Rapid Hop Diffusion of a G-Protein-Coupled Receptor in the Plasma Membrane as Revealed by Single-Molecule Techniques

Suzuki, Kenichi; Ritchie, Ken; Kajikawa, Eriko; Fujiwara, Takeshi; Kusumi, Akihiro

doi:10.1529/biophysj.104.048538

Cited by 239 publications

(282 citation statements)

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“…Even without ligand, the diffusion of OR17-40 was found to be very heterogeneous, with 12% of the receptors diffusing freely in the plasma membrane, 9% being immobile, 30% diffusing inside small domains, and 49% diffusing inside larger domains. The broad distribution (2 decades) of diffusion coefficients exhibits a single population centered at 0.02 m 2 ͞s, a value that is typical for many transmembrane receptors (16,31,32). Diffusion coefficients of Ϸ0.1 m 2 ͞s, measured by fluorescence recovery after photobleaching, have been reported for several GPCRs (33)(34)(35).…”

Section: Discussionmentioning

confidence: 90%

“…Similar domain sizes have been found for NK2R in HEK293 cells (35): Fluorescence recovery after photobleaching experiments revealed domains of radius ϭ 420 Ϯ 80 nm without ligand and 170 Ϯ 50 nm when agonist was bound. The larger domains could arise from confinement by a membrane-skeleton͞cytoskeleton fence structure (32,36) or from long-range protein interactions (31) and might represent a population average of the two larger compartments found here. Because OR17-40 is constitutively internalizing, the smaller domains are likely to correspond to precursors of clathrincoated pits (35,37).…”

Section: Discussionmentioning

confidence: 99%

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

Jacquier

Prummer

Segura

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

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

Jacquier

Prummer

Segura

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…These data also suggest that CX 3 CL1 is not anchored directly to the actin cytoskeleton. A recent model of membrane fluidity proposed that transmembrane proteins might be fenced into discrete regions of the membrane (53). Such a mechanism might account for the limited lateral mobility of CX 3 CL1.…”

Section: Discussionmentioning

confidence: 99%

Expression and Targeting of CX3CL1 (Fractalkine) in Renal Tubular Epithelial Cells

Durkan¹,

Alexander²,

Liu³

et al. 2007

Journal of the American Society of Nephrology

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The chemokine CX 3 CL1 plays a key role in glomerulonephritis and can act as both chemoattractant and adhesion molecule. CX 3 CL1 also is upregulated in tubulointerstitial injury, but little is known about the subcellular distribution and function of CX 3 CL1 in renal tubular epithelial cells (RTEC). Unexpectedly, it was found that CX 3 CL1 is expressed predominantly on the apical surface of tubular epithelium in human renal transplant biopsy specimens with acute rejection or acute tubular necrosis. For studying the targeting of CX 3 CL1 in polarized RTEC, MDCK cells that expressed untagged or green fluorescent protein-tagged CX 3 CL1 were generated. The chemokine was present on the apical membrane and in subapical vesicles. Apical targeting of CX 3 CL1 was not due to signals that were conferred by its intracellular domain, to associations with lipid rafts, or to O-glycosylation but, rather, depended on N-linked glycosylation of the protein. With the use of fluorescence recovery after photobleaching, it was found that CX 3 CL1 is immobile in the apical membrane. However, CX 3 CL1 partitioned with the triton-soluble rather than -insoluble cellular fraction, indicating that it is not associated directly with the actin cytoskeleton or with lipid rafts. Accordingly, disruption of rafts through cholesterol depletion did not render CX 3 CL1 mobile. For exploration of potential functions of apical CX 3 CL1, binding of CX 3 CR1-expressing leukocytes to polarized RTEC was examined. Leukocyte adhesion to the luminal surface was enhanced significantly when CX 3 CL1 was present. These data demonstrate that CX 3 CL1 is expressed preferentially on the apical membrane of RTEC and suggest a novel function for the chemokine in recruitment and retention of leukocytes in tubulointerstitial inflammation.

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“…25,26 It was previously believed that the size of a transmembrane object could not be deduced from mobility measurements, as the theoretical work of Saffman and Dellbrück 27 predicted that, within a layer of incompressible fluid, the diffusion coefficient should be insensitive to the size of the diffusing object. Consequently, studies of lateral mobility have neglected model systems 28 where little information should be obtained, and focused on living cells [29][30][31] to explore the mechanical barriers hindering the movement of proteins. The validity of the Saffman-Dellbrück (SD) model has never been adequately verified becauce of a lack of systematic measurements.…”

Section: Introductionmentioning

confidence: 99%

Variation of the Lateral Mobility of Transmembrane Peptides with Hydrophobic Mismatch

et al. 2010

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A hydrophobic mismatch between protein length and membrane thickness can lead to a modification of protein conformation, function, and oligomerization. To study the role of hydrophobic mismatch, we have measured the change in mobility of transmembrane peptides possessing a hydrophobic helix of various length d π in lipid membranes of giant vesicles. We also used a model system where the hydrophobic thickness of the bilayers, h, can be tuned at will. We precisely measured the diffusion coefficient of the embedded peptides and gained access to the apparent size of diffusing objects. For bilayers thinner than d π , the diffusion coefficient decreases, and the derived characteristic sizes are larger than the peptide radii. Previous studies suggest that peptides accommodate by tilting. This scenario was confirmed by ATR-FTIR spectroscopy. As the membrane thickness increases, the value of the diffusion coefficient increases to reach a maximum at h ≈ d π . We show that this variation in diffusion coefficient is consistent with a decrease in peptide tilt. To do so, we have derived a relation between the diffusion coefficient and the tilt angle, and we used this relation to derive the peptide tilt from our diffusion measurements. As the membrane thickness increases, the peptides raise (i.e., their tilt is reduced) and reach an upright position and a maximal mobility for h ≈ d π . Using accessibility measurements, we show that when the membrane becomes too thick, the peptide polar heads sink into the interfacial region. Surprisingly, this "pinching" behavior does not hinder the lateral diffusion of the transmembrane peptides. Ultimately, a break in the peptide transmembrane anchorage is observed and is revealed by a "jump" in the D values.

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Rapid Hop Diffusion of a G-Protein-Coupled Receptor in the Plasma Membrane as Revealed by Single-Molecule Techniques

Cited by 239 publications

References 78 publications

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

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

Expression and Targeting of CX3CL1 (Fractalkine) in Renal Tubular Epithelial Cells

Variation of the Lateral Mobility of Transmembrane Peptides with Hydrophobic Mismatch

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