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

Abstract

Despite the importance of trafficking for regulating G proteincoupled 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 pulselabeling 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. Singlemolecule 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 Ϸ190 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.cell signaling ͉ G protein-coupled receptors ͉ single-particle tracking ͉ in vivo protein labeling ͉ fluorescence imaging T he sensation of smell is mediated by a specific family of olfactory G protein-coupled receptors (GPCRs), which recognize small volatile molecules (1). Although odorant receptors (ORs) account for the largest mammalian gene family, comprising up to 1,000 members, the mechanism of signal recognition and amplification in olfactory transduction remains elusive (2). This is partly because classical methods for OR detection, based on immunocytochemistry (3, 4) or genetic fusion to GFP (5), have not permitted the simultaneous live-cell imaging of olfactory processes at the cell membrane and in cytoplasmic compartments.Comprehensive functional studies on ORs in a native cellular environment, such as isolated olfactory sensory neurons, adenovirus-infected olfactory epithelia, or genetically engineered animals, are often hampered by practical limitations: (i) olfactory sensory neurons are difficult to maintain in primary culture (6), (ii) virus-mediated gene transfer does not consistently yield functional OR expression (7), and (iii) creating transgenic animals for each OR would be quite expensive. Functional expression of ORs in heterologous cells is, therefore, an important approach for elucidating the molecular mechanism of olfaction and helps to identify specific ligands ...

What do diffusion measurements tell us about membrane compartmentalisation? Emergence of the role of interprotein interactions

“…Moreover, in different systems using different tracking techniques, similar observations were made [50][51][52][53]. Frick et al [54] have recently given additional evidence supporting the previous interpretation by using FRAP on four different proteins.…”

“…As a consequence, assuming that the number of proteins within an assembly is constant, both the microscopic and macroscopic diffusion coefficients remain proportional to L 2 [15]. 40 J Chem Biol (2008) 1:37-48 Moreover, in different systems using different tracking techniques, similar observations were made [50][51][52][53]. Frick et al [54] have recently given additional evidence supporting the previous interpretation by using FRAP on four different proteins.…”

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

“…The dynamics of MHC II-peptide complexes containing fluorescent peptides have been assessed by single molecule microscopy, a technique that provides unprecedented detailed and accurate information on molecular movements on living cells (11,(27)(28)(29)(30). For tracking experiments, we used fluorescent derivatives of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 (SYIPSAEKI).…”

Ligand signature in the membrane dynamics of single TrkA receptor molecules

“…To date, however, the specific influence of different ligands binding the same type of receptor was poorly investigated, and, to our knowledge, limited to a comparison between agonist and antagonist ligands (Jacquier et al, 2006). Here, we investigated TrkA lateral diffusion induced by the addition and consequent binding of four agonist ligands: NGF, 'painless' mutant NGFR100E, proNGF and NT-3 (Figs 2-4).…”