Biomedical subjects

Nicolas Vitale

Publications and source records attributed to Nicolas Vitale.

At least 19 recordsLinked to original sources

PubMed2006

Endogenous ARF6 interacts with Rac1 upon angiotensin II stimulation to regulate membrane ruffling and cell migration.

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.

ADP-Ribosylation Factor 6↗

PubMed2006

Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration.

BACKGROUND: Mammalian Scribble (Scrib) plays a conserved role in polarization of epithelial and neuronal cells. Polarization is essential for migration of a variety of cell types; however, the function of Scrib in this context remains unclear. Scrib has been shown to interact with betaPIX, a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42. Cdc42 controls cell polarity from yeast to mammals during asymmetric cell division and epithelial cell polarization, as well as during cell migration. Cdc42 is, in particular, required for polarization and orientation of astrocytes in a scratch-induced polarized migration assay. Using this assay, we characterized Scrib function during polarized cell migration. RESULTS: Depletion of Scrib by siRNA or expression of dominant-negative constructs inhibits astrocyte polarization. Like Cdc42, Scrib controls protrusion formation, cytoskeleton polarization, and centrosome and Golgi reorientation. Scrib interacts and colocalizes with betaPIX at the front edge of polarizing astrocytes. Perturbation of Scrib localization or of Scrib-betaPIX interaction inhibits betaPIX polarized recruitment. We further show that betaPIX is required for astrocyte polarization and that both the Scrib-binding motif and the GEF activity of betaPIX are essential for its function. Scrib and betaPIX control Cdc42 activation and localization during astrocyte polarization. Thereby, Scrib regulates Cdc42-dependent APC and Dlg1 recruitment to the leading edge to promote cell orientation. CONCLUSION: We conclude that Scrib plays a key role in the establishment of cell polarity during migration. By interacting with betaPIX, Scrib controls localization and activation of the small GTPase Cdc42 and regulates Cdc42-dependent polarization pathways.

Animals↗

PubMed2006

Functional implication of neuronal calcium sensor-1 and phosphoinositol 4-kinase-beta interaction in regulated exocytosis of PC12 cells.

Several studies have shown that the neuronal calcium sensor (NCS-1) and phosphoinositol 4-kinase-beta (PI4K-beta) regulate the exocytotic process of nerve and neuroendocrine cells. The aim of our study was to investigate their possible interaction at rest and during stimulation in living cells and to decipher the role of this interaction in the secretory process. In PC12 cells, we observed a stimulation-induced recruitment of NCS-1 and PI4K-beta from the intracellular compartment toward the plasma membrane. This recruitment was highly correlated to the intracellular Ca(2+) rise induced by secretagogues. Using fluorescence resonance energy transfer between PI4K-beta-ECFP and NCS-1-EYFP, we show that both proteins are interacting in resting cells and that this interaction increases with stimulation. It appears that the membrane insertion of NCS-1 is necessary for the interaction with PI4K-beta, since a mutation that prevented the membrane insertion of NCS-1 abolished NCS-1-PI4K-beta interaction, as revealed by fluorescence resonance energy transfer analysis. Additionally, the overexpression of mutated NCS-1 prevents the stimulatory effect on secretion induced by PI4K-beta, suggesting that the interaction of the two proteins on a membrane compartment is necessary for the secretory function. Moreover, extinction of endogenous PI4K-beta by small interfering RNA inhibits secretion and completely prevents the stimulatory effect of NCS-1 on calcium-evoked exocytosis from permeabilized PC12 cells, showing directly for the first time the functional implication of a NCS-1.PI4K-beta complex in regulated exocytosis.

1-Phosphatidylinositol 4-Kinase↗

PubMed2006

Regulation of neuroendocrine exocytosis by the ARF6 GTPase-activating protein GIT1.

Neuroendocrine cells release hormones and neuropeptides by exocytosis, a highly regulated process in which secretory granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Using chromaffin and PC12 cells, we have recently described that the granule-associated GTPase ARF6 plays a crucial role in exocytosis by activating phospholipase D1 at the plasma membrane and, presumably, promoting the fusion reaction between the two membrane bilayers. ARF6 is activated by the nucleotide exchange factor ARNO following docking of granules to the plasma membrane. We show here that GIT1, a GTPase-activating protein stimulating GTP hydrolysis on ARF6, is the second molecular partner that turns over the GDP/GTP cycle of ARF6 during cell stimulation. Western blot and immunofluorescence experiments indicated that GIT1 is cytosolic in resting cells but is recruited to the plasma membrane in stimulated cells, where it co-localizes with ARF6 at the granule docking sites. Over-expression of wild-type GIT1 inhibits growth hormone secretion from PC12 cells; this inhibitory effect was not observed in cells expressing a GIT1 mutant impaired in its ARF-GTPase-activating protein (GAP) activity or in cells expressing other ARF6-GAPs. Conversely reduction of GIT1 by RNA interference increased the exocytotic activity. Using a real time assay for individual chromaffin cells, we found that microinjection of GIT1 strongly reduced the number of exocytotic events. These results provide the first evidence that GIT1 plays a function in calcium-regulated exocytosis in neuroendocrine cells. We propose that GIT1 represents part of the pathway that inactivates ARF6-dependent reactions and thereby negatively regulates and/or terminates exocytotic release.

