Phospholipid Scramblase 1 Controls Efficient Neurotransmission and Synaptic Vesicle Retrieval at Cerebellar Synapses.

Phospholipids (PLs) are asymmetrically distributed at the plasma membrane. This asymmetric lipid distribution is transiently altered during calcium-regulated exocytosis, but the impact of this transient remodeling on presynaptic function is currently unknown. As phospholipid scramblase 1 (PLSCR1) randomizes PL distribution between the two leaflets of the plasma membrane in response to calcium activation, we set out to determine its role in neurotransmission. We report here that PLSCR1 is expressed in cerebellar granule cells (GrCs) and that PLSCR1-dependent phosphatidylserine egress occurred at synapses in response to neuron stimulation. Synaptic transmission is impaired at GrC Plscr1 -/- synapses, and both PS egress and synaptic vesicle (SV) endocytosis are inhibited in Plscr1 -/- cultured neurons from male and female mice, demonstrating that PLSCR1 controls PL asymmetry remodeling and SV retrieval following neurotransmitter release. Altogether, our data reveal a novel key role for PLSCR1 in SV recycling and provide the first evidence that PL scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.

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

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.

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

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.

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

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.

Polarized secretion of lysosomes at the B cell synapse couples antigen extraction to processing and presentation.

Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II (MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag. Lysosome recruitment and secretion results from the polarization of the microtubule-organizing center (MTOC), which relies on the cell division cycle (Cdc42)-downstream effector, atypical protein kinase C (aPKCζ). aPKCζ is phosphorylated upon BCR engagement, associates to lysosomal vesicles, and is required for their polarized secretion at the B cell synapse. Regulation of B lymphocyte polarity therefore emerges as a central mechanism that couples Ag extraction to Ag processing and presentation.

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

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.

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

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.

Lipid remodelling in neuroendocrine secretion.

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.

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

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.

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

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-ß-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.

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

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

Restoring cellular copper homeostasis in Alzheimer disease: a novel peptide shuttle is internalized by an ATP-dependent endocytosis pathway involving Rab5- and Rab14-endosomes.

CPPs, or Cell-Penetrating Peptides, offer invaluable utility in disease treatment due to their ability to transport various therapeutic molecules across cellular membranes. Their unique characteristics, such as biocompatibility and low immunogenicity, make them ideal candidates for delivering drugs, genes, or imaging agents directly into cells. This targeted delivery enhances treatment efficacy while minimizing systemic side effects. CPPs exhibit versatility, crossing biological barriers and reaching intracellular targets that conventional drugs struggle to access. This capability holds promise in treating a wide array of diseases, including cancer, neurodegenerative disorders, and infectious diseases, offering a potent avenue for innovative and targeted therapies, yet their precise mechanism of cell entry is far from being fully understood. In order to correct Cu dysregulation found in various pathologies such as Alzheimer disease, we have recently conceived a peptide Cu(II) shuttle, based on the αR5W4 CPP, which, when bound to Cu(II), is able to readily enter a neurosecretory cell model, and release bioavailable Cu in cells. Furthermore, this shuttle has the capacity to protect cells in culture against oxidative stress-induced damage which occurs when Cu binds to the Aß peptide. The aim of this study was therefore to characterize the cell entry route used by this shuttle and determine in which compartment Cu is released. Pharmacological treatments, siRNA silencing and colocalization experiments with GFP-Rab fusion proteins, indicate that the shuttle is internalized by an ATP-dependent endocytosis pathway involving both Rab5 and Rab14 endosomes route and suggest an early release of Cu from the shuttle.

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

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-ß-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.

Rho GTPases and exocytosis: what are the molecular links?

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.

Phospholipase D and cancer metastasis: A focus on exosomes.

In mammals, phospholipase D (PLD) enzymes involve 6 isoforms, of which only three have established lipase activity to produce the signaling lipid phosphatidic acid (PA). This phospholipase activity has been postulated to contribute to cancer progression for over three decades now, but the exact mechanisms involved have yet to be uncovered. Indeed, using various models, an altered PLD activity has been proposed altogether to increase cell survival rate, promote angiogenesis, boost rapamycin resistance, and favor metastasis. Although for some part, the molecular pathways by which this increase in PA is pro-oncogenic are partially known, the pleiotropic functions of PA make it quite difficult to distinguish which among these simple signaling pathways is responsible for each of these PLD facets. In this review, we will describe an additional potential contribution of PA generated by PLD1 and PLD2 in the biogenesis, secretion, and uptake of exosomes. Those extracellular vesicles are now viewed as membrane vehicles that carry informative molecules able to modify the fate of receiving cells at distance from the original tumor to favor homing of metastasis. The perspectives for a better understanding of these complex role of PLDs will be discussed.

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

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

Phospholipase D1-generated phosphatidic acid modulates secretory granule trafficking from biogenesis to compensatory endocytosis in neuroendocrine cells.

