Jean-Manuel Segura

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of ≈190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.

CD8+ Cytotoxic T Lymphocyte Activation by Soluble Major Histocompatibility Complex-Peptide Dimers

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10–30-Å) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (≥80 Å) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.

Covalent labeling of cell-surface proteins for in-vivo FRET studies

Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor–acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell‐surface neurokinin‐1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.

Post-translational covalent labeling reveals heterogeneous mobility of individual G protein-coupled receptors in living cells

Keywords: Fluorescent probes ; Membrane proteins ; Neurokinin-1 receptor ; Signal transduction ; Single-molecule studies Reference LCPPM-ARTICLE-2007-003doi:10.1002/cbic.200500477View record in Web of Science Record created on 2007-04-22, modified on 2017-05-12

Increased Mobility of Major Histocompatibility Complex I-Peptide Complexes Decreases the Sensitivity of Antigen Recognition

CD8+ cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2Kd molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.

Reversible Sequential-Binding Probe Receptor–Ligand Interactions in Single Cells

With the reversible sequential (ReSeq) binding assay,we present a novel approach for the ultrasensitive profiling of receptor function in single living cells. This assay is based on the repetitive application of fluorescent ligands that have fast association–dissociation kinetics. We chose the nicotinic‐acetylcholine receptor (nAChR) as a prototypical example and performed ReSeq equilibrium, kinetic, and competition‐binding assays using fluorescent derivatives of the antagonist α‐conotoxin GI (α‐CnTx). Thereby, we determined the binding constants of unlabeled α‐CnTx and d‐tubocurarine. The high selectivity of α‐CnTx for muscle‐type nAChR made it possible to observe specific binding even in the presence of other nAChR subtypes. Imaging of individual nAChRs and ligand‐binding cycles to single cells in microfluidic devices demonstrated the ultimate miniaturization and accuracy of ReSeq‐binding assays even at low receptor‐expression levels. We expect our approach to be of generic importance for functional screening of compounds or membrane receptors, and for the detailed characterization of rare primary cells.

Acetylcholine Receptor Organization in Membrane Domains in Muscle Cells: EVIDENCE FOR RAPSYN-INDEPENDENT AND RAPSYN-DEPENDENT MECHANISMS

Nicotinic acetylcholine receptors (nAChR) in muscle fibers are densely packed in the postsynaptic region at the neuromuscular junction. Rapsyn plays a central role in directing and clustering nAChR during cellular differentiation and neuromuscular junction formation; however, it has not been demonstrated whether rapsyn is the only cause of receptor immobilization. Here, we used single-molecule tracking methods to investigate nAChR mobility in plasma membranes of myoblast cells during their differentiation to myotubes in the presence and absence of rapsyn. We found that in myoblasts the majority of nAChR were immobile and that ∼20% of the receptors showed restricted diffusion in small domains of ∼50 nm. In myoblasts devoid of rapsyn, the fraction of mobile nAChR was considerably increased, accompanied by a 3-fold decrease in the immobile population of nAChR with respect to rapsyn-expressing cells. Half of the mobile receptors were confined to domains of ∼120 nm. Measurements performed in heterologously transfected HEK cells confirmed the direct immobilization of nAChR by rapsyn. However, irrespective of the presence of rapsyn, about one-third of nAChR were confined in 300-nm domains. Our results show (i) that rapsyn efficiently immobilizes nAChR independently of other postsynaptic scaffold components; (ii) nAChR is constrained in confined membrane domains independently of rapsyn; and (iii) in the presence of rapsyn, the size of these domains is strongly reduced.