Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.
Subscribe to Journal
Get full journal access for 1 year
only $13.33 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Cho, W. H. & Stahelin, R. V. Membrane–protein interactions in cell signaling and membrane trafficking. Annu. Rev. Biophys. Biomol. Struc. 34, 119–151 (2005).
Marinissen, M. J. & Gutkind, J. S. G-protein-coupled receptors and signaling networks: emerging paradigms. Trend. Pharm. Sci. 22, 368–376 (2001).
Hopkins, A. L. & Groom, C. R. The druggable genome. Nature Rev. Drug Disc. 1, 727–730 (2002).
Adams, G. P. & Weiner, L. M. Monoclonal antibody therapy of cancer. Nature Biotech. 23, 1147–1157 (2005).
Rees, D. C., Congreve, M., Murray, C. W. & Carr, R. Fragment-based lead discovery. Nature Rev. Drug Disc. 3, 660–672 (2004).
Salamon, Z., Macleod, H. A. & Tollin, G. Surface plasmon resonance spectroscopy as a tool for investigating the biochemical and biophysical properties of membrane protein systems. 2. Applications to biological systems. Biochim. Biophys. Acta 1331, 131–152 (1997).
Lee, A. G. How lipids affect the activities of integral membrane proteins. Biochim. Biophys. Acta 1666, 62–87 (2004).
Fruh, V., Ijzerman, A. P. & Siegal, G. How to catch a membrane protein in action: a review of functional membrane protein immobilization strategies and their applications. Chem. Rev. 111, 640–656 (2011).
Bally, M. et al. Liposome and lipid bilayer arrays towards biosensing applications. Small 6, 2481–2497 (2010).
Holden, M. A. et al. Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording. Nature Chem. Bio. 2, 314–318 (2006).
Dykstra, M. et al. Location is everything: lipid rafts and immune cell signaling. Annu. Rev. Immun. 21, 457–481 (2003).
Sato, T. K., Overduin, M. & Emr, S. D. Location, location, location: membrane targeting directed by PX domains. Science 294, 1881–1885 (2001).
Li, G. Y., Xi, N. & Wang, D. H. Probing membrane proteins using atomic force microscopy. J. Cell. Biochem. 97, 1191–1197 (2006).
Muller, D. J. & Engel, A. Atomic force microscopy and spectroscopy of native membrane proteins. Nature Protocols 2, 2191–2197 (2007).
Groves, J. T., Parthasarathy, R. & Forstner, M. B. Fluorescence imaging of membrane dynamics. Annu. Rev. Biomed. Eng. 10, 311–338 (2008).
Schwarzenbacher, M. et al. Micropatterning for quantitative analysis of protein–protein interactions in living cells. Nature Methods 5, 1053–1060 (2008).
Johnson, A. E. Fluorescence approaches for determining protein conformations, interactions and mechanisms at membranes. Traffic 6, 1078–1092 (2005).
Wallrabe, H. & Periasamy, A. Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotech. 16, 19–27 (2005).
Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774 (2001).
Wang, W. et al. Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy. Nature Chem. 3, 249–255 (2011).
Huang, B., Yu, F. & Zare, R. N. Surface plasmon resonance imaging using a high numerical aperture microscope objective. Anal. Chem. 79, 2979–2983 (2007).
Kadurin, I. et al. Differential effects of N-glycans on surface expression suggest structural differences between the acid-sensing ion channel (ASIC) 1a and ASIC1b. Biochem. J. 412, 469–475 (2008).
Dell, A. & Morris, H. R. Glycoprotein structure determination mass spectrometry. Science 291, 2351–2356 (2001).
Durand, G. & Seta, N. Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring. Clin. Chem. 46, 795–805 (2000).
Liu S. L. et al. Visualizing the endocytic and exocytic processes of wheat germ agglutinin by quantum dot-based single-particle tracking. Biomaterials 32, 7616–7624 (2011).
