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1.
Methods Mol Biol ; 2233: 169-179, 2021.
Article in English | MEDLINE | ID: mdl-33222134

ABSTRACT

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.


Subject(s)
Adrenal Medulla/metabolism , Bodily Secretions/metabolism , Cell Culture Techniques/methods , Chromaffin Cells/cytology , Animals , Cattle
2.
Adv Biol Regul ; 79: 100772, 2021 01.
Article in English | MEDLINE | ID: mdl-33288473

ABSTRACT

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.


Subject(s)
Exocytosis , Neuroendocrine Cells/metabolism , Phosphatidic Acids/chemistry , Phosphatidic Acids/metabolism , Animals , Biological Transport , Cell Membrane/metabolism , Humans , Signal Transduction
3.
Cells ; 9(9)2020 09 09.
Article in English | MEDLINE | ID: mdl-32917016

ABSTRACT

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 Tyr23 is critical for exocytosis. Considering that Tyr23 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.


Subject(s)
Annexin A2/metabolism , Calcium/metabolism , Exocytosis/physiology , Neuroendocrine Cells/metabolism , Humans
4.
Cell Rep ; 32(7): 108026, 2020 08 18.
Article in English | MEDLINE | ID: mdl-32814056

ABSTRACT

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.


Subject(s)
Exocytosis/genetics , Neuroendocrine Cells/metabolism , Phosphatidic Acids/metabolism , Animals , Humans , Mice
5.
IUBMB Life ; 72(4): 533-543, 2020 04.
Article in English | MEDLINE | ID: mdl-31967386

ABSTRACT

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.


Subject(s)
Nuclear Proteins/metabolism , Phosphatidic Acids/metabolism , Animals , Cattle , Cells, Cultured , Chromaffin Cells/metabolism , Endoplasmic Reticulum/metabolism , Exocytosis/physiology , Nuclear Proteins/genetics , PC12 Cells , Rats
6.
IUBMB Life ; 72(4): 544-552, 2020 04.
Article in English | MEDLINE | ID: mdl-31859439

ABSTRACT

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.


Subject(s)
Carrier Proteins/metabolism , Chromaffin Cells/metabolism , Microfilament Proteins/metabolism , Neuroendocrine Cells/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Animals , Calcium/metabolism , Carrier Proteins/genetics , Catecholamines/metabolism , Cattle , Exocytosis/physiology , Growth Hormone/metabolism , Microfilament Proteins/genetics , Mutation , PC12 Cells , Rats , Two-Hybrid System Techniques , Wiskott-Aldrich Syndrome Protein, Neuronal/genetics , src Homology Domains
7.
J Immunol ; 202(10): 2971-2981, 2019 05 15.
Article in English | MEDLINE | ID: mdl-30944160

ABSTRACT

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcγR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcγR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcγR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis.


Subject(s)
ADP-Ribosylation Factors/immunology , Macrophages/immunology , Phagocytosis , Phospholipase D/immunology , Receptors, IgG/immunology , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/genetics , Animals , Isoenzymes/genetics , Isoenzymes/immunology , Macrophages/cytology , Mice , Phagosomes/genetics , Phagosomes/immunology , Phosphatidic Acids/genetics , Phosphatidic Acids/immunology , Phospholipase D/genetics , RAW 264.7 Cells , Receptors, IgG/genetics
8.
Biochim Biophys Acta Mol Cell Res ; 1866(7): 1207-1217, 2019 07.
Article in English | MEDLINE | ID: mdl-30610889

ABSTRACT

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.


