avian peripheral blood monocytes present a dense microtubular network emanating from peripherally located centrosomes. We were interested to study how microtubule and F-actin cytoskeletons cooperate in MNGC to maintain cell shape. Microtubule depolymerization by nocodazole triggered the reorganization of the F-actin cytoskeleton in MNGC that is normally organized into podosomes, cortical actin filaments and membrane ruffles. After nocodazole treatment, F-actin was redistributed into unusual transverse fibers associated with focal adhesion plaques. When microtubules were allowed to repolymerize after nocodazole removal, F-actin appeared transiently, together with the small GTPase Rac, in large membrane ruffles. Using affinity precipitation assays, we show that microtubule depolymerization leads to activation of Rho and inhibition of Rac, whereas microtubule repolymerization induces Rac activation and Rho inhibition. Thus, the level of microtubule polymerization inversely regulates Rho and Rac activity in MNGC. Moreover, using C3 exoenzyme, a known inhibitor of Rho, we demonstrate that both the F-actin fiber formation in response to microtubule depolymerization and the formation of membrane ruffles after microtubule repolymerization occur in C3-treated MNGC, indicating that Rho is not required for these events.
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http://dx.doi.org/10.1078/0171-9335-00255DOI Listing
June 2002
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