ADP-Ribosylation Factor 6↗

PubMed2006

Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis.

Phospholipase D (PLD) produces phosphatidic acid (PA), an established intracellular signalling lipid that has been also implicated in vesicular trafficking, and as such, PLD could play multiple roles during phagocytosis. Using an RNA interference strategy, we show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis in murine macrophages, in line with results obtained with wild-type constructs and catalytically inactive PLD mutants which, respectively, enhance and inhibit phagocytosis. Furthermore, we found that PA is transiently produced at sites of phagosome formation. Macrophage PLD1 and PLD2 differ in their subcellular distributions. PLD1 is associated with cytoplasmic vesicles, identified as a late endosomal/lysosomal compartment, whereas PLD2 localizes at the plasma membrane. In living cells undergoing phagocytosis, PLD1 vesicles are recruited to nascent and internalized phagosomes, whereas PLD2 is only observed on nascent phagosomes. These results provide evidence that both PLD isoforms are required for phagosome formation, but only PLD1 seems to be implicated in later stages of phagocytosis occurring after phagosomal internalization.

Animals↗

PubMed2005

Role of phosphoinositide signaling in the control of insulin exocytosis.

Phosphoinositides (PI) are important signaling molecules involved in the regulation of vesicular trafficking. We found that phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2)] increase the secretory response triggered by 10 mum Ca(2+) in streptolysin-O-permeabilized insulin-secreting INS-1E cells. In addition, nutrient-induced exocytosis was diminished in intact cells expressing constructs that sequester PI(4,5)P(2) and in cells transfected with constructs that reduce by RNA interference the level of two enzymes involved in PI(4,5)P(2) production, type III PI4-kinase beta and type I phosphatidylinositol 4-bisphosphate 5-kinase-gamma. To clarify the mechanism of action of PI, we investigated the involvement in the regulation of insulin exocytosis of three potential PI targets, phospholipase D1, the Ca(2+)-dependent activator protein for secretion 1, and Munc18-interacting protein 1. Transfection of insulin-secreting cells with plasmids that direct the synthesis of small interfering RNAs capable of reducing the endogenous levels of these proteins inhibited hormone release elicited by glucose- and cAMP-elevating agents without affecting basal release. Our data indicate that the production of PI(4,5)P(2) is necessary for proper control of beta-cell secretion and suggest that at least part of the effect of PI on insulin exocytosis could be exerted through the activation of phospholipase D1, Ca(2+)-dependent activator protein for secretion 1, and Munc18-interacting protein 1.

1-Phosphatidylinositol 4-Kinase↗

PubMed2005

The Small GTPase RalA controls exocytosis of large dense core secretory granules by interacting with ARF6-dependent phospholipase D1.

RalA and RalB constitute a family of highly similar Ras-related GTPases widely distributed in different tissues. Recently, active forms of Ral proteins have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. Since RalA is present on the plasma membrane in neuroendocrine chromaffin and PC12 cells, we investigated the potential role of RalA in calcium-regulated exocytotic secretion. We show here that endogenous RalA is activated during exocytosis. Expression of the constitutively active RalA (G23V) mutant enhances secretagogue-evoked secretion from PC12 cells. Conversely, expression of the constitutively inactive GDP-bound RalA (G26A) or silencing of the RalA gene by RNA interference led to a strong impairment of the exocytotic response. RalA was found to co-localize with phospholipase D1 (PLD1) at the plasma membrane in PC12 cells. We demonstrate that cell stimulation triggers a direct interaction between RalA and ARF6-activated PLD1. Moreover, reduction of endogenous RalA expression level interfered with the activation of PLD1 observed in secretagogue-stimulated cells. Finally, using various RalA mutants selectively impaired in their ability to activate downstream effectors, we show that PLD1 activation is essential for the activation of secretion by GTP-loaded RalA. Together, these results provide evidence that RalA is a positive regulator of calcium-evoked exocytosis of large dense core secretory granules and suggest that stimulation of PLD1 and consequent changes in plasma membrane phospholipid composition is the major function RalA undertakes in calcium-regulated exocytosis.

