Nicolas Vitale | Université de Strasbourg (original) (raw)
Papers by Nicolas Vitale
Philosophical Transactions of the Royal Society B: Biological Sciences, 2013
Loss-of-function mutations in the gene encoding for the RhoGAP protein of oligophrenin-1 (OPHN1) ... more Loss-of-function mutations in the gene encoding for the RhoGAP protein of oligophrenin-1 (OPHN1) lead to cognitive disabilities (CDs) in humans, yet the underlying mechanisms are not known. Here, we show that in mice constitutive lack of Ophn1 is associated with dysregulation of the cyclic adenosine monophosphate/phosphate kinase A (cAMP/PKA) signalling pathway in a brain-area-specific manner. Consistent with a key role of cAMP/PKA signalling in regulating presynaptic function and plasticity, we found that PKA-dependent presynaptic plasticity was completely abolished in affected brain regions, including hippocampus and amygdala. At the behavioural level, lack of OPHN1 resulted in hippocampus- and amygdala-related learning disabilities which could be fully rescued by the ROCK/PKA kinase inhibitor fasudil. Together, our data identify OPHN1 as a key regulator of presynaptic function and suggest that, in addition to reported postsynaptic deficits, loss of presynaptic plasticity contributes to the pathophysiology of CDs.
The Journal of cell biology, Jan 31, 2015
Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the fo... more 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-in...
Journal of Neuroscience, 2015
Oligophrenin-1 (OPHN1) is a protein with multiple domains including a Rho family GTPase-activatin... more 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.
![Research paper thumbnail of [The GIT-PIX protein complex: a hub to ARF and Rac/Cdc42 GTPases]](https://attachments.academia-assets.com/42781131/thumbnails/1.jpg)
](Medecine sciences: M/S
We recently described that the tumor suppressor factor Scribble anchors the PIX exchange factor f... more We recently described that the tumor suppressor factor Scribble anchors the PIX exchange factor for Rac/Cdc42 and the ARF-GAP GIT proteins at the plasma membrane. Because it has been postulated that the GIT-PIX proteins dimerize and tightly self-assemble to form a high molecular weight complex, this nexus may be capable of linking together important signalling molecules to control cytosqueleton polymerization and membrane dynamics. To date, most studies that have tempted to unravel the function of these proteins have found their implication in a great variety of cellular functions (receptor recycling, endo-exocytosis, cell migration, synapse formation...) but have mostly neglected to consider the multimeric organization of this hub. There is no doubt that our comprehension of physiopathological disorders such as cancers will be improved when the nature of the complex pathways integrated by the GIT-PIX nodule will be understood.
Methods in cell biology, 2012
In addition to forming bilayers to separate cellular compartments, lipids participate in vesicula... more In addition to forming bilayers to separate cellular compartments, lipids participate in vesicular trafficking and signal transduction. Among others, phosphatidic acid (PA) is emerging as an important signaling molecule. The spatiotemporal distribution of cellular PA appears to be tightly regulated by localized synthesis and a rapid metabolism. Although PA has been long proposed as a pleiotropic bioactive lipid, when and where PA is produced in the living cells have only recently been explored using biosensors that specifically bind to PA. The probes that we have generated are composed of the PA-binding domains of either Spo20p or Raf1 directly fused to GFP. In this chapter, we will describe the expression and purification of GST-fusion proteins of these probes, and the use of phospholipid strips to validate the specificity of their interaction with PA. We will then illustrate the use of GFP-tagged probes to visualize the synthesis of PA in the neurosecretory PC12 cells and RAW 267....
Journal de la Société de biologie, 1999
In neuroendocrine cells, regulated exocytosis is a multistep process that comprises the recruitme... more In neuroendocrine cells, regulated exocytosis is a multistep process that comprises the recruitment and priming of secretory granules, their docking to the exocytotic sites, and the subsequent fusion of granules with the plasma membrane leading to the release of secretory products into the extracellular space. Using bacterial toxins which specially inactivate subsets of G proteins, we were able to demonstrate that both trimeric and monomeric G proteins directly control the late stages of exocytosis in chromaffin cells. Indeed, in secretagogue-stimulated chromaffin cells, the subplasmalemmal actin cytoskeleton undergoes a specific reorganization that is a prerequisite for exocytosis. Our results suggest that a granule-bound trimeric Go protein controls the actin network surrounding secretory granules through a pathway involving the GTPase RhoA and a downstream phosphatidylinositol 4-kinase. Furthermore, the GTPase Cdc42 plays a active role in exocytosis, most likely by providing spec...
