Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot.

HIV-1 Tat is efficiently secreted by HIV-1-infected or Tat-transfected cells. Accordingly, Tat concentrations in the nanomolar range have been measured in the sera of HIV-1-infected patients, and this protein acts as a viral toxin on bystander cells. Nevertheless, assaying Tat concentration in media or sera is not that straightforward because extracellular Tat is unstable and particularly sensitive to oxidation. Moreover, most anti-Tat antibodies display limited affinity. Here, we describe methods to quantify extracellular Tat using a sandwich ELISA or Western blotting when Tat is secreted by suspension or adherent cells, respectively. In both cases it is important to capture exported Tat using antibodies before any Tat oxidation occurs; otherwise it will become denatured and unreactive toward antibodies.

HIV-1 Tat protein perturbs diacylglycerol production at the plasma membrane of neurosecretory cells during exocytosis.

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.

Exocytosis and endocytosis in neuroendocrine cells: inseparable membranes!

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.

Ephrin B1 maintains apical adhesion of neural progenitors.

Apical neural progenitors are polarized cells for which the apical membrane is the site of cell-cell and cell-extracellular matrix adhesion events that are essential for maintaining the integrity of the developing neuroepithelium. Apical adhesion is important for several aspects of the nervous system development, including morphogenesis and neurogenesis, yet the mechanisms underlying its regulation remain poorly understood. Here, we show that ephrin B1, a cell surface protein that engages in cell signaling upon binding cognate Eph receptors, controls normal morphogenesis of the developing cortex. Efnb1-deficient embryos exhibit morphological alterations of the neuroepithelium that correlate with neural tube closure defects. Using loss-of-function experiments by ex vivo electroporation, we demonstrate that ephrin B1 is required in apical progenitors (APs) to maintain their apical adhesion. Mechanistically, we show that ephrin B1 controls cell-ECM adhesion by promoting apical localization of integrin ß1 and we identify ADP-ribosylation factor 6 (Arf6) as an important effector of ephrin B1 reverse signaling in apical adhesion of APs. Our results provide evidence for an important role for ephrin B1 in maintaining the structural integrity of the developing cortex and highlight the importance of tightly controlling apical cell-ECM adhesion for neuroepithelial development.

HIV-1 Tat protein inhibits neurosecretion by binding to phosphatidylinositol 4,5-bisphosphate.

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.

Genetically encoded probes for phosphatidic acid.

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 267.4 macrophages. Interestingly, the two probes show a differential distribution in these cell types, indicating that they may have different affinities for PA or recognize different pools of PA. In conclusion, the development of a broader choice of probes may be required to adequately follow the complex dynamics of PA in different cell types, in order to determine the cellular distribution of PA and its role in various cellular processes.

In vivo delta opioid receptor internalization controls behavioral effects of agonists.

BACKGROUND: GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal. METHODS AND FINDINGS: In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking. CONCLUSIONS: Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs.

ARF6 regulates the synthesis of fusogenic lipids for calcium-regulated exocytosis in neuroendocrine cells.

An important role for specific lipids in membrane fusion has recently emerged, but regulation of their biosynthesis remains poorly understood. Among fusogenic lipids, phosphatidic acid and phosphoinositol 4,5-bisphosphate (PIP(2)) have been proposed to act at various steps of neurotransmitter and hormone exocytosis. Using real time FRET (fluorescence resonance energy transfer) measurements, we show here that the GTPase ARF6, potentially involved in the synthesis of these lipids, is activated at the exocytotic sites in PC12 cells stimulated for secretion. Depletion of endogenous ARF6 by siRNA dramatically inhibited secretagogue-evoked exocytosis. ARF6-siRNA greatly reduced secretagogue-evoked phospholipase D (PLD) activation and phosphatidic acid formation at the plasma membrane and moderately reduced constitutive levels of PIP(2) present at the plasma membrane in resting cells. Expression of an ARF6 insensitive to short interference RNA (siRNA) fully rescued secretion in ARF6-depleted cells. However, a mutated ARF6 protein specifically impaired in its ability to stimulate PLD had no effect. Finally, we show that the ARF6-siRNA-mediated inhibition of exocytosis could be rescued by an exogenous addition of lysophosphatidylcholine, a lipid that favors negative curvature on the inner leaflet of the plasma membrane. Altogether these data indicate that ARF6 is a critical upstream signaling element in the activation of PLD necessary to produce the fusogenic lipids required for exocytosis.