ADP-Ribosylation Factor 6↗

PubMed2005

Thyrotropin receptor trafficking relies on the hScrib-betaPIX-GIT1-ARF6 pathway.

G protein-coupled receptors are regulated by ligand stimulation, endocytosis, degradation of recycling to the cell surface. Little information is available on the molecular mechanisms underlying G protein-coupled receptors recycling. We have investigated recycling of the G protein-coupled thyroid stimulating hormone receptor (TSHR) and found that it relies on hScrib, a membrane-associated PDZ protein. hScrib directly binds to TSHR, inhibits basal receptor endocytosis and promotes recycling, and thus TSHR signalling, at the cell membrane. We previously demonstrated that hScrib is associated with a betaPIX-GIT1 complex comprised of a guanine nucleotide exchange factor and a GTPase-activating protein for ADP ribosylation factors that is involved in vesicle trafficking. We used dominant-negative constructs and small interfering RNA to show that TSHR recycling is regulated by the interaction between hScrib and betaPIX, and by the activity of GIT1. In addition, ARF6, a major target for GIT1, is activated during TSH stimulation of HEK293 and FRTL-5 thyroid cells, and plays a key role in TSHR recycling. Thus, we have uncovered an hScrib-betaPIX-GIT1-ARF6 pathway devoted to TSHR trafficking and function.

ADP-Ribosylation Factor 6↗

PubMed2005

Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles.

Annexin 2 is a calcium-dependent phospholipid-binding protein that has been implicated in a number of membrane-related events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.

Actins↗

PubMed2005

[The GIT-PIX protein complex: a hub to ARF and Rac/Cdc42 GTPases].

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.

Cell Cycle Proteins↗

PubMed2004

Coupling actin and membrane dynamics during calcium-regulated exocytosis: a role for Rho and ARF GTPases.

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 and furious" machinery for secretion remain poorly understood. ARF and Rho GTPases play a central role in coupling actin dynamics to membrane trafficking events in eukaryotic cells. Here, we review the role of Rho and ARF GTPases in supplying actin and lipid structures required for synaptic vesicle and secretory granule exocytosis. Their possible functional interplay may provide the molecular cues for efficient and localized exocytotic fusion.

ADP-Ribosylation Factors↗

PubMed2004

Mammalian Scribble forms a tight complex with the betaPIX exchange factor.

Drosophila Scribble is implicated in the development of normal synapse structure and epithelial tissues, but it remains unclear how it plays a role and which process it controls. The mammalian homolog of Scribble, hScrib, has a primary structure and subcellular localization similar to that of its fly homolog, but its function remains unknown. Here we have used tandem mass spectrometry to identify major components of the hScrib network. We show that it includes betaPIX (also called Cool-1), a guanine nucleotide exchange factor (GEF), and its partner GIT1 (also called p95-APP1), a GTPase activating protein (GAP). betaPIX directly binds to the hScrib PDZ domains, and the hScrib/betaPIX complex is efficiently recovered in epithelial and neuronal cells and tissues. In cerebellar granule cell cultures, hScrib and betaPIX are both partially localized at neuronal presynaptic compartments. Furthermore, we show that hScrib is required to anchor betaPIX at the cell cortex and that dominant-negative betaPIX or hScrib proteins can each inhibit Ca2+-dependent exocytosis in neuroendocrine PC12 cells, demonstrating a functional relationship between these proteins. These data reveal the existence of a tight hScrib/betaPIX interaction and suggest that this complex potentially plays a role in neuronal transmission.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Regulation of phospholipase D1 subcellular cycling through coordination of multiple membrane association motifs.

The signaling enzyme phospholipase D1 (PLD1) facilitates membrane vesicle trafficking. Here, we explore how PLD1 subcellular localization is regulated via Phox homology (PX) and pleckstrin homology (PH) domains and a PI4,5P2-binding site critical for its activation. PLD1 localized to perinuclear endosomes and Golgi in COS-7 cells, but on cellular stimulation, translocated to the plasma membrane in an activity-facilitated manner and then returned to the endosomes. The PI4,5P2-interacting site sufficed to mediate outward translocation and association with the plasma membrane. However, in the absence of PX and PH domains, PLD1 was unable to return efficiently to the endosomes. The PX and PH domains appear to facilitate internalization at different steps. The PH domain drives PLD1 entry into lipid rafts, which we show to be a step critical for internalization. In contrast, the PX domain appears to mediate binding to PI5P, a lipid newly recognized to accumulate in endocytosing vesicles. Finally, we show that the PH domain-dependent translocation step, but not the PX domain, is required for PLD1 to function in regulated exocytosis in PC12 cells. We propose that PLD1 localization and function involves regulated and continual cycling through a succession of subcellular sites, mediated by successive combinations of membrane association interactions.