Cellular and molecular neurobiology, 1997
1. Besides having a role in signal transduction, trimeric G proteins may also be involved in memb... more 1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, G alpha o has been found associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells. 2. Microinjection of GTP gamma S and mastoparan strongly inhibits the amperometric response to either nicotine or high K+. 3. The presence of mastoparan in the cell incubation medium had no effect on K(+)-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane. 4. Microinjection of anti-G alpha o antibodies potentiates by more than 50% the K(+)-evoked secretion, whereas anti-G alpha i1/2 antibodies have no effect. 5. Thus an inhibitory Go protein, probably associated with secretor...
Journal of Biological Chemistry, 2015
Regulated secretion is a central issue for the specific function of many cells; for instance, mam... more Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis but its downstream effectors remain elusive in this process. We combined biochemical, functional and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol (DAG)-induced exocytosis. Results from pull-down assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and GTPγS-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explored the ARF6 signaling cascade that promotes secretion. We demonstrated that ARF6 stimulates a sperm PLD activity, to produce phosphatidic acid, and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). We present, direct evidence showing that active ARF6 increases PLC activity, causing PIP2 hydrolysis, and IP3-dependent intraacrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization.
Biochimie, 2014
Phosphatidic acid (PA) is the simplest phospholipid naturally existing in all-living organisms. I... more 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.
AfCS-Nature Molecule Pages, 2006
Communicative & Integrative Biology, 2013
Human immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator ... more 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.
ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we... more ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we demonstrate that these monomeric G proteins are sequentially activated when HEK 293 cells expressing the angiotensin type 1 receptor (AT 1 R) are stimulated with angiotensin II (Ang II). After receptor activation, ARF6 and Rac1 transiently form a complex. Their association is, at least in part, direct and dependent on the nature of the nucleotide bound to both small G proteins. ARF6-GTP preferentially interacts with Rac1-GDP. AT 1 R expressing HEK293 cells ruffle, form membrane protrusions, and migrate in response to agonist treatment. ARF6, but not ARF1, depletion using small interfering RNAs recapitulates the ruffling and migratory phenotype observed after Ang II treatment. These results suggest that ARF6 depletion or Ang II treatment are functionally equivalent and point to a role for endogenous ARF6 as an inhibitor of Rac1 activity. Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling.
Biochimica Et Biophysica Acta-molecular Cell Research, 2004
Release of neurotransmitters and hormones occurs by calcium-regulated exocytosis, a process that ... more Release of neurotransmitters and hormones occurs by calcium-regulated exocytosis, a process that shares many similarities in neurons and neuroendocrine cells. Exocytosis is confined to specific regions in the plasma membrane, where actin remodelling, lipid modifications and protein–protein interactions take place to mediate vesicle/granule docking, priming and fusion. The spatial and temporal coordination of the various players to form a “fast
Current Chemical Biology, 2007
ABSTRACT Cells have evolved specific intracellular compartments that permit local concentration o... more ABSTRACT Cells have evolved specific intracellular compartments that permit local concentration of macromolecules. These macromolecules are transported from one part of the cell to another and eventually released into the extracellular space to participate in cell-to-cell communication. Neurons and neuroendocrine cells secrete neurotransmitters and hormones by exocytosis, a highly regulated process in which secretory vesicles fuse with the plasma membrane to release their contents in response to a calcium trigger. To date, many proteins that catalyze the formation, targeting and fusion of secretory vesicles have been identified. However, the lipid composition of vesicles and their target membrane is also critical and lipid modifications may be required at several stages of the exocytotic pathway. In this review, we will discuss the latest results suggesting important functions for cholesterol, phosphatidic acid (PA) and phosphatidylinositol 4,5- bisphosphate (PIP2) in the membrane merging process. We propose that exocytotic sites are determined by the local formation of lipid micro-domains, which are potentially important to allow structural and spatial organization of the exocytotic machinery. Among the lipid candidates, our results show that PA plays a decisive role in the late stages of exocytosis, most likely by changing the membrane curvature that may be required for membrane fusion to occur. The spatial and temporal coordination of the various players to form an efficient machinery for secretion now needs to be determined.
New Phytologist, 2014
Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been i... more Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been implicated in the regulation of many cellular events, including cytoskeletal dynamics, membrane trafficking and stress responses. Plant PA shows rapid turnover but the information about its spatio-temporal distribution in plant cells is missing. Here we demonstrate the use of a lipid biosensor that enables us to monitor PA dynamics in plant cells.
