Search

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20 °C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 ± 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 ± 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with β-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30–35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.

The monitoring of the movements of membrane proteins (or lipids) by single-particle tracking enables one to obtain reliable insights into the complex dynamic organization of the plasma membrane constituents. Using this technique, we investigated the diffusional behaviour of a G-protein-coupled receptor. The trajectories of the receptors revealed a diffusion mode combining a short-term rapid confined diffusion with a long-term slow diffusion. A detailed statistical analysis shows that the receptors have a diffusion confined to a domain which itself diffuses, the confinement being due to long-range attractive inter-protein interactions. The existing models of the dynamic organization of the cell membrane cannot explain our results. We propose a theoretical Brownian model of interacting proteins that is consistent with the experimental observations and accounts for the variations found as a function of the domain size of the short-term and long-term diffusion coefficients.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent mitogenic factor which can replace the growth promoting activity of basic fibroblast growth factor (bFGF) on bovine aortic endothelial cells. However, TPA-treated cells lose their strict contact inhibition at confluence, which is a characteristic of cells grown in the presence of bFGF. We have examined whether these changes could be related to modifications of the transbilayer and lateral motions of fluorescent lipids, namely 1-acyl-2-[6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-p hosphatidylcholine (C6-NBD-PC), -phosphatidylserine (C6-NBD-PS), and -phosphatidylethanolamine (C6-NBD-PE) inserted in the outer leaflet of the cell plasma membrane. In TPA-treated cells, the three fluorescent phospholipids remained located in the outer leaflet for at least 1 h at 20 degrees C after their insertion, indicating a blockade of the aminophospholipid translocase activity which is normally present in the plasma membrane of bFGF-treated cells. TPA also induced a large increase in the percentage of C6-NBD-PC and C6-NBD-PE probes which were free to diffuse laterally. The mobile fractions M reached values of approximately 100% for the two lipids, while for bFGF-treated cells they were found around 85 and 75%, respectively. For the C6-NBD-PS probe, M remained unchanged in bFGF and TPA-treated cells, at around 85%. TPA treatment also induced a twofold increase in the lateral diffusion coefficients of C6-NBD-PC and C6-NBD-PE, while that of C6-NBD-PS remained nearly unchanged. These effects of TPA may be related to the observed loss of differentiated properties of vascular endothelial cells and not to its mitogenic properties.

To study the sequential expression of insect epidermal cells during metamorphosis, a library of monoclonal antibodies (MABs) was prepared against the water-soluble proteins from preecdysial pupal cuticle of Tenebrio molitor. Six selected MABs recognizing only larval and pupal cuticular proteins (CPs) in immunoblot analysis were classified into three types. Type 1 recognized a 21.5 and a 22 kDa polypeptide, type 2, a 26 kDa polypeptide, and type 3, three polypeptides of 18.5, 19.5 and 21.5 kDa. They did not immunoreact with any protein of fat bodies or haemolymph from pharate pupae, suggesting that the antigens originate from the epidermis. The stage-specificity was confirmed by electron microscopic immunogold labelling. Type 1 and 3 MABs recognized antigens characterizing larval and pupal preecdysial sclerotized cuticles, while the antigens recognized by type 2 were localized in the first few lamellae of unsclerotized postecdysial cuticle. When the expression of the adult programme was inhibited by application of a juvenile hormone analogue, the larval-/pupal-specific CPs were detected in the supernumerary pupal cuticle. These results suggest that the genes encoding these proteins are juvenile hormone dependent. These MABs should be useful tools to isolate pupal-specific genes whose regulation sems to be different from that of the adult-specific ones.

A library of monoclonal antibodies (Mabs) against adult cuticle of Tenebrio was used to visualize the secretion of cuticular antigens during metamorphosis. Immunoblots of water- and urea-soluble proteins, and high resolution immunogold labelling has shown that, except in one clone, the Mabs recognize antigens in the three developmental stages. However, the MW of larval and pupal antigens are different from the adult ones, though sharing common epitopes. Blots of cuticle proteins (CPs) bound to different lectins shown few water-soluble glycosylated proteins weakly or not recognized by the Mabs, suggesting that the majority of the Mabs do not recognize glycosylated epitopes. The immunolocalization of the different antigens suggests a molecular basis for both developmental and regional variations in cuticular architecture and to the modifications due to sclerotization, which differ between pre- and postecdysial cuticles of the three developmental stages.

We synthesized 3beta-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): K(DOPC)K(POPC)K(DMPC)K(DMPC/15 mol % Chol)K(DMPC/30 mol % Chol). This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I(1)-I(5)) and excimer (I(E)) emission bands. In model membranes, I(1)/I(3) and I(E)/I(3) ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse delta-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse delta-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.

