Tracking receptor motions at the plasma membrane reveals distinct effects of ligands on CCR5 dynamics depending on its dimerization status.

G-protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses type 1 (HIV-1). We used TIRF microscopy and a statistical method to track and classify the motion of different receptor subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on ß-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.

The interplay between α7 nicotinic acetylcholine receptors, pannexin-1 channels and P2X7 receptors elicit exocytosis in chromaffin cells.

Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.

αII-spectrin controls calcium-regulated exocytosis in neuroendocrine chromaffin cells through neuronal Wiskott-Aldrich Syndrome protein interaction.

Besides a fundamental structural role at the plasma membrane, spectrin- and actin-based skeletons have been proposed to participate in various processes including vesicular trafficking. Neuroendocrine cells release hormones and neuropeptides through calcium-regulated exocytosis, a process that is coordinated by a fine remodeling of the actin cytoskeleton. We describe here that calcium-regulated exocytosis is impaired in chromaffin and PC12 cells with reduced αII-spectrin expression levels. Using yeast two-hybrid screening, we show that neuronal Wiskott-Aldrich Syndrome protein (N-WASP) is a partner of the αII-spectrin SH3 domain and demonstrate that secretagogue-evoked N-WASP recruitment at cell periphery is blocked in the absence of αII-spectrin. Additionally, experiments performed with ectopically expressed αII-spectrin mutant unable to bind N-WASP indicated that the interaction between SH3 domain and N-WASP is pivotal for neuroendocrine secretion. Our results extend the list of spectrin interactors and strengthen the idea that αII-spectrin is an important scaffold protein that gathers crucial actin-related players of the exocytic machinery.

CCR5 adopts three homodimeric conformations that control cell surface delivery.

Biophysical methods and x-ray crystallography have revealed that class A G protein-coupled receptors (GPCRs) can form homodimers. We combined computational approaches with receptor cross-linking, energy transfer, and a newly developed functional export assay to characterize the residues involved in the dimerization interfaces of the chemokine receptor CCR5, the major co-receptor for HIV-1 entry into cells. We provide evidence of three distinct CCR5 dimeric organizations, involving residues of transmembrane helix 5. Two dimeric states corresponded to unliganded receptors, whereas the binding of the inverse agonist maraviroc stabilized a third state. We found that CCR5 dimerization was required for targeting the receptor to the plasma membrane. These data suggest that dimerization contributes to the conformational diversity of inactive class A GPCRs and may provide new opportunities to investigate the cellular entry of HIV-1 and mechanisms for its inhibition.

The F-Actin Binding Protein Cortactin Regulates the Dynamics of the Exocytotic Fusion Pore through its SH3 Domain.

Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells.

Pannexin 1 channels: new actors in the regulation of catecholamine release from adrenal chromaffin cells.

Chromaffin cells of the adrenal gland medulla synthesize and store hormones and peptides, which are released into the blood circulation in response to stress. Among them, adrenaline is critical for the fight-or-flight response. This neurosecretory process is highly regulated and depends on cytosolic [Ca(2+)]. By forming channels at the plasma membrane, pannexin-1 (Panx1) is a protein involved in many physiological and pathological processes amplifying ATP release and/or Ca(2+) signals. Here, we show that Panx1 is expressed in the adrenal gland where it plays a role by regulating the release of catecholamines. In fact, inhibitors of Panx1 channels, such as carbenoxolone (Cbx) and probenecid, reduced the secretory activity induced with the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP, 50 µM) in whole adrenal glands. A similar inhibitory effect was observed in single chromaffin cells using Cbx or (10)Panx1 peptide, another Panx1 channel inhibitors. Given that the secretory response depends on cytosolic [Ca(2+)] and Panx1 channels are permeable to Ca(2+), we studied the possible implication of Panx1 channels in the Ca(2+) signaling occurring during the secretory process. In support of this possibility, Panx1 channel inhibitors significantly reduced the Ca(2+) signals evoked by DMPP in single chromaffin cells. However, the Ca(2+) signals induced by caffeine in the absence of extracellular Ca(2+) was not affected by Panx1 channel inhibitors, suggesting that this mechanism does not involve Ca(2+) release from the endoplasmic reticulum. Conversely, Panx1 inhibitors significantly blocked the DMPP-induce dye uptake, supporting the idea that Panx1 forms functional channels at the plasma membrane. These findings indicate that Panx1 channels participate in the control the Ca(2+) signal that triggers the secretory response of adrenal chromaffin cells. This mechanism could have physiological implications during the response to stress.

Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles.

Vesicle fusion with the plasma membrane generates an Ω-shaped membrane profile. Its pore is thought to dilate until flattening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and stimulated emission depletion microscopy imaging of dense-core vesicles, we find that fusion-generated Ω-profiles may enlarge or shrink while maintaining vesicular membrane proteins. Closure of fusion-generated Ω-profiles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within ~1-30 s. Strong calcium influx triggers dynamin-mediated closure. Weak calcium influx does not promote closure, but facilitates the merging of Ω-profiles with the plasma membrane via shrinking rather than full-collapse. These results establish a model, termed Ω-exo-endocytosis, in which the fusion-generated Ω-profile may shrink to merge with the plasma membrane, change in size or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles.