Philosophical Transactions of the Royal Society B: Biological Sciences, 2013
Loss-of-function mutations in the gene encoding for the RhoGAP protein of oligophrenin-1 (OPHN1) ... more Loss-of-function mutations in the gene encoding for the RhoGAP protein of oligophrenin-1 (OPHN1) lead to cognitive disabilities (CDs) in humans, yet the underlying mechanisms are not known. Here, we show that in mice constitutive lack of Ophn1 is associated with dysregulation of the cyclic adenosine monophosphate/phosphate kinase A (cAMP/PKA) signalling pathway in a brain-area-specific manner. Consistent with a key role of cAMP/PKA signalling in regulating presynaptic function and plasticity, we found that PKA-dependent presynaptic plasticity was completely abolished in affected brain regions, including hippocampus and amygdala. At the behavioural level, lack of OPHN1 resulted in hippocampus- and amygdala-related learning disabilities which could be fully rescued by the ROCK/PKA kinase inhibitor fasudil. Together, our data identify OPHN1 as a key regulator of presynaptic function and suggest that, in addition to reported postsynaptic deficits, loss of presynaptic plasticity contributes to the pathophysiology of CDs.
The Journal of cell biology, Jan 31, 2015
Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the fo... more 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-in...
Journal of Neuroscience, 2015
Oligophrenin-1 (OPHN1) is a protein with multiple domains including a Rho family GTPase-activatin... more 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.
Medecine sciences: M/S
We recently described that the tumor suppressor factor Scribble anchors the PIX exchange factor f... more We recently described that the tumor suppressor factor Scribble anchors the PIX exchange factor for Rac/Cdc42 and the ARF-GAP GIT proteins at the plasma membrane. Because it has been postulated that the GIT-PIX proteins dimerize and tightly self-assemble to form a high molecular weight complex, this nexus may be capable of linking together important signalling molecules to control cytosqueleton polymerization and membrane dynamics. To date, most studies that have tempted to unravel the function of these proteins have found their implication in a great variety of cellular functions (receptor recycling, endo-exocytosis, cell migration, synapse formation...) but have mostly neglected to consider the multimeric organization of this hub. There is no doubt that our comprehension of physiopathological disorders such as cancers will be improved when the nature of the complex pathways integrated by the GIT-PIX nodule will be understood.
Methods in cell biology, 2012
In addition to forming bilayers to separate cellular compartments, lipids participate in vesicula... more In addition to forming bilayers to separate cellular compartments, lipids participate in vesicular trafficking and signal transduction. Among others, phosphatidic acid (PA) is emerging as an important signaling molecule. The spatiotemporal distribution of cellular PA appears to be tightly regulated by localized synthesis and a rapid metabolism. Although PA has been long proposed as a pleiotropic bioactive lipid, when and where PA is produced in the living cells have only recently been explored using biosensors that specifically bind to PA. The probes that we have generated are composed of the PA-binding domains of either Spo20p or Raf1 directly fused to GFP. In this chapter, we will describe the expression and purification of GST-fusion proteins of these probes, and the use of phospholipid strips to validate the specificity of their interaction with PA. We will then illustrate the use of GFP-tagged probes to visualize the synthesis of PA in the neurosecretory PC12 cells and RAW 267....
Journal de la Société de biologie, 1999
In neuroendocrine cells, regulated exocytosis is a multistep process that comprises the recruitme... more In neuroendocrine cells, regulated exocytosis is a multistep process that comprises the recruitment and priming of secretory granules, their docking to the exocytotic sites, and the subsequent fusion of granules with the plasma membrane leading to the release of secretory products into the extracellular space. Using bacterial toxins which specially inactivate subsets of G proteins, we were able to demonstrate that both trimeric and monomeric G proteins directly control the late stages of exocytosis in chromaffin cells. Indeed, in secretagogue-stimulated chromaffin cells, the subplasmalemmal actin cytoskeleton undergoes a specific reorganization that is a prerequisite for exocytosis. Our results suggest that a granule-bound trimeric Go protein controls the actin network surrounding secretory granules through a pathway involving the GTPase RhoA and a downstream phosphatidylinositol 4-kinase. Furthermore, the GTPase Cdc42 plays a active role in exocytosis, most likely by providing spec...
