Marie-France Bader

Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells

Phospholipase D (PLD) has been proposed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. We recently described the activation of an ADP ribosylation factor‐regulated PLD at the plasma membrane of chromaffin cells undergoing secretagogue‐stimulated exocytosis. We show here that the isoform involved is PLD1b, and, using a real‐time assay for individual cells, that PLD activation and exocytosis are closely correlated. Moreover, overexpressed PLD1, but not PLD2, increases stimulated exocytosis in a phosphatidylinositol 4,5‐bisphosphate‐dependent manner, whereas catalytically inactive PLD1 inhibits it. These results provide the first direct evidence that PLD1 is an important component of the exocytotic machinery in neuroendocrine cells.

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.

A role for phospholipase D1 in neurotransmitter release

Phosphatidic acid produced by phospholipase D (PLD) as a result of signaling activity is thought to play a role in membrane vesicle trafficking, either as an intracellular messenger or as a cone-shaped lipid that promotes membrane fusion. We recently described that, in neuroendocrine cells, plasma membrane-associated PLD1 operates at a stage of Ca2+-dependent exocytosis subsequent to cytoskeletal-mediated recruitment of secretory granules to exocytotic sites. We show here that PLD1 also plays a crucial role in neurotransmitter release. Using purified rat brain synaptosomes subjected to hypotonic lysis and centrifugation, we found that PLD1 is associated with the particulate fraction containing the plasma membrane. Immunostaining of rat cerebellar granule cells confirmed localization of PLD1 at the neuronal plasma membrane in zones specialized for neurotransmitter release (axonal neurites, varicosities, and growth cone-like structures). To determine the potential involvement of PLD1 in neurotransmitter release, we microinjected catalytically inactive PLD1(K898R) into Aplysia neurons and analyzed its effects on evoked acetylcholine (ACh) release. PLD1(K898R) produced a fast and potent dose-dependent inhibition of ACh release. By analyzing paired-pulse facilitation and postsynaptic responses evoked by high-frequency stimulations, we found that the exocytotic inhibition caused by PLD1(K898R) was not the result of an alteration in stimulus-secretion coupling or in vesicular trafficking. Analysis of the fluctuations in amplitude of the postsynaptic responses revealed that the PLD1(K898R) blocked ACh release by reducing the number of active presynaptic-releasing sites. Our results provide evidence that PLD1 plays a major role in neurotransmission, most likely by controlling the fusogenic status of presynaptic release sites.

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.

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.

Chromogranin a triggers a phenotypic transformation and the generation of nitric oxide in brain microglial cells

Chromogranin A is an ubiquitous 48,000 mol. wt secretory protein stored and released from many neuroendocrine cells and neurons. In human brain, chromogranin A is a common feature of regions that are known to be affected by various neurodegenerative pathologies such as Alzheimers, Parkinsons and Picks diseases. Brain degenerative areas are generally infiltrated by activated microglial cells, the resident macrophage cell population within the central nervous system. Here, we report that both recombinant human chromogranin A and chromogranin A purified from bovine chromaffin granules trigger drastic morphological changes in rat microglial cells maintained in culture. Microglial cells exposed to chromogranin A adopted a flattened amoeboid shape and, this change was associated with an accumulation of actin in the subplasmalemmal region, as observed by immunocytochemistry and confocal laser microscopy. In single microglial cells loaded with indo-1, chromogranin A elicited a rapid and transient increase in [Ca2+]i which preceded the reorganization of actin cytoskeleton. The activity of nitric oxide synthase was estimated by measuring the accumulation of nitrite in the culture medium. Both recombinant human chromogranin A and bovine chromogranin A triggered an important accumulation of nitrite comparable to that induced by lipopolysaccharide, a well-known activator of microglia. The effect of chromogranin A was dose dependent, inhibited by N omega-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase, and by cycloheximide, an inhibitor of protein synthesis. These findings suggest that chromogranin A induces an activated phenotype of microglia, and thus may have a role in the pathogenesis of neuronal degeneration in the brain.

Intersectin-1L nucleotide exchange factor regulates secretory granule exocytosis by activating Cdc42

Rho GTPases are key regulators of the actin cytoskeleton in membrane trafficking events. We previously reported that Cdc42 facilitates exocytosis in neuroendocrine cells by stimulating actin assembly at docking sites for secretory granules. These findings raise the question of the mechanism activating Cdc42 in exocytosis. The neuronal guanine nucleotide exchange factor, intersectin‐1L, which specifically activates Cdc42 and is at an interface between membrane trafficking and actin dynamics, appears as an ideal candidate to fulfill this function. Using PC12 and chromaffin cells, we now show the presence of intersectin‐1 at exocytotic sites. Moreover, through an RNA interference strategy coupled with expression of various constructs encoding the guanine nucleotide exchange domain, we demonstrate that intersectin‐1L is an essential component of the exocytotic machinery. Silencing of intersectin‐1 prevents secretagogue‐induced activation of Cdc42 revealing intersectin‐1L as the factor integrating Cdc42 activation to the exocytotic pathway. Our results extend the current role of intersectin‐1L in endocytosis to a function in exocytosis and support the idea that intersectin‐1L is an adaptor that coordinates exo–endocytotic membrane trafficking in secretory cells.

Phospholipase D in calcium-regulated exocytosis: lessons from chromaffin cells.

Membrane fusion remains one of the less well-understood processes in cell biology. A variety of mechanisms have been proposed to explain how the generation of fusogenic lipids at sites of exocytosis facilitates secretion in mammalian cells. Over the last decade, chromaffin cells have served as an important cellular model to demonstrate a key role for phospholipase D1 (PLD1) generated phosphatidic acid in regulated exocytosis. The current model proposes that phosphatidic acid plays a biophysical role, generating a negative curvature and thus promoting fusion of secretory vesicles with the plasma membrane. Moreover, multiple signaling pathways converging on PLD1 regulation have been unraveled in chromaffin cells, suggesting a complex level of regulation dependant on the physiological context.

Phospholipase D1 is specifically required for regulated secretion of von Willebrand factor from endothelial cells

Endothelial cells regulate thrombosis, hemostasis, and inflammatory responses by supplying the vasculature with several factors that include procoagulant von Willebrand factor (VWF) and fibrinolytic tissue-type plasminogen activator (tPA). Both proteins can be secreted in a Ca(2+)-regulated manner after endothelial activation but exhibit opposing physiologic effects. In search for factors that could modulate endothelial responses by selectively affecting the secretion of procoagulant or anticoagulant proteins, we identify here phospholipase D1 (PLD1) as a specific regulator of VWF secretion. PLD1 is translocated to the plasma membrane upon stimulation of endothelial secretion, and this process correlates with the generation of phosphatidic acid (PA) in the plasma membrane. Histamine-evoked secretion of VWF, but not tPA, is inhibited by blocking PLD-mediated production of PA, and this effect can be attributed to PLD1 and not PLD2. Thus, different mechanisms appear to control the agonist-induced secretion of VWF and tPA, with only the former requiring PLD1.

Regulated Secretion in Chromaffin Cells

Abstract: 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.