Calcium-regulated exocytosis is a multi-step process that allows specialized secretory cells to release informative molecules such as neurotransmitters, neuropeptides, and hormones for intercellular communication. The biogenesis of secretory vesicles from the Golgi cisternae is followed by their transport towards the cell periphery and their docking and fusion to the exocytic sites of the plasma membrane allowing release of vesicular content. Subsequent compensatory endocytosis of the protein and lipidic constituents of the vesicles maintains cell homeostasis. Despite the fact that lipids represent the majority of membrane constituents, little is known about their contribution to these processes. Using a combination of electrochemical measurement of single chromaffin cell catecholamine secretion and electron microscopy of roof-top membrane sheets associated with genetic, silencing and pharmacological approaches, we recently reported that diverse phosphatidic acid (PA) species regulates catecholamine release efficiency by controlling granule docking and fusion kinetics. The enzyme phospholipase D1 (PLD1), producing PA from phosphatidylcholine, seems to be the major responsible of these effects in this model. Here, we extended this work using spinning disk confocal microscopy showing that inhibition of PLD activity also reduced the velocity of granules undergoing a directed motion. Furthermore, a dopamine ß-hydroxylase (DßH) internalization assay revealed that PA produced by PLD is required for an optimal recovery of vesicular membrane content by compensatory endocytosis. Thus, among numerous roles that have been attributed to PA our work gives core to the key regulatory role in secretion that has been proposed in different cell models. Few leads to explain these multiple functions of PA along the secretory pathway are discussed.

Management of Patients with Treatment of Pheochromocytoma: A Critical Appraisal.

The management of pheochromocytomas has significantly evolved these last 50 years, especially with the emergence of new technologies such as laparoscopic procedures in the 1990s. A preoperative blockade using antihypertensive medications to prevent intraoperative hemodynamic instability and cardiocirculatory events is recommended by current clinical guidelines. However, these guidelines are still based on former experiences and are subject to discussion in the scientific community. The aim of this systematic review was to assess the evolution of the management of pheochromocytomas. Laparoscopic procedure is established as the standard of care in current practices. Preoperative medical preparation should be questioned because it does not significantly improve intraoperative events or the risk of postoperative complications in current clinical practice. Current clinical recommendations should be revised and upgraded to current clinical practices.

Development of Cu(ii)-specific peptide shuttles capable of preventing Cu-amyloid beta toxicity and importing bioavailable Cu into cells.

Copper (Cu) in its ionic forms is an essential element for mammals and its homeostasis is tightly controlled. Accordingly, Cu-dyshomeostasis can be lethal as is the case in the well-established genetic Wilson's and Menkes diseases. In Alzheimer's disease (AD), Cu-accumulation occurs in amyloid plaques, where it is bound to the amyloid-beta peptide (Aß). In vitro, Cu-Aß is competent to catalyze the production of reactive oxygen species (ROS) in the presence of ascorbate under aerobic conditions, and hence Cu-Aß is believed to contribute to the oxidative stress in AD. Several molecules that can recover extracellular Cu from Aß and transport it back into cells with beneficial effects in cell culture and transgenic AD models were identified. However, all the Cu-shuttles currently available are not satisfactory due to various potential limitations including ion selectivity and toxicity. Hence, we designed a novel peptide-based Cu shuttle with the following properties: (i) it contains a Cu(ii)-binding motif that is very selective to Cu(ii) over all other essential metal ions; (ii) it is tagged with a fluorophore sensitive to Cu(ii)-binding and release; (iii) it is made of a peptide platform, which is very versatile to add new functions. The work presented here reports on the characterization of AKH-αR5W4NBD, which is able to transport Cu ions selectively into PC12 cells and the imported Cu appeared bioavailable, likely via reductive release induced by glutathione. Moreover, AKH-αR5W4NBD was able to withdraw Cu from the Aß1-16 peptide and consequently inhibited the Cu-Aß based reactive oxygen species production and related cell toxicity. Hence, AKH-αR5W4NBD could be a valuable new tool for Cu-transport into cells and suitable for mechanistic studies in cell culture, with potential applications in restoring Cu-homeostasis in Cu-related diseases such as AD.

Dysfunction of calcium-regulated exocytosis at a single-cell level causes catecholamine hypersecretion in patients with pheochromocytoma.

Neuroendocrine tumors constitute a heterogeneous group of tumors arising from hormone-secreting cells and are generally associated with a dysfunction of secretion. Pheochromocytoma (Pheo) is a neuroendocrine tumor that develops from chromaffin cells of the adrenal medulla, and is responsible for an excess of catecholamine secretion leading to severe clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Surprisingly, while the hypersecretory activity of Pheo is well known to pathologists and clinicians, it has never been carefully explored at the cellular and molecular levels. In the present study, we have combined catecholamine secretion measurement by carbon fiber amperometry on human tumor cells directly cultured from freshly resected Pheos, with the analysis by mass spectrometry of the exocytotic proteins differentially expressed between the tumor and the matched adjacent non-tumor tissue. In most patients, catecholamine secretion recordings from single Pheo cells revealed a higher number of exocytic events per cell associated with faster kinetic parameters. Accordingly, we unravel significant tumor-associated modifications in the expression of key proteins involved in different steps of the calcium-regulated exocytic pathway. Altogether, our findings indicate that dysfunction of the calcium-regulated exocytosis at the level of individual Pheo cell is a cause of the tumor-associated hypersecretion of catecholamines.