Cellular and molecular neurobiology, 1997
1. Besides having a role in signal transduction, trimeric G proteins may also be involved in memb... more 1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, G alpha o has been found associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells. 2. Microinjection of GTP gamma S and mastoparan strongly inhibits the amperometric response to either nicotine or high K+. 3. The presence of mastoparan in the cell incubation medium had no effect on K(+)-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane. 4. Microinjection of anti-G alpha o antibodies potentiates by more than 50% the K(+)-evoked secretion, whereas anti-G alpha i1/2 antibodies have no effect. 5. Thus an inhibitory Go protein, probably associated with secretor...
Journal of Biological Chemistry, 2015
Regulated secretion is a central issue for the specific function of many cells; for instance, mam... more Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis but its downstream effectors remain elusive in this process. We combined biochemical, functional and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol (DAG)-induced exocytosis. Results from pull-down assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and GTPγS-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explored the ARF6 signaling cascade that promotes secretion. We demonstrated that ARF6 stimulates a sperm PLD activity, to produce phosphatidic acid, and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). We present, direct evidence showing that active ARF6 increases PLC activity, causing PIP2 hydrolysis, and IP3-dependent intraacrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization.
Biochimie, 2014
Phosphatidic acid (PA) is the simplest phospholipid naturally existing in all-living organisms. I... more 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.
AfCS-Nature Molecule Pages, 2006
Communicative & Integrative Biology, 2013
Human immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator ... more 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.
ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we... more ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we demonstrate that these monomeric G proteins are sequentially activated when HEK 293 cells expressing the angiotensin type 1 receptor (AT 1 R) are stimulated with angiotensin II (Ang II). After receptor activation, ARF6 and Rac1 transiently form a complex. Their association is, at least in part, direct and dependent on the nature of the nucleotide bound to both small G proteins. ARF6-GTP preferentially interacts with Rac1-GDP. AT 1 R expressing HEK293 cells ruffle, form membrane protrusions, and migrate in response to agonist treatment. ARF6, but not ARF1, depletion using small interfering RNAs recapitulates the ruffling and migratory phenotype observed after Ang II treatment. These results suggest that ARF6 depletion or Ang II treatment are functionally equivalent and point to a role for endogenous ARF6 as an inhibitor of Rac1 activity. Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling.
Biochimica Et Biophysica Acta-molecular Cell Research, 2004
Release of neurotransmitters and hormones occurs by calcium-regulated exocytosis, a process that ... more Release of neurotransmitters and hormones occurs by calcium-regulated exocytosis, a process that shares many similarities in neurons and neuroendocrine cells. Exocytosis is confined to specific regions in the plasma membrane, where actin remodelling, lipid modifications and protein–protein interactions take place to mediate vesicle/granule docking, priming and fusion. The spatial and temporal coordination of the various players to form a “fast
Current Chemical Biology, 2007
ABSTRACT Cells have evolved specific intracellular compartments that permit local concentration o... more ABSTRACT Cells have evolved specific intracellular compartments that permit local concentration of macromolecules. These macromolecules are transported from one part of the cell to another and eventually released into the extracellular space to participate in cell-to-cell communication. Neurons and neuroendocrine cells secrete neurotransmitters and hormones by exocytosis, a highly regulated process in which secretory vesicles fuse with the plasma membrane to release their contents in response to a calcium trigger. To date, many proteins that catalyze the formation, targeting and fusion of secretory vesicles have been identified. However, the lipid composition of vesicles and their target membrane is also critical and lipid modifications may be required at several stages of the exocytotic pathway. In this review, we will discuss the latest results suggesting important functions for cholesterol, phosphatidic acid (PA) and phosphatidylinositol 4,5- bisphosphate (PIP2) in the membrane merging process. We propose that exocytotic sites are determined by the local formation of lipid micro-domains, which are potentially important to allow structural and spatial organization of the exocytotic machinery. Among the lipid candidates, our results show that PA plays a decisive role in the late stages of exocytosis, most likely by changing the membrane curvature that may be required for membrane fusion to occur. The spatial and temporal coordination of the various players to form an efficient machinery for secretion now needs to be determined.
New Phytologist, 2014
Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been i... more Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been implicated in the regulation of many cellular events, including cytoskeletal dynamics, membrane trafficking and stress responses. Plant PA shows rapid turnover but the information about its spatio-temporal distribution in plant cells is missing. Here we demonstrate the use of a lipid biosensor that enables us to monitor PA dynamics in plant cells.