Subject(s)
Annexin A2/metabolism , Calcium/metabolism , Chromaffin Cells/metabolism , Exocytosis , Membrane Microdomains/metabolism , Amino Acid Substitution , Animals , Annexin A2/genetics , Cattle , Membrane Microdomains/genetics , Mutation, Missense , Phosphorylation/genetics , Proto-Oncogene Proteins pp60(c-src)/genetics , Proto-Oncogene Proteins pp60(c-src)/metabolism
9.
J Biol Chem ; 292(10): 4266-4279, 2017 03 10.
Article in English | MEDLINE | ID: mdl-28115519

ABSTRACT

Phosphatidic acid (PA) is the simplest phospholipid naturally existing in living organisms, but it constitutes only a minor fraction of total cell lipids. PA has attracted considerable attention because it is a phospholipid precursor, a lipid second messenger, and a modulator of membrane shape, and it has thus been proposed to play key cellular functions. The dynamics of PA in cells and in subcellular compartments, however, remains an open question. The recent generation of fluorescent probes for PA, by fusing GFP to PA-binding domains, has provided direct evidence for PA dynamics in different intracellular compartments. Here, three PA sensors were characterized in vitro, and their preferences for different PA species in particular lipidic environments were compared. In addition, the localization of PA in macrophages during frustrated phagocytosis was examined using these PA sensors and was combined with a lipidomic analysis of PA in intracellular compartments. The results indicate that the PA sensors display some preferences for specific PA species, depending on the lipid environment, and the localization study in macrophages revealed the complexity of intracellular PA dynamics.


Subject(s)
Biosensing Techniques , Cell Membrane/metabolism , Macrophages/metabolism , Phagocytosis/physiology , Phosphatidic Acids/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Green Fluorescent Proteins/metabolism , Humans , Lipids/analysis , Macrophages/cytology , Second Messenger Systems
11.
J Cell Biol ; 210(5): 785-800, 2015 Aug 31.
Article in English | MEDLINE | ID: mdl-26323692

ABSTRACT

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament-bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2-induced actin bundling is apparently essential for generating active exocytotic sites.


Subject(s)
Annexin A2/metabolism , Cell Membrane/metabolism , Chromaffin Cells/physiology , Exocytosis/physiology , Secretory Vesicles/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Annexin A2/genetics , Catecholamines/metabolism , Cattle , Cells, Cultured , Electron Microscope Tomography , Membrane Fusion/physiology , Membrane Microdomains/metabolism , Nicotine/pharmacology , Protein Structure, Tertiary , beta-Galactosidase/metabolism
12.
J Neurosci ; 35(31): 11045-55, 2015 Aug 05.
Article in English | MEDLINE | ID: mdl-26245966

ABSTRACT

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.


Subject(s)
Chromaffin Cells/metabolism , Cytoskeletal Proteins/metabolism , Endocytosis/physiology , Exocytosis/physiology , GTPase-Activating Proteins/metabolism , Membrane Fusion/physiology , Nuclear Proteins/metabolism , Animals , Cattle , Cell Membrane/metabolism , Mice , Mice, Knockout , Synaptic Vesicles/metabolism
13.
Biochimie ; 107 Pt A: 51-7, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25111738

ABSTRACT

Phosphatidic acid (PA) is the simplest phospholipid naturally existing in all-living organisms. It constitutes only a minor fraction of the total cell lipids but has attracted considerable attention being both a lipid second messenger and a modulator of membrane shape. The pleiotropic functions of PA are the direct consequence of its very simple chemical structure consisting of only two acyl chains linked by ester bonds to two adjacent hydroxyl groups of glycerol, whose remaining hydroxyl group is esterified with a phosphomonoester group. Hence the small phosphate head group of PA gives it the shape of a cone providing flexibility and negative curvatures in the context of a lipid bilayer. In addition, the negatively charged phosphomonoester headgroup of PA is unique because it can potentially carry one or two negative charges playing a role in the recruitment of positively charged molecules to biomembranes. In consequence, PA has been proposed to play various key cellular functions. In the brain, a fine balance between cell growth, migration and differentiation, and cell death is required to sculpt the nervous system during development. In this review, we will summarize the various functions that have been proposed for PA in neuronal development.