Dynamin-2 in nervous system disorders.

Dynamin-2 is a pleiotropic GTPase whose best-known function is related to membrane scission during vesicle budding from the plasma or Golgi membranes. In the nervous system, dynamin-2 participates in synaptic vesicle recycling, post-synaptic receptor internalization, neurosecretion, and neuronal process extension. Some of these functions are shared with the other two dynamin isoforms. However, the involvement of dynamin-2 in neurological illnesses points to a critical function of this isoform in the nervous system. In this regard, mutations in the dynamin-2 gene results in two congenital neuromuscular disorders. One of them, Charcot-Marie-Tooth disease, affects myelination and peripheral nerve conduction, whereas the other, Centronuclear Myopathy, is characterized by a progressive and generalized atrophy of skeletal muscles, yet it is also associated with abnormalities in the nervous system. Furthermore, single nucleotide polymorphisms located in the dynamin-2 gene have been associated with sporadic Alzheimer's disease. In the present review, we discuss the pathogenic mechanisms implicated in these neurological disorders.

Dynamin-2 function and dysfunction along the secretory pathway.

Dynamin-2 is a ubiquitously expressed mechano-GTPase involved in different stages of the secretory pathway. Its most well-known function relates to the scission of nascent vesicles from the plasma membrane during endocytosis; however, it also participates in the formation of new vesicles from the Golgi network, vesicle trafficking, fusion processes and in the regulation of microtubule, and actin cytoskeleton dynamics. Over the last 8 years, more than 20 mutations in the dynamin-2 gene have been associated to two hereditary neuromuscular disorders: Charcot-Marie-Tooth neuropathy and centronuclear myopathy. Most of these mutations are grouped in the pleckstrin homology domain; however, there are no common mutations associated with both disorders, suggesting that they differently impact on dynamin-2 function in diverse tissues. In this review, we discuss the impact of these disease-related mutations on dynamin-2 function during vesicle trafficking and endocytotic processes.

Dynamin-2 regulates fusion pore expansion and quantal release through a mechanism that involves actin dynamics in neuroendocrine chromaffin cells.

Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin's ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.

Phospholipid scramblase-1-induced lipid reorganization regulates compensatory endocytosis in neuroendocrine cells.

Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1⁻/⁻ mice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis.

How important are Rho GTPases in neurosecretion?

Rho GTPases are small GTP binding proteins belonging to the Ras superfamily which act as molecular switches that regulate many cellular function including cell morphology, cell to cell interaction, cell migration and adhesion. In neuronal cells, Rho GTPases have been proposed to regulate neuronal development and synaptic plasticity. However, the role of Rho GTPases in neurosecretion is poorly documented. In this review, we discuss data that highlight the importance of Rho GTPases and their regulators into the control of neurotransmitter and hormone release in neurons and neuroendocrine cells, respectively.

Selective recapture of secretory granule components after full collapse exocytosis in neuroendocrine chromaffin cells.

In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-and-run, cavicapture and full-collapse fusion. During kiss-and-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain. Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-ß-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane.

betaPIX-activated Rac1 stimulates the activation of phospholipase D, which is associated with exocytosis in neuroendocrine cells.

Rho GTPases are crucial regulators of actin cytoskeletal rearrangements and play important roles in many cell functions linked to membrane trafficking processes. In neuroendocrine cells, we have previously demonstrated that RhoA and Cdc42 mediate part of the actin remodelling and vesicular trafficking events that are required for the release of hormones by exocytosis. Here, we investigate the functional importance of Rac1 for the exocytotic reaction and dissect the downstream and upstream molecular events that might integrate it to the exocytotic machinery. Using PC12 cells, we found that Rac1 is associated with the plasma membrane and is activated during exocytosis. Silencing of Rac1 by siRNA inhibits hormone release, prevents secretagogue (high K(+))-evoked phospholipase D1 (PLD1) activation and blocks the formation of phosphatidic acid at the plasma membrane. We identify betaPix as the guanine nucleotide-exchange factor integrating Rac1 activation to PLD1 and the exocytotic process. Finally, we show that the presence of the scaffolding protein Scrib at the plasma membrane is essential for betaPix/Rac1-mediated PLD1 activation and exocytosis. As PLD1 has recently emerged as a promoter of membrane fusion in various exocytotic events, our results define a novel molecular pathway linking a Rho GTPase, Rac1, to the final stages of Ca(2+)-regulated exocytosis in neuroendocrine cells.

Calcium-regulated exocytosis in neuroendocrine cells: intersectin-1L stimulates actin polymerization and exocytosis by activating Cdc42.

Actin cytoskeleton remodeling is a critical step of regulated exocytosis in many secretory cell types, including neuroendocrine cells. While the classical model considers the cortical actin network as a physical barrier preventing the uncontrolled recruitment of secretory granules to the plasma membrane docking sites, recent evidence supports the idea that actin polymerization also plays a more active role in the late stages of exocytosis. However, the molecular machinery underlying this positive function of actin in the course of exocytosis remains largely unknown. Here, we propose that the neuronal guanine nucleotide exchange factor, intersectin-1L, activates the GTPase Cdc42, which in turn provides de novo actin filaments that are important for calcium-regulated exocytosis in PC12 cells.