SNARE-catalyzed fusion events are regulated by Syntaxin1A-lipid interactions.

Membrane fusion is a process that intimately involves both proteins and lipids. Although the SNARE proteins, which ultimately overcome the energy barrier for fusion, have been extensively studied, regulation of the energy barrier itself, determined by specific membrane lipids, has been largely overlooked. Our findings reveal a novel function for SNARE proteins in reducing the energy barrier for fusion, by directly binding and sequestering fusogenic lipids to sites of fusion. We demonstrate a specific interaction between Syntaxin1A and the fusogenic lipid phosphatidic acid, in addition to multiple polyphosphoinositide lipids, and define a polybasic juxtamembrane region within Syntaxin1A as its lipid-binding domain. In PC-12 cells, Syntaxin1A mutations that progressively reduced lipid binding resulted in a progressive reduction in evoked secretion. Moreover, amperometric analysis of fusion events driven by a lipid-binding-deficient Syntaxin1A mutant (5RK/A) demonstrated alterations in fusion pore dynamics, suggestive of an energetic defect in secretion. Overexpression of the phosphatidic acid-generating enzyme, phospholipase D1, completely rescued the secretory defect seen with the 5RK/A mutant. Moreover, knockdown of phospholipase D1 activity drastically reduced control secretion, while leaving 5RK/A-mediated secretion relatively unaffected. Altogether, these data suggest that Syntaxin1A-lipid interactions are a critical determinant of the energetics of SNARE-catalyzed fusion events.

Knockin mice expressing fluorescent delta-opioid receptors uncover G protein-coupled receptor dynamics in vivo.

The combination of fluorescent genetically encoded proteins with mouse engineering provides a fascinating means to study dynamic biological processes in mammals. At present, green fluorescent protein (GFP) mice were mainly developed to study gene expression patterns or cell morphology and migration. Here we used enhanced GFP (EGFP) to achieve functional imaging of a G protein-coupled receptor (GPCR) in vivo. We created mice where the delta-opioid receptor (DOR) is replaced by an active DOR-EGFP fusion. Confocal imaging revealed detailed receptor neuroanatomy throughout the nervous system of knock-in mice. Real-time imaging in primary neurons allowed dynamic visualization of drug-induced receptor trafficking. In DOR-EGFP animals, drug treatment triggered receptor endocytosis that correlated with the behavioral response. Mice with internalized receptors were insensitive to subsequent agonist administration, providing evidence that receptor sequestration limits drug efficacy in vivo. Direct receptor visualization in mice is a unique approach to receptor biology and drug design.

Proteomic consequences of a human mitochondrial tRNA mutation beyond the frame of mitochondrial translation.

Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.

Viability, adhesion, and bone phenotype of osteoblast-like cells on polyelectrolyte multilayer films.

The aim of this study was to develop new biocompatible coatings for bone implants by the alternating deposition of oppositely charged polyelectrolytes. Polyelectrolyte films were built up with different terminating layers on which SaOS-2 osteoblast-like cells and human periodontal ligament (PDL) cells were grown. The terminating layer was made of one of the following polyelectrolytes: poly(ethylene imine) (PEI), poly(sodium 4-styrenesulfonate) (PSS), poly(allylamine hydrochloride) (PAH), poly(L-glutamic acid) (PGA), or poly(L-lysine) (PLL). Cell adherence, viability, stability of osteoblast phenotype, and inflammatory response were studied. Adherence and viability were good on all terminating layers except the PEI-terminating layer, which was cytotoxic. Maintenance of osteoblast phenotype marker expression was observed on PSS- and PGA-terminating films for both cell types, whereas downregulation, associated with the induction of Interleukin-8 (IL-8) secretion, was detected on PEI and PAH for both cell types and on PLL for PDL cells. These results suggested a good biocompatibility of PSS- and PGA-ending films for PDL cells and of PSS-, PGA-, and PLL-terminating films for SaOS-2 cells. As a result, polyelectrolyte multilayer films could emerge as new alternatives for implant coatings.