Subject(s)
Cell Membrane/metabolism , Cytoskeleton/metabolism , Neurons/metabolism , Phosphatidic Acids/metabolism , Animals , Axons/metabolism , Dendrites/metabolism , Humans , Models, Neurological , Neurons/cytology , Signal Transduction
14.
J Neurosci ; 33(50): 19470-9, 2013 Dec 11.
Article in English | MEDLINE | ID: mdl-24336713

ABSTRACT

More than 80 human X-linked genes have been associated with mental retardation and deficits in learning and memory. However, most of the identified mutations induce limited morphological alterations in brain organization and the molecular bases underlying neuronal clinical features remain elusive. We show here that neurons cultured from mice lacking ribosomal S6 kinase 2 (Rsk2), a model for the Coffin-Lowry syndrome (CLS), exhibit a significant delay in growth in a similar way to that shown by neurons cultured from phospholipase D1 (Pld1) knock-out mice. We found that gene silencing of Pld1 or Rsk2 as well as acute pharmacological inhibition of PLD1 or RSK2 in PC12 cells strongly impaired neuronal growth factor (NGF)-induced neurite outgrowth. Expression of a phosphomimetic PLD1 mutant rescued the inhibition of neurite outgrowth in PC12 cells silenced for RSK2, revealing that PLD1 is a major target for RSK2 in neurite formation. NGF-triggered RSK2-dependent phosphorylation of PLD1 led to its activation and the synthesis of phosphatidic acid at sites of neurite growth. Additionally, total internal reflection fluorescence microscopy experiments revealed that RSK2 and PLD1 positively control fusion of tetanus neurotoxin insensitive vesicle-associated membrane protein (TiVAMP)/VAMP-7 vesicles at sites of neurite outgrowth. We propose that the loss of function mutations in RSK2 that leads to CLS and neuronal deficits are related to defects in neuronal growth due to impaired RSK2-dependent PLD1 activity resulting in a reduced vesicle fusion rate and membrane supply.


Subject(s)
Neurites/metabolism , Phosphatidic Acids/biosynthesis , Phospholipase D/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Animals , Cells, Cultured , Coffin-Lowry Syndrome/genetics , Coffin-Lowry Syndrome/metabolism , Mice , Mice, Knockout , Nerve Growth Factor/pharmacology , Neurites/drug effects , Neurons/drug effects , Neurons/metabolism , PC12 Cells , Phosphorylation , Rats , Ribosomal Protein S6 Kinases, 90-kDa/genetics
16.
Commun Integr Biol ; 6(5): e25145, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-24255740

ABSTRACT

Human immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator (Tat) viral protein that is required for efficient HIV gene transcription. We recently reported that extracellular Tat is able to enter uninfected neurosecretory cells. Internalized Tat escapes endosomes to reach the cytosol and is then recruited to the plasma membrane by phosphatidylinositol 4,5-bisphophate (PtdIns(4,5)P 2). Tat strongly impairs exocytosis from chromaffin and PC12 cells and perturbs synaptic vesicle exo-endocytosis cycle through its ability to interact with PtdIns(4,5)P 2. Among PtdIns(4,5)P 2-dependent processes required for neurosecretion, we found that Tat impairs annexin A2 recruitment involved in the organization of exocytotic sites at the plasma membrane. Moreover Tat perturbs the actin cytoskeleton reorganization necessary for the movement of secretory vesicles toward their plasma membrane fusion sites during the exocytotic process.    Here, we investigated whether extracellular Tat affects PtdIns(4,5)P 2 metabolism in PC12 cells. Using a diacylglycerol (DAG) sensor, we found that ATP stimulation of exocytosis triggers the production of DAG at the plasma membrane as seen by the relocation of the DAG probe from the cytosol to the plasma membrane. Exposure to Tat strongly delayed the recruitment of the DAG sensor, suggesting a reduced level of DAG production at the early phase of ATP stimulation. These observations indicate that Tat reduces the hydrolysis rate of PtdIns(4,5)P 2 by phospholipase C during exocytosis. Thus, the neuronal disorders often associated with HIV-1 infection may be linked to the capacity of Tat to interact with PtdIns(4,5)P 2, and alter both its metabolism and functions in neurosecretion.