G-protein-coupled receptor function involves interactions between the receptor, G-proteins and effectors in the cell plasma membrane. The main biochemical processes have been individually identified but the mechanisms governing the successive protein–protein interactions of this complex multi-molecular machinery have yet to be established. We discuss advances in understanding the functional dynamics of the receptor resulting from diffusion measurements, and in the context of the plasma membrane organization.

Previous single-molecule studies have shown a long-term diffusion superimposed to a short-term confinement of the human mu opioid (hMOP) receptors at the surface of heterologous cells. However, additional ensemble average measurements are required to reach a complete understanding of the undergoing process. Here, we analyse, by fluorescence recovery after photobleaching measurements, the lateral diffusion of fully functional T7-EGFP-hMOP receptors in neuroblastoma SH-SY5Y cells naturally expressing a low level of the wild-type receptor. Experiments carried out at variable observation radii demonstrate the restriction of the receptors diffusion to sub-micrometer sized domains. Furthermore, consistently with the long-term single-molecule data, the domains are found permeable.

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20 degrees C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 +/- 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 +/- 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with beta-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30-35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.

Functionalized submicroscopic particles are currently used to label proteins or lipids at the surface of living cells for single particle tracking experiments. In many cases, it can be of crucial importance for the particle to be anchored to a single molecule. We have addressed this question for the labeling at the plasma membrane of NRK cells of the mu-opioid receptor bearing a T7 epitope at the N-terminus. Using biophysical methods we were able to prepare quasi-monovalent anti-T7 antibody conjugated gold colloids (40 nm diameter) leading to stable and specific binding to the receptor. The rational method, we report here, can be extended to design customized probes for the labeling of various tagged molecules.

International audience; We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro. After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis. Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes. ß

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behavior of the receptors compared with the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semipermeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect, which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor.

Normal newborn rat adrenal cells kept in primary culture up to 2½ months respond to ACTH stimulation and produced corticosteroid hormones and smaller amounts of 20α-reduced progesterone metabolites. Cholesterol from the serum complemented culture medium serves as precursor without further addition of exogenous steroid substrates. A long-term qualitative and quantitative study of individual steroid production under the influence of ACTH was performed. ACTH treatment produced a triphasic effect on steroid production: an induction period (up to 3 days), an acute maximum production period (3rd to 6th day) and a chronic production period (to the end of the treatment). The increase in total steroid production resulted from the increase in the production of corticosteroids only. This indicated an increase of cholesterol side-chain cleavage and of the 21- and 11β/18-steroid hydroxylations. Removal of ACTH led to a reversible drop in total steroid production. The response to ACTH was dose dependent, so that a 2.2 mU/ml dose elicited lower steroid production than the 6.6 or 22 mU/ml doses. Increasing the lower dose after a week of treatment to a higher dose brought total steroid production and 1 1β/18-steroid hydroxylation up to the corresponding chronic production levels. The 20α-steroid reduction system was not affected by ACTH. ACTH changes the importance of the two main steroidogenic pathways. With no ACTH there is approximately a 1:1 ratio between corticosteroid synthesis and progesterone reductive metabolism; a low dose of ACTH increases the total steroid production, but since corticosteroid production and 20α-reduced metabolites both increase, the ratio changes little; a high dose of ACTH increases the ratio to more than 30:1. Refractoriness or desensitization to ACTH is postulated to occur through the control of cholesterol availability inside the cell possibly combined with a control of its utilization for steroidogenesis.

International audience; Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.

International audience; To study the sequential expression of the epidermal program in the mealworm Tenebrio molitor, monoclonal antibodies were prepared against the water-soluble proteins from preecdysial adult cuticle. Among the 16 clones obtained, one of them (named K2F6) recognized a 20-kDa antigen, found only in adult extracts but not in the larval or pupal ones, as revealed by immunoblot analysis. Our results strongly suggest an epidermal origin for this protein. The monoclonal antibody K2F6 fails to react with water-soluble proteins from fat body and hemolymph taken during the deposition of the 20-kDa antigen. Electron microscopic immunogold localization of this antigen showed that it is secreted, just after epicuticle deposition, in the 30 first-deposited preecdysial lamellae of sternal and elytral cuticles only. The sclerotizing process, which modifies the physicochemical properties of these cuticles, does not prevent the immunoreaction. When the expression of the adult program was inhibited by application of a juvenile hormone analog (ZR 515), the water-soluble proteins from different pupal-adult intermediates were never recognized by the monoclonal antibody K2F6 using immunoblot analysis. These results support the conclusion that this 20-kDa antigen is a protein specific for the sclerotized cuticle of the adult stage.