Human trafficking | EPFL Graph Search

Human trafficking is the trade of humans for the purpose of forced labour, sexual slavery, or commercial sexual exploitation. Human trafficking can occur within a country or trans-nationally. It is distinct from people smuggling, which is characterized by the consent of the person being smuggled. Human trafficking is condemned as a violation of human rights by international conventions, but legal protection varies globally. The practice has millions of victims around the world. Definition The UN Protocol to Prevent, Suppress and Punish Trafficking in Persons, especially Women and Children, which has 117 signatories and 173 parties, defines human trafficking as: (a) [...] the recruitment, transportation, transfer, harbouring or receipt of persons, by means of threat or use of force or other forms of coercion, of abduction, of fraud, of deception, of the abuse of power or of a position of vulnerability or of the giving or receiving of payments or benefits to achieve the co

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

Valérie Jacquier, Horst Pick, Jean-Manuel Segura, Horst Vogel

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 approximately 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.

2006

Functional Characterization of the Mouse Olfactory Receptor mOR256-17 in Live Cells

Thamani Dahoun

Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system to recognize and discriminate a nearly unlimited number of structurally diverse odorant molecules. The molecular basis of OR-mediated odor signal detection and transduction is poorly understood. This is due in part to difficulties in functional expression of ORs at high yield, preventing structural and biophysical studies at the level of the receptor protein. Here we report on the heterologous expression of the mouse receptor mOR256-17 yielding an average of 106 ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression we employed different fluorescent tags. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and posttranslational fluorescence labeling of a 12 amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. The establishment of a mOR256-17-inducible HEK293S-TetO cell line allowed us to determine by fluorescence spectrofluorometry and fluorescence correlation spectroscopy (FCS) the optimal conditions to solubilize the receptor. By screening a large odorant compound library we discovered a new selective spectrum of potent mOR256-17 specific agonists. Furthermore, by using a reporter assay that couples both Gs and Gq proteins after inhibition of adenylate cyclase by the chemical adenylyl cyclase inhibitors 9-(tetrahydro-2-furanyl)-9H-purin-6amine (SQ22536) and after the inhibition of phospholipase C by the chemical inhibitor 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), we identified one agonist which induced the inositol 1,4,5-trisphosphate (IP3) transduction pathway. We provided further evidence of the involvement of this second messenger (i) by employing another reporter gene specific to the Gq pathway and (ii) by using electrical impedance spectroscopy. Our attempt to create a FRET fluorophore pair system based on fluorescein arsenical hairpin (FlAsH) labeling and on carrier protein tags to study the receptor conformational changes during activation revealed that modifications in mOR256-17 sequences perturbs the correct receptor folding and prohibits its expression at the plasma membrane. Our study indicated that an essential prerequisite for achieving cell surface expression of mOR256-17 in heterologous cells is the co-expression of the accessory protein RTP1 or its short version RTP1S, which are also expressed by olfactory sensory neurons. Elsewhere, Saito and co-workers have proposed that this protein interacts with OR when the OR is expressed heterologously, but they have not provided evidence to prove the role of RTP1 towards OR. To investigate whether direct physical interactions between mOR256-17 and RTP1 occurs during receptor trafficking to the cell membrane, we labeled mOR256-17 and RTP1 orthogonally using the acyl carrier protein (ACP) and the peptidyl carrier protein (PCP) respectively. In addition, cyan fluorescent protein fused to the N-terminus of RTP1 enabled to study RTP1 biosynthesis in presence and absence of the mOR256-17; here we observed that a large portion of RTP1 remains in the endoplasmic reticulum while the mOR256-17 reaches the Golgi apparatus when it is co-expressed with RTP1S. Furthermore, by using two different RTP1S chimera, we could show that the cytosolic part of RTP1S is important for the mOR256-17 folding and trafficking.

EPFL2013

Intracellular trafficking of interleukin-1 receptor I requires Tollip

Harald Hirling, Michel Kropf, Etienne Meylan

Interleukin-1 receptor (IL-1RI) is a master regulator of inflammation and innate immunity. When triggered by IL-1beta, IL-1RI aggregates with IL-1R-associated protein (IL-1RAcP) and forms a membrane proximal signalosome that potently activates downstream signaling cascades. IL-1beta also rapidly triggers endocytosis of IL-1RI. Although internalization of IL-1RI significantly impacts signaling, very little is known about trafficking of IL-1RI and therefore about precisely how endocytosis modulates the overall cellular response to IL-1beta. Upon internalization, activated receptors are often sorted through endosomes and delivered to lysosomes for degradation. This is a highly regulated process that requires ubiquitination of cargo proteins as well as protein-sorting complexes that specifically recognize ubiquitinated cargo. Here, we show that IL-1beta induces ubiquitination of IL-1RI and that via these attached ubiquitin groups, IL-1RI interacts with the ubiquitin-binding protein Tollip. By using an assay to follow trafficking of IL-1RI from the cell surface to late endosomes and lysosomes, we demonstrate that Tollip is required for sorting of IL-1RI at late endosomes. In Tollip-deficient cells and cells expressing only mutated Tollip (incapable of binding IL-1RI and ubiquitin), IL-1RI accumulates on late endosomes and is not efficiently degraded. Furthermore, we show that IL-1RI interacts with Tom1, an ubiquitin-, clathrin-, and Tollip-binding protein, and that Tom1 knockdown also results in the accumulation of IL-1RI at late endosomes. Our findings suggest that Tollip functions as an endosomal adaptor linking IL-1RI, via Tom1, to the endosomal degradation machinery.

Elsevier2006

Functional Characterization of the Mouse Olfactory Receptor mOR256-17 in Live Cells

Thamani Dahoun

EPFL2013