Amino Acid Motifs↗

PubMed2003

Regulation of exocytosis in adrenal chromaffin cells: focus on ARF and Rho GTPases.

Neurons and neuroendocrine cells release transmitters and hormones by exocytosis, a highly regulated process in which secretory vesicles or granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Several stages have been recognized in exocytosis. After recruitment and docking at the plasma membrane, vesicles/granules enter a priming step, which is then followed by the fusion process. Cortical actin remodelling accompanies the exocytotic reaction, but the links between actin dynamics and trafficking events remain poorly understood. Here, we review the action of Rho and ADP-ribosylation factor (ARF) GTPases within the exocytotic pathway in adrenal chromaffin cells. Rho proteins are well known for their pivotal role in regulating the actin cytoskeleton. ARFs were originally identified as regulators of vesicle transport within cells. The possible interplay between these two families of GTPases and their downstream effectors provides novel insights into the mechanisms that govern exocytosis.

ADP-Ribosylation Factors↗

PubMed2002

Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane.

The ADP ribosylation factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore.

ADP-Ribosylation Factor 6↗

PubMed2002

Exocytosis: the chromaffin cell as a model system.

Neurons and neuroendocrine cells release transmitters and hormones by exocytosis of secrctory vesicles or granules. Among the cell models that have provided insight into the molecular machinery underlying the successive steps of exocytosis, adrenal chromaffin cells have taken a prominent place. Thus, most of the molecular players that orchestrate the formation, targeting, docking, and fusion of secrctory granules have been identified in chromaffin cells. By offering the opportunity to combine the use of recent biophysical techniques allowing single-vesicle resolution and specific biochemical modifications in the protein machinery involved in exocytosis, chromaffn cells remain a powerful model to address new and still open questions in the field of secretion.

Actins↗

PubMed2002

Regulated secretion in chromaffin cells: an essential role for ARF6-regulated phospholipase D in the late stages of exocytosis.

ARFs constitute a family of structurally related proteins that forms a subset of the ras GTPases. In chromaffin cells, secretagogue-evoked stimulation triggers the rapid translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD in the plasma membrane. Both PLD activation and catecholamine secretion are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6. ARNO, a potential guanine nucleotide exchange factor for ARF6, is expressed and localized in the plasma membrane of chromaffin cells. Using permeabilized cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits both PLD activation and catecholamine secretion. Chromaffin cells express PLD1 at the plasma membrane. We found that microinjection of the catalytically inactive PLD1(K898R) dramatically reduces catecholamine secretion monitored by amperometry, most likely by interfering with a late postdocking step of calcium-regulated exocytosis. We propose that ARNO-ARF6 participate in the exocytotic reaction by controlling the plasma membrane-bound PLD1. By generating fusogenic lipids at the exocytotic sites, PLD1 may represent an essential component of the fusion machinery in neuroendocrine cells.

ADP-Ribosylation Factor 6↗

PubMedDate not supplied

In vivo studies of the MagScrew total artificial heart in calves.

The purpose of this study was to evaluate the in vivo pump performance of our total artificial heart (TAH), the "MagScrew TAH." The TAH consists of a blood pump and control logic developed at the Cleveland Clinic and the MagScrew actuator and electronic control system developed by Foster-Miller Technologies, Inc. (Albany, NY). MagScrew TAH implantation was performed in two calves. Study durations were 50 and 5 days. The causes of termination were prosthetic valve endocarditis in one case and cable failure in the other. Mean left pump flow ranged from 8.0 to 9.7 L/min, with left atrial pressure of 3.0 to 16.0 mm Hg. Preload sensitivity of the MagScrew TAH demonstrated a Frank-Starling response to preload in automatic mode. The relationship between right and left atrial pressure was well balanced. Mean arterial pressure and mean pulmonary artery pressure were maintained within physiologic ranges over study duration. There were no signs of bleeding, hemolysis, or organ failure. The MagScrew TAH showed physiologic pump performance, and hemodynamics were well maintained without any organ failure. Further development testing will bring the MagScrew TAH to the point of preclinical readiness testing.

Animals↗