Vila-Perello, M., Gallego, R. G. & Andreu, D. A simple approach to well-defined sugar-coated surfaces for interaction studies. ChemBioChem 6, 1831–1838 (2005).
Gingell, D., Todd, I. & Bailey, J. Topography of cell–glass apposition revealed by total internal reflection fluorescence of volume markers. J. Cell Biol. 100, 1334–1338 (1985).
Sato, Y. et al. High mannose-binding lectin with preference for the cluster of 1-2-mannose from the green alga Boodlea coacta is a potent entry inhibitor of HIV-1 and influenza viruses. J. Biol. Chem. 286, 19446–19458 (2011).
Katrlik, J., Skrabana, R., Mislovicova, D. & Gemeiner, P. Binding of D-mannose-containing glycoproteins to D-mannose-specific lectins studied by surface plasmon resonance. Colloid Surf. A 382, 198–202 (2011).
Rathanaswami, P., Babcook, J. & Gallo, M. High-affinity binding measurements of antibodies to cell-surface-expressed antigens. Anal. Biochem. 373, 52–60 (2008).
Troise, F. et al. Differential binding of human immunoagents and herceptin to the ErbB2 receptor. FEBS J. 275, 4967–4979 (2008).
Lehmann, S. et al. An endogenous lectin and one of its neuronal glycoprotein ligands are involved in contact guidance of neuron migration. Proc. Natl Acad. Sci. USA 87, 6455–6459 (1990).
Zieske, J. D., Higashijima, S. C. & Gipson, I. K. Con A-binding and WGA-binding glycoproteins of stationary and migratory corneal epithelium. Invest. Ophthalmol. Vis. Sci. 27, 1205–1210 (1986).
Huppa, J. B. et al. TCR–peptide–MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967 (2010).
Kataoka, M. & Tavassoli, M. Polarization of membrane-glycoproteins during monocyte chemotaxis. Exp. Cell Res. 153, 539–543 (1984).
Russo, V. C. et al. Insulin-like growth factor binding protein-2 binding to extracellular matrix plays a critical role in neuroblastoma cell proliferation, migration, and invasion. Endocrinology 146, 4445–4455 (2005).
Albuquerque, E. X., Pereira, E. F. R., Alkondon, M. & Rogers, S. W. Mammalian nicotinic acetylcholine receptors: from structure to function. Physiol. Rev. 89, 73–120 (2009).
Eaton, J. B. et al. Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells. Mol. Pharmacol. 64, 1283–1294 (2003).
DeFazio-Eli, L. et al. Quantitative assays for the measurement of HER1–HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action. Breast Cancer Res. 13, R44 (2011).
Manz, B. N. & Groves, J. T. Spatial organization and signal transduction at intercellular junctions. Nature Rev. Mol. Cell Biol. 11, 342–352 (2010).
Pick, H. et al. Monitoring expression and clustering of the ionotropic 5HT3 receptor in plasma membranes of live biological cells. Biochemistry 42, 877–884 (2003).
We thank the National Institutes of Health (R21RR026235) for support.
The authors declare no competing financial interests.
About this article
Cite this article
Wang, W., Yang, Y., Wang, S. et al. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells. Nature Chem 4, 846–853 (2012). https://doi.org/10.1038/nchem.1434
Correlating Surface Plasmon Resonance Microscopy of Living and Fixated Cells with Electron Microscopy Allows for Investigation of Potential Preparation Artifacts
Advanced Materials Interfaces (2020)
Non-Invasive Plasmonic-Based Real-Time Characterization of Cardiac Drugs on Cardiomyocytes Functional Behavior
Analytical Chemistry (2020)
Label-Free Imaging of Single Nanoparticles Using Total Internal Reflection-Based Leakage Radiation Microscopy
Angewandte Chemie (2020)
Measuring Stepwise Binding of Thermally Fluctuating Particles to Cell Membranes without Fluorescence
Biophysical Journal (2020)