17.
Front Endocrinol (Lausanne) ; 4: 135, 2013 Oct 02.
Article in English | MEDLINE | ID: mdl-24106488

ABSTRACT

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.

18.
Front Endocrinol (Lausanne) ; 4: 125, 2013 Sep 17.
Article in English | MEDLINE | ID: mdl-24062727

ABSTRACT

The regulated secretory pathway in neuroendocrine cells ends with the release of hormones and neurotransmitters following a rise in cytosolic calcium. This process known as regulated exocytosis involves the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, the synaptic vesicle VAMP (synaptobrevin), and the plasma membrane proteins syntaxin and SNAP-25. Although there is much evidence suggesting that SNARE proteins play a key role in the fusion machinery, other cellular elements regulating the kinetics, the extent of fusion, and the preparation of vesicle for release have received less attention. Among those factors, lipids have also been proposed to play important functions both at the level of secretory vesicle recruitment and late membrane fusion steps. Here, we will review the latest evidence supporting the concept of the fusogenic activity of lipids, and also discuss how this may be achieved. These possibilities include the recruitment and sequestration of the components of the exocytotic machinery, regulation of protein function, and direct effects on membrane topology.

19.
J Neurosci ; 33(8): 3545-56, 2013 Feb 20.
Article in English | MEDLINE | ID: mdl-23426682

ABSTRACT

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.


Subject(s)
Chromaffin Cells/metabolism , Endocytosis/physiology , Neuroendocrine Cells/metabolism , Phosphatidylserines/metabolism , Phospholipid Transfer Proteins/metabolism , Animals , Biological Transport, Active/physiology , Cattle , Cell Membrane/metabolism , Chromaffin Cells/enzymology , Exocytosis/physiology , Female , Lipid Metabolism/physiology , Male , Mice , Mice, Transgenic , Neuroendocrine Cells/enzymology , PC12 Cells , Rats
20.
J Cell Sci ; 126(Pt 2): 454-63, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23178941

ABSTRACT

HIV-1 transcriptional activator (Tat) enables viral transcription and is also actively released by infected cells. Extracellular Tat can enter uninfected cells and affect some cellular functions. Here, we examine the effects of Tat protein on the secretory activity of neuroendocrine cells. When added to the culture medium of chromaffin and PC12 cells, Tat was actively internalized and strongly impaired exocytosis as measured by carbon fiber amperometry and growth hormone release assay. Expression of Tat mutants that do not bind to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] did not affect secretion, and overexpression of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), the major PtdIns(4,5)P2 synthesizing enzyme, significantly rescued the Tat-induced inhibition of neurosecretion. This suggests that the inhibition of exocytosis may be the consequence of PtdIns(4,5)P2 sequestration. Accordingly, expression of Tat in PC12 cells interfered with the secretagogue-dependent recruitment of annexin A2 to the plasma membrane, a PtdIns(4,5)P2-binding protein that promotes the formation of lipid microdomains that are required for exocytosis. In addition Tat significantly prevented the reorganization of the actin cytoskeleton necessary for the movement of secretory vesicles towards plasma membrane fusion sites. Thus, the capacity of extracellular Tat to enter neuroendocrine cells and sequester plasma membrane PtdIns(4,5)P2 perturbs several PtdIns(4,5)P2-dependent players of the exocytotic machinery, thereby affecting neurosecretion. We propose that Tat-induced inhibition of exocytosis is involved in the neuronal disorders associated with HIV-1 infection.


Subject(s)
HIV-1/metabolism , Neurosecretion/physiology , Phosphatidylinositol 4,5-Diphosphate/metabolism , tat Gene Products, Human Immunodeficiency Virus/metabolism , Animals , Cattle , Exocytosis/physiology , Humans , Neurosecretion/drug effects , PC12 Cells , Rats , tat Gene Products, Human Immunodeficiency Virus/pharmacology
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